Essential Oils: Extraction Methods and Applications 9781119829355, 1119829356

Table of contents :
Cover
Title Page
Copyright Page
Contents
Preface
Chapter 1 A Methodological Approach of Plant Essential Oils and their Isolated Bioactive Components for Antiviral Activities
1.1 Introduction
1.2 General Chemical Properties and Bioactivity
1.3 Antiviral Mechanisms
1.3.1 Time of Addition Assay
1.3.1.1 Pretreatment of Host Cells
1.3.1.2 Pretreatment of Virions
1.3.1.3 Co-Treatment of Host/Cultured Cells and Virions During Virus Inoculation
1.3.1.4 Post-Entry Treatment
1.3.2 Thermal Shift Assays
1.3.2.1 Viral Attachment Assay
1.3.2.2 Viral Fusion Assay (Entry Assay)
1.3.3 Morphological Study
1.3.4 Protein Inhibition
1.3.5 Other Metabolic Anti-Viral Mechanisms
1.4 Assessment of Antiviral Activities via In Vitro Assays
1.4.1 Determination of Cytotoxicity (Cytopathogenic Reduction Assay)
1.4.2 In Vitro Activities on Different Viruses
1.4.2.1 Human Herpes Virus
1.4.2.2 Influenza Virus
1.4.2.3 Non-Enveloped Viruses
1.4.2.4 Other Viruses
1.5 Activities of Essential Oils in Relation to Their Bioactive Components
1.6 Antiviral Activities as Compared to the Polarity of Bioactive Components
1.7 In Vivo Studies of Essential Oils for its Antiviral Effect
1.7.1 Herpes Simplex Virus
1.7.2 Influenza Virus
1.7.3 West Nile Virus
1.8 Activities In-Respect to the Available Antivirals
1.9 Antiviral Essential Oils and Their Bioactive Components Loaded in Nanosystems
1.10 Conclusion
References
Chapter 2 Essential Oils Used to Inhibit Bacterial Growth in Food
2.1 Introduction
2.2 Chemistry of Essential Oils
2.3 Essential Oils Against Microorganisms in Food Products
2.4 Application of Essential Oils in the Food Industry
2.5 Essential Oil Extraction Techniques
2.6 Conclusions
References
Chapter 3 Industrial Application of Essential Oils
3.1 Introduction
3.2 Essential Oils
3.2.1 Sources and Chemical Composition
3.2.2 Extraction Methods
3.2.2.1 Conventional Extraction Methods
3.2.2.2 Innovative Extraction Methods
3.2.3 Industrial Applications of Essential Oils
3.2.3.1 Food Preservation and Active Packaging Systems
3.2.3.2 Aromatherapy
3.2.3.3 Pharmaceutical and Medicinal Application
3.2.3.4 Biopesticide in Insect Pest Management
Conclusion
Declaration about Copyright
References
Chapter 4 Influence of Biotic and Abiotic Factors on the Production and Composition of Essential Oils
4.1 Introduction
4.2 Essential Oil Characteristics
4.3 Factors Influencing Essential Oils Production and Composition
4.4 Abiotic Factors
4.4.1 Drought
4.4.2 Salinity
4.4.3 Temperature
4.4.4 Light
4.4.5 Nutrients
4.4.6 Heavy Metals
4.5 Biotic Factors
4.6 Concluding Remarks
Acknowledgements
References
Chapter 5 Investigation of Antiviral Effects of Essential Oils
5.1 Introduction
5.2 Viruses: Structure, Characteristics, and Replication
5.3 In Vitro Antiviral Activity and Mechanism of Action Investigations of Essential Oils and Essential Oil Components
5.3.1 Investigation of In Vitro Antiviral Activities
5.3.1.1 Plaque Reduction Assay
5.3.1.2 The Inhibition of Viral Cytopathogenic Effect
5.3.2 Mechanisms of Action
5.3.2.1 Time-of-Drug-Addition Assay
5.3.2.2 Temperature-Shift Assay
5.3.2.3 Morphological Alteration
5.3.2.4 Protein Inhibition
5.3.2.5 Other Mechanisms of Action
5.3.3 Selectivity Index (SI)
5.4 The Antiviral Efficacy of Essential Oils on Viruses Affecting Different Body Systems
5.4.1 Respiratory System
5.4.1.1 Influenza Virus
5.4.1.2 Adenovirus and Rhinovirus
5.4.1.3 Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-COV-1)
5.4.1.4 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2)
5.4.2 GIT System
5.4.2.1 Coxsackie Virus
5.4.2.2 Dengue Virus
5.4.2.3 Yellow Fever Virus
5.4.2.4 Murine Norovirus Type 1
5.4.3 Nervous System
5.4.3.1 West Nile Virus
5.4.4 Immune System
5.4.4.1 HIV
5.4.5 Reproductive System
5.4.5.1 Human Papilloma Virus (HPV)
5.4.6 Other Viruses
5.4.6.1 Human Herpes Virus
5.4.6.2 Orf Virus
5.5 The Antiviral Efficacy of Essential Oils on Phyto-Pathogenic Viruses
5.6 The Antiviral Efficacy of the Essential Oils on Animal.Infecting Viruses
5.6.1 Virus Affecting Cattle (Bovine Viral Diarrhea Virus)
5.6.2 Virus Affecting Cats (Feline Calicivirus F9)
5.6.3 Virus Affecting Pigs (Porcine Parvovirus)
5.7 Synergistic Effect of Essential Oil Components with Known Antiviral Drugs
5.8 Aromatherapy and its Role as an Antiviral Agent
5.9 Route of Essential Oil Administration
5.10 Nano-Formulated Essential Oils: A Promising Approach to Enhance Antiviral Activity
5.11 Safety of Essential Oils
5.12 Antiviral Essential Oils: Drawbacks versus Future Perspectives
5.13 Summary
References
Chapter 6 Mentha sp. Essential Oil and Its Applicability in Brazil
Introduction
6.1 Ethnobotany of the Mentha in Brazil
6.2 Chemical Constituents of Mentha Oil
6.3 Evaluation of Biological Activities of Mentha Essential Oils
6.4 Toxicity of Essential Oils from Mentha Used in Folk Medicine
6.5 Final Considerations and Perspectives
References
Chapter 7 Microbial Influence on Plants for Enhanced Production of Active Secondary Metabolites
7.1 Introduction
7.2 Classes of Plants Secondary Metabolites
7.2.1 Terpenes
7.2.2 Phenolic Compounds
7.2.3 Nitrogen-Containing Secondary Metabolites
7.2.4 Sulphur Containing Secondary Metabolites
7.3 Secondary Metabolites Production from Plants
7.3.1 In Vivo Production of Secondary Metabolites
7.3.2 In Vitro Secondary Metabolites Production
7.4 Interaction of Microorganisms in the Rhizosphere
7.5 Influence of Bacteria and Fungi on Plants
7.5.1 Plant Growth Promoters
7.5.1.1 Plant Growth-Promoting Bacteria (PGPR)
7.5.1.2 Plant Growth-Promoting Fungi (PGPF)
7.5.2 Production of Plant Biomass
7.5.3 Bacteria and Fungus as Biofertilizers
7.5.4 Role of Bacteria and Fungi as a Phytostimulator
7.5.5 Role of Bacteria and Fungi as a Biopesticides
7.5.6 Stress Tolerant Activity of Bacteria and Fungi
Conclusion and Future Perspectives
References
Chapter 8 Valorization of Limonene Over Acid Solid Catalysts
8.1 Introduction
8.2 Limonene Reactions with Alcohols
8.3 Hydration and Acetoxylation
8.4 Conversion of Limonene into p-Cymene
8.5 Conclusions
References
Chapter 9 Elucidating the Role of Essential Oils in Pharmaceutical and Industrial Applications
9.1 Introduction
9.2 Extraction of Volatile Oils from Various Sources
9.2.1 Terpenes
9.2.2 Hydrocarbons
9.3 Role of Essential Oils in Industry
9.3.1 Role in Cosmetics and Aromatherapy
9.3.1.1 Cosmetic Industry
9.3.1.2 Immortelle Essential Oil
9.3.1.3 Lavender Essential Oil
9.3.1.4 German Chamomile Oil
9.3.1.5 Neroli Essential Oil
9.3.1.6 Peppermint Essential Oil
9.3.1.7 Rosemary Essential Oil
9.3.2 Application in Food Industry
9.3.2.1 Food Preservation
9.3.2.2 Food Packaging
9.4 Pharmacological Effects of Essential Oils
9.5 Concluding Remarks
Acknowledgment
References
Chapter 10 Uses of Essential Oils in Different Sectors
10.1 Introduction
10.2 Food and Beverage
10.3 Packaging
10.4 Cosmetic and Perfumery
10.5 Aromatherapy
10.6 Medical
10.7 Agriculture
10.8 Textile
10.9 Cleaning Household
10.10 Safety of Essential Oils
Conclusion
References
Chapter 11 Chemical Composition and Pharmacological Activities of Essential Oils
11.1 Introduction
11.2 Anticancer
11.2.1 Role of Terpenes in Anticancer Activity
11.2.2 Role of Aromatic Compounds in Anticancer Activity
11.2.3 Mode of Action
11.2.4 The Effect of EOs in Different Types of Cancers
11.2.5 Multi-Drug Resistance (MDR)
11.3 Anti-Inflammatory
11.3.1 Terpenoids for Anti-Inflammatory
11.3.2 Phenylpropanoids for Anti-Inflammatory
11.3.3 Role of Essential Oil for Anti-Inflammatory
11.4 Anti-Viral
11.4.1 Terpenoids for Anti-Viral Activity
11.4.2 Essential Oils for Coronavirus
11.4.3 Essential Oil for Anti-Viral Activity
11.5 Anti-Fungal
11.5.1 Mode of Action
11.5.2 Essential Oil for Anti-Fungal Activity
11.6 Antidiabetic
11.7 Larvicidal Activity
11.8 Anti-Bacterial
Conclusion
Conflicts of Interest
Acknowledgements
References
Chapter 12 Augmented Stability and Efficacy of Essential Oils Through Encapsulation Approach
12.1 Introduction
12.2 Various Strategies for Encapsulation of Essential Oils
12.2.1 Essential Oils Encapsulated in Liposomes
12.2.2 Essential Oils Encapsulated in Cyclodextrin Complexes
12.2.3 Essential Oils Encapsulated in Polymeric Complexes
12.2.4 Essential Oils Encapsulated in Electrospun Fibers
12.2.5 Essential Oils Encapsulated in Microemulsion/Nanoemulsions
12.2.6 Essential Oils Encapsulated in Mesoporous Silica Nanoparticles
12.3 Conclusions
References
Chapter 13 Antimicrobial Effect of Essential Oils for Food Application
13.1 Introduction
13.2 Biotechnological Strategies for Extracting Essential Oils for Food Application
13.3 Methods for Evaluating the EO Inhibitory Activity In Vitro
13.3.1 Factors Affecting Method Susceptibility
13.3.2 Resources Used to Improve Halo Diameter Reading in the Agar Diffusion Method
13.4 Influence of Extraction Methods on the Antimicrobial Compounds in Essential Oils
13.5 Inhibition of Bacteria by Essential Oils in Food
13.6 Use of Essential Oils in Packaging or Food Contact Surfaces
13.6.1 Embedded Films of Nanocapsules with EO
13.6.2 Packaging Reinforced with Nano-Incorporations and Added with EO
13.6.3 Bioactive Films Added EO
13.7 Effect of Encapsulation of Essential Oils on the Inhibitory Activity against Bacteria
13.8 Conclusions
References
Chapter 14 Antioxidant or Antimicrobial Nature of Essential Oils to Minimize Food Waste
14.1 Introduction
14.2 Essential Oils Chemical Composition
14.3 Essential Oils: Their Antimicrobial Activity and Mode of Action
14.4 EO Used in Food Packaging
14.5 Application of EO in Different Food Products
14.5.1 Fruits and Vegetables
14.5.2 Meat and Meat Products
14.5.3 Fish
14.5.4 Dairy Products
14.5.5 Bakery Products
14.6 Legal Aspects of the Use of EO in Food
14.7 Conclusion
References
Chapter 15 Application of Essential Oils to Biofilms
15.1 Introduction
15.2 Definition of Biofilm
15.3 Principles of Biofilm Formation
15.4 Benefits of Biofilm to Microorganism
15.5 Mechanisms of Resistance to Antimicrobial Agents
15.6 Global Threat of Biofilms
15.7 Essential Oils
15.8 Antimicrobial and Antibiofilm Effects of EOs
15.9 Antibacterial Mechanism of Action
15.10 Strategies for Improving the Antibiofilm Efficacy of EOs
15.11 Common Methods for Determination of Antimicrobial and Antibiofilm Activities of EOs
15.12 Limitations of EOs Usage
Conclusion
References
Chapter 16 Biological Applications of Essential Oil
16.1 Introduction
16.2 Sources of Essential Oil
16.3 Extraction of Essential Oil
16.4 Phytochemistry of Essential Oil
16.5 Biological Applications
16.5.1 Applications of Essential Oil on the Treatment of Cancer
16.5.2 Applications of Essential Oil on the Treatment of Respiratory Tract Diseases
16.5.3 Applications of Essential Oil on the Treatment of Cardiovascular Diseases
16.5.3.1 Anti-Inflammatory Activity
16.5.4 Applications of Essential Oil on the Treatment of Obesity
16.5.5 Applications of Essential Oil on the Treatment of Diabetes
16.5.5.1 Antioxidant Activity
16.5.6 Applications of Essential Oils Against Infectious Diseases
16.5.6.1 Antibacterial Activity
16.5.6.2 Antifungal Activity
16.5.6.3 Antiviral Activity
16.5.7 Applications of Essential Oil on Dandruff
16.6 Essential Oil Safety Issue
16.7 Conclusion
Acknowledgment
References
Chapter 17 Current Status and Advancement of Biopesticides from Essential Oil for Agriculture, Food Storage, and Household Applications
17.1 Introduction
17.1.1 Essential Oil Extraction
17.2 Application of Essential Oil Biopesticides in Agriculture
17.2.1 Agriculture Pest
17.2.2 Types of Essential Oils for Agricultural Pest Management
17.3 Application of Essential Oil Biopesticides for Food Storage
17.3.1 Food Storage Pests
17.3.2 Types of Essential Oils for Food Storage Pest Management
17.4 Application of Essential Oil Biopesticides for Household Pests
17.4.1 Household Pests
17.4.2 Types of Essential Oils for Household Pest Management
17.5 Delivery of Biopesticides
17.6 Pesticidal Action of Biopesticides
17.7 Conclusion and Constraints
17.8 Acknowledgement
References
Chapter 18 Essential Oil Used as Larvicides and Ovicides
18.1 Introduction
18.2 Important Aspects of Essential Oils
18.3 Larvicides and Ovicides
18.3.1 Larvicides Against Aedes aegypti
18.3.2 Larvicidal Activity Against Anopheles stephensi
18.3.3 Larvicide Against Aedes albopictus
18.3.4 Ovicidal Activity Against Pediculus humanus capitis
18.3.5 Ovicidal Activity Against Haemonchus contortus
18.3.6 Ovicidal Activity Against Helicoverpa armigera Hubner
18.4 Conclusion
References
Chapter 19 Essential Oil-Based Biopesticides
19.1 Introduction
19.2 Phytochemistry and Sources of Essential Oils
19.3 Biological Activity of Essential Oil Biopesticides
19.3.1 Efficacy of Essential Oils to Insects
19.3.2 Essential Oils as Insect Repellents
19.3.3 Bactericidal Properties of Essential Oils
19.3.4 Antifungal and Anti-Oomycete Properties of Essential Oils
19.3.5 Herbicidal/Weedicide Properties of Essential Oils
19.4 Synergistic Formulations of Essential Oils
19.5 Toxic Effects of Essential Oils on Mammals and Non-Target Organisms
19.6 Advantages, Current Constraints and Long-Term Prospects
19.7 Conclusion
References
Chapter 20 Essential Oils Obtained from Algae: Biodiversity and Ecological Importance
20.1 Introduction
20.2 What are Essential Oils?
20.3 Chemical Structure and Biological Activity from Algal Essential Oils
20.4 Ecological Importance of Essential Oils in Marine System
20.5 Conclusion and Future Perspectives
References
Chapter 21 Gas Chromatography-Olfactometry (GC.O) of Essential Oils and Volatile Extracts
21.1 Introduction
21.2 Historical Aspects
21.3 GC-O Methodologies
21.3.1 Detection Frequency Methods
21.3.2 Dilution Analysis
21.3.2.1 Aroma Extraction Dilution Analysis (AEDA)
21.3.2.2 Combined Hedonic Aroma Response Measurements (CHARM Analysis)
21.3.3 Posterior Intensity Methods (PI)
21.3.4 Time-Intensity Methods
21.3.4.1 Odor-Specific Magnitude Estimation (OSME, Direct Intensity)
21.4 Different GC-O Application to Assess for Essential Oilsf Odorants
21.4.1 Citrus spp. (Rutaceae)
21.4.2 Mentha spp. (Lamiaceae)
21.4.3 Thymus spp. (Lamiaceae)
21.4.4 Foeniculum spp. (Apiaceae)
21.4.5 Coriandrum spp. (Apiaceae)
21.4.6 Pinus spp.
21.4.7 GC-O Applied to Characterize Baccharis dracunculifolia DC. Odorants
Acknowledgements
Funding
References
Chapter 22 In Vitro and In Vivo Methods Used to Assess the Biological Potential of Essential Oils
22.1 Introduction
22.2 Chemistry of EOs
22.3 In Vitro Methods Used to Assess the Biological Potential of EOs
22.4 Evaluation of Antioxidant Potential
22.4.1 What are Antioxidants?
22.4.2 Antioxidant Potential of Botanical Materials
22.4.3 Modes of Action
22.4.4 In Vitro Methods for Antioxidant Activities
22.4.5 DPPH Scavenging Assay
22.4.6 2,2-Azinobis-(3-Ethylbenzothiazoline-6-Sulfonate) Assay
22.4.7 Bleachability of â-Carotene in Linoleic Acid System
22.5 Antimicrobial Activities of Essential Oils
22.5.1 Disk Diffusion Assay
22.5.2 Agar Well Diffusion Method
22.5.3 Determination of Minimal Inhibitory Concentration (MIC)
22.6 Essential Oils as Natural Antimicrobial Agents
22.7 Anticancer Activity of Essential Oils
22.8 Cell Culture and Treatment
22.9 Determination of Cell Viability
22.10 Conclusion
Acknowledgement
References
Chapter 23 Biological Potential of Essential Oils: Evaluation Strategies
23.1 Introduction
23.2 Biological Activities of Essential Oils
23.2.1 EOs as Antibacterial Agents
23.2.2 EOs as Antifungal Agents
23.2.3 EOs as Anti-Inflammatory Agents
23.2.4 EOs as Antioxidants
23.2.5 EOs as Anticancer Agents
23.2.6 EOs as Anti-Diabetic Agents
23.2.7 EOs as Antispasmodics
23.3 In Vitro Assessment of Biological Activities
23.3.1 Antimicrobial Assay
23.3.2 Antibacterial Assay
23.3.3 Antifungal Assay
23.3.4 Antioxidant Assay
23.3.5 Anticancer Assay
23.3.6 Anti-Diabetic Assay
23.4 In Vivo Assessment of Biological Activities
23.4.1 Antimicrobial Assay
23.4.2 Antidermatophytic Assay
23.4.3 Antifungal Assay
23.4.4 Anti-Inflammatory Assay
23.4.5 Antioxidant Assay
23.4.6 Anticancer Assay
23.4.7 Anti-Diabetic Assay
23.4.8 Mosquito Repellent Assay
23.5 Conclusion
References
Chapter 24 Algal Essential Oils and Their Importance in the Ecosystem
24.1 Introduction
24.2 Algal Essential Oils
24.3 Factors Affecting Algae Essential Oil Production
24.3.1 Temperature
24.3.2 Light
24.3.3 Nutrients
24.3.4 Chemical Stress
24.4 Ecological Importance of Algal Essential Oils
24.5 Pheromone Properties of Algal Essential Oils
24.6 Algal Essential Oils in “Beach-Odor”
24.7 Algal Essential Oils in “Off-Odor”
24.8 Antibacterial Activities of Algal Essential Oils
24.9 Antifungal Activities of Algal Essential Oils
24.10 Conclusion
References
Chapter 25 Classical Methods for Obtaining Essential Oils
25.1 Introduction
25.2 Classical Methods for Extracting Essential Oils
25.2.1 Maceration
25.2.2 Mechanical Treatment
25.2.3 Hydro Distillation
25.2.4 Water Distillation
25.2.4.1 Steam Distillation
25.2.5 Cold Pressing Method
25.2.6 Solvent Extraction
25.2.7 Soxhlet Extraction
25.3 Chromatographic Technique for Analysis of Essential Oil
Conclusion
Acknowledgement
References
Chapter 26 A Comprehensive Guide to Essential Oil Determination Methods
Abbreviations
26.1 Introduction
26.2 Chemical Composition of EOs
26.2.1 Hydrocarbons Derived from Terpenes
26.2.2 Oxygenated Compounds
26.3 EO and Its Group
26.4 Biological Activity: Pathway Cell
26.4.1 Osmophores
26.4.2 Trichomes
26.5 Classical Methods for Extraction of Essential Oils
26.5.1 Steam Distillation
26.5.2 Hydro-Distillation Method
26.5.3 Steam Explosion Method
26.5.4 Solvent Extraction Method
26.5.5 Cold Press (CP) Method
26.6 Contemporary Extraction Methods
26.6.1 Supercritical Fluid (SCF) Extraction
26.6.2 High Pressure Extraction
26.6.3 Microwave-Assisted Hydro-Distillation
26.6.4 Hydrodistillation with Pretreatment of Enzyme
26.6.5 Microwave-Assisted Steam Distillation
26.6.6 Ultrasound-Assisted Extraction
26.6.7 Solvent-Free Microwave Extraction
26.6.8 Microwave Hydro-Diffusion and Gravity
26.6.9 Oil Extraction by Solar Energy
26.6.10 Pulse Electric Field
26.7 Conclusion
References
Chapter 27 Encapsulation of Essential Oils
27.1 Introduction
27.2 Encapsulation
27.2.1 Chemical
27.2.1.1 Molecular Inclusion Complexation
27.2.1.2 Interfacial Polymerization
27.2.1.3 In Situ Polymerization
27.2.2 Physical-Chemical
27.2.2.1 Coacervation
27.2.2.2 Emulsification
27.2.3 Physical
27.2.3.1 Spray Drying
27.2.3.2 Freeze Drying
27.2.3.3 Electrospraying and Electrospinning
27.2.3.4 Supercritical Technology
27.3 Process Simulation and Economic Evaluation
Concluding Remarks and Prospects
References
Chapter 28 Encapsulated Essential Oils: Main Techniques to Increase Shelf-Life
28.1 Introduction
28.2 Coating Materials
28.3 Techniques for Essential Oil Encapsulation
28.3.1 Coacervation
28.3.2 Extrusion
28.3.3 Nanoprecipitation
28.3.4 Emulsification
28.3.5 Spray Drying
28.3.6 Thin Film Hydration Method
28.3.7 Supercritical Fluid Technology
28.4 Concluding Remarks
Acknowledgment
References
Chapter 29 Encapsulation Technologies of Essential Oils for Various Industrial Applications
29.1 Introduction
29.2 Encapsulation Technique
29.2.1 Essential Oil as the Core Material
29.2.1.1 Chemical Composition and Physical Properties of EOs
29.2.1.2 Biological Activities of EOs
29.2.2 Wall Materials
29.2.3 Encapsulation Method and Release Mechanism
29.2.4 Applications of Encapsulated EOs
29.2.4.1 Preservative in Foods
29.2.4.2 Baked Foods
29.2.4.3 Beverages
29.2.4.4 Fresh Fruit and Vegetables
29.2.4.5 Raw Meat and Meat Products
29.2.4.6 Milk and Dairy Products
29.2.4.7 Cosmetic and Health Care
29.2.4.8 Cotton and Textile
29.2.4.9 Pharmaceutical
29.3 Conclusions
References
Chapter 30 Extraction of Essential Oils with Supercritical Fluid
30.1 Introduction
30.2 Why Use Supercritical Carbon Dioxide to Extract Essential Oils?
30.3 Commercial Equipment Used for Supercritical Fluid Extraction of Essential Oils: Bench and Industrial Scale
30.3.1 Bench Scale
30.3.2 Pilot and Commercial Scale
30.4 Patent Survey
30.5 Economic Evaluation
30.6 Life Cycle Assessment
30.6.1 Goal and Scope Definition
30.6.2 Life Cycle Inventory
30.6.3 Life Cycle Impact Assessment (LCIA)
30.6.4 Interpretation
30.6.5 Recent Studies on LCA of SFE of Essential Oils and the Main Results
30.7 Current Outlook and Prospects
References
Chapter 31 Advantages of Essential Oil Extraction Using Supercritical Fluid: Process Optimization and Effect of Different Processing Parameters on Extraction Efficiency
31.1 Introduction
31.2 Essential Oils
31.3 Supercritical Fluid Extraction
31.4 Superiorities of SFE over Other Extraction Methods
31.5 Extraction of EOs by Supercritical Fluid
31.5.1 Effects of Temperature
31.5.2 Effect of Pressure
31.5.3 Effect of Particle Size
31.5.4 Effect of Flow Rate
31.5.5 Use of a Co-Solvent
31.5.6 Extraction Time
31.6 Antimicrobial and Antioxidant Properties of Essential Oils Extracted via SFE
31.7 Optimization
31.7.1 Optimization Using RSM and BBD, Taguchi Model
31.7.2 Artificial Neural Networks (ANNs)
31.8 Conclusion
References
Chapter 32 Supercritical Fluid Extraction of Essential Oils from Natural Sources: Mathematical Modeling and Applications
32.1 Introduction
32.2 Essential Oils
32.3 Conventional Extraction Methods
32.4 Supercritical Fluid Extraction
32.4.1 Cosolvent Addition
32.5 Typical Behavior and Mathematical Modeling
32.6 Parameters Affecting the CO2-Supercritical Fluid Extraction
32.6.1 Pressure and Temperature
32.6.1.1 Pressure
32.6.1.2 Temperature
32.6.2 Pre-Treatment – Moisture and Particle Size
32.6.3 Extraction Time and Apparent Solubility
32.6.4 Solvent Flow
32.7 Scale-Up and Economic Analysis
32.8 Applications
32.9 Final Considerations
References
Chapter 33 Fundamentals, Mathematical Models, and Extraction Processes with Supercritical Fluids
33.1 Introduction: Background
33.2 Fundamentals of Supercritical Fluid Extraction
33.2.1 Supercritical Fluids
33.2.2 Solubility and Phase Equilibria
33.3 The Extraction Process
33.3.1 Process Scheme
33.3.2 Process Parameters
33.3.2.1 Temperature and Pressure
33.3.2.2 Flow Rate
33.3.3 Mathematical Modeling of the Extraction Process
33.3.4 Scale-Up
33.4 Separation
33.4.1 Extract Recovery Strategies
33.4.2 Essential Oil Fractionation
33.4.3 Solvent Regeneration and Recycling
33.5 Recent Application of Supercritical Extraction of Essential Oils and Industrial Application of Supercritical Fluid Extraction Processes
33.6 Novel and Future Perspectives of Supercritical Fluid Extraction for Essential Oils
33.6.1 Supercritical Fluid Extraction Coupled with Other Green Extraction Technologies
33.6.2 Future Perspectives
References
Chapter 34 Supercritical CO2 Extraction as a Clean Technology Tool for Isolation of Essential Oils
34.1 Introduction
34.2 Essential Oils
34.3 Applications of EOs
34.3.1 Antibacterial and Antifungal Activity
34.3.2 Antioxidant and Anticancer Activity
34.3.3 Antiviral Activity
34.3.4 Food Preservative and Packaging
34.3.5 Aromatherapy
34.3.6 Dairy Products
34.3.7 Biocontrol Agents
34.4 Extraction Methods
34.4.1 Distillation
34.4.2 Cold Pressing/Expression
34.4.3 Hydrodifussion
34.4.4 Solvent Extraction
34.4.5 Microwave-Assisted Extraction (MAE)
34.4.6 Ultrasound-Assisted Extraction (UAE)
34.5 Supercritical Fluid Extraction (SCFE)
34.6 Parameters Influencing SCFE of EOs
34.6.1 Pressure
34.6.2 Temperature
34.6.3 CO2 Flow Rate
34.6.4 Moisture
34.6.5 Cosolvent
34.6.6 Particle Size of Plant Material
34.6.7 Extraction Time
34.7 Optimization of SCFE Process
34.8 Mathematical Modeling of Extraction Curves
34.9 Coupled or Assisted SCFE
34.9.1 Enzyme-Assisted SCFE
34.9.2 Ultrasound-Assisted SCFE
34.10 Conclusion
References
Chapter 35 Classical Techniques for Extracting Essential Oils from Plants
35.1 Introduction
35.2 Market Value of Essential Oils
35.3 Sources of Essential Oils
35.4 Chemical Nature of Essential Oils
35.5 Extraction of Essential Oils
35.5.1 Distillation Methods
35.5.1.1 Hydrodistillation
35.5.1.2 Steam Distillation
35.5.2 Hydrodiffusion
35.5.3 Solvent Extraction
35.5.4 Soxhlet Extraction
35.5.5 Cold Pressing Method/Expression
35.5.6 Cohobation
35.5.7 Enfleurage
35.5.8 Maceration
35.6 Conclusion
References
Chapter 36 Acquisition of Essential Oils Through Traditional Techniques
36.1 Introduction
36.2 Obtaining Essential Oils
36.2.1 Cold Pressing
36.2.2 Steam Distillation
36.2.3 Hydrodistillation
36.2.4 Enfleurage
36.2.5 Solvent Extraction
36.3 Concluding Remarks
Acknowledgment
References
Chapter 37 Essential Oils: Chemical Composition and Methods of Extraction
37.1 Introduction
37.2 Chemical Assemblage of Essential Oils
37.2.1 Terpenes
37.2.1.1 Monoterpenes
37.2.1.2 Sesquiterpenes
37.2.1.3 Diterpenes
37.2.2 Heteroatomic Metabolites
37.2.2.1 Ketones
37.2.2.2 Acids
37.2.2.3 Aldehydes
37.2.2.4 Alcohols
37.2.2.5 Lactones
37.3 Extraction of Essential Oils Key Factors are Involved in Determining the Extraction Method
37.3.1 Conventional Extraction Methods
37.3.1.1 Hydro Distillation
37.3.1.2 Enfleurage Method
37.3.1.3 Hydro Diffusion
37.3.1.4 Cold Pressing
37.3.1.5 Steam Distillation
37.3.2 Green Extraction Methods
37.3.2.1 Supercritical Fluid Extraction (SFE)
37.3.2.2 Microwave-Assisted Extraction (MAE)
37.3.2.3 Ultrasonication-Assisted Extraction (UAE)
37.3.2.4 Conclusion
37.4 Conclusion
References
Chapter 38 Dental Applications of Essential Oils
38.1 Introduction
38.2 Background
38.3 Preparation of Essential Oils
38.4 Mechanism of Action of Essential Oils
38.5 Methods of Application of Essential Oil for Dental Uses
38.6 Therapeutic Actions of Essential Oil for Dental Uses
38.7 Dental/Oral Conditions Treated by Essential Oils
38.8 Dental Applications of Essential Oils
38.9 Safety Issues in Relation to Use of Essential Oils
38.10 Research
38.11 Conclusion
References
Chapter 39 Essential Oil-Based Therapies
39.1 Introduction
39.2 Essential Oil-Rich Plants
39.2.1 Citronella
39.2.2 Peppermint
39.2.3 Lavender
39.2.4 Tea-Tree
39.2.5 Eucalyptus
39.2.6 Chamomile
39.2.7 Patchouli
39.2.8 Ylang-Ylang
39.2.9 Bergamout
39.2.10 Geranium
39.2.11 Lemon
39.3 How Essential Oil Therapy Works
39.3.1 Cosmetic Aromatherapy
39.3.2 Massage Aromatherapy
39.3.3 Medical Aromatherapy
39.3.4 Olfactory Aromatherapy
39.3.5 Psycho-Aromatherapy
39.4 Essential Oil-Based Therapies
39.4.1 Brainstorming Therapies
39.4.1.1 In the Treatment of Dementia
39.4.1.2 Stress Reduces Therapy Among Adolescents
39.4.2 Anti-Microbial Therapy
39.4.3 In Treatment of Eczema
39.4.4 Anti-Hair Fall Therapy
39.4.5 Anti-Tumor Therapy
39.4.6 Chemopreventive Therapy
39.4.7 Coronavirus Therapeutics
39.4.8 Essential Oil Helps in Epilepsy
39.4.9 Treatment of Cardiovascular Disorders
39.5 How to Use EOs?
Conclusion
References
Chapter 40 Clinical Applications of Essential Oils
40.1 Introduction
40.2 Aromatherapy
40.3 Mode of Action of Essential Oils in Aromatherapy
40.4 Classification of Aromatherapy
40.4.1 Cosmetic Aromatherapy
40.4.2 Massage Aromatherapy
40.4.3 Medical Aromatherapy
40.4.4 Olfactory Aromatherapy and Psycho-Aromatherapy
40.5 Essential Oils from Various Parts of the Plants Used in Aromatherapy
40.6 Essential Oil-Based Therapies
40.6.1 Pain and Inflammation
40.6.2 Hemodialysis
40.6.3 Psychological Disorders
40.6.4 Treatment of Nausea and Vomiting
40.6.5 Managing Menopause Symptoms
40.6.6 Treatment of Dermatological Problems
40.7 Safety Issues Related to Essential Oil-Based Therapy
40.8 Conclusion
References
Chapter 41 Therapeutic Role of Essential Oils
41.1 Introduction
41.2 Uses of Essential Oils
41.2.1 Nontherapeutic Uses of EOs
41.2.1.1 Pesticide
41.2.1.2 Food Preservative
41.2.1.3 Cosmetics and Home Care
41.2.1.4 Mosquito Repellent
41.2.1.5 Others
41.3 Classification of Aromatherapy
41.3.1 Cosmetic Aromatherapy
41.3.2 Massage Aromatherapy
41.3.3 Medical Aromatherapy
41.3.4 Olfactory Aromatherapy
41.3.5 Psycho-Aromatherapy
41.4 Role of Essential Oil in Clinical Practice
41.5 Applications of Edible Essential Oil on Therapy
41.5.1 Almond Oil
41.5.2 Avocado Oil
41.5.3 Canola Oil
41.5.4 Coconut Oil
41.5.5 Flaxseed Oil
41.5.6 Groundnut Oil
41.5.7 Sesame Oil
41.5.8 Sunflower Oil
41.6 Risky EOs to Children
41.6.1 Camphor Oil
41.6.2 Wintergreen Oil
41.7 Side Effects of EOs
41.8 Therapeutic Guidelines and Safety Precautions
41.9 Conclusions
Declaration About Copyright
References
Chapter 42 Plant Essential Oils and Their Constituents for Therapeutic Benefits
42.1 Introduction
42.1.1 Concept and Definition
42.1.2 A Journey Through History
42.1.3 Composition
42.2 Biological Activities
42.2.1 Antimicrobial Activities: Mode of Action and Effects
42.2.1.1 Antibacterial Activities
42.2.1.2 Anti-Fungal Activities
42.2.1.3 Anti-Viral Activities
42.2.2 Antioxidant Activities
42.2.3 Antiphlogistic Activity
42.2.4 Anti-Cancer Activities
42.2.5 Miscellaneous Activities
42.2.5.1 Penetration Enhancement
42.2.5.2 EOs in Food
42.2.5.3 Antinociceptive Effects
42.2.5.4 Insect Repellent Activity
42.3 Conclusion
References
Chapter 43 Essential Oils Used in Packaging: Perspectives and Limitations
43.1 Introduction
43.2 Essential Oils: Definition, Preparation, and Composition
43.3 Essential Oils: Medicinal and Biological Functions
43.4 Functional Application of Essential Oils
43.5 Active Packaging Material Based on Essential Oils
43.5.1 Composite and Nanocomposite Materials Based on Essential Oils
43.5.2 Advantages
43.5.3 Limitations
43.6 Conclusion and Future Perspectives
References
Index
EULA

Citation preview

Essential Oils

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

Essential Oils Extraction Methods and Applications

Edited by

Inamuddin

This edition first published 2023 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2023 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www. wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no rep­resentations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchant-­ability or fitness for a particular purpose. No warranty may be created or extended by sales representa­tives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further informa­tion does not mean that the publisher and authors endorse the information or services the organiza­tion, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data ISBN 9781119829355 Front cover image: Pixabay.com Cover design by Russell Richardson Set in size of 11pt and Minion Pro by Manila Typesetting Company, Makati, Philippines Printed in the USA 10 9 8 7 6 5 4 3 2 1

Contents Preface xxvii 1 A Methodological Approach of Plant Essential Oils and their Isolated Bioactive Components for Antiviral Activities Kunal Sharma, Vivek Mishra, Kumar Rakesh Ranjan, Nisha Yadav and Mansi Sharma 1.1 Introduction 1.2 General Chemical Properties and Bioactivity 1.3 Antiviral Mechanisms 1.3.1 Time of Addition Assay 1.3.1.1 Pretreatment of Host Cells 1.3.1.2 Pretreatment of Virions 1.3.1.3 Co-Treatment of Host/Cultured Cells and Virions During Virus Inoculation 1.3.1.4 Post-Entry Treatment 1.3.2 Thermal Shift Assays 1.3.2.1 Viral Attachment Assay 1.3.2.2 Viral Fusion Assay (Entry Assay) 1.3.3 Morphological Study 1.3.4 Protein Inhibition 1.3.5 Other Metabolic Anti-Viral Mechanisms 1.4 Assessment of Antiviral Activities via In Vitro Assays 1.4.1 Determination of Cytotoxicity (Cytopathogenic Reduction Assay) 1.4.2 In Vitro Activities on Different Viruses 1.4.2.1 Human Herpes Virus 1.4.2.2 Influenza Virus 1.4.2.3 Non-Enveloped Viruses 1.4.2.4 Other Viruses 1.5 Activities of Essential Oils in Relation to Their Bioactive Components 1.6 Antiviral Activities as Compared to the Polarity of Bioactive Components 1.7 In Vivo Studies of Essential Oils for its Antiviral Effect 1.7.1 Herpes Simplex Virus 1.7.2 Influenza Virus 1.7.3 West Nile Virus 1.8 Activities In-Respect to the Available Antivirals

1 1 2 3 4 5 5 5 6 6 6 6 6 7 9 9 9 12 12 15 15 16 16 17 18 18 18 20 21

v

vi  Contents 1.9 Antiviral Essential Oils and Their Bioactive Components Loaded in Nanosystems 1.10 Conclusion References

22 23 24

2 Essential Oils Used to Inhibit Bacterial Growth in Food Luiza Helena da Silva Martins, Sabrina Baleixo da Silva, Adilson Ferreira Filho, Andrea Komesu, Johnatt Allan Rocha de Oliveira and Debora Kono Taketa Moreira 2.1 Introduction 2.2 Chemistry of Essential Oils 2.3 Essential Oils Against Microorganisms in Food Products 2.4 Application of Essential Oils in the Food Industry 2.5 Essential Oil Extraction Techniques 2.6 Conclusions References

31

3 Industrial Application of Essential Oils S. Kiruthika and S. Vishali 3.1 Introduction 3.2 Essential Oils 3.2.1 Sources and Chemical Composition 3.2.2 Extraction Methods 3.2.2.1 Conventional Extraction Methods 3.2.2.2 Innovative Extraction Methods 3.2.3 Industrial Applications of Essential Oils 3.2.3.1 Food Preservation and Active Packaging Systems 3.2.3.2 Aromatherapy 3.2.3.3 Pharmaceutical and Medicinal Application 3.2.3.4 Biopesticide in Insect Pest Management Conclusion Declaration about Copyright References

49

4 Influence of Biotic and Abiotic Factors on the Production and Composition of Essential Oils Sandra Gonçalves, Inês Mansinhos and Anabela Romano 4.1 Introduction 4.2 Essential Oil Characteristics 4.3 Factors Influencing Essential Oils Production and Composition 4.4 Abiotic Factors 4.4.1 Drought 4.4.2 Salinity 4.4.3 Temperature 4.4.4 Light 4.4.5 Nutrients 4.4.6 Heavy Metals

31 32 35 37 40 42 43

49 50 51 52 52 53 54 54 56 57 60 63 63 64 69 69 70 70 72 81 82 83 83 84 85

Contents  vii 4.5 Biotic Factors 4.6 Concluding Remarks Acknowledgements References

86 91 91 92

5 Investigation of Antiviral Effects of Essential Oils 99 Ahmad Mustafa, Dina H. El-Kashef, Miada F. Abdelwahab, Alshymaa Abdel-Rahman Gomaa, Muhamad Mustafa, Nada M. Abdel-Wahab and Alyaa H. Ibrahim 5.1 Introduction 99 5.2 Viruses: Structure, Characteristics, and Replication 101 5.3 In Vitro Antiviral Activity and Mechanism of Action Investigations of Essential Oils and Essential Oil Components 103 5.3.1 Investigation of In Vitro Antiviral Activities 103 5.3.1.1 Plaque Reduction Assay 103 5.3.1.2 The Inhibition of Viral Cytopathogenic Effect 103 5.3.2 Mechanisms of Action 104 5.3.2.1 Time-of-Drug-Addition Assay 104 5.3.2.2 Temperature-Shift Assay 105 5.3.2.3 Morphological Alteration 105 5.3.2.4 Protein Inhibition 105 5.3.2.5 Other Mechanisms of Action 106 5.3.3 Selectivity Index (SI) 106 5.4 The Antiviral Efficacy of Essential Oils on Viruses Affecting Different Body Systems 106 5.4.1 Respiratory System 106 5.4.1.1 Influenza Virus 106 5.4.1.2 Adenovirus and Rhinovirus 111 5.4.1.3 Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-COV-1) 111 5.4.1.4 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) 111 5.4.2 GIT System 113 5.4.2.1 Coxsackie Virus 113 5.4.2.2 Dengue Virus 113 5.4.2.3 Yellow Fever Virus 113 5.4.2.4 Murine Norovirus Type 1 113 5.4.3 Nervous System 113 5.4.3.1 West Nile Virus 113 5.4.4 Immune System 114 5.4.4.1 HIV 114 5.4.5 Reproductive System 114 5.4.5.1 Human Papilloma Virus (HPV) 114 5.4.6 Other Viruses 114 5.4.6.1 Human Herpes Virus 114 5.4.6.2 Orf Virus 115

viii  Contents 5.5 The Antiviral Efficacy of Essential Oils on Phyto-Pathogenic Viruses 5.6 The Antiviral Efficacy of the Essential Oils on Animal‑Infecting Viruses 5.6.1 Virus Affecting Cattle (Bovine Viral Diarrhea Virus) 5.6.2 Virus Affecting Cats (Feline Calicivirus F9) 5.6.3 Virus Affecting Pigs (Porcine Parvovirus) 5.7 Synergistic Effect of Essential Oil Components with Known Antiviral Drugs 5.8 Aromatherapy and its Role as an Antiviral Agent 5.9 Route of Essential Oil Administration 5.10 Nano-Formulated Essential Oils: A Promising Approach to Enhance Antiviral Activity 5.11 Safety of Essential Oils 5.12 Antiviral Essential Oils: Drawbacks versus Future Perspectives 5.13 Summary References 6 Mentha sp. Essential Oil and Its Applicability in Brazil Daniele de Araujo Moysés, Hanna Patricia dos Santos Martins, Margoula Soares Ribeiro, Natasha Costa da Rocha Galucio, Raquel Ribeiro de Souza, Regianne Maciel dos Santos Correa, José de Arimateia Rodrigues do Rego, Maria Fani Dolabela and Valdicley Vieira Vale Introduction 6.1 Ethnobotany of the Mentha in Brazil 6.2 Chemical Constituents of Mentha Oil 6.3 Evaluation of Biological Activities of Mentha Essential Oils 6.4 Toxicity of Essential Oils from Mentha Used in Folk Medicine 6.5 Final Considerations and Perspectives References 7 Microbial Influence on Plants for Enhanced Production of Active Secondary Metabolites Naushin Bano, Mohammad Amir, S. Nabilah Jawed and Roohi 7.1 Introduction 7.2 Classes of Plants Secondary Metabolites 7.2.1 Terpenes 7.2.2 Phenolic Compounds 7.2.3 Nitrogen-Containing Secondary Metabolites 7.2.4 Sulphur Containing Secondary Metabolites 7.3 Secondary Metabolites Production from Plants 7.3.1 In Vivo Production of Secondary Metabolites 7.3.2 In Vitro Secondary Metabolites Production 7.4 Interaction of Microorganisms in the Rhizosphere 7.5 Influence of Bacteria and Fungi on Plants 7.5.1 Plant Growth Promoters 

115 115 115 115 115 116 116 116 117 117 118 118 118 125

126 127 135 139 144 147 148 157 157 159 159 159 160 160 160 161 161 161 164 164

Contents  ix 7.5.1.1 Plant Growth-Promoting Bacteria (PGPR) 7.5.1.2 Plant Growth-Promoting Fungi (PGPF) 7.5.2 Production of Plant Biomass 7.5.3 Bacteria and Fungus as Biofertilizers 7.5.4 Role of Bacteria and Fungi as a Phytostimulator 7.5.5 Role of Bacteria and Fungi as a Biopesticides 7.5.6 Stress Tolerant Activity of Bacteria and Fungi Conclusion and Future Perspectives References

164 164 165 166 166 166 167 168 168

8 Valorization of Limonene Over Acid Solid Catalysts 173 José E. Castanheiro 8.1 Introduction 173 8.2 Limonene Reactions with Alcohols 176 8.3 Hydration and Acetoxylation 177 8.4 Conversion of Limonene into p-Cymene 179 8.5 Conclusions 181 References 181 9 Elucidating the Role of Essential Oils in Pharmaceutical and Industrial Applications Sundaresan Bhavaniramya, Selvaraju Vishnupriya, Kumanan Vijayarani and Ramar Vanajothi 9.1 Introduction 9.2 Extraction of Volatile Oils from Various Sources 9.2.1 Terpenes 9.2.2 Hydrocarbons 9.3 Role of Essential Oils in Industry 9.3.1 Role in Cosmetics and Aromatherapy 9.3.1.1 Cosmetic Industry 9.3.1.2 Immortelle Essential Oil 9.3.1.3 Lavender Essential Oil 9.3.1.4 German Chamomile Oil 9.3.1.5 Neroli Essential Oil 9.3.1.6 Peppermint Essential Oil 9.3.1.7 Rosemary Essential Oil 9.3.2 Application in Food Industry 9.3.2.1 Food Preservation 9.3.2.2 Food Packaging 9.4 Pharmacological Effects of Essential Oils 9.5 Concluding Remarks Acknowledgment References

185 185 186 188 188 190 190 191 191 192 193 194 195 196 196 196 197 198 199 200 200

x  Contents 10 Uses of Essential Oils in Different Sectors Sumeyra Gurkok and Selma Sezen 10.1 Introduction 10.2 Food and Beverage 10.3 Packaging 10.4 Cosmetic and Perfumery 10.5 Aromatherapy 10.6 Medical 10.7 Agriculture 10.8 Textile 10.9 Cleaning Household 10.10 Safety of Essential Oils Conclusion References

207

11 Chemical Composition and Pharmacological Activities of Essential Oils V. Chandrakala, Valmiki Aruna, Gangadhara Angajala and Pulikanti Guruprasad Reddy 11.1 Introduction 11.2 Anticancer 11.2.1 Role of Terpenes in Anticancer Activity 11.2.2 Role of Aromatic Compounds in Anticancer Activity 11.2.3 Mode of Action 11.2.4 The Effect of EOs in Different Types of Cancers 11.2.5 Multi-Drug Resistance (MDR) 11.3 Anti-Inflammatory 11.3.1 Terpenoids for Anti-Inflammatory 11.3.2 Phenylpropanoids for Anti-Inflammatory 11.3.3 Role of Essential Oil for Anti-Inflammatory 11.4 Anti-Viral 11.4.1 Terpenoids for Anti-Viral Activity 11.4.2 Essential Oils for Coronavirus 11.4.3 Essential Oil for Anti-Viral Activity 11.5 Anti-Fungal 11.5.1 Mode of Action 11.5.2 Essential Oil for Anti-Fungal Activity 11.6 Antidiabetic 11.7 Larvicidal Activity 11.8 Anti-Bacterial Conclusion Conflicts of Interest Acknowledgements References

229

207 210 212 213 214 217 219 220 221 221 223 223

229 235 235 235 236 236 238 239 239 239 240 243 244 245 247 248 249 249 253 253 254 255 255 255 255

Contents  xi 12 Augmented Stability and Efficacy of Essential Oils Through Encapsulation Approach Poonam Parashar and Kamla Pathak 12.1 Introduction 12.2 Various Strategies for Encapsulation of Essential Oils 12.2.1 Essential Oils Encapsulated in Liposomes 12.2.2 Essential Oils Encapsulated in Cyclodextrin Complexes 12.2.3 Essential Oils Encapsulated in Polymeric Complexes 12.2.4 Essential Oils Encapsulated in Electrospun Fibers 12.2.5 Essential Oils Encapsulated in Microemulsion/Nanoemulsions 12.2.6 Essential Oils Encapsulated in Mesoporous Silica Nanoparticles 12.3 Conclusions References 13 Antimicrobial Effect of Essential Oils for Food Application Larissa Morais Ribeiro da Silva, Jorge Alberto Sanchos-Burgos, Eveline de Alencar Costa, Maria Jaiana Gomes Ferreira, Cicero C. Pola, Carmen Luiza Gomes and Celli Rodrigues Muniz 13.1 Introduction 13.2 Biotechnological Strategies for Extracting Essential Oils for Food Application 13.3 Methods for Evaluating the EO Inhibitory Activity In Vitro 13.3.1 Factors Affecting Method Susceptibility 13.3.2 Resources Used to Improve Halo Diameter Reading in the Agar Diffusion Method 13.4 Influence of Extraction Methods on the Antimicrobial Compounds in Essential Oils 13.5 Inhibition of Bacteria by Essential Oils in Food 13.6 Use of Essential Oils in Packaging or Food Contact Surfaces 13.6.1 Embedded Films of Nanocapsules with EO 13.6.2 Packaging Reinforced with Nano-Incorporations and Added with EO 13.6.3 Bioactive Films Added EO 13.7 Effect of Encapsulation of Essential Oils on the Inhibitory Activity against Bacteria 13.8 Conclusions References 14 Antioxidant or Antimicrobial Nature of Essential Oils to Minimize Food Waste Dipak Subhash Sali, Vishal Gokul Beldar, Alok Kumar Panda and Manojkumar Jadhao 14.1 Introduction 14.2 Essential Oils Chemical Composition 14.3 Essential Oils: Their Antimicrobial Activity and Mode of Action

269 269 270 270 276 278 279 281 283 284 284 291

291 292 295 295 295 297 299 302 303 303 304 305 306 306 315 315 317 317

xii  Contents 14.4 EO Used in Food Packaging 14.5 Application of EO in Different Food Products 14.5.1 Fruits and Vegetables 14.5.2 Meat and Meat Products 14.5.3 Fish 14.5.4 Dairy Products 14.5.5 Bakery Products 14.6 Legal Aspects of the Use of EO in Food 14.7 Conclusion References

319 322 322 328 328 329 329 330 330 331

15 Application of Essential Oils to Biofilms Sumeyra Gurkok and Selma Sezen 15.1 Introduction 15.2 Definition of Biofilm 15.3 Principles of Biofilm Formation 15.4 Benefits of Biofilm to Microorganism 15.5 Mechanisms of Resistance to Antimicrobial Agents 15.6 Global Threat of Biofilms 15.7 Essential Oils 15.8 Antimicrobial and Antibiofilm Effects of EOs 15.9 Antibacterial Mechanism of Action 15.10 Strategies for Improving the Antibiofilm Efficacy of EOs 15.11 Common Methods for Determination of Antimicrobial and Antibiofilm Activities of EOs 15.12 Limitations of EOs Usage Conclusion References

339

16 Biological Applications of Essential Oil D. Jini 16.1 Introduction 16.2 Sources of Essential Oil 16.3 Extraction of Essential Oil 16.4 Phytochemistry of Essential Oil 16.5 Biological Applications 16.5.1 Applications of Essential Oil on the Treatment of Cancer 16.5.2 Applications of Essential Oil on the Treatment of Respiratory Tract Diseases 16.5.3 Applications of Essential Oil on the Treatment of Cardiovascular Diseases 16.5.3.1 Anti-Inflammatory Activity 16.5.4 Applications of Essential Oil on the Treatment of Obesity 16.5.5 Applications of Essential Oil on the Treatment of Diabetes 16.5.5.1 Antioxidant Activity

361

339 340 340 342 342 344 345 346 349 350 351 352 353 353

361 362 363 363 364 365 367 367 368 368 368 369

Contents  xiii 16.5.6 Applications of Essential Oils Against Infectious Diseases 369 16.5.6.1 Antibacterial Activity 370 16.5.6.2 Antifungal Activity 371 16.5.6.3 Antiviral Activity 371 16.5.7 Applications of Essential Oil on Dandruff 371 16.6 Essential Oil Safety Issue 372 16.7 Conclusion 372 Acknowledgment 372 References 372 17 Current Status and Advancement of Biopesticides from Essential Oil for Agriculture, Food Storage, and Household Applications Masrina Mohd Nadzir, Salfarina Ramli, Farhana Nazira Idris and Faiznur Mohd Fuad 17.1 Introduction 17.1.1 Essential Oil Extraction 17.2 Application of Essential Oil Biopesticides in Agriculture 17.2.1 Agriculture Pest 17.2.2 Types of Essential Oils for Agricultural Pest Management 17.3 Application of Essential Oil Biopesticides for Food Storage 17.3.1 Food Storage Pests 17.3.2 Types of Essential Oils for Food Storage Pest Management 17.4 Application of Essential Oil Biopesticides for Household Pests 17.4.1 Household Pests 17.4.2 Types of Essential Oils for Household Pest Management 17.5 Delivery of Biopesticides 17.6 Pesticidal Action of Biopesticides 17.7 Conclusion and Constraints 17.8 Acknowledgement References 18 Essential Oil Used as Larvicides and Ovicides Gurleen Kaur, Rajinder Kaur and Sukhminderjit Kaur 18.1 Introduction 18.2 Important Aspects of Essential Oils 18.3 Larvicides and Ovicides 18.3.1 Larvicides Against Aedes aegypti 18.3.2 Larvicidal Activity Against Anopheles stephensi 18.3.3 Larvicide Against Aedes albopictus 18.3.4 Ovicidal Activity Against Pediculus humanus capitis 18.3.5 Ovicidal Activity Against Haemonchus contortus 18.3.6 Ovicidal Activity Against Helicoverpa armigera Hubner 18.4 Conclusion References

381 381 383 383 384 385 385 395 396 406 406 407 412 413 415 415 416 427 427 428 430 431 433 434 435 437 438 439 439

xiv  Contents 19 Essential Oil-Based Biopesticides Nishant Sharma, Kunal Sharma, Sachchidanand Soaham Gupta, Kumar Rakesh Ranjan, Vivek Mishra and Maumita Das Mukherjee 19.1 Introduction 19.2 Phytochemistry and Sources of Essential Oils 19.3 Biological Activity of Essential Oil Biopesticides 19.3.1 Efficacy of Essential Oils to Insects 19.3.2 Essential Oils as Insect Repellents 19.3.3 Bactericidal Properties of Essential Oils 19.3.4 Antifungal and Anti-Oomycete Properties of Essential Oils 19.3.5 Herbicidal/Weedicide Properties of Essential Oils 19.4 Synergistic Formulations of Essential Oils 19.5 Toxic Effects of Essential Oils on Mammals and Non-Target Organisms 19.6 Advantages, Current Constraints and Long-Term Prospects 19.7 Conclusion References

443

20 Essential Oils Obtained from Algae: Biodiversity and Ecological Importance Deprá, M. C., Dias, R. R., Nascimento, T. C., Silva, P. A., Zepka, L. Q. and Jacob-Lopes, E. 20.1 Introduction 20.2 What are Essential Oils? 20.3 Chemical Structure and Biological Activity from Algal Essential Oils 20.4 Ecological Importance of Essential Oils in Marine System 20.5 Conclusion and Future Perspectives References

465

21 Gas Chromatography-Olfactometry (GC‑O) of Essential Oils and Volatile Extracts Eduardo Dellacassa and Manuel A. Minteguiaga 21.1 Introduction 21.2 Historical Aspects 21.3 GC-O Methodologies 21.3.1 Detection Frequency Methods 21.3.2 Dilution Analysis 21.3.2.1 Aroma Extraction Dilution Analysis (AEDA) 21.3.2.2 Combined Hedonic Aroma Response Measurements (CHARM Analysis) 21.3.3 Posterior Intensity Methods (PI) 21.3.4 Time-Intensity Methods 21.3.4.1 Odor-Specific Magnitude Estimation (OSME, Direct Intensity) 21.4 Different GC-O Application to Assess for Essential Oils’ Odorants 21.4.1 Citrus spp. (Rutaceae) 21.4.2 Mentha spp. (Lamiaceae) 21.4.3 Thymus spp. (Lamiaceae)

443 445 447 447 449 450 450 452 454 454 455 457 458

465 466 467 470 472 472 477 477 479 481 482 482 483 483 485 485 486 486 487 488 490

Contents  xv Foeniculum spp. (Apiaceae) 491 Coriandrum spp. (Apiaceae) 492 Pinus spp. 493 GC-O Applied to Characterize Baccharis dracunculifolia DC. Odorants 494 Acknowledgements 497 Funding 497 References 497 21.4.4 21.4.5 21.4.6 21.4.7

22 In Vitro and In Vivo Methods Used to Assess the Biological Potential of Essential Oils 501 Syed Ali Raza Naqvi, Sadaf Ul Hassan, Tauqir A. Sherazi, Amjad Hussain, Muhammad Rehan Hasan Shah Gilani and Tanvir Hussain 22.1 Introduction 501 22.2 Chemistry of EOs 502 22.3 In Vitro Methods Used to Assess the Biological Potential of EOs 503 22.4 Evaluation of Antioxidant Potential 504 22.4.1 What are Antioxidants? 504 22.4.2 Antioxidant Potential of Botanical Materials 504 22.4.3 Modes of Action 505 22.4.4 In Vitro Methods for Antioxidant Activities 505 22.4.5 DPPH Scavenging Assay 506 22.4.6 2,2-Azinobis-(3-Ethylbenzothiazoline-6-Sulfonate) Assay 507 22.4.7 Bleachability of β-Carotene in Linoleic Acid System 510 22.5 Antimicrobial Activities of Essential Oils 511 22.5.1 Disk Diffusion Assay 512 22.5.2 Agar Well Diffusion Method 512 22.5.3 Determination of Minimal Inhibitory Concentration (MIC) 513 22.6 Essential Oils as Natural Antimicrobial Agents 514 22.7 Anticancer Activity of Essential Oils 515 22.8 Cell Culture and Treatment 515 22.9 Determination of Cell Viability 515 22.10 Conclusion 515 Acknowledgement 515 References 516 23 Biological Potential of Essential Oils: Evaluation Strategies 521 Santanu Chakraborty, Manami Dhibar, Aliviya Das, Kalpana Swain and Satyanarayan Pattnaik 23.1 Introduction 521 23.2 Biological Activities of Essential Oils 523 23.2.1 EOs as Antibacterial Agents 524 23.2.2 EOs as Antifungal Agents 525 23.2.3 EOs as Anti-Inflammatory Agents 526 23.2.4 EOs as Antioxidants 526 23.2.5 EOs as Anticancer Agents 526

xvi  Contents 23.2.6 EOs as Anti-Diabetic Agents 23.2.7 EOs as Antispasmodics 23.3 In Vitro Assessment of Biological Activities 23.3.1 Antimicrobial Assay 23.3.2 Antibacterial Assay 23.3.3 Antifungal Assay 23.3.4 Antioxidant Assay 23.3.5 Anticancer Assay 23.3.6 Anti-Diabetic Assay 23.4 In Vivo Assessment of Biological Activities 23.4.1 Antimicrobial Assay 23.4.2 Antidermatophytic Assay 23.4.3 Antifungal Assay 23.4.4 Anti-Inflammatory Assay 23.4.5 Antioxidant Assay 23.4.6 Anticancer Assay 23.4.7 Anti-Diabetic Assay 23.4.8 Mosquito Repellent Assay 23.5 Conclusion References

528 528 529 529 529 530 530 532 532 532 532 533 533 533 534 535 535 537 537 537

24 Algal Essential Oils and Their Importance in the Ecosystem S.Z.Z. Cobongela 24.1 Introduction 24.2 Algal Essential Oils 24.3 Factors Affecting Algae Essential Oil Production 24.3.1 Temperature 24.3.2 Light 24.3.3 Nutrients 24.3.4 Chemical Stress 24.4 Ecological Importance of Algal Essential Oils 24.5 Pheromone Properties of Algal Essential Oils 24.6 Algal Essential Oils in “Beach-Odor” 24.7 Algal Essential Oils in “Off-Odor” 24.8 Antibacterial Activities of Algal Essential Oils 24.9 Antifungal Activities of Algal Essential Oils 24.10 Conclusion References

551

25 Classical Methods for Obtaining Essential Oils Syed Raza Ali Naqvi, Hiba Shahid, Ameer Fawad Zahoor, Muhammad Saeed, Muhammad Usman, Ali Abbas, Mamoon Ur Rasheed and Tanvir Hussain 25.1 Introduction 25.2 Classical Methods for Extracting Essential Oils 25.2.1 Maceration

565

551 552 553 553 554 554 554 554 556 557 557 558 558 559 559

565 568 568

Contents  xvii 25.2.2 Mechanical Treatment 25.2.3 Hydro Distillation 25.2.4 Water Distillation 25.2.4.1 Steam Distillation 25.2.5 Cold Pressing Method 25.2.6 Solvent Extraction 25.2.7 Soxhlet Extraction 25.3 Chromatographic Technique for Analysis of Essential Oil Conclusion Acknowledgement References

568 570 570 574 574 576 577 578 578 579 579

26 A Comprehensive Guide to Essential Oil Determination Methods 583 Payel Dhar, Urbashi Neog, Biplab Roy, Nishithendu Bikash Nandi, Sankar Chandra Deka and Pinku Chandra Nath Abbreviations 584 26.1 Introduction 584 26.2 Chemical Composition of EOs 585 26.2.1 Hydrocarbons Derived from Terpenes 587 26.2.2 Oxygenated Compounds 587 26.3 EO and Its Group 588 26.4 Biological Activity: Pathway Cell 588 26.4.1 Osmophores 589 26.4.2 Trichomes 589 26.5 Classical Methods for Extraction of Essential Oils 590 26.5.1 Steam Distillation 590 26.5.2 Hydro-Distillation Method 591 26.5.3 Steam Explosion Method 591 26.5.4 Solvent Extraction Method 592 26.5.5 Cold Press (CP) Method 592 26.6 Contemporary Extraction Methods 592 26.6.1 Supercritical Fluid (SCF) Extraction 592 26.6.2 High Pressure Extraction 593 26.6.3 Microwave-Assisted Hydro-Distillation 593 26.6.4 Hydrodistillation with Pretreatment of Enzyme 593 26.6.5 Microwave-Assisted Steam Distillation 594 26.6.6 Ultrasound-Assisted Extraction 595 26.6.7 Solvent-Free Microwave Extraction 595 26.6.8 Microwave Hydro-Diffusion and Gravity 595 26.6.9 Oil Extraction by Solar Energy 596 26.6.10 Pulse Electric Field 596 26.7 Conclusion 596 References 597

xviii  Contents 27 Encapsulation of Essential Oils Ádina L. Santana and M. Angela A. Meireles 27.1 Introduction 27.2 Encapsulation 27.2.1 Chemical 27.2.1.1 Molecular Inclusion Complexation 27.2.1.2 Interfacial Polymerization 27.2.1.3 In Situ Polymerization 27.2.2 Physical-Chemical 27.2.2.1 Coacervation 27.2.2.2 Emulsification 27.2.3 Physical 27.2.3.1 Spray Drying 27.2.3.2 Freeze Drying 27.2.3.3 Electrospraying and Electrospinning 27.2.3.4 Supercritical Technology 27.3 Process Simulation and Economic Evaluation Concluding Remarks and Prospects References

603 603 605 605 605 606 606 606 606 606 607 607 608 608 609 613 614 614

28 Encapsulated Essential Oils: Main Techniques to Increase Shelf-Life 619 Fernanda Wariss Figueiredo Bezerra, Lucas Cantão Freitas, Vânia Maria Borges Cunha, Giselle Cristine Melo Aires, Rafael Henrique Holanda Pinto and Raul Nunes de Carvalho Junior 28.1 Introduction 619 28.2 Coating Materials 620 28.3 Techniques for Essential Oil Encapsulation 623 28.3.1 Coacervation 623 28.3.2 Extrusion 624 28.3.3 Nanoprecipitation 625 28.3.4 Emulsification 626 28.3.5 Spray Drying 626 28.3.6 Thin Film Hydration Method 627 28.3.7 Supercritical Fluid Technology 628 28.4 Concluding Remarks 629 Acknowledgment 629 References 630 29 Encapsulation Technologies of Essential Oils for Various Industrial Applications Tuyen C. Kha and Phuong H. Le 29.1 Introduction 29.2 Encapsulation Technique 29.2.1 Essential Oil as the Core Material 29.2.1.1 Chemical Composition and Physical Properties of EOs 29.2.1.2 Biological Activities of EOs

635 635 636 637 637 641

Contents  xix 29.2.2 Wall Materials 29.2.3 Encapsulation Method and Release Mechanism 29.2.4 Applications of Encapsulated EOs 29.2.4.1 Preservative in Foods 29.2.4.2 Baked Foods 29.2.4.3 Beverages 29.2.4.4 Fresh Fruit and Vegetables 29.2.4.5 Raw Meat and Meat Products 29.2.4.6 Milk and Dairy Products 29.2.4.7 Cosmetic and Health Care 29.2.4.8 Cotton and Textile 29.2.4.9 Pharmaceutical 29.3 Conclusions References 30 Extraction of Essential Oils with Supercritical Fluid Ádina L. Santana and M. Angela A. Meireles 30.1 Introduction 30.2 Why Use Supercritical Carbon Dioxide to Extract Essential Oils? 30.3 Commercial Equipment Used for Supercritical Fluid Extraction of Essential Oils: Bench and Industrial Scale 30.3.1 Bench Scale 30.3.2 Pilot and Commercial Scale 30.4 Patent Survey 30.5 Economic Evaluation 30.6 Life Cycle Assessment 30.6.1 Goal and Scope Definition 30.6.2 Life Cycle Inventory 30.6.3 Life Cycle Impact Assessment (LCIA) 30.6.4 Interpretation 30.6.5 Recent Studies on LCA of SFE of Essential Oils and the Main Results 30.7 Current Outlook and Prospects References 31 Advantages of Essential Oil Extraction Using Supercritical Fluid: Process Optimization and Effect of Different Processing Parameters on Extraction Efficiency Shaziya Manzoor, Rubiya Rashid, Mudasir Ahmad, F.A. Masoodi, Pir Mohammad Junaid and Sadaf Parvez 31.1 Introduction 31.2 Essential Oils 31.3 Supercritical Fluid Extraction 31.4 Superiorities of SFE over Other Extraction Methods 31.5 Extraction of EOs by Supercritical Fluid

642 643 649 649 649 656 657 657 658 659 660 661 662 663 671 671 672 676 676 677 678 678 679 680 680 681 681 681 681 682

685 685 686 687 688 689

xx  Contents 31.5.1 Effects of Temperature 31.5.2 Effect of Pressure 31.5.3 Effect of Particle Size 31.5.4 Effect of Flow Rate 31.5.5 Use of a Co-Solvent 31.5.6 Extraction Time 31.6 Antimicrobial and Antioxidant Properties of Essential Oils Extracted via SFE 31.7 Optimization 31.7.1 Optimization Using RSM and BBD, Taguchi Model 31.7.2 Artificial Neural Networks (ANNs) 31.8 Conclusion References 32 Supercritical Fluid Extraction of Essential Oils from Natural Sources: Mathematical Modeling and Applications Carina Contini Triques, Edson Antônio da Silva, Kátia Andressa Santos, Elissandro Jair Klein, Veronice Slusarski-Santana, Márcia Regina Fagundes-Klen and Mônica Lady Fiorese 32.1 Introduction 32.2 Essential Oils 32.3 Conventional Extraction Methods 32.4 Supercritical Fluid Extraction 32.4.1 Cosolvent Addition 32.5 Typical Behavior and Mathematical Modeling 32.6 Parameters Affecting the CO2-Supercritical Fluid Extraction 32.6.1 Pressure and Temperature 32.6.1.1 Pressure 32.6.1.2 Temperature 32.6.2 Pre-Treatment – Moisture and Particle Size 32.6.3 Extraction Time and Apparent Solubility 32.6.4 Solvent Flow 32.7 Scale-Up and Economic Analysis 32.8 Applications 32.9 Final Considerations References 33 Fundamentals, Mathematical Models, and Extraction Processes with Supercritical Fluids Facundo Mattea, Nicolás Gañán and Marcelo Ricardo Romero 33.1 Introduction: Background 33.2 Fundamentals of Supercritical Fluid Extraction 33.2.1 Supercritical Fluids 33.2.2 Solubility and Phase Equilibria 33.3 The Extraction Process 33.3.1 Process Scheme

695 695 696 697 697 698 698 699 699 700 701 701 707

707 708 710 711 713 714 723 723 726 728 729 730 732 733 734 734 735 741 741 743 743 745 747 747

Contents  xxi 33.3.2 Process Parameters 33.3.2.1 Temperature and Pressure 33.3.2.2 Flow Rate 33.3.3 Mathematical Modeling of the Extraction Process 33.3.4 Scale-Up 33.4 Separation 33.4.1 Extract Recovery Strategies 33.4.2 Essential Oil Fractionation 33.4.3 Solvent Regeneration and Recycling 33.5 Recent Application of Supercritical Extraction of Essential Oils and Industrial Application of Supercritical Fluid Extraction Processes 33.6 Novel and Future Perspectives of Supercritical Fluid Extraction for Essential Oils 33.6.1 Supercritical Fluid Extraction Coupled with Other Green Extraction Technologies 33.6.2 Future Perspectives References 34 Supercritical CO2 Extraction as a Clean Technology Tool for Isolation of Essential Oils T. P. Krishna Murthy, R. Hari Krishna, M. N. Chandra Prabha, Priyadarshini Dey, Blessy Baby Mathew and C. Manjunatha 34.1 Introduction 34.2 Essential Oils 34.3 Applications of EOs 34.3.1 Antibacterial and Antifungal Activity 34.3.2 Antioxidant and Anticancer Activity 34.3.3 Antiviral Activity 34.3.4 Food Preservative and Packaging 34.3.5 Aromatherapy 34.3.6 Dairy Products 34.3.7 Biocontrol Agents 34.4 Extraction Methods 34.4.1 Distillation 34.4.2 Cold Pressing/Expression 34.4.3 Hydrodifussion 34.4.4 Solvent Extraction 34.4.5 Microwave-Assisted Extraction (MAE) 34.4.6 Ultrasound-Assisted Extraction (UAE) 34.5 Supercritical Fluid Extraction (SCFE) 34.6 Parameters Influencing SCFE of EOs 34.6.1 Pressure 34.6.2 Temperature 34.6.3 CO2 Flow Rate 34.6.4 Moisture 34.6.5 Cosolvent

749 749 751 751 752 753 753 754 755 756 758 758 760 760 767 767 768 769 769 769 770 770 770 771 771 771 772 772 772 772 773 773 773 776 776 777 777 777 777

xxii  Contents 34.6.6 Particle Size of Plant Material 34.6.7 Extraction Time 34.7 Optimization of SCFE Process 34.8 Mathematical Modeling of Extraction Curves 34.9 Coupled or Assisted SCFE 34.9.1 Enzyme-Assisted SCFE 34.9.2 Ultrasound-Assisted SCFE 34.10 Conclusion References

778 778 780 785 786 786 786 786 787

35 Classical Techniques for Extracting Essential Oils from Plants Yogesh Murti, Sonia Singh and Kamla Pathak 35.1 Introduction 35.2 Market Value of Essential Oils 35.3 Sources of Essential Oils 35.4 Chemical Nature of Essential Oils 35.5 Extraction of Essential Oils 35.5.1 Distillation Methods 35.5.1.1 Hydrodistillation 35.5.1.2 Steam Distillation 35.5.2 Hydrodiffusion 35.5.3 Solvent Extraction 35.5.4 Soxhlet Extraction 35.5.5 Cold Pressing Method/Expression 35.5.6 Cohobation 35.5.7 Enfleurage 35.5.8 Maceration 35.6 Conclusion References

795

36 Acquisition of Essential Oils Through Traditional Techniques Lucas Cantão Freitas, Vinicius Sidonio Vale Moraes, Sabrina Baleixo da Silva and Raul Nunes de Carvalho Junior 36.1 Introduction 36.2 Obtaining Essential Oils 36.2.1 Cold Pressing 36.2.2 Steam Distillation 36.2.3 Hydrodistillation 36.2.4 Enfleurage 36.2.5 Solvent Extraction 36.3 Concluding Remarks Acknowledgment References

859

795 796 796 796 803 804 804 806 839 839 843 844 847 848 849 850 851

859 860 861 862 863 864 864 866 866 866

Contents  xxiii 37 Essential Oils: Chemical Composition and Methods of Extraction Arshi Gupta, Kumar Rakesh Ranjan, Nisha Yadav, Deeksha and Vivek Mishra 37.1 Introduction 37.2 Chemical Assemblage of Essential Oils 37.2.1 Terpenes 37.2.1.1 Monoterpenes 37.2.1.2 Sesquiterpenes 37.2.1.3 Diterpenes 37.2.2 Heteroatomic Metabolites 37.2.2.1 Ketones 37.2.2.2 Acids 37.2.2.3 Aldehydes 37.2.2.4 Alcohols 37.2.2.5 Lactones 37.3 Extraction of Essential Oils Key Factors are Involved in Determining the Extraction Method 37.3.1 Conventional Extraction Methods 37.3.1.1 Hydro Distillation 37.3.1.2 Enfleurage Method 37.3.1.3 Hydro Diffusion 37.3.1.4 Cold Pressing 37.3.1.5 Steam Distillation 37.3.2 Green Extraction Methods 37.3.2.1 Supercritical Fluid Extraction (SFE) 37.3.2.2 Microwave-Assisted Extraction (MAE) 37.3.2.3 Ultrasonication-Assisted Extraction (UAE) 37.3.2.4 Conclusion 37.4 Conclusion References

871

38 Dental Applications of Essential Oils Aarati Panchbhai 38.1 Introduction 38.2 Background 38.3 Preparation of Essential Oils 38.4 Mechanism of Action of Essential Oils 38.5 Methods of Application of Essential Oil for Dental Uses 38.6 Therapeutic Actions of Essential Oil for Dental Uses 38.7 Dental/Oral Conditions Treated by Essential Oils 38.8 Dental Applications of Essential Oils 38.9 Safety Issues in Relation to Use of Essential Oils 38.10 Research 38.11 Conclusion References

891

871 873 874 874 874 875 875 875 876 876 876 876 876 879 879 879 880 880 881 881 881 882 883 884 884 885

891 892 892 893 893 894 894 895 897 898 899 899

xxiv  Contents 39 Essential Oil-Based Therapies Syed Ali Raza Naqvi, Vaneeza Javed, Naseem Abbas, Muhammad Rehan Hasan Shah Gilani, Sadaf Ul Hassan, Muhammad Rizwan Javed and Mazhar Hussain 39.1 Introduction 39.2 Essential Oil-Rich Plants 39.2.1 Citronella 39.2.2 Peppermint 39.2.3 Lavender 39.2.4 Tea-Tree 39.2.5 Eucalyptus 39.2.6 Chamomile 39.2.7 Patchouli 39.2.8 Ylang-Ylang 39.2.9 Bergamout 39.2.10 Geranium 39.2.11 Lemon 39.3 How Essential Oil Therapy Works 39.3.1 Cosmetic Aromatherapy 39.3.2 Massage Aromatherapy 39.3.3 Medical Aromatherapy 39.3.4 Olfactory Aromatherapy 39.3.5 Psycho-Aromatherapy 39.4 Essential Oil-Based Therapies 39.4.1 Brainstorming Therapies 39.4.1.1 In the Treatment of Dementia 39.4.1.2 Stress Reduces Therapy Among Adolescents 39.4.2 Anti-Microbial Therapy 39.4.3 In Treatment of Eczema 39.4.4 Anti-Hair Fall Therapy 39.4.5 Anti-Tumor Therapy 39.4.6 Chemopreventive Therapy 39.4.7 Coronavirus Therapeutics 39.4.8 Essential Oil Helps in Epilepsy 39.4.9 Treatment of Cardiovascular Disorders 39.5 How to Use EOs? Conclusion References

903

40 Clinical Applications of Essential Oils Laxmi Tripathi, Praveen Kumar, Kalpana Swain and Satyanarayan Pattnaik 40.1 Introduction 40.2 Aromatherapy 40.3 Mode of Action of Essential Oils in Aromatherapy 40.4 Classification of Aromatherapy

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903 905 905 906 907 908 910 911 912 913 914 915 916 917 918 918 918 918 918 918 921 921 921 921 921 922 922 923 923 923 925 925 925 926

933 934 934 935

Contents  xxv 40.4.1 Cosmetic Aromatherapy 40.4.2 Massage Aromatherapy 40.4.3 Medical Aromatherapy 40.4.4 Olfactory Aromatherapy and Psycho-Aromatherapy 40.5 Essential Oils from Various Parts of the Plants Used in Aromatherapy 40.6 Essential Oil-Based Therapies 40.6.1 Pain and Inflammation 40.6.2 Hemodialysis 40.6.3 Psychological Disorders 40.6.4 Treatment of Nausea and Vomiting 40.6.5 Managing Menopause Symptoms 40.6.6 Treatment of Dermatological Problems 40.7 Safety Issues Related to Essential Oil-Based Therapy 40.8 Conclusion References 41 Therapeutic Role of Essential Oils S. Vishali, E. Kavitha and S. Selvalakshmi 41.1 Introduction 41.2 Uses of Essential Oils 41.2.1 Nontherapeutic Uses of EOs 41.2.1.1 Pesticide 41.2.1.2 Food Preservative 41.2.1.3 Cosmetics and Home Care 41.2.1.4 Mosquito Repellent 41.2.1.5 Others 41.3 Classification of Aromatherapy 41.3.1 Cosmetic Aromatherapy 41.3.2 Massage Aromatherapy 41.3.3 Medical Aromatherapy 41.3.4 Olfactory Aromatherapy 41.3.5 Psycho-Aromatherapy 41.4 Role of Essential Oil in Clinical Practice 41.5 Applications of Edible Essential Oil on Therapy 41.5.1 Almond Oil 41.5.2 Avocado Oil 41.5.3 Canola Oil 41.5.4 Coconut Oil 41.5.5 Flaxseed Oil 41.5.6 Groundnut Oil 41.5.7 Sesame Oil 41.5.8 Sunflower Oil 41.6 Risky EOs to Children 41.6.1 Camphor Oil 41.6.2 Wintergreen Oil

935 935 937 938 939 942 943 946 946 947 947 947 948 948 948 953 953 956 957 957 957 957 958 958 958 958 958 958 959 959 960 964 966 967 968 968 969 969 970 970 970 970 971

xxvi  Contents 41.7 Side Effects of EOs 41.8 Therapeutic Guidelines and Safety Precautions 41.9 Conclusions Declaration About Copyright References 42 Plant Essential Oils and Their Constituents for Therapeutic Benefits Monika Rani, Simran Jindal, Ritesh Anand, Niharika Sharma, Kumar Rakesh Ranjan, Maumita Das Mukherjee and Vivek Mishra 42.1 Introduction 42.1.1 Concept and Definition 42.1.2 A Journey Through History 42.1.3 Composition 42.2 Biological Activities 42.2.1 Antimicrobial Activities: Mode of Action and Effects 42.2.1.1 Antibacterial Activities 42.2.1.2 Anti-Fungal Activities 42.2.1.3 Anti-Viral Activities 42.2.2 Antioxidant Activities 42.2.3 Antiphlogistic Activity 42.2.4 Anti-Cancer Activities 42.2.5 Miscellaneous Activities 42.2.5.1 Penetration Enhancement 42.2.5.2 EOs in Food 42.2.5.3 Antinociceptive Effects 42.2.5.4 Insect Repellent Activity 42.3 Conclusion References

971 972 973 973 973 977 977 977 979 980 983 984 984 987 989 991 994 996 998 998 999 999 999 1000 1000

43 Essential Oils Used in Packaging: Perspectives and Limitations 1009 Khadija El Bourakadi, Abou El Kacem Qaiss and Rachid Bouhfid 43.1 Introduction 1009 43.2 Essential Oils: Definition, Preparation, and Composition 1010 43.3 Essential Oils: Medicinal and Biological Functions 1012 43.4 Functional Application of Essential Oils 1013 43.5 Active Packaging Material Based on Essential Oils 1014 43.5.1 Composite and Nanocomposite Materials Based on Essential Oils 1014 43.5.2 Advantages 1017 43.5.3 Limitations 1018 43.6 Conclusion and Future Perspectives 1019 References 1020 Index 1025

Preface Essential oils have been used by global communities for centuries, for different purposes such as medicinal, flavoring, preservatives, perfumery, aromatherapy, dentistry, cosmetics, insecticide, fungicide, bactericide, among others. Essential oils are natural and biodegradable substances, usually non-toxic or with low toxicity to humans. Essential oils are botanical products with having volatile nature and are known for their special odor and are found effective in the treatment of oxidative stress, cancer, epilepsy, skin allergies, indigestion, headache, insomnia, muscular pain, and respiratory problems, etc. Essential oils principally enhance resistance to abiotic stress and protection against aquatic herbivores. They possess antimicrobial, antifungal, antitumor, and antioxidant activities. However, essential oils are easily lost or degraded under ambient conditions (temperature, air, light, and humidity), resulting in limited applications. So, their encapsulation is one of the proven techniques to successfully protect essential oils and enable various applications. The purpose of this book is to offer current knowledge on essential oils’ chemical structure, therapeutic, and biological activities, to describe their functional uses, and to assess the benefits and drawbacks of their usage in many fields. Essential Oils: Extraction Methods and Applications addresses the topics related to methods of extracting essential oils, biological and therapeutic applications, their uses in different sectors of the industry, and will also address methods and applications of encapsulated essential oils. In addition, we cover issues such as the latest biological applications of essential oils, as well as traditional and modern methods for extracting essential oils. This book should be useful for different industries like pharma, perfumery, flavoring, perfumery, aromatherapy, cosmetics, others and also useful for faculty, researchers, students from academics, and laboratories which are linked to essential oils and their useful properties, applications of the different paradigms. The summaries of the work reported in the following 43 chapters are as follows: Chapter 1 discusses the plant essential oils and their isolated bioactive components for their potential antiviral activities in detail. The fundamental knowledge of antiviral properties of essential oil along with their mechanisms of actions, efficacy, and safety is needed for their targeted drug delivery systems, which are consequential to their further research, new drug design, and further applications. Chapter 2 covers the use of essential oils derived from aromatic plants as a safer and more nutritious alternative to artificial preservatives. Essential oils’ natural properties, extraction procedures, and activity against pathogenic and deteriorating microorganisms, as well as their uses in food preservation, are discussed.

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xxviii  Preface Chapter 3 reviews the sources and composition of various essential oils and the variety of extraction methods. Primarily, it focuses on applying essential oils in different industrial sectors like chemicals, food preservation, pharmaceutical, and pesticides, etc. Chapter 4 focuses on the effect of various abiotic and biotic factors (drought, temperature, salt, heavy metals, UV light, living organisms, etc.) on essential oils production and composition. The importance of these factors to adequate agricultural practices for aromatic plants cultivation and to obtain high-quality essential oils is also discussed. Chapter 5 emphasizes the potential antiviral effect of essential oils by reviewing some recent literature. It illustrates the different methods implemented to investigate the in vitro antiviral activity of EOs and their components along with their mechanisms of action. Furthermore, the effectiveness of EOs against several viral illnesses that impact human body systems, as well as some plants and animals, are also highlighted. Chapter 6 discusses Mentha species and their chemical compositions on account of their biological activity as reported by the scientists, with a focus on microbiological activity. Mentha piperita L. is judged to be the most promising of the species offered to be used as an herbal medication. Chapter 7 describes the plant secondary metabolites that play a significant role as anti-oxidants, anti-cancerous, anti-microbial, and have medicinal properties. The influence of microbes on plants is elaborated in the context of the enhanced production of these secondary metabolites in various stress conditions, with mechanisms of contact briefly elaborated. Chapter 8 details the valorization of limonene (an essential oil) into compounds with high commercial value by different reactions, such as alkoxylation, hydration, and acetalization over heterogeneous catalysts. Also, the conversion of limonene into p-cymene is studied. Different solid materials, like clays, zeolites, heteropolyacids, and silica with sulfonic groups towards the valorization of limonene are discussed. Chapter 9 discusses the role of essential oils in various industrial applications, focusing on pharmaceutical, cosmetic manufacturing, food processing, and preservation industry. The major focus dealt with volatile bioactive compounds in essential oils that are responsible for altering synthetic additives with natural composites in food, cosmetics, and medicines. Chapter 10 discusses the most common uses of essential oils in various sectors. It focuses on the increased use of essential oils in the food, beverage, packaging, cosmetics, perfumery, medical, agriculture, textile, and cleaning industries in line with the increasing awareness and demand of consumers for natural ingredients. Chapter 11 deals with various pharmacological activities of essential oils and their major chemical components. The mechanism of action and pharmacological targets of various essential oils particularly anti-inflammatory, anticancer, antiviral, antifungal, larvicidal, antidiabetic, and antibacterial activities are discussed in the present chapter. Additionally, the potential efficacy of terpenoids and phenylpropanoids in the treatment of cancer, inflammation, and viral infections are illustrated in this chapter. Chapter 12 reveals the stability and efficacy associated challenges that are often encountered by essential oils that led to compromised efficiency. Further, the chapter gives insight to overcome these challenges through various encapsulation techniques, their formulation aspect, and the advantages as per the reported literature. Chapter 13 discusses the antimicrobial effect of essential oils and their food application. Additionally, the biotechnological strategies for extracting essential oils for food application

Preface  xxix and the methods for evaluating the essential oil’s inhibitory activity are discussed. Moreover, the influence of extraction methods on the antimicrobial compounds in essential oils is also presented. Chapter 14 epitomizes the application of essential oils in food packaging and food products. Essential oils sources such as plants, fruit, and flowers and their chemical composition are presented in this chapter. The main focus of this chapter is to highlight the potential application of essential oils as an antimicrobial, antifungal, and antioxidant agent in different food packaging and products. Chapter 15 discusses the use of essential oils against biofilm-forming bacteria. The formation and organization of biofilms and their role in acquiring antibiotic resistance are presented. The main focus is given to provide information on the nature of essential oils, their antimicrobial and antibiofilm activities, and their mechanism of action. Chapter 16 discusses the biological applications of essential oils such as antibacterial, antifungal, antiviral, antioxidant, and anti-inflammatory activities and their usage in the treatment of various ailments such as cancer, respiratory tract diseases, cardiovascular diseases, obesity, and diabetes. Additionally, the sources and extraction process of essential oil are also discussed. Chapter 17 details the various essential oils used as biopesticides in agriculture, food storage, and the household. The delivery and the pesticidal modes of action of biopesticides are discussed in detail. The target pest and the active ingredients responsible for the pesticidal action are also presented. Chapter 18 explicitly describes the larvicidal and ovicidal potential of essential oils with special reference to potent larvicidal activity against mosquito vectors including Aedes aegypti, Anopheles stephensi, A. Albopictus, and the ovicidal activity against human head lice (Pediculus humanus), domestic animal gastrointestinal nematode (Haemonchus contortus) and American bollworm (Armigera Helicoverpa Hubner). Chapter 19 discusses the primary applications of essential oils as pesticides and their biological activity with a different class of organisms and discusses potential directions for the use of essential oils as pesticides of the future. Additionally, the role of essential oils synergistic compositions and toxic effects of essential oils on non-target organisms are also studied. The main aim of this chapter is to explain the present state of knowledge and recent advances in the phytochemistry of plant essential oils, their biological activity in a variety of species, and their potential as biopesticides. Chapter 20 details the scientific advancement and discoveries about the biological potential of essential oils from micro and macroalgae, which has been arousing interest in the most diverse industrial applications. The major focus is attributed to the ecological importance and biodiversity of micro and macroalgae under this new market perspective. Chapter 21 explains how to comprehend the complexity of olfactory responses by developing instrumental ways for objectively analyzing them. Particularly, gas chromatography-­ olfactometry technology has been profusely employed. An overview of data that may be collected using several gas chromatography-olfactometry techniques on essential oils is described, along with the procedures and foundations involved. Chapter 22 covers key in vitro and in vivo methods to assess essential oils with a brief description of different protocols. Essential oils are found effective in the treatment of oxidative stress, cancer, skin allergies, headache, insomnia, muscular pain, and respiratory problems.

xxx  Preface Chapter 23 details the various evaluation strategies adopted to assess the biological potential of different essential oils. This chapter aims at bringing up a summary and critical appraisal of the reported methods, both in vitro and in vivo, for assessment of the biological activities of essential oils. Chapter 24 discusses the importance of algal essentials in the ecosystem. It further details environmental factors affecting the production of essential oils and their organic volatile compounds by algae. Their interesting bioactivity that can offer significant benefits and biotechnological relevance are also presented. Chapter 25 is a good set of classical methods of obtaining essential oils along with their merits and demerits in terms of efficiency, cost, handling, and compatibility. The classical methods, owing to their simplicity, handling, and cost-effectiveness are mostly preferred in all sectors of extracting essential oils. Chapter 26 discusses different techniques for the extraction of essential oils from plantbased materials. The biological activity, different pathways, and chemical constituents are also discussed in the chapter to investigate the suitable treatment for the extraction purpose. Chapter 27 reviews the physicochemical/physical methods used to encapsulate essential oils, and the recent application of capsules. Green and non-thermal methods, such as supercritical fluid-based technologies along with electro-spraying reduce processing time, hence enhance encapsulation efficiency, and prolong the shelf life of encapsulated essential oils when compared to conventional processes. Chapter 28 accounts for general aspects about the techniques of coacervation, extrusion, nano-precipitation, emulsification, spray drying, thin-film hydration method, and supercritical fluid technology and their applications in the essential oils encapsulation. Chapter 29 presents encapsulation technology of essential oils, including preparation of emulsions, the encapsulation methods, and the release of encapsulated products. Several examples of successful applications and recommendations for future investigations of the encapsulated essential oil products into various industries, such as foods, cosmetics, textiles, and pharmaceuticals are also discussed. Chapter 30 reviews the advantages of the supercritical fluid extraction of essential oils and updates the readers on the current efforts to reduce the cost of products and the environmental impact provoked by SFE. Also, this chapter discussed in detail the advances in the manufacture of commercial supercritical fluid extraction equipment, the studies of economic feasibility, and the life cycle assessment of supercritical fluid extraction to improve the sustainability of this process. Chapter 31 discusses the superiority of supercritical fluid extraction of essential oils over other conventional extraction techniques. Furthermore, the effect of different process parameters influencing the efficiency of supercritical fluid extraction is deliberated upon. Optimization of supercritical fluid extraction process is reviewed using different statistical experiments like Box-Behnken design, Central composite design, Taguchi design, and artificial neuron network. Chapter 32 examines the benefits of using supercritical fluid to extract essential oils, including process parameters and their impacts, as well as examples from the literature. Information regarding industrial interest is also exemplified, such as the scale-up and economic analysis. The importance of mathematical modeling along with its applications are also discussed.

Preface  xxxi Chapter 33 summarizes the fundamentals of the extraction of essential oils with supercritical fluids at a laboratory, pilot, and industrial scale. The effects of process parameters are analyzed based on thermodynamics and available mathematical models. Finally, the combination of novel green technologies with supercritical fluids like ultrasound, microwave, or membrane separation is briefly discussed. Chapter 34 provides a basic understanding of supercritical fluids and the role of supercritical CO2 in essential oils extraction. The influence of process parameters in the supercritical fluid extraction process along with optimization using the design of experiments is explained. The applications of various mathematical models for describing extraction curves of supercritical fluid extraction are also presented. Chapter 35 discusses the classical extraction methodologies of essential oils. The selection of extraction method affects the yield of essential oils as well as their effect on the physicochemical properties. The major focus is given to communicate the chemical composition of essential oils and their pharmaceutical applications. Chapter 36 addresses the main traditional techniques for extracting essential oils from plant matrices. The advantages and disadvantages of each method are discussed, mainly in terms of their specificities and process parameters as reported in the specialized literature. Chapter 37 discusses the chemical compounds and their structures in various essential oils from extracted aromatic and medicinal plants. The essential oils can be extracted by classical and green methods, e.g. solvent extraction and supercritical fluid extraction respectively. The contemporary techniques have proved beneficial as they involve little or no solvent, less time, and energy. Chapter 38 focuses on properties and dental applications of essential oils that are being researched as a form of complementary therapy in dentistry, although few are included in the dental practices. Chapter 39 is coverage of key flora used to extract essential oils, their major therapies, and different therapeutic aspects. According to reported literature, the last decades of the 20th century was the blooming era of essential oils-based therapy which now gaining ample intention particularly in the treatment of nervous systems. Chapter 40 details the various clinical applications of essential oils. Therapeutic indications of essential oils obtained from various parts of plants in diverse disease conditions including psychological disorders, cancers, dermatological diseases, pain, and inflammation, etc. are discussed in fair detail. Chapter 41 deals with the role of essential oils and edible essential oils in the therapeutical field. The significance of the essential oils in clinical studies is discussed exhaustively. The side effects of the use of essential oils and the safety precautions to be carried out are also detailed. Chapter 42 discusses various biological activities of essential oils extracted from plants. The emphasis is on the mechanism of action, communicating the benefits, disadvantages, and future viability of various applications of the volatile oils to contribute to citizen science. Furthermore, the history of the benefits of essential oils is discussed. Chapter 43 goals are to afford a summary of current knowledge about essential oils’ chemical structure, therapeutic, and biological activities, to define their functional applications, and to evaluate the possibilities and limitations of their use in the food industry.

xxxii  Preface Highlights: • Provides a broad overview of essential oils • Explores different extraction methods of essential oils • Elaborate potential applications of essential oils in varied fields hence realizing their broad significance • Reveals potential properties of essential oils • Highlights supercritical fluid extraction with CO2 as an innovative method to obtain essential oils Inamuddin Department of Applied Chemistry, Zakir Husain College of Engineering and Technology, Faculty of Engineering and Technology, Aligarh Muslim University, Aligarh, India Tariq Altalhi Department of Chemistry, College of Science, Taif University, Taif, Saudi Arabia Jorddy N. Cruz Department of Pharmaceutical Sciences, Federal University of Pará, Belém, Pará, Brazil

1 A Methodological Approach of Plant Essential Oils and their Isolated Bioactive Components for Antiviral Activities Kunal Sharma1*, Vivek Mishra2†, Kumar Rakesh Ranjan3, Nisha Yadav2 and Mansi Sharma3 Department of Pharmacology, Sri Krishna Medical College, Uma Nagar, Muzaffarpur, Bihar, India 2 Amity Institute of Click Chemistry Research and Studies, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India 3 Department of Chemistry, Amity Institute of Applied Sciences, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India 1

Abstract

The contemporary antivirals are very limited in numbers and also there are high chances of resistance which further demands development of new antivirals. Natural products are still prime sources of innovative therapeutic agents for various illness including viral infections apart from medicinal chemical compounds. The aromatic plant oils and their isolated components are well documented to have numerous pharmacological actions, such as anti-microbial actions (antiviral, antifungal, antibacterial) along with antioxidant, anti-inflammatory, anti-immunomodulatory, and wound healing properties. The available data on essential oils and their chemical complexity confers nonspecific antiviral properties and broad spectrum mechanisms of action which make them potential candidates even for the treatment of drug resistant viral infections. The fundamental knowledge of essential oils and their bioactive components for antiviral properties along with their mechanisms of action, in vitro and in vivo studies, therapeutic targets by in silico study and also their targeted drug delivery system as nano-delivery systems are consequential to their further research, new drug design, and applications. Keywords:  Essential oil derived components, virus, antiviral properties, mechanism of action, nanocarrier delivery systems

1.1 Introduction Viruses infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea, and they grow only inside the host cells. The four general viral properties have been mainly observed for the evolutionary success of the viruses. Such as, *Corresponding author: doctorkunalsharma@gmailcom † Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (1–30) © 2023 Scrivener Publishing LLC

1

2  Essential Oils a) genetic diversity, b) numerous modes of transmission, c) well planned replication process within host cells, and d) the intelligence to persist inside host cells [1]. These properties have made viruses to adapted almost all forms of life, and have engrossed various “ecological niches”, as consequence humans and their livestock, and probably most plants suffer extensively which would further make complex situation to control disease. The genetic diverseness is typically carried through mutations. Due to rapid replication rate of the virus chances of genetic mutations increases multiple fold. The genetic mutation has been observed more in the RNA viruses because of their deficient activity in correctional capabilities i.e. “proofreading” [2]. All the viruses has their own ways of replication, therefore the virus-specific approaches to control viral infections would be more appropriate rather than a general anti-viral approach. The chances of getting mutant viral genomes have been seen more in chronic infections. The state of the persistent infection is undeniably depends on the several other factors such as, cellular and host factors which may results into reactivation of quiescent virus at any time and consequently disease episode [3] and the factors responsible for reactivation are regarded as “stressors” [4]. The imbalance between host and persistent virus infection are resultant of the certain environmental factors such as, pollutants, rise in population density, changing practices in husbandry, etc. Humans, animals, and plants are compelled to live with these increasing numbers of stressors. The notable result to these deliberations is the consciousness that the original infecting virus and reactivated virus may not be genetically identical to each other and their biological properties may differ from parent one [5]. These types of viral mutations could lead to possibilities of ramifications in molding resistance to immune control or to available antivirals, changes in virulence factors and tissue tropisms, and acquisition of immunosuppressive ability, etc. [2]. Unfortunately, the fact that viruses change quickly will always be a challenge for existing vaccines, and as a result, the host may not be entirely protected against a new viral strain. It is critical to continue the quest for effective antivirals for this reason alone. In addition, therapy will almost certainly be required in addition to prevention. Antivirals’ recent relative effectiveness against herpes viruses has inspired optimism for their future. As a result, there are a plethora of potentially helpful essential oils that must be assessed in order to prevent viral infections.

1.2 General Chemical Properties and Bioactivity Essential oils are complex aromatic mixture of various low molecular weights of natural volatile compounds, which are oily and lipid in nature. These are frequently characterized by a strong fragrance [6]. They are usually stored in specialized plant cells (e.g. oil cells or ducts, resin ducts, glands or trichomes) and extracted by various methods such as, solvent and supercritical fluid extraction, expression under pressure, fermentation or enfleurage, either pressurized (low or high) steam distillation or hydro-distillation from the leaves, flowers, buds, seeds, fruits, roots, wood or bark of plants [7]. These oils usually contain 20–60 compounds, but may contain approximately up to 100 different compounds. Chemically, The principle constituents of essential oils are Terpenes, but less frequently it may also contain

Methodological Approach of Essential Oils  3 Few Essential oil components with anti-viral properties CH3

CH3

CH3 H

CH3

H2C H 3C

H3C

CH3

CH3

H3C

OH

2

1

H

OH

4

5

CH2

H

7 CH3

CH3

10

9

OH

CH3

OH

8

CH3

CH3

CH3

O

O O

CH3

CH3

H3C

CH3

O H3C

H3C

6

CH2OH

OH

CH3

CH3

CH3

H CH3 CH

CH3

3

CH3

CH2

O

O

H3C

CH3

12

11

CH3

1.

1-8-cineole

2.

α- Terpineol

3.

α- Thujone

4.

β- Caryophyllene

5.

β- Santalol

6.

ρ- Cymene

7.

Camphor

8.

Carvacrol

9.

Cinnamaldehyde

10. Eugenol 11. Fernesol 12. Germacrone

13

13. Menthol 14. Methyl salicylate

CH3

O

CH3

CH3

CH3

O

CH3

CH3

15. Patchouli alcohol

CH3

16. Piperitenone oxide 17. Pulegone

O OH

14

O

CH3

OH

OH H3C

CH3

15

OH

O

H3C

CH3

16

CH3

CH3

CH3

17

CH3

18

H3C

CH3

19

H3C

CH3

20

18. Terpinen-4-ol 19. Terpinolene 20. Thymol

Figure 1.1  Few proven antiviral essential oil components.

phenylpropanoids and other compounds. Essential oils are mainly composed by monoterpenes and sesquiterpenes and their oxygenated derivatives, such as alcohol, aldehydes, esters, ketones, phenols, oxides, etc. [8–11]. The chemical structure of few proven antiviral essential oil components are shown in Figure 1.1. Essential oils make up a small percentage of the wet weight of plant material, usually less than 1%, and are commonly referred to as secondary plant metabolites. The molecules are typically low molecular weight and have limited water solubility [12, 13]. Many essential oils are known to possess multifunctional properties other than their traditional roles, as various biological agents have been shown to demonstrate antimicrobial (antibacterial, antifungal, and antiviral), anti-inflammatory activities, and anticancer activities [14]. Therefore, the natural chemical constituents of these oils have great potency to inhibit viral attachments, replication, and growth. The essential oils and its compounds are of special interest as valuable resources to control viral infection.

1.3 Antiviral Mechanisms Essential oil and their chemical constituents possess antiviral activity due to their lipophilic nature. The antiviral drugs should have ability produce its anti-viral effect without affecting host cells. The drugs should have potency to inhibit or alter the specific process of the virus replication cycle. There are seven basic steps in the viral replication cycle Figure 1.2 [15, 16]. Viruses have high ability to develop resistance to specific drugs, therefore potent virucidal drugs are being needed, which have capability to disrupt its envelope along with its genetic material which further causes complete loss of virus infectivity.

4  Essential Oils 7. Release

6.

virus atttaches to host cell wall and injects its genetic material into the host cell cytoplasm either Translocation, endocytosis or by fusion of viral envelop.

Attachment

Steps of Viral replication

Maturation

Attachment and penetration

1.

2. Penetration

5.

3.

Assembly

Uncoating

Uncoating, replication, and assembly viral DNA or RNA incorporates itself into genetic material of the host cell and trigger the replication of the viral genome. newly Maturation and Release formed viruses are being matured and then released from the infected host cell, either by cell break, cell death, or cell budding through the cell membrane.

4. Replication

Figure 1.2  Replication cycle of virus.

1.3.1 Time of Addition Assay The viral infectivity during infective phase is inhibited by the essential oils by hampering its infectivity (Figure 1.3). The components of essential oils acts against the virus are commonly determined by the manipulation of time of addition assays. It is determined by the time dependent addition of the maximum non-cytotoxic dose of essential oil to the culture cells before, during, and after virus infection (Figure 1.4). It is the most commonly used

Herpes Simplex Virus Adsorption or binding Of virus to host Cell membrane Uncoating

Genome Replication Release of new progeny viruses

Internalization or penetration

Ef fect of Essential Oil

Nucleus Host cell cytoplasm

Assembly of new progeny viruses

Extracellular space

Figure 1.3  Essential oil activities on specific target sites during the viral life cycle for enveloped viruses [18].

Methodological Approach of Essential Oils  5 Time of Addition Experiment

Intracellular activity

Intercellular activity

Essential oil

Essential oil

HSV 7. Release

Alteration on the virus envelope, or

Incubation for 60 mins at 37°C

+ve

-ve

Af fect on viral No af fect on viral attachment on blocking attachment, adsorption host cell receptor by or entry into the host cell Essential Oils by Essential Oils

Masking the viral proteins

+ve Af fect on viral attachment, adsorption or entry into the host cell by Essential Oils

1. Attachment

2.

Penetration Steps of Viral replication

5.

3. Uncoating

Assembly

iff at d

No af fect on viral attachment on blocking host cell receptor by Essential Oils

6.

Maturation

Indicate interference on free virions either by Expose to virus

Essential oil added

Normal culture cell

+

Pretreated HSV

Cultured cells pretreated with essential oil for 60 mins at 37ºC

-ve

+

60 mins at room temperature

4.

yc le

Normal culture cell

Replication er en nc t st ages of replicatio

By this method the stage of viral replication at which essential oils antiviral activity can be accessed.

Figure 1.4  Time of addition experiment.

procedure to evaluate inter- and intra-cellular antiviral activity of essential oils. The highest antiviral effects were seen when herpes simplex virus was incubated with essential oils before addition to the cultured cells, which indicates direct virucidal action of essential oils [17]. The common methodology by which time of addition assay determined are briefly described below [18, 19].

1.3.1.1 Pretreatment of Host Cells Prior to inoculation with virions, the mono-layered culture cells are pretreated with varying concentrations of the selected antiviral. This assay decides whether selected antiviral could able to prevent attachment of the virus to host the cells.

1.3.1.2 Pretreatment of Virions Virion incubation is done separately from the host/cultured cells at various concentrations of selected antiviral, followed by direct injection of the pretreated virions into the host/ cultured cells. The assay investigates the antivirals virucidal and neutralizing capabilities.

1.3.1.3 Co-Treatment of Host/Cultured Cells and Virions During Virus Inoculation The virions are initially mixed with various selected concentrations of antiviral and then directly inoculated to the host/cultured cells. This assay determines the selected antiviral effects on the steps of viral entry.

6  Essential Oils

1.3.1.4 Post-Entry Treatment In this assay, virions are first inoculated into the host/cultured cells to allow viral penetration, and then virus-infected cells are treated with a various concentrations of selected antiviral. The assays guides whether selected antiviral can interfere intracellularly to the viral replications, such as genome translation and replication, virion assembly and release, etc.

1.3.2 Thermal Shift Assays The thermal shift assays can able examine additional steps of the viral inoculations such as viral attachments and entry or fusion stages, by the temperature shifting during the infection. As, entry/fusion activity of the many enveloped viruses at the host/cultured cell membrane gets inhibited at 4°C and exhibit at 37°C, which does not interfere viral attachment. The following are the brief methodology of Thermal shift assays [20].

1.3.2.1 Viral Attachment Assay At 4°C, a mixture of virions and selected antiviral is incubated with the pre-cooled host/ cultured cells. Subsequently, the host/cultured cells are washed and shifted to 37°C without antiviral to permit viral internalization. Because the chosen antiviral inhibits virus attachment to the host cell membrane, the decrease in plaque development is considered a good result.

1.3.2.2 Viral Fusion Assay (Entry Assay) Virions are inoculated to pre-cooled host/cultured cells at 4°C in this assay, facilitating viral binding to the host/cultured cell membrane, but not viral penetration. The incubated cells of virion are then cleaned to eliminate free (unbonded) viral particles before being treated with a selected antiviral at 37°C. The reduction in plaque formation is considered as positive result of the test, which means that the selected antiviral hinders virus entry (penetration) into host cells [21].

1.3.3 Morphological Study The TEM imaging study was done to observe the direct effect of essential oil on the virus particles, which mainly determined by the structural alteration of the virus. This test is done to differentiate the antiviral effect of essential oil on the stage of adsorption is mainly due to destruction or masking of the viral particles [22]. Many researchers have observed that two major types of structural changes in the virus after treatment with essential oils or their constituents. It characterized either by the (1) swelling or expansion of the diameter of virus alone (Figure 1.5), and/or (2) capsid disintegration when exposed to certain essential oils (Figure 1.6) [23]. The capsid acts as safeguards to the viral-RNA from disintegration and triggers infection via adsorption to host/cultured cells in non-enveloped viruses. In non-enveloped viruses, the capsid protects the viral-RNA from destruction and initiates infection by adsorption to host/cultured cells [24]. Though, studies have been shown that partially degraded capsid in RNA virus may still be infectious and lead to enter normal viral replication cycle which

Methodological Approach of Essential Oils  7

(a) Untreated MNV (20-35 nm)

(b) MNV after exposure to 4.0% allspice oil for 30 min (upto 75 nm)

(c) MNV after exposure to 4.0% lemongrass oil for 24 h (100-500 nm)

(d) MNV after exposure to 4.0% citral for 24 h (350-750 nm)

Figure 1.5  Diagrammatic representation of expansion of the diameter of virus by essential oils in Murine Norovirus (MNV) (not scale) [23].

(a) Untreated MNV (20-35 nm)

(b) (c)

(d) varying degree of MNV capsid disintegration when exposed to antiviral essential oils

Figure 1.6  Diagrammatic representation of capsid disintegration of Murine Norovirus (MNV) by the effect of antiviral essential oil (not scale) [23].

revealed in RNAase I protection assay [25, 26]. Certain enveloped virus like HSV-1, also showed dissolution of its envelope by the treatment with clove (Syzygium aromaticum) and oregano (Origanum vulgare) essential oil [27]. The individual essential oil component such as monoterpenes was also noted to enhance cytoplasmic fluidity and permeability, which further derange the order of embedded proteins of the membrane [28].

1.3.4 Protein Inhibition Influenza virus contains two types of membrane glycoproteins, (1) Hemagglutinin and, (2) Neurominidase. The hemagglutinin protein is present on the envelope on the influenza virus particles and plays important roles in attachment and penetration. It produces agglutination reaction of erythrocytes when viral hemagglutinin protein combines with N-acetyl-neuraminic acid of the mammalian or avian erythrocytes. All these processes make hemagglutinin inhibition assay as a very useful test to detect influenza virus activity. The essential oils show its effects in inhibition of influenza virus activities during adsorption

8  Essential Oils by negative reports on hemagglutinin inhibition study [29, 30]. A study showed inhibition of hemagglutination by the many vapors of the essential oils such as, Citrus bergamia, Cymbopogen flexuosus, Cinnamomum zeylanicum, Eucalyptus globulus, Pelargonium graveolens, Salvia officinals, and Thymus vulgaris [31]. Another membrane glycoprotein is Neuraminidase, which helps virus to release from the host cell membrane after budding. This is very important target sites for the anti-influenza drugs. Zanamivir and Oseltamivir are the synthetic neuraminidase inhibitors. The most of the Essential oils actively inhibits hemagglutinin but only few showed neuraminidase envelope protein inhibition, such as Cinnamomum zeylanicum [31]. The hemagglutinin and neuraminidase inhibition effects are dependent on the types of essential oils and also to their active compounds, such as, cedar leaf essential oil strongly inhibits hemagglutinin but two major components like thujone and pinene were found incapable of its inhibition due to some unknown mechanism [32]. Tat (trans-activator of transcription) protein plays pivotal role in HIV transcription and reactivation of virus from its latent stage. It interacts with TAR (trans-activation region) RNA and forms Tat/TAR RNA complex which required for transcription and further HIV-1 replication [33]. A group of Italian scientists were studied the effects of essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis on Tat/TAR RNA complex by using gel electrophoresis [34]. In another study, the essential oil of Ridolfia segetum and Oenanthe crocata were studied for HIV-1 inhibitory activity. The important viral enzyme reverse transcriptase is associated with DNA polymerase and ribonuclease H activities. Both enzymes are mandatory for reverse transcriptase activities for HIV-1 replication. In further assay, dose dependent inhibition of viral reverse transcriptase associated with RNA-dependent DNA polymerase activity was reported [35]. The constituents of the essential oil of perennial grass Cymbopogon nardus which frequently used in traditional anti-AIDS medications were studied for the anti HIV-1 activity. An in-vitro study of (S)-βcitronellol a monoterpenoid of Cymbopogon nardus oil showed inhibition of HIV-1 reverse transcriptase activity [36]. The in-silico studies (virtual screening) are a vital tool in a field of drug screening and discovery. The findings of the in-silico studies could be utilized further for the in-vitro and in vivo assays. Molecular docking improves the chances of selecting suitable leads for drug discovery in a very short period of time with minimal costs [37]. Many in-silico studies have been carried out to look the potential antiviral targets for essential oils and their components. A computational study on dengue virus proteins was done to look the interactions between the constituents of essential oils and the viral proteins. The findings were suggestive for inhibition of the dengue virus by the most of the constituents. The essential oil constituents bind to their particular binding sites through hydrophobic interactions to the nonpolar domain of the viral proteins [38]. Many molecular docking studies were also done for COVID-19. In a study, out of 171 components of essential oils the EE-farnesol, EE,α- farnesene, and E,β-farnesene were found as best ligands for SARS-CoV-2 endoribonucleoase (Nsp15/NendoU) enzyme. Among all these ligands the E,E-farnesol appeared as most exothermic docking to SARS-CoV-2 ADPribose-1”-phosphatase (ADRP) [39]. In an another recent study on garlic essential oil out of 18 identified compounds 17 have shown inhibition of host ACE-2 (angiotensin converting enzyme) and also to the main SARS-CoV-2’s protease (PDB6LU7). The allyl disulfide and allyl trisulfide show good anti-corona activity in molecular docking study  [40].

Methodological Approach of Essential Oils  9 The  1,8-cineole, a compound from eucalyptus essential oil showed significant binding to SARS-CoV-2 proteinase via ionic interactions, hydrophobic interactions and hydrogen bond [41]. Isothymol, a constituent of essential oil from a western Algerian aromatic medicinal plant Ammoides verticillata, showed blocking of host ACE-2 receptor of SARSCoV- 2 via π- Hydrogen bonding [42]. The chemical constituents of ginger (like 8‑Gingerol, 10‑Gingerol) and black pepper (like Piperdardiine, Piperanine) were also found active against COVID‑19 by hydrogen bonding and hydrophobi interactions in-silico study [43]. However, the polarity of bioactive essential oil components that impacts antiviral activity, as well as interactions that apply to cases involving other viruses and essential oil involvement, must be clarify.

1.3.5 Other Metabolic Anti-Viral Mechanisms Uncoating of Influenza virus occurs when the endosomal and liposomal environment convert to acidic during the infection [44]. Tea tree oil as well as its bioactive component terpinen-4-ol were trialed against the influenza virus replication and reported to suppress viral replication cycle by interfering with the intra-lysosomal acidification process, the major factor for viral uncoating [45]. Many researchers had already proposed theories about the endolysosomal acidification may be good therapeutic target due to its capacity to check the escape of viral genome to the host cell cytoplasm and increase the virus degradation in lysosomes [46]. Further investigations are needed on the alteration of endosomal and/or lysosomal pH of cell and the effect of essential oils or its bioactive components at the stages of the viral replication cycle. There is also intracellular glutathione takes part in various metabolic activity and cellular defense system, depletion of it associated with host cell redox changes were noted in many viral infections [47–50]. The Mentha suaveolens essential oil derived component piperitenone oxide was found to targeting redox signaling pathway and thus inhibiting HSV-1 replication in late stage [51]. There are also available few theories and researches which suggest the genomic targets for essential oils for its antiviral activity [52].

1.4 Assessment of Antiviral Activities via In Vitro Assays When the evaluation of the antiviral activities for essential oils in vitro is concerned, the three methods, such as Cytopathogenic reduction assay, Plaque reduction assay, as well as Viral yield reduction assay are commonly applied [54]. Almost all antivirals showed dose dependent inhibition of the virus. The cytotoxicity study is necessary to establish that essential oils are safe and do not possess toxicity on host/cultured cells at assayed concentrations.

1.4.1 Determination of Cytotoxicity (Cytopathogenic Reduction Assay) Cytotoxicity usually expressed as cytopathogenic effect. The standard neutral red cell cytotoxicity assay is a common in-vitro method to establish cell viability or xenobiotic toxicity. Most of the primary cell lines from different origins may be successfully used for this assay such as, the kidney cell lines (Vero, OK, and RC-37), and human tumor cell line (HeLa) [55]. The assay assesses the xenobiotic concentration-dependent uptake of neutral red dye by the viable cells. The assay quantify the uptake of neutral red dye by the viable cells in

10  Essential Oils concentration dependent manner of xenobiotics. The 50% cytotoxic concentration (CC50) of xenobiotic when the number of the viable cells reduced to its half similarly determines cytotoxicity of essential oils. The potential antiviral selective index (ASI) of essential oil is particularly determined by the ratio of CC50 to 50% viral infectivity concentration (VIC50) i.e. CC50:VIC50, the higher value of therapeutic index determines its selectivity to anti-viral effect [56]. It is consider to have VIC50 values should be lower to 100 μg/mL for mixture of plant extract, 25 μM for isolated pure compounds [57], and acceptable ASI value is >4 [58]. Some common sources of antiviral essential oil are delineated in Table 1.1. Table 1.1  Some common sources of antiviral essential oils. Viruses

Source of essential oils

VIC50

ASI

Mechanisms of action

Ref.

HSV-1

Illicium verum

1 μg/mL

160

Intercellular

[59]

HSV-1

Mentha suaveolens (Apple mint)

5.1 μg/mL

67

Intracellular

[51]

HSV-1

Artemisia kermanensis

0.004%

66.37

ND

[60]

HSV-1

Zataria multiflora

0.004%

55.44

ND

[60]

HSV-1

Rosmarinus officinalis

0.006%

46.12

ND

[60]

HSV-1

Australian tea tree

13.2 μg/mL

43

Intracellular

[51]

HSV-1

Eucalyptus caesia

0.007%

38.81

ND

[60]

HSV-1

Satureja hotensis

0.008%

97.70

ND

[60]

HSV-1

Mexican oregano (Lippia graveolens)

99.6 μg/mL

7.4

Intercellular

[61]

HSV-1

Thymus capitatus

17.6 μg/mL

6.9

Intercellular

[62]

HSV-1

Lallemantia royleana

0.011%

6.4

Intercellular

[63]

HSV-1

Sinapis arvensis

0.035%

1.5

Intercellular

[63]

HSV-1

Pulicaria vulgaris

0.001%

1

Intercellular

[63]

Acyclovir-resistant HSV-1

Mexican oregano (Lippia graveolens)

55.9 μg/mL

13.1

Intercellular

[61]

HSV-2

Thymus capitatus

18.6 μg/mL

6

Intercellular

[62]

Acyclovir-resistant HSV-2

Salvia desoleana

28.57 μg/mL

55.2

Both Inter- and intra-cellular

[64] (Continued)

Methodological Approach of Essential Oils  11 Table 1.1  Some common sources of antiviral essential oils. (Continued) Viruses

Source of essential oils

Bovine herpes virus 2

Mechanisms of action

VIC50

ASI

Ref.

Mexican oregano (Lippia graveolens)

58.4 μg/mL

9.7

Both Inter- and intra-cellular

[61]

Influenza virus-A (H1N1)

Pelargonium graveolens

21

Intercellular

[31]

Influenza virus-A (H1N1)

Lavandula officinalis

8

Intercellular

[31]

Influenza virus-A (H1N1)

Citrus bergamia

5

Intercellular

[31]

Influenza virus-A (H1N1)

Cinnamomum zeylanicum

4

Intercellular

[31]

Influenza virus-A (H1N1)

Cymbopogon flexuosus

4

ND

[31]

Influenza virus-A (H1N1)

Thymus vulgaris

4

Intercellular

[31]

Influenza virus-A (H1N1)

Patchouli

0.088 mg/mL

1.15

ND

[65]

Influenza virus-A (H1N1)

Eucalyptus globulus

0.5

Intercellular

[31]

Avian influenza virus A (H5N1)

Citrus reshni ripe fruit peel

2.5 μg/mL

8.7

ND

[66]

Avian influenza virus A (H5N1)

Fortunella margarita fruit

6.8 μg/mL

ND

ND

[67]

HIV-2

Cymbopogon citratus

0.09 μg/mL

3.6

Intracellular

[34]

HIV-1

Thymus vulgaris

0.3 μg/mL

1.57

Intracellular

[34]

HIV-1

Rosmarinus officinalis

0.65 μg/mL

1.13

Intracellular

[34]

HIV-1

Cymbopogon nardus

1.2 mg/mL

ND

ND

[52]

Coxsackie virus B4

Osmunda regalis (Tunisian fern)

22 μg/mL

789.8

ND

[68]

Coxsackie virus B4

Dysphania ambrosioides

21.7 μg/mL

74.3

ND

[69] (Continued)

12  Essential Oils Table 1.1  Some common sources of antiviral essential oils. (Continued) Source of essential oils

VIC50

ASI

Mechanisms of action

Ref.

Coxsackie virus B3

Eucalyptus bicostata

0.7 mg/mL

22.8

Intercellular

[70]

Coxsackie virus B3

Patchouli

0.081 mg/mL

1.2

ND

[65]

Coxsackievirus B

Teucrium pseudochamaepitys

589.6 μg/mL

1.11

ND

[71]

Bovine viral diarrhea virus

Ocimum basilicum

474.3 μg/mL

3.7

ND

[72]

Bovine viral diarrhea virus

Mexican oregano (Lippia graveolens)

78 μg/mL

7.2

Intracellular

[61]

Respiratory syncytial virus

Mexican oregano (Lippia graveolens)

68 μg/mL

10.8

Intercellular

[61]

Respiratory syncytial virus

Patchouli

0.092 mg/mL

1.1

ND

[65]

Yellow fever virus

Lippia alba

4.3 μg/mL

30.6

Both Inter- and intra-cellular

[73]

Adenovirus-3

Patchouli

0.084 mg/mL

1.2

ND

[74]

Zika virus

Ayapana triplinervis

38 μg/mL

12.5

Intercellular

[75]

Murine norovirus

Thymus capitatus

0.49 μg/mL

ND

ND

[76]

Dengue virus

Lippia alba

0.4-33 μg/mL

4-349

Intracellular

[77]

Japanese encephalitis virus

Trachyspermum ammi

80% in 0.5 mg/mL

ND

Intracellular

[78]

Viruses

Note: HSV, human herpes virus; HIV, human immunodeficiency virus; ASI, antiviral selectivity index. ND, not determined. Intercellular, oil targeting on viral surface during adsorption or preadsorption phase; Intracellular, oil targeting on intracellular activity during virus replication cycle.

1.4.2 In Vitro Activities on Different Viruses 1.4.2.1 Human Herpes Virus Human herpes viruses (e.g., HSV-1 and HSV-2) belong to Herpesviridae family are enveloped and contains double stranded DNA as their genetic material. The most of the antiviral essential oils researches are done for Human herpes virus in last few years. The antiherpes

Methodological Approach of Essential Oils  13 action, preferentially in vitro anti-HSV-1 actions shown by many essential oils, such as Star anise (Illicium verum), Oregano, Eucalyptus caesia, etc., is shown in Table 1.1. Star Anise essential oil found to have most potent anti-HSV-1 intercellular action with VIC50 value of 1 μg/mL and ASI value of 160 [53]. There are also other essential oils from Australian tea tree (Melaeuca alternifolia) and mint plant (Mentha suaveolens) showed having good potency as antivirals to act on intercellular HSV-1 [51]. The wide range of essential oils from Labiatae, Lamiaceae, Myrtaceae, Umbelliferae aromatic plants were screened and found having anti-HSV-1 activity [53, 79]. Among all individual isolated essential oil derived compounds, β-caryophyllene found to have most effective anti-HSV-1 activity with VIC50 of 0.25 μg/mL (~1.2 μM) and ASI values of 140 [59] (Table 1.2). Thus, aromatic plants containing β-caryophyllene, such as Piper nigrum (black pepper), Cinnamomum verum (cinnamon), Syzygium aromaticum (cloves), Origanum vulgare (oregano), and Cannabis sativa (cannabis) may considered as having antiviral activity against HSV-1 [80]. Previous research on commonly encountered essential oil components for their individual anti-HSV-1 activities found that all were found to have strong antiviral activities at concentrations ranging from 0.025 to 0.8 g/mL [53]. Due to substantial cytotoxicity (ASI 4) and inefficacy (VIC50 >25 M), 1,8-cinole [38], Table 1.2  Few common bioactive essential oil components with antiviral properties. Viruses

Components

VIC50

ASI

Mechanisms of action

Ref.

HSV-1

β-Caryophyllene

0.25 μg/mL

140

Intercellular

[59]

HSV-1

β-Eudesmol

6 μg/mL

5.8

Intercellular

[59]

HSV-1

β-Pinene

3.5 μg/mL

24.3

Intercellular

[90]

HSV-1

ρ-Cymene

>0.1%

ND

Intercellular

[63]

HSV-1

Carvacrol

7 μM

43

Intercellular

[91]

HSV-1

Carvacrol

0.037%

1.4

Intercellular

[63]

HSV-1

Carvacrol

48.6 μg/mL

5.1

Intracellular

[61]

HSV-1

Eugenol

35 μg/mL

2.4

Intercellular

[59]

HSV-1

Farnesol

3.5 μg/mL

11.4

Intercellular

[59]

HSV-1

Limonene

5.9 μg/mL

10.2

Intercellular

[90]

HSV-1

Trans-anethole

20 μg/mL

5

Intercellular

[59]

HSV-1

Thymol

7 μM

43

Intercellular

[91]

HSV-2

1,8-cineol

6.9 mg/mL

ND

Intracellular

[59]

HSV-2

menthol

7.2 mg/mL

ND

Intracellular

[39] (Continued)

14  Essential Oils Table 1.2  Few common bioactive essential oil components with antiviral properties. (Continued) Viruses

Components

VIC50

ASI

Mechanisms of action

Ref.

Acyclovirresistant HSV-1

Carvacrol

28.6 μg/mL

8.7

Intracellular

[61]

Bovine herpes virus 2

Carvacrol

663 μg/mL

0.3

Intracellular

[61]

Influenza virus-A (H3N2)

β-Santalol

10–100 μg/mL

Intracellular

[85]

Influenza virus A (H1N1)

Carvacrol

2.6 μg/mL

30% with VIC50 values below 100 μg/mL. The same study showed marjoram (Thymus mastichina), clary sage (Salvia sclarea) and anise (Pimpinella anisum) oils having more than 52% higher anti-influenza activity than oseltamivir without any cytotoxicity [82]. The essential oils from Cinnamomum zeylanicum, Thymus vulgaris, Citrus bergamia, etc. were also observed having significant anti-influenza activity in different studies (Table 1.1) [31]. Germacrone was found to be most effective essential oil derived compound against influenza virus with observed ASI value of more than 41, causing inhibition of multiple steps in viral replication cycle due to its broad spectrum mechanism of action [83]. The results from the studies on germacrone, a principle compound of Rhizoma curcuma (a Chinese medicinal herb) showed broad spectrum antiviral activity and could be considered as reference compound for the drug development of novel antivirals. Few components like 1,8 cineole and eugenol found to have active against both HSV-1 and influenza virus [31, 84]; while other studies revealed β-santalol [85] and terpinen-4-ol [45], the oxygen bearing components were major bioactive compounds against influenza virus. The results suggested that there could be complementary effects of few components of essential oil for anti-HSV and anti-influenza. The results also suggested that oxygenated terpenes are more effective against influenza virus and herpes viruses generally inhibited by non-oxygenated terpene hydrocarbons, but this needs to be verified by further research.

1.4.2.3 Non-Enveloped Viruses The maximum studies for antiviral essential oils were focused on enveloped viruses, while relatively less attention paid to non-enveloped viruses in the past few years. Enveloped and non-enveloped viruses have variations for the essential oil targets. In enveloped viruses, a large portion of essential oils were showed interaction with the viral envelope to have an antiviral impact prior to host cell infection, whereas in non-enveloped viruses have a limited number of active sites are available for essential oils to target. In-vitro study of an essential oil of a Tunisian fern Osmunda regalis was studied against Coxsackie virus B4, which showed VIC50 value of 2.24 μg/mL with very high ASI value of 789.84 [68]. The essential oils from Dysphania ambrosioides and Eucalyptus bicostata also demonstrated significant activities against Coxsackie virus [69, 70]. Carvacrol is a major component of Lippia graveolens (Mexican oregano) essential oil to exhibit strong inhibition of rotavirus and murine norovirus [61]. Germacrone a major active constituent of rhizoma curcuma, a traditional Chinese medicine was studied to determine antiviral efficacy against the multiple strains of a highly contagious feline calcivirus (a non-enveloped RNA virus) which usually involve

16  Essential Oils respiratory tract and oral disease in cats and was found significant dose dependent inhibition of feline calcivirus replication in early stage [86]. All these researches showed effects of essential oils against non-enveloped viruses but not able to describe mechanism of actions in detail and also further studies is needed to elucidate the antiviral actions of individual bioactive compound.

1.4.2.4 Other Viruses The inhibition of reverse transcriptase activity of HIV-1 virus was shown by essential oil of Cymbopogon nardus with VIC50 vaule of 1200 μg/mL and the researcher hypothesized that effect was due to a bioactive compound (S)-β-citronellol, which present particularly in the leaves and stems of this African traditional plant [52]. There are also few other EOs from thyme (T. vulgaris), lemon grass (C. citratus), and rosemary (R. officinalis) were shown effective inhibition of HIV-1 Tat/TAR-RNA interaction with VIC50 value between 0.05 – 0.83 μg/mL along with ASI value below 4 [34]. The effects shown by these studies provide possible options for development of new HIV antivirals. In another study Dengue-2 virus was efficiently inactivated by β-caryophyllene through both inter- and intra-cellular action with VIC50 of 22.5 μM and ASI value of 71.1 [38]. Recently, 1,8-cineole [41] and isothymol [42], the essential oil derivatives, are suggested against COVID-19. There are also few in-vitro studies essential oils showed encouraging results against bovine viral diarrhea virus [61], Zika virus [75], respiratory syncytial virus [32], yellow fever virus [73], and caprine alpha herpes virus [87], etc. The broad spectrum antiviral effect was seen by carvacrol an aromatic compound but the caution is needed before its application as it exhibits very high cytotoxicity which indicated by comparatively low ASI values.

1.5 Activities of Essential Oils in Relation to Their Bioactive Components The bioactivities of essential oils are mainly affected by their chemistry. Still, there are many contradictions regarding the role of the chemistry of the essential oil and its antiviral activity. The few common bioactive essential oil components with antiviral properties are delineated in Table 1.2. There are also role of minor component which may more bioactive then major ones, as 1,8 cineole is present as major component in the oil of both Eucalyptus globulus and Salvia officinalis, but stronger anti-H1N1 activity (VIC50 < 3.1 μ/ml) seen with Salvia officinalis [31]. The essential oil of Cinnamomum zeylanicum contains Eugenol as a major component, which has been found effective for H1N1 infection and, antiviral efficacy present in its principle component. The anise (Pimpinella anisum), marjoram (Origanum majorana) and clary sage (Salvia sclarea) oils were observed to have anti influenza activity and through further analysis, Linalool was found as a common constituents for all three oils, which further supposed to exhibit antiviral effect [31]. A major bioactive component of essential oil of Pogostemon cablin, patchouli alcohol tested for anti-influenza virus –A (H2N2) activity and showed virucidal effect not due to direct inhibition of Hemagglutinin or Neurominidase

Methodological Approach of Essential Oils  17 enzyme which could be speculated due to the destruction of viral envelope. The further study is needed to ascertain the virucidal effect of patchouli alcohol [88]. The essential oil of Lippia graveolens (Mexican oregano) and its bioactive component carvacrol were investigated and compared with each other for their antiviral properties. The highest activity of pure Mexican oregano oil was observed against respiratory syncytial virus, HSV-1, and bovine viral diarrhea virus, while rotavirus was effectively inhibited by carvacrol, but could not be inhibited by the pure Mexican oregano oil even at higher concentrations. Further, Lippia graveolens essential oil could not able to inhibit rotavirus at all tested concentration i.e. 25–3200 g μ/mL [61]. The results indicate that, there are case dependent action of individual components of essential oil and terpenes may not provide equal contribution to antiviral efficacy. There are also few studies which showed higher safety index and a lower toxicity of essential oil mixture than its isolated single components, and this makes value of essential oil mixture differently [61, 74]. But this does not mean that essential oil mixtures are superior to their isolated compound, the antiviral effects always depend on its uses and circumstances. The existing bioactive components, either major or minor, determine the bioactivity of essential oils. The further detailed study is needed for isolation and also for the bioactivity of each individual component to find out precise role of every compound in essential oil.

1.6 Antiviral Activities as Compared to the Polarity of Bioactive Components The polarity of the essential oil derived components is also being hypothesized as one of the important confounding factor for the binding affinity to viral proteins which further influence essential oil for their antiviral properties. The essential oils from the native flowering plant of South America, Glechon spathulata and Glenchon marifolia showed decreased in their HSV-1 efficacy when exposed to light for 20 days. They were also noted to decrease in non-oxygenated monoterpenes hydrocarbons levels, which is considered to have major active anti-viral properties [93]. The components of essential oil of a Sardinian traditional medicinal plant Salvia desoleana studied in-vitro for the anti- HSV-2 activity. The result of bioassay guided fractionation of Salvia desoleana essential oil contained germacrene D (54%) as a major terpene hydrocarbons fraction and the second major was β-caryophyllene (4.8%). The other fractioned was found containing mixed oxygenated terpenoids, which further characterized by the presence of 1,8 cineole, α-terpinyl acetate, linalyl acetate and linalool. Later in the study, the results revealed the anti-HSV-2 activity (for both Acyclovir sensitive and resistant) was only present in the components of terpene hydrocarbons, not in the components of oxygenated terpenoids [64]. Another study on six essential oil derived components phenylpropanoids and sesquiterpene was done to look the effect of polarity and antiviral bioactivity of the essential oil components, which showed increase in polarity reduces antiviral activity [59]. Further, thymol-related monoterpenoids were investigated for anti-HSV-1 effect, and found anti-herpes reduced properties with declining polarity in the order: -hydroxyl (-OH) >-amine (-NH2) > -methyl (-CH3) > -hydrogen (-H) [91]. The Colombian Scientists did a study where the binding affinities of essential oil components were calculated and compared against the structural proteins of Dengue viruses.

18  Essential Oils This in silico study included sesquiterpenes and monoterpenes irrespective of their functional group containing oxygen atoms, like as aldehyde, ketone, carboxylic acid, etc. and further observed that the compounds belong to sequiterpenes family, like α-Copaene, germacrene D, β-caryophyllene, caryophyllene oxide, spathulenol, β-bourbonene and (+)-epi-bicyclosesquifellandrene having the highest affinity [38]. The most of the observed interactions between essential oil components and dengue virus proteins were hydrophobic in nature, which could be further extrapolated as greater interactions with viral proteins showed by the non-polar terpenes. The investigations stated above revealed that germacrene D and β-caryophyllene have broad antiviral properties. The antiviral properties of the oxidized form of germacrene D and β-caryophyllene have not yet been thoroughly investigated, and more research is needed.

1.7 In Vivo Studies of Essential Oils for its Antiviral Effect The preclinical in vivo studies carried out to gain additional information about the compound in laboratory animals. In vivo study is done to confirm the selectivity index estimated by in vitro studies. The usual applied dosage regimens of 7 and 14 days are used in dose range-finding studies in small laboratory animals. Endpoints evaluation includes body weight, clinical signs and symptoms, body fluid testing, histopathological analysis and other pharmacokinetic parameters. The favorable findings of related to antiviral efficacy of the essential oils or its derivatives along absence of animal toxicity need to be established to proceed further for the clinical trials in Humans [94].

1.7.1 Herpes Simplex Virus The anti-herpes effect of eugenol was studied on BALB/c mice herpetic keratitis model. The virus were inoculated by making scar over left cornea and eugenol drops of different concentrations (1 mg/ml and 0.5 mg/ml) were administered in two separate test groups at 24 hours and 2 hours before viral inoculation and the same treatment was continued till fifth post inoculation day. The cornea, iris and lids were further evaluated periodically by using slit lamp till 21 days. The result revealed, 1 mg/ml eugenol treatment of corneal infected mice showed delayed development of keratitis as compared with placebo controlled group but could able to hold disease progression [95]. A placebo controlled randomized, single blinded clinical trial of topical application Melaleuca alternifolia (Tea tree) oil was done on the patient of recurrent herpes labialis, aged between 18 to 70 years. The 6% tea tree oil in an aqueous gel base was advised to apply five times daily and daily swabs were collected for HSV detection PCR and culture till re-­ epithelialization along with two consecutive negative PCR samples for HSV. The observed median time of re-epithelialization for the treatment group was 9 days, whereas 12.5 days in placebo group. The viral titers observed lower in tea tree oil group as numerically compared with placebo group, but not reached its significance level [96].

1.7.2 Influenza Virus Patchouli alcohol a major constituent of the essential oil of Pogostemon cablin (Blanco) an East Indian shrub investigated for its anti-influenza virus A (H2N2) effect in mouse model

Methodological Approach of Essential Oils  19 after proper toxicity study. Mouse model was created by intranasal instillation of H2N2 virus. The efficacy of Patchouli alcohol was evaluated in the basis of their 15 days post infection survival rate and compared with the efficacy of 1 mg/kg/day dose of oseltamivir. The results (outlive/total) showed 7/10 for patchouli alcohol (5 mg/kg/day), 5/10 for oseltamivir and 0/10 for non-treated control mice. The 5 mg/kg/day dose of Patchouli alcohol showed definitive protection against Influenza virus A [88]. Another study in which Kunming mice were divided into different study groups after creating mouse pneumonia model by instillation of intranasal H1N1 virus (IFV-A/PR/8/34). Subsequently, 4 hours after inoculation treatment group received 20–40 μg daily dose of intranasal patchouli alcohol and another group subjected to 10 mg/kg daily dose of oral oseltamivir for 7 days. On the fourth day few mice from each groups were euthanized to assess pulmonary viral titers by plaque reduction assay and remaining were left alive up to 14 days to determine survival rate. The plaque reduction assay on fourth day showed significant reduction in pulmonary viral titers in patchouli alcohol and oseltamivir treated groups as compared to placebo controlled groups. There was also reported 14 days increased in survival time as 100% in 40 μg/day patchouli alcohol treated groups, the effect found superior to oseltamivir groups (90%) in contrast to placebo group (only 30%) [95, 97]. A study done by the scientists of Wuhan Institute of Virology, China to observe the effect of Germacrone on BALB/c mice infected with H1N1 (Swine flu) virus. Germacrone in the daily dose of 50 mg/kg and 100 mg/kg was started 24 hours before virus exposure to the mice and daily intravenous dose was given for 5 days. The other combined (Germacrone 100 mg/kg/day + oseltamivir 1 mg/kg/day) group, positive (oral oseltamivir 50 mg/kg/day) control group and negative (placebo) control groups were treated and also observed for their survival in a similar manner. For the estimation of survival rate, all the treatment group animals were observed for 18 days. The results showed 50% effective survival in germacrone (100 mg/kg/day) group and oseltamivir (1 mg/kg/ day) group along with fourth post infection broncho-alveolar lavage fluid showed significant reductions in viral titer. The combined group showed additive effect and improved survival rate up to 90%. The study was concluded as Germacrone exhibited effective protection and reduced viral titers in lung in swine flu in mouse model. Moreover, the combination of germacrone and oseltamivir revealed additive against influenza virus infection, both in vitro and in vivo [83]. The major monoterpenes of eucalyptus essential oils, 1,8-cineole was evaluated against influenza virus-A in a murine model. There were six study groups (10 each) of BALB/c mice, 1,8-cineole study groups (concentrations of 30.0, 60.0, and 120.0mg/kg + H1N1), positive control study group (oseltamivir 10 mg/kg), placebo control study group (IFV-C + saline + 0.5% Polysorbate 80) and a negative study treatment group (non-infected). The 1,8-cineole and oseltamivir group of mice were received treatment 2 days before intranasal inoculation of virus and further selected treatment maintained up to fifth post infection period to all the groups. On 6th day few mice were sacrificed from each group for the assessment for viral load, cytokine, pathological changes, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and nuclear factor-κB (NF-κB) expression in the lungs and remaining were kept alive at assess 15 days survival rate. The highest 15 days survival rate observed to the 1,8-cineole treated groups (60% in 120 mg/ kg and 50% in 60 mg/kg), whereas only 40% mice were survived for 15 days in oseltamivir treated group; and in placebo control group the average survival time was only 5 days. In addition above results, the tested essential oil compound at dose of 60 and 120 mg/kg

20  Essential Oils were found able to reduce expression of the Nuclear factor-κB, ICAM-1, and VCAM-1 with reduced pro-inflammatory cytokines levels (Interleukin-4, 5, 10, and monocyte chemo­ attractant protein-1) in nasal lavage fluids and Interleukin-1β, Interleukin-6, Tumor necrosis factor-α, and Interferon-γ in mice pulmonary tissues and also alleviate pathology of viral pneumonia. The study further concluded as 1,8-cineole protects mice against influenza virus infection via reduction of pulmonary inflammatory responses [89]. Few researchers explored the anti-avian influenza virus (H9N2) activity of Zataria multiflora essential oils in 21-day virus-infected broiler chicks, and viral replication in the respiratory and gastrointestinal system was investigated using quantitative RT-PCR. Few researchers examined Zataria multiflora essential oils for anti-Avian influenza virus (H9N2) activity in 21 days virus infected broilers chicks, and viral replication in respiratory and gastrointestinal tract was investigated using quantitative RT-PCR. The separate test groups of Zataria oil 20 and 40 μl/kg daily dose of which dissolved in corn oil started 2 days before and after the viral inoculation; another groups were positive control group (amantadine, 4 mg/kg/day) and placebo group (only corn oil). Both, Zataria oil and amantadine treated groups showed significantly decreased viral load due reduction in virus replication as compared to the control group. The group of birds treated with Zataria multiflora oil showed slightly lower H9N2 replication rate in trachea and cecal tonsils when compared with amantadine group, but was not statistically significant. The researchers observed overall anti-avian influenza effect was slightly better than amantadine [98].

1.7.3 West Nile Virus An in vivo study was done to investigate antiviral action of combined monoterpene alcohols against West Nile virus by using 6 weeks knockout (C57BL/6 IRF3-/-/7-/-) male mice model [99]. After determination of daily non-toxic dose i.e. 150 μL of 3.0% combination of monoterpene alcohol was intraperitoneally administered for the duration of 6 days. The results revealed that combined monoterpene alcohols significantly reduce West Nile virus replication in highly sensitive immune-deficient mice resulting low viral titers in brain. Further, the test compound was found to considerably reduce morbidity, as well as less reduction in body weight of the tested mice due to infection [100]. The other in vivo studies were done on murine models of influenza and cytomegalovirus, and found promising results of the Nigella sativa oil (Black-seed oil) for cytomegalovirus [101] and Heracleum spp. oils [102], Cynanchum stauntonii oil [103] and cinnamaldehyde [104] for influenza virus. There are very limited studies in humans are available at present in regard to essential oils as pre-clinical in vivo toxicity studies of many essential oils still need to establish. A small pilot study on human participants was conducted for Tea tree oil ointment against herpes labialis, which showed significant reduction in the time to complete healing as compared to control group [100]. In an another human study, the Backhousia citriodora essential oil was trialed against molluscum contagiosum and found promising results in treatment group while comparing with placebo. The complete healing of the cutaneous lesions observed in 5 out of 16 participants (1 lost follow-up), more than 90% recovery was seen in 4 participants, and 6 were showed less than 90% recovery. Whereas, in placebo group 12 out of 15 participants showed either increase in the numbers of lesions or hardly any change [105].

Methodological Approach of Essential Oils  21 There are also various other studies which showed various essential oil components have antiviral effects against enveloped RNA and DNA viruses. Sometimes, external application of such oils may be cytotoxic to skin and mucous membrane, therefore, it is commonly advised by the physicians that essential oils are used in diluted forms for topical application [28].

1.8 Activities In-Respect to the Available Antivirals Antiviral essential oils are either as standardized plant extracts or as isolated pure compounds provide limitless opportunities for new antiviral agents, since the biochemical diversity provides inimitable availability [106]. According to presently available literatures, essential oils could to be more potent and versatile than marketed antivirals, such as acyclovir resistant HSV-1 efficiently inhibited by the essential oil oregano and carvacrol [61]. Another study on essential oil of Salvia desolena showed inhibitory effect on both acyclovir sensitive and resistant strain of HSV-2, further the essential oil was fractioned and major component of the oil was isolated via gas chromatography. The isolated primary component germacrene D reported overall antiviral effect [64]. The germacrone in a similar way, also found more effective for the inhibition of multiple strains of Influenza virus including amantadine resistant strains than ribavirin. It inhibits early phase of viral replication by directly interfering viral attachment to host cell through blunting protein expression and RNA transcription of the influenza virus. The study suggested that, essential oil components exhibit multiple antiviral mechanisms with possible bypassing actions like M2 ion channels inhibition (e.g., amantadine and rimantadine) and neuraminidase inhibition (e.g. oseltamivir and zanamivir) to override drug resistance. Furthermore, additive effect of the combination of germacrone and oseltamivir observed against inhibition of influenza virus infection in both in vitro and in vivo, which assessed by fractional inhibitory concentration method and/or infected mice survival rate respectively [83, 107]. It has been accepted that the diversified antiviral action of essential oil may be due to its compositional complexity, which further provide concurrence to multiple bioactive components and synergism than virus specific synthetic drugs. The another combination of oseltamivir and Lemon balm (Melissa officinalis) oil was discovered to be more efficient for anti-avian influenza A (H9N2) virus activity. The total reduction (near zero) of avian influenza A virus genome copy number was observed when culture cell was treated with combination of oseltamivir (0.05 μg/mL) and Melissa officinalis oil (0.5 mg/mL), while treatment with 0.05 μg/mL of oseltamivir alone showed 12,000 viral genome copies per milliliter [30]. The 15 μg/mL of piperitenone oxide as principle component of Mentha suaveolens essential oil and 0.05 μg/mL of acyclovir combination were also found to reduce the number of viral titers by ~90%, and exhibited synergistic effects against HSV-1 virus [51]. The additive and synergistic activity of the combination of essential oils or their components could be used as antiviral therapeutic option in future due to its diversified action with higher efficacy and also could be used for the treatment of drug resistant viral strains. Also, as drug resistant viral strains are increasing day by day, more study is needed for the combinational effect of essential oil or its components.

22  Essential Oils

1.9 Antiviral Essential Oils and Their Bioactive Components Loaded in Nanosystems Encapsulation of essential oils or their bioactive components claimed to be a efficacious and appropriate strategy to regulate drug delivery. This provides protection of bioactive components from the environmental interactions, reductions in toxicity and volatility with enhancement of physical stability and their physical stability, which further results into improvement in the patients care, compliance, and convenience [108]. The variety of nanocarriers available at present, like as polymer-based nanocarriers are nanocapsules or nanoparticles, and also lipid-based nanocarriers such as, nano-liposomes, lipid-­nanoparticles, nano-emulsions, etc. These nanocarriers can be framed according to desirable features for therapy [109]. In an earlier research, the antiviral effect of essential oil of Artemisia arborescens in pure form and incorporated with solid lipid nanoparticles were investigated against HSV-1. Additionally, vitro diffusion assay of the selected oil was studied for permeation through and also for accumulation into the skin strata of newborn pig. The solid lipid nanoparticles as nanocarrier for Artemisia oil were found to have high physical stability even after 2 years of storage. The in-vitro assay of skin permeation showed the potentiality of above nano-­ delivery system of Artemisia oil which substantially increases the oil accumulation into the skin while pure oil permeation was not improved. In both scenarios as nano-preparation and pure Artemisia plus almond oil solution (control) showed 36.2 % of reduction in viral infectivity in vero cells (31.1% solid lipid nanoparticles loaded essential oil and 100 μg/ mL pure oil). This indicates nano-delivery system of Artemisia oil did not negatively affect antiviral activity of the oil in vitro assay [110]. A study done to evaluate anti-herpetic activity of prepared hydrogel containing Cymbopogon citratus volatile oil encapsulated in poly (d,l-lactide-co-glycolide) nanoparticles. The free essential oil, essential oil containing hydrogel, essential oil loaded nanoparticles and essential oil loaded nanogel were investigated through in-vitro vero cells assay against the infection of herpes simplex type 1 and type 2 (HSV-1/HSV-2). The simultaneous treatment with antiviral test substance was given for both vero cells and virions. The anti-HSV-1 activity observed as, VIC50 value 11.59 μg/mL and ASI value of 9.99 for, for free essential oil assay; essential oil loaded nanoparticles the value of VIC50 1.32 μg/ mL and ASI 9.83; and for essential oil loaded nanogel assay VIC50 valued 0.32 μg/mL and ASI value 4.67. Whereas for anti-HSV-2 activity was concerned the observations revealed free essential oil (VIC50 6.69 μg/mL, ASI 17.32), essential oil loaded nanoparticles (VIC50 1.34 μg/mL, ASI: 9.67) and essential oil loaded nanogel (VIC50 0.33 μg/mL, ASI 4.50). The value of VIC50 was 0.71 μg/mL and ASI value of 2.31 observed for essential oil containing hydrogel assay for both anti-HSV-1 and anti-HSV-2 activity. The results revealed nanogel preparation of volatile oil strongly inhibited virus at minimal non-cytotoxic concentration which is 42.16 times lower than the free oil preparation. It also highlighted the potential benefit of the nanogel as protection against volatilization, control release nano-delivery system which improves its anti-herpetic properties [111]. In a recent study, the Melissa officinalis essential oil loaded inside glycerosomes compared to pure non-encapsulated Melissa officinalis oil investigated in vitro against HSV-1

Methodological Approach of Essential Oils  23 virus. The glycerosomes preparation of Melissa officinalis essential oil in course of 4 months of storage showed high chemical as well as physical stability. The encapsulation efficiency in glycerosomes was observed to be around 51.3 and 66.0 % for citral and β-caryophyllene, respectively. The 1 hour pre-treatment of vero cells (cell-free HSV-1 virions) with increasing concentrations of pure oil (0-200 μg/mL) and glycerosomes encapsulated oil (0–600 μg/ mL) exhibited reduction in dose dependent viral infectivity [112]. The white spot syndrome virus is highly lethal and contagious viral infection of penaeid shrimps. The effect of prepared feeding pellets absorbed with Litopenaeus vannamei with microencapsulated thyme essential oil in the ratio of 1.05 gram of thyme essential oil mixed with per 100 gram of powder investigated against white spot syndrome virus disease in purpose to protect shrimps. The results analyzed at 72 hours post infection and revealed absence of clinical signs of viral infection on pre-infected shrimps fed with 1% microencapsulated preparation along with significantly higher survival rate than other treatment groups (control and lower dose of Litopenaeus vannamei essential oil) [113].

1.10 Conclusion Essential oils are natural, plant-derived complex aromatic mixtures of monoterpenes, sesquiterpenes, and their oxygenated derivatives and other trace compounds. The published in-vitro studies depicted the major mechanism of these plant aromatic oils and their bioactive components for antiviral actions like direct effects on host cell-free virions, suppression or inhibition of viral attachment, virus penetration and replication, as well as release from the infected host cells and also by inhibiting vital viral enzymes. Over few decades, many studies documented to have antiviral efficacies of essential oils or their components effects against a variety of DNA and RNA viruses through in vitro and in vivo modeling. Most of the documented in vitro experiments done on enveloped viruses, like Herpes simplex virus, Influenza virus, Avian influenza virus and gradually increasing its extent, with SARSCoV-2. There are only a few studies available on the antiviral effects of essential oils or their derivatives against non-enveloped viruses (coxsackievirus and MNV-1). In the present scientific era, new effective antivirals and other virucidal drugs are discovered using in silico analysis, as well as the molecular mechanisms of phytochemical compounds. The molecular docking study is very useful for quantify binding affinity of each individual components of essential oil to the viral or host cell proteins for the prediction of biological effects. In the current situation of COVID-19 pandemic, many researchers have already put their effort to identify a suitable molecules with capability to inhibit attachment or entry and also replication of SARS-CoV-2 in the host cells. In vivo studies in mice or chicks model of influenza virus Zataria oil and essential oil components e.g., 1,8-cineole, germacrone, patchouli alcohol showed prolongation in their survival rate with decrease in tissue viral titers along with inhibition of pro-inflammatory cytokines. Further, the available literatures and studies reported the reduction in the effectivity of essential oils due to their high volatility, the nano-carrier delivery system could be useful to enhance chemical stability, bioavailability, and their overall antiviral effectiveness. Further research is required for the antiviral bio­ activities of essential oils and their components for the identification and future development of novel antivirals.

24  Essential Oils

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Methodological Approach of Essential Oils  27 61. Marciele, R.P., Sydney, H.A., Rudi, W., Sandra, A., Ana, P.C., Luciane, T.L., Antiviral activity of the Lippia graveolens (Mexican oregano) essential oil and its main compound carvacrol against human and animal viruses. Braz. J. Microbiol., 42, 1616, 2011. 62. Toujani, M.M., Rittà, M., Civra, A., Genovese, S., Epifano, F., Ghram, A., Lembo, D., Donalisio, M., Inhibition of HSV-2 infection by pure compounds from Thymus capitatus extract in vitro. Phytother. Res., 32, 1555, 2018. 63. Sharifi-Rad, J., Salehi, B., Schnitzler, P., Ayatollahi, S.A., Kobarfard, F., Fathi, M., Eisazadeh, M., Sharifi-Rad, M., Susceptibility of herpes simplex virus type 1 to monoterpenes thymol, carvacrol, p-cymene and essential oils of Sinapis arvensis L., Lallemantia royleana Benth. and Pulicaria vulgaris Gaertn. Cell. Mol. Biol., 63, 42, 2017. 64. Cagno, V., Sgorbini, B., Sanna, C., Cagliero, C., Ballero, M., Civra, A., Donalisio, M., Bicchi, C., Lembo, D., Rubiolo, P., In vitro anti-herpes simplex virus-2 activity of Salvia desoleana, Atzei & V. Picci essential oil. PLoS One, 12, 2, Article ID e0172322, 2017. 65. Wei, X., Cheng, P., Feng, W., Study on the effect of anti-respiratory viruses of Patchouli oil in vitro. J. Pharm. Clin. Chin. Mater. Med., 6, 65, 2012. 66. Nagy, M.M., Almahdy, D., El-Aziz, O.M.A., Kandil, A.M., Tantawy, M.A., El-Alfy, T.S., Chemical composition and antiviral activity of essential oils from Citrus reshni hort. ex Tanaka (Cleopatra mandarin) cultivated in Egypt. J. Essent. Oil Bear. Plants, 21, 264, 2018. 67. Ibrahim, N.A., El-Hawary, S.S., Mohammed, M.M., Farid, M.A., Abdel-Wahed, N., Ali, M.A., El-Abd, E., Chemical composition, antiviral against avian influenza (H5N1) virus and antimicrobial activities of the essential oils of the leaves and fruits of Fortunella margarita, lour. swingle, growing in Egypt. J. Pharm. Sci., 5, 006, 2015. 68. Bouazzi, S., Jmii, H., El-Mokni, R., Faidi, K., Falconieri, D., Piras, A., Jaïdane, H., Porcedda, S., Hammami, S., Cytotoxic and antiviral activities of the essential oils from Tunisian Fern. Osmunda regalis. S. Afr. J. Bot., 118, 52, 2018. 69. El Mokni, R., Youssef, F.S., Jmii, H., Khmiri, A., Bouazzi, S., Jlassi, I., Jaidane, H., Dhaouadi, H., Ashour, M.L., Hammami, S., The essential oil of Tunisian Dysphania ambrosioides and its antimicrobial and antiviral properties. J. Essent. Oil Bear. Plants, 22, 282, 2019. 70. Ameur, E., Zyed, R., Nabil, A.B.S., Samia, M., Youssef, B.S., Karima, B.H.S., Mahjoub, A., Farhat, F., Rachid, C., Fethia, H.S., Mohamed, L.K., Chemical composition of 8 eucalyptus species’ essential oils and the evaluation of their antibacterial, antifungal and antiviral activities. BMC Complement. Altern. Med., 12, 81, 2012. 71. Hammami, S., Jmii, H., El-Mokni, R., Khmiri, A., Faidi, K., Dhaouadi, H., El-Aouni, M.H., Aouni, M., Joshi, R.K., Essential oil composition, antioxidant, cytotoxic and antiviral activities of Teucrium pseudochamaepitys growing spontaneously in Tunisia. Molecules, 20, 20426, 2015. 72. Kubiça, T.F., Alves, S.H., Weiblen, R., Lovato, L.T., In vitro inhibition of the bovine viral diarrhoea virus by the essential oil of Ocimum basilicum (basil) and monoterpenes. Braz. J. Microbiol., 45, 209, 2014. 73. Gómez, L.A., Stashenko, E.E., Ocazionez, R.E., Comparative study on in vitro activities of citral, limonene and essential oils from Lippia citriodora and L. alba on yellow fever virus. Nat. Prod. Commun., 8, 249, 2013. 74. Astani, A., Reichling, J., Schnitzler, P., Comparative study on the antiviral activity of selected monoterpenes derived from essential oils. Phytother. Res., 24, 673, 2009. 75. Haddad, J.G., Picard, M., Bénard, S., Desvignes, C., Desprès, P., Diotel, N., El-Kalamouni, C., Ayapana triplinervis essential oil and its main component thymohydroquinone dimethyl ether inhibit zika virus at doses devoid of toxicity in zebrafish. Molecules, 24, 19, 3447, 2019.

28  Essential Oils 76. El-Moussaoui, N., Sanchez, G., Khay, E.O., Idaomar, M., Ibn-Mansour, A., Abrini, J., Aznar, R., Antibacterial and antiviral activities of essential oils of northern Moroccan plants. Br. Biotechnol. J., 3, 318, 2013. 77. Ocazionez, R.E., Meneses, R., Torres, F.A., Stashenko, E., Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro. Mem. Inst. Oswaldo Cruz, 105, 304, 2010. 78. Roy, S., Chaurvedi, P., Chowdhary, A., Evaluation of antiviral activity of essential oil of Trachyspermum Ammi against Japanese encephalitis virus. Pharmacogn. Res., 7, 263, 2015. 79. Behl, T., Rocchetti, G., Chadha, S., Zengin, G., Bungau, S., Kumar, A., Mehta, V., Uddin, M.S., Khullar, G., Setia, D., Arora, S., Sinan, K.I., Ak, G., Putnik, P., Gallo, M., Montesano, D., Phytochemicals from plant foods as potential source of antiviral agents: An overview. Pharmaceuticals, 14, 381, 2021. 80. Francomano, F., Caruso, A., Barbarossa, A., Fazio, A., La-Torre, C., Ceramella, J., Mallamaci, R., Saturnino, C., Iacopetta, D., Sinicropi, M.S., β-Caryophyllene: A sesquiterpene with countless biological properties. Appl. Sci., 9, 5420, 2019. 81. Arbeitskreis, B., Influenza virus. Transfus. Med. Hemother., 36, 32, 2009. 82. Choi, H.J., Chemical constituents of essential oils possessing anti-influenza A/WS/33 virus activity. Osong Public Heal. Res. Perspect., 9, 348, 2018. 83. Liao, Q., Qian, Z., Liu, R., An, L., Chen, X., Germacrone inhibits early stages of influenza virus infection. Antiviral Res., 100, 578, 2013. 84. Asif, M., Saleem, M., Saadullah, M., Yaseen, H.S., Al Zarzour, R., COVID-19 and therapy with essential oils having antiviral, anti-inflammatory, and immunomodulatory properties. Inflammopharmacology, 29, 577, 2021. 85. Paulpandi, M., Kannan, S., Thangam, R., Kaveri, K., Gunasekaran, P., Rejeeth, C., In vitro anti-viral effect of β-santalol against influenza viral replication. Phytomedicine, 19, 231, 2012. 86. Wu, H., Liu, Y., Zu, S. et al., In vitro antiviral effect of germacrone on feline calicivirus. Arch. Virol., 161, 1559, 2016. 87. Camero, M., Lanave, G., Catella, C. et al., Virucidal activity of ginger essential oil against caprine alphaherpesvirus-1. Vet. Microbiol., 230, 150, 2019. 88. Wu, H., Li, B., Wang, X., Jin, M., Wang, G., Inhibitory effect and possible mechanism of action of patchouli alcohol against influenza a (H2N2) virus. Molecules, 16, 6489, 2011. 89. Li, Y., Lai, Y., Wang, Y., Liu, N., Zhang, F., Xu, P., 1, 8-Cineol protect against influenza-virus-­ induced pneumonia in mice. Inflammation, 39, 1582, 2016. 90. Astani, A. and Schnitzler, P., Antiviral activity of monoterpenes beta-pinene and limonene against herpes simplex virus in vitro. Iran. J. Microbiol., 6, 149, 2014. 91. Lai, W.L., Chuang, H.S., Lee, M.H., Wei, C.L., Lin, C.F., Tsai, Y.C., Inhibition of herpes simplex virus type 1 by thymol-related monoterpenoids. Planta Med., 78, 1636, 2012. 92. Vimalanathan, S., Anti-influenza virus activities of commercial oregano oils and their carriers. J. Appl. Pharm. Sci., 2, 214, 2012. 93. Venturi, C.R., Danielli, L.J., Klein, F. et al., Chemical analysis and in vitro antiviral and antifungal activities of essential oils from Glechon spathulata and Glechon marifolia. Pharm. Biol., 53, 682, 2015. 94. Szczech, G.M., Preclinical development of antiviral drugs. Clin. Infect. Dis., 22, 355, 1996. 95. Benencia, F. and Courrèges, M.C., In vitro and in vivo activity of eugenol on human herpes virus. Phytother. Res., 14, 495, 2000. 96. Carson, C.F., Ashton, L., Dry, L., Smith, D.W., Riley, T.V., Melaleuca alternifolia (tea tree) oil gel (6%) for the treatment of recurrent herpes labialis. J. Antimicrob. Chemother., 48, 450, 2001.

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2 Essential Oils Used to Inhibit Bacterial Growth in Food Luiza Helena da Silva Martins1*, Sabrina Baleixo da Silva2, Adilson Ferreira Filho2, Andrea Komesu3, Johnatt Allan Rocha de Oliveira4 and Debora Kono Taketa Moreira5 Institute of Animal Health and Production, Federal Rural University of Pará (UFRA), Belém, PA, Brazil 2 Graduate Program in Food Science and Technology, Federal University of Pará, Augusto Corrêa Avenue, Guamá, Belém, Pará, Brazil 3 Department of Marine Sciences (DCMar), Federal University of São Paulo (UNIFESP), Santos, SP, Brazil 4 Institute of Health Sciences, Faculty of Nutrition, Federal University of Pará (UFPA), R. Augusto Corrêa, 01 - Guamá, Belém, PA, Brazil 5 Instituto Federal de Brasília Campus Gama (IFB), Gama, Brasília/DF, Brazil 1

Abstract

Foods are complex matrices with rich carbon sources and good substrates for microorganisms to develop their metabolic activities. According to GRAS, researchers have approved essential oils (EO) and phytochemicals in the food area and both are sources of numerous compounds, such as terpenoids and polyphenols, which can inhibit microorganisms. EOs may be recognized as safe by regulatory agencies such as the US Food and Drug Administration (FDA), and some compounds found in essential oils have already been used as flavoring agents in foods. Some reports of the negative effects that preservatives in ultra-processed foods can have on health have prompted the food industry to look for more sustainable ways. Therefore, the use of EOs from aromatic plants, which have antimicrobial and antioxidant properties, would be an alternative to chemical preservatives, as they could act as natural preservatives that make food safer and more nutritious. The chapter, therefore, deals with essential oils and their use as food preservatives against the deterioration of microorganisms and pathogens that can cause serious diseases in humans. Their availability, their action against bacterial strains, their properties and their potential as a natural product are evaluated. Keywords:  Food conservation, natural products, bioactive compounds, microbiology

2.1 Introduction Microbial contamination of food is clearly one of the top consumer worries, as this element can shorten the shelf life of food and increase the risk of food-borne illnesses. Foods are *Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (31–48) © 2023 Scrivener Publishing LLC

31

32  Essential Oils a highly interesting substrate for microorganisms to carry out their metabolic activities because they are complex matrices and abundant sources of carbon (carbohydrates, lipids, and proteins). In addition, some foods may have a pH close to neutrality, which makes the environment suitable and conducive to microbial growth [1]. Within this theme, there was also an increase in human awareness about health, changing their perceptions of synthetic food additives. As a result, consumers began to avoid more ultra-processed foods, seeking an alternative to their use. Among these compounds, we can mention natural essential oils (EOs), which, due to the numerous benefits they provide, have become an “organic or green strategy.” [2]. Due to the complexity of secondary plant components such as terpenes, phenolic compounds, alcohol, and others, EOs can be characterized as substances from plants that can be isolated and have a variety of beneficial qualities. EOs provide a variety of benefits thanks to their bioactive constituents, which include antioxidants, antimicrobials, and anti-inflammatory agents. As a result, these EOs have antibacterial and preservative qualities that can be used to increase the shelf life of foods and make them safer and more stable. Many EOs have been researched and show considerable promise as food preservatives in a variety of matrices [2]. It is becoming a trend to employ essential oils (EOs) as an alternative to synthetic antimicrobials. EOs can be extracted from plant extracts in a variety of ways, both standard and unconventional. Although EOs are well-known for their aromatic qualities, their antioxidant and antibacterial effects are mostly due to their makeup, which includes phenolic and terpenoid components, as well as aliphatic molecules. Due to their bioactive components, some EOs have even been used to treat human ailments [3]. In the EOs area and phytochemistry, there has been a lot of research recently. Because both sources contain numerous compounds such as terpenoids and polyphenols that can inhibit the growth of a variety of microorganisms, regulatory agencies such as the United States Food and Drug Administration (FDA) (GRAS) may recognize essential oils as safe. Furthermore, some of the compounds found in essential oils have already been used as flavoring agents in foods in Europe, so they can be regarded as safe [1]. Some concerns regarding the detrimental health consequences of preservatives in ultra-processed meals have spurred the food sector to seek out more sustainable methods and consider the future. As a result, the usage of EOs derived from aromatic plants, which have antibacterial and antioxidant capabilities, could be a natural alternative to chemical preservatives, making food safer and more nutritious [4]. As a result, the book’s chapter will examine essential oils and their usage as a food preservative against degrading microorganisms and pathogens that can cause significant diseases in humans, analyzing their availability, antibacterial activity, characteristics, and potential as a natural product.

2.2 Chemistry of Essential Oils EOs are complex compounds with many diverse constituents, roughly 300 of which are commercially important, and one molecule is always the majority (20–95 percent) and the rest is considered trace. Furthermore, around 3,000 essential oils have been developed in recent years [5, 6].

Essential Oils Used to Inhibit Bacterial Growth in Food  33 Table 2.1  Bioactivities attributed to different components in essential oils. Bioactive compounds

Bioactivities

Source

Limonene

Anticancer, antimicrobial

Orange and lemon peels

Pinene

Antimicrobial

Conifers, rosemary, lavender

Eucalyptol

Analgesic, anti-inflammatory, and antimicrobial

Eucalyptus

Myrcene

Analgesic-sedative, anti-inflammatory

Cannabis sativa

Linalool

Sedative, anxiolytic, analgesic, anti-inflammatory, antimicrobial, antioxidant

Lavandula angustifolia, Laurus nobilis, Ocimum basilicum, Coriandrum sativum, Cymbopogon sp., Citrus sp.

Citral

Antimicrobial

Backhousia citriodora, Cymbopogon citratus, Litsea cubeba, Citrus sp

Camphor

Analgesic, antiseptic

Cinnamomum camphora

Menthol

Analgesic, antibacterial, antifungal, antipruritic, anticancer

Mentha canadensis (Mentha arvensis)

Terpineol

Antihypertensive, anticancer, antioxidant, antimicrobial, antifungal and sedative

Tea tree (Malaleuca alternifolia)

Citronellol

Analgesic, anti-inflammatory, antioxidant

Citronella oil, rose oil

Bisabolol

Analgesic, anti-inflammatory, antimicrobial, antioxidant

Matricaria camomilla, Salvia runcinata

β-caryophyllene

Analgesic, anti-inflammatory and anticancer

Origanum vulgare, Cinnamomum zeylanicum, Piper nigrum, Cannabis sativa, Humulus lupulus

Chamazulene

Anti-inflammatory, antioxidant

Matricaria camomilla

Caryophyllene oxide

Analgesic, anti-inflammatory

Didymocarpus tomentosa

Germacrene

Antioxidant

Artemisinic acid

Antimalarial

Patchoulene

Anti-inflammatory, antigastritis

Humulene

Anti-inflammatory, anticancer

Humulus lupulus

Bergamotene

pheromone

Citrus bergamia

Eudesmol

Antimicrobial, antifungal, anticancer, and antiangiogenic

Source: Authors

Artemisia annua

34  Essential Oils EOs are organic compounds that are volatile, aromatic, and low in molecular weight, soluble in organic solvents such as ether, alcohol, and others but insoluble in water and liquid, have no color at room temperature or pressure, have their own flavors, and have a low density [7]. Many plants have already been studied for the extraction of EOs such as Zingiber officinale, Cinnamomum verum, Citrus limon, Syzygium aromaticum, Thymus vulgaris, Origanum vulgare, Lavandula and Mentha piperita, where about 50 aromatic substances have been detected individually, which are responsible for their therapeutic effects and as food preservatives, among others [8]. Table 2.1 shows the various bioactivities attributed to the different constituents of essential oils. Hanif et al. [9] classified EOs into two groups: volatile fraction and non-volatile residue. The first is composed of monoterpenes, sesquiterpenes, and their oxygenated derivatives, aliphatic alcohols, esters, and aldehydes, and accounts for 90-95 percent of the total weight of the oil. Fatty acids, hydrocarbons, sterols, waxes, flavonoids, and carotenoids make up the second category, which accounts for around 1-10% of the overall weight of EO [9]. When it comes to the hydrocarbon backbone of these molecules, two structural families can be used to categorize the primary elements of EOs: terpenoids and phenylpropanoids [10]. Because terpenes and their oxygenated derivatives (terpenoids) make up the majority of EOs, some species may still contain significant amounts of shikimates. These are the phenylpropanoids that give certain plants their distinctive odor and flavor [5]. The substances known as terpenes may also be called isoprenes, terpenoids, or isoprenoids if they have oxygen in their structure. With more than 30,000 structures already consolidated and known, they form the largest group of natural products [11]. These substances are formed by the condensation of a pentacarbonate unit with two unsaturated bonds, isoprene (2-methyl-1,3-butadiene), which is why they are often called isoprenoids [12]. Terpenes are grouped as hemiterpenes (1 unit), monoterpenes (2 units), sesquiterpenes (3 units), diterpenes (4 units), and their oxygenated derivatives, such as alcohols, oxides, aldehydes, ketones, phenols, acids, esters, and lactones, determined by the number of isoprene units present in the structure [5]. Monoterpenes (C10H16) and sesquiterpenes (C15H24) can be considered as the terpenes most abundant in EOs. There are multiple cyclic or linear isomeric structures in these compounds, as well as various degrees of unsaturation, substitutions, and oxygenated derivatives [12]. Although differences in molecular weight exist between the two structures, their structural and functional properties are generally very similar. The diterpenes and triterpenes have higher molecular weight than the monoterpenes and sesquiterpenes and are also found in rare essential oils [8]. Terpenes have antiseptic, anti-inflammatory, bactericidal and antiviral properties. Many of these substances have valuable diverse biological activity used in cosmetic, perfumery, food, and other industries [13]. Monoterpenes are the most common ingredients of essential oils, accounting for around 90% of many of them [12]. Some examples of general monoterpenes are pinene, limonene, eucalyptol, myrcene, linalool, citral, camphene, camphor, menthol, terpineol, citronellol, piperine, and others. Essential oils of coniferous plants (pine), rosemary, lavender, and turpentine include pinenes and bicyclic terpenes [14]. Pinenes-rich oils have been shown to exhibit antibacterial action against bacterial and fungal cells [14]. Limonene and-pinene stop bacteria from growing of the species B. strains, S. aureus, L. monocytogenes, S. enterica, S. cerevisiae,

Essential Oils Used to Inhibit Bacterial Growth in Food  35 Zygosaccharomyces rouxii, Sclerotinia sclerotiorum, and Rhizoctonia solani according to the study of Melkina et al. [15]. Eucalyptus oil is a traditional medication used in the treatment of a variety of ailments and in health care, the ethnomedicinal value of eucalyptus oil for antibacterial and anticancer activities was showed [16]. According to Alipanah et al. [17], lemon and other citrus fruits that contain limonene can be used as an insecticide and with antimicrobial properties, also the limonene showed potential for the treatment of melanoma and breast cancer. Several molecules with interesting properties are found in the sesquiterpenes [13], such as bisabolol, β-caryophyllene, chamazulene, caryophyllene oxide, germacrene, artemisic acid, patchoulene, humulene, bergamotene, farnesene, eudesmol and others. Table 2.1 shows important bioactive properties of some sesquiterpenes. Phenylpropanoids are found in essential oils less frequently and usually in smaller amounts than terpenoids. However, some of the oils in which phenylpropanoids are found contain significant amounts of them [11]. Their main representatives in essential oils include anethole, eugenol and safrole, all of which have a carbon-carbon double bond in the side chain. The α-Asarone, β-asarone, estragole, methyl eugenol, and safrole are all phenylpropanoids that are carcinogenic to rodents [5]. Even though essential oils are made up of phenylpropanoids, terpenes, and terpenoids, they contain a wide range of chemical components and structures that give them their therapeutic effects. Essential oils are therefore commonly employed for medical, pharmaceutical, antioxidant, cardiovascular, cosmetic, and food uses [10].

2.3 Essential Oils Against Microorganisms in Food Products EOs are special because they have antimicrobial activity that can be used in many ways, but only a small fraction is used commercially. Studies state that the chemical components contained in EOs can act on the membrane and cell wall of bacteria, but more research is needed to consolidate this knowledge and verify all the modes of action involved in this action [18]. We can’t attribute antibacterial activity to a single method of action since the bioactive compounds in these EOs have varied structural groups in their composition and so exert different actions in different sections of microbial cells. It is known that EOs can have bacteriostatic effects, i.e. they inhibit bacterial growth only where the bacteria are able to resume their ability to multiply, and we also have a bactericidal effect, which is the death of bacterial cells [2, 19]. It is also known that the antimicrobial effect of EOs against Gramme-positive and Gramme-negative bacteria is not the same [19, 20]. This is because Gramme-negative bacteria restrict the penetration of hydrophobic components more than Gramme-positive ones. When compared the study of Chen et al. [21] with the study employed by Calo et al. [22] with the hydrophobic compounds, Chen et al. [21] investigated a variety of EOs derived from flowers, and these compounds exhibited potential efficacy against microbes as S. aureus, E. coli, Campylobacter, and S. typhimurium. Czaikoski et al. [23] who investigated the EOs extracted from the flowers of Eupatorium intermedium EO in combating the following bacterium types: L. monocytogenes, S. aureus, E. coli and Salmonella typhimurium. The authors demonstrated that the EOs of Eupatorium intermedium were quite effective

36  Essential Oils to avoid the growth of the Gram-positive bacteria L. monocytogenes and S. aureus, but not against E. coli and S. typhimurium. This EO appears to inhibit Gram-positive bacteria more selectively than Gram-negative bacteria. Antimicrobial agents contained in food preservatives are most commonly used, for example, to inhibit the growth and production of toxins produced by E. coli, a pathogenic microorganism, but these preservatives are restricted because of the harm they can cause to human health. Since EOs consist of mixtures of volatile compounds that have odor properties (we can see this in some spices), these compounds are a complex mixture of terpenes, polyphenols, sesquiterpenes, lactones, esters, alcohol, and others [24]. Munekata et al. [24] note that many EOs from various sources have significant antibacterial activity against E. coli, such studies show that membrane morphological changes are the most observed factors, so that cell death occurs because these membranes function in such a way that the metabolic processes vital to the cell, for example, the bacillus form of a bacterium, are transformed into a diffuse and inconsistent form, causing the cells to swell, then an efflux of intracellular components is consolidated and the lysis process occurs in the cell. Mutlu-Ingok and Karbancioglu-Guler [3] employed agar diffusion and broth microdilution methods to examine the antibacterial activity of cardamom, cumin, and dill weed EOs to combat the grow of Campylobacter jejunia and Campylobacter coliby. The composition of EOs was characterized using chromatography techniques (gas CG and with mass spectrometry – GC-MS). It was found that the EOs of cardamom and dill exhibited good antimicrobial activity compared to cumin. By determining the relative electrical conductivity and release of cellular constituents in the supernatant at 260 nm, factors such as permeability and cell membrane integrity can be observed, respectively. Additional effects of the Eos on the cell membrane were investigated in Campilpp. when measuring the extracellular concentration of ATP. The relative electrical conductivity and the release and concentration conditions of ATP in the extracellular components of the medium increased after treatment. In this study, it is concluded that all tested EOs can affect the membrane integrity of Campylobacteres pp. The essential oils of cardamom, cumin and dill weed show potent inhibition of Campylobacteres pp. by damaging the bacterial cell membrane. Aeromonas hydrophila, C. jejuni, C. perfringens, E. coli O157: H7, L. monocytogenes, S.  enterica, and spores of B. cereus and C. botulinum, are the pathogenic bacteria most found in meat and its derivatives, according to Pateiro et al. [19]. Because of the heat that the products are subjected to during processing, they are difficult to eradicate. As a result, several food additives are employed to protect foods from spoiling germs and diseases that may be harmful to human health. However, several of these additives, such as nitrites used to inhibit C. botulinum and L. monocytogenes. Acids like benzoic, sorbic and its derivatives, also parabens and inorganic sulfites, have all been linked to harmful effects (allergies, cancer, etc.), and their use causes health concerns among consumers [21]. EOs such as balsam, basil, cloves, coriander, ginger, marjoram, oregano, rosemary, and thyme are natural preservatives that have been found to be effective against foodborne pathogens in meat products when compared to artificial preservatives [19, 25]. Khaleque et al. [26] studied the effect of EOs extracted from cloves (Syzygium aromaticum), cinnamon (Cinnamomum cassia) and commercial EOs in controlling Listeria monocytogenes in minced meat. Meat was contaminated with L. monocytogenes and then exposed to crude clove EOs at concentrations of 5% and 10% and cinnamon EOs at concentrations of 2.5% and 5.0%. Samples were stored at 8°C, refrigerated at 0°C for 7 days,

Essential Oils Used to Inhibit Bacterial Growth in Food  37 and then frozen at -18°C and stored for 60 days. The results showed that L. monocytogenes was completely inactivated by the third day after inoculation with 10% of EO of raw and commercial cloves. The concentrations of 2.5 and 5% of raw cinnamon EO were not effective in this activation of the microorganism. But the OE of commercial cinnamon at 5% concentration showed a reduction nearly of 4.0 log UF/g of L. monocytogenes growth, and this factor was dependent on storage time and temperature, where in seven days the reduction of 3.5-4.0 log CFU/g of L. monocytogenes was accomplished at freezing, chilling, and refrigeration temperatures. However, this effect was not observed in cloves EO, this is interesting to improve food safety, and it is highly recommended for controlling the pathogenic bacterium L. monocytogenes in meat products. The study conducted by Adjou et al. [27], using the essential oil extracted from Pimenta racemosa at concentrations of 0.5–2 μL/g, was able to improve the technology of production of fermented flour of Galeoides decadactylus, and although the emphasis was on the technological factor, the authors observed that joining EO may indicate a significant reduction in the undesirable microbial population.

2.4 Application of Essential Oils in the Food Industry Essential oils can be extracted from various parts of plants such as fruits, flowers, leaves, stems, bark, and seeds, where they represent defense mechanisms of the plant against the action of microorganisms [4, 22, 28–30]. The composition and chemical quality of essential oils depend on several variables that affect the product, such as the characteristics of the plant, including the stage of development, season, geographical origin, variety, plant region, age, and state of sampling, as well as the methods chosen for extraction, resulting

Essential Oils extracted from different parts of plants

Antimicrobial

Activity

Antioxidant

Insecticidal

Anticancer

Uses

Medicine

Pharmacy Food sector

Figure 2.1  General overview of some essential oil activities and uses (Source: Authors adapted by Pandey et al. [33]).

38  Essential Oils in diversity in the chemical composition of essential oils [29, 31, 32]. The overview of some essential oil is illustrated in Figure 2.1 adapted by [33]. They are, in general, compositions made up of groupings of distinct low molecular weight organic compounds that can be classified into many classes based on their chemical structure. Among the chemicals in these classes are terpenes, terpenoids, aromatic phenylpropanoids), alkaloids, flavonoids, isoflavones, phenolic acids, carotenoids, and aldehydes [34, 35]. Essential oils have a wide range of biological properties that benefit the food industry, including cancer prevention [36], cardiovascular disease prevention [37], obesity prevention [38], anti-inflammatory action, neurological disease prevention, and antimicrobial action, among other things [39–41]. Many of these properties are due to the compounds found in essential oils, which have actions that allow for the capture of reactive oxygen, nitrogen, and peroxide species during decomposition, through the redox potential, the capture of pro-­ oxidant metals, the ability to modulate some enzymatic activities, the possibility of interaction with macromolecules such as proteins, and finally, may inhibit enzymes involved in the generation of free radicals [42–46]. When it comes to antibacterial activity, it’s worth noting that the components found in essential oils can function in a variety of ways. Some terpenes can permeate cellular membranes associated with infections as a result of their amphipathic properties [47, 48]. In the case of phenolics, they can harm the cytoplasmic membrane [49, 50], interfere with nucleic acid synthesis [51], and affect the microorganism’s energy metabolism [52]. Other investigations have found destabilizing and disruptive actions on the membrane [53, 54], damage to membrane proteins [55], suppression of localized metabolic cycles membrane [56, 57], and depletion of the proton motive force [58] in addition to these possibilities, and cytoplasmic components, metabolites, and ions leaking. As a result, the chemicals in essential oils disrupt microbial equilibrium and development, putting the cells of microorganisms under stress [59]. It’s worth noting that the bacteria’s characteristics can influence the effectiveness of EO compounds, according to published studies, lipoteichoic acids in gram-positive bacteria’s cell membrane can enhance the penetration of substances that can inactivate the bacterium.

+ +

Essential Oils

+ Chitosan

+ + + + +

800 600 400 200

+ + + + + - - - + + - + - + - + - + - + - - + + + + +

+

ng coati Fruit

Sodium Tripolyphosphate (TPP)

Figure 2.2  Scheme of the use of Edible films designed to extend the shelf life of foods while also providing antimicrobial protection and improving organoleptic qualities. (Source: Authors adapted by Arabpoor et al. [64]).

Essential Oils Used to Inhibit Bacterial Growth in Food  39 Because gram negative bacteria have an extrinsic membrane around their cell wall, the rate of diffusion of chemicals is limited. Gram positive bacteria are slightly more vulnerable to the effect of essential oil components than gram negative bacteria [30, 60, 61]. Furthermore, the antibacterial efficacy of the chemicals found in essential oil can function by slowing bacterial growth, resulting in a bacteriostatic action, in which the microbial

Table 2.2  Application of essential oils in the food industry. Study

Essential oil

Application

Results

References

Parapenaeus longirostris Lucas 1846

Citrus sinensis (L.) Osbeck

Coating 0, 5%, 1%, 2%

Gelatin film enriched with 2% orange leaf showed good activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans and good antioxidant capacity

[66]

Chitosan films

Eucalyptus globulus

Casting 1%, 2%, 3% and 4%

By incorporating the essential oil to the chitosan film, a reduction in the activity of the Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Candida parapsilosis, highlighting the 4% concentration that obtained better inhibitions

[67]

Film of Quince seed mucilage

Origanum vulgare

Casting 1%, 1.5% e 2%

Antimicrobial and antioxidant properties significantly increased with the incorporation of the essential oil. Good inhibition was demonstrated against S. aureus, followed by Shewanella putrefaciens and Yersinia enterocolitica.

[68]

Strawberries

Thymus vulgaris, Mentha piperita, Cymbopogon citrates and Oreganum compactum

Coating

Thymus vulgaris was the one that presented the best coating for strawberries against antifungal activity during the 14 days studied and preserved the qualities of the sample.

[69]

Source: Authors

40  Essential Oils activity recovers after the agent’s action has ended; this condition occurs at low concentrations of essential oil. Another theory is that the high amounts of bactericide chemicals are to blame [62]. In the food industry, essential oils have been used in research in two ways: directly in the processing or on the fresh sample, or through intelligent packaging that includes the essential oil in the composition, in which case, after the chemical components of essential oils combine in the film, they gradually diffuse through the pores to the film s surface [28, 63]. Edible films are designed to extend the shelf life of foods while also providing antimicrobial protection and improving organoleptic qualities (Figure 2.2). When these coatings are combined with active ingredients, this benefit is amplified, and food safety is ensured. EOs are one of these active ingredients, as they can improve barrier and optical properties while also incorporating antibacterial properties. The antioxidant activity of EOs is mediated by its ability to modulate endogenous antioxidant systems and its ability to eliminate reactive oxygen species. Many terpenoids contained in essential oils, such as ­terpinoleneand-terpinene, among others, have been proven to have excellent antioxidant activities [65]. As indicated in Table 2.2, applications in the literature contain examples of the above-mentioned features [66–69].

2.5 Essential Oil Extraction Techniques EOs are highly volatile and contain over 300 distinct chemicals, including alcohols, aldehydes, ethers, amides, amines, esters, heterocycles, ketones, phenols, and terpenes. There are a variety of terpenes present in essential oils, including cyclic and acyclic terpenes, as well as monoterpenes, sesquiterpenes, and diterpenes. It’s also quite difficult to synthesize many complicated chemicals because of their configuration [18]. Azeotropic distillation (hydrodistillation, hydrodiffusion, and steam distillation) and solvent extraction are the most used methods for extracting EOs commercially. Traditional methods, on the other hand, have some drawbacks, such as a high economic cost due to the use of energy and high-value solvents [70]. Hydrodistillation, steam distillation, or extraction using organic solvents are all common procedures for obtaining EOs from source materials like rosemary. However, because many of the chemicals of interest are volatile, it is usual to lose them throughout this procedure [70]. It is also possible to verify the deterioration of certain of these compounds due to long extraction durations, as well as the subsequent degradation of unsaturated or esterified compounds due to heat or hydrolytic treatment [70]. According to Presti et al. [71], monoterpenes are sensitive to changes in its chemistry at present distillation conditions and during the process using a common solvent. Furthermore, many of these methods take a lot of time and effort. There are now several ways for extracting EOs, some of which are aimed specifically at lowering extraction time and enhancing EO quality. As a result, new extraction techniques like microwave-assisted extraction, pressure solvent extraction, supercritical fluid extraction, and ultrasound-assisted extraction are becoming more popular. And for each of these technologies, the use for a wide range of matrices can already be seen, such as these processes mentioned above, which has already been demonstrated for rosemary oil and bay

Essential Oils Used to Inhibit Bacterial Growth in Food  41 leaves [72, 73], Patchouli (Pogostemon cablin) Leaves [74], wet citrus peel waste [75], and lavender [76] are already studied, as a result, these novel methods for extracting EOs are now competing with older methods, and the use of each will be determined by the quality of the finished product and the production costs. Every day, new ways are produced by combining previously used ones. Microwave-assisted extraction (EP-MAE) enzymolysis-pretreatment method established by Liu et al. [77], which involves pretreatment with enzyme mixtures (hemicellulase and cellulase) followed by microwave irradiation. According to the scientists, the EP-MAE approach for extracting EOs from Cinnamomum burmannii leaves is quite efficient, and it offers a potential paradigm for separating EOs from aromatic plant materials. The ability to reduce extraction time and obtain products with a broader diversity of compounds with many bioactivities is a significant advantage of EOs obtained through innovative technologies, as most of the products acquired serve as an important raw material for making diverse chemical products. As a result, traditional methods of extracting cinnamon oil, such as hydrodistillation (HD), always require a longer extraction duration [21]. Combining techniques, on the other hand, can reinforce and improve the extractive impact, as previously mentioned. When compared to HD extraction, the ultrasound-­ assisted hydrodistillation (UAHDE) extraction technique allows for a shorter extraction time and a higher extraction yield, demonstrating a more valuable EO with a high content of vital trans-cinnamaldehyde, as well as lower electrical consumption and CO2 emissions, making it a more cost-effective method, according to Chen et al. [21]. For each process, distinct product and process features will be illustrated. As a result, the technique has an impact on the yield of EOs, the losses of some volatile compounds, and the concentration of bioactive components [78]. Traditional approaches, on the other hand, are usually criticized for technical shortcomings. Several EO components are susceptible to steam distillation conditions, as previously noted [79], Organic solvents such as hexane and methylene chloride allow for good oil and compound lipid recovery, but they are toxic, which is already a disadvantage of the technique [80]. Another disadvantage is the lengthy extraction time noted with most traditional methods [81]. The new procedures must appear to be cost-effective, environmentally friendly, long-lasting, highly efficient, and capable of producing a high-quality end product. Microwave radiation with a frequency of 2.45 GHz has piqued the interest of analytical chemistry in the case of microwave-assisted extraction since it allows for faster analysis, easier handling and processing, and higher final product purity [82]. Many publications have established the major advantages of microwave assisted EO hydrodistillation, including faster extraction times, higher yields, increased oxygen content, environmental friendliness, and lower cost [78]. One of the uses of EOs is as natural preservatives generated from plant sources, which are being investigated as alternatives to synthetic compounds. The extraction of supercritical carbon dioxide (SC-CO2), which is a sustainable green technology for the extraction of high purity oils with high aromatic content, such as those obtained from the plant, is one of the approaches that has been used. The ginger torch has applications in food, nutraceuticals, and pharmaceuticals [83]. With CO2 as a solvent, the supercritical fluid extraction technique may rapidly extract apolar chemicals from botanical sources [84]. Because gentle extraction conditions have

42  Essential Oils been reached, as well as the fact that CO2 has a lower critical temperature and pressure, supercritical CO2 extraction is suited for a wide range of applications [85]. One of the main methods used for the extraction of EOs is hydrodistillation, however, the drying step can negatively contribute to the process. Thus, to eliminate the drying step and increase the extraction efficiency, enzymatic pretreatment is used [86]. Although it is not an extraction method, it is a vital step that can help some approaches work more efficiently. Enzymatic pre-treatment can increase the hydrodistillation extraction of EOs from Thymus capitatus, Rosmarinus officinalis, Lavandula hybribia flowers by high yield percentages, depending on the enzyme used [87, 88]. Steam explosion is one of the novel ways for collecting EOs, which involves self-evaporation and results in the displacement of a major portion of the plant’s volatile molecules. According to Golmohammadi et al. [89], steam explosion pretreatment can increase extraction yield and kinetics. Since cell swelling allows for better kinetic extraction under diffusivity and early accessibility circumstances. For industrial testing, steam explosion investigations can be carried out on industrial scale equipment. Because it can recover citrus by-products, this technology has a lot of potential as a way to help the business. Additionally, mathematical models based on veneered particle descriptions were compared to predict the behavior of steam explosion and water distillation processes [89]. The technology of using Green and Solvent-free simultaneous ultrasonic-microwaveassisted EO extraction could be a great environmentally friendly option. Non-ionizing electromagnetic waves with a frequency of 300 MHz to 300 GHz are employed in this technology. In this way, microwaves instantly heat polar molecules like H2O inside the plant cell, while the evaporation process exerts tremendous pressure on the cell walls. This pressure exerts a pushing effect on the cell wall, stretching it until it ruptures, resulting in an increase in phytochemical leaching from the broken cells [90]. Ultrasonic Assisted Extraction (UAE) can also be used to separate biologically active plant components, according to Sahin et al. [91]. This technology creates micropores in plant cell walls using high-frequency sound waves (20-50 kHz), and high-intensity mechanical and thermal effects cause plant components to be liberated [92]. The search for more efficient ways is far from over; EO extraction varies depending on available resources and the intended product’s focus.

2.6 Conclusions The use of EOs to protect food against hazardous and pathogenic bacteria could reduce the usage of chemical additives, which can be harmful to human health and the environment in some situations. As a result, research and applications in this subject are required. Many researchers that employed various EOs extracted from diverse sources of plant origin revealed high potential against dangerous and pathogenic bacteria in their results, which can be applied in the food business in the future, according to this book chapter. Extraction techniques may also become more accessible, affordable, and environmentally friendly, allowing for the widespread use of EOs, a reality that is not far from our daily lives and that provides people with better and safer food.

Essential Oils Used to Inhibit Bacterial Growth in Food  43

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Essential Oils Used to Inhibit Bacterial Growth in Food  45 39. Santangelo, C., Filesi, C., Varì, R., Scazzocchio, B., Filardi, T., Fogliano, V., D’Archivio, M., Giovannini, C., Lenzi, A., Morano, S., Masella, R., Consumption of extra-virgin olive oil rich in phenolic compounds improves metabolic control in patients with type 2 diabetes mellitus: A possible involvement of reduced levels of circulating visfatin. J. Endocrinol. Invest., 39, 1295, 2016. 40. Casamenti, F. and Stefani, M., Olive polyphenols: New promising agents to combat aging-­ associated neurodegeneration. Expert Rev. Neurother., 17, 345, 2017. 41. Rehman, A., Jafari, S.M., Aadil, R.M., Assadpour, E., Randhawa, M.A., Mahmood, S., Development of active food packaging via incorporation of biopolymeric nanocarriers containing essential oils. Trends Food Sci. Technol., 101, 106, 2020. 42. Cheynier, V., Compostos fenólicos: Das plantas aos alimentos. Phytochem. Rev., 11, 153, 2012. 43. Bartwal, A., Mall, R., Lohani, P., Guru, S.K., Arora, S., Role of secondary metabolites and brassinosteroids in plant defense against environmental stresses. J. Plant Growth Regul., 32, 216, 2013. 44. Kapravelou, G., Martínez, R., Andrade, A.M., López Chaves, C., López-Jurado, M., Aranda, P., Arrebola, F., Cañizares, F.J., Galisteo, M., Porres, J.M., Improvement of the antioxidant and hypolipidaemic effects of cowpea flours (Vigna unguiculata) by fermentation: Results of in vitro and in vivo experiments. J. Sci. Food Agric., 95, 1207, 2015. 45. Martins, N., Barros, L., Ferreira, I.C., In vivo antioxidant activity of phenolic compounds: Facts and gaps. Trends Food Sci. Technol., 48, 1, 2016. 46. Granato, D., Mocan, A., Câmara, J.S., Is a higher ingestion of phenolic compounds the best dietary strategy? A scientific opinion on the deleterious effects of polyphenols in vivo. Trends Food Sci. Technol., 98, 162, 2020. 47. Zhao, Y.C., Xue, C.H., Zhang, T.T., Wang, Y.M., Saponins from sea cucumber and their biological activities. J. Agric. Food Chem., 66, 7222, 2018. 48. Góral, I. and Wojciechowski, K., Surface activity and foaming properties of saponin-rich plants extracts. Adv. Colloid Interface Sci., 279, 102145, 2020. 49. Tsuchiya, H. and Iinuma, M., Reduction of membrane fluidity by antibacterial sophoraflavanone G isolated from Sophora exigua. Phytomedicine, 7, 161, 2000. 50. Ding, L., Xiao, S., Liu, D., Pang, W., Effect of dihydromyricetin on proline metabolism of Vibrio parahaemolyticus: Inhibitory mechanism and interaction with molecular docking simulation. J. Food Biochem., 42, e12463, 2018. 51. Plaper, A., Golob, M., Hafner, I., Oblak, M., Solmajer, T., Jerala, R., Characterization of quercetin binding site on DNA gyrase. Biochem. Biophys. Res. Commun., 306, 530, 2003. 52. Haraguchi, H., Tanimoto, K., Tamura, Y., Mizutani, K., Kinoshita, T., Mode of antibacterial action of retrochalcones from Glycyrrhiza inflata. Phytochemistry, 48, 125, 1998. 53. Lambert, R.J., Skandamis, P.N., Coote, P.J., Nychas, G.J., A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. J. Appl. Microbiol., 91, 453, 2001. 54. Neto, N.J.G., Magnani, M., Chueca, B., García-Gonzalo, D., Pagán, R., Souza, E.L., Influence of general stress-response alternative sigma factors σS (RpoS) and σB (SigB) on bacterial tolerance to the essential oils from Origanum vulgare L. and Rosmarinus officinalis L. and pulsed electric fields. Int. J. Food Microbiol., 211, 32, 2015. 55. Ultee, A., Kets, E.P.W., Alberda, M., Hoekstra, F.A., Smid, E.J., Adaptation of the food-borne pathogen Bacillus cereus to carvacrol. Arch. Microbiol., 174, 233, 2000. 56. Cox, S.D., Mann, C.M., Markham, J.L., Determining the antimicrobial actions of the tree oil. Molecules, 6, 87, 2001. 57. Picone, G., Laghi, L., Gardini, F., Lanciotti, R., Siroli, L., Capozzi, F., Evaluation of the effect of carvacrol on the Escherichia coli 555 metabolome by using 1 HNMR spectroscopy. Food Chem., 141, 4367, 2013.

46  Essential Oils 58. Ultee, A., Kets, E.P.W., Smid, E.J., Mechanism of action of carvacrol on the food-borne pathogen Bacillus cereus. Int. J. Food Microbiol., 64, 373, 2001. 59. Alves, A.G., Stamford, T.L.M., Queiroz, F.R.C.B., Leite, S.E., The cytotoxic effect of essential oils from Origanum vulgare L. and/or Rosmarinus officinalis L. @ on Aeromonas hydrophila. Foodborne Pathog. Dis., 9, 298, 2012. 60. Tongnuanchan, P., Benjakul, S., Prodpran, T., Physico-chemical properties, morphology and antioxidant activity of film from fish skin gelatin incorporated with root essential oils. J. Food Eng., 117, 350, 2013. 61. Rodriguez-Garcia, I., Silva-Espinoza, B., Ortega-Ramirez, L., Leyva, J.M., Siddiqui, M.W., Cruz-Valenzuela, M.R., Ayala-Zavala, J.F., Oregano essential oil as an antimicrobial and antioxidant additive in food products. Crit. Rev. Food Sci. Nutr., 56, 1717, 2016. 62. Swamy, M.K., Akhtar, M.S., Sinniah, U.R., Antimicrobial properties of plant essential oils against human pathogens and their mode of action: An updated review. Evid. Based Complementary Altern. Med., 2016, 1, 2016. 63. Li, J., Ye, F., Lei, L., Zhao, G., Combined effects of octenylsuccination and oregano essential oil on sweet potato starch films with an emphasis on water resistance. Int. J. Biol. Macromol., 115, 547, 2018. 64. Arabpoor, B., Yousefi, S., Weisany, W., Ghasemlou, M., Multifunctional coating composed of Eryngium campestre L. essential oil encapsulated in nano-chitosan to prolong the shelf-life of fresh cherry fruits. Food Hydrocoll., 111, 106394, 2021. 65. Vianna, T.C., Marinho, C.O., Júnior, L.M., Ibrahim, S.A., Vieira, R.P., Essential oils as additives in active starch-based food packaging films: A review. Int. J. Biol. Macromol., 182, 1803, 2021. 66. Alparslan, Y., Yapici, H.H., Metin, C., Baygar, T., Günlü, A., Baygar, T., Quality assessment of shrimps preserved with orange leaf essential oil incorporated gelatin. LWT, 72, 457, 2016. 67. Hafsa, J., Ali Smach, M., Khedher, M.R.B., Charfeddine, B., Limem, K., Majdoub, H., Rouatbi, S., Physical, antioxidant and antimicrobial properties of chitosan films containing Eucalyptus globulus essential oil. LWT, 68, 356, 2016. 68. Jouki, M., Yazdi, F.T., Mortazavi, S.A., Koocheki, A., Quince seed mucilage films incorporated with oregano essential oil: Physical, thermal, barrier, antioxidant and antibacterial properties. Food Hydrocoll., 36, 9, 2014. 69. Vu, K.D., Hollingsworth, R.G., Leroux, E., Salmieri, S., Lacroix, M., Development of edible bioactive coating based on modified chitosan for increasing the shelf life of strawberries. Food Res., 44, 198, 2011. 70. Bousbia, N., Vian, M.A., Ferhat, M.A., Petitcolas, E., Meklati, B.Y., Chemat, F., Comparison of two isolation methods for essential oil from rosemary leaves: Hydrodistillation and microwave hydrodiffusion and gravity. Food Chem., 114, 355, 2009. 71. Presti, M.L., Ragusa, S., Trozzi, A., Dugo, P., Visinoni, F., Fazio, A., Dugo, G., Mondello, L., A comparison between different techniques for the isolation of rosemary essential oil. J. Sep. Sci., 23, 273, 2005. 72. Kosar, M., Tunalier, Z., Ozek, T., Kürcüglu, M., Can Baser, K.H., A simple method to obtain essential oils from Salvia triloba L. and Laurus nobilis L. by using microwave-assisted hydrodistillation. Z. Naturforsch. C. J. Biosci., 60, 501, 2005. 73. Moradi, S., Fazlali, A., Hamedi, H., Microwave-Assisted Hydro-Distillation of essential oil from rosemary: Comparison with traditional distillation. Avicenna J. Med. Biotechnol., 10, 22, 2018. 74. Kusuma, H.S. and Mahfud, M., Microwave-assisted hydrodistillation for extraction of essential oil from patchouli (Pogostemon cablin) leaves. Period. Polytech. Chem. Eng., 61, 82, 2016. 75. Bustamante, J., Van Stempvoort, S., García-Gallarreta, M., Houghton, J.A., Briers, H.K., Budarin, V.L., Clark, J.H., Microwave assisted hydro-distillation of essential oils from wet citrus peel waste. J. Cleaner Prod., 137, 598, 2016.

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3 Industrial Application of Essential Oils S. Kiruthika and S. Vishali* Department of Chemical Engineering, College of Engineering and Technology, SRM Institute of Technology, Kattankulathur Tamil Nadu, India

Abstract

Consumers have become increasingly interested in natural commodities as solutions to artificial ingredients or pharmaceutically relevant chemicals in recent years. Essential oils have gained prominence in the food, cosmetics, and pharmaceutical industries, among others. Food safety and sustainability during preparation, transportation, and storage are now required for modern food management. The bioactivities of essential oils (EOs), which are significant fragrant components of spices and herbs, have been known and used in medication, food preservation, seasoning, and perfume since ancient times. This chapter introduces the origin, composition, extraction techniques, and commercial applications of essential oils. Antimicrobial property, food security, extension of shelf-life, pharmaceuticals, aromatherapy, and pest control management are among the applications. Essential oils are mentioned for their therapeutic benefits in massage treatments as well as their application as natural skin permeation enhancers for drug administration systems. Recent breakthroughs in the application and alternative fruit and food degradation methods were highlighted, particularly secondary metabolites of plants as fruit conservatives, pharmaceutical use of essential oils, and their antimicrobial scavenger properties. Essential oil components and derivatives are thought to be a viable alternative for controlling a variety of dangerous insects. Their quick disintegration in the environment has improved selectivity, favoring helpful insects. Keywords:  Essential oils, antimicrobial, food packaging, pharmaceuticals, aromatherapy, pest control management

3.1 Introduction The expanding demand and consumer interest in aromatic and therapeutic plants for medical, culinary, and human-made purposes have increased these plants’ appeal. Consumers are discovering the beneficial effects of herbal and medicinal plants and their byproducts to learn more about health, food, and nutrition issues. Rich on the Mediterranean periphery, aromatic plants are closely linked to the origins of Western civilization. Since ancient times, they have been mentioned for their use as spices, potted herbs, or medicinal plants. They  improved the taste of foods and helped preserve them, were used to embalm the *Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (49–68) © 2023 Scrivener Publishing LLC

49

50  Essential Oils dead, or were incorporated into balms healing ointments. In addition, these plants generate essential oils, which are a type of secondary metabolite. The chemical composition of essential oil is complex. Essential oils’ constituent components are helpful in various sectors, including agronomy, the environment, and human health. Essential oil is derived from the medication Qunita Essentia, which Paracelsus von Hohenheim named in Switzerland in the 16th century [1]. The nineteenth century is generally considered the beginning of the modern phase of essential oil in industrial use. As a result, they are familiar with a broad range of industrial services, including perfumes, cosmetics, and detergents, pharmaceuticals, pest control, and fine chemicals, as well as flavorings for the food industry. They are used extensively in the perfumery, aromatherapy, and cosmetic industries. The essential oil also acts as a chemical signal to the plant, allowing it to regulate its surroundings, such as resisting pests, enticing pollinators, limiting seed development, and communicating with other species. Interest in the application of essential oils in various fields is continuously increasing, and with their significant pharmacological activities, these aromatic medicinal herbs are used in the pharmaceutical industry as antibacterial, anti-inflammatory, antiviral, and antifungal agents. Several types of research were carried to study the antibacterial benefits of essential oils against several bacteria [2]. In addition, psychiatrists use essential oils for their mental patients because of the pleasant smell created by the aromatic plant oils [3]. In addition, the need for essential oils for edible preservatives is growing because they are biologically active against food-borne pathogens like Salmonella  typhosa, Pseudomonas putida, Clostridium welchii, Listeria monocytogenes, and Staphylococci aureus [4]. The problem of pathogens in food and meat products was solved by the low dosage of essential oils, as numerous studies have shown [5]. Furthermore, due to the development of drug-resistant bacterium and the risk of transference from animals to humans, there is an increased demand for natural plants as an alternative to conventional antibiotics in animal nutrition. The European Union has banned the use of synthetic antibiotics in animal feed to promote the healthy growth of animals [6]. In the pharmaceutical industry, medicines in capsules, syrups, ointments, creams, and sprays contain some compositions of essential oil, and their production numbers are constantly increasing. For example, the uses of essential oils in numerous areas such as the food, pharmaceutical and cosmetic industries are still being explored in order to formulate and manufacture more efficient essential oil-based products.

3.2 Essential Oils Essential oils (EO), as defined by the European Pharmacopeia 7th edition [7], as “Odorant products, which have a complex composition and are obtained from raw plant extract, either extracted by the steam of water, dry distillation or a suitable mechanical method without heating. Generally, a physical method is used to separate the essential oil from the aqueous phase, which has no significant change in its chemical composition”. Multiple segments of the oil plants such as peels, barks, leaves, flowers, buds, seeds, and others are used to produce these aromatic oily liquids. Various extraction techniques are applied for the extraction process [2].

Industrial Application of Essential Oils  51 Essential oils are soluble in organic solvents but insoluble in inorganic solvents. Except for vetiver, sassafras, and cinnamon, EO’s are volatile with a distinct odor and a density of less than one. The extraction yields are dependent on the type of species and plant segments used, but a meager result like 1% can make them precious rare components [1]. Among the plethora of aromatic plant species, only 10% out of 17,000 plant species are considered aromatic [8]. Various fields in applying essential oils are drug, cosmetics, and food production, whereas their potential as anticancer, antibacterial, antiviral, and anti-inflammatory agents has been studied and evaluated. In addition, many human diseases have been reported to be treated with essential oils due to their numerous biological activities.

3.2.1 Sources and Chemical Composition Essential oils can be found in the roots, peels, leaves, seeds, fruits, barks, and other parts of aromatic plants. Essential oils are found in various plants; however, the portions of plants that act as the principal origin of essential oil may differ (Table 3.1). The differences between the chemical properties of essential oils are based not only on their molecular number and type but also on their steric structures, which the extraction process can influence. In addition, the quality, quantity, and constitution of the essential oils can change depending on the plant organ, age, soil composition, climate change, and life cycle stages [9]. Table 3.1  Plant parts that contain essential oils [4, 7, 9]. Plant part

Plants

Leaflets

Basil, Melaleuca, Bay leaf, Thyme, Cinnamon, Wintergreen, Peppermint, Eucalyptus, Lemongrass, Oregano, Pine, Rosemary

Peel

Bergamot, Tangerine, Lemon, Lime, Orange, Tea tree, Mandarin, Grapefruit, spearmint

Floweret

Palmarosa, Chamomile, Jasmine, Clay Sage, Immortelle,Clove, Hyssop, oregano, Orange, Cumin, Geranium, Baccharises, Rosalina, Blue Tansy, Ajowan, Sylvestris, Tarragon, Neroli, Geranium, Lavender, Rose

Kernel

Carrot Celery, Nutmeg, Fennel, Almond, Ylang-Ylang, Anise, Celery, Cumin, Nutmeg Oil, Cardamom, Caraway, Coriander, Parsley

Lumber (woods)

Sandalwood, Guaiac Wood, Camphor, Cedar, Rosewood, Amyris, Myrtle

Plant shell (Bark)

Cinnamon, Cassia

Resins

Myrrh, Frankincense

Rhizome

Ginger

Fruits

Black pepper, Xanthoxylum, Nutmeg

Roots

Ginger, Plai, Turmeric, Valerian, Vetiver, Spikenard, Angelica

52  Essential Oils Each oil has over a hundred components on average, though the quantity varies depending on the oil. A majority of the active components are terpenoids (monoterpenoids and sesquiterpenoids) and phenylpropanoids. These two groups are produced by distinct metabolic pathways and emerge from different stages of primary metabolism. Essential oils, like all organic chemicals, are made up of hydrocarbon molecules and are categorized as phenols, esters, ketones, terpenes, alcohols, esters, and aldehydes, etc. [10, 11]. Aldehydes, esters, coumarins, phenols, ketones, monoterpene and sesquiterpene alcohols, alcohols, lactones, and oxides are some of the other oxygenated chemical compounds in essential oils [12].

3.2.2 Extraction Methods Several aromatic plants can be extracted and form essential oils that subsequently have many cosmetics, pharmaceutical, and food safety fields. The main factor in ensuring the quality of essential oils is the extraction method used since inappropriate extraction procedures may cause the destruction and vary the action of phytochemicals present in aromatic oils. Such extraction techniques can be categorized into conventional and innovative approaches.

3.2.2.1 Conventional Extraction Methods The most effective processes used in this extraction method include steam distillation, freeze-drying, hydrolyzation, rotary evaporation, and GC chromatography assays. The traditional techniques for obtaining essential oils from various plants are depicted in Figure 3.1.

DISTILLATION ROTARY EVAPORATOR

HYDROLYZATION

SOLVENT EXTACTION

ESSENTIAL OILS

GC CHROMATOGRAPHY

FLORAL EXTRACTION

COLD PRESS FREEZE DRYING

Figure 3.1  Oil extraction methods from different plant sources.

Industrial Application of Essential Oils  53 Steam distillation separates components that disintegrate at high temperatures by distilling them with steam. Steam distillation extracts 93 percent of essential oils, with the remaining 7% can be extracted further using various processes [13]. During the hydrolysis process, plant components are immersed in water and then boiled to release their properties [14]. The technique is used explicitly for citrus family oils like orange, lemon as the essential oils are stored in the skin or epicarp of citrus. The solvent extraction process isolates essential oils from plant material using food-grade solvents such as hexane and ethanol. It›s best for plant materials that produce little essential oil and are primarily resinous, or are fragile aromatics that can›t endure the pressure and strain of steam distillation. These traditional extraction processes can result in the decomposition of some unsaturated molecules and the loss of specific components due to heat impacts or hydrolysis. In addition, these conventional extraction procedures could often extract EOs from plants with concentrations ranging from 0.005 to 10 %, based on the temperature, time, and pressure of the distillation, and most crucially, the type and quality of raw plant materials. These drawbacks have sparked new research, prompting the improvement, optimization, and advancement of current and emerging “green” technology processes.

3.2.2.2 Innovative Extraction Methods In recent years, innovative techniques based on the green extraction principles and methodologies for getting natural extracts of equivalent or superior quality from conventional procedures have repeatedly emerged. Reducing the number of operating units, the quantity of energy consumption, and the amounts of CO2 emissions are some of the advantages of innovative extraction methods. Green extraction can be defined as extraction technology innovation and development that lowers energy usage, enables the utilization of alternate solvents and innovative plant sources, and ensures intact and superior quality extracts without using petroleum-based solvents [15]. The following are the different types of green extraction methods. 1. Turbo distillation – The turbo distillation concedes for substantial stirring and blending, as well as a cutting and damaging impact on plant components, resulting in a distillation time reduction of 2 to 3 times. 2. Ultrasound-Assisted Extraction – Micro-jets are created when cavitation bubbles created during ultrasonication collide, destroying EO glands and allowing for mass transfer and production of essential oils. 3. Microwave-Assisted Extraction – Depending on the experimental methods, plant components are separated in a microwave unit under varied conditions. 4. Instantaneous Controlled Pressure Drop Technology – It removes volatile compounds by evaporation at elevated pressure and temperature for a short period and self-vaporization from plant materials due to a rapid pressure drop in several cycles. The other emerging techniques for EOs extraction with high purity and less energy consumption are Supercritical fluid extraction, and Pulsed charge field sustained extraction, Simultaneous distillation extraction, and Subcritical water extraction [15].

54  Essential Oils

3.2.3 Industrial Applications of Essential Oils Interest in the application of EO’s in various fields is increasing continuously. These aromatic medicinal plants are being implemented in pharmaceutical industries as antibacterial, anti-inflammatory, antiviral, and antifungal agents with their significant pharmacological activities. Researchers have investigated EO’s antimicrobial properties against diverse bacteria through various studies [16, 17]. Additionally, essential oils are commonly used by psychiatrists as they have antipsychotic effects when used with their psychic patients [18, 19]. Numerous researchers have documented the beneficial effects of essential oils in treating pathogenic bacteria in food products and meat products [20]. To encourage the healthy growth of animals, the European Union has banned chemotherapeutic agents in feeding livestock [21]. In pharmaceutical industries, medicines in capsules, syrups, ointments, creams, and sprays contain some composition of essential oils, and their production numbers are constantly growing. Thus, the applications of essential oils in numerous fields; food, pharmaceutical, and cosmetic industries are still being investigated to formulate and produce more efficient essential oil-based products. This chapter covers the extraction process involved in producing essential oils and their compositions, their pharmaceutical and therapeutic potentials as antimicrobial, antiviral, anticancer, and skin permeation enhancer agents. The various application of essential oils in industries is depicted in Figure 3.2.

3.2.3.1 Food Preservation and Active Packaging Systems The food industry is constantly evolving to meet customer requirements by using innovative techniques and ingredients to create more efficient, safer, more environmentally sustainable solutions. For example, essential oils are currently being incorporated into various biodegradable materials such as edible foils. As a result, active packaging technologies with improved preservation properties are being developed to help both the food and packaging industries minimize food waste and eliminate waste. EOs can be incorporated into packaging materials as free or encapsulated molecules, with the latter having shown particular promise. The addition of these lipophilic components to Agriculture

Aromatherapy Bio pesticide

Skin care

Preservatives

Bio-insecticide

Pharmaceutical Industry

Cosmetics

Pest Management

Food Industry Medicine

Figure 3.2  Schematic representation of EO’s in industries.

Industrial Application of Essential Oils  55 the final product offers various exciting bioactivities that can help increase the product’s shelf life by preventing the food from deteriorating. In addition, integrating biodegradable packaging with essential oils derived from natural agro-industrial waste can create a more sustainable food sector. Therefore, EOs are becoming increasingly common in products, since they can serve a number of functions, including facilitating lower water vapor penetration and enhancing antioxidant and antibacterial activity [22]. Biofilms are typically produced with hydrophilic matrices (to increase water vapor permeability), which essentially act as a platform for other substances, such as lipids, to be absorbed into the biofilm [23]. The materials selected for this purpose should ideally have a lower viscosity, high hygroscopicity and emulsifiability, lower reactivity and lower costs, and no influence on the organoleptic properties of the processed food [24, 25]. Homogenization emulsification is used to incorporate lipophilic chemicals such as EO into hydrophilic compounds [26]. As a result, the combination of EOs with biodegradable films is gaining popularity, particularly in the active packaging sector. The US Food and Drug Administration has classified EOs as GRAS (Generally Recognized as Safe) antimicrobial additives in foods. They are rich in biologically active components with proven antibacterial and antioxidant activities of interest as food additives [27, 28]. However, its use as a food supplement is gaining popularity [29]. The primary strategy for ensuring food security is to lower the baseline microbial loading and employ active packaging to prevent germs from growing in post-process usage such as processing and transportation [30]. Because of their numerous uses, cinnamon essential oils are considered the most widely used essential oils in the food and cosmetics industries, especially as antibacterial agents [31]. For example, Cinnamon oil encapsulated in cyclodextrin nanosponges could be applied as an antibacterial food packaging ingredient [32]. In addition, garlic essential oil nanophytosomes have demonstrated their potential as a naturally occurring food preservation agent using yogurt by efficiently showing good physicochemical characteristics, especially in acidic foods. Essential oils have an excellent antibacterial effect against food-borne bacteria and can be used as preservatives or antimicrobial agents in food packaging in the food industry [33, 34]. Functional edible coatings that contain natural antioxidants can increase the shelf life of meat products and thus find application in the food sector [35]. Biomaterials such as lipids, polysaccharides, and proteins are often used by themselves or in edible films and coatings [36]. The buyer’s sensory acceptance of the final product should be considered when using and selecting EOs. Their direct use is often limited due to their pungent taste. As a result, EO can improve food safety and the quality of the indulgence layers proposed as an alternative food packaging option [37]. Applying oregano and thyme-based edible coatings to fresh beef steaks may have the ability to reduce pathogens and increase color stability while maintaining good sensory properties [38]. Avoiding perishable bacteria associated with fresh foods and their products is a significant challenge in the food sector. Bioactive bundling systems are considered to be a promising innovation that has a decisive influence on the extension of shelf life and product safety. As a result of microbial and enzymatic inactivation, the products lose their organoleptic and nutritional properties. It is crucial to develop effective storage methods and alternative technologies to increase food quality and shelf life. As natural antibacterial agents, essential

56  Essential Oils oils (EOs) and their components have much potential to inhibit the growth of pathogenic and decomposing microorganisms in food [38]. Essential oils and their usage in food and products to prevent the spread of food-borne diseases and germs have been achieved by directly adding EO to the product as an ingredient or consolidation to edible coating films and material to secure the surface of the product. For example, chitosan and thymol in a coating on fresh fig fruits can help to maintain their freshness and extend their shelf life [39]. Furthermore, the combination of Thymol EO and Chitosan demonstrated a considerable antibacterial effect. As a result, functional coatings are a robust platform that could extend the life of fresh meat while preserving its quality and safety [40]. The following essential oils are utilized as preservative agents in food and fruits. 1. Rosemary and Oregano – Beef steak with edible coatings that reduce fatty acid oxidation, water loss, and shear force and improve the recognition of smell, taste, and general consumer acceptance. 2. Lemon essential oil–chitosan – Strawberry storage quality as measured by a reduced respiratory rate and increased antifungal activity in vitro and during cold storage. 3. Quince seed mucilage film (QSMF) with essential oils of thyme or oregano Extension of the life of chilled rainbow trout with low bacterial growth and a significant extension of the shelf life by up to 11 days. 4. Lemongrass essential oil is mixed into an edible coating made of alginate – Freshly cut pineapple shelf life extension and quality retention during storage at low temperature with significantly lower respiration rate, total plate count, weight loss, yeast and mold counts. 5. Oregano oil – In Fresh lettuce, the oil reduces Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli. 6. Carvacrol/Eugenol – Washing spinach leaves and green beans with carvacrol or eugenol resulted in decreased E. coli and S. enterica. 7. Lemongrass – Apple with immediate E. coli inactivation that went undetected in the refrigerator for two weeks.

3.2.3.2 Aromatherapy Essential oils have been valued for generations as an aroma with healing properties for the body, mind, and soul. These scent molecules are potent organic plant chemicals that eliminate disease, bacteria, viruses, and fungi from the environment [41, 42]. Many scientists have proven their multifaceted character as follows [43]: vv Antiviral, antibacterial, and anti-inflammatory properties vv Immune-boosting system with hormonal, endocrine, psychological, cardiovascular, and relaxing effects as well as a boost for the mind and alertness Since their effectiveness does not deteriorate with time or age, these oils are known for their energy-specific properties. The stimulating properties of these oils are due to their structures, which are very similar to hormones [44].

Industrial Application of Essential Oils  57 One of the most demanding issues of this therapy is its capability to penetrate the subcutaneous tissue. Because of their intricate structure and chemical properties, their effects are also complex and subtle. Essential oils are incorporated into a biological signal from the recipient cells in the nose when breathed in, which is the mechanism by which they work. The signal is sent from the olfactory bulb to the limbic and hypothalamus regions of the brain. These impulses allow the brain to generate neurotransmitters like serotonin and endorphin, which connect our nerves and other systems in the body, offer positive change, and induce relaxation. The calming, euphoric, and energizing oils release serotonin, endorphin, and norepinephrine, which have the desired effects on the mind and body [45]. Aromatherapy is nature’s non-invasive, natural gift to humanity. The use of perfume not only eliminates symptoms of illness it also rejuvenates the whole body. Aromatherapy promotes physical, psychological, and spiritual well-being for a new stage of development. This treatment is effective not only as a preventative measure but also in the acute or chronic phases of the disease.

3.2.3.3 Pharmaceutical and Medicinal Application The function of essential oils as an antimicrobial, skin permeation enhancer, antiviral, and anticancer find its application in pharmaceutical fields.

3.2.3.3.1 Anticancer Agent

Essential oils were first discovered and used to treat inflammatory and oxidative diseases. It reacts with oxygen species related to the cause of inflammation and oxidation, both of which can contribute to cancer, suggesting that they can act as anticancer agents as well [46]. In addition, studies have shown the application of various essential oils as chemotherapeutic agents of different types of cancer. Table 3.2 shows the species of essential oils used to treat the different types of cancer.

3.2.3.3.2 Essential Oils as Antibacterial Agent

The use of aromatic plants as antibacterial agents to inhibit bacterial growth and prevent putrefaction has been used since ancient times. Various studies have scientifically reported that the potential of essential oils to prevent bacterial growth in many areas. 1. Eucalyptus globules essential oil - The chemical constituents found in eucalyptus extract may be the major cause of the essential oil’s ability to inhibit bacterial growth. Because this essential oil has high antibacterial potential, it has been suggested as a natural antibiotic for treating various infectious diseases. However, more clinical research is needed and recommended [53]. 2. Artemisia annua can inhibit H. influenza, S. pneumoniae, M. luteus, and C. krusei microbial strains [54]. 3. Curcuma mangga, Curcuma aeruginosa, and Zingiber cassumunar essential oils possess antimicrobial activity against bacteria [55].

58  Essential Oils Table 3.2  Essential oils used in cancer treatment. Type of cancer

Essential oil

Cell line

Reference

Human prostate cancer

Hypericumhircinum L.

✓✓ Human ✓✓ squamous carcinoma (A431)

[47]

Leaves of Xylopia frutescence

✓✓ NCI-H358M (lung carcinoma cell)

[48]

Solanium erianthum, of fruit and leaf volatile essential oils

✓✓ HS 578T

[49]

Lavender angustifolia essential oil

✓✓ PC-3, Human prostate cancer cell line

[50]

Seed essential oils from Scorodophloeus zenkeri and Afrostyrax lepidophyllus

✓✓ A 375 human malignant melanoma ✓✓ Human glioblastoma ✓✓ MDA-MB 231 Human breast adenocarcinoma ✓✓ Carcinoma cell lines ✓✓ HCT116 human colon

[50]

Cedrelopsis grevei

✓✓ MCF-7 ✓✓ Human breast cancer cell

[51]

Xylopia frutescens Aubl. (Annonaceae)

✓✓ NCI-H358M (human lung cancer cell lines)

[48]

Artesimia indica

✓✓ A-549 (lung) ✓✓ THP-1 (leukemia) ✓✓ Caco-2 (colon) ✓✓ HEP-2 (liver)

[52]

Human breast cancer

Lung cancer

3.2.3.3.3 Essential Oils as Antiviral Agent

Various infectious diseases are caused by some agents called viruses, and meanwhile, there are many kinds of drugs available to be antiviral agents. Vegetable essential oils have been evaluated for their antiviral performance, along with essential oils widely used in cooking [56]. Research has illustrated the ability of eucalyptus essential oils to prevent viral infections, and the action of the essential oils studied may occur due to synergies among major and minor elements [57]. Lippia graveolens (Mexican oregano) Essential Oil - This essential oil is a plant of the family group of Verbenaceae, which are frequently known as Mexican oregano and used in Mexico as a food flavoring and a folk remedy [10]. The antibacterial activity of this oil has been revealed, and the inhibition activity of the essential oil was due to its main constituents, named carvacrol [11].

Industrial Application of Essential Oils  59

3.2.3.3.4 Essential Oils as Skin Penetration Enhancers for Transdermal Therapeutics

Research has been done to see if these aromatic herbs can help to increase the percutaneous absorption of certain medications when administered topically through the lower layer of the skin. The following are some of the essential oils used for the drug delivery 1. Turpentine Essential Oil was proved to be an excellent natural penetration enhancer for hydrogel formulations [58]. 2. Eucalyptus Essential Oil is incorporated with chlorhexidine digluconate (CHG) as a skin antiseptic to enhance its efficacy during topical application as an antiseptic skin drug [59]. 3. Eryngium bungei Essential Oil – There are nine species comprised under the genus Eryngium (Umbelliferae), usually found in many regions of Iran. Their leaves are traditionally used in salad or food and also for traditional medicine as a diuretic agent. The use of this essential oil and its methanolic extract to enhance the permeation of Piroxicam, and nonsteroidal anti-inflammatory drugs (NSAID) used as an antipyretic agent, on Franz diffusion cells [59]. The results illustrated that the most remarkable permeation rate could be achieved when extract and essential oil concentrations increased. The enhancement of drug permeation was due to increased lipid disruption in the stratum corneum and existing of E. bungei methanol extract and essential oil.

3.2.3.3.5 Essential Oils as Anti-Inflammatory

Tea tree oil reduced the histamine reactivity of weal and flare in humans. After 10 minutes, topical applications of 100 percent tea tree oil can lessen the irritation caused by histamine diphosphate. In addition, data on several essential oils suggests that non-cytotoxic amounts increase interleukin-10 production, which has an anti-inflammatory effect [60].

3.2.3.3.6 Essential Oils as Anti-Lice and Anti-Dandruff

The tea tree oil is found in the majority of head lice treatments. Tea tree oil has insecticidal properties due to its anticholinesterase properties [61]. Shampoos with 5% tea tree oil were found in single-blind and parallel-group research to be efficient and well-tolerated for patients with mild to moderate dandruff, with an improvement of at least 41% being seen [62]. On the Culex pipiens molecules, the essential oils of Nepeta parnassica showed promising insect repellent/toxicity responses [63].

3.2.3.3.7 Essential Oils as Antioxidant

In vitro, the Nigella sativa L. essential oil is a powerful antioxidant with radical-scavenging solid ability. Antibacterial and antioxidant activities are found in Leptospermum petersonii, Manuka (Leptospermum scoparium), and Kanuka (Kunzea ericoides). The essential oil of Melaleuca armillaris has strong antioxidant properties; it changes the superoxide dismutase variables and boosts vitamin C and E levels [64]. Gene mutations and post translational

60  Essential Oils changes of numerous proteins can be caused by free radicals created during inflammation. If not, removing it risks introducing harmful radicals into the system as a whole. Antioxidant properties of substances usually counteract this mechanism. Cupressus sempervirens, Euphorbia globules, Thymus vulgaris, and Citrus limon have all been proven to have anti-inflammatory properties in animal studies [65].

3.2.3.4 Biopesticide in Insect Pest Management Biopesticides are non-toxic parasite-controlling substances from bacteria, fungi, microalgae, viruses, worms, and parasites or materials from organic sources that come from life forms, their products (phytochemical, microbes), and their derivatives (semiochemical). Biopesticides are often less harmful by nature. It usually only attacks the target pests, and highly associated species are effective in small amounts and decomposes quickly. However, to employ biopesticides effectively, users must have a thorough understanding of pest management. Integrated pest control (IPM) is becoming more popular. However, the disadvantages of using synthetic insecticides have increased interest in goods based on essential oils as longterm insect control and management options beneficial for society and the environment. As they evolved, aromatic plants developed various chemical repellants against insect herbivores and other plant infections. Many aromatic plant combinations of mono- and sesquiterpenes exhibit promise spatial and contact insecticides and repellant capabilities that could be used in domestic and agriculture insect management. The outstanding benefits of essential oil-based insecticides are as follows: vv Insect nervous systems have multiple mechanisms of action. vv Low toxicity to mammals – Lethal dose ( LD50) between the range of 800 to 3000 mg/kg can be used as pure compounds in rodents. vv Environmental persistence is low - less than 24 hours on the surface, soil, and water. vv Due to their shortened half-life on vegetation, they can be used in conjunction with other biocontrol agents, reducing the risk to honey bees and forage pollinosis. The following are the different types of biopesticides: vv Pesticides from microbes vv Pesticides from horticulture vv Plant-Incorporated Protectants (PIP’s) vv Semiochemicals

3.2.3.4.1 Pesticides from Microbes

These contain microorganisms such as fungi, viruses, bacteria, and protozoa as bioactive components and are used to manage plant diseases, pest insects, and weeds biologically. The  insect pathogenic bacteria Bacillus thuringiensis is the most extensively employed

Industrial Application of Essential Oils  61 microbe in the creation of biopesticides. For most Diptera, Lepidoptera, Coleoptera this bacterium acts as a pesticide [66]. During the production of Bacillus thuringiensis spores, the bacteria create protein crystals or the toxic potential to rupture intestinal tissue when fed by a specific or sensitive insect [67]. Baculoviruses are DNA viruses with two strands that are found in arthropods, primarily insects. Baculoviruses are very harmful to a variety of pest insects. Baculoviruses only cause death in the larval stage of Lepidoptera, the primary group from which they were isolated. Baculoviruses are divided into two groups: granuloviruses (GVs) and nucleopolyhedroviruses (NPVs). Microbial pesticides are also made from entomopathogenic fungi and associated metabolites [68]. Metarhizium anisopliae is entomopathogenic hyphomycete fungi that are commonly used to control pest insects and are found worldwide [69]. This fungus contains a large variety of distinct strains and isolates from different geographic locations and hosts.

3.2.3.4.2 Pesticides from Horticulture

Natural pesticides, also known as herbal pesticides, are naturally occurring compounds used to manage pests using a safe technique. It can be crucial to determine whether a biopesticide can regulate a pest using a safe approach. The Environmental Protection Agency [70] has formed a commission to examine if a pesticide satisfies the biochemical pesticides requirements. Plants that produce bioactive substances are classified as biopesticides as well. Over 6000 plant species with insecticidal characteristics have been found. Various plant compounds produced from pyrethrum, tobacco, jamaica apple, neem, and other plants have been employed as safer pesticides in insect pest management [71]. Botanical pesticides offer environmentally beneficial properties such as a volatile nature and a minimal environmental danger when opposed to existing synthetic pesticides. Table 3.3 shows the different plants products used as pesticides.

Table 3.3  Some crop products used as bio-pesticides [71, 78]. Target pests

Plant products as biopesticides

Chewing and sucking insects

Azadirachta indica

Fleas, ants, ticks, aphids, flies, and roaches

Chrysanthemum cinerariaefolium 

Fleas and lice on mammals, as well as leaf-feeding insects, some beetles and caterpillars

Rotenoids

Thrips and caterpillars

Ryania speciosa

Thrips, leaf hoppers, Squash bugs, stink bugs and caterpillars

Schoenocaulon officinale

House crickets, paper wasps, fire ants, a number of different varieties of flies and fleas

Limonene and Linalool

62  Essential Oils

3.2.3.4.3 Plant-Incorporated-Protectants (PIP)

Phyto-protectors are biopesticide products made by plants from genetically engineered material that has been inserted into their gene makeup. A good illustration is the Bacillus thuringiensis protein is used to manufacture plant-incorporated protectants. Bacillus thuringiensis toxins are host-specific and can kill in a short amount of time, approximately 48 hours [72]. It is harmless for constructive living things, humans, and the environment and has no adverse effects on vertebrates.

3.2.3.4.4 Semiochemicals

Semiochemicals are chemical signals generated through one species, most common pests that cause another member of the same or other species to change their behavior. The most extensively utilized semiochemicals for crop protection are pheromones, which can operate as communication to connect in their variety and are created for pest management by disrupting mating, trying and enticing the pest, and catching masses [73]. Pheromone is a chemical formed and excreted by a species, particularly mammals or insects that affects the behavior or physiology of other organisms. Pheromones have various impacts and are named after the reaction they elicit, for example, alarm pheromones, aggregation pheromones, sex pheromones, etc. A few pheromones act as sexual attractants, helping individuals search and identify their mate, while others cause congeners to follow, oviposition, and aggregate. Pheromones have become indispensable instruments for controlling and monitoring agricultural pest infestations, and a massive collection of around 1,500 sex attractants and pheromones were recorded as a result [74]. Pheromones and some other semiochemicals are being used on millions of hectares to monitor and manage pests. Cost reduction, ease of use, high sensitivity, and specificity are just benefits of using pheromones for pest monitoring [75]. Monitoring pest insects with pheromone decoys can lead to better management decisions, such as applying insecticides. Pheromones are highly species-specific odors emitted by insects. For example, insects such as the wood beetle invading the woods emit aggregating pheromones to signal the existence of a good food supply to other insects [76].

3.2.3.4.5 Significance of Biopesticides in Insect Pest Management

Agricultural production traditionally depends on manmade synthetic insecticides, but due to new rules and restrictions and the increase of insect resistance, their supply is shrinking. Therefore, the pest control plan should be replaced. Biopesticides, based on living microorganisms or natural ingredients, are the best replacement for synthetic insecticides. Biopesticides are effective in pest control and are now used all over the world. There is advanced scope for studying biopesticides in conjunction with post-genomic technologies, ecological research, and integrated pest management [77]. In this context, the usage of biological insecticides rather than synthetic chemical pesticides has gained prominence as a critical element of integrated pest management (IPM) due to their environmental friendliness and economic feasibility. IPM programs use biopesticides instead of synthetic chemical pesticides because they are compostable, self-perpetuating, less toxic to desirable insects, and shorter life cycles [78]. In integrated pest control, baculovirus biopesticides replace chemical insecticides;

Industrial Application of Essential Oils  63 nevertheless, they have many limitations for commercialized use, including delayed lethality, limited shelf life, high costs, and present norms and regulations controlling biocontrol entities [79]. Utilizing rDNA technology, a variety of methods for increasing the destructive action of wild-type baculoviruses have been devised, involving the introduction of genes that code for insect hormones or enzymes, as well as unique pest poisons [80].

Conclusion Essential oils are discussed in this chapter, including their components, extraction methods, and application in industry. EOs have been used in various industries, including the pharmaceutical, food, pharmaceutical, cosmetic industries, and their presence has resulted in a more vital ability to battle food-borne pathogens and other microbes. The following conclusions can arrive: vv Essential oils can be used as a preservative to improve the shelf life and prevent postharvest degradation in vegetables and fruits, allowing for food security and sustainability. vv By using active packaging in the food industry, essential oils can reduce the initial microbial load and restrict the development of remaining microbes throughout processing and storage. vv The research and development of edible coatings and films for food processing., as fragrance and tissue repair and maintenance in the cosmetics, as medicinal herbs and drug enhancers in the pharmaceutical sector, as well as other plant extract emulsions and formulations, are the finest tools for understanding the many benefits of essential oils in food processing and security. Because EOs are safe, natural, and GRAS-recognized, they can be used in small or large amounts, following the effects of the bioactive chemicals identified to enhance safety and reliability while avoiding nutritive or sensory impairments. vv Essential oils can act as anticancer, antiviral, antibacterial, and skin penetration enhancers, particularly in the pharmaceutical sector. vv A more diverse application of essential oils, such as developing their application in the pest management industry, could be beneficial to both the economy and the environment. vv As a result, in the future, new technological techniques to producing better delivery mechanisms for essential oils may be developed to assure good biocompatibility and bioactive components. Essential oils can be used as preservatives in the food sector to counteract microbial degradation and ensure food safety to the greatest extent possible.

Declaration about Copyright All the figures given in this manuscript are self-drawn and not copied from any other articles.

64  Essential Oils

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Industrial Application of Essential Oils  65 21. Graha, J.P., Boland, J.J., Silbergeld E., Growth promoting antibiotics in food animal production: An economic analysis. Public Health Rep., 122, 1, 79, 2007. 22. Kouhi, M., Prabhakaran, M.P., Ramakrishna, S., Edible polymers: An insight into its application in food, biomedicine and cosmetics. Trends Food Sci. Technol., 103, 248, 2020. 23. Atares, L. and Chiralt, A., Essential oils as additives in biodegradable films and coatings for active food packaging. Trends Food Sci. Technol., 48, 51, 2016. 24. Blanco, P.A., Soto, K.M., Hernandez, I.M., Mendoza, S., Food antimicrobials nanocarriers. Sci. World J., 2014, 837215, 2014. 25. Zhu, G., Xiao, Z., Zhou, R., Yi, F., Fragrance and flavor microencapsulation technology. J. Adv. Mater. Res., 2, 440, 2012. 26. Burgos, N., Mellinas, A.C., Garcia, S.E., Jimenez, A., Nanoencapsulation of flavor and aromas in food packaging, in: Food Packaging, p. 567, Academic Press, Romania, 2017. 27. Chung, S.K., Seo, J.Y., Lim, J.H., Park, H.H., Yea, M.J., Park, H.J., Microencapsulation of essential oil for insect repellent in food packaging system. J. Food Sci., 78, 709, 2013. 28. Wrona, M., Bentayeb, K., Nerín, C., A novel active packaging for extending the shelf-life of fresh mushrooms (Agaricus bisporus). Food Control, 54, 200, 2015. 29. Bhavaniramya, S., Vishnupriya, S., Al, A.M.S., Vijayakumar, R., Baskaran, D., Role of essential oils in food safety: Antimicrobial and antioxidant applications. GOST, 2, 49, 2019. 30. Yildirim, S., Rocker, B., Pettersen, M.K., Nilsen, N.J., Ayhan, Z., Rutkaite, R., Radusin, T., Sumisnska, P., Marcos, B., Coma, V., Active packaging applications for food. Compr. Rev. Food Sci. Food, 17, 165, 2018. 31. Haddi, K., Faroni, L., Oliveira, E.E., Cinnamon oil, in: Green Pesticides Handbook: Essential Oils for Pest Control, p. 117, CRC Press, Boca Raton, 2017. 32. Simionato, I., Domingues, F.C., Nerín, C., Silva, F., Encapsulation of cinnamon oil in cyclodextrin nanosponges and their potential use for antimicrobial food packaging. Food Chem. Toxicol., 132, 110647, 2019. 33. Clemente, I., Aznar, M., Silva, F., Nerin, C., Antimicrobial properties and mode of action of mustard and cinnamon essential oils and their combination against food-borne bacteria. Innov. Food Sci. Emerg. Technol., 36, 26, 2016. 34. Nazari, M., Ghanbarzadeh, B., Kafil, H.S., Zeinali, M., Hamishehkar, H., Garlic essential oil nanophytosomes as a natural food preservative: Its application in yogurt as food model. Colloid Interface Sci. Commun., 30, 100176, 2019. 35. Vital, A.C.P., Guerrero, A., Monteschio, J.D.O., Valero, M.V., Carvalho, C.B., Filho, B.A.D.A., Madrona, G.S., Prado, I.N.D., Effect of edible and active coating (with rosemary and oregano essential oils) on beef characteristics and consumer acceptability. PLoS One, 11, 0160535, 2016. 36. Mannozzi, C., Cecchini, J.P., Tylewicz, U., Siroli, L., Patrignani, F., Study on the efficacy of edible coatings on quality of blueberry fruits during shelf-life. LWT - Food Sci. Technol., 85, 440, 2017. 37. Acevedo, F.A., Salvia, T.L., Rojas, G.M.A., Martin, B.O., Edible films from essential-oilloaded nanoemulsions: Physicochemical characterization and antimicrobial properties. Food Hydrocoll., 47, 168, 2015. 38. Ruiz, N.Y., Viuda, M.M., Sendra, E., Perez, A.J.A., Fernandez, L.J., In vitro antibacterial and antioxidant properties of chitosan edible films incorporated with Thymus moroderi or Thymus piperella essential oils. Food Control, 30, 386, 2013. 39. Yemiş, G.P. and Candogan, K., Antibacterial activity of soy edible coatings incorporated with thyme and oregano essential oils on beef against pathogenic bacteria. Food Sci. Biotechnol., 26, 1113, 2017. 40. Saki, M., ValizadehKaji, B., Abbasifar, A., Shahrjerdi, I., Effect of chitosan coating combined with thymol essential oil on physicochemical and qualitative properties of fresh fig (Ficus carica L.) fruit during cold storage. J. Food Meas. Charact., 13, 1147, 2019.

66  Essential Oils 41. Hernander, H.E., Lira, M.C.Y., Guerrero, L.I., Wild, P.G., Di, P.P., Effect of nanoemulsified and microencapsulated Mexican oregano (Lippia graveolens Kunth) essential oil coatings on quality of fresh pork meat. J. Food Sci., 82, 1423, 2017. 42. Ali, B., Al, W.N.A., Shams, S., Ahamad, A., Khan, S.A., Essential oils used in aromatherapy: A systemic review. Asian Pac. J. Trop. Biomed., 5, 601, 2015. 43. Agatonovic, K.S., Chan, C.K.Y., Gegechkori, V., Morton, D.W., Models for skin and brain penetration of major components from essential oils used in aromatherapy for dementia patients. J. Biomol. Struct. Dyn., 38, 2402, 2020. 44. Michalak, M., Aromatherapy and methods of applying essential oils. Arch. Phys. Glob. Res., 22, 25, 2018. 45. Bharkatiya, M., Nema, R.K., Rathore, K.S., Panchawat, S., Aromatherapy: Short overview. Int. J. Green Pharm., 2, 13, 2008. 46. Manion, C.R. and Widder, R.M., Essentials of essential oil. Am. J. Health-Syst. Pharm., 74, 153, 2017. 47. Bayramoglu, B., Sahin, S., Sumnu, G., Solvent-free microwave extraction of essential oil from oregano. J. Food Eng., 88, 535, 2008. 48. Quassinti, L., Lupidi, G., Maggi, F., Sagratini, G., Papa, F., Vittori, F.S., Bianco, A., Bramucci, M., Antioxidant and antiproliferative activity of Hypericum hircinum L. subsp. majus (Aiton) N. Robson essential oil. Nat. Prod. Res., 27, 862, 2013. 49. Ferraz, R.P.C., Cardoso, G.M.P., Silva, T.B.D., Antitumour properties of the leaf essential oil of Xylopia frutescens Aubl. (Annonaceae). Food Chem., 141, 196, 2013. 50. Essien, E.E., Ogunwande, I.A., Setzer, W.N., Ekundayo, O., Chemical composition, antimicrobial, and cytotoxicity studies on S. erianthum and S. macranthum essential oils. Pharm. Biol., 50, 474, 2012. 51. Zhao, Y., Chen, R., Wang, Y., Qing, C., Wang, W., Yang, Y., In vitro and in vivo efficacy studies of Lavender angustifolia essential oil and its active constituents on the proliferation of human prostate cancer. Integr. Cancer Ther., 16, 215, 2017. 52. Fogang, H.P.D., Maggi, F., Tapondjou, L.A., Womeni, H.M., Papa, F., Quassinti, L., Bramucci, M., Vitali, L.A., Petrelli, D., Lupidi, G., In vitro biological activities of seed essential oils from the cameroonian spices Afrostyrax lepidophyllus Mildbr and Scorodophloeus zenkeri harms rich in sulfur containing compounds. Chem. Biodivers., 11, 161, 2014. 53. Afoulous, S., Ferhout, H., Raoelison, E.G., Valentin, A., Moukarzel, B., Couderc, F., Bouajila, J., Chemical composition and anticancer, anti-inflammatory, antioxidant and antimalarial activities of leaves essential oil of Cedrelopsisgrevei. Food Chem. Toxicol., 56, 352, 2013. 54. Rashid, S., Rather, M.A., Shah, W.A., Bhat, B.A., Chemical composition, antimicrobial, cytotoxic and antioxidant activities of the essential oil of Artemisia indica Willd. Food Chem., 138, 693, 2013. 55. Bachir, R.G. and Benali, M., Antibacterial activity of the essential oils from the leaves of Eucalyptus globulus against Escherichia coli and Staphylococcus aureus. Asian Pac. J. Trop. Biomed., 2, 739, 2012. 56. Avar, S., Maksimovi, M., Vidic, D., Pari, A., Chemical composition and antioxidant and antimicrobial activity of essential oil of Artemisia annua L. from Bosnia. Ind. Crops Prod., 37, 479, 2012. 57. Kamazeri, T.S.A.T., Samah, O.A., Taher, M., Susanti, D., Qaralleh, H., Antimicrobial activity and essential oils of Curcuma aeruginosa, Curcuma mangga, and Zingiber cassumunar from Malaysia. Asian Pac. J. Trop. Med., 5, 202, 2012. 58. Jassim, S.A.A. and Naji, M.A., Novel antiviral agents: A medicinal plant perspective. J. Appl. Microbiol., 95, 412, 2003. 59. Elaissi, A., Rouis, Z., Ben Salem, N.A., Mabrouk, S., Ben Salem, Y., Salah, K.B.H., Aouni, M., Farhat, F., Chemli, R., Harzallah, S.F., Chemical composition of 8 eucalyptus species’

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4 Influence of Biotic and Abiotic Factors on the Production and Composition of Essential Oils Sandra Gonçalves*, Inês Mansinhos and Anabela Romano† MED - Mediterranean Institute for Agriculture, Environment and Development & CHANGE - Global Change and Sustainability Institute, Faculdade de Ciências e Tecnologia, Universidade do Algarve, Campus de Gambelas, Faro, Portugal

Abstract

Since they are sessile, plants are frequently subjected to different environmental stresses including biotic and abiotic factors. The response of plants against these stresses is particularly relevant in the actual scenario of climate change conditions. To cope with these constraints plants developed many defense mechanisms that involve, among others, the synthesis of a vast diversity of secondary metabolites. Essential oils (EOs) produced by aromatic plant species are complex mixtures of several secondary metabolites that have many applications in the agriculture, health, cosmetics, and food industries. Nowadays, the demand for EOs is still increasing, nevertheless, their quality and potential applications are very dependent on several factors. Knowledge about the impact of biotic and abiotic factors on EOs is important to better adequate agricultural practices used in aromatic plants cultivation and to obtain high-quality EOs for industrial applications. Hence, this chapter is an overview of the impact of various abiotic and biotic factors like drought, temperature, salt, heavy metals, UV light, and living organisms, on EOs production and composition. Keywords:  Drought, essential oils, fungi, temperature, salinity, heavy metals, light, nutrients

4.1 Introduction Plants are exposed to many environmental stresses and developed several mechanisms as an adaptive response to many biotic and abiotic factors. The expression of genes implicated in secondary metabolites pathways is changed by various environmental stresses and one of those mechanisms involves the synthesis of a broad range of secondary metabolites produced from primary metabolites [1]. Secondary metabolism affects the adaptation capacity of plants in response to environmental factors and the ecological relationships between plants and other organisms. The antioxidant defense system and several metabolites are crucial for plant survival when exposed to unfavorable conditions [2]. Although the effect of secondary metabolites in plant growth and development is not direct, they are involved *Corresponding author: [email protected] † Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (69–98) © 2023 Scrivener Publishing LLC

69

70  Essential Oils in various important functions, namely in the protection against biotic and abiotic stress factors and herbivores, in the attraction of beneficial insects and pollinators, etc. [3]. Under stressful environmental conditions secondary metabolites have a prolonged impact on the growth, development and survival of the plant [4]. Among the different types of secondary metabolites produced by plants, there are three main groups according to their biosynthetic pathways: terpenoids, phenolic compounds, and nitrogen-containing compounds [5]. Aromatic plants produce a mixture of volatile odoriferous compounds known as essential oils (EOs) [6]. EOs components are secondary metabolites and, therefore, their production is also affected by many environmental factors. Essential oils have been traditionally used for centuries and due to their pluripotent biological effects, they have many industrial applications (e.g., cosmetics, perfumery, personal health care and household cleaning products, food preservation, pharmacology, agrochemicals, and textile products). When aromatic plants are cultivated for EOs extraction, a slight improvement in EO production together with enhanced plant growth may allow obtaining a valuable increase in EO yield [7]. Thus, many investigations have been performed in the last years to evaluate the impact of several factors on EOs production of a great variety of aromatic plants, particularly Mediterranean species extensively used for their outstanding properties. The aim of this chapter is to provide an overview of recent findings about the impact of the multiple biotic and abiotic factors on EO production and composition.

4.2 Essential Oil Characteristics Essential oils, synthesized by aromatic and medicinal plants, are complex mixtures of more than 300 volatile organic compounds generally of low molecular weight that represent a small portion of the plant composition [8]. They can be stored in different plant structures (e.g., epidermal cells, glandular trichomes) and are obtained from distinct parts of the plant including leaves, flowers, stems, roots, and seeds. Essential oils are produced through mevalonic acid and 1-deoxy-D-xylulose-5-phosphate pathways, and involve different enzymatic reactions [6]. The main components of the EOs are terpenes, terpenoids, and aromatic phenols. They contain around 20–60 single components (or more), at different concentrations and among those two or three are the main components that are present in great amounts (20–70%), whilst the remaining are present in small quantities [8]. The percentage of EO produced and differences in its composition are attributed to the biosynthetic pathways, but also to different factors like growing conditions and agronomic practices. Many EOs exhibit a wide array of biological effects namely antimicrobial, antioxidant, anti-inflammatory, neuroprotective, cardioprotective, and insecticidal, and therefore are useful as cosmetics and hygienic products, agrochemistry products, food preservatives, etc. [8–10]. The EOs bioactivity is the sum of its constituents that can act synergistically or antagonistically. The EOs production and chemical composition (including both qualitative and quantitative changes) varies depending on several factors that can influence their bioactivity and potential applications.

4.3 Factors Influencing Essential Oils Production and Composition Biosynthesis and accumulation of plant secondary metabolites as well as their distribution are greatly influenced by ontogenic, genetic, morphogenetic, and environmental

Biotic and Abiotic Factors on Essential Oils  71 factors [11]. Likewise, the yield and composition of the EO are influenced by many intrinsic/­ endogenous and extrinsic/exogenous factors [12]. The exogenous factors include light, precipitation, growing site, and soil. On the other hand, the endogenous factors are closely linked to the structural and physiological attributes of the plants [13]. Environmental

Biotic

Abiotic

Bacteria

Drought

Fungi

Heavy metals

Parasites

Light

Virus

Nutrients Salinity Temperature

P K As

N Ca

Cd Pb

Figure 4.1  Environmental factors affecting the production of secondary metabolites in plants.

72  Essential Oils factors, which can be separated into biotic and abiotic (Figure 4.1), are decisive for the biosynthesis and accumulation of plant secondary metabolites [14]. Thus, plants belonging to the same species grown in distinct environmental conditions may have variations in secondary metabolites profile [11]. The effects of different abiotic and biotic factors on EOs production and composition are discussed below.

4.4 Abiotic Factors Plants interact with their surrounding environment including several abiotic factors, such as water availability, radiation (light, UV), temperature (heat and cold), soil characteristics, minerals, gaseous toxins, pollutants, pesticides, metals, growth regulators and salts, etc. [15]. These factors can induce changes in EO production and in the levels of different components in many aromatic plants as reported by many authors (Table 4.1). Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. Factor/treatment details

Plant species

Common name

Plants recorded at different phenological stages: full flowering (May), post fruiting stage, in the driest period (August)

Thymus vulgaris L.

50% (moderate water deficit; 50 ml/pot/3 days), 25% (severe water deficit, 50 ml/pot/week) of field capacity (FC) 50% (moderate water deficit; 50 ml/pot/3 days), 25% (severe water deficit, 50 ml/pot/week) of FC

Effect(s)

Reference

Garden thyme

Dry period: ↑ 1,8-cineole

[16]

Lavandula angustifolia Mill.

Lavender

No significant differences in EO yield; ↑ 1,8-cineole, borneol; ↓ α-pinene, β-pinene, D-limonene

[17]

Salvia fruticosa Mill.

Greek sage

↑ EO yield; ↑ α-thujone and camphor; ↓ α-pinene, β-myrcene, D-limonene, terpinolene, trans-sabinol and α-caryophyllene

[17]

Drought

(Continued)

Biotic and Abiotic Factors on Essential Oils  73 Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. (Continued) Plant species

Common name

Irrigation interval (4, 8, 12 and 16 days)

Thymus vulgaris L.

80, 60%, 40% of Class A pan evaporation

Factor/treatment details

Effect(s)

Reference

Garden thyme

> Interval days ↓ EO yield; ↑ EO content and individual constituents (thymol, α-thujene and β-caryophyllene)

[19]

Foeniculum vulgare Mill.

Fennel

↑ EO content and individual constituents contents (limonene, fenchone); ↓ EO yield

[20]

Two irrigation levels: rain fed, 100% ETo using a drip irrigation system

Origanum vulgare ssp. hirtum

Greek oregano

↑ Thymol; ↓ p-cymene

[21]

Low stress (irrigation after depletion (IAD) of 20–25% of FC, mild stress (IAD of 35-40% of FC), severe stress (IAD of 55–60% of FC)

Thymus daenensis Celak

Avishan-edenaii

↓ EO content

[80]

Low stress (IAD of 20–25% of FC), severe stress (IAD of 55–60% of FC)

Thymus vulgaris L.

Garden thyme

↑ EO content

[80]

70% irrigation

Lavandula latifolia Medik

Spike lavender

↓ EO content

[87]

(Continued)

74  Essential Oils Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. (Continued) Factor/treatment details

Plant species

Common name

Effect(s)

Reference

Heavy metals Cadmium (0, 5, 10, 20 mg kg-1 soil)

Ocimum basilicum L.

Sweet basil

↑ EO yield and individual constituents (linalool, octanol, nerol, neryl acetate, γ-cadinene, caryophyllene oxide, farnesol and neophytadiene); ↓α-thujene, α-pinene, 1,8-cineole, octanal, geranial, methyl eugenol, β-selinene, cisα-bisabolene, humulene epoxide II and phthalic acid

[64]

Lead (100, 200, 400 mg kg-1 soil)

Ocimum basilicum L.

Sweet basil

↑ EO yield and individual constituents (estragole, octanol, linalool, nerol, neryl acetate, caryophyllene oxide, neophytadiene and oxabicyclododeca); ↓α-pinene, 1-octan-3-ol, 6-methyl-5-hepten, β-pinene, 1,8-cineole, octanal, geranial, geranyl acetate, methyl eugenol, caryophyllene, β-selinene, germacrene D, β-bisabolene, cis-αbisabolene and phthalic acid

[64]

Cadmium (10, 20, 40 ppm)

Mentha x piperita L.

Peppermint

↑ Menthofuran and pulegone; ↓ menthol

[65]

Cadmium (10 mg/L)

Mentha x piperita L. cv. Mitchum

Peppermint

↑ α-Pinene, limonene, 1,8-cineole and β-caryophyllene; ↓ menthofuran and menthyl acetate

[66]

(Continued)

Biotic and Abiotic Factors on Essential Oils  75 Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. (Continued) Factor/treatment details

Plant species

Common name

Effect(s)

Reference

Lead (100 mg/L)

Mentha x piperita L. cv. Mitchum

Peppermint

↑ α-Pinene, limonene, 1,8-cineole, menthyl acetate, β-caryophyllene and pulegone; ↓ menthone, neomenthol and menthol

[66]

Copper (100 mg/L)

Mentha x piperita L. cv. Mitchum

Peppermint

↑ Limonene; ↓ α-pinene, menthone, menthofuran, neomenthol and menthol

[66]

Copper (5, 25 mg kg-1 soil), zinc (0, 10, 50 mg kg-1 soil)

Mentha pulegium L.

Pennyroyal

↑ Pulegone, cisisopulegone, α-pinene, sabinene, 1,8-cineol and thymol

[67]

Arsenite (10, 25, 50, 100 µM)

Ocimum basilicum L.

Sweet basil

25 µM: ↑ Eugenol, methyl eugenol, β-caryophyllene and β-ocimene; Up to 50: ↑ linalool and methyl chevicol 100 µM: ↓ Eugenol, methyl eugenol, β-caryophyllene, β-ocimene

[68]

Chromium (10, 20 mg kg-1 soil), cadmium, lead and nickel (25, 50 mg kg-1 soil)

Ocimum basilicum L.

Sweet basil

Cr, Cd and Pb: ↑ methyl chavicol and methyl eugenol; ↓linalool Ni: ↑ methyl eugenol; ↓ linalool and methyl chavicol

[68]

Chromium (10, 20, 50, 100 µM)

Ocimum tenuiflorum L.

Holy basil or tulsi

↑ Eugenol

[69]

(Continued)

76  Essential Oils Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. (Continued) Factor/treatment details

Plant species

Common name

Effect(s)

Reference

Arsenite (10, 25, 50, 100 µM)

Ocimum gratissimum L.

Clove basil

Up to 50 µM: ↑ eugenol, β-ocimene and germacrene-D, and 1,8-cineole 100 µM: ↓ eugenol, β-ocimene, germacrene-D and 1,8-cineole

[70]

Arsenite (10, 25, 50, 100 µM)

Ocimum tenuiflorum L.

Holy basil or tulsi

Up to 25 µM: Eugenol, carvacrol, methyl eugenol, methyl chavicol and β-caryophyllene; 100 µM: eugenol, methyl eugenol, methyl chavicol, β-caryophyllene, β-ocimene and carvacrol

[70]

Nickel (10, 20, 30, 50 mg kg-1 soil)

Tagetes minuta L.

MusterJohnHenry

↓ Dihydrotagetone, ocimene and tagetone

[71]

25%, 50%, 75% solar irradiance

Ocimum basilicum L.

Sweet basil

↑ Methyl eugenol; ↓ linalool and eugenol

[41]

Light intensity: 100%, 50%, 25% natural sunlight

Rosmarinus officinalis L.

Rosemary

25%: ↓ EO yield and individual constituents (thuja-2,4-diene and 3-octanone) 50%: ↑ EO yield and individual constituents (α-pinene, camphene, β-pinene, myrcene and β-caryophyllene)

[44]

Elevated UV-B (ambient ± 9.6 kJ m-2 d-1)

Curcuma caesia Roxb.

Black turmeric

↑ EO content and individual constituents (epiglobulol, germacrene and 4-terpineol)

[46]

Light

(Continued)

Biotic and Abiotic Factors on Essential Oils  77 Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. (Continued) Factor/treatment details

Plant species

Common name

Effect(s)

Reference

Elevated UV-B (ambient ± 9.6 kJ m-2 d-1)

Curcuma longa L.

Turmeric

↑ EO content and individual constituents (α-terpinolene, β-caryophyllene and β-sesquiphellandrene)

[46]

Ultraviolet-B (3.6 kJ m-2 day-1 above ambient)

Coleus forskohlii (Willd.) Briq

Forskohlii

↓ EO content and individual constituents (β-pinene, myrcene, γ-terpinene, nonanal, α-cis-bergamotene, β-transbergamotene and intermedeol); ↑ pinene, d-Camphene, borneol, decanal and sclareol

[49]

Darkness

Rosmarinus officinalis “Arp”

Lightgrown rosemary

↑ Caryophyllene oxide; ↓ α-pinene, camphene, β-terpinene, 1,8-cineole and camphor

[50]

Nitrogen (150, 175, 200, 225, 250 mg/L), phosphorus (30, 40, 50, 60, 70 mg/L)

Lavandula angustifolia (Mill.)

Lavender

N: ↑1,8-Cineole, camphor and borneol and myrtenal P: ↑1,8-Cineole, pinocarvone and α-terpineol

[17]

Nitrogen fertilizer (50, 100, 150 kg/ha)

Anethum graveolens L.

Dill

↑ EO yield and individual constituents (α-phellandrene, carveol, p-cymene, limonene, γ-terpinene and dihydrocaevone); ↓ myristicin and thymol

[52]

Organic foliar fertilization

Ocimum basilicum L.

Sweet basil

↑ EO content and individual constituents (eucalyptol, methyl chavicol and β-elemene)

[53]

Nutrients

(Continued)

78  Essential Oils Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. (Continued) Factor/treatment details

Plant species

Common name

Effect(s)

Reference

Chemical fertilizer, nano chelated fertilizer

Mentha x piperita L.

Peppermint

↑ EO content, yield and individual constituents (menthol, 3-octanol, terpinene-4-ol and neo-iso-menthol); ↓ germacrene D and (E)-caryophyllene

[54]

Biowastes

Kunzea robusta de Lange & Toelken

Kānuka

↑ EO content

[55]

Biowastes

Leptospermum scoparium J.R. Forst & G. Fors

Mānuka

↑ EO content

[55]

Fertilizers (urea, vermicompost)

Mentha piperita L.

Peppermint

↑ EO content and individual constituents (menthol and menthone)

[56]

Nitrogen (60, 90, 120 kg/ha) and sulfur (20, 40, and 60 kg/ha)

Tagetes minuta L.

Wild marigold

N: ↑ Z-β-ocimene, dihydrotagetone, z-tagetone, Z-ocimenone and E-ocimenone S: ↑ Z-β-ocimene, dihydrotagetone, E-tagetone and Z-tagetone

[59]

Magnesium (2 and 4 mM), manganese (50 and 150 mM)

Tanacetum parthenium L. Schulz Bip.

Feverfew

Low Mg and high Mn: ↑ monoterpenes High Mg and low Mn: ↑ sesquiterpenes

[60]

Zinc (0.2, 0.15, 0.10, 0.095, 0.09 mg/L)

Ocimum basilicum L.

Sweet basil

↑ EO yield and linalool

[61]

(Continued)

Biotic and Abiotic Factors on Essential Oils  79 Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. (Continued) Factor/treatment details

Plant species

Common name

Effect(s)

Reference

Salinity 50, 100 and 200 mM NaCl

Coriandrum sativum L.

Coriander

50 and 100 mM: ↑ EO yield and α-pinene; ↓ n-nonane 200 mM: ↓ EO yield and individual constituents (n-decanal, 2E-dodecanal and 2E-tridecen-1-al); ↑ 2E-decanal and dodecanal

[28]

4.2 g/L NaCl

Rosmarinus officinalis L.

Rosemary

↓ EO yield

[29]

2.5, 5, 7.5, 10 and 12.5 g/L NaCl

Rosmarinus officinalis L.

Rosemary

7.5 and 10 g/L: ↑ EO yield; > 5g/L: ↑ phelladrene > 7.5: ↓ dill ether

[30]

NaCl, KCl, MgSO4, MgCl2, Na2SO4, CaCl2 (50, 100, 15 and 200 mM)

Salvia officinalis L.

Sage

NaCl: ↑ α-pinene, camphor, camphene and β-thujone; ↓ α-thujone; KCl: ↑ α-pinene, camphor, camphene, β-thujone and 1,8-cineole, and ↓ α-thujone; MgSO4: ↑ 1,8-cineole, α-pinene, camphor, camphene, β-thujone and α-thujone; MgCl2: ↑ α-pinene, camphor, camphene and α-thujone, and ↓ 1,8-cineole; Na2SO4: ↑ 1,8-cineole, α-pinene, camphor, camphene, β-thujone, α-thujone; CaCl2: ↑α-thujone, α-pinene, camphor, β-thujone and α-thujone, and ↓1,8-cineole

[31]

(Continued)

80  Essential Oils Table 4.1  Effect of different abiotic factors on essential oils production of some aromatic plants. (Continued) Factor/treatment details

Plant species

Common name

Effect(s)

Reference

40, 80 and 120 mM NaCl

Ocimum basilicum cv. Keshkeni luvelu

Sweet basil

40 mM: ↑ monoterpene hydrocarbons and sesquiterpene hydrocarbons 80 and 120 mM: ↑ sesquiterpene hydrocarbons; ↓ monoterpene hydrocarbons,

[32]

3, 6 and 9 dS/m Caspian Sea water mixture

Mentha x piperita L.

Peppermint

↓ EO content

[33]

25, 50 and 100 mM NaCl

Mentha spicata L.

Spearmint

No significant effect on EO yield; 100 mM: ↑ limonene; ↓ carvone

[34]

150 mM NaCl

Mentha spicata L.

Spearmint

↑ Limonene; β-ocimene, myrtenal, cis-dihydro carvone, cis-carveol, pulegone and β-elemene; ↓ carvone

[35]

Different geographical conditions (max 28 ºC; min-4 °C)

Thymus fedtschenkoi Ronnige

Avishan

> Thermal amplitude: ↑ EO yield

[36]

Free air temperature increment (2.5–3.0 ºC above ambient)

Salvia sclarea L

Clary sage

↑ Linalyl acetate, spathulenol; ↓ ocimene (Z) beta, linalool, linalyl propionate and geraniol

[37]

Different geographical conditions

Lavandula angustifolia cv. Etherio

Lavender

↑ Linalool

[39]

Temperature

Biotic and Abiotic Factors on Essential Oils  81

4.4.1 Drought Drought (water stress), which occurs when the disponibility of water in the soil diminishes, is one of the main abiotic stresses encountered by plants that induce dramatic changes in productivity and growth [3]. Water management is particularly important in arid and also in semi-arid regions like the Mediterranean basin. Drought stress induces alterations at physiological and metabolic levels including a decrease in plant growth, stomatal closure and reduction in photosynthesis rate. The increase in EOs components has been reported as a strategy adopted by many aromatic plants to respond to water stress conditions [3]. The results available demonstrated that the effect of drought stress on EO yield and composition is variable with the species. Llorens-Molina and Vacas [16] observed that the content of 1,8-cineole, a valuable metabolite that is the main component of Thymus vulgaris chemotype, was significantly higher in samples collected in the drought period. In Greek sage (Salvia fruticosa Mill) both moderate and severe water deficit increased EO yield but did not considerably influence this parameter in Lavandula angustifolia Mill [17]. In addition, drought stress impacted the EO composition in both species; a trend was observed of decreasing total monoterpene hydrocarbons and increasing oxygenated hydrocarbons with increasing water stress. This impact is mainly related to changes in the proportion of components than in their presence/absence. Findings also revealed that deficit irrigation can alter the biological properties of the EOs. The biocidal properties of lavender EO from plants growing under medium water stress were higher than that from plants growing under regular irrigation. On the other hand, in the case of sage, water stress induced no changes in EO biological activity. The results suggest that manipulation of agricultural practices, like management of irrigation, is important to modulate EOs quality and their potential uses. Ramezani et al. [18] investigated the impact of water stress on EOs composition of three genotypes of Salvia spp. and studied the expression analysis of monoterpene synthases. The authors’ findings indicated that water stress had a positive effect on monoterpenes accumulation but negatively influenced the production of α and β-thujone. Moreover, the relative expression of three genes enhanced in water stress conditions as well as their respective metabolites. A greenhouse experiment conducted by Ahl et al. [19] revealed that water stress improves EO content in T. vulgaris. However, since this stress decreases biomass production, EOs yield also decreased. Coban et al. [20] also studied the impact of deficit irrigation on EO production of fennel (Foeniculum vulgare Mill) and observed that there was a tendency to improve its content under increased water deficit conditions, however, the opposite occurred for EO yield. Overall, reviewed results demonstrated that plant response to water stress varied considerably depending on the plant species and growth stage, but also with the experimental conditions including the intensity and duration of stress. The impact of irrigation and nitrogen (N) fertilization on the production of EO from Greek oregano (Origanum vulgare ssp. hirtum) was recently investigated and it was noticed that irrigation did not affect EO content and compounds concentration, possibly due to the adequate precipitation that occurred in the growing period [21]. On the other hand, the EO content in inflorescences and leaves increased with increasing N-supply. Some studies indicate that arbuscular mycorrhiza fungi (AMF) and plant growth-promoting bacteria (PGPB) can counteract the

82  Essential Oils adverse effects induced by drought stress in aromatic plants. This is linked to the benefic effects of AMF on the uptake and transport of water and some nutrients as phosphorus, inducing growth promotion [22–24].

4.4.2 Salinity Salt stress, arising from the excess of inorganic salts and weak quality of water for irrigation, is a key impediment to improving plants growth all over the world. The elevated concentration of salts in the soil leads to reduced soil porosity, aeration and water conductance. As a consequence of greater amounts of soluble salts and osmotic pressure in the cytosol, plant growth in saline soils is limited. Salinity induces several morphological, biochemical and physiological variations in plants like the stimulation of plant secondary metabolites production [3]. There are some reports describing the effect of salinity on EOs production and composition, and the results are highly variable and very dependent on the salinity level. Salinity decreased EO content in mint plants and induced changes in its chemical composition mainly pulegone and menthone contents [25]. A recent investigation was carried out to study the effects of different salinity levels on Iranian mint ecotypes growing in greenhouse conditions and it was noticed that the content of EO enhanced in response to salinity [26]. In curly-leafed parsley, moderate salinity levels also induced a positive effect on EO yield and in the contents of certain of its constituents [27]. Recent investigations also showed that low and moderate salt stress (50 and 100 mM NaCl) improved EO yield in coriander plants (Coriandrum sativum L.) [28]. On the other hand, Sarmoum et al. [29] observed that saline treatment induced lower EO yield in rosemary plants (Rosmarinus officinalis L.), but increased the contents of D-verbenone camphene, caryophyllene oxide and α-pinene compounds, three compounds with biocidal effects. In the same species the percentage of EO augmented with increasing salinity concentrations until 10 g/L NaCl but decreased thereafter [30]. This decrease in EO percentage at the highest salt level was ameliorated when plants were inoculated with PGPB. The content of one of the main compounds, phellandrene, improved with increasing salt strength. Lately, Kulak et al. [31] evaluated the impact of various salt compounds (NaCl, KCl, MgSO4, MgCl2, Na2SO4, and CaCl2) at distinct levels (0, 50, 100, 150, and 200 mM) on EO composition of sage (Salvia officinalis L.) and observed that it was strongly affected by the two factors since each salt treatment induced different chemotypes. Salt stress particularly improved the percentage of α-pinene and camphor, and also of camphene in almost all salt treatments. According to Farsaraei et al. [32] the stimulation of EO production in sweet basil (Ocimum basilicum cv. Keshkeni luvelu) under moderate salinity level (until 80 mM NaCl) could be related to the elevated oil gland density and an enhancement in the ideal number of generated glands to leaf emergence. In addition to the inoculation with PGPB, other treatments have been used to ameliorate the adverse effects of salt stress on aromatic plants. High salt stress (200 mM) reduced EO yield in coriander but this was alleviated by the foliar application of silicon [28]. Khalvandi et al. [33] found that fungal symbiosis and methyl jasmonate treatment alleviated the adverse effects induced by salinity on EO amount from peppermint (Mentha piperita L.). A study conducted with hydroponically grown spearmint plants (Mentha spicata L.) showed that salinity did not affect EO yield but reduced the content of the main compound, carvone, the responsible for the quality of the EO from this species [34]. However, this negative

Biotic and Abiotic Factors on Essential Oils  83 effect of salinity was counteracted by foliar application with K, Zn, and Si, and at the same time increased limonene content. The addition of Cu could be also used as a strategy to improve the amount of certain components of the spearmint EO under salt stress without compromising EO yield [35].

4.4.3 Temperature Temperature is a crucial physical factor affecting the ontology and development of plants. High and low temperatures can induce changes in several physiological, biochemical and molecular processes in plants and can also influence secondary metabolites production. There are few investigations about the impact of temperature on EO production and chemical composition. Delazar et al. [36] observed that altitude, temperature and soil greatly affected the volume of production of EO from Thymus fedtschenkoi Ronniger but had few effects on its chemical composition. Elevated temperature (2.5–3.0 °C above ambient temperature) significantly improved the linalool content in the EO from Salvia sclarea L. [37]. Also, the increase in temperature enhanced the production of several terpenes [38]. Lowered temperatures had a negative effect on EO production of Lavandula angustifolia Mill. as a consequence of EO gland cells breaking or modification of the terpene biosynthesis [39]. In Chamomilla recutita (L.) Rausch Fahlen et al. [40] reported the greatest amount of α-bisabolol when 21−3 h photoperiod and 25−18 °C thermoperiod were used.

4.4.4 Light Light, including quality, quantity and photoperiod, is another physical factor that strongly affects plant growth and different processes [14]. Light is a vital factor in the synthesis of several plant compounds since it provides the energy required to fix carbon and had a great impact on the biosynthesis of secondary metabolites as well. Light intensity and photoperiod might have different impacts on the production of EOs. Some studies showed that high light intensities improved EO yield but the opposite situation is also reported. The effect of four levels of irradiance on EO content and chemical profile of basil plants (Ocimum basilicum L.) was investigated [41]. It was reported that heavy shading reduced EO content and also the contents of the major compounds i.e. linalool and eugenol. The amount of these compounds, which are important for the typical odor of the plant, was improved by high daily light integrals, while methyl eugenol was improved by lower daily light integrals. Moreover, light treatments did not affect the amount of 1,8-cineole, an important compound of Ocimum species. In Flourensia cernua (DC.) Benth. & Hook.f. (tarbush) higher contents of several compounds (camphene, sabinene, b-pinene, borneol, bornyl acetate and Z-jasmone) were obtained from plants subjected to partial shade compared with totally irradiated plants [42]. The effect of three light periods (8, 16 and 24 h) and two light intensities (100 and 200 μmol m-2 s-1) on EO composition of Japanese mint (Mentha arvensis var. piperascens) was investigated [43]. It was observed that the use of 16 h light periods together with the high light intensity produced the highest contents of 1-menthone and L-menthol. Raffo et al. [44] investigated the influence of light intensity and water disponibility on rosemary EO production and composition. Results demonstrated the necessity of a prudent control of both light and irrigation for the standardization or modification of EO composition depending on different potential uses. This is because variations in light intensity and

84  Essential Oils water availability appeared to induce an inverse impact on the relative abundance of EO components produced via the action of two different classes of enzymes (pinene synthases and bornyl diphosphate synthases). Ultraviolet (UV) radiation promotes the production of defense secondary metabolites, particularly phenolics. Although UV-B is a minor portion of the incident sunlight it greatly influences plant growth and several physiological and metabolic processes. Climate change conditions are increasingly exposing plants to new combinations of UV-B radiation [45]. The elevated amounts of biologically effective UV-B radiation reaching the Earth are mainly related to the high concentrations of ozone-depleting substances and changing climatic conditions [45]. The effects of elevated UV-B on EO production of two Curcuma species (C. caesia Roxb. and C. longa L.) were recently investigated [46]. It was observed that elevated UV-B improved EO contents and changed EO chemical composition in both species, but in general, a greater proportion of compounds were more improved in C. longa than in C. caesia. Authors suggest that improved EO content is probably related to the upregulation of the terpene biosynthetic pathway, which is the most important biosynthetic pathway in species of this genus, and that elevated UV-B could disrupt the pathway and portion of some terpenes [47]. As previously reported in O. basilicum [48] the content of 1,8-cineole, the major active compound of C. caesia with a recognized insecticidal effect, is increased with UV-B treatment. Also, in agreement with Chang et al. [48] increases in alpha-pinene and beta-pinene contents and non-considerable changes in camphor content were observed under elevated UV-B. Contrarily to the above reported EO content of Coleus forskohlii (Willd.) Briq was reduced with supplemental UV-B and EO composition was substantially changed [49]. In terms of EO composition, in addition to changes in the quantities of some compounds with elevated UV-B treatment, some new compounds not found in control plants were detected and the opposite also occurred. The above described variations in the results might be related to several factors like the intrinsic characteristics of the plant species, climatic conditions, planting conditions, dose and duration of UV-B exposure, among others [48]. Also the type and age of the organ greatly influenced the EO composition, thus these factors need to be taken into account when plants are subjected to low light [50]. Both light and developmental stages have an important function in plant secondary metabolism.

4.4.5 Nutrients Plant growth and development are greatly affected by several nutrients that are important for the production and development of plant structural components and metabolic regulation [3]. Nutrient’s stress occurs as a consequence of a mixture of other stressful conditions like water and saline stresses which can disrupt the availability, partitioning and transport of nutrients. Nutrients influence the production of many secondary metabolites including EOs components since affect the amounts of enzymes involved in terpenoids biosynthesis. Indeed, nutrient management has been considered a crucial practice in aromatic plants cultivation with a great impact on the yield and quality of EOs. Thus, in the last years several investigations have been conducted to evaluate the effects of various nutrient levels [51, 52] and fertilization practices on EO yield and quality of different aromatic plants [53–55]. A field experiment was performed to evaluate the impact of different fertilizer sources on EO

Biotic and Abiotic Factors on Essential Oils  85 production by Mentha x piperita L. [54]. The greatest EO content and yield, and the best quality of the EO, in terms of menthol content, were obtained using 50 % chemical fertilizer + nano chelated fertilizer. Additionally, a good association was found between the dry mass yield and EO amount and yield. Keshavarz-Mirzamohammadi et al. [56] also observed that dry matter weight and EO content of peppermint (M. piperita) enhanced with increasing vermicompost in fertilizer treatments which improved nutrient availability. Availability of nutrients as is the case of N and P or the influence of some factor affecting the uptake of these nutrients is important for terpenoids biosynthesis, the main EO components, and can affect their contents in the EO [57]. Studies from Chrysargyris et al. [51] revealed that EO yield from Lavandula angustifolia Mill. was not affected by the different N and phosphorous (P) levels tested, but induced changes in the main constituents of the EO (1,8-cineole, borneol, camphor, alpha-terpineol, and myrtenal). Increased N level did not affect the percentage of EO recovered from Anethum graveolens L., however caused important changes in EO composition, the contents of some compounds increased (α-phellandrene, limonene and dihydrocaevone) whereas of other decreased (β-phellandrene, thymol and myristicin) [52]. Amooaghaie and Golmohammadi [58] observed that using high amounts of micro (Zn, Fe, Mn, and Cu) and macronutrients (N and P) considerably improved the EO productivity in T. vulgaris. A field experiment carried out by Walia and Kumar [59] revealed that greater N (120 kg ha-1) and moderate S (40 kg ha-1) levels are ideal to improve the biomass production and EO yield of wild marigold (Tagetes minuta L.) with the required quality. Together with macronutrients, the concentrations of micronutrients can also induce changes in EOs profile. Results from Farzadfar et al. [60] showed that Mn leads to the biosynthesis of monoterpenes, whereas Mg can redirect biosynthetic pathways to produce sesquiterpenes. Zn is also an indispensable micronutrient for crop growth and several processes. Different Zn concentrations improved EO yield and biological properties (antioxidant and antifungal activities) of basil EO [61]. The available results concerning the impact of nutrients on EO production and composition are variable, and for the same nutrients, there are conflicting results. Although increased concentrations of nutrients can be advantageous in some cases, the moderate use of chemical fertilizer can also decrease the content of EO and certain major components by improving the gland size, diluting the concentration of EO in plant organs [62].

4.4.6 Heavy Metals Heavy metals stress is one of the most important abiotic constraints faced by plants as a result of its high bioaccumulation and toxicity. The increment of several anthropogenic activities like intensive agriculture, metal industries and mining, increase the amounts of some toxic heavy metals (e.g., arsenic, mercury, lead, cadmium, etc.) above recommended amounts in the soil but also in the water and air [3]. The uptake of these metals by plants can alter several metabolic processes including the accumulation of secondary metabolites by modifying the expression of genes implicated in their biosynthetic pathways [63]. Many secondary metabolites act as antioxidant agents and, thus, their enhanced production is a mechanism implemented by plants to counteract the effects of free radicals in heavy metals stress conditions.

86  Essential Oils Cadmium (Cd) and lead (Pb) are two of the most toxic heavy metals affecting crop productivity and also human health. In sweet basil (Ocimum basilicum L.), although the growth was adversely affected by Cd and Pb, these heavy metals affected positively EO yield and composition [64]. A recent study by Azimychetabi et al. [65] showed that Cd considerably affected the EO composition of peppermint (M. piperita): improved the contents of menthofuran and pulegone but reduced the contents of menthol and menthone. Though, these changes did not compromise the antioxidant properties of the EO that increased in Cd stress. Zheljazkov et al. [66] observed that Cd, Pb and copper (Cu) applied in combination decreased EO amount in basil, and Cu alone at the highest concentration tested (150 mg L−1) also decreased this parameter in dill. These authors showed that the heavy metals were not removed from plant tissues during EOs recovery and, thus aromatic plants are proposed as an alternative to extracting heavy metals from mildly contaminated soils. Lajayer et al. [67] studied the impact of different concentrations of Cu and zinc (Zn) on different parameters of Mentha pulegium L. plants. They reported that the highest EO amount and yield were attained in plants exposed to Cu and Zn (5 and 10 mg kg-1, respectively). Moreover, major components of the EO such as pulegone, cis-isopulegone, α-pinene, sabinene, 1,8-cineol, and thymol increased with Cu and Zn treatments. Increasing levels of metals [Chromium (Cr), Cd, Pb, and Nickel (Ni)] caused changes in EO contents and EO composition of sweet basil that varied with the heavy metal and its concentration [68]. Furthermore, the authors noticed that arbuscular mycorrhizal fungi symbiosis can be used as a strategy to improve EO yield and maintain its quality under metal-contaminated soils. Chomium treatment induced the production of eugenol, a key compound of the EO, by Ocimum tenuiflorum [69]. Essential oil yield and the main EO constituents enhanced at smaller arsenic amounts in Ocimum spp. and the metal was not detected in the EO [70]. Additionally, studies from Chand et al. [71] showed that the treatment with Ni and vermicompost had a considerable effect on the quality of T. minuta EO. Studies about the effects of several heavy metals on aromatic plants indicated that certain plants can absorb and accumulate some metal contaminants in their tissues without contaminating the EO with these metals and, thus, can be considered a viable alternative for remediation of contaminated sites and, at the same time, be used as a source of valuable EOs for many applications [66, 70–72].

4.5 Biotic Factors Biotic stress includes the attack of plants by biological organisms such as bacteria, viruses, fungi, parasites, nematodes and herbivores [11]. Since plants are sessile, they cannot move to defend themselves from the attack of these organisms, however they developed a passive first-line defense system that can include physical barriers (e.g., cuticles), wax, and trichomes, and the production of chemical compounds. The defense against other organisms is one of the main functions of plant secondary metabolites. In response to the pathogen attack plants activate several metabolic pathways leading to increases in the amounts of secondary metabolites and/or the synthesis of new compounds [2]. Besides their antimicrobial properties, certain secondary metabolites are involved in the formation of polymeric obstacles to pathogen penetration. In this respect, plants contain advanced recognition and signaling mechanisms that allow prompt detection of the

Biotic and Abiotic Factors on Essential Oils  87 pathogen attack and can instigate an active defensive reaction. Plants can also produce various types of secondary metabolites as a response to virus infestation (i.e., alkaloids, phenol, flavonoids, and terpenes, etc.) as recently reviewed by Mishra et al. [73]. The formation of mycorrhizal symbioses between plant roots and AMF can modulate the production of secondary metabolites by plants and, at the same time, improve plant production, decreasing the requirements for chemical fertilizers and pesticides [74, 75]. AMF can improve growth and yield by enabling the uptake of nutrients and water [75]. Table 4.2  Effect of biotic factors on essential oils production of some aromatic plants. Treatment

Plant species

Common name

Effect(s)

Reference

Arbuscular mycorrhizal fungi Rhizophagus intraradices

Ocimum tenuiflorum L.

Holy basil or Tulsi

↑ EO content and individual constituents (eugenol, β-elemene, β-caryophyllene, germacrene A and germacrene D)

[76]

Rhizophagus irregularis

Anethum graveolens L.

Dill

↑ EO content and individual constituents (cisdihydrocarvone, transdihydrocarvone and iso-dihydrocarveol)

[77]

Rhizophagus irregularis

Coriandrum sativum L.

Coriander

↑ EO content and individual constituents (γ-terpinene, linalool, camphor, α-terpineol, geraniol, geranyl acetate and β-Caryophyllene)

[77]

Glomeromycota

Matricaria chamomilla L.

Chamomile

↑ EO content

[78]

(Continued)

88  Essential Oils Table 4.2  Effect of biotic factors on essential oils production of some aromatic plants. (Continued) Treatment

Plant species

Glomus etunicatum, G. lamellosum

Origanum onites L.

Glomus etunicatum, G. lamellosum

Common name

Effect(s)

Reference

Oregano

↑ α-Terpinene, cissabinene hydrate, terpinen-4-oil, carvacrol ↓ thymol, trans-sabinene hydrate, linalool, linalyl acetate,-thujene and caryophyllene oxide

[78]

Mentha viridis L.

Mint

↑ Sabinene, limonene, 1,8-cineole, transocimene, cissabinene hydrate, dehydrocarvone, carvone, eugenol, (E)-methyl cinnamate, bourbonene; ↓ linalyl acetate, carvacrol, trans-4thujanol, linalool, cis-pinocamphenone, α-terpineol, β-caryophyllene and germacrene D

[79]

Rhizophagus intraradices, Funneliformis mosseae

Thymus daenensis Celak

Avishan-edenaii

↓ EO content

[80]

Rhizophagus intraradices, Funneliformis mosseae

Thymus vulgaris L.

Garden thyme

No effect in EO content

[80]

Glomus intraradices

Ocimum gratissimum L.

Basil

↑ EO content

[81]

Glomus intraradices, G. mosseae

Ocimum basilicum L.

Sweet basil

↑ EO content and individual constituents (linalol, methyl chavicol, transgeraniol, camphor and limonene)

[85]

(Continued)

Biotic and Abiotic Factors on Essential Oils  89 Table 4.2  Effect of biotic factors on essential oils production of some aromatic plants. (Continued) Treatment

Plant species

Glomus intraradices, G. mosseae

Satureja hortensis L.

Common name

Effect(s)

Reference

Satureja

↑ EO content and individual constituents (carvacrol, thymol, p-cymene, α-terpinene and γ-terpinene)

[85]

Mentha piperita L.

Peppermint

↑ EO content and individual constituents (menthol, menthone, 1,8-cineole, caryophyllene and trans-sabinene hydrate)

[33]

Cuscuta campestris Yunck.

Mentha piperita L.

Peppermint

↑ EO yield and individual constituents (menthone, transsabinol, piperitone, δ-selinene, viridiflorol, δ-cadinene); ↓ menthol, pulegone, (E)-β-ocimene and γ-terpinene

[86]

Cuscuta campestris Yunck.

Chamomilla recutita (L.) Rausch.

Chamomile

↓ EO yield and individual constituents (α-bisabolol oxide A and B, α-bisabolene oxide A and (Z)-en-yn-dicycloether)

[86]

Peppermint

↑ EO yield and individual constituents (-)-menthone, (-)-menthol, linalool, 1,8 cineole and (+)-pulegone

[83]

Endophytic fungi Piriformospora indica

Parasitism by field dodder

Plant growth-promoting rhizobacteria Bacillus subtilis GB03, Pseudomonas fluorescens WCS417r, P. putida SJ04

Mentha piperita L.

(Continued)

90  Essential Oils Table 4.2  Effect of biotic factors on essential oils production of some aromatic plants. (Continued) Treatment

Plant species

Sinorhizobium meliloti Rm1021, Bacillus subtilis BS119 m, Bradyrhizobium sp. USDA 4438, Pseudomonas fluorescens WCS417 r

Ocimum basilicum L.

Sinorhizobium meliloti Rm1021, Bacillus subtilis BS119 m, Bradyrhizobium sp. USDA 4438, Pseudomonas fluorescens WCS417 r

Satureja hortensis L.

Common name

Effect(s)

Reference

Sweet basil

↑ EO content and individual constituents (linalol, methyl chavicol, transgeraniol, camphor and limonene)

[85]

Satureja

↑ EO content and individual constituents (carvacrol, thymol, p-cymene, α-terpinene and γ-terpinene)

[85]

Arbuscular mycorrhiza fungi can also modify terpenoid metabolism and shikimate pathway improving the production of many secondary metabolites [74]. Inoculation of plants with a group of bacteria called PGPR can also significantly enhance growth and secondary metabolites production. PGPR usually exerts an impact on plant through the improvement of its nutritional status and/or phytohormone production. Indeed, the use of fungal and bacterial inoculants has been considered an efficient alternative for promoting plant secondary metabolism. In the particular case of aromatic plants, the literature available describing the positive effect of AMF on EO yield and composition is vast while the effects of PGPR are less investigated (Table 4.2). For instance, Thokchom et al. [76] noted that inoculation of Ocimum tenuiflorum L. plants with AMF (Rhizophagus intraradices) enhanced EO concentration and EO quality, by increasing the contents of eugenol and other important bioactive compounds (β-elemene, β-caryophyllene and germacrene A, and germacrene D), as well as antioxidant activity. Inoculation with R. intraradices fungi also improved EO production in coriander and dill although the response depended on plant species and pool of AMF in the soil [77]. Similar results were also observed for other important aromatic species like chamomile [78], mint and oregano [79]. Arbuscular mycorrhiza fungi have been suggested as a good strategy to improve plant growth and EO production in low fertility soil reducing fertilizer inputs [79]. Furthermore, several studies indicated that AMF inoculation could be a good approach to relieve the negative impacts of water stress in plants [79, 80]. Khalvandi et al. [33] observed that inoculation of peppermint plants with Piriformospora indica, a beneficial endophytic fungus, and methyl jasmonate treatment alleviated the adverse effects of salinity on EO amount and that the main EO constituents (menthol, menthone and 1,8-cineole) enhanced.

Biotic and Abiotic Factors on Essential Oils  91 Inoculation of PGPRs caused the induction of monoterpene pathways in Origanum × majoricum [82]. The inoculation of peppermint (M. piperita) plants with bacterial strains induced qualitative and quantitative variations in monoterpene amount and increased glandular trichome density [83]. More recently, Mentha × piperita plants were also inoculated with bacteria and then sprayed with methyl jasmonate and salicylic acid solutions to investigate the interaction of these two signaling molecules and rhizobacteria [84]. Inoculated plants treated with methyl jasmonate revealed the highest increase in EO production suggesting a synergistic effect between PGPR and methyl jasmonate. The increase in EO production was also associated with a rise in glandular trichome density. Recently, Khalediyan et al. [85] showed that both AMF and PGPR considerably improved the EO contents in basil and satureja (Satureja hortensis L.) as well as the contents of several EOs components (Table 4.2). In addition to the impact of the aforementioned biotic and abiotic factors on EO production, weed competition is one the main limiting factors on commercial cultivation of aromatic species since they are capable to decrease EO amount and also biomass yield. Recent studies showed that parasitism by field dodder (Cuscuta campestris Yunck.), a parasitic weed species, on peppermint and chamomile plants induced changes in EO yield and composition in both species [86].

4.6 Concluding Remarks The synthesis of secondary metabolites is one of the plants mechanisms to cope with abiotic and biotic constraints. Secondary metabolites may assist the plant to avoid injuries induced by these stresses and play an important role in their adaptation to the local environment. Essential oils, mainly produced by aromatic plants, are complex mixtures of several secondary metabolites that gained interest as promising alternatives in many industries due to their wide array of biological effects and the recent increasing tendency to the application of natural and low toxicity products. The production and composition of EOs are depended on different factors like the genotype, climate, soil type, cultivation practices and plant development stage. From the available literature, there are numerous examples that the quantity and quality of EOs change in response to biotic and abiotic factors such as drought, light, salinity, nutrient deficiency, heavy metals, fungi and bacteria, etc. In some cases, these changes can implicate the increase of the biological properties of the EO and their value for certain uses. The reviewed results are relevant to improve predictions of aromatic plant’s responses to environmental changes and to provide tools to assist the sustainable production of aromatic plants. In a climate change scenario, particularly in the Mediterranean basin, it is particularly relevant the adequate water management adopting suitable and alternative agricultural practices and cultivation strategies (e.g., application of biowastes, arbuscular mycorrhizal fungi).

Acknowledgements This research was funded by National Funds through FCT—Foundation for Science and Technology under the under the Projects UIDB/05183/2020 and LA/P/0121/2020.

92  Essential Oils Inês Mansinhos (Grant SFRH/BD/145243/2019) and Sandra Gonçalves (CEECINST/00052/2021) are funded by national funds through FCT.

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96  Essential Oils 67. Lajayer, H.A., Savaghebi, G., Hadian, J., Hatami, M., Pezhmanmehr, M., Comparison of copper and zinc effects on growth, micro- and macronutrients status and essential oil constituents in pennyroyal (Mentha pulegium L.). Braz. J. Bot., 40, 379, 2017. 68. Prasad, A., Kumar, S., Khaliq, A., Pandey, A., Heavy metals and arbuscular mycorrhizal (AM) fungi can alter the yield and chemical composition of volatile oil of sweet basil (Ocimum basilicum L.). Biol. Fertil. Soils, 47, 853, 2011. 69. Rai, V., Vajpayee, P., Singh, S.N., Mehrotra, S., Effect of chromium accumulation on photosynthetic pigments, oxidative stress defense system, nitrate reduction, proline level and eugenol content of Ocimum tenuiflorum L. Plant Sci., 167, 1159, 2004. 70. Siddiqui, F., Krishna, S.K., Tandon, P.K., Srivastava, S., Arsenic accumulation in Ocimum spp. and its effect on growth and oil constituents. Acta Physiol. Plant, 35, 1071, 2013. 71. Chand, S., Kumari, R., Patra, D.D., Effect of nickel and vermicompost on growth, yield, accumulation of heavy metals and essential oil quality of Tagetes minuta. J. Essent. Oil Bear. Plants, 18, 767, 2015. 72. Lajayer, B.A., Ghorbanpour, M., Nikabadi, S., Heavy metals in contaminated environment: Destiny of secondary metabolite biosynthesis, oxidative status and phytoextraction in medicinal plants. Ecotoxicol. Environ. Saf., 145, 377, 2017. 73. Mishra, J., Srivastava, R., Trivedi, P.K., Verma, P.C., Effect of virus infection on the secondary metabolite production and phytohormone biosynthesis in plants. 3 Biotech., 10, 547, 2020. 74. Agnolucci, M., Avio, L., Palla, M., Sbrana, C., Turrini, A., Giovannetti, M., Health-promoting properties of plant products: The role of mycorrhizal fungi and associated bacteria. Agronomy, 10, 1864, 2020. 75. Kaur, S. and Suseela, V., Unraveling arbuscular mycorrhiza-induced changes in plant primary and secondary metabolome. Metabolites, 10, 335, 2020. 76. Thokchom, S.D., Gupta, S., Kapoor, R., Arbuscular mycorrhiza augments essential oil composition and antioxidant properties of Ocimum tenuiflorum L. – A popular green tea additive. Ind. Crops Prod., 153, 112418, 2020. 77. Rydlová, J., Jelínková, M., Dušek, K., Dušková, E., Vosátka, M., Püschel, D., Arbuscular mycorrhiza differentially affects synthesis of essential oils in coriander and dill. Mycorrhiza, 26, 123, 2016. 78. de Almeida, D.J., Alberton, O., Otênio, J.K., Carrenho, R., Growth of chamomile (Matricaria chamomilla L.) and production of essential oil stimulated by arbuscular mycorrhizal symbiosis. Rhizosphere, 15, 100208, 2020. 79. Karagiannidis, N., Thomidis, T., Lazari, D., Panou-Filotheou, E., Karagiannidou, C., Effect of three Greek arbuscular mycorrhizal fungi in improving the growth, nutrient concentration, and production of essential oils of oregano and mint plants. Sci. Hortic., 129, 329, 2011. 80. Arpanahi, A.A., Feizian, M., Mehdipourian, G., Khojasteh, D.N., Arbuscular mycorrhizal fungi inoculation improve essential oil and physiological parameters and nutritional values of Thymus daenensis Celak and Thymus vulgaris L. under normal and drought stress conditions. Eur. J. Soil Biol., 100, 103217, 2020. 81. Hazzoumi, Z., Moustakime, Y., Elharchli, E., Joutei, K.A., Effect of arbuscular mycorrhizal fungi (AMF) and water stress on growth, phenolic compounds, glandular hairs, and yield of essential oil in basil (Ocimum gratissimum L). Chem. Biol. Technol. Agric., 2, 10, 2015. 82. Banchio, E., Bogino, P.C., Santoro, M., Torres, L., Zygadlo, J., Giordano, W., Systemic induction of monoterpene biosynthesis in Origanum × majoricum by soil bacteria. J. Agric. Food Chem., 58, 650, 2010. 83. Cappellari, L.R., Santoro, M.V., Reinoso, H., Travaglia, C., Giordano, W., Banchio, E., Anatomical, morphological, and phytochemical effects of inoculation with plant growthpromoting rhizobacteria on peppermint (Mentha piperita). J. Chem. Ecol., 41, 149, 2015.

Biotic and Abiotic Factors on Essential Oils  97 84. Cappellari, L.R., Santoro, M.V., Schmidt, A., Gershenzon, J., Banchio, E., Induction of essential oil production in Mentha x piperita by plant growth promoting bacteria was correlated with an increase in jasmonate and salicylate levels and a higher density of glandular trichomes. Plant Physiol. Biochem., 141, 142, 2019. 85. Khalediyan, N., Weisany, W., Schenk, P.M., Arbuscular mycorrhizae and rhizobacteria improve growth, nutritional status and essential oil production in Ocimum basilicum and Satureja hortensis. Ind. Crops Prod., 160, 113163, 2021. 86. Sarić-Krsmanović, M., Dragumilo, A., Umiljendić, J.G., Radivojević, L., Šantrić, L., ĐurovićPejčev, R., Infestation of field dodder (Cuscuta campestris Yunck.) promotes changes in host dry weight and essential oil production in two aromatic plants, peppermint and chamomile. Plants, 9, 1286, 2020. 87. García-Caparrós, P., Romero, M.J., Llanderal, A., Cermeño, P., Lao, M.T., Segura, M.L., Effects of drought stress on biomass, essential oil content, nutritional parameters, and costs of production in six Lamiaceae species. Water, 11, 573, 2019.

5 Investigation of Antiviral Effects of Essential Oils Ahmad Mustafa1,2*†, Dina H. El-Kashef 3†, Miada F. Abdelwahab3†, Alshymaa Abdel-Rahman Gomaa3†, Muhamad Mustafa4,5†, Nada M. Abdel-Wahab3† and Alyaa H. Ibrahim5,6† General Systems Engineering, Faculty of Engineering, October University for Modern Sciences and Arts (MSA), 6th of October City, Giza, Egypt 2 Center of Excellence, October University for Modern Sciences and arts (MSA), 6th of October City, Giza, Egypt 3 Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, Egypt 4 Department of Medicinal Chemistry, Faculty of Pharmacy, Deraya University, New-Minia, Minia, Egypt 5 IBMM, Univ. Montpellier, CNRS, ENSCM, Montpellier, France 6 Department of Pharmacognosy, Faculty of Pharmacy, Sohag University, Sohag, Egypt 1

Abstract

Essential oils (EOs) represent one of the most interesting natural products obtained from various aromatic plants. Their effective use in many industries including pharmaceuticals, agriculture, perfumes and food is predominantly attributed to their distinctive aroma and intriguing bioactivities. The chemical profile of these agents exhibited the presence of a variety of volatile constituents that are mainly classified into terpenes and oxygenated compounds. In fact, most of the EOs and essential oil components (EOCs) have received considerable attention in the last years, owing to their promising biological activities, for instance antioxidant, antibacterial, antifungal, antiviral, antispasmodic, insecticidal, anti-inflammatory and cytotoxic activities. This chapter emphasizes particularly the potential antiviral effect of EOs by reviewing some recent literature. It illustrates the different methods implemented to investigate the in vitro antiviral activity of both EOs and EOCs, as well as their mechanisms of action. In addition, the efficacy of EOs against several viral infections affecting human body systems, some plants and animals is also highlighted. Besides, this chapter gives insights into the application of nanoencapsulation technology to improve EOs bioavailability and hence their antiviral efficacy. Keywords:  Essential oils, antiviral activity, COVID-19, Aromatherapy

5.1 Introduction Essential oils, also called ethereal or volatile oils, are very complicated mixtures of low molecular weight organic molecules, characterized by their strong aroma [1]. They are highly concentrated lipophilic, limpid, rarely colored volatile liquids synthesized as secondary metabolites by various aromatic plants. EOs can be obtained from leaves, buds, *Corresponding author: [email protected]; [email protected] † These authors have contributed equally to this work. Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (99–124) © 2023 Scrivener Publishing LLC

99

100  Essential Oils flowers, twigs, roots, rhizomes, seeds, fruits, and bark, wood or entire plants. They are normally stored in oil cells or ducts, secretory cavities, resin ducts, epidermal cells or glandular trichomes [2, 3]. EOs contribute significantly in the protection of plants against bacteria, viruses, fungi and insects and also against herbivores, in addition, they play an important role in attraction of some insects in order to promote pollens and seeds dispersion [4]. Although EOs are mainly plant generated, few of them are animal derived or produced by certain microorganisms [5, 6]. The chemical profile of any essential oil differs according to the geographical location, soil composition, climate, plant part and age, harvest time and extraction methods. Interestingly, unsuitable extraction procedure can result in loss of bioactivity and physical changes such as increased viscosity, discoloration and off-odor [7, 8]. Extraction of EOs can be performed through a variety of techniques depending on the botanical material utilized as well as its form and state. Steam distillation, hydro-­ distillation and hydro-diffusion constitute the main types of distillation implemented for EOs extraction. On the other hand, conventional solvent extractions are carried out in case of delicate flower that cannot tolerate heat (using petroleum ether, hexane, acetone, methanol or ethanol). Supercritical carbon dioxide extraction is considered more economically viable due to higher yield and lower energy consumption. Further introduced procedures include the application of subcritical water, which provides quick extraction of more valuable essential oil with considerable saving in energy and plant material. Furthermore, solvent-free microwave offers substantially fast and efficient extraction with reduced environmental impact [7, 9, 10]. There are approximately 3000 EOs obtained from different plant species, of which only 300 are economically important for perfumes, food, agronomical, sanitary and pharmaceutical industries, due to their characteristic fragrance and interesting pharmacological actions [11, 12]. EOs are gaining extensive attention for their potential and diverse bioactivities including antibacterial, antifungal, antiviral, antispasmodic, antioxidant, antiparasitic, anthelmintic, insecticidal, anti-inflammatory and cytotoxic activities [5, 13]. The tremendous value of each volatile oil is attributed to its complex nature, constituted by dozens of different components which may possess potential influence or synergistic effect [8, 14]. The composition of EOs is accomplished in details through analysis by gas chromatography and mass spectrometry. EOs can comprise from 60 to 300 constituents at varied concentrations, with 2 or 3 major components at relatively higher concentrations (20%–80%). Generally, the constituents in EOs can be arranged into 2 large classes: terpene hydrocarbons and oxygenated compounds [5, 7, 15]. Terpenes are considered the most prevalent group of organic compounds detected in EOs, despite the fact that they contribute to odor and taste only to a limited extent. They are made from combination of isoprene units (C5H8) with a head-to-tail repeated way. Both monoterpenes (C10H16) and sesquiterpenes (C15H24) constitute the major hydrocarbons found in EOs, whereas diterpenes (C20), triterpenes (C30) and tetraterpenes (C40) are usually found at low concentrations [15, 16]. Oxygenated compounds present in volatile oils can be derived from terpenes, termed “terpenoids” or “isoprenoids”. Numerous EOs are predominantly composed of monoterpenes and sesquiterpenes along with their oxygenated derivatives [2]. Other common oxygenated compounds found in plant EOs include alcohols, aldehydes, ketones, phenols, acids, oxides, lactones, esters, ethers and peroxides [7]. Moreover, some EOs may also comprise nitrogen or sulfur compounds [1]. The chemical structures of some selected compounds from various EOs are illustrated in Figure 5.1.

Antiviral Effects of Essential Oils  101 Monoterpene hydrocarbons Acyclic monoterpene

Alicyclic monoterpene

p-Menthane

Ocimene

Aromatic monoterpene

Fenchane

p-Cymene

Sesquiterpene hydrocarbons

Diterpene hydrocarbons Cyclic sesquiterterpene

Acyclic sesquiterpene

p-Camphorene Farnesene

Zingibrene

Alcohols

Phenols OH

OH Linalol

Aldehydes O

OH

Acids

O

OH

Menthol

Thymol

Oxides

Ketones

O

O Eugenol

O

Citronellal

Peroxides

Benzaldehyde

Phellandral

Lactones

Esters

Ethers

O O

OH

O O

O

O

O

O

O

Menthone

O

O

2-Methyl-2-pentenoic acid 1,8-Cineol

Ascaridole

Geranyl acetate Matricarin

Anethol

Nitrogen and sulfur containing compounds S N H Skatole

Allyl sulphide

N

C

S

Allyl isothiocyanate

Figure 5.1  Chemical structures of some selected compounds from different essential oils.

5.2 Viruses: Structure, Characteristics, and Replication Viruses are not considered free-living organisms as they are unable to carry out metabolic processes and they cannot replicate or multiply without depending on a host cell. They are sub-microscopic, obligate intracellular parasites, complex organic particles with variable size and structure [17]. The virus reproduction cycle is divided into 2 definite phases: an intracellular phase where the virus adjust the invaded cell to produce viral particles “virions”, and an extracellular phase during which virions are released out of the infected cells and continue to exist in the extracellular environment [18]. A true virion incorporates RNA or DNA, which is frequently enclosed by a protein shell, termed capsid (non-enveloped or naked viruses), while most virions are also enveloped by a bilayer lipid membrane (enveloped viruses). Enveloped viruses usually possess matrix proteins which connect the capsid

102  Essential Oils to the envelope. Each virus (either non-enveloped or enveloped) also have a virus attachment protein implanted in its outer most layer to enable docking of the virus to the host cell plasma membrane, which facilitates its entry into the cell [19, 20]. The specific surface proteins of viruses allow them to selectively attach to receptors on the corresponding host cells [20]. For instance, human immunodeficiency virus (HIV) preferentially infects helper T lymphocytes as they express primary receptor CD4 and one of two co-receptors (CXCR4 or CCR5) [21]. Viral replication cycle includes a series of steps [17, 20, 22] as shown in Figure 5.2; Virus Nucleic acid (DNA or RNA)

1. Viral attachment/endocytosis

Host cell

2. Viral penetration

3. Uncoating

4. Replication

Nucleus 5. Assembly

6. Release

Figure 5.2  Viral replication [28].

Antiviral Effects of Essential Oils  103 1. Attachment: The first phase of viral replication cycle starts when the virus recognizes the target host cells followed by attachment to the surface receptors present on the cell membrane of those cells. 2. Penetration: Following recognition of the target cells, viral particles penetrate though cell membranes mainly via endocytosis. 3. Uncoating: Represents the complete removal of the protective viral capsid releasing the genomic nucleic acid material of the virus into the cytoplasm of host cells. 4. Replication: Viral gene expression and replication starts after uncoating of viral genome. Since viruses are obligate pathogens, they take control of the host cells’ organelles and cellular proteins to support in replication. Therefore, some viruses replicate in the cytoplasm whereas some others replicate inside the nucleus. 5. Assembly: The packaging of the newly replicated viral nucleic acid genome with essential viral proteins within capsids after transcription and translation forming new virions which are ready for release from host cells. 6. Virion release: The release of newly formed virions from host cells takes place by different mechanisms including; lysis of the infected host cells, such as enveloped and non-enveloped viruses, or budding, such as enveloped viruses.

5.3 In Vitro Antiviral Activity and Mechanism of Action Investigations of Essential Oils and Essential Oil Components 5.3.1 Investigation of In Vitro Antiviral Activities The most frequently used methods for examining the antiviral activity (in vitro) of both EOCs and EOs are inhibiting the viral cytopathogenic effect and plaque formation. Other methods include; the inhibition of specific immunofluorescence and DNA hybridization, reduction in the virus yield, in addition to, the reduction in viral functions within cell cultures. Generally, antiviral agents can inhibit viruses in a dose-dependent manner (Figure 5.3) [17, 23].

5.3.1.1 Plaque Reduction Assay This assay typically uses the cultured host cells in a monolayer which are permitted to bind to the virus. The layer of medium that is thickened with any thickening agent such as agar is added to allow plaque formation by stopping mixing cussed by currents in the medium. Finally, the formed plaques are counted microscopically using low power. The exact test procedure may vary according to the virus under investigations and the host cell lines so many modifications could be applied [24–26].

5.3.1.2 The Inhibition of Viral Cytopathogenic Effect The principal of this assay is that the viral infection causes host cell death and as a result cell viability is the alternate readout for viral infection. Cell viability may be measured with a

104  Essential Oils Assays and Mechanisms of Action

Investigation of in vitro antiviral activities

Mechanisms of action

Plaque reduction assay.

Time-of-drug-Addition assay

The inhibition of viral cytopathogenic effect

Temperature-shift assays

Reduction in the virus yield Inhibition of specific immunofluorescence

Morphological Alteration Protein Inhibition Other mechanisms of action

Figure 5.3  In vitro assays for investigation of antiviral activities of EOs and EOCs and their mechanism of action.

variety of assays. The antiviral agents are those which have the potential to protect the host cells from viral cytopathogenic effect [27]. 

5.3.2 Mechanisms of Action The commonly used methods for determination of the mechanism of antiviral action are time-of-drug-addition assay, temperature-shift assay, morphological alteration, and protein inhibition (Figure 5.3). These methods are manipulated to detect a potential antiviral agent can inhibit the viral replication cycle in which step such as entry stage (i.e. viral attachment and uncoating), replication stage (i.e. viral genome replication and protein translation), and virion exit (i.e. assembly and release) (Figure 5.2) [17, 20, 28].

5.3.2.1 Time-of-Drug-Addition Assay Antiviral EOs and EOCs possess adverse action on certain targets in the virus infection cycle (Figure 5.2) consequently, by manipulating of time-of-drug-addition assay, EOs and EOCs antiviral mechanisms can be determined. The first manipulation involves the pretreatment of cultured host cells with the EOs for 1 h before virus inoculation. A positive result point out that EOs can inhibit viral attachment to host cell by blocking certain receptors. In the second manipulation, viruses are pre-treated for 1 h with EOs prior to simultaneous incubation with cultured host cells and this is known as pretreatment of cell-free viruses. A positive result shows that EOs can interact with the free virus and can alter the structure of virus envelope or the viral proteins. Another method is the cotreatment of virus and host cells throughout virus inoculation, where the virus incorporated with various EOs

Antiviral Effects of Essential Oils  105 or EOCs concentrations are inoculated to the cells directly. A positive result demonstrates that the substance under-investigation inhibits the entry stage of virus replication. Finally, post-entry treatment method where, the infected host cells were treated with the EOs at certain time intervals of the viral replication cycle to detect the stage of the infection cycle which EOs can inhibit. Hence, the time-of-drug-addition assay is commonly employed to study and determine the mechanism of viral inhibitory activity of EOs, either intercellular or intracellular. Usually, EOs and EOCs act directly on the free virus with the intercellular mechanism of action, while sometimes multiple mechanisms of action may further exist [17, 28, 29].

5.3.2.2 Temperature-Shift Assay Involving two main methods; the first one is viral attachment assay where, EOs are mixed with the virus then inoculated to the prechilled host cells at 4°C then the cultured host cells are washed followed by shifting temperature to 37°C. Positive result indicates that EOs inhibits the attachment of the virus to the host cell membrane. The second method is viral entry/fusion assay where, the virus is inoculated at 4°C to the prechilled cultured host cells allowing the virus to only bind to not to enter the host cell membrane. Afterwards, the cultured cells are washed and treated with EOs at 37°C. Obtaining a positive result points that EOs inhibit virus entry to the host cells [17].

5.3.2.3 Morphological Alteration Studying structural changes of the virus caused after treating with EOs or EOCs to determine whether the test substance can inhabit viral replication by destroying, distorting, or masking the virus. This could be done using transmission electron microscopy imaging to visualize these changes [30].

5.3.2.4 Protein Inhibition Several proteins are potential targets for antiviral agents like EOs. Influenza virus, as an example, contains a membrane protein which allows the virus entering and exiting the host cell i.e., hemagglutinin. This protein leads to red blood cells agglutination; thus, hemagglutination inhibition assay is applied to study the influence of EOs on the membrane protein and, as a result, viral adsorption, and entry to host cells. The nonexistence of agglutination registers inhibition of hemagglutinin activity. Another essential surface protein in the influenza virus is a neuraminidase. EOs can inhibit these two target proteins by varying degrees according to the type of the EOs or EOCs [28, 31]. Another example is HIV, where Tat/TAR-RNA complex is formed by the reaction of Tat (trans-activator of transcription) with TAR (trans-activation-responsive region) RNA and is regarded as an important target of HIV-1 inhibitors. EOs of Thymus vulgaris, Rosmarinus officinalis, and Cymbopogon citratus were found to destabilize Tat/TAR‐RNA complex [32, 33]. Finally, several in silico investigations were recently done to investigate the effect of EOs and EOCs on SARS-CoV-2 (coronavirus 2 with severe respiratory syndrome, 2019). Da Silva and coworkers hypothesized that several EOCs may interact with key active sites of

106  Essential Oils SARS-CoV-2 i.e. the spike protein which is the binding domain of SARS-CoV-2 rS, the main protease, ADP-ribose-1”-phosphatase, RNA-dependent RNA polymerase (SARSCoV-2 RdRp), endoribonucleoase, and human angiotensin-converting enzyme (hACE2) [34]. Also, it was reported that some monoterpenes, phenyl propanoids, and terpenoid phenols for example, geraniol, anethole, cinnamaldehyde, cinnamyl acetate, carvacrol, pulegone and thymol from EOs collected from medicinal herbs affiliated to certain families such as Myrtaceae, Lamiaceae, Geraniaceae, Lauraceae,  and Fabaceae can inhibit the viral spike protein and are considered a promising antiviral agents against SARS-CoV-2 [35, 36].

5.3.2.5 Other Mechanisms of Action This includes, for example, interfering with cellular endosomal/lysosomal pH which will inhibit viral replication cycle at early stages. Tea tree EO in addition to terpinen-4-ol as the major component inhibited replication of influenza virus by interrupting virus uncoating through interfering with acidification of intralysosomal compartment [37]. The lipophilic nature of EOs and EOCs has been proposed to be the cause of the inhibitory activity on SARS-CoV-2. EOs and EOCs have the affinity to penetrate the lipophilic viral membrane leading to membrane disruption and hence inhibition of viral replication [5].

5.3.3 Selectivity Index (SI) It indicates feasibility of certain EOs as a therapeutic antiviral agent by determining the window between its cytotoxicity and antiviral activity. Selectivity index of a substance is calculated by dividing 50% cytotoxic concentration (CC50) value by the 50% infection concentration (IC50) value according the equation; (SI = CC50/IC50). Where CC50 represents the EO concentration which decreases 50% of cell viability, while IC50 is the EO concentration that reduces viral infection by 50% [38]. Consequently, a potential therapeutically acceptable antiviral substance would have good antiviral effect at deficient concentrations and would show cytotoxicity only at extremely high concentrations expressed as having an SI value of 4 or more [39].

5.4 The Antiviral Efficacy of Essential Oils on Viruses Affecting Different Body Systems There are many studies have investigated the effect of Eos potent anti-viral activity against viruses as shown in Table 5.1. Detailed explanation of their effect and working mechanisms is also shown below:

5.4.1 Respiratory System 5.4.1.1 Influenza Virus Several studies revealed that EOs exhibited potent anti-viral activity against influenza virus including marjoram, clary sage, anise, Thymus vulgaris, Cinnamomum zeylanicum, and

Antiviral Effects of Essential Oils  107 Table 5.1  Effect of EOs and their derivatives on different viruses. System

Plant essential oil

Virus

Reference

Respiratory

Clary sage, marjoram anise, Thymus vulgaris, Cinnamomum zeylanicum and Citrus bergamia, the bioactive constituents of essential oils germacrone, 1,8‐cinole, eugenol β‐santalol and terpinen‐4‐ol

influenza virus

[31, 40]

Australian tea tree

influenza virus

[41]

Patchouli alcohol, the major component of oil derived from the aerial part of Pogostemon cablin

IFV‑A (H2N2)

[42]

β-Santalol was isolated from the essential of Santalum album L

influenza A/HK (H3N2)

[43]

Eugenol is the major active compound of Syzygium aromaticum

IAV

[44]

Germacrone is a major component of the essential oil extracted from Curcuma rhizome

the H1N1 and H3N2 influenza A viruses and the influenza B virus

[45]

1,8-Cineol, the major monoterpene principal derived from eucalyptus essential oils

influenza A virus

[40]

The tea tree oil and eucalyptus oil

influenza A viruses

[46]

Citrus bergamia, Eucalyptus globulus, citronellol, eugenol

influenza virus

[31]

Melissa officinalis L.

Avian influenza virus (H9N2)

[47]

Zataria multiflora

Avian influenza virus (H9N2)

[48]

Cedar leaf oil

Adenovirus and rhinovirus

[49]

Laurus nobilis, Juniperus oxycedrus ssp. oxycedrus, Thuja orientalis, Cupressus sempervirens ssp. pyramidalis, Pistacia palaestina, Salvia officinalis, Satureja thymbra

SARS-CoV-1

[50]

(Continued)

108  Essential Oils Table 5.1  Effect of EOs and their derivatives on different viruses. (Continued) System

GIT

Plant essential oil

Virus

Reference

(In silico) Isothymol in Ammoides verticillata (Desf.) Briq-allyl disulfide and allyl trisulfide in garlic volatile oil-E,E)-α-farnesene, (E)-β-farnesene, (E,E)-farnesol – the Melaleuca cajuputi essential oil components guaiol, terpineol, linalool, β-selinenol, 1,8-cineole, α-eudesmol, and γ-eudesmol – (In vitro) citronellol, limonene, geraniol in. Geranium oil and lemon oil

SARS-COV-2 (ACE2 active site)

[34, 51–54]

Eucalyptol, menthol, transpinocarveol, linalool, methyl salicylate, α-pinene

SARS CoV-2 (spike protein)

[56]

(In silico) EOC carvacrol (Origanum vulgare L. Thymus vulgaris) 1,8-cineole (Eucalyptus globulus Labill) – jensenone (Eucalyptus jenseni) – guaiol, terpineol, 1,8-cineole, linalool, α-eudesmol, β-selinenol, and γ-eudesmol (Melaleuca cajuputi Powell)

SARS CoV-2 (main protease)

[52, 53, 58–60]

Osmunda regalis, Eucalyptus bicostata and Dysphania ambrosioides essential oils

Coxsackie virus

[61, 62]

Eucalyptus camaldulensis

Coxsackievirus B4

[63]

Lippia alba and Lippia citriodora

Dengue viruses (DENV 1–4)

[64]

β-caryophyllene

DENV-2

[65]

Lippia alba, Lippia origanoides, Origanum vulgare and Artemisia vulgaris

Yellow fever virus

[66]

Citral and limonene (Lippia alba and Lippia citriodora)

Yellow fever virus

[67]

oregano oil and its primary active principle, carvacrol and Artemisia princeps

Murine norovirus

[68, 69]

(Continued)

Antiviral Effects of Essential Oils  109 Table 5.1  Effect of EOs and their derivatives on different viruses. (Continued) System

Plant essential oil

Virus

Reference

Nervous

Monoterpene alcohols (CMA) derived from Melaleuca alternifolia

West Nile Virus

[70]

Immune

Cymbopogon nardus, Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis

HIV‐1

[32]

Reproductive system

Myrtle (Myrtus communis)

Human Papilloma virus

[71]

Other Viruses

β caryophyllene in Star Anise (Eucalyptus caesia)

Human Herpes Virus

[72]

Melissa officinalis

Human Herpes Virus

[73]

Thymol and carvacrol

Human Herpes Virus

[74]

Chamomile oil

Herpes simplex virus 2

[75]

Achillea fragrantissima

ORF Virus

[76]

Virus affecting plants

Melaleuca alternifolia

Tobacco Mosaic Virus

[77]

Virus affecting cattle

Lippia graveolens Kunth

Bovine viral diarrhea Virus

[78]

Virus affecting cats

Germacrone (curcuma rhizome) and α-thujone (Artemisia princeps)

Feline calicivirus F9

[69, 79]

Virus affecting pigs

Germacrone

Porcine parvovirus

[80]

110  Essential Oils Citrus bergamia oils in comparison with oseltamivir. Furthermore, among the bioactive constituents of Eos, germacrone, 1,8‐cinole and eugenol are reported to display significant anti‐influenza activities. This effect was probably attributed to their effective broad‐ spectrum modes of action through multiple steps of viral replication inhibition. In addition, components of oxygen terpene such as terpinen‐4‐ol and β‐santalol owned high anti‐­influenza activities via the inhibition of IFV‑A replication and viral RNA synthesis when examined against IFV‑A [31, 40]. The essential oil, Melaleuca alternifolia concentrate, is originated from the Australian tea tree. It was examined for its antiviral activity in the influenza virus. The results focused on a combination of terpinen-4-ol, the main bioactive compound of the concentrate, with the viral hemagglutinin’s membrane fusion site. The results also confirmed that the concentrate has a remarkable inhibitory activity on influenza virus infection by blocking the viral entrance to the host cells by disrupting the standard procedure of the viral membrane fusion [41]. Patchouli alcohol, a significant component of patchouli oil, is derived from Pogostemon cablin’s aerial part. It displayed considerable antiviral properties against influenza‑A virus (H2N2), showing IC50 value of 4.03 µM [42]. β-Santalol, a bioactive component separated from the essential sandalwood oil (Santalum album L.), was examined for its anti-viral activity in influenza A/HK (H3N2). It possessed a strong anti-viral activity (86%) at a 100 μg/ml concentration compared to oseltamivir (83%) at the same concentration. Furthermore, β-Santalol demonstrated a complete inhibitory effect on the viral RNA synthesis [43]. Eugenol is the major active compound of Syzygium aromaticum L. and was reported to have a promising anti-influenza A virus property together with its ability to inhibit the replication of influenza A virus and viral autophagyin, in addition to its inhibition of the release of cytokines induced by influenza A virus [44]. Germacrone, a major component of the essential oil extracted from curcuma rhizome, showed antiviral activity against the H1N1 and H3N2 influenza A viruses and influenza B virus in a dose-dependent manner. Furthermore, it was found to possess an inhibitory effect on the viral replication. Germacrone also showed an effective protection of mice from lethal infection of influenza [45]. 1,8-Cineol, an essential monoterpene principal originating from eucalyptus EO, was screened for its anti-influenza A virus properties. The obtained results revealed the ability of 1,8-cineol to protect against the infection of influenza virus in mice. Besides, it significantly decreased IL-4, IL-5, IL-10, and MCP-1 levels in nasal lavage fluids and the level of IL-1β, TNF-α, IFN-γ, and IL-6 in the mice lung tissues infected with influenza virus [40]. The tea tree oil and eucalyptus oil were investigated for their antiviral activity aerosols in range of concentrations against influenza A virus. The results revealed that both tested oils aerosols show potent antiviral activity and are able to inactivate the model viruses with efficiency of more than 95% within 5–15 min of exposure [46]. Many EOs and their major components were evaluated for their anti-influenza virus activity in vapor and liquid phases. Among the tested oils, Citrusbergamia, Eucalyptus globulus, and the isolated components eugenol and citronellol were highly potent against influenza virus for only 10 min exposure, in vapor phase. Additionally, Cinnamomum zeylanicum, Pelargonium graveolens, Cymbopogon flexuosus possessed an important antiviral activity with 30minutes exposure. On the other hand, in liquid phase, Citrus bergamia,

Antiviral Effects of Essential Oils  111 Cinnamomum zeylanicum, Thymus vulgaris, and Cymbopogon flexuosus showed 100% inhibitory activity at 3.1 μL/mL concentration [31]. The Melissa officinalis L. essential oil was reported to have a prominent antiviral efficiency at various concentrations (0.5-0.005 mg/mL) against avian influenza virus (H9N2) [47]. The effect of Zataria multiflora essential oil on replication rate of the H9N2 virus was screened. The results showed that the EO had a positive effect on reducing viral replication in both the intestine and trachea of H9N2 influenza infected broiler chickens [48].

5.4.1.2 Adenovirus and Rhinovirus The antiviral efficacy of cedar leaf oil vapor was tested against rhinovirus and adenovirus. The results demonstrated that adenovirus and rhinovirus were inactivated by exposure to cedar leaf oil vapor, in addition to the ability of the vapor to inhibit rhinovirus virus-­ induced cytokine IL-6 [49].

5.4.1.3 Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-COV-1) The anti-viral efficacy of Laurus nobilis, Juniperus oxycedrus ssp. oxycedrus, Thuja orientalis, Cupressus sempervirens ssp. pyramidalis, Pistacia palaestina, Salvia officinalis, and Satureja thymbra essential oils were screened for their inhibitory activity against SARS-CoV-1 using the visual scoring of the virus-induced cytopathogenic post-infection effect. The L. nobilis oil possessed a promising potency against SARS-CoV-1 (IC50 = 120 μg/ml) [50].

5.4.1.4 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) Since the emergence of the novel corona virus SARS-COV-2 from Wuhan, China and its rapid spread throughout the world causing the pandemic coronavirus disease 2019 (COVID-19), an urgent need has risen to find powerful antiviral medications to combat this virus. Researchers investigated EOs and EOCs to find potential anticoronavirus agents, yet the results available depend mainly on computer assisted molecular docking in addition to few in vitro studies [5, 17]. Targets of Drug Design for SARS-COV-2 Antivirals from EOs (interrupting virus-host cell interactions) A Inhibition of Virus Attachment and Entry to Host Cell via: a) Inhibition of ACE2 Receptor in the Host Cell Five compounds present in Ammoides verticillata (Desf.) Briq. essential oil namely isothymol, thymol, p-cymene, limonene, and γ-terpinene were studied for their binding to ACE2 receptor via molecular docking. The results displayed that isothymol had the strongest binding with ACE2 receptor proteins [51]. Thuy and coworkers investigated the in silico ACE2 receptor blocking action of 17 organosulfur compounds present in garlic volatile oil. The compounds allyl disulfide and allyl trisulfide showed binding interaction with the amino acid units of the host ACE2 receptor [52].

112  Essential Oils An in silico docking study was performed on 171 essential oil components to investigate their anticoronavirus activity. The compounds (E,E)-α-farnesene, (E)-β-farnesene, (E,E)farnesol displayed high binding affinity to ACE2 protein [34]. Melaleuca cajuputi essential oil components were tested for their inhibition of ACE2. The in silico simulation revealed that compounds, guaiol, terpineol, linalool, β-selinenol, 1,8-cineole, α-eudesmol, and γ-eudesmol have potent ACE2 inhibitory activities [53]. In vitro ACE2 inhibitory activity of 30 EOs was examined by Kumar et al. Geranium oil and lemon oil exhibited significant downregulation of the ACE2 receptor in HT-29 epithelial cells at concentrations 50 μg/mL and 25 μg/mL respectively. Moreover, the EOCs citronellol, limonene, and geraniol displayed ACE2 receptor down regulation in HT-29 epithelial cells [54]. b) Blocking the Spike Protein of the Virus The viral spike protein (S glycoprotein) of SARS CoV-2 is comprised of two subunits. The S1 subunit, known as the receptor binding domain (RBD), is responsible for the viral attachment to ACE2 of the host cell. The S2 subunit accounts for fusion between the host cell membrane the viral envelope [55]. Therefore, the S1 subunit represents an important target for drugs because blocking of the S1 subunit (RBD) prevents binding of the virus to the host cell. Hence, 25 monoterpenes and phenylpropanoids were screened by molecular docking to test their binding activity to S1 subunit of SARS CoV-2 virus. The results showed that the compounds, cinnamaldehyde, anethole, carvacrol, cinnamyl acetate, geraniol, thymol, α-terpineol, and pulegone exhibited remarkable binding to RBD of SARS CoV-2 virus [35]. c) TMPRSS2 which is Important for the Attachment of the Host ACE2 Receptor to the Virus Spike Protein Molecular docking analyses revealed that the EOCs eucalyptol, menthol, trans-­pinocarveol, linalool, methyl salicylate, and α-pinene exhibited effective binding to the human serine protease TMPRSS2. Interestingly, methyl salicylate was oriented within TMPRSS2 active site the same as the serine protease inhibitor (camostat) [56]. B Inhibition of Virus Replication via Targeting the Main Viral Protease Involved in the SARS CoV-2 Replication Cycle With the aim of halting SARS CoV-2 viral replication inside host cell, attention has been drawn to the viral proteolytic enzyme Mpro/3CLpro as a key target to discover anti SARS CoV2 drugs [57]. Molecular docking investigation on the EOC carvacrol found in Origanum vulgare L. and Thymus vulgaris revealed its effective binding affinity to the viral main protease Mpro/3CLpro [58]. Likewise, the compounds 1,8-cineole and jensenone, obtained from Eucalyptus globulus Labill. and Eucalyptus jenseni Maiden, respectively, were found to bind to the viral main protease Mpro/3CLpro [59, 60]. Interestingly, the in silico studies displayed that garlic EO organosulfur derivatives allyl disulfide and allyl trisulfide, as well as Melaleuca cajuputi Powell EO characteristic compounds guaiol, terpineol, 1,8-cineole, linalool, α-eudesmol, β-selinenol, and γ-eudesmol, showed potential binding to the viral protease Mpro/6LU7 in addition to their ACE2 inhibitory activity discussed earlier in this chapter [52, 53].

Antiviral Effects of Essential Oils  113

5.4.2 GIT System 5.4.2.1 Coxsackie Virus The antiviral activity of essential oil obtained from Osmunda regalis was determined against Coxsackie virus. The oil showed potent anti‐coxsackie activity (IC50 = 2.24 μg/mL). Moreover, Dysphania ambrosioides EOs and Eucalyptus bicostata demonstrated their strong anti‐coxsackie potency [61, 62]. The leaf essential oil of Eucalyptus camaldulensis showed a promising effect against Coxsackie virus B4 with percentage of reduction 53.3% [63].

5.4.2.2 Dengue Virus The inhibitory effect of Lippia alba Eos and Lippia citriodora was estimated against four dengue viruses serotypes (DENV 1–4). The results revealed that the essential oil separated from Lippia alba and Lippia citriodora possessed virucidal activity with IC50 values range of 1.9–33.7 μg/mL for Lippia citriodora and 0.4–32.6 μg/mL for Lippia alba [64]. The biological examination was performed on β-caryophyllene to assess the in vitro inhibition of DENV-2. It was found that β-caryophyllene possessed critical anti-DENV-2 activity leading to potent virus replication inhibition at an early stage of the virus replication cycle [65].

5.4.2.3 Yellow Fever Virus The inhibitory activity of the EOs obtained from Lippia origanoides, Lippia alba, Artemisia vulgaris, and Origanum vulgare was determined against yellow fever virus (YFV). The minimum inhibitory concentration of the examined essential oils was estimated. The MIC of L alba, L origanoides, O. vulgare was essential oils 3.7 μg/mL essential oils, whereas for A. vulgaris was 11.1 μg/mL. The results concluded that the in vitro YFV infectivity inhibition is based on a direct inactivation of virion, which prevents the adsorption to host cells [66]. The antiviral activity of limonene, citral, and EOs (Lippia alba and Lippia citriodora) on the replication of YFV was investigated. EOs and citral showed strong activity before and after virus adsorption on cells with IC50 values of 4.3-25 μg/mL [67].

5.4.2.4 Murine Norovirus Type 1 The antiviral efficacy of oregano oil and its primary active principle, carvacrol, against murine norovirus was examined. The results demonstrated that carvacrol possessed an effective role in inactivating the virus within 1 h of exposure by acting directly on the viral capsid and subsequently the RNA. On the other hand, α-thujone and Artemisia princeps var. orientalis essential oil showed a strong inhibitory effect on murine norovirus -1 [68, 69].

5.4.3 Nervous System 5.4.3.1 West Nile Virus The combination of monoterpene alcohols derived from Melaleuca alternifolia owned a distinctive virucidal activity against west Nile virus infection at the maximum non-cytotoxic

114  Essential Oils concentration of 0.0075%. Moreover, the combination of monoterpene reduces the virus titer and percentage of infected cells [70].

5.4.4 Immune System 5.4.4.1 HIV The essential oil of Cymbopogon nardus as found to possess a characteristic antiviral activity against HIV‐1 reverse transcriptase activity, showing IC50 of 1.2 mg/mL. This outcome can be illustrated by the presence of (S)‐β‐citronellol as one of the major bioactive compounds of Cymbopogon oil. Also, Cymbopogon citratus, Thymus vulgaris, and Rosmarinus officinalis EOs substantially inhibited HIV‐1 (IC50 = 0.05–0.83 μg/mL) [32].

5.4.5 Reproductive System 5.4.5.1 Human Papilloma Virus (HPV) A clinical study was conducted to examine the antiviral action of vaginal suppositories containing 0.5% myrtle (Myrtus communis) EO and 10% myrtle aqueous extract in patients with HPV infection. The myrtle EO containing suppository displayed increased viral clearance rate (92.6%) in comparison to placebo (68%) [71].

5.4.6 Other Viruses 5.4.6.1 Human Herpes Virus EOs from star anise, Australian tea tree (Eucalyptus caesia), showed potent antiviral activity when screened against herpes simplex virus (HSV), among them, the star anise essential oil possessed the most potent antiviral efficacy showing an IC50 value of 1 μg/mL. This action is due to the presence of the efficient antiviral component β caryophyllene [72]. Using a plaque reduction assay, the antiviral activity of lemon balm oil (the essential oil of Melissa officinalis) was examined on herpes simplex virus. The IC50 value of the balm oil for herpes simplex virus plaque formation was made at high dilutions of 0.0004% and 0.00008% for HSV-1 and HSV-2, respectively. The results revealed that lemon balm oil has a direct antiviral effect on herpes viruses. The lipophilic nature of lemon balm essential oil enables it to penetrate the skin leading to the availability of topical treatment of herpetic infections [73]. Thymol and carvacrol, which are the major constituents of several EOs, were tested for their anti-herpes simplex virus type 1 activity. The obtained results showed that carvacrol and thymol are considered effective agents for topical therapeutic application to decrease herpes simplex virus transmission [74]. Anise, ginger, hyssop, chamomile, thyme, and sandalwood EOS were evaluated for their potency in herpes simplex virus type 2. All the tested EOs reduced the virus infection which may be attributed to their direct interaction with the viral envelope and glycoproteins. The results revealed that among the examined oils, the chamomile oil possessed a remarkable antiviral activity [75].

Antiviral Effects of Essential Oils  115

5.4.6.2 Orf Virus The essential oil extracted from Achillea fragrantissima was found to exhibit a potent antiviral activity against orf virus (a parapox virus). This effect attributed to the ability of essential oil to block the viral infection by blocking the cell membrane receptor for orf virus or induce internal changes in the host cells, which in turn affect the virus replication cycle or due to production cytokines which blocked viral infection [76].

5.5 The Antiviral Efficacy of Essential Oils on Phyto-Pathogenic Viruses Virus Affecting Plants (Tobacco Mosaic Virus) Tobacco mosaic virus is a widespread plant pathogen, is found in tobacco as well as in many other plants. The Essential oil obtained from Melaleuca alternifolia was found to have a remarkable antiviral potential against tobacco mosaic virus that resulted in suppressing the lesion formation of Nicotiana glutinosa within 10 days of administration [77].

5.6 The Antiviral Efficacy of the Essential Oils on Animal‑Infecting Viruses 5.6.1 Virus Affecting Cattle (Bovine Viral Diarrhea Virus) Bovine viral diarrhea Virus (BVDV) is a serious pathogen correlated to respiratory, gastrointestinal, and reproductive diseases of cattle. The Mexican oregano EO (Lippia graveolens Kunth) and its main principle carvacrol were examined for their antiviral effect against BVDV. The results showed that the oil and carvacol exert a pronounced antiviral effect [78].

5.6.2 Virus Affecting Cats (Feline Calicivirus F9) Feline calicivirus (FCV) F9 is an important cause of upper respiratory infections and oral disease in cats. Germacrone is one of the essential compounds of essential oil separated from curcuma rhizome was as a strong replication inhibitor of FCV at low concentrations. It exerted potent FCV inhibition in the early phase of the viral life cycle. Besides, α-thujone, one of the bioactive constituents of Artemisia princeps var. orientalis essential oil, possessed a promising inhibitory effect in FCV-F9 [69, 79].

5.6.3 Virus Affecting Pigs (Porcine Parvovirus) Porcine parvovirus (PPV) is considered the main cause of reproductive disorders in pigs. Germacrone, is one of the main bioactive components in volatile oils that were investigated for its antiviral activity on PPV in swine testis (ST) cells. It was found that germacrone can protect cells from PPV infection and suppresses the synthesis of viral mRNA and protein. Furthermore, it inhibits PPV replication at an early stage in a dose-dependent manner [80].

116  Essential Oils

5.7 Synergistic Effect of Essential Oil Components with Known Antiviral Drugs Combination of the EOC eupalyptol (1,8-cineole) with the known antiviral medication oseltamivir demonstrated synergistic effect against influenza A virus (H3N2) infection in mice. This in vivo experiment performed by Lai and co-workers showed that this combination therapy resulted in higher number of survivor in addition to longer mean survival time compared to monotherapy [81]. In the same context, Melissa officinalis EO combined with oseltamivir had synergistic activity against avian influenza virus (AIV) subtype H9N2 as proved through an in vitro study [47].

5.8 Aromatherapy and its Role as an Antiviral Agent Aromatherapy is a natural method of healing based on the use of EOs in therapeutic (i.e. psychological or physical) purposes. The name is formed from two parts aroma meaning fragrance or scent and therapy which mean treatment or cure. Historically, EOs have gained an important position as fragrances with a therapeutic benefits on the body, mind and soul [82]. Aromatherapy is a rapidly growing complementary medicine technique worldwide for instance The National Institutes of Health and National Center for Complementary and Integrative Health reported that Americans spend every year more than $30.2 billion on it [83]. Numerous studies demonstrated that essential oils obtained from many herbs showed a strong antiviral activity. Reports showed that the aromatherapy is considered a safe method to prevent of herpes simplex viral infection [54]. Other reports illustrated that some volatile components found in household aromatherapy product, namely; α-pinene, eucalyptol, and methyl salicylate demonstrated favorable binding to spike proteins and ACE2 when docked to several key targets in SARS-CoV-2 [56].

5.9 Route of Essential Oil Administration EOs and EOCs can be used by four routes: topical, inhalation, internal (e.g., suppositories, gargles, douches), and oral (e.g., capsules). A variety of essential oils formulations are available for body application, including lotions, soaps, shower gels, scrubs, and bath solubles. The preferred method for topical application on the skin includes dilution of EOs using many oils such as jojoba oil and coconut oil. Patients should use essential oil internally or orally only under the guidance of a certified aromatherapist [84]. Inhalation is a simple, rabid and effective method of EOs administration. The simplest method of inhalation is by adding one or two drops of EO on a tissue or cloth and breathing in. another method is diffusion which is the process that disperses EOs into the air by three main ways i.e. the use of heat, water, and atomizing (which is the preferred method). Diffusion allows better absorption of microdroplets through the nasal mucosa [84, 85].

Antiviral Effects of Essential Oils  117

5.10 Nano-Formulated Essential Oils: A Promising Approach to Enhance Antiviral Activity There are many problems that limit therapeutic use of EOs and EOCs such as, high volatility, and susceptibility for oxidation which decreases their stability, in addition to, lipophilicity and partial solubility in water that reduces their bioavailability. So, nanoencapsulated is largely used to resolve these problems. The main goal of such a technique is to stabilize the active ingredient, to augment bioactivity, to mask unpleasant taste. Also this technique can improve the release, and/or achieve controlled release of the active ingredient [86, 87]. Encapsulation is the process of entrapping one substance within another one to produce a delivery carrier in micro or nano diameter. Nanoencapsulation is the technique used to encapsulate the active substance in the nanometric size to modify its physicochemical characters and to enhance bioactivity as well as to produce a sustained release effect [86]. Nanoencapsulated EOs are considered a successful approach in nanodrug delivery systems used for controlling various viral infections. The effect of solid lipid nanoparticle (SLN) formulation of Artemisia arborescens essential oil on transdermal delivery and in vitro inhibitory activity on Herpes simplex was evaluated and the results showed that SLN formulation is capable of entrapping the essential oil in high yields in addition to improving the skin accumulation of the oil [88]. Cymbopogon citratus EO encapsulated in polymeric nanoparticles as a hydrogel was tested for in vitro inhibitory potency on Herpes simplex virus. The Nanogel substantially inhibited virus 42-folds lower than free oil. Furthermore, the nanogel has potential to prevent the EO volatilization and to obtain controlled release [89]. EO of Melissa officinalis L. was formulated in glycerosomes and was evaluated for its inhibitory activity on HSV type 1. The results showed that glycerosomes of the EO were highly effective in inhibiting in vitro HSV type 1 human cell infection in concentrations causing no cytotoxic effects [90].

5.11 Safety of Essential Oils EOs are usually described as safe and have minor adverse effects. Many of them are considered as food additive. According to Food and Drug Administration in U.S.A, they belong to the category of generally recognized as safe [91]. The most common adverse effects include irritation and sensitization of eye, mucous membrane and skin usually with EOs that contain high amount of phenols and aldehydes. Photo toxicity of EOs containing furocoumarins, such as, Citrus bergamia is also reported. Inconvenient storage conditions  of EOs may lead to oxidation of some of its constituents for instance monoterpenes which can cause contact sensitization [84]. Sometimes, cross-sensitization might occur among EOs as well as foods. In rare occasions allergic reactions may occur due to inhaled EOs nevertheless, there is no clear data about concentration and level of exposure. Airborne contact dermatitis was reported in a single case after receiving aromatherapy [91]. Moreover, topical administration of EO caused allergic conditions only in rare cases. Henley and coworkers reported a reversible prepubertal gynecomastia due to repeated topical administration  of lavender and tea tree oils [83, 92]. Usually most adverse reactions are cased after use of lesser quality

118  Essential Oils EOs which are subjected to oxidization reaction with age and change in their chemical composition on long time storage. To sum up, despite being generally considered safe, EOs may be dangerous and toxic. They may sometimes cause skin dermatitis, chemical burn, and oral toxicity. At the same time they may be phototoxic and some of them are flammable [83]. Using low quality EOs and lack of understanding the safety guidelines may negatively affect clinical outcomes of their use [84].

5.12 Antiviral Essential Oils: Drawbacks versus Future Perspectives Although there are diverse in vitro preliminary studies of EOs and their significant antiviral potential, but so far, the exact mechanisms of the antiviral action have not yet been fully illustrated. For this reason, various studies should be performed to understand the mode of antiviral action of EOs clearly [17]. Also, the high volatility of these oils that discourage their use without a suitable pharmaceutical formulation is considered a great limitation. Moreover, the hydrophobicity of EOs, as well as, the very low solubility in biological fluids is a major obstacle that decreases their absorption and lead to a very low bioavailability. Consequently, all these aspects restrict the utility of EOs as potential therapeutic agents, which provokes us toward performing more pharmaceutical research [86].

5.13 Summary Nowadays there is an increased awareness and more recommendation for natural medicine techniques and environmental friendly pharmaceutical industries. Many EOs and EOCs have shown potential as effective antiviral agents and could inhibit several viral infections. EOs and EOCs usually act synergistically and also, may potentiate other antiviral agents. From the previously mentioned literature survey on antiviral actions of EOs and EOCs we could conclude that, they may be potential drug leads as antiviral agents as they are capable of targeting vital phases in viral life cycle. Although the direct interaction of EOs with free viruses which causes viral membrane disruption is probably the most common mechanism of the antiviral activity, they also can inhibit virus attachment, replication, and release. More research should focus on the field of synergism between EOs and EOCs to obtain the best efficacy with minimal side effects.

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6 Mentha sp. Essential Oil and Its Applicability in Brazil Daniele de Araujo Moysés1, Hanna Patricia dos Santos Martins2, Margoula Soares Ribeiro3, Natasha Costa da Rocha Galucio1, Raquel Ribeiro de Souza4, Regianne Maciel dos Santos Correa5, José de Arimateia Rodrigues do Rego6, Maria Fani Dolabela2 and Valdicley Vieira Vale7* Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Pará, Belém, Brazil 2 Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Pará, Belém, Brazil 3 Programa de Pós-Graduação em Ciências Biológicas, Botânica Tropical, Universidade Federal Rural da Amazônia and Museu Paraense Emilio Goeldi, Belém, Brazil 4 Programa de Pós-Graduação em Assistência Farmacêutica, Universidade Federal do Pará, Belém, Brazil 5 Curso de Farmácia, Escola Superior da Amazônia, Belém, Brazil 6 Programa de Pós-Graduação em Química, Universidade Federal do Pará, Belém, Brazil 7 Programa de Pós-Graduação em Inovação Farmacêutica, Universidade Federal do Pará, Belém, Brazil 1

Abstract

Mentha is a plant species widely used for different purposes, including culinary and medicinal. This work aimed to identify the popular medicinal uses in Brazil, as well as the biological activities scientifically. Several species are attributed to the genus, four of them are found in Brazil: Mentha piperita L., M. pulegium, M. spicata and M. suaveolens, but the hybridization between the species makes botanical identification difficult, with other species being described in the national territory. Different culture media also influence the chemical composition of these oils with different compositions between species. As for popular uses, respiratory diseases, anti-inflammatory and gastrointestinal problems stood out, but in the biological activities performed, antimicrobial and antioxidants stand out, showing a gap between studies and popular uses. The tests are mostly in vitro, justified by their low yield, but selection criteria for them are still scarce. Correlating the studies of biological activity and toxicity, in addition to its wide popular use, M. piperita stands out as the most promising species in the genus to be used for pharmacological purposes. Keywords:  Mentha, chemical compounds, biological activity, toxicity

*Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (125–156) © 2023 Scrivener Publishing LLC

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126  Essential Oils

Introduction The genus Mentha L. is a tribute to a Greek goddess. The story tells that god Hades was married to Persephone and had the nymph Minthe as his lover. Her mother-in-law, Demeter, discovered the extramarital affair and told Persephone. Then, the betrayed wife beats Minthe until her disintegration and from her remains came the goddess, who lent the name to the Mint plant [1]. The Egyptians, Hebrews, Greeks, Medievals, Romans and Americans related this genus to myths, having been reported in the bible as a tithe [2]. In addition, the genus Mentha has been known since the early days by the Chinese, who already advocated its soothing and antispasmodic properties. In addition, Hippocrates considered the species of the genus aphrodisiacs, while Pliny, an Italian philosopher, attributed analgesic property [3]. Other utility of this genus was reported by the Arabs, who watered the banquet tables with mint before the parties and cleaned the floor with the herb to stimulate the guests’ appetite [2]. The oil has wide application in food pharmaceutical and cosmetic industries [4], in which it is widely used in candies, sweets and liqueurs [5]. Mentha, popularly known as Mint, is a genus belonging to the Lamiaceae family, and consists of herbaceous, perennial and aromatic plants. The classifications of each species in this genus are based on leaf morphology, flowering and habit. The shape of leaves, their size, veins and petioles vary greatly. The size and colors of the leaves vary in shades of green, as well as their hairiness and leaf blade shape. The crispness, roughness and variegations are of fundamental importance for the taxonomy [6, 7]. The essential oil of Mentha is produced and stored in pelted glandular trichomes, which are mainly present in the leaves and flowers and, in a lower density, in the stems, [4]. There are approximately 25 species distributed around the world [8]. In Brazil, four species are officially registered: Mentha piperita L., Mentha pulegium L., Mentha spicata L., Mentha suaveolens Ehrh [9]. It is native to the Middle East and arrived in Brazil through Japanese immigrants who cultivated in small quantities in the interior of São Paulo at the beginning of the 20th century [8]. The climate and soil have a direct influence on the plant, acting in the formation of oil and its components. The soil for peppermint (Mentha piperita) must be sandy, fertile, permeable, fresh, rich in organic matter, protected from cold winds and frost. Organic soils best receive the mint, commonly found in forest clearing. The very clayey and compact soils, on the other hand, are not suitable for cultivation, unless they have their physical properties improved through the massive incorporation of organic fertilizers [10]. In view of the spontaneous crossing between species, there is a great variety of hybrids of this genus, making the identification and classification of species difficult, even among specialists [11]. It is noteworthy that the genus Mentha is characterized by presenting a large chemical polymorphism between species due to frequent hybridization [12], and in some species the main constituent is menthol (Figure 6.1-1) [4]. Other metabolites identified in essential oils of different species were: menthone (Figure 6.1-2); Carvone (Figure 6.1-3); limonene (Figure 6.1-4); pulegone (Figure 6.1-5); linalool (Figure 6.1-6); linalyl acetate (Figure 6.1-7); menthyl acetate (Figure 6.1-8); 1,8-cineole (Figure 6.1-9); menthofuran (Figure 6.1-10); piperitone (Figure 6.1-11); piperitenone (Figure 6.1-12); piperitenone oxyde (Figure 6.1-13); β-caryophilylene (Figure 6.1-14);

Mentha sp. Essential Oil and Its Applicability in Brazil  127

O

HO

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OH

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19

Figure 6.1  Major chemical constituents identified in Mentha essential oils (1: Menthol; 2: Menthone; 3: Carvone; 4: Limonene; 5: Pulegone; 6: Linalool; 7: Linalyl acetate; 8: Menthyl acetate; 9: 1,8-cineole; 10: Menthofuran; 11: Piperitone; 12: Piperitenone; 13: Piperitenone oxyde; 14: β-caryophylene; 15: Gamamuurolene; 16: Muurolene; 17: Cis-cis-p-menthenolide; 18: β-elemene; 19: Terpinolene); Self drawn.

Gamma-muurolene (Figure 6.1-15); Muurolene (Figure 6.1-16); cis-cis-p-menthenolide (Figure 6.1-17); β-elemene (Figure 6.1-18); terpinolene (Figure 6.1-19) [13–21]. It is noteworthy that the production of essential oils occurs during photosynthesis. Plants produce a wide variety of organic compounds (secondary metabolites) that apparently have no direct role in their growth and development, however, are related to plant defense, protecting plants from herbivory and against infection by pathogenic microorganisms, in addition to acting as attractants for pollinating animals, seed dispersers and as agents in plant-plant competition [23]. Due to the historical importance of the genus, the aim of this work was to carry out a narrative review about the Mentha genus, including studies that addressed the biological activities, and ethnobotanical and chemical aspects of the plant.

6.1 Ethnobotany of the Mentha in Brazil Mentha species, according to are scientific studies and popular knowledge, present medicinal properties acting as analgesics, anesthetics, sedatives, antiparasitic, antibiotics, expectorants, antiseptics, anti-inflammatory and decongestants. In cooking, fresh mint is widely used as a condiment and flavoring in meat, salads, juices and refreshing teas. In the North and Northeast regions of Brazil, ethnobotanical studies are almost exclusive to knowledge associated with indigenous or Quilombola ethnic groups. Among the Brazilian population, the leaves are the most used parts of mint, mainly in the form of teas for infusion, or as a flavoring for citrus juices, to increase the sensation of freshness, as shown in Table 6.1, which presents the main popular uses of Mentha species, by traditional Brazilian communities (Table 6.1).

128  Essential Oils Table 6.1  Species of the Mentha genus used by traditional Brazilian peoples and communities. Community

Scientific name

Indications/ Pathologies

Várzea in GaranhunsPernambuco (PE) [23]

Mentha piperita L.

Flu

Mentha pulegium L.

Flu

Sobral, Ceará (CE) [24]

Mentha piperita

Colic, worms, headache, flu, poor digestion

Infusion of leaves for tea preparation

Quilombola community Sítio Arruda in Araripe, Ceará (CE) [25]

Mentha villosa Huds

Headache, flu, fever

Leaves in the form of tea

Quilombola community Barra II, Bahia (BA) [26]

Mentha gentilis L

Pressure

Infusion of leaves for tea preparation

Mentha viridis L Mentha spicata

Amoeba; regulation of menstruation flow; uterus; stomach; throat; tonic; flu; worm; insomnia

Decoctions or juice are made from leaves to treat the aforementioned diseases. The use is for teas, juices, porridge or syrups

Mentha pulegium L

Bellyache, chilblain, flu, cough, uterine cleaning, stomach, throat, fortifying

The leaves are ingested as teas by decoction, steeped in alcohol or infusion. It can be used in the form of teas, syrups, cachaça, topical or porridge.

Kantaruré Ethnicity Baixa das Pedras, Bahia (BA) [27]

Mentha piperita L.

Antiparasitic action

Leaves in the form of tea

Quilombo Sangrador in Presidente Juscelino, Maranhão (MA) [28]

Mentha aquatica L.

-

-

Mentha piperita L.

Diseases associated with the respiratory system

Infusion of leaves for tea preparation

Preparation and use Used in the form of tea. The leaves are most used part

(Continued)

Mentha sp. Essential Oil and Its Applicability in Brazil  129 Table 6.1  Species of the Mentha genus used by traditional Brazilian peoples and communities. (Continued) Community

Scientific name

Indications/ Pathologies

Preparation and use

Mentha spicata L.

Diseases associated with the respiratory system

Leaves ingested in natura

Mentha suaveolens L.

Nervous System Diseases

Infusion of leaves for tea preparation

Mentha sp.

Fever, flu, cough

Leaves in the form of teas or rubbing the leaves and taking it to the nose to inhale the smell (in natura)

Mentha sp

Hoarseness, flu

The leaves are used to prepare teas

Mentha arvensis L

Tummy ache, bloated tummy, indigestion, sinusitis

Leaf tea by decoction or fresh consumption

Mentha pulegium L.

Abdominal pain, bloated stomach, indigestion, diarrhea

Leaf tea by decoction or infusion

Mentha villosa Huds.

Pain, postpartum colic

Leaf tea by decoction or infusion

Palmeira in Cuitegi, Paraíba (PB) [31]

Mentha piperita L.

Malaise, inflammation, amoeba, hemorrhage, stroke, high blood pressure, menstrual cramps, worm, tummy bloating, tummy ache, decrease menstrual flow, anxiety, cough, earache

Leaf tea by decoction or infusion or consumption in the form of juice (in natura)

São Benedito, em Tutóia, Maranhão (MA) [32]

Mentha arvensis L.

Sinusitis, body pain

-

Mentha villosa

Flu, stomachache, vomiting, convulsion, worm, amoeba, colic, diarrhea, gastritis

-

Comunidades Quilombolas Palmeira dos Negros e Sapé em Igreja Nova, Alagoas (AL) [29]

Comunidade do Catu, em Canguaretama, Rio Grande do Norte (RN) [30]

(Continued)

130  Essential Oils Table 6.1  Species of the Mentha genus used by traditional Brazilian peoples and communities. (Continued) Indications/ Pathologies

Community

Scientific name

São Luís, Maranhão (MA) [33]

Mentha piperita L.

Analgesic, liver, against seasickness

-

Mentha sylvestris Mentha arvensis L.

Catarrh, flu, cough, colds

-

Mentha Sylvestris

Flu, colic and stomach pains, fever and cough

Leaves ingested in the form of tea, licker or in natura (juice)

Mentha spicata L.

Flu, pain in general, cough and hoarseness

Leaves ingested in the form of tea or licker

Mentha piperita L.

Diseases associated with the digestive tract

Leaves in the form of tea

Mentha suaveolens L.

Diseases associated with the digestive tract; infectious and parasitic diseases

Leaves ingested in the form of tea or licker

Mentha aquatica L.

Integumentary system diseases

Leaves in the form of tea

Mentha spicata L.

Diseases associated with the digestive tract and respiratory system

Leaves in the form of tea or juice

Mentha x villosa Huds.

Pain, headache, fever, worms; intestinal gases, bellyache, expectorant, bronchodilator, flu; cough, sinusitis, nausea, vomiting, breaking, evil eye

Tea (infusion), syrup and tincture from the leaves and flowers

Mentha arvensis L.

Headache, flu, colds, asthma, tiredness in the chest

Leaves ingested as teas or syrups

Caxias, Maranhão (MA) [34]

Traditional community of the settlement Pedra Suada, in Cachoeira Grande, Maranhão (MA) [35]

Quilombo Tiningú in Santarém, Pará (PA) (CARVALHO; OLIVEIRA, 2015) [36]

Preparation and use

(Continued)

Mentha sp. Essential Oil and Its Applicability in Brazil  131 Table 6.1  Species of the Mentha genus used by traditional Brazilian peoples and communities. (Continued) Community

Scientific name

Indications/ Pathologies

Preparation and use

Itaituba, Pará (PA) [37]

Mentha spicata L.

Colic, stomachache, gas, expectorant

Tea or syrup from leaves

Quilombola Community TaueráAçu in Abaetetuba, Pará (PA) [38]

Mentha pulegium L

Wind pain; headache; dentition; diarrhea; Flu; Bellyache

Tea from leaf branches or fresh consumption

Boa Vista, Roraima (RR) [39]

Mentha spicata

Flu; analgesic

Leaves in the form of tea

Mentha piperita L

Worm

Leaves in the form of tea

Amazon Riverside Communities in Santo Afonso and Santa Luzia, Amazonas (AM) [40]

Mentha villosa

Abdominal pain, worm, colic, headache and soothing

Leaves in the form of tea

Mentha piperita

Gas

Mentha piperita var. citrata

Child colic, amoeba and diarrhea, digestive, soothing stomach pain; colic and soothing for children

Rural communities of Itacoatiara in Itacoatiara, Amazonas (AM) [41]

Mentha villosa Huds

Throat

Leaves in the form of tea

Mentha pulegium L.

Child’s disease

Leaves in the form of tea

Mentha arvensis L.

Sinusitis

Leaves in the form of tea or in natura

Mentha X villosa Huds.

Gastrointestinal problems

Leaves in the form of tea

Mentha X piperita L.

Analgesic, digestive, soothing and anti-inflammatory

Mentha pulegium L.

Cold, cough, bronchitis, stomach ache

Quilombo Mata Cavalo in Nossa Livramento, Mato Grosso (MT) [42]

(Continued)

132  Essential Oils Table 6.1  Species of the Mentha genus used by traditional Brazilian peoples and communities. (Continued) Indications/ Pathologies

Community

Scientific name

Preparation and use

Quilombola Community of Piracanjuba, in Piracanjuba, Goiás (GO) [43]

Mentha canadensis L.

Flu and asthma

Infusion of leaves for tea preparation

Mentha pulegium L.

Heal navel and jaundice

Infusion of leaves for tea preparation

Mentha spicata L.

Soothing; gases

Leaf tea by infusion, maceration and in syrup

Traditional communities (quilombolas and indigenous) of Araçuaí, Minas Gerais (MG) [44]

Mentha arvensis L

Bronchitis

The leaves are used to prepare syrup

Mentha spicata L.

Fever, Flu and Cough

The leaves are used to treat fever in the form of tea by decoction. For flu, the leaves are infused and for the treatment of cough, the complete plant is used in decoction

Quilombola communities in Sapê do Norte, Espírito Santo (ES) [45]

Mentha arvensis

Headache, gas

Tea from leaves and flowers by decoction and infusion for inhalation or ingestion

Mentha viridis

Cefaleia e intestino

Tea from leaves and flowers by decoction (Continued)

Mentha sp. Essential Oil and Its Applicability in Brazil  133 Table 6.1  Species of the Mentha genus used by traditional Brazilian peoples and communities. (Continued) Indications/ Pathologies

Community

Scientific name

Preparation and use

São Paulo, São Paulo (SP) [46]

Mentha sativa

Use as an expectorant, in fevers, flu, rash diseases, in sore throat and eyes (external use), to improve digestion, bitter taste in the mouth, reduce digestive gas, colic and fat intolerance

-

Mentha piperita

Healing (external), analgesic, intestinal antiparasitic, diarrhea, bronchitis, stomach pain, cough, as a sedative. Appetite stimulant for children. For expelling intestinal parasites

Leaves ingested in the form of juices, macerated in water, teas by infusion or decoction, or fresh (in natura)

Mentha pulegium L.

Edible as a condiment. For expelling intestinal parasites (mainly Ascaris lumbricoides, Entamoeba hystolitica and Giardia lamblia), kidney stones, fever, flu, bronchitis, stomach ache, use in oral affections, coughing, in colic and intestinal gas

Leaves ingested fresh or infused teas

Mentha spicata L.

Cough, abortifacient, flu, expulsion of intestinal parasites (mainly Ascaris lumbricoides), as analgesic

Leaves ingested in the form of tea by infusion or decoction, syrup

Mentha arvensis

Diseases associated with the respiratory system

Leaves and branches ingested in the form of teas (Continued)

134  Essential Oils Table 6.1  Species of the Mentha genus used by traditional Brazilian peoples and communities. (Continued) Indications/ Pathologies

Community

Scientific name

Preparation and use

Sertão do Ribeirão in Florianópolis, Santa Catarina (SC) [47]

Mentha pulegium L.

flu, cold, cold, cough, phlegm, sinusitis, bronchitis, asthma, throat, hoarseness

-

Mentha sp

worms, chilblains, ainpingi (fungi), scabies, Measles, Malaria, Bowel stomach, stomach pain, stomach ache, diarrhea, gastritis, nausea, induce vomiting, gas, poor digestion, congestion, stomach burning, purgative, laxative

-

Some species deserve special attention, as there are some ethnobotanical studies that demonstrated several claims for use, being the oil of M. piperita an important example. This species is used in different regions of Brazil (North and Northeast) to treat influenza [23, 24]. In addition, in these regions there are reports about its use to treat colic [24, 37], headache [23], gastrointestinal problems (TGI) [24, 30, 37, 40], as analgesic [38], against worms [24, 40], among others claims (Table 6.1). Due to the similarities between the claims presented by studies, it is important to validate these activities and their toxic potential [48, 49]. A very interesting study of M. piperita oil, carried out in a community from Paraíba, Brazil, found the claim of anti-hemorrhagic properties, reduction of menstrual flow and blood pressure and for the treatment of cerebrovascular accident (Table 6.1) [31]. Currently, several drugs are available for the treatment of cardiovascular and hemorrhagic problems [26, 31], However, the search for low cost therapeutic alternatives, with high activity and low toxic potential is still important [50]. Plants with claims of popular use are a promising source of new active molecules [51]. Another species, M. arvensis, has some popular use very similar to M. piperita. The oil of M. arvensis is used in the treatment of gastrointestinal problems [30], influenza [36], inflammatory diseases [41], respiratory diseases [46], among others (Table 6.1). This may be related to the similarities between the oils, as it is common for oils from different species of the same genus to have chemical similarities [12] and this may justify similar therapeutic actions [52]. Quilombola communities in Pará, Goiás and Bahia use M. pulegium oil, however, the claims are not always similar, as examples are: navel cure, jaundice treatment [43], teething

Mentha sp. Essential Oil and Its Applicability in Brazil  135 [38] and asthma [36]. Also, there are similarities between the use of the two species mentioned above as for the treatment of gastrointestinal problems [26], flu [26, 36], inflammatory diseases [42], etc. (Table 6.1). Once again, the oil obtained from Mentha has as its alleged uses the treatment of flu [36, 44], gastrointestinal problems [42], and inflammatory diseases [42]. The oil obtained from M. spicata seems to be the most widely used for medicinal purposes in Brazil. Its use was registered in the states of Pará, Roraima, Maranhão, Minas Gerais, Goiás and São Paulo [28, 37, 39, 43, 44, 46]. None of the Mentha species is endemic throughout Brazil, it is concentrated in only a few States, in the case of M. spicata, it can be found in 18 Federative Units (Amazonas, Pará, Roraima, Alagoas, Bahia, Ceará, Pernambuco, Piauí, Sergipe, Distrito Federal, Goiás, Espírito Santo, Minas Gerais, Rio de Janeiro, São Paulo, Paraná, Rio Grande do Sul and Santa Catarina) and together with M. suaveolens are the ones with the smallest distribution and consequently a smaller report of popular use [9]. For the use of M. spicata oil, we observed that in two or more states there was a report, highlighting the use of this oil to treat gastrointestinal problems [35, 37] respiratory diseases [28, 35], flu [46] and as analgesic (Table 6.1) [39, 46]. It is quite clear that, regardless the species and the location in Brazil, some allegations of use appear with high frequency. Used by Quilombola communities in Alagoas and Ceará, rural communities in Amazonas and also in São Paulo, different from previous studies, the allegations of the use of M. villosa oil is very diverse and not always confluent, with its use being reported for the treatment of headache [25], flu [36], sore throat [41], worms [40] and gastrointestinal problems [40]. However, when comparing the use of this oil with the oils from other species, the points of similarity of popular medicinal use are clear. Although different species are reported in different regions of the national territory, all the species of Mentha analyzed here have common points in terms of popular use, highlighting flu and respiratory diseases (asthma, cough, chest congestion), digestive problems, headaches, intestinal colic, as antiparasitic and anthelmintic. It is important to carry out studies that validate the popular use of oils, since in all cases the fresh leaves are used to prepare a tea or a syrups (licker), which guarantee the chemical constituents of essential oils.

6.2 Chemical Constituents of Mentha Oil The peppermint essential oil is a liquid substance, light dense and bright, ranging from colorless to pale yellow, with a very characteristic and refreshing odor [53]. It can be obtained in several ways, highlighting the steam drag as the most widespread technique. When evaluating the chemical constituents of the most used species in Brazil, there are several differences between the major chemical compounds, as shown in Table 6.2. The chemical characterization of five species of Mentha showed that the major compounds for M. x villosa are: carvone (51.20%) and limonene (18.98%); for M. arvensis: menthol (83.91%) and menthone (3.41%); for M. citrata: linalyl acetate (35.32%) and linalool (44.01%); for M. spicata: peperitenone oxide (55.30%) and piperitone oxide (17.38%), and for M. piperita: pulgenone (25.70%) and menthone (24.65%), also a study found concentrations of menthofuran (23.31%), menthol (6.68%), linalyl acetate (4.49%) and carvone (3.75%) [54].

136  Essential Oils Table 6.2  Characterized compounds from Mentha sp. Species/Reference M. piperita

M. spicata

M. pulegium

M. suaveolens

[15]

[16]

[17]

[18]

[17]

[19]

[20]

[21]

Compound

Area %

α-Pirene

0.73

-

0.32

0.51

0.51

0.4

0.36

0.56

Sabinene

0.22

-

0.33

0.14

0.64

0.1

0.53

0.26

β-Pirene

0.72

2.08

0.61

0.69

0.89

0.2

0.65

0.76

β-Myrcene

0.40

1.22

-

2.08

-

-

-

0.43

Limonene

1.85

-

6.13

14.34

4.29

0.9

-

3.10

Eucalyptol

0.20

-

-

-

-

-

-

-

Linalool

0.17

0.39

0.21

-

-

-

-

1.37

Isopulegol

1.35

-

-

-

-

-

-

-

Menthone

9.48

24.56

-

-

19.24

-

-

-

Isomenthone

8.36

-

-

-

6.09

0.5

-

-

Menthol

67.98

36.02

-

-

0.30

13.4

-

-

α-Terpineol

0.46

-

0.16

0.07

-

-

0.71

-

Pulegone

0.40

1.35

0.22

-

38.81

70.4

-

46.52

2-Hexenyl isovalerate

0.49

-

-

-

-

-

-

-

Piperitone

0.85

-

-

-

6.35

0.2

-

18.29

Menthyl acetate

2.37

8.95

-

-

-

0.1

0.08

-

β-Bourbonene

0.49

-

2.80

1.18

0.056

-

-

-

β-Caryophyllene

0.58

-

2.96

1.76

0.16

0.5

1.68

-

Germacrene

0.69

-

-

-

-

-

-

-

β-Germacrene

0.11

-

-

-

-

-

-

-

β-Phellandrene

-

1.52

-

-

-

-

-

-

1.8-cineole

-

5.13

-

-

0.06

-

-

1.91

Terpinolene

-

2.02

0.098

-

0.06

-

-

0.09

Menthofuran

-

6.88

-

-

-

-

-

0.08

Trans-carveol

-

1.69

-

-

-

-

-

(Continued)

Mentha sp. Essential Oil and Its Applicability in Brazil  137 Table 6.2  Characterized compounds from Mentha sp. (Continued) Species/Reference M. piperita

M. spicata

M. pulegium

M. suaveolens

[15]

[16]

[17]

[18]

[17]

[19]

[20]

[21]

Compound

Area %

Cis-carveol

-

3.49

1.18

-

-

-

-

-

Cubenol

-

0.56

-

-

-

-

-

-

Spathulenol

-

0.10

-

-

-

-

-

-

Eugenol

-

0.30

-

-

-

-

-

-

Carvone

-

2.30

59.40

67.08

-

-

-

-

β-elemene

-

1.30

0.84

0.38

-

-

0.16

-

Oct-len-3-ol

-

-

0.12

-

-

-

-

0.49

Myrcene

-

-

0.38

-

0.21

-

-

-

Octan-3-ol

-

-

0.30

-

0.85

0.7

-

-

1.8-cineole

-

-

3.80

-

-

-

-

-

(Z)-β-ocimene

-

-

0.33

0.11

-

-

-

1.08

(E)-β-ocimene

-

-

0.12

0.03

-

-

-

0.13

Y-terpinene

-

-

0.36

-

0.05

-

-

0.05

Cis-hydrate de sabinene

-

-

0.97

-

-

-

-

0.03

Delta-terpineol

-

-

0.20

-

-

-

-

-

Trans-dihydrocarvone

-

-

1.55

1.03

-

-

-

-

Neoiso-dihydro carveol

-

-

0.22

0.79

-

-

-

-

Acetate dedihydroiso carveol

-

-

0.37

0.06

-

-

-

-

Piperitenone

-

-

0.14

-

16.52

0.2

1.17

18.29

Piperitenone oxide

-

-

-

-

-

74.69

-

Acetate de cis carvyle

-

-

0.61

-

-

-

-

-

(Z)-jasmone

-

-

0.63

-

-

-

-

-

β-copaene

-

-

0.35

0.09

-

-

-

-

α-cadinol

-

-

0.47

-

-

-

-

-

Oxyde de caryophyllene

-

-

0.65

-

0.18

-

-

(Continued)

138  Essential Oils Table 6.2  Characterized compounds from Mentha sp. (Continued) Species/Reference M. piperita

M. spicata

M. pulegium

M. suaveolens

[15]

[16]

[17]

[18]

[17]

[19]

[20]

[21]

Compound

Area %

Spatulenol

-

-

0.66

-

-

-

-

-

Octan-3-one

-

-

-

-

0.66

0.3

-

-

Para cymene

-

-

-

-

0.07

-

0.13

-

Isopulegone

-

-

-

-

0.55

1.0

-

2.89

α-humulene

-

-

-

0.63

0.59

1.0

-

-

Neo Menthyl acetate

-

-

-

-

-

3.5

-

-

Santolinyl acetate

-

-

-

0.34

-

-

-

-

α-Gurjunene

-

-

-

0.21

-

-

-

-

β-Gurjunene

-

-

-

0.19

-

-

-

-

Muurolene

-

-

-

2.29

-

-

0.09

-

α-Mentha-1(7),8-diene

-

-

-

-

-

-

0.02

-

α-Terpinene

-

-

-

-

-

-

0.13

0.15

Nopinone

-

-

-

-

-

-

0.05

-

Borneol

-

-

-

-

-

-

0.29

0.95

p-Cymen-8-ol

-

-

-

-

-

-

0.12

-

Daucene

-

-

-

-

-

-

0.11

-

Gama-Muurolene

-

-

-

-

-

-

5.53

-

Gama-Amorphene

-

-

-

-

-

-

0.30

-

Camphene

-

-

-

-

-

-

-

0.28

Cis-cis-p-menthenolide

-

-

-

-

-

-

-

12.23

Menthol is an organic compound made synthetically or obtained from peppermint or mint oils with local anesthetic and flavoring properties. When added to pharmaceuticals and foods, menthol works as a tonic for peppermint flavors. It also has a counter-irritant effect on the skin and mucous membranes, thus producing an analgesic or local anesthetic effect [55]. Carvone is a p-menthane monoterpenoid consisting of cyclohex-2-enone with methyl and isopropenyl substituents at the 2 and 5 positions, respectively. It has the function of an

Mentha sp. Essential Oil and Its Applicability in Brazil  139 allergen, it is a member of carvones and a botanical antifungal agent [56]. The limonene is a natural cyclic monoterpene, and the (4R)-limoneme is an optically active form of limonene with (4R) configuration. It is a plant metabolite, enantiomer of a (4S)-limonene [57]. Linalool is an octa-1,6-diene monoterpenoid substituted by methyl groups at positions 3 and 7, and a hydroxyl group at position 3. It was isolated from plants such as Ocimum canum. It has the function of a vegetable metabolite, volatile oil component, antimicrobial agent and fragrance. It is a tertiary alcohol and monoterpenoid [58]. Mentone is a cyclohexanone substituted by a methyl group and an isopropyl group at the 5 and 2 positions, respectively (the 2R, 5S stereoisomer). It is a monoterpenoid, enantiomer of a (-) - mentone [59]. Mint acetate is a natural terpenoid that contributes to the smell and taste of peppermint. It is the menthol acetate ester. Menthyl acetate constitutes 3 to 5% of the volatile oil of mentha piperita, contributing to its smell and flavor [60, 61]. Eucalyptol is a naturally produced cyclic ether and monoterpenoid. Eucalyptol is an ingredient in many brands of mouthwashes and cough suppressants. It controls airway mucus hypersecretion and asthma through the inhibition of anti-inflammatory cytokines. Eucalyptol is an effective treatment for non-purulent rhinosinusitis. Eucalyptol reduces inflammation and pain when applied topically. It kills leukemia cells in vitro. Mentha piperita has menthol (Figure 6.1-1) and menthone (Figure 6.1-2) as major oil composition and perhaps they are responsible for its biological activity in the most reported popular use: for respiratory diseases. In an in vitro study on rat tracheal rings, the oil was able to relax the tracheal muscles showing antispasmodic activity in the rat trachea with involvement of prostaglandins and nitric oxide synthase, which had their concentrations increased in the presence of the essential oil of Mentha piperita [62]. For M. spicata, its major components were limonene (Figure 6.1-4), β-Caryophyllene (Figure 6.1-14) and Carvone (Figure 6.1-4). The most popular use for this species includes the treatment of fever, respiratory diseases and as antispasmodic. Biological activities related to these popular uses were not found in the literature, only studies about its antimicrobial and antioxidant activities, therefore, studies are needed to validate the popular use. From M. pulegium were characterized as majority compounds menthone (Figure 6.1-2, pulegone (Figure 6.1-5) and piperitenone (Figure 6.1-11). The popular use for this species includes the treatment for flu, stomach pain, ringworm, cough, stomach problems, diarrhea and headaches (Table 6.1). About the biological activities, there is an absence of studies that justify the popular use, highlighting also antimicrobial and antioxidant activities. The main chemical compounds found in M. suaveolens were puleone (Figure 6.1-5), menthyl acetate (Figure 6.1-8), piperitenone (Figure 6.1-12) and piperitenone oxide (Figure 6.1-13). The popular use of this species includes diseases of the nervous and digestive system and parasitic diseases. Once again, the absence of biological activities that ratify popular use is verified. Therefore, it is crucial for all species to carry out studies about biological activities that validate popular use.

6.3 Evaluation of Biological Activities of Mentha Essential Oils Based on ethnobotanical studies of different oils obtained from Mentha, it seems to be interesting to evaluate the activities for different gastrointestinal problems, respiratory

140  Essential Oils diseases including flu and different inflammatory diseases. However, most of these assessments require the use of animals and therefore a considerable amount of oil is required. This is a limiting factor for carrying out these studies, as oil yields are reduced, ranging from 0.348% in summer to 0.177% in winter [63]. As a result, most studies with these oils are performed in in vitro models, which provide preliminary results. A problem with this type of model is the assessment of activity only, or cytotoxicity, with no criteria being observed for what should be considered active or cytotoxic. In this context, a flowchart to guide the process of evaluating the biological activities of Mentha and other oils is providing in Figure 6.2. The choice of the in vitro model for preliminary evaluation is very important. There are several in vitro models available, with different sensitivities and specific goals. Models using enzymes with cyclooxygenase, lipoxygenase and other mediators of inflammation may be suitable for screening for anti-inflammatory activity (Figure 6.2) [64]. For respiratory problems, in vitro methodologies can be used. The ex-vivo tissues of the respiratory tract, for example, can be used to assess the potential for contraction and relaxation. Also, the model of cough induced by citric acid in Japanese quail are useful to determine mucociliary transport velocity [65]. To assess cytotoxicity, the ideal is to use normal strains from different embryological and tumor origins. Due to the high cost, liver strains have been used, especially HepG2, and fibroblasts, manly in cell viability assays (MTT or sulphorodamine assay). For genotoxicity, classical methods such as the micronucleus and Ames test are still recommended. After this screening protocol, only the most promising oil will be submitted to in vivo studies, reducing the number of animals, as well as making it necessary to obtain a greater quantity of only the promising sample(s). In order to defend against pathogens, the host activates signaling pathways that lead to the production of reactive oxygen species (ROS): superoxide (O-2), hydrogen peroxide (H2O2) and hydroxyl radicals (• OH). However, excessive or uncontrolled production of

Obtaining the oil Oil characterization Evaluation of in vitro biological activity Enzymes, cells, etc.: IC50

Cytotoxicity assessment (cell viability assay): use at least 2 cell lines - CC50

Criterion: consider active oil with IC50 < 10μg/mL

Consider oil cytotoxic with CC50 10

Genotoxicity and Mutagenicity: Micronucleus and Ames Test - Select non-mutagenic sample Obtaining a greater amount of the selected oil and in vivo studies of acute oral toxicity and activity

Figure 6.2  Flowchart for the evaluation of the biological activities of Mentha oils; Self drawn.

Mentha sp. Essential Oil and Its Applicability in Brazil  141 ROS can cause cell damage and has been implicated in a variety of pathological conditions (Silva, Cerchiaro & Honório, 2011 https://doi.org/10.1590/S0100-40422011000200024), among these we can mention: emphysema, bronchopulmonary dysplasia, pneumoconiosis, toxicity by bleomycin, paraquat, butylhydroxytoluene, mineral fibers and tobacco, asthma and coronary heart disease [66]. Products of natural origin, such as Mentha vegetable oils, may contain antioxidant agents, such as vitamins C, E and A [67], phenolic compounds capable of restricting the propagation of reactions and injuries caused by free radicals [64–66]. The benefits of these compounds are their ability to scavenge free radicals [67]. In a DPPH (2,2-diphenyl-1-picrylhydrazyl) assay performed with M. spicate, the essential oil had results for antioxidant activity, being inactive in one study (IC50 of 3450 ± 172.5μg/ mL) [68] and active in other studies Egypt (81.66, 77.40 and 63.80 μg/mL) [69–71]. These divergences between the studies and activities may be related to several factors such as the time of collection of the plant material and the place where the plant was collected. These parameters can influence, in qualitative and quantitative terms, the chemical composition of the oil, impacting on the activity (Table 6.2) [72]. The M. spicata essential oil exhibited only 6% inhibition of DPPH radicals, this is due to the turbidity observed at higher concentrations, which led to the use of a concentration 10 times lower than planned. Contrasting results with the ABTS method (2,2-azinobis 3-ethylbenzothiazoline-6-sulfonic acid) were observed, where it was found 53.2% inhibition [73]. The different results can be explained by differences in the relationships between the radical and the antioxidant [74]. For M. rotundifolia, the antioxidant activity in the DPPH assay resulted in IC50= 26.11 and 29.52 μg/mL, collected from two different locations [75], the antioxidant capacity of this species is superior to other Mentha species such as M. spicata. A study carried out with M. pulegium showed IC50=321.41 μg/mL in the DPPH method, while for the FRAP method (iron-reducing antioxidant power) the IC50 was 58.27 μg/mL, demonstrating considerable antioxidant activity [76]. Thus, the antioxidant effect of these oils can be justified by the popular use, due to the oxidative stress responsible for lung diseases such as asthma, chronic obstructive pulmonary disease, obstructive sleep apnea and acute respiratory distress syndrome [66]. Several in vitro studies have evaluated the activity of oils obtained from different species of Mentha for the following activities: antibacterial, antifungal, antioxidant, trypanocide, larvicidal, antiproliferative, antiviral and for the treatment of asthma. In addition, antiinflammatory potential and repellent capacity were evaluated as well as. Some studies evaluated the antibacterial activity, through the agar diffusion method, of M. piperita oil in different species of bacteria. This oil was active in gram positive species, such as Staphylococcus epidermidis, Staphylococcus aureus and MRSA. Also, a significant inhibitory effect was observed against gram negative bacteria (Table 6.3). The major limitation of the studies was the use of a qualitative methodology: the agar diffusion test. It is important to use a quantitative method to allow the determination of IC50 using methods as the microdilution test. Some studies relate the IC50 range to antimicrobial activity, allowing us to suggest whether the sample is active or inactive. In a in vivo study with rats infected with Helicobacter pylori, the activity of M. piperita was compared to Amoxicillin. The histological examination of the stomach mucosa of

142  Essential Oils infected rats showed the essential oil reached 80% of H. pylori eradication (Table 6.3). In this study, the authors concluded this oil had high anti-Helicobacter pylori activity [77]. In addition, the antifungal activity of M. piperita oil was evaluated, showing potential against Candida albicans, and it seems to be promising for dermatophyte fungi, as Fusarium moniliforme, Aspergillus niger, and Aspergillus fumigatus (Table 6.3). Table 6.3  Biological antimicrobial activities of Mentha sp. Species

Methods

Active in species (MIC e IC50)

Reference

M. piperita

Broth microdilution method

Bacillus cereus, Bacillus subtilis, Staphylococcus aureus e MRSA, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa

[15]

M. piperita

Agar diffusion assay

Staphylococcus aureus, Micrococcus flavus, Bacillus subtilis, Staphylococcus epidermidis, Salmonella enteritidis

[16]

M. piperita

-

Fusarium moniliforme, Aspergillus niger e Aspergillus fumigatus

[51]

M. piperita

In vivo

Helicobacter pylori: atividade similar a amoxicilina

[77]

M. piperita

Agar diffusion assay

S. mutans

[78]

M. piperita

Broth microdilution method

Candida albicans MIC= 0.6 mg/mL

[79]

M. piperita

Poisoned food technique

Fungos dermatófitos

[80]

M.piperita

Virus-infected HepG2 cell cultures

Ethanol extract from leaves containing high levels of phenolic acid and flavonoid: active in RSV (Respiratory Syncytial Virus) and significantly decreased the production of NO, TNF-α, IL-6 and PGE2 in RAW 264.7 cells stimulated by lipopolysaccharide

[81]

M. arvensis

Agar diffusion assay

S. mutans

[78]

M. arvensis

Broth microdilution method

Candida albicans MIC= 1.1 mg/mL

[79]

(Continued)

Mentha sp. Essential Oil and Its Applicability in Brazil  143 Table 6.3  Biological antimicrobial activities of Mentha sp. (Continued) Species

Methods

Active in species (MIC e IC50)

Reference

M. arvensis

Broth microdilution method

Pseudomonas fluorescens: 0.567 mg/mL inibiu 100%

[82]

M. arvensis

Broth microdilution method

S. agalactiae MIC= 0.018 mg/mL

[83]

M. arvensis

Agar diffusion assay

Candida albicans: efeito sinérgico with cetoconazol

[83]

M. pulegium

Agar diffusion assay

P. mirabilis

[76]

M. pulengium

Broth microdilution method

Candida albicans MIC= 0.74 mg/mL

[79]

M. spicata

Broth microdilution method

Vibrio spp MIC= 0.023 mg/mL

[89]

M. spicata

Broth microdilution method

Candida albicans MIC= > 2.0 mg/mL

[79]

M. spicata

Antibiofilm activity

Inhibit biofilm formation of methicillinresistant Staphylococcus aureus (MRSA)

[80]

M. spicata

Poisoned food technique

Dermatophyte fungi

[80]

M. spicata

agar diffusion assay

Diminuição da formação de biofilme em Vibrio spp.

[84]

M. rotundifolia

Trypanocidal in vitro activity

Trypanosoma cruzi epimastigotes- IC50 = 5 μg/Ml

[85]

Essential oils from Mentha species also showed trypanocidal activity, especially M. rotundifolia, which exhibited 50% inhibition of Trypanosoma cruzi epimastigote growth at a concentration of 5 μg/Ml. In this same study, the major compound of M. rotundifolia, identified as piperitenone oxide, showed activity inhibiting growth in 80% at a concentration of 100 μg/Ml. Similar result was found for the essential oil of M. rotundifolia (85.3% in 100 μg/Ml) [85]. The antiproliferative activity of M. spicata was tested in breast (MCF-7 and T47D) and colon (HCT-116) cancer strains. The essential oil concentrations that reduced survival by 50% (LD50) were 324 ± 81, 279 ± 52 and 975 ± 156 μg/Ml for T47D, HCT-116 and MCF-7.

144  Essential Oils The action observed in the study is related to the antioxidant effect of M. spicata essential oil, which would neutralize free radicals in these cell lines [68]. The antiproliferative activity against MDA-MB-231 breast carcinoma and A375 human melanoma strains was also evaluated and at the highest concentration, 150 μ g/Ml, the tested extracts had a weak effect [86]. A study revealed the interesting potential of M. arvensis essential oil in the treatment of asthma, in which it was possible to observe an increase between the pre-convulsion period possibly caused by smooth muscles relaxation of the bronchi, as well as a decrease in inflammatory cells [87]. In addition to biological activities, essential oils can be allies in the control of certain diseases when in the formulation of repellants, representing safe alternatives and less harmful to the environment than repellents with toxic compounds [88]. Contributing to this idea, a study evaluated the repelling action of M. arvensis essential oil, which showed dosedependent activity and protection time of 165 minutes at 100% oil concentration. Also, this study showed that only 25% of the oil with 5% vanillin extended the repellent activity, increasing from 45 to 120 minutes, so even when using a lower concentration of the essential oil, this combination generated a very interesting protection time [89]. Other studies observed the larvicidal effect of the essential oil of M. arvensis, M. piperita and M. spicata against Aedes aegypti, in which the respective LC50 values were 78.1 ppm [46], 98.7 ppm [47] and 56.1 ppm [90]. In aromatherapy, the essential oil of mint is widely used to increase confidence, inhibit negative thoughts, fear and selfishness. However, some side effects can happen if it is used in large amounts in children and infants, with dyspnea and asphyxia as the main problems [91].

6.4 Toxicity of Essential Oils from Mentha Used in Folk Medicine M. piperita essential oil was preselected to assess neurotransmitter receptor binding and acetylcholinesterase inhibition in a double-blind, placebo-controlled, balanced crossover study. For this, 24 participants (mean age: 25.2 years) consumed single doses of placebo encapsulated with 100 µl of the essential oil. This dose enhanced modulated performance in demanding cognitive tasks and attenuated the increase in mental fatigue associated with the performance of extended cognitive tasks in healthy adults. These results were correlated with the presence of high levels of menthol/menthone and cholinergic inhibitory properties, regulating calcium and binding to the GABA A/nicotinic receptor [92]. Several beneficial biological effects have been described for Mentha, associated with the widespread historical benefits of plants in flavoring ingredients, hypertension, liver disease and diabetes, however, few researchers have performed toxicological tests [93]. Thus, it is important to know the security regarding its use. Toxicity is a harmful effect that causes damage or death to the exposed environment, which may vary according to the administered dose, exposure time and frequency, in addition to depend on the route of administration to be used. Many studies with alcoholic and aqueous extracts of medicinal plants demonstrate the cytotoxic and teratogenic effects these substances can cause [48, 49]. The mediation of acute toxicity occurs through adverse effects observed within a short period of time after administration of a single dose or multiple doses within 24 hours, thus,

Mentha sp. Essential Oil and Its Applicability in Brazil  145 it is possible to evaluate the mechanism of action, identify target organs and systems and characterize the median lethal dose (LD50%) for the purposes of studies and development of medicines or inputs based on plant drugs and other compounds [110, 111]. The LD50 for Mentha is shown in Table 6.4. These relatively high LD50 values obtained for oral and intraperitoneal administration of the plant extract demonstrate the plant extract is not toxic to mice [96]. The oral LD50 of M. longifolia essential oil was found at 470 mg/kg in rats, and it is moderately toxic to oral medications [97]. The acute toxicity of aqueous leaf extract from M. spicata was determined in fasting rats and the LD50 was ˃ 1500 mg/kg non-toxic, suggesting a promising use for the treatment of diabetes [98]. In the acute toxicity investigation, there was no mortality and the LD50 in rats and mice of both sexes was greater than 2,000 mg/kg. The results indicated that the acute toxicity of M. mozaffarianii essential oil in mice and rats was of low order and revealed mild tissue damage to various organs when administered subchronically at a dose of 100 mg/kg [99]. An important point in the evaluation of substances is related to studies that investigate cytotoxicity (metabolic changes in cells that can lead to their death) and genotoxicity (induction of damage to genetic material, especially DNA). The investigation of cytotoxic and genotoxic effects of essential oils from Mentha species are of extreme importance due to the increased use in cosmetics, herbal medicines, aromatherapy and other industrial and self-use inputs [101]. Recent studies showed the antioxidant capacity of phenolic compounds found in M. pulegium, M. piperita L, M. longifolia L. against several cancer cell lines, as shown in Table 6.4 [102–104]. In a study to evaluate the cytotoxicity of M. pulegium essential oil on colon cancer cell line (HT29) by the MTT method, the results showed that as the concentration increases, cell viability decreases. In this study, the most cytotoxic concentration tested was 200 mg/ mL and the smallest was 1mg/mL [102]. The cytotoxic activities of essential oils from the leaves of four species of Mentha - M. arvensis, M. piperita, M. longifolia and M. spicata - affected by the harvest season was evaluated in breast cancer cell lines (MCF-7) and prostate cancer (LNCaP) using the MTT assay. The essential oils tested exhibited good cytotoxicity potential as shown in Table 6.5 [106]. M. spicata methanolic extract showed a good cytotoxic effect, as shown in Table 6.5, on HepG2 (Human Hepatocellular Carcinoma) and HTC-116 (Human Colon Carcinoma) evaluated by MTT, having a great antioxidant and anticancer agent potential [107]. Table 6.4  Lethal dose (LD50) of Mentha in different studies. Study

Species

LD50

Study model

[50]

Mentha piperita

4.800 mg/kg

mice

[97]

Mentha longifolia L.

470.0 mg/kg

Wistar rats (200–230 g)

[98]

Mentha spicata 

1500 mg/kg

Wistar rats (200–220g)

[99]

Mentha mozaffarianii

>2.000 mg/kg

Wistar rats (150–200 g) and mice NMRI (18–25 g)

[105]

Mentha longifólia Huds.

> 3200 mg/kg

mice

146  Essential Oils Table 6.5  Inhibitory or cytotoxic concentration that kills 50% of cells or microorganisms (50%) induced by treatment with mentha in several studies. Study

Species

IC50% (µg/mL)

Study model

[106]

Mentha longifolia

45.02 e 50.6

MCF-7

[106]

Mentha arvensis

55.3 e 59.7

MCF-7

[106]

Mentha piperita

75.2 e 80.8

MCF-7

[106]

Mentha spicata

80.0

MCF-7

[106]

Mentha longifolia

43.5 e 52.0

LN-CaP

[106]

Mentha piperita

90.4 e 95.7

LN-CaP

[106]

Mentha arvensis

50.2 e 55.7

LN-CaP

[106]

Mentha spicata

75.8 a 90.0

LN-CaP

[109]

Mentha piperita 

10.89

SPC-A1

[109]

Mentha piperita 

16.16

K562

[109]

Mentha piperita 

38.76

SGC-7901

[107]

Mentha spicata 

25.2 ± 3.6

HepG2

[107]

Mentha spicata 

62.1 ± 4.9

HTC-116

[108]

Mentha mozaffarianii

26.5 ± 6.7

MOLT-4

[108]

Mentha mozaffarianii

26.5 ± 2.3

HL-60

[108]

Mentha mozaffarianii

22.3 ± 5.4

MCF-7

The cytotoxic potential of essential oils from M. mozaffarianii were evaluated in three human cancer cell lines: breast adenocarcinoma (MCF-7), acute lymphoblastic leukemia (MOLT-4) and acute promyelocytic leukemia (HL-60) cells) using 3-(4-, 5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay (MTT). The results indicate oils obtained at different phenological stages of the plant showed relatively high cytotoxic activity, attributed to the presence of oxygenated monoterpenes with higher amounts of piperitone oxide and its derivatives [108]. The essential oil from the leaves of M. piperita showed activity against SPC-A1 human lung carcinoma cells (IC50% = 10.89), human leukemia K562 (IC50% = 16.16) and human gastric cancer SGC-7901 (IC50) % = 38.76 µg/ml). This study may provide an experimental basis for future systematic research, rational development and clinical use of peppermint resources [109]. About genotoxicity, few studies report both in vitro and in vivo activities. As an example, the study about the treatment of Swiss albino mice with aqueous extract from leaves of M. piperita resulted in a significant reduction in the number of lung adenomas, and reduced the frequency of chromosomal aberrations and micronuclei in bone marrow cells.

Mentha sp. Essential Oil and Its Applicability in Brazil  147 The results indicate that the aqueous extract from leaves of M. piperita is chemopreventive and antigenotoxic when administered after an initial dose of benzo[a]pyrene in newborn Swiss albino mice. The chemopreventive action and the antigenotoxic effects observed in this study may occur due to antioxidant properties [110]. In a study to evaluate the antigenotoxic action, infusions of M. piperita and M. pulegium were tested using the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster, where hydrogen peroxide was an inducer of genotoxicity. None of these infusions showed significant genotoxicity, but showed antigenotoxic activity, as the phenolic compounds present in these infusions can act as protectors against damage induced by free radicals [111]. However, In a another study with human lymphocytes, the oil from M. piperita (0.20 μl/ml) induced chromosomal aberration (16.0 ± 2.3%) when compared to the control (2.0 ± 0.6%), inhibited mitotic activity at a concentration of 0.10 μl/ml and induced dose-independent sister chromatid exchange [112]. Therefore, Mentha’s toxicological evaluation indicates that it is safe for consumption in low dosages. Regarding cytotoxicity, concentrations greater than 10.0 µg/mL may have great potential for antineoplastic activity, and genotoxicity may be related to some compounds present in the product obtained from the extraction, which varies with the type of product (aqueous or essential oil) requiring further studies to better understand the genotoxic potential and safety of Mentha.

6.5 Final Considerations and Perspectives Mentha oils have several claims of use for medicinal purposes, in different states and regions of Brazil, especially in the Northeastern states and some studies report their use by quilombola communities [23–25, 27, 29, 30]. This may occur due to the maintenance of medicinal culture in these communities, or it may be related to the difficulty of access to medicines, therefore, the maintenance of medicinal culture is important, as well as the access of communities to medicines [113, 114]. Among the claims of popular use stand out the treatment of gastrointestinal and respiratory problems. In addition to these activities, studies report others mainly related to menthol and its derivatives, which are some of the main constituents of essential oils of Mentha, presenting anesthetic [115, 116], anti-inflammatory activity [117], potential for gastroprotection [118] and treatment of respiratory diseases [119]. However, it is important to assess whether these activities are more pronounced when using oils or whether fractionation contribute to their expansion. Despite all claims of use, studies that evaluated biological activities are still limited and results are preliminary, since most have been performed in in vitro models. Therefore, further studies in animal models are needed to confirm the pharmacological activity and clarify the mechanisms of action, in addition to acute oral toxicity tests, acute repeated doses, subchronic and chronic and the evaluation of carcinogenic and teratogenic potential [120]. We noted that most studies focused their assessment on antimicrobial activities and not on claims of use. Some oils were active for different species of microorganisms [133, 134] and to assess the oil’s potential in infections caused by resistant multidrug bacteria. For assessing cytotoxicity, it is ideal to use non-neoplastic and tumor strains, such as the hepatocellular carcinoma strain HepG2 [123] and VERO (kidney) of normal adult African

148  Essential Oils green monkey [124] and fibroblasts such as MRC-5 (derived from normal lung tissue of a male fetus) [125], being frequently used the cell viability assay (MTT or sulphorodamine assay) [126]. In genotoxicity, classical methods such as the micronucleus and Ames test are still recommended [127, 128]. These tests follow the recommendation for biological evaluation of substances according to the OEDC [129]. After these recommendations for screening substances, only the most promising oil will be submitted to in vivo studies, reducing the number of animals in the study, as well as making it necessary to obtain a greater quantity of only the promising sample(s). A major difficulty faced in conducting in vivo studies is the low yield of oils and in this context, in vitro assays can be used as a strategy for screening and selecting the most promising oil, as it has shown better activity (IC50 100 µg/mL). After analyzing all available information, it can be suggested that Mentha piperita oil seems to be the most promising, since it has a higher lethal dose (4800.0 mg/kg), greater cytotoxicity in cancer cells (10.89 µg/mL) and it presents antimicrobial activity in a wide range of bacteria and its highest value of MIC = 9.10 µg/mL.

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154  Essential Oils 105. Tafrihi, M., Imran, M., Tufail, T., Gondal, T.A., Caruso, G., Sharma, S., Sharma, R., Atanassova, M., Atanassov, L., Valere Tsouh Fokou, P., Pezzani, R., The wonderful activities of the genus mentha: Not only antioxidant properties. Molecules, 26, 1118, 2021. 106. Hussain, A.I., Anwar, F., Nigam, P.S., Ashraf, M., Gilani, A.H., Seasonal variation in content, chemical composition and antimicrobial and cytotoxic activities of essential oils from four mentha species. J. Sci. Food Agric., 90, 1827, 2010. 107. Abdel-Hameed, E.S., Salman, M.S., Fadl, M.A., Elkhateeb, A., El-Awady, M.A., Chemical composition of hydrodistillation and solvent free microwave extraction of essential oils from Mentha piperita L. growing in taif, kingdom of saudi arabia, and their anticancer and antimicrobial activity. Orient. J. Chem., 34, 222, 2018. 108. Jassbi, A.R., Mirzaie, Y., Firuzi, O., Asadollahi, M., Composition and cytotoxic activity of the essential oils of mentha mozaffarianii jamzad at different phenological stages. Curr. Bioact. Compd., 14, 191, 2018. 109. Sun, Z., Wang, H., Wang, J., Zhou, L., Yang, P., Chemical Composition and Anti-Inflammatory, Cytotoxic and Antioxidant Activities of Essential Oil from Leaves of Mentha piperita Grown in China. PLoS One, 9, e114767, 2014. 110. Samarth, R.M., Panwar, M., Kumar, A., Modulatory effects of Mentha piperita on lung tumor incidence, genotoxicity, and oxidative stress in benzo[a]pyrene-treated swiss albino mice. Environ. Mol. Mutagen., 47, 192, 2006. 111. Romero-Jiménez, M., Campos-Sánchez, J., Analla, M., Muñoz-Serrano, A., Alonso-Moraga, A., Genotoxicity and anti-genotoxicity of some traditional medicinal herbs. Mutat. Res., 585, 147, 2005. 112. Lazutka, J.R., Mierauskiene, J., Slapsyte, G., Dedonyte, V., Genotoxicity of dill (Anethum graveolens L.), peppermint (Mentha×piperita L.) and pine (Pinus sylvestris L.) essential oils in human lymphocytes and drosophila melanogaster. Food Chem. Toxicol., 39, 485, 2001. 113. WHO global report on traditional and complementary medicine 2019, World Health Organization, Geneva, https://apps.who.int/iris/bitstream/handle/10665/312342/9789241515 436-eng.pdf?sequence=1&isAllowed=y, 2019. 114. Agra, M.F., Freitas, P.F., Barbosa-Filho, J.M., Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. Rev. Bras. Farmacogn., 17, 114, 2007. 115. Ghelardini, C., Galeotti, N., Di Cesare Mannelli, L., Mazzanti, G., Bartolini, A., Local anaesthetic activity of B-caryophyllene. Farmaco, 56, 387, 2001. 116. Watt, E.E., Betts, B.A., Kotey, F.O., Humbert, D.J., Griffith, T.N., Kelly, E.W., Veneskey, K.C., Gill, N., Rowan, K.C., Jenkins, A., Hall, A.C., Menthol shares general anesthetic activity and sites of action on the GABA(A) receptor whith the intravenosus agent, propofol. Eur. J. Pharmacol., 590, 120, 2008. 117. Rozza, A.L., Meira de Faria, F., Souza Brito, A.R., Pellizzon, C.H., The gastroprotective effect of menthol: Involvement of anti-apoptotic, antioxidant and anti-inflammatory activities. PLoS One, 9, e86686, 2014. 118. Rozza, A.L., Hiruma-Lima, C.A., Takahira, R.K., Padovani, C.R., Pellizzon, C.H., Effect of menthol in experimentally induced ulcers: Pathways of gastroprotection. Chem. Biol. Interact., 206, 272, 2013. 119. McKay, D.L. and Blumberg, J.B., A review of the bioactivity and potential health benefits of peppermint tea (Mentha piperita L.). Phytother. Res., 20, 619, 2006. 120. ANVISA, Agência nacional de vigilância sanitária. Guia para a condução de estudos não clínicos de toxicologia e segurança farmacológica necessários ao desenvolvimento de medicamentos, Gerência de Avaliação de Segurança e Eficácia – GESEF. 2º Versão, Brasília, 2013, http:// antigo.anvisa.gov.br/documents/33836/2492465/Guia+para+a+Condu%C3%A7%C3%A3o+ de+Estudos+N%C3%A3o+ Cl%C3%ADnicos+de+Toxicologia+e+Seguran% C3%A7a+

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7 Microbial Influence on Plants for Enhanced Production of Active Secondary Metabolites Naushin Bano, Mohammad Amir, S. Nabilah Jawed and Roohi* Protein Research Laboratory, Department of Bioengineering, Integral University, Lucknow, UP, India

Abstract

Secondary metabolites (SM) are not crucial for a cell’s livelihood but play a significant role in better adaptation to its environment. Secondary metabolites being antiviral, antibiotic and antifungal, can guard plants from pathogens. Spices secondary metabolites such as black pepper, cardamom, ginger, turmeric, etc. are extremely important in today’s world because they have antioxidant, anti-cancerous, antimicrobial and other medicinal properties. The secondary plant metabolites modules, namely phenolics, alkaloids, saponins, and terpenes, are briefly discussed here in connection with their applications and advancements. There are numerous instances where plants’ secondary metabolites can be used to advance various fields, including medicine, dyes, pigments, insecticides, pesticides, etc. Secondary metabolites are being made in both in vitro and in vivo tests. The influence of microbes on plants is described in the context of the enhanced manufacturing of these secondary metabolites in various stress conditions, with mechanisms of contact briefly elaborated. This chapter gives a detailed outline of the plant secondary metabolites, their classifications, secondary metabolite production and applications, and microorganisms’ influences on plants and their interactions. Keywords:  Secondary metabolites, PGPR, PGPF, microbial interactions, symbiosis, plant growth promoters

7.1 Introduction Sessile organisms such as plants can have a significant impact on conventional as well as western medicines. The phytochemical elements of plants are classified into two groups according to their function in fundamental metabolic processes: primary and secondary metabolites. Primary metabolites play a direct role in healthy implementation, reproduction, and development, while Secondary metabolites are the byproducts of secondary metabolic processes such as the shikimic acid pathway [1]. Secondary metabolites are organic compounds synthesized by an organism that is not essential to facilitate growth and life. They can’t perform primary functions like growth, photosynthesis, reproduction, and energy. Secondary metabolites are the main sponsors of specific odor, color, and taste *Corresponding author: [email protected]; ORCID: 0000-0003-4296-6105 Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (157–172) © 2023 Scrivener Publishing LLC

157

158  Essential Oils of plant parts [2]. Plant secondary metabolites are extremely valuable commercially [3]. About 50% of modern drugs are from natural products [4]. Secondary metabolites of plants have many important applications, which include antibacterial [5], antifungal [6], antiviral [2], against plants insects, and anticancer too [7]. They are used in the pharmaceutical industry as antiseptics, carminatives, stimulants, expectorants, diuretics, rubefacients, and counterirritants, etc. [8], as shown in Figure 7.1. This chapter encompasses an immense description of terpenes, phenols, nitrogen, and sulphur-containing compounds, which are examples of plant’s secondary metabolites, as well as their uses and examples. One of the essential activities that occur in the terrestrial environment is microbial interaction. The interaction of microorganisms is happening in the rhizosphere in which plants, bacteria, and fungus interact with each other and forms a symbiotic relationship. Their consequences occur on plant growth and performance [9]. This chapter is entirely dedicated to displaying the impact of bacteria and fungi on plant development and growth, biomass production, and the role of bacteria and fungi as biofertilizers, phytostimulators, biopesticides, and stress tolerance activity plants.

PHARMACEUTICALS

DYE AND PIGMENTS

ANTI TUMER

AGRO CHEMICALS

SECONDARY METABOLITE

COSMETICS

IMMUNO SUPPRESSANT

FLAVOURING AGENTS ECOLOGICAL ROLE

Figure 7.1  Applications of plants secondary metabolites.

Plant-Microbe Interaction for Secondary Metabolite Production  159

7.2 Classes of Plants Secondary Metabolites Plant’s secondary metabolites are categorized into major classes based on their chemical structures. Secondary metabolites include phenolics, terpenes, nitrogen-comprising chemicals, and sulfur-comprising compounds [10]. The secondary metabolites groups with examples are designated in Figure 7.2.

7.2.1 Terpenes Terpenes are among the major and most diversified secondary metabolite classes. They are generally polymers of 5 carbons unit called isoprenes. The majority of the different terpene plants create secondary metabolites (toxins and nutrients) to build complex structures as a defense mechanism, and these structures are thought to serve as a defense and food source for insects and mammals [3, 11]. Terpenoids are industrially the crucial aroma and flavoring compounds. Terpenoids’ potential as pharmacological agents with anticancer and antibacterial properties are still being researched [12].

7.2.2 Phenolic Compounds Phenols are the major group of secondary plant metabolites. Phenols are utilized as Coloring agents. These are synthesized by plants from phenylalanine amino acids. Secondary metabolites covering a group of phenol, a useful hydroxyl group on a fragrant ring, are formed by plants in an extensive range of methods. Phenols are used by plants to defend themselves against pests and illnesses, such as core parasitic nematodes [13]. They are the most abundant secondary plant modules produced by teas, vegetables, cocoa, fruits, and additional plants with strength benefits. These exhibit antioxidant, anti-inflammatory, and anticarcinogenic capabilities, as well as other beneficial biological qualities, and might be protective against oxidative strain and certain illnesses [14].

PLANT SECONDARY METABOLITES

Terpenes

Monoterpenes e.g., geraniol, Sesquiterpenes e.g., humulene, Diterpenes e.g., cafestol, Sesterterpenes e.g., geranylfarnesol, Triterpenes e.g., squalene, Sesquarterpenes e.g., ferrugicadiol, Tetraterpenes e.g., lycopene, Polyterpenes e.g., gutta-percha.

Phenolics

Coumarin e.g., hydroxycoumarins, Furano-coumarins e.g., psoralen, Lignin e.g., resveratol, Flavonoids e.g., quercitin, Isoflavonoids e.g., genistein, Tanins e.g., tannic acid.

N containing compounds

Alkaloids e.g., cocaine, Cyanogenic glucosides e.g., dhurrin, non-protein amino acids e.g., canavanin.

S containing compounds

Glutathione, Glucosinolates, Phytoalexins, Thionins, Defensins, Allinin

Figure 7.2  Classes of plants secondary metabolites and their examples.

160  Essential Oils

7.2.3 Nitrogen-Containing Secondary Metabolites Amino acids (non-protein), cyanogenic glucosides, and alkaloids are examples of secondary metabolites of plants that encompass nitrogen in their construction. Because of their therapeutic characteristics, they are of great interest [15]. Many alkaloids have been synthesized from mutual amino acids like tryptophan, lysine, tyrosine, and aspartic acid. These are nitrogen-containing compounds and play a self-protective role against herbivores and pathogen attacks due to their property of bitterness and toxicity. Cyanogenic glycosides are nitrogen-containing secondary metabolites established in plants that produce volatile poisons or toxins when crushed. An extensive range of plants could be originated, along with legumes, grasses, and Rosaceae family plants [16]. Non-protein amino acids are also found in secondary metabolites that contain nitrogen. Apart from the 20 amino acids that make up proteins in plants, there are around 200 amino acids that exist in free form in plant cells and are not integrated into proteins. Nonprotein amino acids are free amino acids that were not found in proteins. Their primary purpose appears to be herbivore defense. Non-protein amino acids may obstruct protein synthesis or absorption, or they may be incorrectly integrated into proteins, rendering them non-functional [17].

7.2.4 Sulphur Containing Secondary Metabolites Sulfur-containing compounds have a wide number of chemical structures and forms of action. They offer plants a flexible range of chemical roadblocks over a vast spectrum of possible competitors due to their wide variety of chemical structures [18]. Glucosinolates and Phytoalexins are the Sulphur containing secondary metabolites. Glucosinolates (GSL) are nearly exclusively found in Brassicales plants. A sulphur-linked-D-glucopyranose moiety and an amino acid-derived side sequence make up glucosinolates [19]. Jasmonic acid encourages metabolites that are conjugated and free methods of antioxidants and glucosinolates. Phytoalexins are antimicrobial compounds that have unique essentials of pathogendefeating mechanisms in plants. Plants produce phytoalexins as a reply to abiotic and biotic stressors. Since resulting a part of a complex shield mechanism that allows plants to keep invading microbes under control [20].

7.3 Secondary Metabolites Production from Plants There are many ways for the synthesis of secondary metabolites in plants, such as in vivo and in vitro methods [21]. In in vivo, a whole living organism is used, while in in vitro dead organisms or isolated cellular components are used. In vitro is performed under controlled laboratory conditions, and it is less expensive and less time-consuming than in vivo. Many biotechnological techniques for secondary metabolite synthesis from therapeutic herbs have been proposed and tested. Cell-processing screening, media manipulation, feeding of precursors, evocation, large-scale cultivation within a bioreactor system, hairy root culture, immobilization of plant cells, biotransformation are few names amongst them [22].

Plant-Microbe Interaction for Secondary Metabolite Production  161

7.3.1 In Vivo Production of Secondary Metabolites The methods of in vivo culturing are callus culture and suspension culture. The first step to establishing cell suspension culture is to raise callus from any explant part of the plant. Any plant part may act as an explant for culture technique such as pieces of healthy leaf, stem, hypocotyl, cotyledons, etc. Saad and Elshahed in 2012 [23] use explant for inducing the callus and for regeneration of the plant. The callus can be maintained for a long time for subculturing or transferring it to a fresh medium of the same composition. Some calluses are heavily lignified, hard in texture, and difficult to break, while some are fragile and can be broken easily into fragments (friable culture). The steepness of the curve indicates the rate of increase, which is highest when the population density hits half of the carrying capacity. Media components used are macronutrients, micronutrients, carbon and energy sources, vitamins and myoinositol, amino acid, growth regulators, etc. [24]. Environment, seasonal variation, insect and microbial diseases, and geographic constraints have no effect on culture systems. Because the product is made by a specific cell line, the quality will be consistent. Product recovery will be simple. Plant cultures are beneficial for plants that are difficult or expensive to grow in the field. Production time is reduced, and labor costs are kept to a minimum by using this technique [25].

7.3.2 In Vitro Secondary Metabolites Production Several medicinal plants have been reported for in vitro secondary metabolite production using plant cell suspension cultures. Transgenic hair crops have transformed tissue’s function cultivation for the generation of secondary metabolites. Secondary metabolites of A. rhizogenes produced by hairy roots are identical to those of the intact roots, with the like or greater yields of the same [26]. The symbiotic relationship and impacts of plants and endophytes on one another must be examined during the development of additional significant pharmacologically bioactive natural products such as camptothecin, vinblastine, and podophyllotoxins [27]. Recent research articles on secondary metabolites production proposed studies improving production by introducing chemical/drug alterations to cell walls or with modified quantities of the Boron as a micronutrient and a quasi-essential element. Attention was drawn to well-known medicinal plants, such as Artemisia species. Artemisia  species are shown to have antimicrobial, antineoplastic, antioxidant, antiviral, antipyretic, anticoagulant, antithrombotic, anticoagulant, antihypertensive, antithrombotic, anti-inflammatory, antimicrobial, anticonvulsant, and anti-inflammatory effects [2]. The researchers surveyed the making of caffeine and artemisinin in heterologous hosts, as well as mentioned in using glycosyltransferases as an example; biotechnological tactics used to improve the bioactivity of secondary metabolites of the plant [10].

7.4 Interaction of Microorganisms in the Rhizosphere The rhizosphere is the zone around plant roots where chemical reactions between plants, microorganisms, and soil take place. The interactions of plant roots with their microbiota are done by the secretion of a large number of metabolites of the plant into the rhizospheric zone.

162  Essential Oils Even a small plant can secrete more than hundreds of different metabolites through the root exudates [28]. There are three types of interactions that occur in the rhizosphere region, (1) Fungus and plant interactions involve the appearance of a fungal spore that emerges when it detects plant root secretions. The mycelium will then grow in the root direction, adhere to it, colonize the surface of the root, and enter it. (2) Bacterium with plant interactions, here the impact of a certain bacteria can fluctuate the soil and environmental conditions like when significant volumes of chemical nitrogen fertilizer are given to the soil, a bacterium that stimulates growth by giving plants with fixed nitrogen, gets activated and performs its function [29]. (3) the third type of interaction includes Bacterium and Fungus interactions in which some bacteria attach themselves to fungal hyphae to feed them. Their interactions are vital for the health of plants and animals and are important drivers of many ecological activities [30]. In recent years, advances in our understanding of plant-beneficial microbial interactions have commanded the creation and marketing of microbial inoculation for the purpose of enhancing plant health [9]. Inter and intraspecific microbial interactions occur in the rhizosphere. Interspecific interactions occur between creatures of different species. Intraspecific interactions, on the other hand, occur between organisms of the same species [31]. Selim and Zayed, in the year 2017, described the intraspecific and interspecific microbial interactions, with their types [32] are shown in Table 7.1. Microorganisms can also form long-term associations with each other, known as Ectosymbiosis, in which the microbes remain outside the other organism. And in Endosymbiosis, the microbes are located within another organism. The types of interactions with host and microorganisms and their characteristics were defined by Malcolm in the year 1966 [33], as shown in Table 7.2.

Table 7.1  Types of microbial interaction. Intraspecific interaction Positive Interaction

Mutualism Commensalism Protocooperation

Negative Interaction

Amensalism Parasitism Predation Competition

Neutral Interaction

Neutralism

Interspecific interaction Positive Interaction

Cooperation

Negative Interaction

Competition

Plant-Microbe Interaction for Secondary Metabolite Production  163 Table 7.2  Types of interaction between microorganisms and hosts. S. no. Name

Characteristic

Host

1

Neutralism

Neither population Not Not affected affects the other affected

The tarantulas and cacti living in a desert [34].

2

Mutualism and symbiosis

Interaction is required for survival in the habitat, but only specific species are admitted.

Interaction between protozoa and archaea in digestive tracts of some animals [35].

3

Commensalism One is benefited and other is not affected

Not Benefited affected

At the same place, facultative and obligatory anaerobes grow, facultative anaerobes help obligate anaerobes to grow by consuming the oxygen from the environment [32].

4

Amensalism or Antagonism

Products of one impact another

Not Harmed affected

Bacillus subtilis, Streptomyces sp. etc. are described as producers of drugs that inhibit microbes reduce plant infections [36].

5

Parasitism and predation

Host is usually compromised

Harmed

Benefited

Chytrids parasitize algae and other fungi by invading the host [32].

6

Synergism

Growth of one is improved by another

Benefited

Benefited

Microbial interactions with plants in the rhizosphere [37].

7

Competition

Organism in the environment attempts to acquire limiting nutrients

Harmed

Harmed

Soil bacteria and fungi, compete for nutrients [32].

Benefited

Micro-organisms Examples

Benefited

164  Essential Oils

7.5 Influence of Bacteria and Fungi on Plants Plants are extremely well adapted to a wide range of environmental surroundings, yet certain biotic and abiotic stress may reduce plant development significantly [38]. In plants, roots communicate with living organisms in the rhizosphere using a chemical signal like Nematodes, fungi, bacteria, arthropods, etc. [39]. Bacteria, fungus, actinomycetes, protozoa, and algae are amongst the many types of microorganisms found in soil, where bacteria are the most prevalent [40]. Because many soil bacteria interact with plant roots, the bacterial concentration in the rhizospheric region is substantially higher than the number of bacteria in the soil. The rest of the soil reflects the high levels of nutrients released by most plants’ roots, which are then needed to promote bacterial development and metabolism [29]. Plant growth is supported by microorganisms that collect nutrition in the soil, manufacture many regulators of plant growth, by dominating or inhibiting phytopathogens, plants can be protected, soil structure improvement, and bioremediate impure soils and degrade xenobiotic compounds, like pesticides [41].

7.5.1 Plant Growth Promoters  Some bacteria and fungi present in the soil help directly and indirectly in plant growth. These microorganisms are commonly found near the roots because of the presence of root exude, which serves as a food source for them and which they rely on for their survival [42].

7.5.1.1 Plant Growth-Promoting Bacteria (PGPR) The bacteria which help in the promotion of plant development are present in the rhizosphere, which colonized plant roots. They assist in the promotion of plant growth through mobilizing nutrients present in soil and also helps to reduce the world’s reliance on dangerous agricultural chemical compounds. These organisms offer the plant a variety of services and benefits in exchange for the plant providing carbon and other metabolites to the microbial diversity [43, 44]. Auxin biosynthesis, ACC deaminase activity, cytokinin, gibberellin biosynthesis, nitrogen fixation, phosphorus solubilization, and iron sequestration by bacteria are examples of direct processes. Indirect mechanisms are bacterial traits that prevent one or more plant pathogenic organisms, both fungi, and bacteria, from functioning. Antibiotics, cell wall degrading enzymes, competition, hydrogen cyanide, systemic resistance produced by antibiotics, quorum quenching, and siderophores are examples of indirect mechanisms [45]. The Direct and indirect mechanisms of action of plant growth-promoting Rhizobacteria are shown in Figure 7.3. Examples of plant growth-promoting rhizobacteria are Agrobacterium, Azotobacter, Bacillus, Flavobacterium, Micrococcus, Mesorhizobium, Pseudomonas, Serratia, etc.

7.5.1.2 Plant Growth-Promoting Fungi (PGPF) PGP is advantageous fungi that live in the rhizosphere of plants [46]. The term PGPF refers to the group of rhizosphere fungi that colonize plant roots and promote the growth of the plant. PGPF is a miscellaneous community of nonpathogenic fungi that advantage their

Plant-Microbe Interaction for Secondary Metabolite Production  165 Direct plant growth promotion (Biofertilizer Activity)

Nitrogen fixation Phosphate solubilization

MECHANISM OF ACTION OF PGPR

Potassium solubilization Siderophore production Phytohormone production

Cytokinin, GA

IAA

Ethylene

In-Direct plant growth promotion (Biocontrol Activity)

Antibiotic production Hydrolytic enzyme production Induced Systemic Resistance (ISR) Exopoly saccharide production

Figure 7.3  Direct and indirect mechanism of action of plant growth-promoting Rhizobacteria.

host plants in many ways. The need for plant growth-promoting fungi has been acknowledged as an environment-friendly method of increasing production of the crop. Through either a direct or indirect process, these fungi have been shown to improve crop yields by enhancing germination, seedling vigor, plant growth, photosynthesis, and flowering [47]. Chemical control will be reduced to a minimum with the usage of PGPF, and plants will be protected from biotic and abiotic challenges. Many PGPF species have been documented to augment plant growth, escalate innate immunity and other essential secondary metabolites in plants [48]. Examples of plant growth-promoting Fungi are Trichoderma, Talaromyces, Fusarium, Phytophthora, Penicillium, Rhizoctonia, Gliocladium, Phoma, etc.  

7.5.2 Production of Plant Biomass The presence of microbes for the preservation of soil function is tremendously significant since they play a key role in disintegration, nutrient cycling, elimination of toxins, and soil structure improvement. A number of investigations demonstrate that these bacteria play an important function in improving plant development with agricultural yields [49].  Plant root-associated rhizospheric bacteria can increase crop productivity by a number of direct and indirect processes. These plant growth-enhancing rhizobacteria (PGPR)

166  Essential Oils can only boost crop productivity and/or in combination with other microorganisms under stress situations. The example of bacterial species that helps in production of plant biomass is Bacillus amyloliquefaciens, Brachypodium distachyon, B. amyloliquefaciens B. cereus, Bacillus subtilis, Klebsiella variicola, Burkholderia Phytofirmans, etc. [42].  The new bioeconomy includes fungi and fungal enzymes. Filamentous fungal enzymes can unleash the potency of refractory lignocellulose structures in plant cell walls, and yeast can create bioethanol from the sugars released following enzyme treatment. An example of fungus species that help in the manufacture of plant biomass is Trichoderma reesei, Aspergillus oryzae, Aspergillus nidulans, Aspergillus niger, etc. [50].

7.5.3 Bacteria and Fungus as Biofertilizers Biofertilizer is the biological element that consists of living microbes that assist in plant development and productivity in a number of ways. Biofertilizer is a microbial substance that colonizes the rhizospheric region or inside the plant and increases the availability or supply of nutrients, hence enhancing plant development. Biofertilizers improve the texture of the soil and increase plant yield. They prevent infections from flourishing. They are environment friendly and economical too [51, 52]. Biofertilizers have the capability of nitrogen fixation, Sulphur oxidization, production of plant hormones, or decay of organic compounds. Examples of bacterial strains as biofertilizers are Pseudomonas fluorescens, Azotobacter chroococcum, A. vinelandii, Bacillus megaterium, Thiobacillus thiooxidans, Delfia acidovorans, Bacillus sp. etc. The example of fungal strains are Piriformospora indica, Epichloe coenophiala, Trichoderma viride, and some species of Fusarium, Sclerotium, Rhizoctonia, Pythium, etc. [53].

7.5.4 Role of Bacteria and Fungi as a Phytostimulator Phytostimulation directly boosts plant growth. Phytostimulators help in plant growth promotion, usually through phytohormones such as indole-3-acetic acid [54]. Different processes are used by PGPR to improve plant growth and development. The creation of plant growth regulators (PGRs), also known as phytohormones, is one of the well-known and prevalent methods used by PGPR. The PGPR employs several growths and developmentpromoting processes. The creation of plant growth regulators (PGRs), also known as phytohormones, is one of the well-known and prevalent methods used by PGPR [55]. Ascorbic acid, auxins, abscisic acid, gibberellins, and cytokinins are five of the most significant plant-growth-promoting hormones. Synthetically created PGPRs are equally suitable for encouraging plant growth, but it is more cost-effective and environmentally preferable to use microbial-derived PGPRs [56]. Example of bacterial species that helps in phytostimulation are Azospirillum brasilense, Pseudomonas fluorescens, etc. and the examples of fungal strains are Piriformospora indica, T. harzianum, Brassica campestris, etc. [57]. 

7.5.5 Role of Bacteria and Fungi as a Biopesticides The new sustainable agriculture products can be made with biopesticides. Some of the most dangerous pesticides that are now in use are likely the most probable replacements [42].

Plant-Microbe Interaction for Secondary Metabolite Production  167 The most frequent form of microbial pesticides are bacterial-dependent biopesticides that functions in different ways. Because of their broad range of usage, many farmers will use neem on their farmlands to kill insects or control plant pathogenic microorganisms. As an insecticide, they are only effective against specific species of moths, butterflies, and beetles or specific species of flies, mosquitoes, and beetles. Pest control strategies that are considered effective must make contact with the target pest and could involve ingestion. In insects, bacteria produce endotoxins that are species-specific [58]. Bacterial biopesticides include B. thuringiensis, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. subtilis, P. fluorescence, P. aeruginosa, P. syringae, Agrobacterium radiobacter, Streptomyces lydicus, Coniothyrium minitans, etc. [42]. Insect and plant diseases by fungi, bacteria, nematodes, and weeds can be controlled with fungal biopesticides. In terms of the mode of action, the pesticide fungus and the target pest have varying effects. The eco-friendly, affordable, and effective qualities of biocontrol agents like Trichoderma are used to eliminate the harmful effects of pesticides [59]. These fungal biopesticides, which are effective against plant infections such as Rhizoctonia, Pythium, Fusarium, and other soil-borne pathogens, include T. harzianum, which blocks the growth of Rhizoctonia, Pythium, Fusarium, etc. Trichoderma viride has proven to be effective against plant parasite fungi that occur in the soil [60].

7.5.6 Stress Tolerant Activity of Bacteria and Fungi Plants are exposed to two forms of environmental stress: (1) abiotic stress and (2) biotic stress. Radiations, salts, floods, drought, temperature extremes, heavy metals, and other abiotic stresses contribute to the destruction of significant crop plants around the world. Biological stress, on either side, comprises threats by pathogens such as fungi, nematodes, bacteria, and herbivores [61]. Dryness, saltiness, heavy metals, temperature variations, and ultraviolet (UV) radiation are all abiotic stressors that inhibit crop yield, resulting in a drop in total crop productivity [62]. The rhizosphere/endo-rhizosphere colonized by growth-promoting rhizobacteria protects the roots from abiotic stress and supports plant growth through a variety of direct and indirect strategies. Endophytic microorganisms, such as bacteria and fungus, live within healthy plant tissues and aid plant growth in stressful situations. Pressures from the environment such as drought or flooding affect plant growth in arid and semiarid areas by affecting a number of physiological systems. These pressures can be biotic (pathogen and disease stress) or abiotic (salinity, dryness, temperature, and heavy metal stress). Stress causes a variety of physiological problems in plants, including nutritional deficiency, hormonal imbalance, an increase in the concentration of specific osmolytes, and greater ethylene levels [63]. Various bacteria from different genera, such as Achromobacter, Methylobacterium, Bacillus, Pantoea, Paenibacillus, Pseudomonas, Variovorax, Burkholderia, Azospirillum, Microbacterium, Rhizobium, and Enterobacter, have been reported in the last decade to provide tolerance to host plants under various environmental conditions [64]. Mucor, Microsphaeropsis, Phoma, Peyronellaea, Alternaria, Aspergillus, Steganosporium, and other fungal pathogens are amongst them [47]. 

168  Essential Oils

Conclusion and Future Perspectives The synthesis of valuable natural products has stimulated improvement in synthetic biology. Many of the obstacles are overcome by microbial synthesis obstructing traditional chemical synthesis and plant metabolic engineering. It is anticipated that biotechnology is the most influential tool which would stimulate the production of novel medications from plant sources in a much quicker and more controlled manner. It will help to increase the number of drugs in the pipeline of discovery and development. The discovery of drugs with rhizospheric microbes are the leading topic of research at the present time. The literature shows that the microbes which are present in the rhizosphere are helpful for plants as well as they have a lot of industrial applications. Microbes interact with plants in many ways, which are briefly described in this book chapter. Because of the exudates produced by their roots, plants are the primary cause of the majority of these interactions. They have a substantial impact on plant development, as well as on ecological fitness, plant resilience to a variety of biotic and abiotic stresses in soils, among other things. The microbial interaction in rhizosphere helps in plant growth, and plant helps microorganisms symbiotically to provide them nutrition. The present article throws light on the production and application of secondary metabolites from plants and the influence of bacteria and fungi on plants. The field application of these plant growth-promoting bacteria and fungi are limited, despite the fact that numerous studies have demonstrated the effectiveness of PGPR and PGPF for increasing plant growth and development. It is possible that this is related to a lack of awareness as well as a lack of accessibility to methodologies, sources, and applications. It will take considerable thought and investigation to create useful applications for this topic.

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Plant-Microbe Interaction for Secondary Metabolite Production  169 8. Zachariah, T.J. and Leela, N.K., Spices: Secondary metabolites and medicinal properties, in: Indian Spices, pp. 277–316, Springer, New York, 2018. 9. Guerriero, G., Berni, R., Munoz-Sanchez, J.A., Apone, F., Abdel-Salam, E.M., Qahtan, A.A., Alatar, A.A., Cantini, C., Cai, G., Hausman, J.F., Siddiqui, K.S., Production of plant secondary metabolites: Examples, tips and suggestions for biotechnologists. Genes, 9, 309, 2018. 10. Jamwal, K., Bhattacharya, S., Puri, S., Plant growth regulator mediated consequences of secondary metabolites in medicinal plants. J. Appl. Res. Med. Aromat. Plants, 9, 26, 2018. 11. Gershenzon, J. and Croteau, R., Terpenoids, in: Herbivores: Their Interactions with Secondary Plant Metabolites, Vol. 1: The Chemical Participants, second edition, pp. 165–219, Academic Press, United States, 1992. 12. Desmedt, W., Mangelinckx, S., Kyndt, T., Vanholme, B., A phytochemical perspective on plant defense against nematodes. Front. Plant Sci., 11, 602079, 2020. 13. Shahidi, F. and Ambigaipalan, P., Phenolics and polyphenolics in foods, beverages and spices: Antioxidant activity and health effects - A review. J. Funct. Foods, 18, 820, 2015. 14. Jan, R., Asaf, S., Numan, M., Kim, K.M., Plant secondary metabolite biosynthesis and transcriptional regulation in response to biotic and abiotic stress conditions. Agronomy, 11, 31, 2021. 15. Hyde, K.D., Xu, J., Raptor, S., Jeewon, R., Luong, S., Niego, A.G.T., Abeywickrama, P.D., Aluthmuhandiram, J.V., Brahamanage, R.S., Brooks, S., Chaiyasen, A., The amazing potential of fungi: 50 ways we can exploit fungi industrially. Fungal Divers., 97, 136, 2019. 16. Puri, S.K., Habbu, P.V., Kulkarni, P.V., Kulkarni, V.H., Nitrogen-containing secondary metabolites from endophytes of medicinal plants and their biological/pharmacological activities -a review. Syst. Rev. Pharm., 9, 22, 2018. 17. Burow, M., Wittstock, U., Gershenzon, J., Sulfur-containing secondary metabolites and their role in plant defense, in: Sulfur Metabolism in Phototrophic Organisms, pp. 201–222, Springer, New York, 2008. 18. Nguyen, V.P., Stewart, J., Lopez, M., Ioannou, I., Allais, F., Glucosinolates: Natural occurrence, biosynthesis, accessibility, isolation, structures, and biological activities. Molecules, 25, 4537, 2020. 19. Jeantet, P., Phytoalexins: Current progress and future prospects. Molecules, 20, 2770, 2015. 20. Khan, T., Khan, M.A., Karam, K., Ullah, N., Mashwani, Z.U.R., Nachman, A., Plant in vitro culture technologies; A promise into factories of secondary metabolites against COVID-19. Front. Plant Sci., 12, 610194, 2021. 21. Sharrar, A.M., Crits-Christoph, A., Méheust, R., Diamond, S., Starr, E.P., Banfield, J.F., Bacterial secondary metabolite biosynthetic potential in soil varies with phylum, depth, and vegetation type. MBio, 11, 00416, 2020. 22. Fehér, A., Callus, dedifferentiation, totipotency, somatic embryogenesis: What these terms mean in the era of molecular plant biology. Front. Plant Sci., 10, 536, 2019. 23. Saad, A.I.M. and Elshahed, A.M., Plant tissue culture media preparation. Recent Adv. Plant Vitr. Cult., 2, 29, 2012. 24. Isah, T., Stress and defense responses in plant secondary metabolites production. J. Biol. Res., 52, 0716, 2019. 25. DiCosmo, F. and Misawa, M., Plant cell and tissue culture: Alternatives for metabolite production. Biotechnol. Adv., 13, 425, 1995. 26. Hussain, M.S., Fareed, S., Ansari, S., Rahman, M.A., Ahmad, I.Z., Saeed, M., Current approaches toward production of secondary plant metabolites. J. Pharm. Bioallied Sci., 4, 10, 2012. 27. Fiorilli, V., Catoni, M., Lanfranco, L., Zabet, N.R., Interactions of plants with bacteria and fungi: Molecular and epigenetic plasticity of the host. Front. Plant Sci., 11, 274, 2020.

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8 Valorization of Limonene Over Acid Solid Catalysts José E. Castanheiro

*

MED - Instituto Mediterrâneo para a Agricultura, Ambiente e Desenvolvimento, Chemistry Department, Universidade de Évora, Portugal

Abstract

Limonene has generally been recuperated from orange peels. It is an essential oil with different appli­ cations. The limonene is a compound with high importance on biorefineries. Limonene’s commer­ cial value can be improved by different reactions, like alkoxylation, acetoxylation, and hydration reactions. The products of these reactions have high commercial value. Usually, these reactions are performed using inorganic acids, like sulfuric acid. However, these acids have some drawbacks, such as its reutilization and its separation from reaction mixture. To overcome these problems, solid materials (heterogeneous catalysts) have been developed to replace the mineral acids (homogeneous catalysts). Zeolites, heteropolyacids, acid resins and silica can be used as material for limonene val­ orization. This work is a revision about the valorization of limonene using heterogeneous materials. Keywords:  Essential oil, limonene, heterogeneous catalysts, fragrances

8.1 Introduction From the waste of the citrus industry, it is obtained as an essential oil. The main compound (about 68–98% w/w) is limonene [1]. The limonene has been used in different applications such as, a perfume, and solvent [1–3]. The limonene is a very important compound on biorefineries. However, the limonene molecule can be valorized by different reactions, such as alkoxy­ lation, hydration and acetoxylation. The alkoxylation of limonene is a reaction between limonene and an alcohol (methanol, ethanol, propan-1-ol and butan-1-ol) in presence of a catalyst. Figure 8.1 shows the limonene alkoxylation reaction. However, it is also obtained other terpenes products, by isomerization of limonene and other ethers. Also, the limonene can be converted into alpha-terpineol by hydration of the alkene using an acid catalyst. Figure 8.2 shows the scheme of limonene hydration. It can be also obtained byproducts from isomerization of limonene. Another reaction to improve the commercial value of the limonene is the acetoxylation of limonene with acetic acid. Figure 8.3 shows the scheme of limonene acetoxylation. In this reaction, it can be observed the formation of isomerization products. Email: [email protected]

*

Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (173–184) © 2023 Scrivener Publishing LLC

173

174  Essential Oils

R-OH

O-R

Figure 8.1  Alkoxylation reaction of limonene (R is an alkyl group of the alcohol).

H2O

OH

Figure 8.2  Scheme of reaction between limonene and water.

Valorization of Limonene  175

O

OH

O

O

Figure 8.3  Scheme of the limonene acetoxylation.

Another important limonene reaction is the conversion of this olefin on p-cymene. Limonene reaction to p-cymene is shown on Figure 8.4. It can be also formed other compounds. The products of these reactions can be used in pharmaceutical, cosmetic industry, as fragrance and perfume. Traditionally, these reactions have been performed using homoge­ neous catalysts, like sulfuric acid. However, this process has some problems. The separation of the catalyst from the reaction mixture is difficult and the effluent must be treated [4]. However, the homogenous catalysts should be replaced by solid catalysts (heterogeneous catalysts). By this change in the process, these reactions become more environmentally friendly. The heterogeneous catalysts can be easily separated from the reaction mixture, and it can be reused [4]. This chapter focuses on different reactions to valorization the limonene.

+

Figure 8.4  Scheme of limonene transformation into p-cymene.

H2

176  Essential Oils

8.2 Limonene Reactions with Alcohols Zeolites, like Y, ZSM-5 and mordenite, have in its structure tetrahedra of AlO4- and SiO4. These materials exhibit a high-level surface area, a structure crystalline, with micropo­ rous, which have a pore with regular size. Also, the zeolite materials show selectivity to the reactants, selectivity to products and selectivity to the transition state. They have acid-base character. Zeolites have been used, as heterogenous catalysts in different processes [5–15]. Also, these materials have been used to convert compound obtained from biomass to yield chemicals and biofuels, which are an alternative to the fossil sources [16–18]. Limonene reactions using methanol, ethanol, propanol, butanol, and pentanol was per­ formed over zeolites (beta, H-USY or H-mordenite) as catalysts [19]. The main product was an ether. These reactions were carried out using different reactors (batch and continuous). The authors observed that yield to ether was 85% (limonene conversion: 93% and selectiv­ ity: 92%), using methanol as alcohol and with both reactors. Also, the authors observed that some zeolites like H-USY showed low limonene conversion. In this study, it was observed that when the reaction temperature changed from 25°C to 40°C, the limonene conversion also changed from 40% to 90%. However, the selectivity to ether was almost 95% [19]. The limonene alkoxylation with methanol and isopropanol was carried out using beta zeolite, as catalyst [20]. After 2 hours, the yield to ether is 87.5% at selectivity 89.7%. After the reaction, beta zeolite was regenerated by calcination at 500°C. Other solid acid materials have been used as catalysts in a reactor [20]. Heteropolyacids (HPAs) are materials that have used as catalysts in different reactions. These materials have some advantages, such as, are strong acid catalysts (near superacid region), are oxidants (can be used redox reactions). Due these properties, the heteropolyac­ ids can be used in acid and oxidation reaction. These materials can also be immobilized on silica, activated carbons, alumina, zeolites, to improve the surface area [21–23]. The reaction of limonene with alcohols (methanol, ethanol, 1-propanol and 1-butanol) to ether was performed using H3PW12O40 immobilized on silica [21]. The reactions were carried out with 30 cm3 of alcohol, 0.5 cm3 of limonene, and 1.0 g of H3PW12O40 immo­ bilized on silica at 60°C. The material activity reduces when the number of carbons of the alcohols used increase. A possible explanation is the presence of some internal diffusion and/or sterical hindrance. The selectivity to the main product (α-terpinyl alkyl ether) decreased when the number of carbon atoms of alcohol increased (65% (1-butanol) 20,000 mg/L; SI = 33), and replication (EC50 = 2400 mg/L; CC50 > 20,000 mg/L; SI = 8.3). As per authors, Table 11.4  Plants/terpenes and their derivatives tested against virus infected models and their mode of action. Type of virus/ experimental model

Inhibitory concentration (IC50)

Selective Index (SI) Mode of action

Glycyrrhizin

SARS-CoV infected Vero cells

600mg/L 300mg/L 2400mg/L

>33 >67 >8.3

[143] Inhibition of absorption, penetration and replication of SARS-CoV with an increase of NO synthase activity

18β-Glycyrrhetin acid

SARS-CoV infected Vero cells

>20µM

1

Anti SARS-CoV activity by neutralization test

[144]

3β-Fridelanol

Hepatitis B virus/ 88.5µM HepG2.2.15 cells

-

Inhibit 50% of HBeAg secretion

[145]

Hornstedtia bella Škorničk

vaccinia virus (VV)

-

-

[146]

Plant/terpene

-

Reference

(Continued)

246  Essential Oils Table 11.4  Plants/terpenes and their derivatives tested against virus infected models and their mode of action. (Continued) Type of virus/ experimental model

Inhibitory concentration (IC50)

Selective Index (SI) Mode of action

Reference

Eucalyptus globulus bicostata

Coxsackie virus B3

0.7mg/mL

22.8

Intracellular mechanism of action

[147]

Maxican oregano (Lippia graveolens)

Bovine viral diarrhea virus

78µg/mL

7.2

Intracellular mechanism of action

[114]

Betulinic acid

Hepatitis C virus/Ava5 cells

40µM

-

Reduces hepatitis [119] C virus replication by suppressing the expression of COX-2

Glycyrrhizin

Dengue virus-2/ Vero E6 cells

8.1µM

-

Inhibited virus cytopathic effect and infectivity by 50%

Betulinic acid

Influenza A (PR/8-virus)/ A549 cells

50µM

-

Attenuated [121] necrosis, number of inflammatory cells and pulmonary edema induced by virus

Glycyrrhizin

Varicella-zoster virus/human embryonic fibroblast

0.71mM

-

Reduced the number of loci by 50%

Plant/terpene

[127]

[124]

HSV: Human herpes Viruses, IFV: Influenza virus, SARS CoV: Severe Acute Respiratory Syndrome Coronavirus

the anti-CoV activity of glycyrrhizin is related with the induction of nitrous oxide synthase, leading to inhibit the SARS-CoV replication in Vero cells and an increase of the intracellular levels of nitric oxide [123] and also glycyrrhizin shows antiviral activity against varicella-­zoster virus [124], herpes simplex virus [125], hepatitis C virus [126], and dengue virus [127]. With an IC50 value of 120 g/mL and a selectivity index (SI) of 4.16 for Laurus nobilis, this EO had the capability to inhibit the replication of CoV-1 and showed inhibitory

Chemical Composition of Essential Oils  247 activity against SARS and HSV-1. Laurus nobilis oil showed activity against CoV-SARS. The major parts of this oil were 1, 8-cineol, β-pinene and α-pinene (Table 11.4) [128]. An EO’s antiviral selectivity index (SI), is a proportion of its healing efficacy, it can be measured as the proportion of CC50 - 50% cytotoxic concentration to IC50 - 50% infection concentration [129]. To describe anti-viral ability in natural products, the amount of IC50 ought to be >100µg/mL for complexes and for pure compounds it should be 25 µM [130]. SI value should be in > 4 [131].

11.4.3 Essential Oil for Anti-Viral Activity In recent years, EO of several plant extracts has antiviral properties. Different viruses, including the human pathogen herpes simplex, have been demonstrated to be very sensitive to the inhibitory movement of EO. These outcomes support the utilization of EO from medicinal plants as specialists for the treatment of viral infections and propose the use of this type of natural products such as antiviral drugs [132]. In general, the effects of all antivirals are dose-dependent. For example, the EO of E. cinerea and E. maidenii showed an antiviral activity at a concentration of 150 mg/mL. This activity was dose-dependent, when decrease of EO concentration the antiviral activity also decreases [133]. The EOs extracted from clove and oregano has shown significant antiviral efficacy towards on different types of viruses like coxsackie virus B-1, polio virus and adeno virus type-3 [134, 135]. EO of thyme, eucalyptus and tea, and their mono terpenes composition has been identified by in vitro study and it shows anti-viral activity against HSV-1. For HSV-1the essential oils and the monoterpenes shows inhibition against the virus of 96% and 80% respectively [136]. EOs isolated from the Artemisia douglasiana and Eupatorium patens shows effective anti-virus activity against dengue virus [137] for Junin virus the EOs of Lippia junelliana and Lippia turbinate showing potent antiviral activity. The EO components of Pogostemoncablin shows effective anti-viral efficacy against H2N2 influenza-A virus [138]. The essential oils extracted from Colombian MAPs like Ocimum campechianum, Hyptismutabilis, Lepechinia vulcanicola, Minthostachysmollis and Lepechinia salviifolia, have anti-viral activity against the type-1 and type-2 herpes virus [139] and also the early stages of viral infections are inhibited by EOs. From all these benefits, the plant based EOs may be suitable for developing antiviral agents against different viral infections in humans. They may also replace some of the widely used antiviral drugs. As per researchers, Isoborneol have anti-viral activity against the HSV but the antiviral properties of terpenes, inhibition of the viral replication cycle and their modes of actions are still in research condition [140]. The Eucalyptus essential oil has significant antiviral activity against HSV-1 and HSV-2 herpes simplex viruses (Table 11.5) [141]. The healing properties of garlic oil have been used for thousands of years to treat various ailments, such as influenza and colds. By using GC-MS to analyze garlic oil, 18 compounds were identified. The major constituents are allyl methyl trisulphide, dial tetracycline, allyl (e)-1-propane disulphide, allyl disulphide and allyl trisulphide. Of the 18 constituents, 17 virus major proteins Mpro/6LU7 and SARS-CoV-2 were examined for activity against ACE-2 protein. It is an enzyme that causes the virus to invade the Mpro and host cell helps in the virus response. The seventeen constituents were examined and all are showed

248  Essential Oils Table 11.5  Plants and their main components for anti-virus activity. Plants

Viruses

Typical

Major components

Reference

Laurus nobilis L.

SARS-CoV

RNA enveloped virus

1,8-cineole, α-terpinyl acetate, Sabinene, and α-pinene terpinen-4-ol

[148]

Lipia junelliana

Junin virus

RNA enveloped virus

Piperitenone oxide, limonene, Myrcenone, and ρ-Cymene, α-pinene

[149, 150]

Eucalyptus maidenii F

Junin virus

RNA enveloped virus

1,8-cineole, α-pinene, β-phellandrene, ρ-Cymene and Limonene

[151]

Pinus mugo Turra

HSV-1/ HSV-2

RNA enveloped virus

Α-terpineol, (E)-caryophyllene, α-cadinol dehydroabietal

[152, 153]

Terminalia ivorensis A. Chev

E7, E19

RNA enveloped virus

δ-3-carene, α-pinene, β- caryophyllene, Cedrol, and terpinolene

[154, 155]

Eucalyptus bicostatamaidin, Blakely and Simmonds

Coxsakie virus B3

RNA enveloped virus

1,3-cineole, α-pinene, α-terpineol, Globulol, and aroma dendrene

[156]

significant interactions with ACE-2 and Mpro thus disclosed active power of garlic oil to evaluate Coronavirus [142].

11.5 Anti-Fungal A fungal infection generally results from a eukaryotic organism, which makes its detection and appropriate therapeutic treatment more complex than its bacterial counterpart. The chitin structure in the cell wall of fungi makes it the prime target for selectively toxic antifungal agents. Human cells are devoid of chitin. Chemical treatments are available and highly effective, but these species and strains are resistant to them. Fungal infections vary in severity and intensity based on the host’s immune status, inoculum charge, and resistance. EOs are the most promising natural products for inhibiting fungal growth [157, 158]. Plant pathogenic fungi cause a number of plant diseases that contribute considerably to overall agricultural yield reductions [159]. The plant fungal pathogens are generally classified into two classes: biotrophic pathogens that interact closely with plants and living tissues (biotrophs) and non-biotrophic pathogens that do not interact with plants and necrotrophic pathogens, this type of pathogens, kill the tissue to extract nutrients (necrotrophs) [160].

Chemical Composition of Essential Oils  249 For example, B. cinerea may spoil up to 235 plants widespread over the world; it lead to grey mold [161] as well as M. fructicola can infects Rosaceae family plant members worldwide, causing brown rot [162]. At the post-harvest stage, the fruits and plants are affected by the fungal diseases. The effects of these phytopathogens are responsible for the changes in shapes, odor, taste, and less nutritional values. It leads to the reduction of the commercial value of these products [163]. For the losses of agriculture, the farmers decided to use the chemical treatments to control the fungus spread, but it causes toxic effects on living species and environment and also its increases the production costs. The researchers used essential oils to avoid these effects. Also it had excellent resistant against fungus [164]. The antifungal efficacy of EO mostly depends on the properties of terpenes and terpenoids. The antifungal effects of EOs mostly trust on the properties of terpenes and terpenoids. As a result of their highly lipophilic nature and low molecular weight, so its capability to disrupt the cell wall, they can cause the death of cells as well as reduce the development of fungi responsible for food spoilage. EO and its components are used against a wide range of plant pathogens. A wide variety of flora pathogens have been shown to be successfully eliminated by the EOs isolated from a few plants species like citrus, rosemary, oregano, lemongrass, thyme, basil, and fennel [165]. Ultee and Smid [166] reported the oregano and thyme essential oils are act as a best inhibitor of fungal pathogens, because it contains carvacrol and thymol as a main constituent as well as its destroy the fungal cell membranes. Researchers have recently shown that Eugenol (from clove) causes a stable injury to C. albicans cells and has been shown to be a cost-effective antifungal agent.

11.5.1 Mode of Action The EOs penetrates the fungal cell membrane and destroys protoplasmic cell walls through a permeabilization method, which triggers the destruction of the mitochondrial cell membranes [167]. Iminazole is highly toxic to fungus and these cell membranes are packed with unsaturated fatty acids; the interplay of these compounds leads to the loss of cell viability. In flora and micro-organism cells, EOs inhibits the production of RNA, proteins, polysaccharides, and DNA [168, 169]. Ketoconazole is a commercial drug for the treatment of antifungal infections caused by Trichophyton. For using this drug gives side-effects of stomach pain, skin irritation, nausea and its harmful limits controls the therapeutic application in some cases and the therapeutic reactions can be little slow, and this therapy is inappropriate for some patients with acute or quickly progressing mycoses, and ketoconazole has low efficacy in patients with immunological disorders or those with infectious disorders. EOs are a good resource for developing effective antifungal drugs; despite their low rate of absorption from the human intestine and consequently powerful antifungal properties, these compounds could ultimately be limited in their medicinal uses in the treatment of common plant diseases [170].

11.5.2 Essential Oil for Anti-Fungal Activity Antifungal activities of rosemary EO were studied and it was active against Fusarium oxysporum [172, 173], F. proliferatum [172] and F. verticillioides [171], and also the antifungal activities tested against Aspergillus oryzae, and Mucor pusillus [173], Alternaria alternata [174] and Botrytis cinerea [174]. However, the sensitivity of corresponding EOs is different.

250  Essential Oils For example, at 20 µ/mL Asp. niger shows 93% inhibition [173], at 600 µg/mL inhibition of F. verticillioides was 67% [171]. Elsewhere, the expansion of B. cinerea was fully hindered by rosemary EO at 25.6µg/mL [174]. Antifungal activity of EOs from Thymus species has been the most commonly investigated. Research has shown that EOs derived from Thymus species can enhance the activities. This is due to its high carvacrol content and thymol content. The EO possesses strong antifungal properties against Asp. parasiticus [180], Asp. flavus [176, 178], Asp. niger [177– 179] and Asp. carbonarius [176]. At last, Botrytis cinerea [181], and F. solani [177] were also investigated on Thymus species. The final results of these studies showed some differences in threshold values for minimum inhibitory concentrations (MICs) (Table 11.1). From the literature study, there have been many studies on clove essential oil, and the clove EO have the main constituent of Eugenol its shows the potential antifungal activity [182]. However, there is some inhibition of Asp. niger was done at 200 µL/L [179], at 100 µL/L, the conidial growth of Asp. flavus, the rate of inhibition was 87% [183]. The potency of clove EO shows active antifungal efficacy against Rhizopus nigricans [186], Asp. oryzae, Penicillium citrinum, Aspe. ochraceus [184] and Fusarium oxysporum [185], as well as the clove EO shows anti-fungal efficacy to secure gray molds in some fruits like pomegranate and strawberries [179, 187]. The EO isolated from cumin has a broad spectrum of antifungal activity. It is most popular spices because of its strong characteristic flavor [188]. It shows antifungal property against Botrytis cinerea [194], Penicillium spp. [192, 193] and Aspergillus spp. [189–191]. According to Montenegro et al. [195], Mentha pulegium EOs and their major compounds offered antifungal activity against M. fructicola and B. cinerea in potato dextrose agar (PDA) growth [196]. An essential oil was identified as having twelve components: 50.11% terpenyl ketones, 1.81% terpenes, 46.89% terpenyl alcohols and derivatives, 0.37% linear alcohols, and 0.82% other components. The estimation of Mentha pulegium EO against fruit diseases is accountable for a significant portion of postharvest losses. They disclose that EO and its major constituents were generally hindering the growth of plants. As a result, Mentha pulegium EO, some terpenes have to inhibit infectious diseases in plants it is confirmed by their inhibitory properties. Generally, Mentha pulegium oil appeared to inhibit microfungi growth in the same way as EOs used previously, which reported that the inhibitory effect of EOs increases with the main component: -OH > -CHO > =O > R-O-R’ [197]. Moumni et al. investigated the in vitro studies of seven EOs and their activity against the two fungi like A. alternata and S. cucurbitacearum. The most of these fungi are seedborne, like gummy stem blight (with foliar symptoms) and black rot (with fruit symptoms), which are produced by Stagonosporopsis cucurbitacearum, and which represent serious diseases that are a significant requirement to cucurbit production around the world as well as it responsible for less cucurbit production [198–200] and the genus Alternaria infects the plant seedlings, leaves, stems, flowers and fruit. By using GC/MS study more than 41 compounds were isolated from the seven different essential oils (lemongrass, lavender, Lavandin, Tea tree, Bay laurel, Marjoram). In vitro study for the antifungal efficiency of these seven EOs on mycelial growth of two fungi showed that the lemongrass EO was the most effective. The mycelial growth of A. alternata was completely inhibited by application of lemongrass at a moderate concentration,

Chemical Composition of Essential Oils  251 Table 11.6  Essential oils for anti-fungal activity. Test method used

Inhibition concentration

Reference

Asp. parasiticus, Asp. Flavus, Penicillium expansum, Penicillium citrinum, Asp. ochraceus, F. moniliforme, F. graminearum,

Macrodilution

1.25–2.5 µL/mL

[202]

Carum carvi L.

F. oxysporum, F. verticillioides, Asp. fumigatus

Agar

1–3.6 µL/mL

[203]

Carum spp. (caraway)

Asp. ochraceus

Macrodilution

0.625 L/mL

[189]

Cinnamon sp. (cinnamon)

Aspergillus ochraceus

Macrodilution

0.078 L/mL

[189]

Fusarium verticilloides

Semisolid agar antifungal susceptibility technique

60 L/L

[204]

Cinnamon sp. (cinnamon)

Clodosporium spp., Penicillium spp.

Disc diffusion

100% inhibition at 20 L

[205]

Citrus limonia

Asp. fumigatus, Botrytis cinerea,

Microdilution

312 to >2500 g/mL

[206]

Mentha suaveolens

Candida albicans

Agar diffusion method

-

[207]

Hypericum hyssopifolium and Hypericum Heterophyllum

Fusarium species

Agar diffusion method

100 µL

[208]

Rosmarinus ocinalis L. (rosemary)

Asp. Niger, Asp. Flavus

Broth macrodilution

1 L/mL

[209]

Rosmarinus ocinalis (rosemary)

Botrytis cinerea, Penicillium expansum

Broth microdilution

2500–5000 g/mL

[181]

Rosmarinus ocinalis (rosemary)

Alternaria alternate

Microdilution

1000 g/mL

[175]

Plant/EO

Test culture used

Anacyclus valentinus

(Continued)

252  Essential Oils Table 11.6  Essential oils for anti-fungal activity. (Continued) Test method used

Inhibition concentration

Reference

Fusarium verticillioides

Microdilution

150 g/mL

[171]

Rosmarinus ocinalis L.

Botrytis cinerea

Volatile phase assay

100% inhibition at 1.6 g/mL air

[174]

Rosmarinus ocinalis

Aspergillus niger

Microdilution

1000 g/mL

[210]

Rosmarinus ocinalis

Aspergillus flavus

Macrodilution

500 g/mL

[211]

Thymus algeriensis

Aspergillus niger, Fusarium soloni -

-

1–2 L/mL

[177]

Thymus daenensis

Aspergillus flavus, Alternaria alternata, Fusarium oxysporum

Broth microdilution

1–4 g/mL

[212]

Thymus capitatus

Asp. parasiticus, F. moniliforme

Disc diffusion

77.7–91.2% inhibition at 500 L/L

[180]

Thymus broussonnetii subs. hannonis

Penicillium italicum, G. citri-aurantii, Penicillium digitatum

Agar dilution

>4000 L/L

[212]

Zingiber ocinale

Asp. flavus, Asp. fumigates, Asp. niger, Penicillium citrinum, F. verticillioides, Asp. ochraceus, Penicillium chrysogenum

Micro dilution

1250–2500 g/mL

[192]

Plant/EO

Test culture used

Rosmarinus ocinalis (rosemary)

while S. cucurbitacearum was fully inhibited at the higher lemongrass concentration, with fungicidal activity seen in both cases. In lemongrass essential oil contains citral, -myrcene, and geraniol as main constituents it’s controlled these fungi most effectively as well as the marjoram essential oil containing terpinen-4-ol or linalool shows good antifungal activity (Table 11.6) [201].

Chemical Composition of Essential Oils  253

11.6 Antidiabetic High levels of oxidative stress often facilitate diabetes development; pancreatic beta-cells are particularly susceptible to ROS, resulting in decreased insulin excretion and elevated blood sugar levels [213]. A common symptom of type-2 diabetes is high blood glucose levels, which induce the generation of ROS. ROS attack pancreatic beta-cells, resulting in the insufficiency of beta-cells and insulin resistance. A rise in blood sugar levels impairs beta-cell function and causes a decrease in insulin secretion. This process ultimately leads to hypertension, diabetic nephropathy, neuropathy, and cardiovascular diseases. The EOs are extracted from the two plants namely Afromomum melegueta and Afromomum danielli and they have 38 constituents, it was confirmed by using GC/MS analysis. The EOs and their components have a significant difference in inhibitory activity (p0.5mm) and LMV (large unilamellar vesicles; >100 nm) [18]. The liposomes are smart choice as nanocarriers ascribing to improved pharmacokinetic, bio-efficacy, stability and release profile [16]. Past few decades reported that encapsulation of essential oils into liposomes is gaining great interest of food and pharmaceutical scientist crediting to augmented physicochemical stability (sensitivity towards light, oxygen, temperature, and volatility) bioavailability and aqueous solubility [19, 20]. The various methods employed for preparation of liposomes are thin film hydration (with/without sonication), modified thin film hydration method, freeze-thaw method, RESS technique (rapid expansion of supercritical solution) and PGSS drying of emulsions (precipitation from gas saturated solution) as shown in Table 12.1. Table 12.1  Various techniques for encapsulating essential oils.

S. no.

Essential oil

Encapsulation method/carrier used

Inferences

Reference

Liposomes 1

Clove essential oil

Liposomal encapsulation

Improved stability

[24]

2

Atractylodes macrocephala Koidz

Modified RESS technique (rapid expansion of supercritical solutions)

The modified RESS yields stable narrow size vesicles liposomes that can efficiently incorporates essential oil

[25]

3

Eucalyptus citriodora

Solid nanoliposomes encapsulation

Stable liposomes with average 266.56 nm size were formed, having 22.47% entrapment efficiency and controlled release.

[26]

4

Lavandin

Thin film Hydration

Yields stable multivesicular liposomes having 0.4-1.3 μm size and entrapment efficiency of 66%.

[27]

(Continued)

272  Essential Oils Table 12.1  Various techniques for encapsulating essential oils. (Continued)

S. no.

Essential oil

Encapsulation method/carrier used

5

Rose essential oil

Inferences

Reference

Supercritical process

Liposomes having double-layered vesicles of size 2 years

[40]

Polymeric nanoparticles 11

Turmeric and lemongrass

Alginate and chitosan

Enhanced antiproliferative property and stability

[44]

12

Rosemary

Zein and polylactic acid

Controlled release and utility as natural pesticide

[45]

(Continued)

Stability and Efficacy of Essential Oils  273 Table 12.1  Various techniques for encapsulating essential oils. (Continued)

S. no.

Essential oil

13

Sweet orange and bergamot

14

Lavandula angustifolia

Encapsulation method/carrier used

Inferences

Reference

Eudragit® RS 100

Enhanced antimicrobial activity of encapsulated oils and improved shelf life of orange juice

[46]

Whey protein based polymer

Enhanced thermal stability, release and antibacterial activity

[47]

Nanofibers 15

Chrysanthemum

Chitosan nanofibers

Improved antibacterial and antioxidant properties

[55]

16

Moringa oil

Gelatin nanofibers

Improved antibacterial activity over 4 days

[56]

17

Peppermint and chamomile

Gelatin nanofibers

Enhanced bioactivities and stability

[57]

18

Tea tree

Plasma treated poly(ethylene oxide) nanofibers

99.9% antibacterial activity against E. coli. over a period of 7 days and extended shelf life.

[58]

19

Cinnamon

β-CD complexed polylactide nanofibers

Enhanced antimicrobial activity and stability encapsulated oils

[59]

20

Eugenol

β-CD complexed polymeric nanofibers

Enhanced stability of encapsulated oils

[61]

(Continued)

274  Essential Oils Table 12.1  Various techniques for encapsulating essential oils. (Continued)

S. no.

Essential oil

Encapsulation method/carrier used

Inferences

Reference

Microemulsion/Nanoemulsion 21

Cinnamon, oregano and rosemary essential oil

High frequency ultrasound assisted microemulsion

Stable microemulsions to deliver natural antimicrobials

[64]

22

Anise, ajowain and sea fennel

Microemulsion employing surfactant, Polysorbate 80,glycerol and ethanol

Enhanced larvicidal activity in C. quinquefasciatus, in low concentrations and no negative impact on nontarget species

[69]

23

Nisin

Reverse micelles containing essential oils of rosemary, thyme, oregano, and dittany

Improved antimicrobial property crediting to synergistic effect attributed by essential oils

[71]

24

Pimpinella L anisum Trachyspermum L, ammi Sprague and Crithmummaritimum

Microemulsion employing ethyl oleate and Polysorbate 80 as surfactant

Improved antibacterial and antifungal activity

[72]

25

Thyme oil

Essential oil loaded microemulsions/ nanoemulsions based calciumalginate biopolymer films

Enhanced antimicrobial activity

[73]

26

H. cordata

Microemulsions

Enhanced antiviral activity and stability

[75]

(Continued)

Stability and Efficacy of Essential Oils  275 Table 12.1  Various techniques for encapsulating essential oils. (Continued)

S. no.

Essential oil

Encapsulation method/carrier used

Inferences

Reference

Mesoporous silica particles 27

Carvacrol, eugenol, thymol and vanillin

Immobilization on mesoporous silica matrix

Enhanced antibacterial activity and stability

[77]

28

Cinnamon essential oil

Alginatemesoporous silica nanocomposite

Enhanced antibacterial activity and faster seed germination

[78]

29

Carvacrol, cinnamaldehyde, eugenol and thymol

MCM-41 and β-CD complex

Enhanced antifungal activity over a period of 30 days

[79]

30

Eugenol

Mesoporous silica nanoparticles loaded PHBV films

Enhanced and prolonged antimicrobial activity

[80]

Sherry et al. reported that encapsulation of essential oils into liposomes enhances the thermal stability, and there was no change in vesicle size over a period of size of the liposomes 390 days [16]. Sivropoulou et al. reported enhanced antimicrobial activity of carvacrol and thymol when encapsulated in liposomes [21]. Similarly Moghimipour et al. formulated Eucalyptus camaldulensis loaded liposomes [22]. The results suggested significantly enhanced antibacterial and antifungal activity when compared to free essential oil as well as efficiently improved its stability over an extended period of time. Another study by Wu et al. demonstrated formulation of laurel essential oil and silver nanoparticles loaded liposomes for food packaging [23]. Further, the liposomes were mixed with chitosan for polyethylene films coating to be used for packaging pork. The results demonstrated approximately 29.30% of laurel essential oil and 11.79% was released from liposomes over a period of 7 days at 25 °C which was much sustained to that of free oil. The essential oil/silver nanoparticles loaded liposomes demonstrated substantial antioxidant and antimicrobial properties. Also, the liposomes coated films maintained the quality of pork over a period of 15 days at 4 °C which was significantly higher when compared with 9 days wrapped in uncoated polyethylene films. This study accomplishes a probability of extending storage period utilizing such methods. Likewise, Sebaaly et al. encapsulated clove essential oil (eugenol) into liposomes [24]. The liposomes displayed stability over a period of 2 months at 4°C in terms of insignificant change in vesicle size, shape, encapsulation efficiency, and PDI values. The liposomes

276  Essential Oils prevent UV exposure induced degradation as well as exhibited enhanced DPPH scavenging property of essential oil (eugenol). Literatures have reported enhanced antimicrobial property of Artemisia afra, Eucalyptus globulus and Melaleuca alternifolia when encapsulated in polymeric liposomes [25]. The liposomes were coated with chitosan and a comparison of antimicrobial activity was done between encapsulated and free essential oils. The results demonstrated the encapsulated oil exhibited significant antimicrobial activity at relatively lower concentration when compared to free oils and uncoated liposomes respectively (except for Artemisiafra). Also, a synergistic to additive interactions was observed with FIC (fractional inhibitory concentration) values of 0.25–0.45, 0.26–0.52, and 0.26–0.52 for Eucalyptus globulus, Melaleuca alternifolia, and Artemisia afra, respectively. Further, it was perceived that polymeric coating played insignificant role in enhancing antimicrobial activity but significantly enhance the membrane stability, shelf life and facilitate controlled release of encapsulated essential oil. Literature reports the encapsulation of lavandin, rose, and eucalyptus citriodora essential oils into liposomes employing various techniques viz. thin film hydration, supercritical process and solid nanoliposomes encapsulation [26–28]. The above mentioned studies gives a supplementary evidence of competence of liposomes in enhancing stability and therapeutic/preservative properties of encapsulated essential oils that are of great application for food and pharmaceutical industries and enhancing commercial status of dermatocosmetic products.

12.2.2 Essential Oils Encapsulated in Cyclodextrin Complexes Cyclodextrins have attained extensive consideration as pharmaceutical excipient owing to capability to interact with poorly aqueous soluble drugs/phytopharmaceuticals that lead to an increase in their aqueous solubility [29]. The basic mechanism comprises of formation of non-covalent dynamic inclusion complexes and/or the aggregates and allied domains formation and cyclodextrins capability of forming and stabilizing supersaturated drug solutions. In addition to the above attributes, cyclodextrin has been stated as GRAS according to US FDA [30]. Recent studies have reported inclusion of essential oils into cyclodextrins that can considerably combats the limitations associated with essential oils [31, 32]. Literature has evidenced improved properties of garlic oil, cinnamon and thyme essential oils when complexed with cyclodextrins [33, 34]. Working on same perspective, Zavala et al. attempted encapsulation of cinnamon and garlic essential oil into cyclodextrin complexes [32]. The cyclodextrin encapsulated oils were demonstrated hydrogen bonding between oil and β-CD and exhibited considerably higher antifungal activity when compared with free essential oil. Also, the complex displayed prominent stability, improved solubility, and bioavailability. One more study by Raileanu et al. established encapsulation of lavender and mint oil into silica and β-CD complex [35]. The resultant complexes were remarkably stable against environmental condition and hold promising application in agricultural, food, cosmetic and pharmaceutical industries. Similarly Arana et al. encapsulated Lippia graveolens in β-CD complex [36]. The results of experiments revealed improved antioxidant and antimicrobial activity of the encapsulated oil as well as encapsulation provides stability to the essential oils. Another study performed by Kfoury et al. reported encapsulation of estragole essential oil (component of basil and tarragom) within various grades of cyclodextrin viz. α-CD,

Stability and Efficacy of Essential Oils  277 β-CD, γ-CD, HP-β-CD (hydroxypropyl-beta-cyclodextrin), randomly methylated-­ βcyclodextrin, and a low methylated-β-cyclodextrin. The findings suggested improved antioxidant activity, controlled release and photostability of encapsulated oils [37]. Comparable to the above study, Das et al. encapsulated four different essential oils and their active components thyme oil (thymol), lemon balm oil (citral), lavender oil (linalool) and peppermint oil (menthol and borneol) into RAMEB (randomly methylated β cyclodextrin) [38]. The outcomes advocated significantly improved antioxidant potential, antifungal activity and antimicrobial property against pathogens (S. pombe, E. coli, and S. aureus). Additionally improved aqueous solubility and stability of encapsulated moieties was perceived when compared to free essential oils. Similarly, Babaoglu et al. encapsulated clove essential oil in HP-β-CD employing kneading method [39]. The study demonstrated enhanced stability, antioxidant properties and controlled release profile when compared with the free essential oils ascribing to increased aqueous solubility of essential oil due to inclusion complex formation. Geraniol is a fragrant essential oil used for food preservation as well as in treatment of infectious diseases and ascrining to these properties Kayaci et al. encapsulated geraniol essential oil into various kinds of cyclodextrin i.e. α-CD, β-CD, and γ-CD [40]. The findings revealed highest compatibility and complexation of geraniol essential oil with γ-CD, further this complex was incorporated into PVA nanofibers employing electrospinning. The resultant product demonstrated greater thermal stability and no loss of essential oil due to volatility when compared with uncomplexed PVA/geraniol nanofibers. Also, only 10% loss of geraniol essential oil was observed over a period of two years when formulated as γ-CD-PVA complexed nanofibers which was only one day when formulated as uncomplexed PVA nanofibers. Thus, it can be concluded that such complex have potential application in the food packaging owing to above mentioned improved attributes of essential oil. Another study performed by Hill et al. demonstrated encapsulation of cinnamon essential oil (trans-cinnamaldehyde) and eugenol (clove essential oil) into β-CD [41]. The mixture of trans-cinnamaldehyde and eugenol 2:1 were encapsulated through freeze-drying method and were assessed for thermal stability and antimicrobial activity against Salmonella enterica serovar Typhimurium and Listeria innocua. The results revealed improved thermal stability and enhanced antimicrobial activity of encapsulated essential oils when compared with in their free form. Baicalein (extracted from Scutellariabaicalensis) is bestowed with anti-­ inflammatory, antioxidant, and antitumor activity but is often less utilized because of poor physiochemical challenges (poor stability and low aqueous solubility). Thus, Zhou et  al. attempted encapsulation of baicalein into DM-β-CD(2,6-di-O-methyl-β-cyclodextrin) to encounter aforesaid challenges [42]. The results of study revealed that encapsulated oils exhibited higher solubility and stability when compared with free essential oils witnessing significance of cyclodextrin in modifying physiochemical properties of encapsulated oils. The aforementioned studies suggested that encapsulation with cyclodextrins conceivably an efficient tool in overcoming the limitations associated with essential oils further enhancing application of essential oils in pharmaceutical, food, aromatherapy, cosmetic areas. Moreover, if the controlled release is achieved for essential oils holding antimicrobial attribute, then it could be employed as efficacious packaging material for food industry that can preserve flavors and aroma and provide stability to food stuff over a prolonged period during transportation.

278  Essential Oils

12.2.3 Essential Oils Encapsulated in Polymeric Complexes Polymeric nanoparticles specifically biodegradable polymers (PLGA, PCL, chitosan, dextran etc.) have fuelled an exponentially growing interest in novel drug delivery various drugs [43]. These polymers are bestowed with numerous properties viz. biodegradability, biocompatibility, facile tailoring for surface engineering, controlled release of encased drug/molecule and good stability and have improved stability and efficacy of various encapsulated oils such as rosemary, turmeric lemon grass etc. [44, 45]. Thus, it is worthwhile to encapsulate essential oil into these polymers, so as to get modified physiochemical properties and/or therapeutic efficacy so as to improve their application as shown in Figure 12.2. In the same framework Froiio et al. encapsulated sweet orange and bergamot essential oil essential oil into Eudragit® RS 100 nanoparticles [46]. The researchers investigated the antibacterial activity of the encapsulated oils against E. coli. The findings revealed improved antibacterial potential of oils as evidenced through prolonged shelf-life of orange juice for a period of one week. The study suggests that encapsulation of essential oil into edible polymer could have promising application in food preservation. In another study Rashed et al. encapsulated Lavandula angustifolia essential oil into biodegradable whey protein based polymeric nanoparticles [47]. The essential oil loaded nanoparticles exhibited significantly enhanced antibacterial assets, better release of bioactive constituents and long-term storage stability at variable temperature conditions of 5°C, 25°C, and 45°C maintained at neutral pH indicating thermal stability of encapsulated oil [44]. Essential oil have been reported to replace synthetic hazardous pesticides and are being utilized as a natural pesticide in crop management ascribing to antimicrobial activity to combat weeds, pathogens and insects. The use such essential oils in greenhouse cultures appears hopeful, but is often challenged owing to rapid release and their high volatility. In the same context Stramarkou et al. encapsulated rosemary oil into biodegradable polymers (zein and polylactic acid) in order to enhance their efficiency and to achieve control release behavior. The experimental findings revealed biphasic release pattern comprising initial rapid release for 1-10 days followed by sustained release, over a period of 30 days. These

POLYMER CROSSLINKERS/ SURFACTANT ESSENTIAL OIL

AGRICULTURE

APPLICATIONS COSMETIC NANOCAPSULATION TECHNIQUES

FOOD INDUSTRY NANOEMULSION

NANOGEL

NANOCAPSULES/ NANOPARTICLES

PHARMACEUTICALS

Figure 12.2  Representation of various nanoencapsulation techniques for essential oils and their applications.

Stability and Efficacy of Essential Oils  279 results perceived the potential of encapsulated nanoparticles for replacement of agrochemicals and their utilization in greenhouse as pesticides. Turmeric and lemongrass essential oil containing arturmerone and citral respectively are well recognized for their antifungal, antibacterial, antioxidant, anti-mutagenic, and antitumor properties. However, challenges such as instability, volatility, and poor aqueous solubility limit their clinical outcome. Consequently Natrajan et al. attempted encapsulation of these essential oils into alginate and chitosan nanocapsules [44]. The findings demonstrated good stability and hemocompatibility of essential oils loaded nanocapsules. Furthermore, the essential oil-loaded nanocapsules were assessed for their antiproliferative activity through MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in A549 cell (lung cancer cell lines). The results revealed that both the nanoformulations displayed significant antiproliferative properties when compared with unentrapped oils, suggesting their biomedical and pharmaceutical application in future. In the same prospect Liakos et al. formulated nanocapsules of Peppermint, Cinnamon and lemongrass essential oil utilizing cellulose acetate [48]. The nanocapsules displayed enhanced antimicrobial activity against four microbial strains namely S. aureus, P. aeruginosa, E. coli, and C. albicans. The findings suggested that encapsulation of essential oils into nanocapsules offer a promising application in medical, pharmaceutical, and household products for prevention and eradication of biofilms (microbial colonization). Recently, essential oils have gained potential utility as an antioxidant for cosmetic products. In the same context Jummes et al. formulated Cymbopogon martinii Roxb loaded nanoparticles [49]. The Cymbopogon martinii Roxb loaded nanoparticles exhibited significantly improved antioxidant potential against DPPH free radical as well as superior antibacterial activity against S. aureus and E. coli when comparatively to that of essential oil emulsion. In another work study, Badri et al. co-encapsulated Nigella sativa L. essential oil for enhancement of anti-inflammatory and analgesic property of indomethacin [50]. The nanoformulation demonstrated considerably improved penetration across stratum corneum to dermis layer in addition to superior anti-inflammatory and analgesic property ascribing to synergistic effect when compared with indomethacin nanoparticles. Another study by Wattanasatcha et al. reported equivalent preservative potential of thymol in 12-52fold lower concentrations in lotion, gel and cream formulations when compared with methylparaben (usually used preservative) [51]. The nanoformulation displayed minimal bactericidal concentration values against E. coli, P. aeruginosa and S. aureus comparable to unencapsulated thymol but over prolonged period of three months in comparison to 2-3 weeks of unencapsulated thymol.

12.2.4 Essential Oils Encapsulated in Electrospun Fibers Electrospinning is a patented technology since 1902 that produces micro- and nanofibers [52]. Last decade has evidenced that this technique has wide application in encapsulation of essential oils for antimicrobial packaging of food materials [53]. Owing to the challenging attributes (instability, hydrophobicity, and volatility), the essential oils cannot be directly added to electrospinning solution thus, some carrier such as cyclodextrins, liposomes are usually necessary for generation of nanofibers as represented in Figure 12.3. Various biopolymers such as PVA, chitosan, polycarbonate, polyethylene oxide etc. have been employed for encapsulating essential oils (peppermint, chamomile, moringa, tea

280  Essential Oils Essential oil

Carrier

Encapsulation of essential oil

Syringe Pump

CD

Polymer

Nanofibres

Figure 12.3  Schematic representation of fabrication of essential oils-loaded electrospun nanofibers (CD: cyclodextrin).

tree, chrysanthemum etc.) through electrospinning ascribing to attributes that result in production of desirable nanofibers [54–58]. Taking into account above advantages Conn et al. reported encapsulation of cinnamon essential oil in β-CD, further incorporating complex to form polylactide nanofibers [59]. The findings indicated improved solubility and diffusion into agar medium, when compared with uncomplexed nanofibers (nonβ-CD) ascribing to reduction in contact angle. Further, a significant increase in antimicrobial activity was observed against both gram-positive and gram-negative bacteria. The nanofibers effectually extended the shelf life of strawberries and displayed promising application as suitable food packaging. Another study performed by Rieger and Schiffmann suggested considerably improved antimicrobial property of cinnamon essential oil when fabricated as chitosan/polyethylene oxide complexed nanofibers against E. coli and P. aeruginosa [53]. The enhanced antimicrobial property could be credited to swift release of essential oils (cinnamaldehyde) that causes rapid inactivation of strains leading to exponential mortality. Alike, Wen et al. reported fabrication of β-CD-PLA complexed nanofibers of cinnamon essential oil. The nanofibers displayed superior antimicrobial activity against E. coli and S. aureus when compared to only PLA nanofibers [60]. Comparable study by Kayaci et al. suggested improved efficiency of nanofibers encapsulated menthol, vanillin, eugenol, geraniol, and allyl isothiocyanate. The experimental demonstrated formulation of cyclodextrin encased essential oils that were further incorporated into polymeric matrix to form nanofibers [61]. The nanofibers displayed improved stability of encapsulated essential oils. Another study executed by Tang et al. revealed encapsulation of peppermint and chamomile essential oil into gelatin nanofibers [57]. The nanofibers encased essential oils exhibited enhanced antioxidant potential and antibacterial attribute against E. coli and S. aureus proportionate to free essential oils. Also, the combination of these two oils exhibited enhanced bioactivities when compared with either oil individually. The above reports established that potential of electrospinning technology in enhancing bioactivity of essential oils and a promising platform for encapsulation of essential oils in nanofibers that can be developed at industrial scale.

Stability and Efficacy of Essential Oils  281

12.2.5 Essential Oils Encapsulated in Microemulsion/Nanoemulsions Microemulsions are thermodynamically stable isotropically clear systems encompassing two immiscible liquids i.e. oil and water that acquire stability through formation of interfacial film of surfactant molecules [62]. Studies have suggested that o/w microemulsions facilitates solubility enhancement, improve bioavailability and are preferable nanocarriers for drug potential delivery of hydrophobic/poorly aqueous soluble drugs [63]. Literatures have reported improved stability and successful delivery of cinnamon, oregano and rosemary essential oil when encapsulated into microemulsion [64]. The microemulsion encapsulated has been reported to have wide application in agricultural fields as fungicide, herbicide, and insecticide for crop protection [65–68]. Taking into account these attributes of microemulsion scientists have successfully encapsulated of essential oils in order to combat the challenges offered by the same (Figure 12.4). Essential oils of Anise, (Pimpinella anisum) ajowain (Trachyspermumammi) and sea fennel (Crithmummaritimum) are well reported to possess mosquito larvicidal effects. Thus, Pavella et al. developed eco-friendly microemulsion of aforementioned essential oils and investigated their larvicidal property against Culex quinquefasciatus larvae and non-target invertebrates [69]. Further toxicity of microemulsions was tested against non-target species Daphnia magna and Eisenia fetida. The results displayed that all the microemulsions exhibited toxicity in mosquito larvae with LC50 values ranging from 1.45–4.01 ml L−1. The microemulsions showed significant larval mortality and decline in hatched adults subsequent to short-term exposure to sublethal concentrations. On the contrary an insignificantly low or no mortality was noticed in Daphnia magna and Eisenia fetida species. These results indicated the new perceptions of employing plant-based essential oils as novel and eco-friendly larvicidal products in future. Another study demonstrated encapsulation of Nigella sativa into microemulsion [70]. The prepared microemulsion was investigated for its antibacterial activity against six pathogenic strains (S. aureus, B. cereus, S. typhimurium, L. monocytogenes, P. aeruginosa and E. coli) and compared with well-established antimicrobials specifically eugenol (natural antimicrobial) and Ceftriaxone® (synthetic antimicrobial). The results displayed that the microemulsion holds superior antimicrobial activity than eugenol against all bacterial strains Apiaceae Essential Oils MICROEMULSIONS Apiaceae Essential Oils

M. canis

C. albicans

Antimicrobial activity

S. aureus

E. coli

Figure 12.4  Schematic representation of enhanced antimicrobial activity of microemulsion encapsulated essential oils. Reproduced with permission [72].

282  Essential Oils except P. aeruginosa. Also, the essential oil microemulsion displayed comparable antimicrobial activity against E. coli and higher antimicrobial activity against S. typhimurium to that of Ceftriaxone® in low concentration. However, poor or no antimicrobial activity of microemulsion was identified against L. monocytogenes and P. aeruginosa. Concomitantly, a higher antimicrobial activity against all pathogens except P. aeruginosa was observed in case of essential oil loaded N. sativa microemulsion when compared to eugenol microemulsion. The results suggested potential application of microemulsion in preventing bacterial growth of aforementioned pathogenic strains. Parallel to this study another investigation performed by Chatzidaki et al. reported formulation of reverse micelles of nisin into microemulsion containing rosemary, thyme, oregano, and dittany essential oil [71]. The micelles were employed as nanocarriers for nisin and investigated for their antimicrobial property against foodborne pathogens viz. L. lactis, S. aureus, L. monocytogenes, and B. cereus. The encapsulated nisin displayed satisfactorily enhanced antibacterial activity when loaded into essential oils based reverse micelles ascribing to microemulsions structure and synergistic effect of essential oils. Likewise Pavoni et al. attempted encapsulation of three essential oils (Pimpinella anisum L., Trachyspermumammi (L.) Sprague and Crithmummaritimum L.,) belonging Apiaceae family and assessed antimicrobial activity against fungal Microsporumcanis and Candida albicans, and bacterial strains E. coli and S. aureus [72]. The results revealed substantially improved antibacterial and antifungal activity against the fungal and bacterial strains when compared un-encapsulated essential oil ascribing to stabilization, improved dispersibility and enhanced interaction with the pathogen cell wall. Another study performed by Almasi et al. reported fabrication of microemulsions/ nanoemulsions encapsulated essential oil based antimicrobial calcium alginate biopolymer films for food packaging purpose [73]. Essential oil thyme along with acetic or propionic acid was formulated as microemulsion and was further fabricated as film employing calcium alginate. The microemulsion based biopolymer films were transparent with sufficient mechanical strength and high internal porosity. The results revealed that highest antimicrobial activity was observed with microemulsion-films. Further, microemulsion-films exhibited significantly higher antimicrobial efficiency when applied to ground meat as compared to plain calcium-alginate films against S. aureus, yeast, coliforms, mold, and lactic acid bacteria. Another study performed by Pavela et al. demonstrated formulation of Carlina acaulis and carlina oxide loaded microemulsion and nanoemulsions and compared their larvicidal property against Culex quinquefasciatus [74]. The maximum larvicidal activity was displayed by microemulsion containing 0.5% essential oil. The sublethal effect were assessed at LC16, LC30, LC50 and LC90 levels by exposing mosquito larvae to each concentration for a period of 1-7 h followed by 18 days monitoring. The findings suggested that even at a low LC16 concentration the microemulsion displayed 100% mortality at 18 days. Additionally the formulation were found to have no toxicity in HaCaT (human keratinocytes) and NHF A12 (human fibroblast) cell lines. Further acute toxicity in vivo studies in animal model showed non-toxic behavior subsequent to oral administration in rats at a high dose of 1098 mg kg−1 and 5000 mg kg−1. This study evidences the potential of microemulsion of C. acaulis as effective and safe larvicide against C. quinquefasciatus with a long-lasting effect. In the same framework Pang et al. reported formulation of microemulsion encapsulating Houttuynia cordata Thunb. (H. cordata) essential oil through high pressure homogenization and investigated its antiviral potential [75]. The prepared system

Stability and Efficacy of Essential Oils  283 displayed 94.7% encapsulation efficiency and an average globule size of 179.1nm. Further, H. cordata loaded microemulsion showed promising antiviral activity and safety validated through in vitro antiviral testing.

12.2.6 Essential Oils Encapsulated in Mesoporous Silica Nanoparticles Mesoporous silica nanoparticles are widely employed for delivery of numerous drug, biological molecules and proteins owing to substantial stability, well-defined porous architecture, rigid structure, easily modifiable morphology, tuneable surface chemistry and biocompatibility [76]. Therefore, they are often employed for encasing various moieties to enhance activity, improve stability, modulate release, and delivery of entrapped molecule. Taking into consideration these attributes of mesoporous silica nanoparticles Rico et al. attempted immobilization of carvacrol, eugenol, thymol and vanillin essential oil into three different mesoporous silica with variable surface areas, morphology and chemical reactivity [77]. The various formulations were assessed for inhibition potential against Listeria innocua and E. coli strains. The immobilized carvacrol retards growth up to 2-6 logarithmic cycles of L. innocua in a very low concentration. The immobilized eugenol inhibited microbial growth at a significantly lower concentration when compared to free carvacrol whereas immobilized thymol displayed most noteworthy boosted antimicrobial effect in a lower concentration of 0.004–0.012 mg/ml. The study revealed that immobilization of above-mentioned essential oils ensures preservation of functional hydroxyl moiety responsible of antibacterial effect, mask their characteristic odor/taste as well as improved antibacterial potential of the same when compared with free essential oils. A study executed by Cadina et al. demonstrated encapsulation of cinnamon essential oil into mesoporous silica nanoparticles and were further incorporated into a sodium alginate seed coating in a concentration of as low as 100%), F. oxysporum (59.6%), P. parasitica (>100%), P. aphanidermatum (NA) A. brassicae (>100%), C. mycophilum (23.9%) and T. aggressivum (NA)

[70]

(Continued)

390  Essential Oils

Table 17.1  Types of essential oil in agricultural biopesticide, active components, target pests and their respective effectiveness. (Continued) Types of plants

Active components Mastic thyme: eucalyptol (43.26%), linalool (36.72%) linalyl acetate (5.58%) Thyme: eucalyptol (37.48%), camphor (12.42%), camphene (7.38%) Tasmanian blue gum: eucalyptol (84.27%), cymene (7.52%) Rosemary: eucalyptol (23.75%), camphor (20.94%), α-Pinene (17.02%)

Target pests

Effectiveness

Ref.

ED50 (lavender cotton) = B. cinerea (NA), S. sclerotiorum (24.6%), F. oxysporum (17.7%), P. parasitica (17.3%), P. aphanidermatum (NA) A. brassicae (>100%), C. mycophilum (23.9%) and T. aggressivum (NA) ED50 (sweet orange) = B. cinerea (NA), S. sclerotiorum (78.2%), F. oxysporum (31.6%), P. parasitica (27.4%), P. aphanidermatum (NA) A. brassicae (68%), C. mycophilum (32%) and T. aggressivum (NA) ED50 (patchouli) = B. cinerea (NA), S. sclerotiorum (5.4%), F. oxysporum (11.4%), P. parasitica (40.6%), P. aphanidermatum (NA) A. brassicae (15.1%), C. mycophilum (0.6%) and T. aggressivum (11.7%) ED50 (mastic thyme) = B. cinerea (NA), S. sclerotiorum (14.87%), F. oxysporum (58%), P. parasitica (22%), P. aphanidermatum (NA) A. brassicae (>100%), C. mycophilum (14.1%) and T. aggressivum (NA) ED50 (thyme) = B. cinerea (NA), S. sclerotiorum (18%), F. oxysporum (36.3%), P. parasitica (13.1%), P. aphanidermatum (NA) A. brassicae (67.7%), C. mycophilum (9.3%) and T. aggressivum (NA) ED50 (Tasmanian blue gum) = B. cinerea (NA), S. sclerotiorum (8.9%), F. oxysporum (>100%), P. parasitica (>100%), P. aphanidermatum (NA) A. brassicae (>100%), C. mycophilum (27.4%) and T. aggressivum (NA) ED50 (rosemary) = B. cinerea (6.3%), S. sclerotiorum (23%), F. oxysporum (3.4%), P. parasitica (15.2%), P. aphanidermatum (NA) A. brassicae (>100%), C. mycophilum (25.7%) and T. aggressivum (3.4%) (Continued)

Biopesticides from Essential Oils  391

Table 17.1  Types of essential oil in agricultural biopesticide, active components, target pests and their respective effectiveness. (Continued) Types of plants

Active components

Target pests

Effectiveness

Ref.

Hop (Humulus lupulus L.)

Myrcene (17.7%), transcaryophyllene (14.9%), α-humulene (32.5%)

Zymoseptoria tritici

IC50 = 0.36 g/L

[71]

Lemon verbena (Aloysia citriodora), lemongrass (Cymbopogon winterianus), bushy mat grass (Lippia alba) and American basil (Ocimum americanum)

A. citriodora: β-citral (27.66%), cis-citral (20.68%) and limonene (19%) C. winterianus: cis-geraniol (32.85%), β-linalool (29.33%), citronellal (14.50%) L. alba: of β-linalool (66.57%) O. Americanum: cineole (35.71%), alcanfor (12.50%), p-eugenol (11.42%), β-linalool (11.26%)

Colletotrichum gloesporioides, Botrytis cinerea and Monilinia fructicola

EC50 (C. gloesporioides) = 0.41 mL/L (A. citriodora), 0.86 mL/L (C. winterianus), 0.76 mL/L (L. alba), 0.64 mL/L (O. Americanum) EC50 (B. cinerea) = 0.63 mL/L (A. citriodora), 0.52 mL/L (C. winterianus), 0.64 mL/L (L. alba), 0.74 mL/L (O. Americanum) EC50 (M. fructicola) = 0.21 mL/L (A. citriodora), 0.23 mL/L (C. winterianus), 0.41 mL/L (L. alba), 0.41 mL/L (O. Americanum)

[72]

Mountain germander (Teucrium montanum)

Germacrene D (12.8%), (E)-caryophyllene (8.0%), epi-α-cadinol (4.5%), α-pinene(3.1%), bicyclogermacrene (3.1%), cubebol (3.0%), and epi-cubebol (3.0%)

Spodoptera littoralis

LD50(90) = 56.7 (170) μg/larva

[73]

(Continued)

392  Essential Oils

Table 17.1  Types of essential oil in agricultural biopesticide, active components, target pests and their respective effectiveness. (Continued) Types of plants

Active components

Target pests

Effectiveness

Ref.

Marsh rosemary (Ledum palustre)

Ascaridole (35.3%), p-cymene (25.5%)

Spodoptera littoralis

LC50 = 117.2 μg/larva

[74]

Mandarin orange (Citrus reticulata Blanco)

limonene (60.74%), γ-terpinene (10.04%), β-myrcene (7.43%), α-pinene (3.93%), β-pinene (2.16%), terpinolene (1.30%), α-thujene (1.07%), linalool (2.92%), octanal (2.34%), decanal (1.73%)

Penicillium italicum and Penicillium digitatum

ED50 = 1.30 μL/mL (P. italicum), 21.30 μL/mL (P. digitatum)

[75]

Neem (Azadirachta indica A. Juss.), Citronella (Cymbopogon nardus (L.)

N/A

Rhizoctonia solani and Sclerotium rolfsii

R. solani: ED50 = 13.67 mg/L (neem), 25.64 mg/L (citronella) S. rolfsii: ED50 = 14.71 mg/L (neem), 20.88 mg/L (citronella)

[76]

Oregano (Origanum vulgare)

Methyleugenol (16.5%), myristicin (15.6%), carvacrol (15.0%), thymol (9.8%), apioline (9.4%), and (Z)-βfarnesene (8.7%)

Botrytis cinerea

EC50 = 52.92 mg/L (essential oil), 112.43 mg/L (methyleugenol), 17.56 mg/L (thymol), 26.22 mg/L (carvacrol)

[77]

(Continued)

Biopesticides from Essential Oils  393

Table 17.1  Types of essential oil in agricultural biopesticide, active components, target pests and their respective effectiveness. (Continued) Types of plants

Active components

Target pests

Effectiveness

Ref.

O. vulgare: Carvacrol (67.67%), o-cymene (11.60%) and γ-terpinene (7.45%)

Fusarium oxysporum

EC50 = 134.5 μg/mL (O. vulgare essential oil), 62.6 μg/mL (carvacrol)

[78]

True cinnamon tree (Cinnamomum zeylanicum)

C. zeylanicum: Eugenol (78.16%), eugenyl acetate (3.60%)

Fusarium oxysporum

EC50 = 171.79 μg/mL (C. zeylanicum essential oil), 187.46 μg/mL (eugenol)

[78]

Sphaeranthus amaranthoides

D-Carvone (89.7%), trans-2-Caren-4-ol (3.50%), 2-Cyclohexen1-ol (6.50%), Copane (6.42%), (+)-epiBicyclosesquiphellandrene (3.30%)

Spodoptera litura

LC50 = 2.71-2.74 ppm

[79]

Stemless Carline Thistle (Carlina acaulis) root

Carlina oxide (94.6%)

Lobesia botrana

LC50 = 7.299 µL/mL

[80]

Stevia (Stevia rebaudiana) leaf

Spathulenol (14.9%), (E)-nerolidol (8.0%), phytol (9.2%)

Metopolophium dirhodum

LC50(90) = 5.1 and 10.8 mL/L LC50(90) (phytol) =  1.4(4.2) mL/L LC50(90) (E)-nerolidol =  3.5(9.3) mL/L LC50(90) (spathulenol) =  4.3(7.5) mL/L

[81]

(Continued)

394  Essential Oils

Table 17.1  Types of essential oil in agricultural biopesticide, active components, target pests and their respective effectiveness. (Continued) Types of plants

Active components

Target pests

Effectiveness

Ref.

Senecio glaucus subsp. coronopifolius

α-pinene (26.2%), myrcene (11.4%), p-cymene (9.9%), β-pinene (7.7%), γ-muurolene (4%), deoxynivalenol (3.1%), α-phellandrene (2.7%)

Botrytis cinerea, Meloidogyne javanica, Tetranychus urticae

IC50 = 0.77 μL/mL air (B. cinerea), LC5 = 9973 ppm (M. javanica), LD50 = 5843 ppm (T. urticae)

[82]

Tea tree (Melaleuca alternifolia)

Terpinen‐4‐ol (40.09%), γ‐terpinene (21.85%), α‐terpineol (6.91%), α-terpinene (11.34%), 1,8‐cineole (1.83%)

Helicoverpa armigera

LD50 = 50.28 μg/ larva (24 hr)

[83]

Toothache plant (Acmella oleracea L.)

(E)-caryophyllene (20.8%), β-pinene (17.3%), myrcene (17.1%), caryophyllene oxide (10.0%)

Spodoptera littoralis

LD50 = 68.1 µg/larva LD90 = 132.1 µg/larva

[84]

White wild basil (Ocimum gratissimum L.)

Thymol (50.0%), p-cymene (16.8%)

Spodoptera littoralis

LD50 = 30.2 μg/larva

[85]

LC50,90 (Lethal concentration) = Concentration of a chemical which kills 50% (or 90%) of the sample population. LD50,90 (Lethal dose) = Amount of material which kills 50% (or 90%) of the sample population. IC50 (Inhibitory concentration) = Half maximal inhibitory concentration. ED50 (Effective dose) = Amount of material which produces the desired effect in 50% of a population. EC50 (Effective concentration) = Half maximal effective concentration. MIC = minimal inhibitory concentrations. IRMG = inhibition percentage of radial mycelial growth.

Biopesticides from Essential Oils  395 Deterioration of stored food and agricultural commodities in terms of their quantity, quality, nutritional, and commercial value due to infestation of storage pests causing significant losses that range from 5-10% in temperate zones and 20-30% in the subtropical and tropical regions, mainly in developing countries [87, 90]. There are nearly 70 species of Lepidopterous pests, 600 species of pest beetles, and 355 mites that cause qualitative and quantitative losses in storage commodities [91]. Globally, 5 to 10% of stored product beetles are found in the temperate zone, whereas 20–30% are in the subtropical and tropical zone [90]. In the whole world, these insect pests cause 10–40% of losses annually [92]. Under ideal circumstances, where insect pests directly access stored food, these losses could reach 50%. Indirect damages such as accumulation of insect carcasses, insect webbing, and chemical secretion could also increase the losses [90].

17.3.1 Food Storage Pests The rice weevil, S. oryzae Linnaeus 1763 (Coleoptera: Curculionidae), is a severe pest of stored grains found worldwide. It is the most destructive stored grain pest which feeds upon rice, maize, sorghum, wheat, barley, etc. [93, 94]. It causes substantial economic losses, especially to countries with agriculture as their primary economic sources, since large quantities of stored grains, legumes, etc., are being destroyed. The pests infest the grains and make them unsuitable for consumption [93]. The adult female rice weevil feeds the rice grain, and about 1 mm deep hole in the kernel is bored with her snout, laying a single oval white egg per day per grain. About 150-400 eggs are laid during their whole life and incubated for 4-9 days. The eggs become larvae, which live inside the kernel for 19-35 days and feed on the endosperm within the kernel, thus removing the proteins and vitamins of the grain. Pupation occurs inside the grain, which lasts for 5-7 days. The adult beetles make a hole in the grain to come out of the rice kernel by chewing their way out. The adult beetle can live for 6–8 months to 2 years, 5 to 6 generations per year [93]. Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) is a maize weevil found abundantly in warm temperate to tropical regions [95] and infest rice, wheat, and sorghum in storage [96]. Besides lowering the nutritional value, seed viability, food security, and market value, the infestation of maize weevil cause the moisture content and temperature to increase, thus encourage the growth of fungi such as Aspergillus flavus [96]. Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) is a confused flour beetle that attacks stored products and several dried foods, including fruits, chocolate, flours, and grains [97]. This pest causes significant damage to stored grains, especially when the kernels are damp or broken, even though they cannot penetrate intact kernels. The infestation causes the weight of stored grains and products to decrease, simultaneously affecting their quality and marketability. Besides, Tribolium spp. can produce quinones, carcinogens that can cause dermatitis and allergies and dermatitis, among other illnesses [98]. Tribolium castaneum (Coleoptera: Tenebrionidae) is a red flour beetle that causes significant losses to stored grain products [31, 99]. It is the most malicious and diverse pest in which the pest is nomadic, carrying along with the parasites, thus increases the risks of spreading resistance genes and contamination [31]. It has developed resistance against phosphine, a synthetic insecticide, in various places globally, especially in developing countries [88].

396  Essential Oils Callosobruchus maculatus F. (Coleoptera: Bruchidae) is a pest of stored pulses including chickpea (Cicer arietinum L.), cowpea (Vigna unguiculata (L.) Walp.), haricot beans (Phaseolus vulgaris L.), soy (Glycine max Mer.), and lentil (Lens culinaris Medik). The larvae penetrate and feed within the grains, which eventually cause the weight and nutritional value of the food to decrease besides affecting the cleanliness and encourage germination [100]. Zabrotes subfasciatus (Coleoptera: Bruchidae) is a bean weevil that infests stored grains and can be found in cold and temperate climates. The pests cause significant losses to stored grains by attacking the cotyledons, opening their galleries, and wrecking the kernels. The eggs, larvae, and adults of Z. subfasciatus can disturb the grains and product quality [101]. Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), also known as the lesser grain borer, is a severe and widespread coleopteran pest of stored cereals and stored pharmaceuticals [102, 103]. The kernels’ endosperm and germ are fed by the larvae and adults, in which the adults are the most lethal instar, and they can live for 25-65 days based on environmental conditions. The infestation of R. dominica adults usually occurs during the first weeks of storage [103, 104]. It is common to use synthetic pesticides to avoid a massive loss of food grains due to the infestation by grain pests. For decades, phosphine fumigants and methyl bromide have been used to control and protect food storage from pests [87, 105]. In Oklahoma, wheat is put away at relatively high ambient temperature for a long time, encouraging the storage facilities to be fumigated at least once a year [89]. Phosphine fumigant was applied for food storage in Morocco at 4-6 g/ton of grain for 5-7 days. The fumigation is carried out once or twice for short-term storage, while more extended storage required 4 or 5 fumigations of phosphine [86]. There are also chemical pesticides like organophosphates (malathion), organochlorines (lindane), pyrethroids (deltamethrin), and carbamates (carbaryl). Although the efficiency of phosphine fumigants is good in terms of killing the insects, prolonged and unwarranted use of phosphine will cause it to become ineffective [89, 105]. Many studies on phosphine resistance against significant insect pests have been reported in Morocco, Australia, Mexico, India, Brazil, and United States [86, 89, 91, 95, 102]. Methyl bromide has been found to cause nature ozone depletion, and phosphine disturbs the biological system, which causes pest resistance [87]. Regular application of organophosphates and pyrethroids have also cause phosphine resistance in stored grain pests [106].

17.3.2 Types of Essential Oils for Food Storage Pest Management Due to environmental and health concerns, biopesticides have become more popular than synthetic pesticides. Many plant species have been valued as antifeedants, toxicants, and repellents against many pests of stored grain and their products [88, 100]. Chaubey [107] reported that the most commonly used essential oils are from the plant family Apiaceae, Myrtaceae, Compositae, Cupressaceae, Labiatae, Asteraceae, Poaceae, Lauraceae, Piperaceae, Zingiberaceae, and Rutaceae. Coriander, Eucalyptus, and pine are among the plants extracted for their oils as biopesticides [87]. Monoterpenoids in the essential oils are found to have fumigant activity against stored-product insects [87, 91, 108–110]. On the other hand, Kout et al. [111] reported substituted phenols (eugenol, isoeugenol, methyl eugenol, isosafrole, and safrole) are better toxicants and repellents than monoterpenes. Other groups of compounds such as alkaloids, terpinolene, sulphur compounds, cyanohydrins,

Biopesticides from Essential Oils  397 and cyanates also present insect toxicity in the vapour phase. The compounds have high volatility, which is suitable as fumigants to control stored product insects. Eucalyptus sp. oil contains metabolic compounds such as monoterpenes and sesquiterpenes, aromatic phenols, esters, ethers, aldehydes, oxides, alcohols, and ketones [87, 93]. Each species of Eucalyptus contains different composition and amount of compounds. Gusmão et al. [112] attested Eucalyptus citriodora contained 89.59% citronellal, 3.34% citronellyl acetate, and 2.87% 1,8-cineole, in which the oil is a good candidate for use as repellents and toxic to stored product pests (Coleoptera) [113]. Eucalyptus dundasii contained 1,8-cineole (54.15%), p-cymene (12.41%), α-thujene (11.37%), and E-caryophyllene (6.7%), which showed repellency against grain R. dominica and Oryzaephilus surinamensis [110]. Compounds in Eucalyptus oils such as α-pinene, α-terpineol, alloocimene, aromadendrene, 1,8-cineole (eucalyptol), citronellal, citronellyl acetate, citronellol, eucamalol, p­­cymene, limonene, linalool, and γ-terpinene are found to have pesticidal activity [112, 113], whereby, 1,8-cineole, α-pinene, and terpineol are the principal constituent for fumigant activity against major stored grains [87, 91]. Based on the species of Eucalyptus, the oil has been demonstrated to have antifungicidal, antimicrobial, antibacterial, and repellent activities. Eucalyptus maculata oil showed repellency against S. oryzae and can be used as an insecticide in controlling rice weevil in postharvest rice grains [93]. The pesticide activity is through synergistic of Eucalyptus oil compounds activities [93]. A study by Aref et al. [110] showed that E. dundasii oil does not cause toxicity when consumed with food. Coriandrum sativum L. (Apiaceae) is a herb commonly grown in the Mediterranean region, Central Europe, North Africa, and Asia. The essential oil from ripe coriander fruit range from 0.03 to 2.6% [109]. Islam et al. [109] reported a total of 65 constituents were found in C. sativum, with esters (52.65%) being the most numerous, followed by aldehydes (15.38%), hydrocarbons (14.52%) and aromatic compounds (9.08%). The compounds responsible for the odor were E-2-decenal, Z-2-decenal, E-2-decen-1-ol, a co-eluting odor-cluster (E-2-dodecen-1-ol, E-2-dodecenal, and 1-dodecanol), beta-ionone, and eugenol. The bioactivity of C. sativum is dependent on a high concentration of eugenol, beta-­ ionone, and Z-2-decenal found in the leaves [109]. Oil from coriander was found to cause toxicity and repellent against Corcyra cephalonica, C. chinensis, T. confusum, T. castaneum, S. oryzae and R. dominica [87, 99]. The essential oil contains camphor, linalool, limonene, linolenic acid, γ-terpinene, 1,8-cineole, carvone, α-pinene, phenolic acids, triacontane, quercetin, protocatechuic acid, caffeic acid, and geraniol which exhibited volatile toxicity against stored product insects [87, 100, 109]. Ocimum basilicum (sweet basil) is a plant that majorly consists of a blend of linalool and methyl chavicol. There is also 1,8 cineole (+limonene), eugenol, methyl, eugenol geraniol and geranial [100]. Da Silva Moura [96] reported 85% of O. basilicum was estragole, and 12% was linalool. Kout et al. [111] reported methyl chavicol makes up 75% of the oil. Linalool, eugenol, and methyl eugenol are known as insect repellents [100]. Variations of the flower, leaf, chemotypes, and plant origin affect the variety of compounds in the essential oil of O. basilicum [96]. Bacopa caroliniana (Walt.) B.L. Robins. is a herb contained 18 volatile compound in which the highest compound was α-terpinolene. The majority of the compounds found in B. caroliniana were terpenes and terpenoids. Compounds such as limonene, α-terpinolene, and α-pinene possessed a good acetylcholinesterase (AChE) inhibitory activity [114].

398  Essential Oils Azadirachta indica A. Juss (neem seed) oil consists of azadirachtin and other potentially bioactive compounds that effectively control pests of stored grains. Various compounds in neem seed, mainly the tetranortriterpenoid azadirachtin, have insecticidal activity in deterring feeding and oviposition. Furthermore, the oil also can be used as a repellent and inhibit insect growth, especially bean weevil, Zabrotes subfasciatus [101]. Croton rudolphianus Müll. Arg., is a widely grown plant in Brazil, and 31% of the plant oil consists of compounds such as eugenol, (E)-caryophyllene, and, methyl chavicol which are related to the fumigant and repellent effect against Sitophilus zeamais [115]. Mentha piperita L. or peppermint is a perennial aromatic plant found chiefly in Asia, Europe, North Africa, and North America. The major compounds in M. piperita are menthone, menthol, and menthofuran with antifeedant and insecticidal activities against numerous pests [116]. Giunti et al. [103] reported M. piperita had intense repellent activity against stored pests in which the oils consist of menthol (36.72%), menthone (18.23 %), iso-menthone (13.56%), menthyl acetate (5.79%) and eucalyptol (4.73%). Nenaah [90] reported that Asteraceae Egyptian plants (i.e. Achillea biebersteinii, Ageratum conyzoides, and Achillea fragrantissima) could control three major pests of stored grains worldwide (R. dominica, S. oryzae, and T. castaneum). The oil of A. fragrantissima contained cis-thujone (28.4%), 2,5-dimethyl-3-vinyl-4-hexen-2-ol (Santolina alcohol) (16.1%), 3,3,6-trimethyl-1,5-heptadien-4-one (Artmisia ketone) (14.8%), and trans-thujone (12.5%). The main compounds found in A. conyzoides were demethoxyageratochromene (precocene I) (68.3%), β-caryophyllene (10.4%) and α-humulene (4.6%). The crude oil precocenes I showed anti-juvenile hormonal activity resulting in a delayed larvae metamorphosis, sterile, moribund, and dwarfish adults. Twenty-six compounds were detected in A. biebersteinii oil, in which 96.7% were identified. The major compounds were cis-ascaridol (33.8%), p-cymene (22.4%), camphor (8.6%), 1,8-cineole (6.3%), piperitone (5.4%) and carvenone oxide (4.3%).

Table 17.2  Plant essential oil and its use against storage pests. Types of plant

Active components

Target pest

Baccharis reticularia

Limonene (14.60%), β-myrcene Tribolium (12.60%), bicyclogermacrene, castaneum β-caryophyllene (12.50%), β– eudesmol (10.40%), spathulenol (6.90%), globulol (4.20%), β-pinene (2.60%), β-elemene (0.90%), aromadendrene (0.70%), α-humulene (0.70%), germacrene D (1.70%), viridiflorene (1.00%), viridiflorol (1.00%), 1-epicubenol (0.90%), 13-epi-manoyl oxid (1.60%), α-pinene (0.90%), kaurene (0.90%)

Effectiveness

Ref.

100% repellency = 17.6 µg/cm3

[31]

(Continued)

Biopesticides from Essential Oils  399 Table 17.2  Plant essential oil and its use against storage pests. (Continued) Types of plant

Active components

Target pest

Effectiveness

Ref.

Coriandrum sativum

Linalool, limonene, γ-terpinene, linolenic acid, 1,8-cineole, α-pinene, carvone, triacontane, phenolic acids, quercetin, caffeic acid, protocatechuic acid, and geraniol

Sitophilus oryzae Contact: LC50 = 36.68 μg/cm3 Fumigation: LC50 = 18.11 μg/cm3

[87]

Achillea biebersteinii

Cis-ascaridol (33.80%), p-cymene (22.40%), camphor (8.60%), 1,8-cineole (6.30%), piperitone (5.40%), carvenone oxide (4.30%), 2-hexanal (0.30%), α-pinene (0.30%), camphene (1.10%), germacrene D (0.80%), sabinene (0.20%)

Callosobruchus chinensis

Contact: LC50 = 27.76 μg/cm3 Fumigation: LC50 = 16.25 μg/cm3

Corcyra cephalonica

Contact: LC50 = 47.93 μg/cm3 Fumigation: LC50 = 18.25 μg/cm3

Sitophilus oryzae LC50 = 31.6 mg/g grains Rhyzopertha dominica

LC50 = 22.8 mg/g grains

Tribolium castaneum

LC50 = 39.7 mg/g grains

Achillea Cis-thujone (28.40%), 2,5-dimethyl- Sitophilus oryzae LC50 = 48.3 fragrantissima 3-vinyl-4-hexen-2-ol mg/g grains (Santolina alcohol) (16.10%), 3,3,6-trimethyl-1,5-heptadien-4one (Artmisia ketone) (14.80%), trans-thujone (12.50%), transpinocarveol (4.70%), 1,8-cineol (3.70%), β-caryophyllene (2.30%), lavandulol (2.40%), bicyclogermacrene (1.60%), trans-sabinyl acetate (1.20%), terpinene-4-ol (1.10%), bornyl acetate (0.70%), spathulenol (0.30%)

[90]

[90]

(Continued)

400  Essential Oils Table 17.2  Plant essential oil and its use against storage pests. (Continued) Types of plant

Ageratum conyzoides

Active components

Demethoxyageratochromene (Precocene I) (68.30%), β-caryophyllene (10.40%), α-humulene (4.60%), bicyclogermacrene (2.80%), epiα-muurolol (2.40%), p-cymene (2.30%), germacrene D (1.30%), linalool (0.40%), bornyl acetate (0.30%), α-cubebene (0.30%)

Target pest

Effectiveness

Rhyzopertha dominica

LC50 = 36.9 mg/g grains

Tribolium castaneum

LC50 = 78.6 mg/g grains

Sitophilus oryzae LC50 = 35.4 mg/g grains

Ref.

[90]

Rhyzopertha dominica

LC50 = 27.1 mg/g grains

Tribolium castaneum

LC50 = 47.8 mg/g grains

Limonene (59.3%), terpineol (8.31%), linalool (6.56%), citronellol (6.21%), cyclopentane propyl (2.34%), geraniol (2.31%), terpinene-4-ol (2.07%), carvone (1.99%), decanal (1.40%), citral (1.15%), 3-carene (1.11%)

Callosobruchus maculatus

LC50 = 15.91 μL/L air

Sitophilus zeamais

LC50 = 80.01 μL/L air

Ocimum basilicum

Estragole (85.00%), linalool (12.00%)

Sitophilus zeamais

20 µL/L air (30%), linalool (>15%)

Sitophilus zeamais

LD50 = 202.32 ppm

[120]

Sitophilus oryzae LD50 = 169.23 ppm

Lobularia maritima

Azeleonitrile (39.70%), trans3-pentenenitrile (36.30%), 4-isothiocyanato-1-butene (10.90%), 5-hexenenitrile (3.70%), benzyl cyanide (0.40%), ethyl benzene (0.40%), benzaldehyde (0.20%), octanal (0.10%), eucalyptol (0.10%), cubenol (0.10%), hexanal (0.10%), furfural (0.10%)

Callosobruchus maculatus

LD50 = 72.12 ppm

Tribolium castaneum

LD50 = 348.47 ppm

Callosobruchus maculatus

LC50 = 7.48 μL/L

Tribolium castaneum

LC50 = 59.94 μL/L

[121]

Sitophilus oryzae LC50 = 35.37 μL/L

(Continued)

Biopesticides from Essential Oils  405 Table 17.2  Plant essential oil and its use against storage pests. (Continued) Types of plant

Active components

Target pest

Effectiveness

Ref.

Mentha longifolia

Pulegone (29.93%), 1,8-cineole (25.46%), menthone (17.85%), β-pinene (3.49%), isomenthone (2.89%), α-pinene (2.51%), trans-sabinene hydrate (2.28%), germacrene D (1.61%)

Sitophilus zeamais

Contact: 100% mortality = 0.50 µL/g grain Fumigation: 70% mortality = 32 µL/L air

[122]

Tagetes minuta

Butanoic acid (20.68%), cis-βocimene (12.85%), limonene (12.33%), cis-tagetone (5.64%), cis-ocimenone (4.28%), α-terpinolene (3.36%), decane (2.47%), sabinene (1.76%), alloocimene (0.60%)

Sitophilus zeamais

100% mortality = 0.375 µL/g grain

[122]

Ocimum basilicum

Methyl chavicol (86.30%), transα-bergamotene (5.90%), ocimene (2.50%), β-elemene (1.90%), 1,8-cineole (1.30%), methyleugenol (1.10%), δ-cadinene (1.20%)

Sitophilus zeamais

LD50 = 0.43 µL/ kg LD99 = 1.52 µL/ kg

[123]

Zanthoxylum rhoifolium

β-elemeno (31.26%), D-germacrene (18.16%), β-caryophyllene (12.09%), δ-elemene (7.63%), β-cedrenus (6.69%), bicyclogermacrene (4.57%), E-caryophyllene (3.63%), α-cadinol (2.09%), seicheleno (1.91%), phytol (1.26%), geranyl acetate (0.75%), D-cadinol (0.25%), hexadecanal (0.24%)

Bemisia tabaci

100% mortality = 5% emulsifiable concentrate

[124]

LC50,90 (Lethal concentration) = Concentration of a chemical which kills 50% (or 90%) of the sample population. LD50,90,99 (Lethal dose) = Amount of material which kills 50%, 90% or 99% of the sample population. RD50 ,90 (Repellent dose) = Amount of material which produces the 50% (or 90%) repellency.

It is vital to ensure the food stored is free from pests. The losses caused by the infestation of pests hinder the food from having a good quality to be marketed. Various biopesticides have been used to control the storage pests since they are eco-friendly, readily biodegradable, economical, and less toxic. Types of plants and the targeted pests are summarized in Table 17.2.

406  Essential Oils

17.4 Application of Essential Oil Biopesticides for Household Pests Seasonal or weather changes such as storms and floods can result in unhygienic environments that attract pests to find food from the ruin or rotten remains and later increased the pest population. Maintaining a clean home has been the key to prevent household pest infestation. Household pests are despised because they cause discomfort, property damage, and most importantly, they can transmit pathogenic microorganisms. Other than rats, insects including termites (Rhinotermitidae), clothes moth (Tineola bisselliella), silverfish (Lepisma saccharinum), bed bugs (Cimex lectularius), house flies (Musca domestica), ants (Hymenoptera: Formicidae), cockroaches and mosquitoes are common household pests. Pathogens have been isolated from the body of flies and ants, which indicates the pest’s possibility to contaminate food and water source when in contact. The pest contact with surfaces of kitchens can also result in foodborne illnesses. Some of the pest, such as the bed bug, is a nocturnal feeder that bites only at night. During the day, the bed bug hides in secluded areas, away from direct light. This insect’s bite can cause skin lesion inflammation or trigger allergy but are not competent as a vector to transfer pathogens to human [125].

17.4.1 Household Pests Most household pests are insects that can be divided into two groups. The first groups are those causing nuisance or property damage such as clothes, books and furniture because the pests feed on cellulose. In contrast, the second group can transmit infectious pathogens such as mosquitoes, flies, ants, and cockroaches. Mosquitoes than any other diseases vector, cause more deaths since many of the mosquitoes’ species can transmit diseases. Blood-feeding female mosquitoes transmit chikungunya, dengue, encephalitis, filariasis, malaria, and yellow fever. The transmission of these diseases caused serious human health problems and hindered developing countries’ socioeconomic development. Most mosquito-borne diseases do not have a vaccine, and there is no specific treatment for the viruses except for yellow fever. Therefore, it is necessary to control mosquito-borne diseases. The housefly (M. domestica) is a common household pest that feeds on fecal and decaying matters, and wounds and sores’ discharges, among others. House flies have been mechanically transmitting diseases to humans through the action of regurgitation and excretion wherever they land. Pathogens are transported through the house flies’ feces, mouthparts, saliva, gut, and the surface of their body [126]. The prominent cockroaches’ species are German Cockroach, Blattella germanica; American Cockroach, Periplaneta americana; and Oriental Cockroach, Blatta orientalis. Cockroaches can harbor and carry human disease-causing pathogens (e.g. bacteria, helminthes, protozoa and viruses), and induce asthma. Therefore, cockroaches are considered essential hygiene indicators. Ants (Hymenoptera: Formicidae) show great dominance and diversity in tropical environments. This insect can carry and transfer pathogenic microorganisms to food. Enterobacter spp. and Escherichia coli are the severe intestinal disorders’ causative agents and among the pathogens carried by ants. Conventional insecticide may result in environmental

Biopesticides from Essential Oils  407 contamination, lead to colonies’ fragmentation, and increase nests’ number, leading to the increased ants’ population. Approaches to control household pests begin with identifying an infestation, then applying insect repellent and appropriate pesticides. The key components of successful eradication efforts rely on follow-up evaluation and risk management procedures to prevent the reintroduction of the bugs. Insect repellent is applied to the skin mainly to prevent bites. Prophylactic use of insect repellents is significant in areas with widespread mosquito-borne diseases. The active ingredient of insect repellent is of synthetic or natural origin. Examples of synthetic active ingredients include DEET (N,N-diethyl-3-methylbenzamide), Picaridin, and IR3535; a synthetic compound based on the amino acid alanine, whereas those derived from natural compounds include lemon eucalyptus oil, or active compound p-menthane-3,8-diol, and citronella oil, which is extracted from Cymbopogon nardus. Catnip oil extracted from Nepeta cataria consist of nepetalactone, and 2-undecanone is extracted from the wild tomato (Lycopersicon hirsutum Dunal f. glabratum C. H. Müll) plants [127]. Permethrin is from the class of Pyrethrins, is from the chrysanthemum flower extract. It is an insect repellent not suitable for skin but meant to be applied on clothes and books to repel moths and silverfish from destroying clothes and books, respectively. Pyrethrins break down very quickly in sunlight, so they should be stored in darkness and suitable for repelling insects in closed areas such as closets or bookshelf [128]. A practical and selective insect control method in a household setting is the food-based baits. A conventional bait consists of a carrier from grain or animal protein, a toxicant such as organophosphates, carbamates, Bacillus thuringiensis (Bt), parasitic nematodes or fungi, and sometimes an additive such as oil, sugar, or water. Many baits function as an arrestant instead of attracting insects. The base material does not attract the insect but is palatable enough to consume it when encountered. Distribution of baits is done by either ground or aerial broadcast for wide dispersion. Toxic baits are often environmentally safe and cheap methods to control insects [129]. Undisputedly, the unhygienic environment impedes the control of household pests, particularly in the kitchen, bathroom areas, drain, and dumpster. The extensive use of synthetic insecticides results in insects’ resistance to pyrethroids, organophosphates, carbamates spinosad, indoxacarb, spiromesifen, neonicotinoids, and resurgence in pests, which may negatively affect humans and the environment. Furthermore, there is no denying the lessons learned from areas that have recorded high cases of dengue fever. Cooperation from everyone in the neighborhood is necessary to ensure the neighborhood is free from mosquito breeding grounds.

17.4.2 Types of Essential Oils for Household Pest Management Efforts have been made to find biopesticides with a novel mechanism of action, safe for humans and the environment. Essential oils contain bioactive compounds’ mixtures that can be used in isolation or in combination to manage pests. Many studies on essential oils derived from edible or medicinal plants have been directed to control mosquitoes and flies. Some of the properties of essential oils include adulticidal, larvicidal, ovicidal, ovipositional, and repellence. However, the effectiveness of essential oil is heavily dependent on the sensitivity or the susceptibility of the pest towards the essential oil. For instance,

408  Essential Oils the integument of the pest and larval cuticle could hamper the ability of the essential oil or the constituent to reach the target site in adult pest and larvae, respectively, to result in the neurotoxicity effect. The non-motility of insect’s eggs makes them easier to target than the mobile adult. Deleterious effects of essential oil on the development and growth of pests have been elucidated from the investigation carried out towards the insect’s eggs. However, it is not practical in a household situation, or its application is limited to pests such as mosquitoes. Nevertheless, advances in nanoemulsion formulation could enhance penetration, thus delivering the essential oil or penetrating the insect integument effectively [130]. Pogostemon cablin Benth. (Lamiaceae) is extensively cultured for its essential oil (patchouli oil). The toxicity and repellency properties of the patchouli oil were investigated on three ant’s species: Camponotus melanoticus, Camponotus novogranadensis, and Dorymyrmex thoracicus. The percentage of mortality was the same between the ants’ species. However, an intoxication effect, which generally precedes the insect’s death, was observed immediately after applying the compound. Additionally, curved or paralyzed legs and tremors were exhibited. These symptoms suggested the patchouli oil’s neurotoxic effect on the ants. The repellency of this oil was greater for C. melanoticus. In contrast, approximately 88% and 86% of the C. novogranadensis and D. thoracicus avoided half of the petri dish treated with the essential oil of P. cablin, respectively. Therefore, P. cablin essential oil as repellent was suggested to be more practical to control the ants [131]. Analysis of dead M. domestica treated with Eucalyptus cinerea essential oil indicated that flies absorbed the essential oil’s constituents and their possible metabolites. Among the constituents, the intoxication was caused mainly by the 1,8-cineole compared to α-pinene. A reduced proportion of 1,8-cineole and the appearance of 2,3-dehydro-1,8-cineole and possibly the addition of α-terpineol suggested that M. domestica metabolized 1,8-cineole to these terpenes, which indicates the possible transformation by the insect’s oxidative detoxification pathway. The essential oil and 1,8-cineole’s toxicity increased when the piperonyl butoxide, a cytochrome P450 inhibitor, was used in combination with either of them. Thereby, the improved toxicity of deltamethrin/1,8-cineole combination was likely due to the ability of 1,8-cineole to act as a P450 inhibitor-like compound. Using the E. cinerea essential oil and or 1,8-cineole as fumigants or synergistic pyrethroid insecticides is an exciting alternative to control flies in human habitats, especially against resistant flies, to decrease the doses of synthetics insecticides [132]. Eugenol, eugenol acetate, and β-caryophyllene are Piper betle essential oil constituents with a higher cidal activity than the P. betle essential oil. It was also found that eugenol had 4.5-fold higher ovicidal activity on M. domestica eggs than P. betle essential oil. Furthermore, P. betle essential oil, eugenol, and β-caryophyllene increased the carboxylesterase enzyme activity in both larval and adult stages of M. domestica, whereas both eugenol acetate and β-caryophyllene caused inhibition of glutathione-S-transferase (GST) enzyme levels in larvae and β-caryophyllene alone cause inhibition of GST enzymes in adults. The effects of P. betle essential oil constituents on both enzymes responsible for insecticide resistance, rationalizing the toxic effect of the compounds towards M. domestica. Additionally, hyperactivity, tremors, and uncoordinated movements of flies exposed to P. betle essential oil and

Biopesticides from Essential Oils  409 its constituents were also observed, indicating the neurotoxicity property of the essential oil [133]. Allium sativum L., Azadirachta indica A. Juss., Cinnamomum cassia (L.), Eucalyptus camaldulensis Dehnh., Piper nigrum L., and Thevetia peruviana (Pers.) are widely cultivated for food and medicine. The essential oils’ toxicity activity was evaluated against larvae, pupae, and adults of M. domestica, along with oviposition deterrence and repellency against adult M. domestica. It was revealed in all the studied parameters that T. peruviana and A. indica essential oils were the most potent, suggesting that these oils can effectively control M. domestica in different life cycle activities. Moreover, the speed of mortality caused by A. indica and T. peruviana essential oils were faster than the rest of the essential oils [134]. The Amomum subulatum-derived essential oil has acute toxicity to the third instar mosquito larvae, which is the ultimate stage of larval development of mosquitoes. This oil’s toxicity is primarily due to 1,8-cineole and α-terpineol presence as the essential oil’s primary constituents. The concern over the biopesticide’s harmful effect on the environment was also addressed. The results demonstrated that A. subulatum-derived essential oil was toxic for Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus with LC50 less than 50 μg/mL, but safe for four aquatic organisms typically used as biocontrol agents against mosquito young instars; Anisops bouvieri, Diplonychus indicus, Gambusia affinis, and Poecilia reticulata, with LC50 values were found to be more than 3000 μg/mL [135]. Essential oils from various parts of four plant species; Cymbopogan citrates (leaves), Cinnamomum zeylanicum (bark), Rosmarinus officinalis (shoot), and Zingiber officinale (rhizome), showed significant repellency in human-bait technique against C. tritaeniorhynchus than A. subpictus. Test on volunteers using the essential oils did not cause skin irritation, hot sensations, or rashes on the arms. Larvacidal activity to the late third instar showed larvicidal potential in the essential oils as Z. officinale > R. officinalis > C. zeylanicum> C. citrate. The yields of C. citrates, C. zeylanicum, R. officinalis, and Z. officinale were 4.5, 3.1, 2.4, and 1.9 mL/kg, respectively. From the yield, C. citrates can likely be a sustainable source of essential oil [136]. The fumigant and contact toxicity of Artemisia sieberi Besser, E. camaldulensis Dehn. and Thymus persicus (Ronniger ex Rech. f.) essential oils against the first instar nymphs and adult German cockroach or B. germanica (L.) (Blattodea: Blattellidae) were compared with Eruca sativa (Miller) cold press oil. The essential oils were significantly more toxic than cold press oil in both fumigant and dipping methods. Still, the cold press oil’s fumigant activity was proven in contact toxicity assay, although the toxicity was lower than the essential oils. Among the two life stages of B. germanica, there was no significant difference in the essential oils’ fumigant toxicity between first instar nymphs and adults. On the contrary, the first instar nymphs were more prone to contact toxicity than the adult stage. The A. sieberi essential oil and E. sativa cold press oil’s fumigant and contact actions may be necessary to manage German cockroaches in the cockroaches’ inaccessible and enclosed hiding spaces [137]. Some of the previously reported studies of the essential oils against household pests, their active compounds, and their effectiveness are listed in Table 17.3.

410  Essential Oils Table 17.3  Types of essential oil in household biopesticide, active components, target pests and their respective effectiveness. Types of plants

Active compounds

Target pest

Effectiveness

Ref.

Teucrium montanum subsp. jailae

Germacrene D (12.8%), epi-α-cadinol (4.5%), α-pinene (3.1%), bicyclogermacrene (3.1%), cubebol (3.0%), epi-cubebol (3.0%)

Musca domestica adult female

LD50 = 154.9 μg/ adult LD90 = 268.7 μg/ adult

[73]

Culex quinquefasciatus larvae (Southern house mosquito)

LC50 = 180.5 mg/L LC90 = 268.7 mg/L

Linalool (10.7%), (E)-nerolidol (13.3%), (E)-β-ocimene (3.8%) germacrene D (5.0%), neral (4.6%), hedycaryol (4.0%), geraniol (3.9%), linalool acetate (3.6%), neryl acetate (3.4%), terpinen-4-ol (3.3%), geranyl acetate (3.1%)

Musca domestica adult female

LC50 = 103.7 μg/ adult LC90 = 223.9 μg/ adult

Culex quinquefasciatus larvae (Southern house mosquito)

LC50 = 221.1 mg/L LC90 = 297.1 mg/L

α-bulnesene (13.95%), α-guaiene (11.96%), patchoulol (36.60%)

Three species of urban ants: Camponotus melanoticus

LD50 = 2.31 μg of oil/mg insect LD90 = 3.32 μg of oil/mg insect

Camponotus novogranadensis

LD50 = 3.23 μg of oil/mg insect LD90 = 13.79 μg of oil/mg insect

Dorymyrmex thoracicus

LD50 = 5.02 μg of oil/mg insect LD90 = 10.21 μg of oil/mg insect

Thymus alternans

Pogostemon cablin Benth. (Lamiaceae)

Eucalyptus cinerea

1,8-cineole (74%), Musca domestica α-pinene (0.1%), (Housefly) α-terpineol (24.7%) and 2,3-dehydro-1,8-cineole (1.2%)

[73]

[131]

LC50 of 1,8-cineole [132] = 3.3 mg/dm3, α-pinene = 11.5 mg/dm3, and α-terpineol = 36.8 mg/dm3 (Continued)

Biopesticides from Essential Oils  411 Table 17.3  Types of essential oil in household biopesticide, active components, target pests and their respective effectiveness. (Continued) Types of plants

Active compounds

Target pest

Effectiveness

Piper betle L.

Safrole (44.25%), eugenol acetate (9.77%), eugenol (5.16%), β-caryophyllene (5.98%), β-selinene (5.93%), α-selinene (5.27%)

Musca domestica adult

LC50 of eugenol [133] acetate = 73.50 mg/dm3, LC50 of eugenol = 88.38 mg/dm3, LC50 of P. betle essential oil = 213.76 mg/dm3

Allium sativum L. (Alliaceae)

Not reported

Musca domestica larvae Pupae Adult

LC50 = 169.72 ppm [134] LC50 = 150.56 ppm LC50 = 166.69 ppm

Eucalyptus camaldulensis Dehnh. (Myrtaceae)

Not reported

Musca domestica Larvae Pupae Adult

LC50 = 182.23 ppm [134] LC50 = 164.84 ppm LC50 = 139.15 ppm

Thevetia peruviana Not reported (Pers.) (Apocynaceae)

Musca domestica Larvae Pupae Adult

LC50 = 277.01 ppm [134] LC50 = 164.87 ppm LC50 = 302.75 ppm

Amomum subulatum Roxb. (Zingiberaceae)

The third instar mosquito larvae Anopheles subpictus

LC50 = 41.25 mg/ ml LC90 = 80.29 mg/ ml

Aedes albopictus

LC50 = 44.11 mg/ ml LC90 = 85.6 mg/ml

Culex tritaeniorhynchus

LC50 = 48.12 mg/ ml LC90 = 89.30 mg/ ml

Artemisia sieberi Besser

1,8-cineole (39.8%), α-terpineol (11.5%)

Not reported

German cockroach or LC50 = 2202.8 mg/L Blattella germanica LC50 = 2703.9 (L.) mg/L Nymph Adult

Ref.

[135]

[137]

(Continued)

412  Essential Oils Table 17.3  Types of essential oil in household biopesticide, active components, target pests and their respective effectiveness. (Continued) Types of plants

Active compounds

Target pest

Effectiveness

Ref.

Formulations of combined essential oils from Cymbopogon citratus (Stapf.), Myristica fragrans (Houtt.), Illicium verum Hook. f.

C. citratus: geranial (45.4 %); M. fragrans: α-pinene (21.6 %); I. verum:trans-anethole (94.0 %)

Musca domestica L.

Combination of 0.5 % I. verum essential oil + 0.5 % geranial) insecticidal activity was more effective than 1% w/v cypermethrin

[138]

LC50,90 (Lethal concentration) = Concentration of a chemical which kills 50% (or 90%) of the sample population. LD50,90 (Lethal dose) = Amount of material that kills 50% (or 90%) of the sample population.

17.5 Delivery of Biopesticides Although the efficiency of the essential oils’ biopesticides is well documented, their performance is still inadequate for large-scale applications. It requires effective and innovative strategies to manage volatility, stability, and solubility problems in water. Encapsulation of essential oils has been proposed to solve these limitations since this strategy can retain essential oils longer by interaction with a matrix. The ideal biopesticide’s release consists of rapid initial release between the toxicity and efficiency scale at the middle concentration, followed by a long and constant release [18]. The most recent essential oils encapsulation strategies for biopesticide production utilized nanotechnology. The essential oils can be encapsulated in nanodroplets created by emulsion in surfactant, nanoparticles generated by a matrix, and nanocapsules (Figure 17.2). Essential oil nanoemulsion is one of the actively researched formulations and has shown excellent efficacy against various agriculture, stored product, and household pest [61, 103, Nanoemulsion

Nanocapsule

Essential oil

Essential oil

Surfactant monolayer

Shell

Figure 17.2  Illustration of nanoencapsulation of essential oils.

Nanoparticle

Essential oil

Matrix

Biopesticides from Essential Oils  413 139, 140]. Nanoemulsion of Mentha spicata essential oil was prepared by ultrasonic emulsification of mixture of the essential oil and Tween 80 at ratio 1:1 (v/v) by Mohafrash et al. [139] for larvicidal application against Culex pipiens and M. domestica. It was shown that the essential oil nanoemulsion has high larvicidal activity and toxicity against C. pipiens and M. domestica compared to regular M. spicata oil and the active ingredients of commercial synthetic pesticide, lambda-cyhalothrin. The significant larvicidal activity and toxicity could be due to the high surface area of the nanoemulsion, which leads to the increase in the contact area, penetration, and fast delivery to the action’s site compared to the standard emulsion. Similarly, Geranium maculatum L. nanoemulsion demonstrated twofold essential oil’s insecticidal efficacy against larvae of C. pipiens and Plodia interpunctella [141]. On the other hand, essential oil nanoparticles can be prepared from a mixture of essential oil and with a polymer such as polyethylene glycol (PEG). Comparing citrus essential oil emulsions and essential oil-PEG nanoparticles showed an insignificant difference in mortality and progeny production, despite the nanoparticles having a tenth part of essential oil compared to the emulsion [142]. The formulation could influence biopesticide toxicity, which is critical in the active substance’s stability and gradual release [143]. Using natural biopolymers as the raw material for the nanoparticles could eliminate hazardous effects on the non-target organism. This is clearly shown from the neem oil-loaded zein nanoparticles’ non-toxic effect towards the non-target organism Caenorhabditis elegans and the unchanged genes’ number that encodes the soil’s nitrogen-fixing enzymes and denitrifying enzymes exposed to the neem oil-loaded zein nanoparticles [144]. Nanocapsules biopesticide typically consists of a shell with a space to place the essential oils. Nanocapsules akin to nanoemulsion and nanoparticles offer a larger surface area per unit volume, improved solubility, physical stability, and the bioavailability of essential oils. This is apparent from a study conducted by Kala et al. [145] on the larvicidal performances of pectin-cedarwood essential oil nanocapsule, which found entirely damaged epithelial cells, muscles, and adipose tissue of Anopheles culicifacies larvae. This indicates that the encapsulation of cedarwood essential oil in pectin nanocapsules promotes the penetration of essential oil into the larval body and directly acted upon the epithelial cell, muscles, and adipose tissue of A. culicifacies larvae.

17.6 Pesticidal Action of Biopesticides Plant essential oils consist of several constituents with many modes of action. Against various pests, the oils displayed toxicity, repellence, and growth inhibitory properties [90]. The presence of oxygenated terpenes or phenolic structure might cause antifungal activity of essential oil [146]. It was reported that the lipid fraction of the cell’s plasma membrane was affected by several monoterpenes (i.e. linalyl acetate, (+) menthol and thymol), causing leakage of the intracellular materials. However, this effect seems to be influenced by the microbial membranes’ lipid composition and net surface charge [147]. The action of essential oils against fungi occurs at the cell membrane or cell wall level and within the cell. The integrity of the cell membrane and cell wall is disrupted by inhibiting ergosterols’ biosynthesis and blocking of β-glucan’s formation, respectively. Moreover, the essential oil can impede the function of the mitochondrial transport chain and obstruct the efflux pump in the cell [148].

414  Essential Oils The essential oil’s mechanism of action against bacteria includes the destruction of the inner and outer membrane, inhibition of ergosterol biosynthesis and disruption of membrane integrity, damage to the cell wall, and leakage of intercellular material [149]. The essential oil not only inhibits the growth or kills bacteria but also inhibits glucose-­ dependent respiration and activates the leakage of intracellular K+ [150]. The essential oil’s insecticidal activity is due to the penetration of the insect’s body by the essential oil via the respiratory system that leads to asphyxiation and, finally, the insect’s death [151]. The insect’s exposure to this biopesticide also breaks down the insect’s nervous system by targeting the octopaminergic system [152]. Essential oil at high concentration or long exposure times can reduce overall detoxification enzymes (i.e. esterases, epoxide hydrolase, oxidases, reductases, and group transferases), which are responsible for eliminating toxic phytochemicals activities in adult insects, leading to the insect’s death [153, 154]. It was reported that the AChE enzyme’s activity in insects was inhibited by the essential oil, which leads to paralysis due to the nerve impulses’ blockage followed by the insect’s death [155]. The essential oil insecticidal activity also might be related Na+/K+-ATPase activities. The enzyme, Na+/K+-ATPase is necessary to transmit the nerve impulses in insects. Therefore, inhibition of Na+/K+-ATPase cause the imbalance of ion and disrupts nerve impulse transmission in insects [110]. The population of insects can also be reduced at the beginning of an insect’s lifecycle by inhibiting its ovipositional behavior using essential oil through the oxygen and surface tension alteration within the eggs, which resulted in the failure of egg hatching [156]. Furthermore, the essential oil’s penetration inside the egg inhibits the embryonic development of the egg, which ultimately caused the death of the embryo [156, 157]. In contrast to the other pest, the essential oil is typically used as a repellent against rodents. The repellent effect of some essential oil against rodents could be a valuable means for protecting food commodities in the field and during storage [158, 159].

Fungus • • •

• • • • •

Leakage of intracellular materials Disruption of cell membrane and cell wall’s integrity Impede the function of mitochondrial transport chain

Destruction of inner and outer membrane Disruption of membrane integrity Damage to the cell wall Leakage of intercellular material Inhibit glucosedependent respiration

Nematode •

Bacteria

Insect

Pesticidal action of essential oil biopesticide

• • •

Mite Rodent •

•

Disrupt the cell membrane and change its permeability

•

Breaks down the insect’s nervous system Reduce overall detoxification enzymes Alteration in the oxygen and surface tention within the eggs Inhibits the embryonic development of the egg

Elimination through action on the GABA receptor and octopamine receptor

Repellent effect

Figure 17.3  Possible pesticidal action of essential oil biopesticides.

Biopesticides from Essential Oils  415 Although several types of research have been published on essential oil’s acaricidal and nematicidal activity, the mechanism of action against these two pests is still unclear. It has been suggested that eliminating mites by essential oil happens through action on the gamma-aminobutyric acid (GABA) receptor and octopamine receptor [160], whereas the essential oil may disrupt the nematode’s cell membrane and change its permeability [161]. A schematic representation of the summary of the possible pesticidal action of essential oil biopesticides against various pests is shown in Figure 17.3.

17.7 Conclusion and Constraints The evidence in this chapter showed that essential oils effectively control crops, stored food, and household pests. Still, essential oils as biopesticides have several constraints, such as poor water solubility and decreased protection time due to their highly volatile nature and rapid degradation. Although nano formulated biopesticide exhibited great potential for resolving these problems, information on the long-term assessment is necessary to validate the efficacy of the biopesticide in the Integrated Pest Management (IPM) program. Furthermore, there is a possible reduction of treatment efficacy due to the alteration of pest behavioral responsiveness. The decline of responsiveness to biopesticides occurs after repeated exposure to the given cue (i.e. essential oil biopesticide) [103]. It is due to the non-associative learning characterized by specific features involving neurological and molecular mechanisms [162]. Although the target organism typically regains responsiveness when it is no longer exposed to the given cue [103], failing to investigate this phenomenon can impair the potential efficacy of the essential oils biopesticide in its target application. This decline in responsiveness needs to be considered when designing the IPM programs. Another critical issue is that the low adoption rate of biopesticides compared to synthetic pesticides, particularly in the agriculture industry can hamper the market’s growth. To date, Bhutan is the only country that has entirely banned synthetic pesticides. A Switzerland’s June 13, 2021 referendum on the ban of synthetic pesticides was rejected by voters, reflecting strong opposition to the proposal from the Swiss farming sector [163, 164]. Potential, possible mechanism of action and sustainable sources of essential oils as biopesticides are presented. However, the method available to control the pest depends on the pest’s susceptibility towards the agent with more minor side effects to human health and are more compatible with the environment. Penetration enhancer or improved delivery system is vital to allow the essential oil to effectively reach the target site in the insect and do the intended purpose. Nevertheless, other strategies such as physical and biological control in agriculture, grain cleaning and monitoring stored grain for signs of pest infestation, maintaining a hygienic household environment are also essential to effectively control agriculture, food storage, and household pests.

17.8 Acknowledgement The financial support by Universiti Sains Malaysia through the Research University Grant (RUI) (1001/PJKIMIA/8014156) is gratefully acknowledged.

416  Essential Oils

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18 Essential Oil Used as Larvicides and Ovicides Gurleen Kaur, Rajinder Kaur and Sukhminderjit Kaur* Department of Biotechnology, Chandigarh University, Mohali, Punjab, India

Abstract

Essential oil, naturally concentrated oil elicited from herbs and shrubs are indispensably renowned due to the embellishment of eminent applications associated with essential oil which are beneficial to mankind. The distribution of separate chemical compounds within specific essential oil engenders miscellaneous qualities such as antifungal, antimicrobial, anticancer, antibacterial, larvicidal, and ovicidal activities. Larvicidal and ovicidal activities hold special attention due to the acceleration of various pests and insects that are pernicious to substantial economical crops and human beings. Malaria is one of the severe as well as sometimes deadly vectors borne disease, known to infect millions of people universally. Worldwide, about millions of people are infected by the head lice, wingless insect causing scalp infection and irritation. Considerable crops like cotton, sorghum, legumes maize, and tomato suffer extensively due to enormous economical crop yield loss by American bollworm. To eradicate such harmful pests and insects, crucial focus is on utilization of chemical based products consequently insurgence resistance by pest and insects towards synthetic pesticides originates. Essential oil benefits overpower the use of synthetic chemical based products. The chapter elucidates about the essential oil acting as larvicide against mosquito vectors comprising of Aedes aegypti, Anopheles stephensi, Aedes Albopictus and the ovicidal activity against human head lice (Pediculus humanus), domestic animal gastrointestinal nematode (Haemonchus contortus) and American bollworm (Armigera Helicoverpa Hubner). Keywords:  Larvicides, ovicides, essential oil, natural extract, mosquitoes, head lice, pesticides

18.1 Introduction Essential oils extracted from various plants are known to have multifarious medicinal uses owing to their antiviral, antifungal, antimicrobial, antibacterial, anticancer, larvicidal, and ovicidal applications. Commonly, essential oils are extensively obtained from different segments of herbs and shrubs such as aerial parts, leaves, flower buds, roots and seeds. Essential oils are the secondary metabolites which are reserved within cavities, glandular trichomes, epidermal cells, and secretary cells. Phenylpropanoids and terpenes, the dominant constituent of essential oils are accountable for the fragrance and biological aspects of essential oil [1]. Many researchers have focused their attention upon essential oils as these are endowed *Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (427–442) © 2023 Scrivener Publishing LLC

427

428  Essential Oils with miscellaneous beneficial applications. Out of all these benefits of essential oils, larvicidal and ovicidal properties holds special attention because insects affects humans widely, both destroying economical crops and causing diseases in humans. Worldwide the spread of diseases by mosquitoes has caused an immense burden on human wellbeing. The eradication of mosquito borne disease has always been a challenging situation. Among all infectious diseases, vector borne disease has been recorded with greater than 17 % of infection. One of the most common parasitic infections, malaria is known to infect approximately 219 individuals around the globe whereas dengue is known to infect over 3.9 billion people [2]. Major diseases that have been reported in human include malaria, yellow fever, zika virus, dengue, chikungunya. Taking agricultural crops into consideration, the major biotic stress accountable for economical crop loss is produced by insects. The proportion of dry matter consumption is the direct loss created by pests whereas the infection of destructed seeds is the indirect agricultural loss. The various pests which infect the agricultural crops are American bollworm, fruit fly, wheat aphid, pyrilla, gall midge and many more [3]. American bollworm larvae cause huge destruction to leguminous plants, flowers and develop holes in soft leaves. One of the commonly used methods to control the spread of harmful insect species is the use of synthetic larvicides and ovicides. The chemical based control interventions aims to scale down the spread of insects but unlocks the risk of people wellbeing and miscellaneous environmental problems. The persistent implementation of synthetic or unnatural products on insects supports development of drug resistance in insects [1]. Such problems need an immediate solution with maximum efficiency and stability of the formulated larvicide and ovicide. Essential oil based larvicides and ovicides typify as natural control measure against specific species. Different research studies have reported about the ketones, hydrocarbons, aldehyde, phenols, alcohol which forms the fundamental composition of chemical compounds in essential oil. For example, caryophyllene is the major component present in Vitex negundo which act against third instar stage of Aedes aegypti [4]. The absolute preference of essential oil over synthetic products is their biodegradable characteristic which puts an end to environmental issues and concerns. Another imperative subject of development of resistance to synthetic pesticide is figured out by the presence of various principal components in the essential oil of plant species which provides larvicidal and ovicidal effect against particular species [1]. This chapter discusses about the use of essential oil as potential larvicide and ovicide against specific species of mosquitoes (Aedes aegypti, Anopheles stephensi, Aedes albopictus), human head lice (Pediculus humanus capitis), domestic animals gastrointestinal nematode (Haemonchus contortus), American bollworm (Helicoverpa armigera) which is harmful to human race.

18.2 Important Aspects of Essential Oils A significant issue that alarms and threatens people all over the map is multidrug resistance development to antibiotics. Consequently, it shifts the interest of researchers to the field of medical plants having antibacterial properties for the efficacious and proficient antibiotic treatment. Since old ages, an annual herb and a folk medicine referred to as Nigella sativa L. or black cumin is known for its broad-spectrum activity. Dalli et al. [5] investigated the in-vitro antibacterial activity of five samples of essential oil of Nigella sativa L. species

Essential Oil Used as Larvicides and Ovicides  429 against clinically isolated multidrug resistant strains from patients. Researchers reported that monoterpenes are the major chemical constituents of essential oil. Pinene, phellandrene, thymol, and cymene were identified and related to antibacterial activity that would manifest curation and prevention against uncontrolled emanation of antibiotic resistance [5]. Taking into consideration the antimicrobial properties of essential oil, Ghavam et al., 2021 assessed the antimicrobial aspects of essential oil extracted from Dracocephalum kotschyi. Limonene and cyclohexylallene were potent components of wild D. kotschyi whereas as limonene, geraniol, neral, pinene and methylgeranate were found in cultivated strains. The components from both wild and cultivated D. kotschyi essential oil were investigated against 12 strains of microbes which demonstrated its capableness to cure microbial infection [6]. In another literature study, Huang et al. [7] reviewed the collegial antimicrobial competence of plant essential oil against food borne pathogen for the preservation of sea food. Edible coating, films, and physical methods such as ultraviolet radiation, hydrostatic pressure, and thermal treatment were synergistically employed with essential oil for the increased antimicrobial efficiency. Regardless of the current progress in development of anticancer compounds, plant synthesized products are reckoned as leading and fundamental health management means in certain parts of world. Essential oil derived from Eucalyptus is profused with phytochemicals that is known to treat various ailments. Eucalyptus essential oil has wide spectrum activity against malignancies demonstrating in vivo and in vitro proficiency for deceleration or inhibition of cancer cell lines [8]. Black Piper nigrum L. essential oil was studied by Zhang et al., 2021 to examine antifungal, antioxidant and hepatoprotective abilities [9]. It was analyzed that essential oil was propertied with proanthocyanidins, flavonoids, and phenolics contributing to free radical and lipid peroxidation scavenging ability. Human liver damage is associated with lipid peroxidation. Intake of P. nigrum L. essential oil demonstrated shielding ability against lipid peroxidation indicating its hepatoprotective and antioxidant proficiency. Even the maize fungal growth was inhibited by the utilization of P. nigrum L. essential oil. Such essential oil caused mitochondrial dysfunction and destroyed the cell membrane integrity of Aspergillus flavus therefore inhibited the fungal growth [9]. Plant derived essential oils are also effective against mosquitoes by acting as larvicides and ovicides. Wangrawa et al., 2021 studied the oviposition deterrence, excito repellency and larvicidal activity of four essential oil including Ocimum. canum Sims, Lantana. camra L., Hyptis. spicigera Lam, Hyptis. suaveolens Poit against malaria vectors Anopheles coluzzii and A. gambiae. Such essential oils were reported with competent repellent ability to control larvae or as a larvicide against mosquito [10]. Rutaceae family comprises of Atlantia monophylla L. that is useful for the treatment of skin disease, rheumatoid pain, glandular swelling. Helicoverpa armigera is a cotton bollworm and a crop pest affecting the yield of plants. Essential oils from A. monophylla L. have demonstrated larvicidal, ovicidal (Figure 18.1) as well as antifeedant properties against H. armigera [11]. In another literature study, larvicidal and ovicidal effect of garlic and asafoetida essential oil was assessed against west Nile virus vectors. The results illustrated that out of 52 eggs only 19 eggs and 14 eggs hatched but the larvae died that were exposed to asafoetida and garlic essential oil respectively. Allyl disulfide was the predominant component for the inhibition of egg hatch thus making both the essential oil competent enough for mosquito

430  Essential Oils Anti fugal (Black Piper nigrum)

Anticancerous (Eucalyptus)

Antimicrobial (Dracocephalum kotschyi)

Antibacterial (Nigella sativa)

Larvicidal (Eugenia calycina cambes)

Essential oil

Ovicidal (Atlantia monophylla)

Figure 18.1  Important aspects of essential oils.

vector control [12]. This chapter mainly describes about larvicidal and ovicidal aspect of various essential oil.

18.3 Larvicides and Ovicides Larvicides are a sort of pesticides which assist in domiciliary and outdoor mosquito control by killing the larvae stage so that it does not mature into adult stage where as ovicides work to kill the insect at the egg stage. Numerous larvicides are well known such as organophosphates which further include temephos, diazinon, malathion, parathion, and many more [13]. Organophosphates obstruct with activity of enzyme cholinesterase eminent for the functioning of nervous system therefore disturbing the working of mosquito’s central nervous system. Pyripoxyfen and methoprene are other larvicides that are classified as insect growth regulator (IGR). IGR intervene with the complete metamorphosis of pest where modification takes place when insect or pest verge upon from pupal stage to adult phase [14]. One more category of larvicides consists of microbial larvicides. Bacillus sphaericus and B. thuringiensis israeliensis suppresses development of larval stage by producing crystal toxin which when ingested by mosquito gets activated by proteolytic cleavage and solubilization thereby interacting with epithelium midgut consequently causing larval death. The essential oil which works as larvicide includes Bunium persicum, Eucalyptus globulus, Piper aduncum, Ocotea quixos, Nepeta cataria, Zingiber cassumunar and many more. Essential oil of Blumea lacera, Neanotis montholonii, Cyathocline purpurea, Neanotis lancifolia has ovicidal effects (Figure 18.2) [15].

Essential Oil Used as Larvicides and Ovicides  431 Essential oil as Ovicides 1)

Allium sativum L.

2)

Ferula asafoetida

3)

Life Cycle of Mosquito

Essential oil as Larvicides 1)

Bunium persicum

2)

Syzygium aromaticum

Lavandula augustifolia

3)

Eucalyptus globulus

4)

Blumea lacera

4)

5)

Zingiber cassumunar

Neanotis montholonii

5)

Eucalyptus nitens

Larva Stage

Egg Stage

Pupa Stage

6)

Cyathocline purpurea

6)

Piper aduncum

7)

Neanotis lancifolia

7)

Ocotea quixos

8)

Ocimum basilicum

8)

Nepeta cataria

Adult Stage

Figure 18.2  Essential oils as larvicides and ovicides.

18.3.1 Larvicides Against Aedes aegypti The pervasiveness of Aedes vector in all corners of the world has led to worldwide rise of vector borne viral infections causing dengue, zika infection, and chikungunya. Mostly in the tropical region, dengue has been investigated as the preponderant and rapidly spreading mosquito borne viral disease among all the disease. Aedes aegypti is the causative mosquito of dengue which breeds in clean water, in and around human habitations. Female adult Aedes aegypti prefer human as host which is its distinctive nature and utilizes human made water reservoir for laying of eggs and raising of immature stages of its two phased life cycle [16]. One of the basic methods to deal with or manage the vector borne viral infections is the use of larvicides that function to inhibit the larval growth. However, the mosquitoes become resistant to synthetic larvicides and also pose threat to environment. Such issue is overcome by the use of safe, nontoxic, environment friendly and promising essential oil from plants as larvicides against mosquitoes. In a literature study, essential oil was extracted from three plants comprising of carom seeds (Trachyspermum ammi L.), star anise (Illicium verum) and clove (Syzygium aromaticum L.) as they are extensively known for their culinary and medicinal properties [16]. Major components found in T. ammi L. were thymol, anethole and terpinen-4-ol whereas p-anisaldehyde, p-allyl-anisole, trans-anethole, phenol 4-(2pro-penyl)-acetate were reported in I. verum. S. aromaticum L. was endowed with ­α-copaene, cocaryophyllene, o-allylguaiacol, humulene, eugenol components. The essential oil extracted from three different samples was synergistically used and illustrated equal toxicity and larvicidal effect against late third stage of Aedes aegypti mosquito [16]. Essential oil extracted from the leaves of Eugenia calycina cambes leaves is another natural larvicide against A. aegypti. E. calycina is also known for its antifungal and antibacterial and

432  Essential Oils oral properties. β-caryophyllene, caryophyllene oxide compounds were found in E. calycina cambes demonstrating potent larvicidal effect against Aedes aegypti [17]. Kurniasih et al. (2021) examined larvicidal effect and toxicity of essential oil extracted from Cymbopogon nardus L. (lemongrass) and Citrus sinesis L. (orange) against Aedes aegypti suggesting both as green candidate of insecticides. The prime compounds found in essential oil of lemongrass were limonene, methyl salicylate, citronellal, α-pinena, α-carene whereas linalyl acetate, myrcene, geranial, sabinene, citronellal, linalool, α-terpineol, limonene and α-pinena were present in C. sinesis L. Citronellal compound demonstrated repellant and antifeedant activity against A. aegypti which reveals the larvicidal property of orange and lemongrass essential oil [Table 18.1] [18]. Table 18.1  Different essential oils and their components which act as larvicide by targeting specific stage of A. aegypti. Essential oil

Components

Target stage

Reference

Psidium guajava L. (guava)

Caryophyllene oxide, (E)-caryophyllene

Fourth instar L4 stage

[19]

Vitex gardineriana Schauer

Caryophyllene oxide, cis-calamenene, 6,9-guaiadiene

Third Instar Larvae stage

[20]

Artemisia vulgaris

α- humulene, β-caryophyllene, caryophyllene oxide

Third and fourth Larvae stage

[21]

Trachyspermum ammi L.

Thymol, anethole, terpinen-4-ol

Third stage

[16]

Illicium verum

p-anisaldehyde, p-allyl-anisole, trans-anethole, phenol 4-(2pro-penyl)-acetate

Syzygium aromaticum L.

α-copaene, cocaryophyllene, o-allylguaiacol, humulene, eugenol

Ocimum campechianum

Bicyclogermacrene, 1,8-cineole, α-caryophyllene, cis-ocimene, β-caryophyllene, eugenol

Late third, early fourth stage

[22]

Ocotea quixos

α-pinene, terpinen-4-ol, 1,8-cineole, sabinene, β-caryophyllene

Piper aduncum

β-caryophyllene, trans-ocimene, dillapiole

Vitex trifolia

Caryophyllene, eucalyptol, sabinen

[4]

Vitex negundo

Caryophyllene

Third instar stage

Amomum rubidum

Limonene, β-phellandrene, δ-3-carene

Fourth instar larvae stage

[23] (Continued)

Essential Oil Used as Larvicides and Ovicides  433 Table 18.1  Different essential oils and their components which act as larvicide by targeting specific stage of A. aegypti. (Continued) Essential oil

Components

Target stage

Reference

Origanum majorana

Iso-menthone, iso-menthone, verbenone, 3-octanol, pulegone, isopulegone, trans-p-menthan-2-one

Third stage

[24]

Leptospermum scoparium

Calamenene, caryophyllene oxide, ladene, leptospermone, cubenol, isoleptospermone

Late third instar larvae

[25]

Mentha arvensis L.

Limonene, menthone, menthol, menthyl acetate

Third and fourth instar larvae

[26]

Peganum harmala

Thymol, limonene

[27]

Nepeta cataria

Thymol, neptalactone

Early fourth instar stage

Phellondendron

Eugenol, eudesmol

Cymbopogon nardus L. (lemongrass)

Limonene, methyl salicylate, citronellal, α-pinena, α-carene

Third instar larvae stage

[18]

Citrus sinesis L. (orange)

linalyl acetate, myrcene, geranial, sabinene, citronellal, linalool, α-terpineol, limonene and α-pinena

Eugenia calycina cambes

β-caryophyllene, caryophyllene oxide

Third instar larvae development stage

[17]

Macherium acutifolium

Trans-stilbene, indene

Third instar larvae

[28]

Tridax procumbens

Thymol, γ-terpinene

Third stage of larvae development

[29]

18.3.2 Larvicidal Activity Against Anopheles stephensi Within South East Asia, Anopheles stephensi is unavoidable malarial vector. A. stephensi is considered as a powerful competent vector of Plasmodium vivax and P. falciparum as well as an adept urban malaria vector having resistance to high temperature and numerous types of insecticides [30]. In year 2012, Djibouti city of Africa was initially reported with uncommon outbreak of urban malaria which was progressively recorded annually and identified as an Asian Anopheles stephensi A. stephensi usually breeds in clean water cistern and acclimate expeditiously to cryptic habitat. In a literature study, researchers worked upon the mosquito repellant species of camphor tree; Cinnamomum camphora L. to investigate its

434  Essential Oils larvicidal properties against A. stephensi. Early fourth instar larvae stage was targeted for larvicidal properties. It was reported that the adeptness of essential oil was dose dependent as increased concentration of essential oil increased the mortality of larvae. C. camphora L. is endowed with eucalyptol, pinene, and terpineol compounds that caused larvicidal effects against A. stephensi. Eucalyptol is characteristic anti-inflammatory compound and is used for the treatment of airway disease. The killing proficiency of C. camphora represented it as an ideal biolarvicide [31]. The Apiaceae family comprises of a perpetual plant, Bunium persicum widely grown in Iran. Itis reported to have flatus relieving and antiseptic properties. The larvicidal ability of essential oil of B. persicum was assessed against late third early fourth instar larvae of Anopheles stephensi. The major components were investigated to be cuminaldehyde and terpinene that demonstrated larvicidal activity against A. stephensi [32]. In a recent study, mosquito protective textile was developed by employing nanoemulsification technique on essential oil of Syzygium aromaticum and Eucalyptus globulus and its effectiveness was assessed against A. stephensi. Mosquito repellent textile aids in prevention of human beings from mosquito bite. The major components reported in essential oil of S. aromaticum were phenol, alpha-caryophyllene, beta cadinene, transcaryophyllene and 2- methoxy-3-(2-propenyl) while alpha-pinene, spathulenol, 1,8 cineol, benzene and globulol were present in essential oil of E. globulus. E. globulus carry out acetylcholinesterase inhibition activity and has oviposition repellent, insecticidal, larvicidal and insect repellent properties while S. aromaticum possessed mosquito repellent property. Researchers examined that both essential oil had protection efficiency that can be used to control mosquito disease [33].

18.3.3 Larvicide Against Aedes albopictus A deadly vector of arbovirus and the causative agent of yellow fever, dengue fever and chikungunya fever is an Asian tiger mosquito species Aedes albopictus which is indigenous species of tropical and subtropical southeast Asia. A. albopictus is similar to A. aegypti and is distinguished by white marks on its thorax and legs. A. albopictus is combative biter that not only feeds daytime on human but on other mammals too. A. albopictus causes high fever, bone pain, rashes, headache, bruises on skin and pain in muscles (Figure 18.3). In comparison to A. aegypti, A. albopictus is much resistant to cold and possess expertise to undergo diapauses as to prevent its egg hatch during winter season [34]. Researchers have worked upon essential oil extracted from different plant species such as Zingiber cassumunar, Z. castaneum, Z. collinsii, Z. zerumbet, Eucalyptus nitens, P. nigrum L., P. Kadsura and Ocimum basilicum that act as larvicide against A. albopictus. An essential mosquito repellent and an eminent plant derived traditional medicine regarded as Z. cassumunar is known to demonstrate larvicidal and adulticidal activities against A. albopictus. Z. cassumunar is well endowed with miscellaneous potentials such as antimicrobial, antioxidant, anticancerous, anti-inflammatory, anti-aging, antihistaminic and pain reliever applications. Terpinen-4-ol was the major component identified in Z. cassumunar essential oil. The researchers concluded that Z. cassumunar essential oil demonstrated moderate larvicidal efficiency against first instar larvae stage of A. albopictus. The essential oil also showed adulticidal activity due to the presence of terpinen-4-ol [35]. In another study, Z. castaneum rhizome essential oil was evaluated against fourth instar larvae stage of A. albopictus. The results displayed complete mortality against A. albopictus. Z. castaneum can be used for the development of

Essential Oil Used as Larvicides and Ovicides  435 Larvicide against Aedes albopictus 1. Bone Pain

Muscle Pain

High fever 2.

Family: Zingiberaceae (Zingiber cassumunar) (Zingiber castaneum) (Zingiber collinsii) (Zingiber zerumbet) Family: Myrtaceae (Eucalyptus nitens)

Aedes albopictus Headache

Rashes

3.

Bruises on skin

Family: Piperaceae (Piper nigrum L. - Black pepper) (Piper Kadsura)

4. Family: Lamiacea (Ocimum basilicum)

Figure 18.3  Various essential oils that are useful as larvicide against mosquito A. albopictus.

novel formulation against A. albopictus [36]. Z. collinsii is another species belonging to the family Zingiberaceae which is known for its insect or pest repellant properties. The essential oil of Z. collinsii was investigated against fourth instar larvae stage of A. albopictus. The major components examined were α-pinene, β-pinene, β-caryophyllene, humulene oxide II and camphene. Competent and concentration dependent larvicidal activity was depicted by Z. collinsii making it as an alternative source to synthetic insecticide [37]. Another important species of Zingiberaceae family is Z. zerumbet. It is known to possess antinociceptive, antimicrobial, antioxidant, anti-inflammatory, antimycotoxin, and antifungal larvicidal and insecticidal properties. Zerumbone was the principal component present in the essential oil of Z. zerumbet. Researchers reported 97.5% in 24 hours and 100% mortality after two days against A. albopictus which demonstrate excellent proficiency of Z. zerumbet essential oil as an effective larvicide [38]. Hence, it can be concluded that the essential oil examined from Zingiberaceae family have the potential to act as efficient larvicide against A. albopictus (Figure 18.3).

18.3.4 Ovicidal Activity Against Pediculus humanus capitis De Gee referred to as Pediculus humanus captis belongs to the family of Pediculicidae is one of extremely prevailing human head louse obligate ectoparasite causing head hair and scalp infection. P. humanus captis is tiny wingless parasite and its size ranges from 2mm-4mm, eminently host specific in nature and feeds on human scalp. Generally, the female P.  Humanus captis is larger in size than male parasite. The life cycle begins with the egg (nit) stage that are white or grey in color that hatches to nymph requiring approximately 10 days. Afterwards the nymph matures into adult by eliciting blood from scalp

436  Essential Oils of human. About 14–20 days would be required by adult P. humanus captis for its reproduction. Neurotoxic chemical synthetic insecticides were used for the control of human lice but after exposure the lice would usually developed resistance to such insecticides. Therefore, ongoing research takes safe and effective options and alternatives into consideration such as use of essential oil. The adequacy of essential oil from Zingiberaceae family including Z. zerumbet, C. zedoaria, and C. xanthorrhiza were determined against P. humanus captis [39]. The essential oil of Eucalyptus globulus was used as an augmenting compound for enhanced potency of all three essential oil. The leading components present in Z. zerumbet were 1,8-cineole, camphene, zerumbone, camphor, α-humulene whereas C. zedoaria consisted of isobornel, camphene, zingiberene, camphor and 1,8-cineole. Curcumene, xanthorrhizol, bisabolol and zingiberene were the major components found in C. xanthorrhiza. High ovicidal activity was showed by essential oil when used in combination with Eucalyptus globulus. Out of all three essential oil, C. zedoaria essential oil demonstrated higher ovicidal activity (100%) when synergistically used with E. globulus. The ovicidal activity significantly increased from 5% to 100%. The egg hatching was inhibited for an incubation period of 7-14 days. More than 50% of inhibition rate was demonstrated by each essential oil when used in combination with Eucalyptus globulus. E. globulus was endowed with α-pinene, β-pinene, terpineol and 1,8-cineole compound. Such study indicates the use of essential oil as herbal ovicide to control P. humanus captis infestation [39]. In another literature study, the in-vitro adulticidal efficaciousness of five essential oil Monarda fistulosa (wild bergamot), Eugenia caryophyllus (clove), Lavandulaaugustifolia (lavender), Melaleuca alternifolia (tea tree), Litsea cubeba (yunnan verbena) was examined against P. humanus captis. Approximately 1239 live P. humanus captis from human scalp were taken for the adulticidal assessment. The main components present in five essential oil were eugenol, geraniol, linalol, limonene,

Essential oil act as ovicide against Human Head lice

Zingiber zerumbet

Eucalyptus globulus

Eugenia caryophyllus

Pediculus human captis (human head louse) Family: Pediculicidae Causes: Scalp infection Melaleuca alternifolia Lavandula augustifolia

Litsea cubeba

Figure 18.4  Essential oil acting as ovicide against P. humanus captis (Human head lice).

Essential Oil Used as Larvicides and Ovicides  437 neral, linalyl acetate, terpinen-4-ol. The researchers concluded that E. caryophyllus when diluted in Cocos nucifera (coconut) or Helianthus annuus (sunflower) indicated more than 90% mortality within 120 minutes whereas significant potential was also reported from L. cubeba oil dilution with C. nucifera. However, the remaining essential oil illustrated least potential activity against the human lice. Hence, Eugenia caryophyllus owing to eugenol as its predominant compound can be used as an eminent ovicide and Litsea cubeba also have the potential and its efficacy can be further enhanced by research studies to be used as an ovicide against P. humanus captis (Figure 18.4) [40].

18.3.5 Ovicidal Activity Against Haemonchus contortus Gastrointestinal nematode specifically H. contortus is the originator and root cause of the widespread disease of helminthiasis prevailing among domesticated animals. Within ruminants, abrupt vomiting and bloody diarrhea, blood deficiency, lower protein level in blood, chronic gauntness and sudden death are the characteristic features of gastrointestinal parasitic disease. Accordingly, around the globe it has become eminent to restrict and manage the spread of helminthes in farming of livestock animals. Certain drugs are known to treat helminthes but it creates the problem of non-availability to rural sustenance animal keeper and is mostly high priced. Another issue associated with the use of drugs is emergence of multiple resistances towards helminthes. Consequently, essential oil extracted from various herbs and shrubs act as better and efficient ovicide with cost effective approach, to control the spread of H. contortus [41]. The family of Rutaceae comprise of Zanthoxylum that is considered as a medicinal herb and is extensively used as traditional medicine in china. The essential oil isolated from Zanthoxylum genus is persuasively used for the treatment of infectious diarrhea, tooth pain, abdomen illness, stomachic parasites and inflammatory ailments. Amide, alkaloid, lignan, flavanoid and coumarin are the constituent of Zanthoxylum. In a literature study, the ovicidal activity of a spiky shrub referred to as Z. simulans was determined against Haemonchus contortus. The major components as analyzed by gas chromatography in Z. simulans were borneol and β-elemene. Ovicidal activity of Z. simulans was examined on the eggs that were taken from sheep, were infected experimentally with the third larvae stage of H. contortus. Results indicated that egg hatching was inhibited by Z. simulans at every tested concentration. Consequently, to control gastrointestinal nematodes of sheep, Z. simulans is a possible choice [41]. The family of Asteraceae consists of one of the far extending genus in Asia, Africa, Europe and Northern America known as Artemisia. During the use of indigenous medicine period, various species of Artemisia were considered to prevent helminthes including A. maritima, A. vulgaris, A. monosperma, A. absinthium and A. judaica. Many diseases could be prevented with the application of Artemisia species such as bacterial infection, viral infection fungal infection, malaria, hypersensitivity, cancer, inflammation, and jaundice. Researchers have examined the in-vitro ovicidal activity of essential oil from persistently odoriferous and aromatic shrub known as Artemisia lancea as it has been reported to possess anthelmintic properties against Haemonchus contortus. Camphor and 1,8-cineole were the major components of A. lancea. The essential oil extracted from A. lancea demonstrated effective ovicidal activity as the egg hatch was inhibited in all the tested concentration in dose dependent manner. The inhibition of egg hatch (93.6%) was observed at 10mg/ml of concentration [42].

438  Essential Oils

18.3.6 Ovicidal Activity Against Helicoverpa armigera Hubner H. armigera is considered as an ancient bollworm having the ability to feed on arboricultural crops of extensive taxonomic diversification. H. armigera causes immense loss to different crops consisting cotton, sorghum, maize, tomato and various leguminous plants. It is also characterized by its potential to sustain on substitute hosts, possess higher mobility and ability to give rise to larger number of offspring. Various insecticides have been used as pest control but H. armiger has developed resistance against them. To prevent the repeated utilization of chemical pesticides, pest management studies lay the foundation for the plant extracted essential oil for the prevention of pest in eco-friendly manner [43]. H. armigera is comparatively secured from common attackers due to the obscure or mysterious sustaining tendency of H. armigera larvae inside the cotton bolls. Ovicidal properties of essential oil extracted from Cheilocostus speciosus rhizome were investigated against H. armigera in a concentration dependent manner where α-humulene, zerumbone and camphene were the principal compounds. Camphene demonstrated maximum ovicidal activity against H. armigera indicating the potential of C. speciosus essential oil to be used as ovicide against pest management [43]. Researchers investigated ovicidal property of Duranta erecta Linn leaf extract against H. armigera and observed that ethyl acetate extracts of H. armigera showed maximum ovicidal effect [44]. Research was conducted on the ovicidal and larvicidal potential of essential oil of Origanum vulgare L. which has recognized applications in the drug industry, comestible and cosmetic industry. O. vulgare exhibits antifungal, antibacterial, antigenotoxic, antimicrobial and antioxidant properties. O. vulgare possess superior capableness to control agronomical insect pest, mosquitoes and houseflies. The principal components present in essential oil of O. vulgare were determined to be p-cymene, carvacrol and ϒ-terpinene. The essential oil of O. vulgare demonstrated ovicidal Table 18.2  Essential oils as ovicide against different insect and pest species. Essential oil

Ovicidal activity against

Reference

Artemisia lancea

H. contortus

[42]

Zanthoxylum simulans

H. contortus

[41]

Monarda fistulosa, Eugenia caryophyllus, Lavandula augustifolia, Melaleuca alternifolia, Litsea cubeba

P. humanus captis

[40]

Zingiber zerumbet, Curcuma zedoaria, Curcumaxanthorrhiza

P. humanus captis

[39]

Atlantia monophylla

H. armigera

[11]

Origanum vulgare

H. armigera

[45]

Lippia alba

Oligonychus coffeae

[46]

Thymus vulgaris

Spodoptera littoralis

[47]

Helicteres velutina

Aedes aegypti

[48]

Cymbopogon winterianus

Cochliomyia hominivorax

[49]

Essential Oil Used as Larvicides and Ovicides  439 effect in a dose dependent manner against H. armigera. The compound carvacrol showed best ovicidal effect indicating O. vulgare as an advanced ecologically acceptable safe insecticide against H. armigera [Table 18.2] [45].

18.4 Conclusion The naturally volatile substances extracted from various herbs and shrubs are indispensable because of the presence of characteristic phytochemicals. Essential oil are adorned by incredible qualities like antipathogenic, antimicrobial, antibacterial, antifungal, anticancerous, larvicidal, ovicidal, biological and pharmaceutical applications. There are numerous pest and insects which pose threat to human life. Mosquitoes are the leading cause of malaria, dengue and chikungunya causing risk to human life. If the subject of agronomics is taken into consideration, ancient bollworm specifically H. armigera causes immense loss to various crops like cotton, tomato, maize, sorghum and different legume plants. Human head lice is the another pest causing human head scalp infection. Therefore, it becomes eminent to destroy the larvae or egg stage of such pests and insects before they reach the adult phase to prevent human and environment from their infection. Miscellaneous synthetic larvicides and ovicides are available commercially but they cause risk to environment and human life. Plant based extracted components or essential oil overcomes all the cons associated with the use of chemical based products against specific insects and pests. No toxic behavior, environment friendly and biodegradable behavior of essential oil encourages its acceptance to a greater extent in different applications. But the only disadvantage associated with essential oil is the volatile nature of its compounds which limits its use as an insecticide or repellent in certain cases. However, such dilemma is conquered by the employment of nanotechnology. Essential oil is formulated into nanoformulations which enhances its stability as well as efficiency. Hence, nanotechnology proves to be an eminent approach to improve potency of essential oil so that it can be used in different aspects to prevent human health and environmental problems.

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440  Essential Oils l. seeds against multidrug-resistant bacteria: A comparative study. Evid. Based Complementary Altern. Med., 2021. 6. Ghavam, M., Manconi, M., Manca, M.L., Bacchetta, G., Extraction of essential oil from Dracocephalum kotschyi Boiss.(Lamiaceae), identification of two active compounds and evaluation of the antimicrobial properties. J. Ethnopharmacol., 267, 113513, 2021. 7. Huang, X., Lao, Y., Pan, Y., Chen, Y., Zhao, H., Gong, L., Xie, N., Mo, C.H., Synergistic anti­ microbial effectiveness of plant essential oil and its application in seafood preservation: A review. Molecules, 26, 307, 2021. 8. Abiri, R., Atabaki, N., Sanusi, R., Malik, S., Abiri, R., Safa, P., Shukor, N.A.A., Abdul-Hamid, H., New insights into the biological properties of eucalyptus-derived essential oil: A promising green anti-cancer drug. Food Rev. Int., 38, 598–633, 2022. 9. Zhang, C., Zhao, J., Famous, E., Pan, S., Peng, X., Tian, J., Antioxidant, hepatoprotective and antifungal activities of black pepper (Piper nigrum L.) essential oil. Food Chem., 346, 128845, 2021. 10. Wangrawa, D.W., Badolo, A., Guelbéogo, W.M., Nebie, R.C.H., Borovsky, D., Sanon, A., Larvicidal, oviposition-deterrence, and excito-repellency activities of four essential oils: An eco-friendly tool against malaria vectors Anopheles coluzzii and Anopheles gambiae (Diptera: Culicidae). Int. J. Trop. Insect Sci., 41, 1771, 2021. 11. Shiragave, P.D., Effect of crude extract of Atalantia monophylla L. @ on ovicidal activity against Helicoverpa armigera Hubner (Lepidoptera: Noctuidae) and preliminary phytochemical study. J. Entomol. Zool. Stud., 6, 1744–1746, 2018. 12. Muturi, E.J., Ramirez, J.L., Zilkowski, B., Flor-Weiler, L.B., Rooney, A.P., Ovicidal and larvicidal effects of garlic and asafoetida essential oils against West Nile virus vectors. J. Insect Sci., 18, 43, 6, 1744–1746, 2018. 13. Medscape, Organophosphate toxicity, 2020, https://emedicine.medscape.com/article/ 167726-overview. 14. Choi, L., Majambere, S., Wilson, A.L., Larviciding to prevent malaria transmission. Cochrane Database Syst. Rev., 8, CD012736, 2019. 15. Torawane, S., Andhale, R., Pandit, R., Mokat, D., Phuge, S., Screening of some weed extracts for ovicidal and larvicidal activities against dengue vector Aedes aegypti. J. Basic Appl. Zool., 82, 1, 2021. 16. Pandiyan, G.N., Mathew, N., Munusamy, S., Larvicidal activity of selected essential oil in synergized combinations against Aedes aegypti. Ecotoxicol. Environ. Saf., 174, 549, 2019. 17. Silva, M.V., Silva, S.A., Teixera, T.L., De Oliveira, A., Morais, S.A., Da Silva, C.V., Espindola, L.S., Sousa, R.M., Essential oil from leaves of Eugenia calycina Cambes: Natural larvicidal against Aedes aegypti. J. Sci. Food Agric., 101, 1202, 2021. 18. Kurniasih, N., Nuryadin, W., Harahap, M.N., Supriadin, A., Kinasih, I., Toxicity of essential oils from orange (Citrus sinesis L. Obbeck) and lemongrass (Cymbopogon nardus L. Rendle) on Aedes aegypti a vector of Dengue Hemorrhagic Fever (DHF). J. Phys. Conf. Ser., 1869, 012015, April 2021. 19. Mendes, L.A., Martins, G.F., Valbon, W.R., de Souza, T.D.S., Menini, L., Ferreira, A., Da Silva Ferreira, M.F., Larvicidal effect of essential oils from Brazilian cultivars of guava on Aedes aegypti L. Ind. Crops Prod., 108, 684, 2017. 20. Pereira, E.J.P., Silva, H.C., Holanda, C.L., de Menezes, J.E.S.A., Siqueira, S.M.C., Rodrigues, T.H.S., Fontenelle, R.O., do Vale, J.P.C., da Silva, P.T., Santiago, G.M.P., Santos, H.S., Chemical composition, cytotoxicity and larvicidal activity against Aedes aegypti of essential oils from Vitex gardineriana Schauer. Bol. Latinoam. Caribe Plantas Med. Aromat., 17, 3, 2018.

Essential Oil Used as Larvicides and Ovicides  441 21. Balasubramani, S., Sabapathi, G., Moola, A.K. et al., Evaluation of the leaf essential oil from Artemisia vulgaris and its Larvicidal and repellent activity against dengue fever vector Aedes aegypti an experimental and molecular docking investigation. ACS Omega, 3, 15657, 2018. 22. Scalvenzi, L., Radice, M., Toma, L., Severini, F., Boccolini, D., Bella, A., Guerrini, A., Tacchini, M., Sacchetti, G., Chiurato, M., Romi, R., Larvicidal activity of Ocimum campechianum, Ocotea quixos and Piper aduncum essential oils against Aedes aegypti. Parasite, 26, 23, 2019. 23. Huong, L.T., Sam, L.N., Giang, C.N., Dai, D.N., Ogunwande, I.A., Chemical composition and larvicidal activity of essential oil from the rhizomes of Amomum rubidum growing in Vietnam. J. Essent. Oil-Bear. Plants, 23, 405, 2020. 24. Chaves, R.D.S.B., Martins, R.L., Rodrigues, A.B.L. et al., Evaluation of larvicidal potential against larvae of Aedes aegypti (Linnaeus, 1762) and of the antimicrobial activity of essential oil obtained from the leaves of Origanum majorana L. PLoS One, 15, e0235740, 2020. 25. Muturi, E.J., Selling, G.W., Doll, K.M., Hay, W.T., Ramirez, J.L., Leptospermum scoparium essential oil is a promising source of mosquito larvicide and its toxicity is enhanced by a biobased emulsifier. PLoS One, 15, e0229076, 2020. 26. Manh, H.D. and Tuyet, O.T., Larvicidal and repellent activity of mentha arvensis L. essential oil against Aedes aegypti. Insects, 11, 198, 2020. 27. Yang, S., Bai, M., Yang, J., Yuan, Y., Zhang, Y., Qin, J., Kuang, Y., Sampietro, D.A., Chemical composition and larvicidal activity of essential oils from Peganum harmala, Nepeta cataria and Phellodendron amurense against Aedes aegypti (Diptera: Culicidae). Saudi Pharm. J., 28, 560, 2020. 28. Melo, S.J., Sousa, J.P.B., Sa, M.G., Morais, L.S., Magalhaes, N.M., Gouveia, F.N., Albernaz, L.C., Espindola, L.S., Machaerium acutifolium compounds with larvicidal activity against Aedes aegypti. Pest Manage. Sci., 77, 1444, 2021. 29. Brandao, L.B., Santos, L.L., Martins, R.L., Rodrigues, A.B., Rabelo, E.D.M., Galardo, A.K., Almeida, S.S.D.S., Larvicidal evaluation against Aedes aegypti and antioxidant and cytotoxic potential of the essential oil of tridax procumbens L. Leaves. Sci. World J., 2021. 30. World Health Organisation, 2019, https://www.who.int/news/item/26-08-2019-vectoralert-­anopheles-stephensi-invasion-and-spread. 31. Xu, Y., Qin, J., Wang, P., Li, Q., Yu, S., Zhang, Y., Wang, Y., Chemical composition and larvicidal activities of essential oil of Cinnamomum camphora (L.) leaf against Anopheles stephensi. Rev. Soc. Bras. Med. Trop., 53, e20190211, 2020. 32. Vatandoost, H., Rustaie, A., Talaeian, Z., Abai, M.R., Moradkhani, F. et al., Larvicidal activity of Bunium persicum essential oil and extract against malaria vector, Anopheles stephensi. J. Arthropod Borne Dis., 12, 85, 2018. 33. Sheikh, Z., Amani, A., Basseri, H.R., MoosaKazemi, S.H., Sedaghat, M.M., Azam, K., Yousefpoor, Y., Amirmohammadi, F., Azizi, M., Development of mosquito protective textiles using nanoemulsion of Eucalyptus globulus and Syzygium aromaticum essential oils against malaria vector, Anopheles stephensi (Liston). Malar. J., 2021. 34. Debboun, M., Nava, M.R., Rueda, L. (Eds.), Mosquitoes, Communities, and Public Health in Texas, Academic Press, Netherlands, 2019. 35. Li, M.X., Ma, Y.P., Zhang, H.X., Sun, H.Z., Su, H.H., Pei, S.J., Du, Z.Z., Repellent, larvicidal and adulticidal activities of essential oil from Dai medicinal plant Zingiber cassumunar against Aedes albopictus. Plant Divers., 43, 317–323, 2020. 36. Huong, L.T., Huong, T.T., Bich, N.T., Viet, N.T., Ogunwande, I.A., Larvicidal efficacy of essential oils from the rhizomes of Zingiber castaneum against Aedes albopictus. Am. J. Essent. Oil Nat. Prod., 8, 23, 2020.

442  Essential Oils 37. Huong, L.T., Huong, T.T., Huong, N.T., Hung, N.H., Dat, P.T., Luong, N.X., Ogunwande, I.A., Mosquito larvicidal activity of the essential oil of Zingiber collinsii against Aedes albopictus and Culex quinquefasciatus. J. Oleo Sci., 69, 153, 2020. 38. Huong, L.T., Chinh, H.V., An, N.T., Viet, N.T., Hung, N.H., Thuong, N.T., Giwa-Ajeniya, A.O., Ogunwande, I.A., Zingiber zerumbet rhizome essential oil: Chemical composition, antimicrobial and mosquito larvicidal activities. Eur. J. Med. Chem., 30, 1–12, 2019. 39. Soonwera, M., Wongnet, O., Sittichok, S., Ovicidal effect of essential oils from Zingiberaceae plants and Eucalytus globulus on eggs of head lice, Pediculus humanus capitis De Geer. Phytomedicine, 47, 93, 2018. 40. Candy, K., Nicolas, P., Andriantsoanirina, V., Izri, A., Durand, R., In vitro efficacy of five essential oils against Pediculus humanus capitis. Parasitol. Res., 117, 603, 2017. 41. Qi, H., Wang, W.X., Dai, J.L., Zhu, L., In vitro anthelmintic activity of Zanthoxylum simulans essential oil against Haemonchus contortus. Vet. Parasitol., 211, 223, 2015. 42. Zhu, L., Dai, J.L., Yang, L., Qiu, J., In vitro ovicidal and larvicidal activity of the essential oil of Artemisia lancea against Haemonchus contortus (Strongylida). Vet. Parasitol., 195, 112, 2013. 43. Benelli, G., Govindarajan, M., Rajeswary, M., Vaseeharan, B., Alyahya, S.A., Alharbi, N.S., Kadaikunnan, S., Khaled, J.M., Maggi, F., Insecticidal activity of camphene, zerumbone and α-humulene from Cheilocostus speciosus rhizome essential oil against the Old-World bollworm, Helicoverpa armigera. Ecotoxicol. Environ. Saf., 148, 781, 2018. 44. Chennaiyan, V., Sivakami, R., Jeyasankar, A., Effect of Duranta erecta Linn (Verbenaceae) leaf extracts against Armyworm Spodoptera litura and Cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae). Int. J. Adv. Res. Biol. Sci., 3, 311, 2016. 45. Xue, G. and Yujian, R., Larvicidal and ovicidal activity of carvacrol, p-cymene, and γterpinene from Origanum vulgare essential oil against the cotton bollworm, Helicoverpa armigera (Hubner). Environ. Sci. Pollut. Res. Int., 27, 18708, 2020. 46. Deka, B., Pandey, A.K., Babu, A., Baruah, C., Sarkar, S., Acaricidal and ovicidal properties of Lippia alba essential oil and its chemical constituents against red spider mite, Oligonychus coffeae Nietner (Acari: Tetranychidae) infesting tea crops. Arch. Phytopathol. Plant Prot., 54, 1738–1752, 2021. 47. El-Aw, M.A.M., Draz, K.A., El Naggar, E.A., Abd Elsalam, F.H., Ovicidal, larvicidal and biochemical effects of Thyme, Thymus vulgaris, on the cotton leafworm, Spodoptera littoralis (boisd.). J. Plant Prot. Pathol., 12, 389, 2021. 48. Fernandes, D.A., Rique, H.L., de Oliveira, L.H.G., Santos, W.G.S., de Souza, M.D.F.V., da Cruz Nunes, F., Ovicidal, pupicidal, adulticidal, and repellent activity of Helicteres velutina K. Schum against Aedes aegypti L. (Diptera: Culicidae). Braz. J. Vet. Med., 43, e102120, 2021. 49. Bricarello, P.A., de Barros, G.P., Seugling, J., Podesta, R., Velerinho, M.B., Mazzarino, L., Ovicidal, larvicidal and oviposition repelling action of a nanoemulsion of citronella essential oil (Cymbopogon winterianus) on cochliomyia hominivorax (Diptera: Calliphoridae). J. Asia Pac. Entomol., 24, 724–730, 2021.

19 Essential Oil-Based Biopesticides Nishant Sharma1, Kunal Sharma2, Sachchidanand Soaham Gupta1, Kumar Rakesh Ranjan1*, Vivek Mishra3† and Maumita Das Mukherjee1 Department of Chemistry, Amity Institute of Applied Sciences, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India 2 Department of Pharmacology, Sri Krishna Medical College, Uma Nagar, Muzaffarpur, Bihar, India 3 Amity Institute of Click Chemistry Research and Studies, Amity University Uttar Pradesh, Noida, Uttar Pradesh, India 1

Abstract

The excessive and ever-increasing use of synthetic pesticides as a consequence of the rush to enhance agricultural output has had enormous negative consequences. They have grown as a major contaminant of soil and water, are very hazardous to humans and animals. Past few years have seen a significant increase in the usage of essential oils (EOs) derived from aromatic plants and are generally considered as low risk pesticides, owing to their popularity among organic growers and ecologically concerned customers. EO-based pesticides benefit also from the ongoing decline in the use of conventional pesticides as a result of regulatory action and rising pest resistance. The purpose of this chapter is to summarize the primary applications of essential oils as pesticides and its biological activity with different class of organisms such as insects, bacteria, fungi, Acari etc., as documented in the scientific literature. The chapter concludes with a section summarizing the major findings of the literature and discussing potential directions for the use of Essential oils as pesticides of the future Keywords:  Essential oils, insecticides, biopesticides, pest resistance, biological activity

19.1 Introduction Pesticide is a broad term that encompasses a diverse range of substances with a variety of distinct activities (for example, herbicidal agents, insecticides, nematicides, rodenticidal agents, algicides, fungicides, and bactericidal agents) [1]. While the adoption of synthetic pesticides in agronomic practices resulted in an increase in agricultural productivity, the continued demand for more efficient crop production resulted in an overuse of these compounds to the point where they became a key contaminant of agricultural soil and surface as well as ground water, posing a significant hazard to humans and animals [2]. In recent years, environmental dangers associated with pesticide use have sparked concerns among *Corresponding author: krranjan@amityedu † Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (443–464) © 2023 Scrivener Publishing LLC

443

444  Essential Oils scientists and the general public. There are two prominent reasons for this: (1) the elevated toxicity levels and nonbiodegradability of pesticides; and (2) their bioaccumulation in topsoil, aquifers, and crops has been shown to be detrimental to human health. In order to mitigate the risk of long-term damage to animals, one must find new highly selective and biodegradable pesticides, while on the other side, to lower pesticide use while preserving crop yields, one must look for environmentally benign pesticides and devise methods to employ such pest management methods. Biological systems are tremendously complex, yet they are also extremely accurate and efficient. Biological control is a well-established concept and has recently gained a great deal of attention for its application in the integrated management of crop pests [3]. Secondary metabolites from plants have evidently had an influence insect behavior, growth, and procreation. Recognizing these compounds is a fundamental first step toward comprehension plant-insect interactions. Numerous active phytochemicals have been identified as a result of ongoing research into plant-based pest control agents. In India, the concept of biocontrol of plant pests has been in practice dating back thousands of years and often much longer [4]. The neem tree (Azadirachta indica A. juss), including leaf extract, seed cake, and oil have been used as fertilizers and to help minimize post-harvest loss in stored cereals [5]. Essential oils (EOs) are found in all natural substances derived from plants and are thus regarded potential biocontrol agents. Introducing EOs as pest biocontrol agents may be more advantageous, according to previous study, since their toxicity is lower, they have a lesser environmental impact, and they have a better degree of specificity of action [6–8]. EOs demonstrated considerable anti-insect activity against a wide variety of stored grain insects, working both via ingestion and contact. Furthermore, they exhibited fumigant, repellent, and anti-feedant properties, among other things. EOs are also effective instruments in the management of bacterial and fungal plant diseases, as well as the management of microbial populations in stored products [9]. Pest insects respond differently to different EOs. The most significant effect is that they close up the air openings (spiracles) via which insects breathe, resulting in their death by asphyxiation. Additionally, EOs may have poisonous effects when they mix with the fatty acids of the insect and interfere with the insect’s regular metabolic process. EOs pose few risks. Generally, oils have low dangers to people or most species of plants and animals that are beneficial to control insect infestations. This is important because it encourages EOs to blend effectively with other biological controls agents [10]. In comparison to certain other plant phytochemicals, plant EOs have a significant history of human usage beyond pest control, most notably as fragrances, natural flavors, sauces, as well as spices, and also therapeutic purposes. EOs, on the other hand, have a bright future in the biopesticide industry; it is paradoxical, therefore, that commercial pesticides derived from plant essential oils have only been developed in the past few decades. Although essential oil-based insecticides have a number of advantages, they also have some disadvantages (for example, limited physical stability, poor water solubility, and rapid degradation in the environment, as well as a scarcity of the raw material from which they are derived) that are associated with the extensive flexibility in their chemical composition. These barriers make it harder to achieve their goals. This chapter will discuss the present state of knowledge and recent advances in the phytochemistry of plant EOs, their biological activity in a variety of species, and their potential as biopesticides.

Essential Oil-Based Biopesticides  445

19.2 Phytochemistry and Sources of Essential Oils EOs are described as any volatile oil(s) that include significant aromatic components that provide a plant’s characteristic odor, flavor, or fragrance. They are present as droplets of fluid in the leaves, stems, bark, flowers, roots, and/or fruits of various plants. They can also be found in glandular hairs or secretory cavities of the plant cell wall [11]. From a chemical standpoint, EOs are a complex and dynamic combination of compounds that are unique to each plant and extraction technique. They are lipophilic and volatile, with a near-insoluble in water [12]. These oils usually contain 20-60 compounds but may contain approximately up to 100 different compounds. EOs are utilized by florae to defend themselves from pathogens, viruses, and fungal infections alongside herbivores, hence essential oils have a significant impact on plant defense. Additionally, they may attract some insects to aid in pollen and seed dispersal, while repelling others. EOs are yielded by approximately 17,500 odorous species of vascular plants, the greater part of which are classified in a handful of families, including the myrtle family (Myrtaceae), the laurels (Lauraceae), the Labiatae (Lamiaceae), and Asteraceae (Compositae). Essential oils account up a minuscule portion of the wet mass of plant material, intricate secretory ducts and orifices [13]. Table 19.1 summarizes the most frequently used essential oils in pesticide formulations, and key components. Essential oils (EOs) are products derived through volatile phytochemicals, hydrodistillation uses indirect steam from outside the still and the plant contents are heated in three times the weight of water, as opposed to direct steam, which is created in the still and used in steam distillation. Hydrodiffusion replaces plant volatiles with osmotic action or CO2 Table 19.1  Some common plant essential oils and primary constituents. Plant essential oil (EO)

Primary constituents (in %)

Ref.

Cinnamon EO

Eugenol (5–18%); Cinnamaldehyde (55–76 %)

[14]

Citronella EO

Citronellol (10–16%); Citronellal (27–33%); Geraniol (24–40%)

[14]

Clove EO

Eugenol (89%)

[14]

Eucalyptus EO

1,8-Cineole (67–84%)

[14]

Lemongrass EO

Neral (5–51%); myrcene (9–25%); Geranial (34–45%)

[14]

Mint EO

Menthone (10–40%); Menthol (30–55%)

[14]

Orange EO

d-Limonene (91–97%)

[14]

Peppermint EO

Menthone (20–46%); Menthol (7–48%)

[15]

Rosemary EO

α-pinene (8%); Camphor (9%); 1,8-Cineole (52 %)

[16]

Tea tree EO

α-terpinene (14–28%); Terpinen-4-ol (35–48%)

[17]

Thyme EO

p-cymene (33%); Thymol (50%)

[18]

446  Essential Oils Terpenes

p.cymene

HO

Betapinene

Sabinene

Alpha-pinene

Citronellol HO HO Carvacrol

HO

Thymol

Geraniol

Sesquiterpenes

H

H HO Farnesol

Caryophyllene

Aromatics

O

OH

Cinnamaldehyde

Cinnamyl alcohol

HO

HO

O Eugenol

p-Allylphenol O

O

O

O Estragole

Anethole

Safrole

Terpenoids (Isoterpenoids)

O O Ascaridole

HO Menthol

Figure 19.1  Chemical structures of selected components of essential oils.

Essential Oil-Based Biopesticides  447 extraction; cold pressing is a particular approach for extracting Citrus (Rutaceae) peel oils from fresh or dried out plant material [19]. Modern extraction techniques encompass a wide range of routes such as microwave-assisted as well as supercritical fluid extraction, which offer a number of advantages, particularly decreased chemical oxidation, which is vital for flower scents. These procedures necessitate fractionation of the extracts to clean up, as they also yield higher-molecular-weight (MW) lipids. Alternatively, the original steam distillation process extracts a fairly pure auxiliary metabolite portion from plants, composed largely of low MW volatile phytochemicals of terpenoids and phenolic origin, and discounting the majority of principal metabolites and high MW auxiliary derivatives with widely varying structures and modes of action [20]. While commercial botanical pesticides frequently allude to a solitary natural product as the effective/active component, the majority of chemical defense in plants is composed of compendiums of natural chemicals produced from a unique biosynthetic route. The majority of EO constituents are terpenes (low MW monoterpenes and sesquiterpenes) and, to a certain degree, phenylpropanoids. Terpenoids are the primary components of essential oils. Terpenes form structurally and functionally diverse classes. They are composed of different arrangements of multiple 5-carbon-base (C5) units known as isoprene. Monoterpenes are generated by combining two isoprene molecules (C10); 90% of essential oils are made up of these molecules, which come in a wide variety of structures. Sesquiterpenes are generated by assembling three isoprene molecules (C15). Aromatic molecules, which are acquired from phenylpropane, are found in far lower abundance than terpenes (Figure 19.1) [11]. Clove oil (Syzygium aromaticum, eugenol), Thymus vulgaris oil (thymol, carvacrol), Mentha oil (menthol, pulegone), Cymbopogon (lemongrass) oil (citronellal, citral), Cinnamomum verum oil (cinnamaldehyde), rosemary oil (1,8-cineole), and oil of oregano are among the most widely recognized EOs with bioactive components showing activity against insects and other pests.

19.3 Biological Activity of Essential Oil Biopesticides According to a literature review, the anti-insecticide effects of plants from the Myrtaceae, Lamiaceae, Asteraceae, Apiaceae, and Rutaceae families have been carefully researched against arthropods, especially particular insect orders including but not limited to Lepidoptera, Coleoptera, Diptera, Isoptera, and Hemiptera [21]. EOs and their elements are predominantly lipophilic chemicals that behave as toxic substance, deterrents to feeding, repellents, oviposition deterrents, and all the more so attractants to a broad array of insect pests. Additionally, these EOs have a long history of usage for preserving stored products. This section highlights the mode of action and efficacy of EOs against various classes of pests and other organisms.

19.3.1 Efficacy of Essential Oils to Insects Insecticides are one of the most significant types of pesticides because they can diminish the damage caused by pests and increase agricultural output. At the same moment, insects can cause a variety of significant ailments, including yellow fever and act as vector of viruses

448  Essential Oils like dengue and chikungunya [22]. The desired application is what distinguishes insecticides from insect repellents (insect repellents are typically intended to safeguard humans, whilst insecticides are intended for agrarian use), as well as by the pesticide-targeted pest interaction route: primarily, insecticides function through direct contact, whereas insect repellents are a class of compounds, which creates an atmosphere within 4 cm of the skin and thus thwart the direct interaction of the insect/skin [22]. The instant onset of intoxication in insects and other arthropods from EOs or their components suggests a neurotoxic mechanism of action. Octopamine, a neurotransmitter acts directly when it interacts with at least two distinct kinds of receptors, octopamine-I, and octopamine-II. Thus, when octopamine’s function is disrupted, the nervous system of insects becomes completely dysfunctional. Consequently, the insect octopaminergic system is indeed a biorational target for pest management. The absence of octopamine receptors in vertebrates presumably explains the EOs’ strong selectivity in mammals as insecticides. Numerous elements of essential oils have been shown to act on insects’ octopaminergic system [23]. This effect also includes the neuro-immunogenic toxic action of EOs via inhibiting acetylcholinesterase (AChE), which is also a neurotransmitter (Figure 19.2). Additionally, the γ-aminobutyric acid (GABA) receptor has been postulated as one mechanism by which EOs produce noxiousness in insects. They could interfere with GABA-gated chloride channels in insects. Some monoterpenes, such as thujone, can cause neurotoxicity in insects by binding to GABA receptors in their nervous systems. On top of this, effects on the pheromone systems and hormone as well as cytochrome P450 monooxygenase have been observed in the presence of EOs on insect populations (Figure 19.3) [24]. Physical effects, for instance dissolution and disruption of cell membranes, as well as blocking of the tracheal system, could perhaps occur in insects as alternative mechanisms of toxicity. Research has established that a monoterpenoid (linalool) affects the nervous system of insects by disrupting ion transport and acetylcholine esterase release [25]. Enan demonstrated that eugenol toxicity was increased in mutant D. melanogaster lacking octopamine production, implying that the toxicity is arbitrated via the octopaminergic system, but with geraniol, that wasn’t the case. It was hypothesized that the insecticidal activities of eugenol are attributable to the cellular alterations it causes [26]. A separate research group arrived at a similar conclusion, suggesting that eugenol may have the ability to activate octopaminergic receptors. They discovered that even at low doses, there were substantial impacts. in Helicoverpa armigera’s abdominal epidermal tissue [27]. Govindarajan et al. [28] conducted an intriguing study in 2016 suggested that EOs could be used to effectively control harmful three mosquito species larvae. According to Lopez et al. [29], Coriandrum sativum oil is extremely poisonous to S. oryzae, R. dominica and C. pusillus, whereas camphor

OH NH2

O

O O

N+

H2N

OH

HO Octopamine

Acetylcholine

Figure 19.2  Octopamine, acetylcholine, and GABA neurotransmitter.

γ−Aminobutyric Acid

Essential Oil-Based Biopesticides  449 EOS and/or their constituents Direct Toxicity

Indirect Toxicity

Blocking

Inhibition of AChE Octopamine receptor GABA receptors

Hormones

IGR

Pheromone

Cytochrome P450

Neurotoxicity Biochemical and Physiological dysfunction Symptoms DEATH

Hyperactivity Convulsions

Tremors

Paralysis

Figure 19.3  Mechanism of action of EOs in essential oils in insects. (Adapted from [24]).

components are extremely poisonous to R. dominica and C. pusillus. Given that the majority of monoterpenes are harmful to animal and plants tissues, several researchers attribute the primary insecticidal activity of EOs to these molecules [29]. The mechanism by which EOs function as insecticides or repellents varies according to the route of application.

19.3.2 Essential Oils as Insect Repellents The compounds that act locally or distally to repel insects are known as insect repellents. Since EOs have a distinct smell and are of a volatile nature, they appear to have strong repellent potential. However, there is no agreement as to how repellents function in different arthropods, as there are conflicting observations, such as different insects having distinct organs but responding to the same repellent. Mosquito antennae hairs are also known to be sensitive to temperature and moisture but the receptors responsible for the cockroach’s repulsion reaction are not well understood. Unfortunately, study in this area has produced few results that have been recognized. Jiang et al. [30] investigated the insect repellent and insecticidal properties of EOs derived from Cinnamomum camphora L. Presl leaves, twigs, and seeds versus the cotton aphid (Aphis gossypii Glover). The seeds’ EO produced the highest results (LC50 observed at 146.8 mg/L after 2 days, 89.9 % repellence at 20 ml/L concentration of EO after 1 day). Another group, Kerdudo et al. [31] has also examined the repellent and insecticidal activity of the EO of P. carolinensis (Jacq.) against the yellow fever vector mosquito (Aedes aegypti), getting excellent outcomes.

450  Essential Oils In a laboratory setup, certain EO compounds repel cockroaches. Marketable pest-­control solutions based on EOs, and especially insect sprays, are likely to work to some extent since these compounds flush roaches from their hiding places and discourage their return [32]. Another possible use of EOs that has yet to be realized is impregnating repellents into packaging to keep insects out. It is critical to keep in mind that repellents may also be beneficial in integrated pest management (IPM) of agrarian crops, especially when used in conjunction with a stimulodeterrent diversionary strategy (SDDS) or “push-pull” method [33]. A huge number of research studies where plant EOs were evaluated and reported for anti-­ insect efficacy [30, 34–50]. When the entire available data is analyzed together, a variety of plant species arise as frequently effective against insects, even if their relative effectiveness varies significantly amongst different pest types. The essential oils of thyme, oregano, basil, rosemary, and mint are among the most active, but much more experiential research with less familiar plant species and a broader selection of pest species will likely discover exceptionally beneficial biological activity.

19.3.3 Bactericidal Properties of Essential Oils Bacteria that cause plant diseases hold the potential to impart a significant commercial blow to the harvest. For instance, consider the bacterial infections produced by Xanthomonas spp. impact a wide variety of plant species, wreaking havoc on them and resulting in yield and crop quality losses [51]. While interest in EO as antibacterial in a plant pathogen viewpoint has increased, it is still insignificant (Table 19.2), and the majority of research on EO’s antibacterial activities focus on food preservation or health issues. Essential oils have been shown to have antibacterial activity against Gram-negative and Gram-positive bacteria in both their motile and sessile stages. While the bulk of EOs are considered to act on bacteria’s cell wall and membrane, additional study is required to ascertain the mechanism of action of particular oils. Pathogens exhibit a wide range of responses and susceptibility to EO or its primary constituents. For example, it has been found that the effect of basil EO on diverse microorganisms elicited a variety of inhibitory responses, against a broad spectrum of infections. It was shown to be very effective against P. tolaasii but had no effect on B. nigrifluens. Additionally, X. citri and R. fascians were kept inhibited, albeit to a greater extent than P. tolaasii. On the other hand, O. onites has been shown to be effective versus C. michiganensis and Xanthomonas spp. with reliable inhibitory zones [61].

19.3.4 Antifungal and Anti-Oomycete Properties of Essential Oils Many plant EOs along with its components have shown antifungal action versus a variety of plant infective fungi, comprising those accountable for both pre- and post-crop harvest diseases. According to Freiesleben and Jager [61], EO based antifungal agents can render the fungus inactive by affecting the structure and function of the fungal cell’s membranes or organelles and/or by suppressing nuclear material or protein production. Isman and Machial [62], concur that their action mechanism is undetermined but is likely related to their ability to dissipate the integrity of fungal membranes and cell walls. Numerous studies have shown that the response of phytopathogenic fungi to EOs differs; for example, black caraway and fennel essential oils inhibit Botrytis cinerea, but peppermint

Essential Oil-Based Biopesticides  451 Table 19.2  Examples of EOs exhibiting antibacterial activities against phytopathogenic bacteria. Essential oil source

Targeted bacteria

Disease caused

Refs

C. michiganensis

Ring rot

[52]

R. fascians

Leafy gall

[53]

C. michiganensis

Ring rot

[54]

A. biebersteinii

Pseudomonas spp.

Bacterial canker

[52]

C. aurantium L.

A. tumefaciens

Crown gall

[56]

E. amylovora

Fire blight

C. scolymus (stems)

E. amylovora

Fire blight

E. carotovora

Soft rot

E. africanus L.

A. tumefaciens

Crown gall

D. solani

Black leg and soft rot

E. amylovora

Fire blight

E. carotovora

Soft rot

A. vitis

Crown gall

B. nigrifluens

Cankers

Pseudomonas spp.

Bacterial canker

Xanthomonas spp.

Blights and bacterial spots

E. amylovora

Fire blight

X. vesicatoria

Bacterial leaf spot

P. syringae

Bacterial canker

E. carotovora

Soft rot

Pseudomonas spp.

Bacterial spots and blights

[55]

E. amylovora

Fire blight

[57]

E. carotovora

Soft rot

A. tumefaciens

Crown gall

Pseudomonas spp.

Bacterial canker

A. millefolium O. ciliatum O. heracleoticum T. fallax

Gram positive

A. biebersteinii

J. regia L. (shells) O. ciliatum O. onites

O. vulgare S. scandens S. hortensis S. aromaticum Tanacetum aucheranum

Gram negative

Xanthomonas spp.

[57]

[58]

[57]

[53]

[53]

[57]

[59]

[60]

452  Essential Oils does not. The addition of phenolic (fennel EO) or aromatic (black caraway) compounds to B. cinerea seems to have a greater antifungal impact. Similarly, P. digitatum was shown to be sensitive to thyme and summer savory essential oils but not to fennel or sweet basil essential oils, while Aspergillus sp. was found to be sensitive to EOs from clove, lemongrass, thyme, and oregano but not to ginger and cinnamon [61]. Furthermore, these studies show that EO can neutralize fungi that cause plant harm both during farming and during post-harvest diseases, such as Penicillium species, G. citri-aurantii, and R. stolonifer.

19.3.5 Herbicidal/Weedicide Properties of Essential Oils Weeds are an important concern in agriculture. Owing to the invasion of weeds, crop production is significantly affected in terms of quality and quantity, resulting in significant losses for crop farmers. EOs may possibly be a potential control technique for unwanted plants (weeds) in organic farming and integrated pest management systems. Whilst synthetic herbicides are used extensively, they can have a wide variety of hazardous impacts on both the flora and fauna [50]. Although EO-based herbicides have the potential to alleviate many of the drawbacks of synthetic herbicides, certain of the chemical and physical features of EOs, for instance their high volatility and limited water solubility, can prove to be a limitation [63]. EOs has been synthesized by a number of researchers in the past, and their herbicidal and phytotoxic effects have been tested (Table 19.3). Tworkoski [77] conducted laboratory and greenhouse studies with EOs from various plants in order to determine the herbicidal effect and identify the active constituent with herbicide activity. EOs at aqueous strengths ranging from 5% to 10% (v/v) were utilized to new growth shoots of common lambsquarters Chenopodium album, common ragweed Ambrosia artemisiifolia, and johnsongrass Sorghum halepense in the greenhouse; shoot death was reported with initial inception within 1 hour of application and effecting even 1 day after application. Cinnamon EO (1 %, v/v) was one of the most phytotoxic, causing electrolyte leakage and cell death. Eugenol was confirmed to be the active ingredient in the investigated EOs. At a dosage of 0.4 mL/L, C. citratus was the most effective, successfully reducing weed populations by 100% compared to the leading herbicides, which were glyphosate (36.5% effectiveness at 1 mL/L) and 2.4-D (100% at 2 mL/L). Selectivity is one of the most important factors to consider when contemplating the use of EOs as a weedicide/herbicide, because the formulation should primarily target weeds rather than crops, as Sharma et al. shown [78]. The modulation of mitochondrial respiration is thought to be the most common mechanism by which EOs act as herbicides, followed by damage to membrane integrity, and oxidative stress, which changes pH homoeostasis and inorganic ion balance [79]. In terms of herbicidal use, it is apparent that cellular metabolic processes contribute to the phytotoxic effects of EOs. The research establishment is achieving progress in understanding the affected biological functions, such as photosynthesis and respiration, and in discovering molecular mechanisms. However, given the numerous interconnected pathways involved, no clear differentiation among the different chemical classes of EOs’ components has yet been established. A bulk of them is included within a single EO, which complicates the task of identifying the appropriate mode of action.

Essential Oil-Based Biopesticides  453 Table 19.3  Examples of EOs exhibiting herbicidal activities against various plants. Plant source of the EOs

Weeds/plant species tested against

References

Achillea gypsicola

A. retroflexus

[64]

L. serriola R. crispus Achillea biebersteinii

A. retroflexus R. crispus

Angelica glauca

L. minor

[65]

Citrus aurantiifolia

A. fatua

[66]

P. minor Coriandrum sativum L.

C. album

[67]

E. crus-galli Eucalyptus spp.

12 Mediterranean species

A. ryegrass

[68]

E. crus-galli

[69]

P. minor

[70]

L. sativa

[71]

R. sativus Origanum acutidens

R. crispus

[72]

C. album Origanum vulgare L.

H. vulgare

[73]

T. aestivum Rosmarinus officinalis

R. sativus

[74]

S. marianum

[75]

T. incarnatum Syzygium aromaticum

C. lambsquarters

[76]

R. pigweed Tanacetum aucheranum

A. retroflexus C. album R. crispus

Tanacetum chiliophyllum

A. retroflexus

[60]

454  Essential Oils

19.4 Synergistic Formulations of Essential Oils Synergists are also referred to as activators or adjuvants. The term “synergistic” or “synergistic” refers to the act of cooperating/working together. These terms originate from the Greek word synergid, which implies cooperation (syn = “together,” ergon = “work”). Synergists are compounds that have little or no insecticidal activity of their own but significantly increase the toxicity of an insecticide when combined with it. Thus, the objective of the synergistic formulation or combination is to reduce the dose of insecticide used, thereby lowering the danger of resistance development and environmental contamination. Now, owing to the commercialization of standardized industrial solutions, the use of handcrafted products is rapidly reducing. Cotton damage caused by the bollworm, Helicoverpa armigera, can be diminished by mixing extracts of three local plants with standard insecticides at 50% of the recommended strength (Azariracthta indica, Khaya senegalensis, and Hyptis sauveolens). This blend or formulation outperforms the efficacy of the pesticide when used alone. The synergistic reason for combining chemicals is to create a dynamic product with several pathways of activity, while adhering to the premise that the blended/combination’s overall activity of the product exceeds the sum total of its known and unknown active ingredients. EOs can work together to create a synergy that can then be negated by the base product used to create the synergy [21]. Low pH and salinity have been shown to have a substantial effect on the action of essential oils such as thyme, anise, and saffron [80]. When combined with other monoterpenes, Hummelbrunner and Isman [81] proved that they had a synergistic impact on mortality and produced a monoterpene combination that included 0.9 % active component to be used against the foliar feeding pests.

19.5 Toxic Effects of Essential Oils on Mammals and Non-Target Organisms As a whole, the extensive use of plant EOs in medicines and foods and therapeutics raises the likelihood that EOs are not harmful to mammals in most cases. While the biological impacts of specific chemical constituents of EOs are well understood, determining the toxicokinetics of their combinations is to a greater extent vastly challenging. Moreover, one of the most distinctive aspects of EOs is their low-risk profile. Their toxicity to mammals is low, and they have been subjected to extensive experimental and clinical research as a result of their usage as therapeutic medicines. A large percentage of EOs have an oral LD50 of between 2,000 and 5,000 mg/kg in rats, notably chamomile, anise, lavender, clove, citronella, eucalyptus, and marjoram. A classification of EO components’ toxicological potential has been established, with three structural classes being distinguished [82]. Class I substances, such as the aliphatic compound limonene, have an oral toxicity that is on the lower side. Intermediates are Class II substances that have some functionality. Due to their reactive nature, Class III substances have a significant potential for toxicity. These classes are related to the NOEL (no observed effect level), the level at which the substances have no observable impact. A system for

Essential Oil-Based Biopesticides  455 assessing the safety of essential oils has been devised on the basis of this classification and other factors [83]. Some EOs, or at least the monoterpenes contained within, were found to be dermal irritants. Wintergreen, clove, eucalyptus, and sage essential oils, for example, are well-identified for their irritating capabilities. D-limonene generates higher irritant transdermal absorption. Photosensitivity is caused by the essential oils of bergamot and angelica (Angelica archangelica). Tea tree oil, for example, can be irritating to the skin. Thujone for instance is neurotoxic. Despite the fact that the long-term toxicity of essential oils is poorly understood, the literature contains some instances of deleterious consequences associated with long-term animal and human use. EOs are employed in veterinary treatment, and some EOs have exhibited toxicity following repeated administration. When cats and dogs are given essential oils of wintergreen, sassafras, tea-tree oil, or pennyroyal, they develop disease symptoms [84]. Despite the fact that the vast majority of essential oils are not especially hazardous, some must be handled with utmost care. Some essential oils (EOs) have toxicities that are distinct from those of the plant from which they are derived, despite the fact that the plant’s safety is well acknowledged. It is important to remember that risk encompasses both hazard and exposure. Exposure can occur through skin contact or inhalation in the perspective of plant protection with an EOIn terms of ecological toxicity, EOs are quite benign for use, although they do come with certain concerns.

19.6 Advantages, Current Constraints and Long-Term Prospects The application of EOs is a rapidly growing field of research, with particular emphasis on their potential insecticidal, herbicidal, antifungal, and antimicrobial properties (covered in

EOs and/or their constituents Insects, acari, nematodes Direct Action

AChE inhibition, octopamine and GABA receptors blocking

Neurotoxicity

Indirect Action Effects hormone, pheromone systems, cytochrome P450, insect growth regulator

plants (weeds) fungi/oomycetes Cell membrane disruption Inhibition of cell wall formation Dysfunctions of the fungal mitochondria Inhibition of ef flux pumps

Effect on photosynthesis, respiration, cell division, enzyme function and activity, action on the molecular level and gene expressions; inhibition of mitochondrial respiration, oxidative stress

Dysfunctions at biochemical and physiological levels

DEATH

Figure 19.4  General pathways of EOs’ pesticide action discussed in this chapter.

456  Essential Oils this chapter; Figure 19.4), as well as their antiviral properties (not covered in this chapter), based on the properties of constituent compounds. However, despite the fact that the biological properties of EO against a diverse range of species have been widely investigated in vitro, their mechanisms of action have received little attention. Our understanding of the antibacterial mechanism of the EO is exceedingly restricted as a result of the lack of investigations on its antibacterial activity. Many of the commodity essential oils are relatively well documented and understood in terms of their safety for humans. Because of their short field lifespan, they are able to cohabit with biocontrol agents and pollinators. Similarly, their minimal bioaccumulation improves farmers’ safety and implies that foods have little or no residues. Finally, because several EOs are affordable in abundance, bioinsecticides based on these active components are economically viable, at least at current pricing. EO based pesticides offer numerous advantages, a few of them are listed below • Broad-spectrum pesticides possess insecticidal, antifeedant, repellent, oviposition deterrent, growth regulatory, and antivector activities. • They are useful in foodstuffs and stored foods. • Reduced-risk pesticides are nontoxic to mammals and fish. • They are widely used as flavoring agents in beverages and foodstuffs. • Commercialization is possible due to abundant availability. • Green pesticides are largely used against home and garden pests. • There is slow pest resistance due to complex mixtures of several compounds. • There is a unique impact on integrated pest management. • There is limited persistence and high volatility. • There is no harm to predators, parasitoids, and pollinators. Even though EOs offer numerous advantages for application as sustainable biocontrol agents, there are certain limitations, which needs to be addressed. All oils, regardless of origin, have the potential to be phytotoxic when applied in excessive amounts to plants, EOs are no different. To determine the risk of phytotoxicity associated with a particular crop application, items must be empirically tested. Additionally, while it is predicted that minimal or no residues would remain on food after the spraying of essential oil-based pesticides, it is uncertain whether or not this approach will have an impact on the organoleptic characteristics of food. Few other limitations of EO-based insecticides are as follows: • • • •

Few pest control products are available on the market. A greater application rate and frequent reapplication are required. There is poor specificity due to the presence of several compounds. There is a lack of sufficient supply, protection technology, and regulatory approval. • There is inconsistency in raw material composition obtained from plants grown in different geographical, genetic, climatic, and seasonal areas. • There is minimum involvement from low-budget companies. • Oil can have an adverse effect on the germination power of the treated seeds. Numerous new researches have come up which has transformed the way EOs have been and will be used in the future such as nanoemulsions, EO encapsulation etc. These methods

Essential Oil-Based Biopesticides  457 have proven to be more effective than the conventional methods of fumigation, spraying etc. Incorporating EO active chemicals into nanoemulsion formulations can be used to generate biodegradable coatings that increase the quality and biological characteristics of the active compounds used in the coatings. In essence, EO emulsions, particularly nanoemulsions, may be of considerable importance for assuring either controlled release of the EO components and improved product stability or enhanced biological qualities. Several findings have already proven that when EO is produced in nanoemulsions, its efficiency, stability, and even bioavailability is increased. Another developing technique is the encapsulation of EOs, with the ability to improve the product’s stability and enable a controlled release. Encapsulation has emerged as a novel way for enhancing the stability and efficacy of EO by addressing some of the disadvantages inherent in raw EO use. In particular, the problem of minimizing the EO impacts in fields can be handled by employing EO encapsulation and regulated release [61]. Numerous articles evaluated discussed the materials’ selectivity. The suggested pesticides (whether herbicides, insecticides, or other activities) should have a high selectivity for the targeted organisms, harming as few nontarget organisms as is reasonably possible in the process. Future studies should consider the advantages that biotechnology can provide, from cocultures which can be utilized for pesticidal activity probing to engineering plants with superior EO content or higher concentrations of biologically active terpenoids [85].

19.7 Conclusion There are several practical applications for the enthralling field of EOs in a variety of fields. Within those areas, the use of essential oils (EOs) to replace synthetic pesticides that are currently in use has the potential to result in a significant improvement in the overall quality of life while also serving as an effective tool for foiling evolution of targeted pest resistance. This is largely because of increased public demand for pest management products that have less adverse effects on human health and the environment, jointly in agronomy in societal wellness and urban pest management. In this chapter, we have discussed in detail, with the help of current literature, various activities of EOs as pesticides and their mode of action. EO has shown effective actions against insect-pests, bacteria, weeds, and fungi, both as pre- and post-harvest biocontrol agents. They also support different modes of application – spraying, emulsification, fumigation etc. It must please keep in mind that biocontrol does not come without hazards. In this situation, the environmental danger associated with EO is substantially lower than that associated with synthetic pesticides, owing to EO’s volatile nature, which results in a large reduction in persistency when compared to commercial (synthetic inorganic and organic) pesticides. A variety of new technologies, such as EO formulation by means of emulsions or encapsulations, may permit EO to be applied more widely, as a process of enhancing simultaneously their biological activity and  chemical stability. When employing these approaches, however, it is necessary to consider economic concerns. Use of EO, which has been preferentially formulated in accordance with a few considerations, in conjunction with synthetic pesticides, in a traditional pesticide crop management system, could be a feasible and gradual transition, allowing for a reduction in pesticide use and the implementation of an IPM system in place of traditional pesticide crop management systems. Furthermore,

458  Essential Oils authorizations for the commercialization of biopesticides (and even more so for the commercialization of synthetic pesticides) are issued only after a lengthy and complicated process. Toxicological assessments are either unavailable or prohibitively expensive and time consuming for small and medium-sized enterprises, such as local manufacturers, and hence not available at times. One of the biggest hindrances to the commercialization of EO compounds as biopesticides may possibly therefore be a result of regulatory and governing obstacles. Nonetheless, the present trend toward a reduction in the use of synthetic pesticides may make it possible for EO products to be produced and utilized in a variety of locations around the world. Recent industry forecasts predict strong growth in markets for biopesticides over the next five years, and manufacturers of pesticides derived from plant essential oils should be well positioned to capitalize on expanding market opportunities and deliver on the promise of these products first predicted more than two decades ago.

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20 Essential Oils Obtained from Algae: Biodiversity and Ecological Importance Deprá, M. C., Dias, R. R., Nascimento, T. C., Silva, P. A., Zepka, L. Q. and Jacob-Lopes, E.* Bioprocess Intensification Group, Federal University of Santa Maria, UFSM, Santa Maria, RS, Brazil

Abstract

Historically, essential oils have been used mainly under the view of popular medicine. However, with the advancement of science, discoveries about its biological potential aroused interest in the most diverse industrial applications. Since then, not only from plants, new matrices of essential oils, such as micro and macroalgae, have emerged as potential sources to further leverage the market for natural products, in addition to trying to meet the demand of consumers thirsty for healthy lifestyles. In light of this, this chapter had an overview of essential oil conception from microorganisms such as micro and macroalgae. In addition, the chapter discusses volatile compounds from a structural and biological point of view, which is attributed to essential oils. Subsequently, a literature review is deeply described the ecological importance and biodiversity of micro and macroalgae under this new market perspective. Finally, future perspectives associated with these products in latent progress will be examined in order to provide a close look at this new industrial niche. Keywords:  Chemical structure, volatile organic compounds, microalgae, essential oil, biological activity

20.1 Introduction The market for natural products in the world seems to have advanced into an exponential growth that is difficult to stop. As is well known, this trend is sustained, above all, by a change in consumer behavior, which has inevitably driven the market to address and adapt to increasingly organic processes [1]. Under the same premise, the essential oils market has also gained prominence in this niche, and it seems to have taken off in the last five years. Proof of this is the market demand for these products. Currently, it is estimated that the production of essential oils reached a volume of 247.08 kilotons in 2020, generating a market value of approximately USD 18.62 billion. In addition, revenue forecasts project values up to USD 33.26 billion for 2027, under a compound growth rate (CAGR) of 7.5% [2].

*Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (465–476) © 2023 Scrivener Publishing LLC

465

466  Essential Oils Apparently, the rise of the essential oils market is associated with the potential health benefits correlated to its biological properties [3]. Thus, the inclusion of these compounds in the food, pharmaceutical, and medical sectors, in addition to personal care related to aromatherapy, has increasingly received the attention of decision-makers in research, development, and innovation (RD&I) groups in order to include them in the most diverse industrial segments [4]. In practice, industries, in general, have used essential oils from secondary plant metabolisms. However, due to the high metabolic versatility of micro and macroalgae, its potential to generate volatile compounds, such as essential oils, has been verified and, therefore, has become an important subfield of the chemistry of micro and macroalgae bioproducts [5]. In this sense, this chapter addresses the basic concepts of the chemistry of essential oils from micro and macroalgae. In addition, throughout the chapter, they are deeply discussed under the chemical-structural aspect and their relationship to the biological activities of these compounds. The chapter also takes a critical approach to the ecological relevance of essential oils from the point of view of the marine system. Finally, some future perspectives associated with these products in latent progress will be examined in order to provide a close look at this new industrial niche.

20.2 What are Essential Oils? Essential oils can be understood as colorless liquid substances (at room temperature), which have a strong characteristic aroma associated with the extraction matrix. They are volatile in nature, have low water solubility, and are unstable at high temperatures, light and oxygen, and soluble in ether, fixed oils, and alcohol [6]. Due to the range of their characteristics, essential oils are applied in various industrial sectors, such as pharmaceuticals, food, cosmetics, and perfumery. In the point of cosmetic products, fragrances, and household products, they are mainly incorporated for their pleasant odor. In the food sector, it is used for presenting antimicrobial properties and food preservatives resulted from their variety of active constituent present (for example, terpenes, terpenoids, carotenoids, coumarins, curcumins) [7]. The mainstream components of essential oils are based on molecules as terpenes, whose organic compounds consist of multiples units of isoprene (comprising five carbon atoms). However, essential oils can contain considerable amounts of straight-chain, aromatic, or heterocyclic compounds [8]. Also, hydrocarbons and oxygenated compounds, such as alcohols, aldehydes, ketones, acids, esters, oxides, lactones, acetals, and phenols, are responsible for the characteristic odors and flavors [9]. Thus, essential oils from distinct plants exhibit a wide spectrum of biological and antibacterial activities that are attributed, in some cases, to the phenolic compound attendances [10]. Reports indicate that the essential oil trade arose from ancient India, Persia, Egypt, Greece, and Rome, which had extensive trade in aromatic oils and ointments in nations of the East. In the past, these products were prepared by placing flowers, roots, and leaves in fatty acids [11]. Thus, it is suggested that the technique of distilling essential oils was only developed in the golden age of Arab culture. In this way, it was the Arabs who pioneered the distillation of ethyl alcohol from fermented sugar, providing an innovative solvent for the extraction of essential oils in place of the fatty acids that were certainly used in the past [8].

Essential Oils Obtained from Algae  467 However, with the advances in research towards obtaining more and more natural products, it resulted in an increase in the options of matrices for obtaining essential oils. Thus, it is believed that today, more than 300 essential oils are used in various industrial plants [12]. Consequently, these substances already constitute derivatives of the most diverse chemical classes [13]. Otherwise, due to the expansion and substantial demand, the search for new matrices was necessary. Associated with this, seaweed essential oils have gained great relevance to the natural-based essential oils market. This is because the chemical arrangement of essential oils and volatile fractions of micro and macroalgae includes compounds commonly recognized in conventional matrices. At the level of exemplification, chemical structures as hydrocarbons, oxygenated hydrocarbons, and terpenes were found. Besides, the volatile fractions of micro and macroalgae develop a spectrum portfolio of biological activities, including antibacterial, antifungal, anticancer, and antibiotic activities [14]. Today, at the industrial level, the incorporation of these compounds is often used to extend the shelf of minimally processed foods [15]. Therefore, although recent researches have gone deeper on the promising effects related to essential oil structures, to date, small has been published about the real biological potential attributed to essential oils from micro and macroalgae. Thus, more details about the chemical structure and biological activity of these matrices will be compiled and discussed in the next section.

20.3 Chemical Structure and Biological Activity from Algal Essential Oils For many years, research related to algal oils has focused on the fraction of essential fatty acids, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [16, 17]. However, other more unusual algal compounds, under the denomination “essential oil”, make up a complex mixture of odoriferous organic compounds with several important bioactive properties [14, 18]. Although the terminology is similar, essential fatty acids and the compounds that make up the essential oil are not analogous, as they have distinct structures and properties. For example, from a physicochemical point of view, the components of essential oils, unlike essential fatty acids, have a lower boiling point, greater volatility, low to moderate hydrophilicity and low molecular weight [19]. As can be seen in Figure 20.1, the essential oil algae constituents are a complex mixture of different volatile chemical compounds, including terpenes, alkanes, alkenes, some fatty acids, esters, phenols, alcohols, aldehydes, ketones, nitrogen and sulfur derivatives and halogenated compounds [14, 18, 20, 21]. Among the various chemical classes, terpenes, organic molecules formed by isoprene units (C5H8) are present in all types of essential oils, including algae. In general, in algae essential oil, monoterpenes and sesquiterpenes are the most abundant terpenes [14]. These structures can be classified according to the presence or absence of oxygen in the molecule in hydrocarbons or oxygenates [22].

468  Essential Oils O OH

OH

O

Br Br

OH 1,8-cineole (1)

α-pinene (2)

Linalool (3)

Geraniol (4)

Aplysin (5)

Aplysinol (6)

β-elemeno (7)

O β-ionone (8)

n-pentadecane (9)

2, 4,6-trimethyldecane (10)

Dictyopterene (11)

1,3,5-underatriene (12)

O O

O Palmitic acid (13)

O

O

OH

Methyl eicosa-5,8,11,14,17-pentaenoate (14)

Pentadecanal (15)

Octan-3-one (16)

Cl OH

O OH

1-Octen-3ol (17)

2,4-Bis(1,1-Dimethylethyl)phenol (18)

OH

O S

Br

Br

OH

NH2 2-phenylethylamine (19)

C11 sulfur compound (20)

Elatol (21)

2-bromophenol (22)

Figure 20.1  Examples of chemical structures described in the algal essential oils.

Structurally, monoterpenes can be acyclic, monocyclic or bicyclic [18]. Among the cyclic structures, 1,8-cineole (1) (oxygenated) and α-pinene (2) (hydrocarbon) are the most common examples of monocyclic and bicyclic algal monoterpenes, respectively. In contrast, myrcene (hydrocarbon), linalool (3) and geraniol (4) (oxygenated) are among the most reported acyclic structures [18]. Likewise, algal sesquiterpenes can be cyclic or bicyclic and still contain halogens in the structure. Conversely, they are less volatile and constitute a more diverse group than monoterpenes [23, 24]. In general, its structure consists of a linear or cyclic terpenic part and an aromatic [25]. The structural diversity of this compounds occurs due the assembly of the C15 skeletons that constitute the central terpenic structure, together with the stratification of functional groups and substituents in distinct positions [26]. Aplysin (5), aplysinol (6) (halogenated), β-elemene (7) (hydrocarbon) and β-ionone (8) (oxygenated) are examples of the structural diversity of algal sesquiterpenes [21, 27]. Although some of these structures have been identified in brown algae (Nizamuddinia zanardinii), red algae of the genus Laurencia are the principal sources [14, 21]. Among the various chemical classes, algae alkanes (saturated) and alkenes (unsaturated) are fewer complex hydrocarbons with linear or branched carbon chains. The carbon chain of these alkanes ranges from 7 to 36 carbon atoms, while that of alkenes ranges from 8 to 19 with 1 to 4 installations [14]. n-pentadecane (9) and 2,4,6-trimethyldecane (10) are examples of alkane and alkene often identified in algal essence oil, respectively [28]. In addition to these hydrocarbons, compounds with 11 acyclic and cyclic non-isoprenoid C atoms such as dictyopterene A (11) and 1,3,5-undecatriene (12) are specific to algal organisms, especially in brown algae of the genus Dictyopteris [29, 30].

Essential Oils Obtained from Algae  469 Saturated and unsaturated fatty acid structures with 3 to 18 carbon atoms have been reported in the algae volatile oil, the palmitic acid (13) has been most frequently described [20]. In addition, fatty acid methyl esters (e.g., methyl eicosa-5,8,11,14,17-pentaenoate (14) commonly appear in the volatile oil of numerous seaweeds [20, 31]. Aldehydes, ketones, alcohols and phenolics organic functions are regularly reported in the volatile composition of all algae and consequently in the essential oil. Pentadecanal (15), octan-3-one (16), 1-octen-3-ol (17) and 2,4-Bis(1,1-Dimethylethyl)phenol (18) are frequently described molecules [20, 28, 32]. On the other hand, compounds derived from nitrogen and sulfur are less frequent, but they are marked by an unpleasant odor [14]. While amino compounds such as 2-phenylethylamine (19) are common in red algae (Ceramium rubrum, Cystoclonium purpureum, Desmarestia aculeate, Dumontias incrassate), sulfur compounds are common in brown algae, especially sulfur metabolites C11 (20), which are restricted to the genus Dictyopteris [14, 33]. Referring to halogenated compounds, these are found abundantly in algal organisms (mainly red algae) and rarely in higher plants [18]. Although chlorine and iodine have been described in algae essential oil, bromine is the most common halogen and is generally linked to terpenoid compounds, phenols, carboxylic compounds, and fatty acid derivatives [18, 34]. While halogenated terpenes such as sesquiterpene elatol (21) appear to be restricted to red algae, less complex structures such as 2-bromophenol (22) are reported in several classes, including green and brown algae [14, 18]. According to El Hattab [14], among all these chemical structures reported, C11 hydrocarbons, sulfur compounds, and halogenated terpenes are exclusive compounds of brown algae in general, Dictypteris and red algae, respectively. In contrast, the other compounds are usual to the essential oil of most marine algae. In addition to playing ecologically essential roles [35], the varied chemical composition gives algal essential oil several bioactive properties, including antimicrobial, antioxidant, anti-inflammatory and cytotoxic activity [36–39]. The antimicrobial effect against numerous strains of bacteria and fungi is the most explored biological activity in several algae. The essential oil of Dictyopteris polypodioides (Dictyopteris membranacea) showed an inhibitory effect against strains of Staphylococcus aureus, Agrobacterium tumefaciens, Salmonella typhimurium, Bacillus cereus, Micrococcus luteus and Escherichia coli, with the first two being the most affected [39]. Likewise, this antibacterial effect has been demonstrated against Pseudomonas aeruginosa [40]. Furthermore, fungi such as Macrophomina phaseolina, Rhizoctonia solani and Fusarium solani were also inhibited [41]. Likewise, volatile oil from red algae such as Laurencia obtus was found to be potent against strains of Staphylococcus aureus, Escherichia coli (O157: H7) and Candida albicans [42]. Most marine algae compounds that make up the essential oil demonstrate some antioxidant potential, especially polyphenols and their derivatives such as bromophenols and terpenoid substances [43]. This biological activity is usually measured by in vitro tests. Essential oils extracted from the Laurencia genus, for example, exhibited free radical scavenging activity in a DPPH assay, bleaching activity of the ABTS radical cation and also inhibited β-carotene bleaching [42]. Anti-inflammatory agents of marine origin are under constant evaluation. It is believed that the sesquiterpene fraction strongly contributes to this activity [44]. Several terpenoids

470  Essential Oils have been shown to inhibit inflammatory biomarkers such as iNOS, COX-2, superoxide anion production, and elastase release [44, 45]. The anticancer activity of algal essential oils is commonly described for red algae and refers to their halogenated organic compounds, especially sesquiterpenes [46]. The potent cytotoxic effects of sesquiterpenes have been demonstrated for several tumor cell lines, including leukemia cell line, mammary adenocarcinoma derivative, prostate adenocarcinoma, cervical adenocarcinoma and carcinoma epidermoid [47, 48]. Finally, the oxygenated structures of essential oils are responsible for most biological properties [49]. For example, the essential oil’s antioxidant and antimicrobial capacity has been directly attributed to the phenolic structure attendances [50]. In addition, antimicrobial activity has been associated with the presence of sulfur compounds, as well as terpenes content. Additionally, antimicrobial activity may increase due to synergism between the different components [39]. Otherwise, cytotoxicity was associated mainly with the presence of halogenated organic compounds [36], while anti-inflammatory effects were related to the sesquiterpene fraction [44].

20.4 Ecological Importance of Essential Oils in Marine System The ecological importance of volatile compounds in terrestrial ecosystems is well documented. These compounds are generally found to function as infochemicals, which play a central role in the language of life on earth. On the other hand, in relation to aquatic chemical ecology, its functions are essentially unknown. However, although they remain little investigated, there is evidence of their involvement in several processes that guide the behavior of aquatic food webs. This is the case of volatile compounds produced by aquatic primary producers such as algae and cyanobacteria. The volatile compounds of these organisms can exert crucial roles in intraspecific (pheromone) and interspecific (allelochemical) relationships and in chemical defenses [51–54]. In intraspecific relationships, the pheromones perform an important function in the reproduction. These substances are actively excreted and are involved in synchronization of the copula of gametes of different sexes and in enhancement of the copula efficiency by attraction. The macroalgae Ectocarpus siliculosus and Fucus serratus are well-known examples of volatile pheromones producers that act as sexual attractants. These macroalgae produce the sexual attractants identified as ectocarpene and fucoserratene, respectively. Therefore, Table 20.1 shows the algae pheromones most widely known [14]. With regard to the interspecific chemical communication that corresponds to interactions between individuals belonging to different species, the volatile compounds function especially as kairomones. Kairomones are allelochemicals relevant to the biology of an organism that, when in contact with an individual of another species, favors the recipient species. In other words, the one who benefits from the chemical signal transmitted by this allelochemical is the signal receiver and not the emitter. That would be, for example, the case of the bouquet of volatiles emitted by the algae Ulothrix fimbriata and Achnanthes biasolettiana when injured, which function as chemical clues to find foods. These chemical cues attract the herbivore Radix ovata. In relation to these volatiles, it is important to mention that there are several that are attractive to organisms only when present in mixture, that is, not effective as an isolated substance. Furthermore, there are differences in the

Essential Oils Obtained from Algae  471 Table 20.1  Pheromones from algae species. Pheromones

Algae species

Ectocarpene

Scytosiphon sp., E. fasciculatus, E. siliculosus, A. tricularis, S. rigidula

Hormosirene

H. banksia, X. chondrophylla, X. gladiata, D. antarctica, D. potatorum, D. willana, C. peregrina, C. bullosa, A. mirabilis, M. simplex, S. lomentaria

Ectocarpene/Hormosirene

A. japonicus, A. utricularis, S. rigidula

Desmarestene

D. acculeata, D. viridis, C. spongiosus, D. firma

Dictyotene

D. dichotoma, D. diemensis, D. prolifera

Lamoxirene

L. angustata, L. japônica, L. saccharina, L. sinclarii, L. digitata, L. hyperborean, P. gardneri, A. crassifólia, A. esculenta, A. marginata, E. radiata, E. arbórea, P. californica, U. pinnatifida, D. reticulata, L. variegata, L. littoralis, M. integrifólia, M. pyrifera, N. luetkeana, P. porra, A. cribrosum, C. triplicata, H. sessile, K. gyrata

Cystophorene

C. siliquosa

Finavarrene

D. foeniculaceus, A. nodosum, P. callitricha

Pre-ectocarpene

E. siliculosus

Multifidene

C. multifida, Z. angustata, C. tomentosa

Caudoxirene

P. caudata, D. foeniculaceus

Viridiene

Syringoderma sp., S. phinneyi

Fucoserratene

F. serratus, F. spiralis, F. vesiculosus

Adapted from El Hattab (2020); Pohnert and Boland (2002) [14, 55].

bouquet of volatile substances from one species of alga to another and this difference may, for the same organism, not show any attractive activity [56, 57]. Until the moment, independent of these insights, it is suggested that the volatile compounds of algae in marine and freshwater habitats may have a driver role as kairomone, as well as determined for terrestrial organismic interactions. Still, it is noteworthy that the perception of volatile allelochemicals from algae and cyanobacteria by semi-aquatic and terrestrial organisms may be involved in regulating food web interactions between the marine and terrestrial ecosystems [58]. Besides the ecological functions of volatile compounds in intraspecies and interspecies communication, they may also be linked to the activated chemical defense of algae and cyanobacteria. There is evidence for the release of odorous compounds that act as repellents for herbivores grazers. It has been evidenced that algae lipoxygenase products, such as volatile aldehydes, can not only serve as an allelochemical, but also as an ecological defensive.

472  Essential Oils This because they reduce the proliferation of marine copepods due to their embryogenic toxicity. About this, the release of volatile aldehydes, which occurs after cell damage, impairs the reproductive success of herbivores, but does not prevent herbivores from grazing [55, 59]. Experiments carried out by Jüttner [60] confirm the repellency of volatile aldehydes from benthic diatoms. The compound 2(E), 4(E), 7(Z)-Decatrienal was identified as the most active repellent against crustaceans as a target organism. In addition, in this study, the concomitant formation of EPA was observed. This leads to a possible advanced repellent action, once EPA is toxic to shepherds. In this sense, when produced by algae and activated cyanobacteria, they work as a synonym, because both the sending and receiving organism benefit from the chemical signal. The herbivores that initiate algae and cyanobacteria grazing cause the release of these compounds, which serves as a warning clue to the recipient, to move away from toxic EPA, protecting consequently algae and cyanobacteria from herbivory. Primary producers living in colonies also benefit from this chemical defense, taking into account that the repellent reaction driven by victim-cells primary would be advantageous for the others, i.e., for the survival of the group. Finally, it is known that terrestrial and marine organisms emit and receive volatile compounds for communication and defense purposes. However, it cannot be said that ecological interactions mediated chemically by these substances are holistically understood. The significance of ecological interactions mediated by volatile compounds is, sometimes, conflicting. Clearly, more research needs to be done to elucidate the effectiveness and importance of these interactions so that clear conclusions that will help define their role in maintaining biodiversity and ecosystems functionality can be drawn [58].

20.5 Conclusion and Future Perspectives In recent times, and even more recently, in the pandemic era we are experiencing, the search for natural and sustainable products has become a must. This is the case of essential oils, which has become a substantially promising topic, especially regarding their applicability in the pharmaceutical and food industries. Therefore, it is no surprise that more and more research groups are focused on the use of substances extracted from new potential sources as, undeniably, the successful application of compounds extracted from micro and macroalgae has been happening. However, although this matrix has already been proven and recognized, there are still several gaps to be deeply investigated, in order to make them really applicable to industrial scales. Thus, finding the best strain for extracting specific compounds, in addition to determining the best technique for extracting and recovering, are still paths that scientific research will have to follow. Furthermore, information about the stability of the use of these ingredients should be encouraged for different scopes and, therefore, to improve their application in the most diverse industrial segments.

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Essential Oils Obtained from Algae  475 39. Riad, N., Zahi, M.R., Trovato, E., Bouzidi, N., Daghbouche, Y., Utczás, M., Mondello, L., El Hattab, M., Chemical screening and antibacterial activity of essential oil and volatile fraction of Dictyopteris polypodioides. Microchem. J., 152, 104415, 2020. 40. Ozdemir, G., Horzum, Z., Sukatar, A., Karabay-Yavasoglu, N., Antimicrobial activities of volatile components and various extracts of Dictyopteris membranaceae and Cystoseira barbata from the coast of Izmir, Turkey. Pharm. Biol., 44, 183, 2006. 41. Sultana, V., Ehteshamul-Haque, S., Ara, J., Athar, M., Comparative efficacy of brown, green and red seaweeds in the control of root infecting fungi and okra. Int. J. Environ. Sci. Technol., 2, 129–132, 2005. 42. Demirel, Z., Yilmaz-Koz, F.F., Ulku, N.K.-Y., Ozdemir, G., Sukatar, A., Antimicrobial and antioxidant activities of solvent extracts and the essential oil composition of Laurencia obtusa and Laurencia obtusa var. pyramidata. Rom. Biotechnol. Lett., 16, 5927, 2011. 43. Nogueira, C.C.R., Paixão, I.C.N.D.P., Teixeira, V.L., Antioxidant activity of natural products isolated from red seaweeds. Nat. Prod. Commun., 9, 1031, 2014. 44. Cheung, R.C.F., Ng, T.B., Wong, J.H., Chen, Y., Chan, W.Y., Marine natural products with anti-inflammatory activity. Appl. Microbiol. Biotechnol., 100, 1645, 2016. 45. Jean, Y.H., Chen, W.F., Duh, C.Y., Huang, S.Y., Hsu, C.H., Lin, C.S., Sung, C.S., Chen, I.M., Wen, Z.H., Inducible nitric oxide synthase and cyclooxygenase-2 participate in anti-inflammatory and analgesic effects of the natural marine compound lemnalol from Formosan soft coral Lemnalia cervicorni. Eur. J. Pharmacol., 578, 323, 2008. 46. Wang, B.G., Gloer, J.B., Ji, N.Y., Zhao, J.C., Halogenated organic molecules of rhodomelaceae origin: Chemistry and biology. Chem. Rev., 113, 5, 3632–3685, Chem Ver., 2013. 47. Iliopoulou, D., Mihopoulos, N., Vagias, C., Papazafiri, P., Roussis, V., Novel cytotoxic brominated diterpenes from the red alga Laurencia obtusa. J. Org. Chem., 68, 7667, 2003. 48. Kladi, M., Vagias, C., Papazafiri, P., Furnari, G., Serio, D., Roussis, V., New sesquiterpenes from the red alga Laurencia microcladia. Tetrahedron, 63, 7606, 2007. 49. Ferreira, D., de, F., Nora, F.M.D., Lucas, B.N., de Menezes, C.R., Cichoski, A.J., Giacomelli, S.R., Wagner, R., Barin, J.S., Oxygen introduction during extraction and the improvement of antioxidant activity of essential oils of basil, lemon and lemongrass. Cienc. Rural, 47, 1–7, 2017. 50. Ribeiro-Santos, R., Andrade, M., Sanches-Silva, A., de Melo, N.R., Essential oils for food application: Natural substances with established biological activities. Food Bioprocess Technol., 11, 43, 2018. 51. Steinke, M., Malin, G., Liss, P.S., Trophic interactions in the sea: An ecological role for climate relevant volatiles? J. Phycol., 38, 630, 2002. 52. Hay, M.E., Marine chemical ecology: Chemical signals and cues structure marine populations, communities, and ecosystems. Annu. Rev. Mar. Sci., 1, 193, 2009. 53. Ianora, A., Bentley, M.G., Caldwell, G.S., Casotti, R., Cembella, A.D., Engström-Öst, J., Vaiciute, D., The relevance of marine chemical ecology to plankton and ecosystem function: An emerging field. Mar. Drugs, 9, 1625, 2011. 54. Schwartz, E.R., Poulin, R.X., Mojib, N., Kubanek, J., Chemical ecology of marine plankton. Nat. Prod. Rep., 33, 843, 2016. 55. Pohnert, G. and Boland, W., The oxylipin chemistry of attraction and defense in brown algae and diatoms. Nat. Prod. Rep., 19, 108, 2002. 56. Fink, P., Von Elert, E., Jüttner, F., Oxylipins from freshwater diatoms act as attractants for a benthic herbivore. Arch. Hydrobiol., 167, 561, 2006a. 57. Fink, P., Von Elert, E., Jüttner, F., Volatile foraging kairomones in the littoral zone: Attraction of an herbivorous freshwater gastropod to algal odors. J. Chem. Ecol., 32, 1867, 2006b.

476  Essential Oils 58. Fink, P., Ecological functions of volatile organic compounds in aquatic systems. Mar. Freshw. Behav. Physiol., 40, 155, 2007. 59. Ianora, A., Miralto, A., Poulet, S.A., Carotenuto, Y., Buttino, I., Romano, G., Smetacek, V., Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom. Nature, 429, 403, 2004. 60. Jüttner, F., Evidence that polyunsaturated aldehydes of diatoms are repellents for pelagic crustacean grazers. Aquat. Ecol., 39, 271, 2005.

21 Gas Chromatography-Olfactometry (GC‑O) of Essential Oils and Volatile Extracts Eduardo Dellacassa1* and Manuel A. Minteguiaga1,2 1

Laboratorio de Biotecnología de Aromas, Departamento de Química Orgánica, Facultad de Química, Universidad de la República (DQO-FQ-UdelaR), Av. General Flores Montevideo, Uruguay 2 Espacio de Ciencia y Tecnología Química, Centro Universitario Regional Noreste, Sede Tacuarembó, Universidad de la República, Ruta Tacuarembó, Uruguay

Abstract

Volatile stimuli can be detected by specific protein receptors from olfactory epithelium and transmitted to the brain where finally are interpreted as the sense of smell. The interpretation of odor response complexity provokes the continuous development of instrumental methods for objectively analyze them. Gas chromatography-olfactometry (GC-O) introduction was a milestone in the odor research of, coupling a resolution technique of volatiles mixtures (gas chromatography) with the selectivity and sensitivity of the human olfactory system. GC-O generates a list of the odor descriptor of each peak in a GC run (“aromagram”), indicating which GC peaks (chromatographic resolved components) have or not given aromas/odors associated. The GC-O technique has been profusely employed for the evaluation of aromas in food matrices, but its application to essential oil study is still scarce for the characterization of specific odor active compounds which contribute to the overall olfactory impression. In this chapter an overview of data able to be obtained applying different GC-O methodologies to essential oils are presented, including the procedures and fundaments involved. The information presented does not intend to be exhaustive, but provides examples available in the literature and references to further reading. Keywords:  Gas chromatography-olfactometry, essential oils

21.1 Introduction From a practical and operational point of view, an odor can be defined as the sensations or impressions perceived by the nervous central system caused by the interaction of small molecules (the odorant) over generalist and specific protein receptors located in the olfactory epithelium [1, 2]. The odorants are able to provoke a cascade of events which are transmitted from the epithelium to the brain, but up to date, no clear correlations between the physicochemical properties of odorants and the associated aroma (or sensations) have been *Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (477–500) © 2023 Scrivener Publishing LLC

477

478  Essential Oils established [3]. Furthermore, structurally related odorants (such as the limonene enantiomers) have different aromatic notes, while other compounds which do not have structural similarities between them could cause analogous responses on the olfactory system [3, 4]. Thus, the odor detection threshold concept, the minimum concentration of an odorant which can be regarded as an aroma and provokes a sensorial stimulus, is key when working with olfaction [3, 4]. Being olfaction the most appropriate way to perceive the presence of odorants, the human nose was early selected as detector for gas chromatographs through evaluation at real time of its effluents, and consequently, the gas chromatography-olfactometry (GC-O) was the tool found [5]. At GC-O, at the end of the analytical column (in general capillary more than packed) the gas flow is divided into two streams: the first carries the analytes (odorants or not) to an instrumental detector (e.g., FID, MS, TCD, ECD, NPD); while the other one transports them to an olfactometric port where sensorial judges sniff and finally determine which of the eluted compounds actually are odorants (Figure 21.1). In the beginning, GC-O was conceived to give sensorial description of the gaseous effluents, aiming to assess if the compounds eluting as peaks were odorants or not, and to primarily evaluate its aromatic attributes [3, 4]. To have a realistic evaluation, the extract composition to be analyzed by GC-O must be so much representative as possible of the vapor phase that come out from the original product during its olfaction. In the flavor and fragrance field, that means that the aroma of the raw vegetal source needs to be trapped during extraction, and this goal can be reached faithfully by solid phase microextraction (SPME) and solvent-assisted flavor evaporation (SAFE), and in a lesser extension by steam distillation (SD), simultaneous distillation extraction (SDE) and supercritical fluid extraction (SFE) (among others) [3, 4]. However, from the named techniques only SD is industrially scalable at a non-onerous budget, and thus, the correspondent products (essential oils) are usually those evaluated by GC-O when real applications are advised. Essential oil composition also includes odorants originated in the physical process of distillation (artifacts), and thus, given such dependence with experimental conditions in sample preparation, the comparison of GC-O results is a challenging task when not an impossible one [3, 4].

olfatometric port

Figure 21.1  Olfatometric port.

GC-O of Essential Oils  479 It is important to consider that GC-O evaluations of essential oils and volatile extracts are not free of bias in the survey of the relevance of the characterized odorants, and in their correlation with the raw material sensorial properties [3–5]. The concentration of each odorant may be corrected applying the concept of odor threshold, as the minimal concentration that can be detected by human nose in the raw material or matrix. Thus, the differences in volatilities which originated the named bias in GC-O are totally or at least partially corrected (when no matrix-specific odor threshold is available for the compounds and the correction is made with this value in a so similar matrix as possible). In consequence, for an individual component is defined the ratio: concentration in the sample/concentration threshold in the matrix, which is named odor activity value (OAV) which provides a more objective (but not necessarily precise) ranking of the odorants in the essential oils or volatile extracts from food origin (see section 21.3) [3–5]. For applying GC-O evaluations, are needed extracts from the matrix that accomplish a series of requirements, among them: –– a level of concentration enough to detect and ideally identify all its key impact odorants –– a high representativeness of the “real aroma” of the matrix which is perceived naturally for the sense of smell In the search of chemicals responsible of findings on key impact odorants, GC-O represents a relevant approach in flavor and fragrance research, as the number of vegetal matrices and its fractions is continuously increasing. Beyond its application in flavor and fragrance field, GC-O methods are of outermost importance for food matrices and its volatile extracts (i.e., in cheese and dairy products, honey, tea, coffee, beer, wine, fruits, etc.), providing invaluable information to define chromatographic regions of odor activity, giving an ever fresh-look even in well-known matrices [3, 4]. Databases built so descriptors are listed for different compounds make easier the interpretation of experimental results. However, two people smelling the same thing can have remarkably different reactions, depending on their cultural background, transferring misinterpretations to odor databases.

21.2 Historical Aspects Gas chromatography, the technique to analyze mixtures by the transportation of their vapor phase by a carrier gas through a solid or liquid stationary phase, was originated in the Cremer and Prior [6] pioneer work in 1945-1947, despite most of the researchers attributed the initial milestone in James and Martin [7] classical paper, as mentioned by Jennings and Poole [8]. In the 60s and 70s, the advent of GC-O was a breakthrough in the flavor and fragrance field. Fuller et al. [9] described for the first time the conjunction of a gas chromatograph (GC, or a vapor phase chromatograph, as known at such time) with the human sense of olfaction, in an attempt to employ and develop the perfumer’s skills. The authors named the dispositive where panelists smelt the vapor phase exiting from the GC as “perfumer detector” or “human sensor”, connected in parallel to a TCD detector. At such time, Fuller et al. [9]

480  Essential Oils were aware that in many laboratories was a common practice to smell the odor eluting from a GC equipment, but there was not publication regarding the employment of a trained panelist (as a professional perfumer) in the olfactory assessments. More than only pretending to use the specialist knowledge on different aromatic matrices, the authors aimed also to stimulate the creative thinking of perfumers by exposing them to situations where the relationships between major and minor components’ concentrations were relevant. The design of the olfactory dispositive was a challenge, and two options were originally tested to isolate the panelist from the surrounding environment and to obtain and adequate ergonomics (a plastic personal hood, and a telephone booth). However, both options were in common an interface composed by a tube heated by a resistance wire which exited the GC and reached the “human sensor”. A limitation that authors found was the sensitivity of the noise not only to the aromas but to the heat itself. The heat of the interface was crucial for preventing the condensation of the eluates when exiting the GC system, avoiding a temperature drop between the analytical column and the interface tube. In this pioneer work, the panelists transmitted their impressions on the smells they perceived in two ways: 1) simultaneously writing and registering the time of the events 2) by tape recording. The panelists were trained by smelling in pure injections 150 common components in perfumery, and after that, their abilities to identify and distinguish single compounds in known mixtures was evaluated. For mixtures of fifteen compounds belonging to different functional groups (whose peaks were perfectly resolved), the specialists were able to correctly recognize in average ten of them, but even in the cases of erroneous identifications, the components named by the panelists were close in odor (for example, for the group composed by borneol, bornyl acetate, camphor and iso-borneol). Also in this first work, Fuller et al. (1964) [9] determined thresholds limits for several odorants. They did not work specifically on pure essential oils, but employed for olfactory purposes some rectified fractions as citral and impure anethole (from pine oil). Since this first milestone, gas chromatography in parallel to human olfaction for the analysis of plant fragrance extracts was almost forgotten in the literature. In fact, since the works of Dravnieks et al. [10], it was employed mostly for detection of airborne compounds. By 1971, Dravnieks & O’Donell [11] stated “…It has become rather common to equip a gas chromatograph with a sniffing port in parallel with the hydrogen flame ionization detector…”. Olfaction of vapor eluates exiting GC was employed at such time to identify the chromatographic peaks which mostly represent the odor complex of the samples, and to locate minor components of the extracts in regions of the chromatograms where olfactory perception was evidenced but no peaks were detected. In such a contribution Dravnieks & O’Donell [11] introduced a key improvement in the conception of GC-O with the aim to reduce the desiccating effects on the nasal membrane. A more ergonomic interface was designed through the utilization of a humidified air stream which mixed with the gaseous effluents eluting from a packed GC column [11]. This concept was able to be extended by the work performed by Acree et al. [12], who incremented the humidified air volumes together with their linear velocities to preserve the separation efficiency when reaching the olfaction port, which ultimately allows the transition from packed to capillary and narrow bore columns.

GC-O of Essential Oils  481

21.3 GC-O Methodologies The adoption of GC-O for the identification of compounds in complex matrices which in turns can trigger the sensation of smell and the subsequent quantification of odor activity, was only possible due to the improvement of extraction techniques and the development of GC-O methodologies aiming to standardize the sensorial evaluation procedures [3, 4]. Furthermore, GC-O was conceived since its origin as a quantitative bioassay which allowed recognizing and identifying in the 70s around 1500 compounds as odorants in food, flavor, and fragrance fields [13, 14]. But this number was boosted by the new approaches and technologies in separation science and synthesis, which led currently to have a pallet of odorants surpassing 7000 compounds, with a high proportion being developed confidentiality by the industries [4]. As above mentioned, the odor detection threshold and OAV values are key concepts when evaluating the real contribution of a single volatile compound to the overall aroma of a product, as essential oils, or food matrices. In 1963, Rothe et al. [15] working on bread odorants (furfurol, iso-butyraldehyde, iso-valeraldehyde, among others) derived the OAV meaning (also known in literature as odor units and/or odor values), but at such time the determination of odorants’ concentrations was performed by paper chromatography and colorimetric reactions.. Simply, OAV is a measure of the potency of a specific odorant in an also specific sample. As in its definition is implicated the aroma/odor detection threshold, a priori humans are not able to perceive volatiles with OAV values lower that one, while if these are greater than one they may be detected unless other phenomena as odor suppression (arising from compound-matrix interaction) are potentially involved [3, 4, 15]. Frequently some of the most potent odorants in food, flavors and fragrances (such pyrazines and some aliphatic short-chain oxygenated compounds) exhibit low-concentration odor detection thresholds [3, 4, 15]. Thus, OAV values, is employed to have an estimation of the odor potency. Undoubtedly, to gain in OAV accuracy it is crucial to work in both components of the ratio: the concentrations of the odorants must be accurately determined (which is fortunately possible when working with GC), and the thresholds must be studied in a so-similar medium to the raw matrix as possible (ideally, the matrix itself). From this point of view, it can be uncovering an important limitation in the general employment of OAV values for ranking odorants potency: if the compound of interest is present at a concentration level below the instrument detection limits (a common situation in GC-O analyses), no OAV can be calculated [3, 4, 15]. Another relevant limitation from OAV approach in odorant rankings is these values do not present linear correlation with the perceived intensity, and even do not predict the intensity when mixtures (as essential oils) are evaluated [3, 4]. There are main four “traditional” methods for processing the GC-O data to estimate the odor activity of single odorants in the extracts being analyzed [16]: 1) detection frequency, 2) dilution analysis, 3) posterior intensity, and 4) time-intensity methods.

482  Essential Oils In all of them the actual value of GC-O data recorded depends directly on their individual fundamentals (related to the way every method interprets the sensorial information) in conjunction with the previous step chose of sample preparation, and the simultaneous GC analytical conditions selected [17]. These techniques have been previously reviewed [3, 4, 17–19], so they will be only briefly commented here.

21.3.1 Detection Frequency Methods Detection Frequency allows to detect aroma/odors simply by calculating the frequency (named detection frequency) that a group of assessors sniffed such stimulus, and it is employed as an estimate of the odor’s potency/intensity. Meaning it is needed a panel composed by trained or untrained people who analyze the same sample, with the aim of calculate the percentage who perceived the odorants at certain retention time or index [20]. For each one of the aroma/odor time events, the values of the parameter Nasal Impact Frequency (NIF) are calculated, generally as percentages (or, in the 0-1 scale) between the people who sensed the odorants over the total people integrating the panel. This NIF value varies between zero or 100% (alternatively: 0 to 1), being the former case when no individuals detected the aroma/odor and the latter case when all of them did it [21]. Another option is available by calculating the Surface of Nasal Impact Frequency (SNIF) parameter, which in turns represents the peak areas obtained by adding the individual values of aroma/odor duration (in time or index units) multiplied per one (as it is considered for every assessor), which allows plotting aromagrams [22]. Detection frequency method was first formalized and standardized by Pollien et al. [22]. This detection frequency-based method simplicity includes the possibility of working with unexperienced assessors in the sensorial panel, thus not being time-consuming in training. The main drawback is associated with the fact that the detection frequency is directly related to the perceived intensity, but it cannot be obtained correlations between NIF or SNIF values and the real odorant concentration in the extracts (given that all the panel evaluate the same sample, the odorant concentration is a constant for all its members) [3, 22, 23]. Consequently, as concentration of a single odorant is not a variable, and diverse odorants are present at different concentrations levels in the samples (all above the aroma detection threshold), the method may result in aromagrams composed by peaks with equal or similar intensity for every odorant, despite its real concentration [23].

21.3.2 Dilution Analysis Dilution analysis methods are applied using two options [19]: –– giving aroma potency values following step by step dilution of the sample to the detection threshold of every odorant: aroma extraction dilution analysis (AEDA) –– combining hedonic response measurement (Charm Analysis)

GC-O of Essential Oils  483

21.3.2.1 Aroma Extraction Dilution Analysis (AEDA) In 1983 Schieberle and Grosch [24] coined the initials AEDA for Aroma Extract Dilution Analysis, pretending to be a GC-O method with the purpose to work with serial dilutions of the aromatic samples (lemon essential oil in the original publication). It can be considered as a quantitative GC-O method aiming to determine the potency of odorants in the samples of interest [24]. The serial (stepwise) dilutions of the samples are analyzed by GC-O until no aroma is perceived by the sensorial judges, being the factor of flavor dilution (FD) calculated for each of the odorant as the inverse of the dilution (for example, if a compound is olfactory perceived up to a dilution of 1/500, then its FD is 500) [3, 4, 17–19, 24, 25]. In practice, since its origin this methodology has been closely associated to OAV values, because in general, in GC-O the FD concentration overlaps with the aroma detection threshold. However, it should be considered that the threshold is calculated in the analytical conditions and not the correspondent value in the original product or raw material). Once the most potent odorants are identified with their FDs, the real concentrations of them in the matrix are determined, and the OAV values are calculated [3, 4, 24]. Usually, the performed dilutions are combined with solvent-employing extraction techniques [among others liquid-liquid extraction (LLE), simultaneous distillation/extraction  (SDE), and ­solvent-assisted flavor evaporation (SAFE)] [3, 25–28].

21.3.2.2 Combined Hedonic Aroma Response Measurements (CHARM Analysis) Almost in parallel with AEDA, in 1984 Acree et al. [13] introduced the CHARM analysis as a human bioassay to found quantitatively the most significant (intense) flavors in food matrices based as AEDA on sample dilutions and on graphical representations. The participants record the instant each odor is detected and the instant it disappears. Each odor stimulus is identified and characterized by its specific presentation time. Analyses are repeated at several dilutions of the stimulus mixture until no odors are detected. Response data for each odor are combined over all dilutions to yield odor-activity units, which values are proportional to the amount of stimulus and inversely proportional to the participant’s threshold [13]. Thus, being sniffers focused only on the aroma attributes, and not in intensity, the quality of the results are better. The CHARM analysis procedure implies that the sensorial panelists reactions are software recorded during the aroma assessment, registering the observation they made (sensory descriptors of odorants eluting the GC column at specific times) [13]. To a practical level, when an odorant is eluting and its correspondent aroma is perceived, the panelists strike the space bar of the computer’s keyboard, and the time starts counting. The aroma descriptors are recorded through previously coded keys that the panelists strike when they identified a specific odor (for example, F = floral). When the aroma is no longer perceived, the panelists strike another pre-established key, and thus, the end of such an aroma band is defined [13]. The information (initial and final times, and descriptors) is processed through a dedicated software, which also calculate specific retention indices of the odorants after the later analysis of a lineal hydrocarbons mixture at identical GC conditions as the samples. The software also records the “reaction time”, that is, the time that the panelist took to choose an aroma descriptor after it started to be detected [13].

484  Essential Oils At the end of the process, the software gives two different graphical results: –– a coincident response aromagram (consensus aromagram), or –– a CHARM response aromagram. The former is obtained simply as the algebraic sum of the obtained records (from several assessment of the same panelist, or from individual assessment of all the members of the sensorial panel), giving values of 1 and 0 if the panelists detect or not the odorants at specific times (or preferably, retention indices) being reasonable equivalent to SNIF aromagrams, (section 3.1) (Figure 21.2). Thus, the most intense odorants will obtain the higher summed values (many sums of 1 values), and the opposite, if a slight aroma is detected the algebraic sum will include many 0 values and some 1 values. If the sum of sensorial responses gives zero, it means that no odorant at all is eluting at such specific time (Figure 21.2). Furthermore, the area of the so-generated peaks depends on the relative frequency of detection of the odorants at such specific time region [13]. As in direct frequency aromagrams, the limitation of coincident response aromagrams is that they only detect the odors above the specific odorants’ detection thresholds. Thus, to have a complete picture of the aromas of a given sample, a serial dilution approach must be followed, justifying the introduction of charm response aromagrams [13]. Charm response aromagrams are constructed for one individual or the sensorial panel, considering the ratio between the total amount of an odorant eluting at a specific time and its threshold value (being equivalent to the OAV concept as previously discussed) [13]. Such a ratio can be estimated by serial diluting the sample and recording the olfactometry responses of the panelists until no longer odor is perceived, being the last detected dilution (a) Concensus Aromagram

(b) Charm Aromagram

Evaluation 1 Sensorial response

Evaluation 3

Sensorial response

Evaluation 2

Dilution 1

Algebraic sum

Dilution 3

Dilution 4 Retention Index

Charm = dn-1 Charm

Coincident response

Evaluation 4

Dilution 2

Retention Index

Retention Index

Figure 21.2  Differences between the consensus aromagram and Charm aromagram for the evaluation of odorants in GC-O. See Acree et al. 1984 [13] for further mathematical details.

GC-O of Essential Oils  485 the odorant threshold in the analytical conditions. The dilution factor (d) to which such an odorant is olfactorily detectable (with n as the number of coincident responses for the stimulus) defines a new parameter, the “instantaneous charm value”, with c = d n-1 [13]. CHARM response aromagrams are obtaining by plotting c against the retention indices, and the area under the peaks is known properly as “charm value” which depends on the aroma intensities of the considered odorant (Figure 21.2). This value has the advantage to be free of intensity estimations compared to the posterior intensity and time-intensity methods, thus being more reliable [13].

21.3.3 Posterior Intensity Methods (PI) This method measures aroma/odor intensities using a pre-defined scale which the sensorial panel employs to rank odorants once their elution was accomplished from the GC-O system [3, 4]. The large variabilities found between the assessors stimulated, Dravnieks [29] to introduce the modified frequency parameter (MF) by combining frequency and intensity values, improving detection frequency methods.



M = F (%)x I[%]

In this formula, F (%) represents the detection frequency of an aromatic event in GC-O expressed as percentage of the maximum frequency that can be obtained (that is, the number of panelists, as 100%). While I (%) is the percentual average intensity calculated from the maximum intensity that can be recorded for and odorant (the maximum of the intensity scale multiplied by the number of panelists, as 100%). This method differs from time-intensity one in the fact that the aroma ranking is performed after elution of the odorant (without considering truly the elution time interval but the maximum intensity perceived) instead a real-time manner [3]. So, aromagrams built on PI approach do not present peaks but lines (unless time-intervals could also be evaluated) (see section 4.7). As closely related to the detection frequency method (modulated by the intensity), PI assumes that the quantity of panelists who sensed the odorants correlates to their concentration or importance in the sample. Furthermore, PI is advantageous given its simplicity to implement at laboratory conditions and to perform valuable evaluations, requiring relatively little training to perform the sensorial panel (being also timesaving when compared to other GC-O methods) [30].

21.3.4 Time-Intensity Methods This type of methods aims to directly estimate the intensity of the panelists’ perception recording simultaneously the time interval of an aroma/odor event and the intensity, through the aid of specific devices such a sliding scale connected to a resistor [3, 4, 31, 32]. This approach allows obtaining aromagrams similar in appearance to the conventional chromatograms of a GC detector. The most consolidated of these methods, and the only that will be briefly discussed here is OSME (odor-specific magnitude estimation) [3, 4, 31, 32].

486  Essential Oils

21.3.4.1 Odor-Specific Magnitude Estimation (OSME, Direct Intensity) In 1992, Miranda López et al. [32] formally introduced the OSME GC-O method (acronym of the Greek word to name “smell”), working on odorants from Pinot Noir varietal wines elaborated with grapes harvested at different phenological (maturation) stages. This method aimed to circumvent criticisms related to the odor threshold methods/dilution methods (AEDA and CHARM analysis), because the latter assume lineal intensity/concentration functions which in fact are rarely or not observed for the most common odorants [33, 34]. OSME methodology implies that the assessors describe quantitatively the intensity they perceived during GC-O analyses, determining the so-called odor peaks with their respective aroma descriptors (which are tape-recorded using a microphone). Employing a time-intensity device (such a slide bar), the panelist “construct” the odor peaks while simultaneously describes verbally their aromatic attributes [32]. To obtain the best representative results, the selected panelists must be trained sniffing in GC-O conditions known samples (aroma standards) which eventually could be present in the real-world samples to be evaluated or are so similar olfactory as possible [3, 4, 17, 32]. Besides, repetitions of the same assessors are advised to obtain average odor peaks for every member of the panel [3]. For each odorant, OSME provides: –– the odor peak, obtained by graphing retention time vs. odor intensity values, –– the odor duration time, which is the total time that the panelist sensed the compound in the GC-effluent –– the maximum odor intensity –– the area under the odor peak –– the linear retention index based on panelists responses –– the odor quality (descriptors) OSME technique also has many advantages: –– it is strongly related to psychophysical views because it directly collects each compound’s odor intensity as it is present in the extract rather than estimating it by indirect approaches based on a diluting series as done in Charm and AEDA, –– OSME is less time consuming since it does not require a dilution series (once the panel is correctly trained), –– it provides one aromagram which, like the GC, represents the exact sensory phenomena occurring during the compound elution [3, 17]. In summary, the four described methods have been used extensively to identify odorants in diverse matrices and products. However, the foundations and implications of such methodologies are still areas for research and development.

21.4 Different GC-O Application to Assess for Essential Oils’ Odorants As derived of aromatic plants obtained by non-conventional agriculture or from direct collection in nature, essential oils represent an almost homogenous group of industrial

GC-O of Essential Oils  487 products that found applications in several industries as formulation components in beverages, cosmetics, foods, perfumes, and pharmaceuticals, among others. To exemplify applications of GC-O on essential oils, it will consider the most relevant essential oils for the world market [35]. Some essential oils, produced predominantly for industrial and domestic purposes, were selected for exemplify the relative importance of GC-O on their added value: citrus, mint, thymus, coriander, fennel, pine. In addition, some commercially minor important but academic interesting examples will be further discussed. For a better understanding of the GC-O importance for the essential oils’ valorization, it must be highlighted that their particular aroma are originated from a complex combination of odorants, in general, hundreds to thousands volatiles at different concentration levels. These compounds eventually present olfactory synergy or additive effects in the mixtures, following a priori unpredictable rules, but being the basis of the sensation that we know as smell [36]. From a practical point of view, in this chapter we will discuss mainly some results on GC-O with the extracts obtained through steam distillation, hydro-distillation, and simultaneous distillation-extraction (SDE), and in few cases with supercritical fluid extraction (SFE), purge and trap (P&T), solvent-assisted flavor evaporation (SAFE), and cold-­expression (for the Citrus oil); while solid phase microextraction (SPME) will not be discussed.

21.4.1 Citrus spp. (Rutaceae) As occurs for all essential oil samples, it is doubtless the utility of GC-MS to analyze their compositions, but the compounds’ profiles obtained through this technique do not correlate exactly with aroma’ profiles in GC-O. Moreover, both of the following cases are frequently found when comparing GC-MS to GC-O: 1) a peak is observed in the former and it is not detected olfactorily in the latter analyses (perhaps, such a peak corresponds to a non-odor active compound, or is present below its aroma/odor detection threshold), and 2) no peak is observed in the former but at such specific time is detected an aroma/odor by GC-O (the correspondent odorant concentration is below the limit of detection of the GC-MS instrument) [3, 4, 17]. To know the characteristic Citrus oils odorants of and to make an organoleptic evaluation of it by explaining odor typicity, GC-O is a fundamental tool to be applied which justify the reason these oils are one of the most relevant examples and models of assessment through GC-O. Up today, the identification of aroma components and the key impact odorants which contribute to the aromatic characteristics of Citrus essential oils, is a strong demand for explaining the citrus flavor particularities searched. The focus of the subsequent work must include deep studies for identification of unknown odorants and investigation of the synergistic and inhibitory effects between them. GC-O has been widely used for the analysis of cold-pressed peel oil for many species, including C. sinensis, C. reticulata, C. grandis, C. paradisi, C. aurantifolia, C. limon, C. bergamia and other minor ones [37]. Moreover, Tranchida et al. [38] have reviewed the gas chromatographic analysis of Citrus essential oil, including a specific section for

488  Essential Oils GC-O assessment. Thus, in this section we will discuss some select examples published later the named review, as examples of the information available in the literature. A good example are the studies performed on grapefruit (C. paradisi) essential oil, where two components, namely, nootkatone and p-1-menthene-8-thiol were suggested to have a key role in the juice aroma attributes [39]. By application of AEDA analysis on a volatile extract obtained from fresh grapefruit juice it was possible to confirm the significant contribution of p-1-menthene-8-thiol to C. paradisi typical aroma. Nevertheless, the results clearly showed that other minor components also are necessary to induce such aroma to the human nose [39]. However, in further studies it appears that the sulphur compounds are either not present in peel oil at high threshold levels or were decomposed during processing and storage to concentrations below their aroma thresholds [40]. The fragrant mandarin essential oil is widely employed for confectionary products. Thus, sensorial assessments are relevant to define specific applications for sweet confections and for genuineness delimitation, since the complex taxonomic situation of mandarins is not a trivial issue and cannot be underestimated [38, 41]. Minteguiaga et al. [41] studied the aroma compounds from C. deliciosa Tenore cv. Caí mandarin by GC-MS, GC-FID and GC-O (PI method with a panel of five trained assessors) through the analysis of commercial steam distilled (SD) and cold-pressed essential oils (CP) (with the fruit harvested at different maturation stages). The chemical profiles of the of SD and CP oils were slightly different, and interestingly, the aromatic profiles were remarkably divergent. Some of the most potent odorants were characterized mostly in CP “green” oils, such as p-menth-2-en1-ol, cis-­sabinene hydrate, and δ-cadinene; while in the SD sample highlighted olfactorily β-pinene, decanal and thymol [41]. The authors also discussed about the role of artifacts potentially generated in the distillation procedure which might be responsible for some unpleasant notes in SD oils [41]. Goh et al. [42] obtained two pomelo varieties [C. grandis L. (Osbeck), var. Hongxin and Shatian] CP peel essential oils, and performed GC-MS and GC-O experiments (AEDA with four experienced assessors) in the search of the compounds responsible for the appealing aromas of such fruits. In addition, the original essential oil samples were olfactory evaluated through sensory analyses by other panelists provided with a structured six-point scale [42]. In the two-varieties’ essential oils, 2-(E)-dodecanal was highlighted as the most potent odorant (FD: 10,000-100,000), but the contribution of limonene, trans-carvyl acetate, perilla acetate, and nerolidol (among others) was also important. Most important, some odorants allowed to discriminate by olfactometry both varieties: decanal, nonanal, trans-ocimene and nootkatone for Shantian variety; and citronellal and (E,E)-farnesol for Hongxin [42].

21.4.2 Mentha spp. (Lamiaceae) Mentha L. genus bears some of the most popularly known herbs all along the globe, characterized by their distinctive refreshing aroma originated in the fragmented aerial parts, which can be employed as raw material for essential oil production. Herbal infusions and decoctions are maybe the preferred uses of Mentha spp. aerial parts by people according to ethnopharmacological indications. However, the utilization as flavoring agent, n gastronomy and food industry, perfumery, pharmacy, and cosmetology, is the main reason behind the economic importance of those species. Around 30 currently cosmopolitan Mentha spp. can be found, spanning several natural hybrids and many cultivars, some of which

GC-O of Essential Oils  489 are commercially important. Some examples are M. aquatica L. (water mint), M. arvensis L. (corn mint), M. longifolia (L.) L. (horse mint), M. x piperita L. (peppermint), M. pulegium L. (pennyroyal or squaw mint), (M. x rotundifolia) (L.) Huds. [hybrid created by the cross-pollination between M. longifolia (L.) L and M. suaveolens Ehrh], and M. spicata L. (spearmint); among others [43]. Due to the economic importance of the genus, it is not surprisingly that many papers in the literature deal with the identification of the main odorants in Mentha spp. essential oils, particularly considering that minor differences in cultivation and post-harvest practices and geographical origin can modify the chemical composition and aromatic quality of the oils [44, 45]. Moreover, the utilization of GC-O is a clever step to ensure the genuineness of the species, hybrids and cultivars giving the fact that most of the aerial parts and their essential oils are commercialized indistinctively with the generic terms “mint herbs” or “mint oils”, without proper taxonomic identifications [45]. Díaz Maroto et al. [44] focused their work on the aromatic profile of three M. pulegium (pennyroyal) commercial samples; because this species is widely employed as spice in many culinary recipes, and its essential oil has several applications in flavor and fragrance industry. The extraction step was performed by a micro-scale SDE, followed by identification (GC-MS) and an olfactory description (GC-FID-O, by an intensity rating five-point scale frequency method) of the volatile components. As expected, pulegone being the main compound of the volatile extracts (41.1–42.3%) displayed the most intense pungent, minty, and balsamic aromas. Chemically and biosynthetically pulegone related compounds such as iso-pulegone and piperitol exhibited also intense minty notes. Other olfactory remarkable intense M. pulegium compounds were: methional (baked potato), 2,5-diethyltetrahydrofuran (roasted nuts), 3-octanol (mushroom), 1,8-cineole + limonene (eucalyptus + citric), carvacrol (spicy), and isopulegol (herbaceous) [44]. Díaz Maroto et al. [45] also studied the chemical composition of SDE volatile extracts from M. spicata and Mentha x piperita from commercial samples available in Spain. Authors followed a GC-O protocol (intensity description through a rating scale) finding the typical minty/balsamic aroma impact compounds of Mentha x piperita were trans-p-menthone, L-(-)-menthol (this being the responsible for the typical refreshing sensation), neo-­menthol, iso-menthone and 1,8-cineole. While compounds such as piperitone, 1-octen-3-ol, trans-2nonenal, α-terpinene, β-bourbonene, epi-bicylosesquiphellandrene, 2,5-diethyltetrahydrofuran and methional (among others) exhibited a great diversity of aromas including earthy, floral, fruity, and toasty/baked potato notes. In the case of M. spicata, R-(-)-carvone was the main component of the volatile extract, which conferred the typical sweet spearmint aroma; although it was also identified the contribution of cis-dihydrocarveol, 1-octen-3one and most of the above-mentioned compounds to the aromatic profile of this species [45]. Kelley and Cadwallader [46] studied the odorants of the essential oils from three Mentha spearmint species and different cultivars: US Farwest native (Mentha spicata), Macho mint cultivar (Mentha spicata cv. Macho mint), and Scotch type (Mentha gracilis L.). The authors identified the most potent odorants by GC-O (AEDA) and precisely quantified them through three gas chromatographic protocols: a stable isotope dilution assay (SIDA) using GC-MS, a non-isotopic internal standardization by GC-FID after a flash chromatography fractionation, and a GC-O dilution analysis. R(-)-carvone was the component which presented the highest FD in AEDA (most potent odorant) independently of the spearmint sample analyzed, and accordingly presented the highest abundance in the oils and OAV

490  Essential Oils values. Other important odorants for the three cultivars were eugenol, trans-β-damascenone, and (3-trans, 5-cis)-1,3,5-undecatriene. Finally, the authors discussed that several aromatic impact compounds in spearmint essential oils such as eugenol, some aliphatic C3 to C9 aldehydes, and noisoprenoids are really artifacts produced by distillation, for example, by the breakdown of amino acids following the inverse Strecker reaction [46]. Recently, Gabetti et al. [47] working on peppermint EOs (Mentha x piperita) combined the potential of GC-O (AEDA) with the powerful GCxGC-TOF/MS platform (targeted and untargeted analysis) to identify (or tentatively identify) minor key aromatic compounds, as well as to separate geographical origins. The main odorants identified in the oils were 1,8-cineole, menthone, and menthofuran; but with the aid of GC-O/GCxGC-TOF combination the aromatic role of the menthofurolactone diastereomers, ethyl-3-methylbutanoate, 2-(trans)-hexenal, myrtenyl methyl ether, and propyl-2-methylbutanoate was highlighted. Moreover, the abundances of these compounds and others, together with some unknowns from 131 peak-regions, were informative enough to separate Piedmont Italian samples (var. Italo-Mitcham) from American samples (var. Black Mitcham) by clustering analysis [47].

21.4.3 Thymus spp. (Lamiaceae) Thymus L. is a large genus in Lamiaceae with more than 350 species, including species widely known in culinary, as the common Mediterranean region-native thyme (T. vulgaris L.), which possess many industrial cultivars especially prized by their essential oils [48, 49]. Particularly, T. vulgaris has been demonstrated to be a largely plastic species, a fact that can be evidenced by the number of chemotypes or chemical races (at least seven) classified according to their main volatile components. Typical thyme aroma is considered herbaceous, pungent, and slightly sweet [48]. Díaz-Maroto et al. [48] extracted commercial T. vulgaris dried leaves by SDE and SFE to obtain its volatile profile and used GC-FID-O to assess for the aromatic impact compounds with a panel of six sensorial judges who evaluated the odor intensities with a five-point structured scale. The composition of both extracts was substantially different, with thymol and p-cymene being the most abundant components in SFE and SDE (respectively) and being SFE the technique that more closely caught the aroma of the spice. From the GC-FID-O aroma evaluation, the simple phenols thymol and carvacrol were classified as thyme key-odorants since both presented the highest perceived intensity regardless of the extraction technique employed [48]. The descriptors associated with those simple phenols were spicy, oregano, and thyme, to which aroma also contributed the isomers of methylthymyl ether. Different aromatic attributes were conferred to the volatile extracts by linalool and its oxides (floral), methional (baked potato), 1-octen-3-ol/1-octen-3-one (mushrooms), 1,8-cineole (minty, balsamic), camphor/borneol (camphoraceous), and other twenty-five odorants identified [48]. Goodner et al. [49] extracted the essential oils of cultivated clones of T. hyemalis Lange (winter thyme) and T. vulgaris subsp. vulgaris at five different stages of development to complete the assessment along all the life cycle of the plants. GC-MS-O employing a rating scale of aroma intensities was included as an analytical tool to provide organoleptic information related to the optimal harvesting period of both thyme species, assessing in parallel the chemical identity of the essential oil components through mass spectrometry. Twenty-seven consistent aromas were highlighted for T. hyemalis essential oil along the five

GC-O of Essential Oils  491 stages and, between them, the most intense perceived compounds were: thymol/­carvacrol (typical thyme green aroma), β-damascenone (sweet, honey-like), borneol (earthy, like menthol), linalool (floral), bornyl acetate (herbaceous), and p-cymene (engine oil-like, or green-menthol). Thymol/carvacrol intensities (as assessed by olfactometry) were at the same level independently of the phenological stage considered, but p-cymene level notoriously increased in the fruit maturation period. A different olfactory profile was found for T. vulgaris essential oil, being the olfactive most relevant components 1,8-cineole (sweet, menthol-like), terpinyl acetate (spice/pepper), β-damascenone (sweet, honey-like), borneol (herbal, menthol-like), linalool (floral), and eugenol (clove) among others. Despite most of the named compounds kept their aroma intensities all along the plant life cycle, the case of eugenol was remarkable, decreasing dramatically the GC-O intensity with the fruit maturation process surely due to a concomitant concentration variation [49]. Sonmezdag et al. [50] extracted the aroma-active volatile components of the wild thyme T. serpyllum L. from Turkey, employing P&T with N2 as purging gas and ethyl-vinyl benzene/­ divinyl benzene composed adsorbent cartridges; being the volatiles eluted by CH2Cl2. After concentration, the organic extract was checked in relation to their representativeness of the original herbal aroma through a sensorial evaluation performed by a panel of 10 experienced and trained assessors. The similarity score between the P&T extract aroma and the authentic herbal aroma was found to be 75.1/100, thus considered an acceptable level by the authors [50]. Finally, the extracts were analyzed by GC-MS-O (AEDA), diluting the P&T extract in CH2Cl2 in the 1:1 to 1:1024 range; in the runs participated two experienced sniffers as assessors. In total, 24 components were identified and quantified in the P&T extract from T. serpyllum; but only 12 odorants were detected (two of them not chemically identified): thymol (thyme odor; FD: 1024), sabinene hydrate (herbal note; FD: 512), and δ-3-carene (mint-like; FD: 256). Other relevant aroma-active compounds were the monoterpenoids myrcene, γ-terpinene, α-pinene, carvone, and terpinen-4-ol [50].

21.4.4 Foeniculum spp. (Apiaceae) Fennel (Foeniculum vulgare Mill.) is one of the most relevant spices in the commercial market, including several subspecies and varieties, especially in the Mediterranean countries where it is practically omnipresent in the diet and culinary [48]. Two subspecies of F. vulgare are recognized: F. vulgare capillaceum (with the varieties azoricum, dulce and vulgare) and F. vulgare piperitum. F. vulgare L. subsp. capillaceum var. dulce (sweet fennel) is the most cultivated and culinary employed fennel herb due to its delicate aroma [48]. In fact, such fennel seeds have an anise-like aroma, with their essential oils are rich in phenylpropanoids (trans-anethole and its positional isomer estragole) and fenchone, exhibiting wide contents variation according to the origin and phenological stage [48]. Díaz-Maroto et al. [48] performed the first study on aroma-active compounds from fennel seeds volatile extracts (obtained through SDE and SFE), employing GC-FID-O with a structured sensorial scale. Independently of the technique of choice, the most abundant compounds found in the extracts were trans-anethole (49.7–63.8%), estragole (25.8–20.3%) and fenchone (19.3–12.7%). Accordingly, these compounds together with the trace-level component 1-octen-3-ol, were the olfactorily most intense odorants (labeled as “strongly perceptible” by the panelists). The characteristic anise-like sweet aroma was assigned to trans-anethole and estragole, but other minor components such as cis-anethole and

492  Essential Oils p-anisaldehyde also contributed to such typical aroma. Moreover, while fenchone influence the overall aromatic profile, its descriptors were not anise-like but mint, camphoraceous and warm, while 1-octen-3-ol exhibited a characteristic mushroom odor. Other twenty-­ three odorants were characterized as F. vulgare aroma-active compounds, including the monoterpenoids α-pinene, α-terpinene, 1,8-cineole, and limonene [48].

21.4.5 Coriandrum spp. (Apiaceae) The annual herb coriander, cilantro, or Chinese parsley (Coriandrum sativum L.), is also a world-known spices, being native of the Mediterranean region, the Near East, North Africa and Russia; but currently cultivated beyond seas [51, 52]. The fresh leaves of pre-flowering stage immature individuals are edible and broadly employed in culinary as condiment due to the remarkably pungent aromatic notes with fatty/aldehydic overtones of its essential oil [51]. Differently, C. sativum dried seeds render an oil (coriander oil) with typical floral notes originated in an abundant composition of linalool. This seeds also are employed as condiment, and beyond, coriander oil (and the species itself) is considered as medicinal [52]. The work performed by Eyres et al. [51] aimed to characterize the odorants of the leaves of C. sativum (true coriander) from Fiji Islands, and to compare them with the correspondents from Eryngium foetidum (wild coriander), which sometimes is employed as a substitute of the former. For such a purpose, the authors obtained steam-distilled essential oils from the leaves of both species and performed GC-FID-O runs of diluted samples with two experienced assessors, who provided duration and description of the aromas to a software (CharmwareTM). According to the CHARM analysis procedure, for every panelist the perceptions obtained for sequential cyclohexane-diluted samples were integrated, calculated the Charm values (as peak areas), and generated the respective aromagrams. To further identify and elucidate the aroma compounds, the authors conducted GCxGC-TOFMS (with cryogenic modulation) runs. Therefore, 81 and 55 compounds were identified in the C. sativum and E. foetidum oils (respectively), mainly C10, C12, C13 and C14 linear aliphatic aldehydes and alcohols. For C. sativum, 33 consensus odorants were determined, where 2-(cis)-decenal (odor notes: coriander, spicy, pungent, and aldehydic) shows the higher value of total Charm value (TCV: 18.3%) in despite of being 0.16% of the sample. The most abundant compound of the oil, 2-(trans)-decen-1-ol (26.0% of FID area), contributed only with 0.39% of TCV demonstrating that there is not an evident relationship between FID and olfactory response [51]. The second most intense aroma of C. sativum was due to an odor cluster composed by 2-(trans)-dodecen-1-ol, 2-(trans)-dodecenal and 1-docecanol (TCV, 14.0%) with characteristic olfaction descriptors such coriander, floral, citrus, pungent and spicy. Other important aroma-active compounds for the true coriander essential oil were: β-ionone, eugenol and other C13 and C14 aldehydes and alcohols. In the case of the wild coriander, 2-(trans)-dodecenal was both the most abundant (FID area, 63.5%) and the most intense odorant (TCV, 52.9%) displaying a typical coriander aroma with pungent and spicy notes. Other important odorants of E. foetidum essential oil were eugenol, dodecanal, 4-(cis)-dodecenal, β-ionone, and 2-(trans)-tetradecenal [51]. After the first publication, Eyres et al. [53] conducted a subsequent and deeper work on C. sativum essential oil, employing MDGC-O (heart-cut multidimensional gas chromatography-olfactometry with a cryogenic trap connecting 1D and 2D-GC dimensions)

GC-O of Essential Oils  493 as a complimentary approach to GCxGC-TOFMS in order to separate the aroma clusters evidenced in the previous work. In particular, authors focused on the cluster generated in GC-O by the co-elution of 2-trans-dodecen-1-ol, 2-trans-dodecenal and 1-docecanol. With the aid of MDGC-O, base-line separation of such compounds was obtained, and finally, confirmed that the aroma attributes (floral, coriander-like) only corresponded to 2-trans-dodecenal [53]. Differently from Eyres et al. [51, 53], Ravi et al. [52] focused on C. sativum aroma-­ compounds from seeds harvested in different regions of India. The authors obtained the coriander essential oil by hydro-distillation, and then conducted a quantitative descriptive analysis (sniff test) for obtaining its odor profile (profilogram) by providing to the sensorial panelists a structured scorecard. The main descriptors associated with coriander oil were floral, pleasant, turpentine, spicy, sweet, and rose-like; but high level of variation was evidenced between different seed oil samples. The instrumental analysis was carried out by GC-FID, GC-MS, e-nose, and GC-O. For this last technique, the sensorial panel (ten assessors) was asked only to provide odor descriptors for the major compounds of the oil (­ frequency method) [52], instead a complete run evaluation with intensity assessment. Linalool was the main component of the oil independently of the considered sample (57.5%-75.1%) which conferred floral, pleasant, grassy, and citric notes in GC-O evaluation. Other abundant components of the coriander oils also exhibited aromatic importance: geranyl acetate (rose-like, pleasant, herbal) α-pinene (woody, spicy, oily), terpineol (sweet, lilac-like), and geraniol (rose-like, sweet, fresh); among others. Finally, to discriminate between samples and to explain the observed composition variability, the obtained GC-MS and e-nose results were subjected to principal component analysis [52].

21.4.6 Pinus spp. Pinus L., is represented by at least 100 species that are distributed, as native or cultivated, in practically all the world regions. Beyond the importance of those species as construction materials (building and furniture fabrication), the typical aroma of pines is extremely pleasant and relaxing, being attractive, for example for toys manufacture [54]. Despite the economic importance of pines, very few works have been conducted to identify the aroma active compounds that confer the aroma of natural pine wood. Schreiner et al. [54] aimed to elucidate the wood smell of Scots pine (P. sylvestris), being this the first study that characterized the aroma active components in Pinus wood. They obtained crude CH2Cl2 extracts from P. sylvestris shavings, and then extracted the aromas from them at vacuum condition by SAFE. The authors ensured through sensorial analysis that the so-obtained aroma extracts were representative of the P. sylvestris wood aroma. In parallel, wood splints were also submitted to sensorial tests by trained and untrained panelists to rate their intensity and pleasantness using a structured scale, and to recognize special attributes (descriptors) of the aroma. In general, the wood splints were characterized as having intense aroma (7.8/10) and being pleasant (6.2/10), and as expected, the main attributes were resin-like (7.2/10), sawdust-like (2.7/10), and frankincense-like among others. The SAFE extracts were injected on-column to perform GC-FID-O assessments, which were conducted through AEDA approach by serial dilution of the samples. The most intense odorants identified were then further identified employing sophisticated instrumental set-ups: GC-MS/O and 2D-GC-MS/O (with a cryogenic trap as interface

494  Essential Oils between the two GCs) [54]. A total of 44 odorants were detected (39 of them being chemical identified) spanning FDs from 9 to 729, and some intensity differences were observed when comparing wood from different trees, which could be attributable to differences in plant material age or environmental conditions influences. The most intense odorants (FD: 729) identified independently of the samples were (in order of odor importance): vanillin (aromatic descriptor: vanilla), phenylacetic acid (honey-like), α-pinene (woody, resinous), 2,4-(trans,trans)-nonadienal (fatty), δ-octalactone (coconut-like), 3-phenylpropanoic acid (vomit-like or fruity), and heptanoic acid (pepperoni or plastic-like). The results of sensorial analysis and GC-O correlated closely according to the authors, demonstrating the validity of the followed procedure despite some descriptors could not be associated to any odorant [54].

21.4.7 GC-O Applied to Characterize Baccharis dracunculifolia DC. Odorants Even not being one of the most important essential oils in commerce, Baccharis spp. L. oils, and their volatile extracts, possess great potential for the flavor and fragrance industries [55, 56]. This has been the reason why this genus has been the research focus of our group in the last years. Baccharis L. contains more than 440 species, mainly herbs and shrubs which are naturally distributed exclusively in the American continent [55, 56]. Inside Baccharis L. genus, a standout place must be dedicated to the Latin American bush species B. dracunculifolia DC. (vernacular names: “alecrim-do-campo”, “chilca blanca”, “vassoura”, among others), which essential oil is widely used for perfumery owing to their floral, spicy and grassy aromatic notes [55, 57]. Here will briefly described the research conducted aiming to characterize the aromatic profile of B. dracunculifolia essential oils from Uruguayan origin through GC-O. Previously, the typical odorants of the Baccharis spp. were reported including B. anomala DC. [58]; B. articulata (Lam.) Pers. [59], and B. dentata (Vell.) G.M. Barroso, and B. uncinella DC. [60]. B. dracunculifolia was evaluated sampling aerial parts at full flowering stage from a natural growing population in Paysandú Province (32°03′55″S 57°19′38″O). Separated male and female branches were allowed to dry protected from the sunlight and essential oils submitted to hydro-distillation during 90 minutes with the employment of a Clevenger type apparatus [55]. The pale-yellowish and floral scented oils were studied by GC-MS [59]. To differentiate the aromatic profile of male and female individuals of B. dracunculifolia the essential oils were analyzed by GC-O following the analytical conditions described by Minteguiaga et al. [59] using an SGE ODO-1 phaser sniffing port. A panel composed by five trained individuals was asked to provide descriptor for such an odor. The raw data was processed by calculating the modified frequency values (Dravnieks’ formula) according to the PI method [11]. Figure 21.3 shows the aromagrams obtained from “male” and “female” B. dracunculifolia essential oils, being plotted modified frequency vs. retention time as an indicative of the sensorial response to all the components of the oils. The compositions of the male and female individuals’ essential oils were found to be practically the same (Table 21.1) [56], but the GC-O profiles were different (Figure 21.3, Table 21.1), being nonanal and the sesquiterpene alcohols (trans)-nerolidol and viridiflorol not detected as odorants in female plants (OAV values below 1).

GC-O of Essential Oils  495 90,0

B. dracunculifolia "male" EO

2

Modif ied Frequency

80,0

12 13

70,0 60,0

7

1

4 3

50,0

8 5

11 10

9 6

40,0 30,0

3,0

10,0

17,0

24,0

31,0

38,0

45,0

52,0

59,0

Retention time (min) 90,0

B. dracunculifolia "female" EO

Modif ied Frquency

80,0

13

2 7

70,0

3

60,0 50,0

5

1

6

8 9

12

40,0 30,0

3,0

10,0

17,0

24,0

31,0

38,0

45,0

52,0

59,0

Retention time (min)

Figure 21.3  Aromagrams obtained through GC-O (posterior intensity method) for the essential oils obtained from B. dracunculifolia male and female plants. Odorants identified: 1. α-pinene, 2. β-pinene, 3. (trans)-βocimene, 4. nonanal, 5. α-cubebene, 6. β-bourbonene, 7. linalool, 8. pinocarvone, 9. β-elemene, 10. (trans)nerolidol, 11. viridiflorol, 12. spathulenol, and 13. ζ-cadinol.

Table 21.1 also shows the odor descriptors associated with the essential oils and their constituents, which could, eventually, conduct to fractionate the samples with the aim to enrich in select specific notes (or components) for several applications, including perfumery. In brief, the results here presented for an aromatic plant demonstrate the utility of GC-O as a powerful tool to differentiate essential oils extracted from male and female plants, which is important for exploitable species by the flavor and fragrance industry. Meaning that essential oils from both sexes could have different aromatic qualities and consequently, different applications. Furthermore, GC-O eventually could be employed to differentiate male and female individuals outside flowering period, which is a challenging task in the current research. The data above presented indicate there is not linear correspondence between compound concentration in essential oils and olfactory modified frequency. Both, the concentration equilibrium between the original odorants from a matrix and their specific

496  Essential Oils Table 21.1  Odorants identified through GC-O (posterior intensity method) in B. dracunculifolia essential oils from both sexes. Non polar LRIs1

Polar LRIs1

Components

% Comp. % Comp. EO EO MF3 MF3 Aromatic 2 2 (♂) (♀) (♂) (♀) descriptors4

931

1011

α-pinene

4.5

3.9

60.6

51.6

Herbaceous

977

1094

β-pinene

10.5

10.9

85.6

77.5

Fuel, chemical, herbaceous

1047

1234

(trans)-βocimene

0.4

0.4

48.3

65.8

Herbaceous, spicy

1105

1382

nonanal

0.1

0.08

57.7

n.d.5

Fruity, floral, fatty

1349

1443

α-cubebene

0.1

0.1

51.6

51.6

Herbaceous, cooked vegetables

1386

1496

β-bourbonene

0.08

0.1

44.7

54.8

Herbaceous, chemical

1100

1528

linalool

0.4

0.2

63.2

70.7

Floral, microbiological, almonds

1160

1545

pinocarvone

0.3

0.4

57.7

60.6

Floral

1399

1572

β-elemene

0.4

0.3

51.6

60.6

Herbaceous, bug-like

1556

2013

(trans)-nerolidol 16.7

17.3

54.8

n.d.5

Herbaceous, dried fruits

1595

2055

viridiflorol

1.7

1.7

57.7

n.d.5

Floral, fruity, chemical

1583

2093

spathulenol

5.5

5.2

77.5

57.7

Wood, floral, humidity

1645

2156

ζ-cadinol

0.4

3.0

70.7

81.6

Clove, caramel, honey

Lineal retention indices in non-polar (95%-polydimethyl-5%-polyphenilsiloxane) and polar (100% polyethylene glycol) stationary phases; 2modified frequency [30]; 3abundance percentage expressed as peaks areas in GC-MS analyses with non-polar stationary phase; 4aromatic descriptors named by the panelists for every odorant; 5not odor detected. 1

physicochemical properties contributes to the “real picture” in olfactory analyses. The synergistic effects among odorants are common when dealing with GC-O evaluations and operate in the sense of smell in an unpredictable way, even influenced by previous cultural experiences. These observations demonstrate that our understanding in odor science can

GC-O of Essential Oils  497 only be possible through innovations in GC-O-based analytical approaches [61–65], which also requires multidisciplinary approaches.

Acknowledgements The authors want to acknowledge to Uruguayan National System of Researchers [SNI-ANII].

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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500  Essential Oils 50. Sonmezdag, A.S., Kelebek, H., Selli, S., Characterization of aroma-active and phenolic profiles of wild thyme (Thymus serpyllum) by GC-MS-Olfactometry and LC-ESI-MS/MS. J. Food Sci. Technol., 53, 1957, 2016. 51. Eyres, G., Dufour, J.-P., Hallifax, G., Sotheeswaran, S., Marriott, P.J., Identification of ­character-­impact odorants in coriander and wild coriander leaves using gas chromatography-olfactometry (GCO) and comprehensive two-dimensional gas chromatography–time-offlight mass spectrometry (GCxGC-TOFMS). J. Sep. Sci., 28, 1061, 2005. 52. Ravi, R., Prakash, M., Bhat, K.K., Aroma characterization of coriander (Coriandrum sativum L.) oil samples. Eur. Food Res. Technol., 225, 367, 2007. 53. Eyres, G., Marriott, P.J., Dufour, J.-P., The combination of gas chromatography-olfactometry and multidimensional gas chromatography for the characterisation of essential oils. J. Chromatogr. A, 1150, 70, 2007. 54. Schreiner, L., Bauer, P., Buettner, A., Resolving the smell of wood identifcation of odour-active compounds in Scots pine (Pinus sylvestris L.). Sci. Rep.-UK, 8, 8294, 2018. 55. Minteguiaga, M., Mercado, M.I., Ponessa, G.I., Catalán, C.A.N., Dellacassa, E., Morphoanatomy and essential oil analysis of Baccharis trimera (Less.) DC. (Asteraceae) from Uruguay. Ind. Crops Prod., 112, 488, 2018a. 56. Minteguiaga, M., González, A., Cassel, E., Umpierrez, N., Fariña, L., Dellacassa, E., Volatile constituents from Baccharis spp. L. (Asteraceae): Chemical support for the conservation of threatened species in Uruguay. Chem. Biodivers., 15, 1800017, 2018b. 57. Groom, N., The perfume handbook, Chapman & Hall, London, 1992. 58. Xavier, V.B., Vargas, R.M.F., Minteguiaga, M., Umpierrez, N., Dellacassa, E., Cassel, E., Evaluation of the key odorants of Baccharis anomala DC essential oil: New applications for known products. Ind. Crops Prod., 49, 492, 2013. 59. Minteguiaga, M., Umpiérrez, N., Fariña, L., Falcão, M.A., Xavier, V.B., Cassel, E., Dellacassa, E., Impact of gas chromatography and mass spectrometry combined with gas chromatography and olfactometry for the sex differentiation of Baccharis articulata by the analysis of volatile compounds. J. Sep. Sci., 38, 3038–3046, 2015. 60. Xavier, V.B., Minteguiaga, M., Umpierrez, N., Vargas, R.M.F., Dellacassa, E., Cassel, E., Olfactometry evaluation and antimicrobial analysis of essential oils from Baccharis dentata (Vell.) G.M. Barroso and Baccharis uncinella DC. J. Essent. Oil Res., 29, 137, 2017. 61. Hattori, S., Takagaki, H., Fujimori, T., Evaluation of Japanese green tea extract using GC/O with original aroma simultaneously input to the sniffing port method (OASIS). Food Sci. Technol. Res., 9, 350, 2003. 62. Hallier, A., Courcoux, P., Serot, T., Prost, C., New gas chromatography-olfactometric investigative method, and its application to cooked Silurus glanis (European catfish) odor characterization. J. Chromatogr. A, 1056, 201, 2004. 63. Williams, R.C., Sartre, E., Parisot, F., Kurtz, A.J., Acree, T.E., A gas chromatograph-pedestal olfactometer (GC-PO) for the study of odor mixtures. Chemosens. Percept., 2, 173, 2009. 64. Burseg, K. and de Jong, C., Application of the olfactoscan method to study the ability of saturated aldehydes in masking the odor of methional. J. Agric. Food Chem., 57, 9086, 2009. 65. Villière, A., Le Roy, S., Fillonneau, C., Prost, C., InnOscent system: Advancing flavor analysis using an original gas chromatographic analytical device. J. Chromatogr. A, 1535, 129, 2018.

22 In Vitro and In Vivo Methods Used to Assess the Biological Potential of Essential Oils Syed Ali Raza Naqvi1*, Sadaf Ul Hassan2, Tauqir A. Sherazi3, Amjad Hussain4 Muhammad Rehan Hasan Shah Gilani5 and Tanvir Hussain6 Department of Chemistry, Government College University, Faisalabad, Pakistan Department of Chemistry, School of Sciences, University of Management and Technology, Lahore Campus, Pakistan 3 Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan 4 Department of Chemistry, University of Okara, Okara, Pakistan 5 Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, Pakistan 6 Department of Chemistry, The College of Art and Science, Sialkot, Pakistan 1

2

Abstract

EOs are the botanical product having volatile nature and known for its special odor. These are found effective in treatment of oxidative stress, cancer, epilepsy, skin allergies, indigestion, headache, insomnia, muscular pain, and respiratory problems. The EOs such as ginger oil, cinnamon oil, clove bud oil, mustard oil, eucalyptus oil, sweet oranges oil, and rosemary oil recently also reported as effective remedy against COVID-19 infection. The biological assays, either in vitro or in vivo are the backbone of developing phytochemical based pharmaceutical industry. In this chapter, the in vitro assays for assessing antioxidant, antibacterial and anticancer activities of EOs will be explored. The data cited in this draft was collected from electronic database sources including PubMed, and Google search engine. Keywords:  EO, antioxidants, antibacterial agents, anticancer, PubMed data

22.1 Introduction EOs (EOs), also termed as ethereal-oils, are defined in different ways, however the plant extracts having specific aroma and volatile in nature obtained through distillation processes is a common myth. The aroma of EOs are blends of fragrant substances which is pure volatile compound and can be valuable to society due to its odor. Aromatherapy is technique used to apply EOs in a holistic environment, to maintain or improve physical, emotional, and mental well-being [1]. Aromatic herbs and their EOs (EOs) have been shown to be effective against a wide range of viruses, hepatitis B virus, and even SARS.CoV-1 [2]. *Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (501–520) © 2023 Scrivener Publishing LLC

501

502  Essential Oils The procedures for extracting the EO from naturally occurring pant materials were initially devised by Arabs. In the eleventh century, Avicenna, the Arab physician presented the protocol for extracting the EOs from flowers through distillation [3]. He extracted the perfume from rose flowers in the form of oil or attar and made rose water. The term “rose water” in history was first time reported by Ibn-e-Khaldun, who was an Arab historian. The fame of EOs over the globe was due to their specific aroma and therapeutic effect against numerous diseases and activation in emotional behaviors. The history of EO usage as therapy of diseases which commonly known as aromatherapy begins over 3500 years BC. EOs are thought to have both physical and psychological advantages and dominantly, being used in fixing different disease processes of human beings. These are used as main ingredients in cosmetic industry for their antioxidant, antimicrobial, anti-allergic, and anti-aging potential. Similar to other phytochemicals, EOs are also evaluated using in vitro and in vivo methods for their medicinal or therapeutic potential. The next sections will discuss the in vitro and in vivo methods used to assess the biological potential of EOs.

22.2 Chemistry of EOs Although the term “aromatic” is now used to denote the attribute of emitting a fragrance which is either pleasant or unpleasant. Aromatic hydrocarbon compounds have a chemical structure that results in electron delocalization, resulting in increased molecular stability. As a result, EOs can contain both aromatic and aliphatic (non-aromatic) molecules [4]. Essential Oils are usually liquid and colorless at normal pressure and temperature. With the exception of a few cases (cinnamon, sassafras, clove, and vetiver), they have a distinct odor and a density less than one. They also have a strong optical activity and a refractive index [4]. The flavor contribution of individual compounds, on the other hand, is governed by the unique odor, rather than by their concentration [4, 5]. Regarding the chemistry of EOs; EO components may be short chain and long chain, hydrocarbons or oxygenated hydrocarbons, phenylpropanoid and lipophilic terpenoids. These include terpenes, aldehydes, fatty acids, phenols, ketones, esters, alcohols, oxides phenol, and compounds containing nitrogen and Sulphur. The isoprene units combined and make the long chains called monoterpene, sesquiterpenes, diterpenes, and big terpenes. Monoterpenes are the major part of EOs and further classified as acyclic, monocyclic, bicyclic, alcohols, ethers, peroxides, esters; bicyclic, monocyclic, aldehydes and phenols [6]. Monoterpene hydrocarbons are less valuable in terms of contributing to the EO’s smell than oxygenated molecules, which are extremely odoriferous. In addition to their chemical properties, EOs can be seen to vary greatly in their physical properties in terms of their refractive index. For instance, the EOs of different parts of Siphonochilus ethiopicus (e.g. limbo, foliar sheath, and rhizomes) showed significant difference in their physical properties [7]. Typically, EOs are analyzed by GC-MS. The beauty of the technique exists in its identification and quantification, almost all the components present in the sample. For example Origao EO contain two main compounds, i.e. thymol (27%) and caracole (30%) while limonene (31%) was found main compound the oil of Anethum graveolens; β-thujone (57%) in Artemisia herba-alba EO; linalool (68%) in Coriandrum sativum EO; menthone (19%) and menthol (59%) in Mint EO [8, 9].

Biological Potential of Essential Oils  503 CH3

CH3

CH3 OH

OH H3C

CH3

H3C

CH3

Carvacrol

p-Cymene

CH3

Thymol

CH2 H3C

H3C

CH2

H3CO

O

HO Estragole

Eugenol CH3

CH3

CH3 CH3OH

OH

HO

CH3 H3C

CH3

Menthol

H3C

CH3

H3C

Geranoil

CH3 Farnesol

Figure 22.1  Chemical structures of key compounds present in EOs.

The nature and knowledge of the function groups of EO components can be accessed using FTIR, Raman, and FTIRMS techniques. These finding show that the unique Raman intensities are linked to specific terpenes, allowing FT-Raman to distinguish between EOs whose major components belong to different classes of chemicals. Figure 22.1 shows the chemical structure of some major components of EOs having variety of biological activities with their functional groups [10].

22.3 In Vitro Methods Used to Assess the Biological Potential of EOs Essential Oils and EO components from different plants are gaining interest in the food, cosmetics, nutraceuticals, and pharmacological applications [11]. They are evaluated by variety of different assays; however antioxidant, antimicrobial and anticancer potential of EO make it promising ingredient to use in food, beverage, cosmetic, and pharmaceutical industries. The following section will emphasize on in vitro antioxidant, antimicrobial and anticancer assays to evaluate the medicinal potential of EOs [1, 6]. Whereas in vivo assay

504  Essential Oils concerned for the evaluation of EOs; they vary from animal model to model. Therefore, main focus was put on in vitro studies [12].

22.4 Evaluation of Antioxidant Potential 22.4.1 What are Antioxidants? Antioxidants compounds plays a defensive role by preventing the generation of free radicals and they have been characterized in biological terms as substances “that can slow down the process of oxidation, even in small concentration” [13, 14]. The numerous reports on antioxidant study of EO, All have found more or less valid to screen the EO. Then one follows methods which are predominantly used to assess the antioxidant activity of EO [15].

22.4.2 Antioxidant Potential of Botanical Materials Since the start of 21st century, there has been widespread public concern regarding the hazardous effect of synthetic anti-oxidants such as butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT), tert-Butylhydroquinone (TBHQ) Ascorbic acid, used in different functional foods, as food preservative and antioxidant supplements. The chemical structures of synthetic antioxidants are shown in Figure 22.2 [16]. On food systems and humans, these synthetic antioxidants are proven to be harmful and carcinogenic Synthetic antioxidants e.g. BHA, BHT, TBHQ etc. are not generally recognized as safe and cause cerebrovascular and other disorders [17]. Exploration of natural antioxidant [18] prompted food scientists to exploit novel sources of natural antioxidants

(a)

(b)

O

OH

OH

(c)

(d) HO H

HO

OH

HO

HO

O

O

OH

Figure 22.2  The chemical structures of BHA (a), BHT (b), TBHQ (c), and ascorbic acid (d).

Biological Potential of Essential Oils  505 from plant and animal origins which are thought to be naturally safe. Many antioxidant sources have been investigated, and more study is ongoing. EOs obtained from leaves, barks, roots, flowers, and stems is known to show antioxidant activity [17]. Many recently published reports reflect tremendous potential of EOs as strong antioxidant blend [14]. Different classes of biomolecules having different functional groups, involve in antioxidant potential of EOs due to which variety of in vitro antioxidant assays were developed and reported [19].

22.4.3 Modes of Action A number of in vitro studies have been demonstrated the antioxidant properties of botanical EOs and their extracts. They are rich in polyphenolics and can terminate oxidation chain by direct chain-breaking action either by reduction, hydrogen/proton donation or single oxygen quenching, in addition to being capable of scavenging non-physiological free radicals such as ABTS and DPPH [20]. They can scavenge a variety of reactive oxygen species in vivo such as peroxyl radicals, hydroxyl radicals [21], hypochlorous acid and superoxide with significance. Polyphenols suppress the generation of free radicals by inhibiting the oxidative enzymes including lipoxygenases [22] and cyclooxygenases.

22.4.4 In Vitro Methods for Antioxidant Activities Antioxidant activity and free radical scavenging capacity in biological systems and foods are depending on number of factors. The antioxidant capacity of natural products including EOs has been evaluated using various methods that are based on the radical scavenging methods and measurement of reducing potential assays [23]. In literature it is stated that a standardized antioxidant testing method must fulfill the following requirements: • • • • •

Adaptable for high-throughput approach analyses, Good accuracy and acceptable reproducibility, Reasonable cost, Simplicity in performance, Employing a biologically relevant source of radicals.

Typically, not a single reported assays fulfill all these requirements; however, if the proposed method for the antioxidant activity fulfill all the above mention requirements then the method would be called as standard method. In general trend, the new developed reported methods not reflect its demerits, however to approach standard characteristics, both strengths and weaknesses must be mentioned. Due to various modes of action, multiple tests are mandatory for testing antioxidant activity. Each assay used to test antioxidant potential is performed with different set of reaction conditions. Sometime the kinetics and rate of reaction should also be monitored. A reliable antioxidant methodology has ability of assess key properties which are important for food or biological systems. As a result, to reduce the discrepancies in methodology,

506  Essential Oils antioxidant testing should be standardized. Sometime general but mostly specific methods are used to access the antioxidant status especially in the food and biological systems. The common in vitro antioxidant assays used are DPPH free radical scavenging assay and inhibition of lipid peroxidation [23]. Among these methods, DPPH radical scavenging method, ABTS radical scavenging assay, and β-carotene bleaching (BCB) assay are used by majority of researcher as method of choice to evaluate the antioxidant and free radical scavenging ability of natural products and EOs [15, 24]. The following sections will explain these assays.

22.4.5 DPPH Scavenging Assay In DPPH scavenging assay, the hydrazyle upon receiving the proton produces hydrazine, which is a chromophore quenched product (Figure 22.3) [15]. The process was also studied by electron spin resonance spectroscopy to assess the ability of botanical sample to make stable DPPH molecule. The process reflects the DPPH• absorbance strength is related to antioxidant inversely and reaction time [14]. The DPPH° scavenging assay is also termed as discoloration reaction. The discoloration assay, which measures the decrease in absorbance at 515–525 nm produced by the addition of antioxidant to DPPH°, is a more frequently used method. The antioxidant activity is measured by monitoring the decline in the absorbance at different concentration while comparing the absorbance of the control sample. Not only the antioxidant activity of botanical and natural products is determined by this method in common, but the antioxidant potential of synthetic substances such as ascorbic acid, BHT, BHA, TBHQ, has been assessed using the DPPH method [25]. The DPPH method provides a high throughput, sensitive, accurate, quick, easy, and cost-effective way to assess the scavenging activity of antioxidants in fruits and vegetable juices or extracts, as well as plant EOs; however, it needs to take precautions to use DPPH. The assay shows reproducible. ABTS assay, is another approach to study antioxidant potential of plant materials [26]. The assay, in practice is a time taking protocol to scavenge free radical. This drawback make the DPPH° scavenging assay superior as it is one step assay. The advantage is that in this method the rate of reaction can also be accessed and the time required to consume half concentration of radical is also calculated.

NO2 N

NO2

N

NO2

+

NO2

H

H

N

NO2 +

N

H

DPPH Free Radical (Purple at 517 nm) R

R

R

NO2

DPPH-H (Colorless at 517 nm)

Antioxidant

Figure 22.3  DPPH free radical scavenging principle (redraw and amended the mechanism path) [15].

Biological Potential of Essential Oils  507 However, the disadvantage of the method is nonlinearity and the reactivity of DPPH with alkyl radicals [27, 28]. In the presence of light, DPPH° is not stable which is because of the direct n reaction of oxygen with DPPH° and in this way this interference can disturb the accuracy of the method and enhance the random error in the results [29]. Literature revealed that DPPH free radical scavenging assay is one of the most commonly used assays for evaluating the antioxidant potential of EOs [31]. The general protocol to conduct this assay is as follow; ØØ Prepare a 2.7 µM DPPH free radical solution in DMSO or ethanol. ØØ Took 1 mL of DPPH free radical solution in a tube followed by equal volume of EO of various concentrations and vortex. ØØ Incubation of the reaction mixture in dark for 30 min. ØØ Note the absorbance at 517 nm. ØØ Repeat all these steps for standard using some synthetic antioxidant. ØØ Conduct experiment in triplicate to reach maximum level of confidence. ØØ Calculate the % inhibition of DPPH free radical using the following equation:



Percentage scavenging of EOs =

Abscontrol − Abssample ×1000 Abscontrol

The antioxidant activities of most commonly used EOs determined by DPPH° is shown in Table 22.1.

22.4.6 2,2-Azinobis-(3-Ethylbenzothiazoline-6-Sulfonate) Assay The 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS•+) is a green-blue stable radical cation. The ABTS•+ assay is used to determine the antioxidant potential of plant extracts and EOs. It is also termed as trolox equivalent antioxidant capacity (TEAC) assay. In contrary to the DPPH° scavenging assay, it measures the presence of water soluble antioxidants in sample material such as rutin, coumarins, chlorogenic acid, and tannins [32]. These compounds which are also known as member of elite class of compounds, not only give antioxidant activity but also exhibit strong anti-inflammatory potential. The ABTS•+ on accepting a proton when it is mixed with strong antioxidants produce ABTS [32]. The reduction of ABTS•+ to ABTS is measured by spectroscopically, therefore the extent of reduction is recorded by monitoring the suppression of its characteristic long wave absorption spectra. The ABTS•+ assay is a common antioxidant evaluator method of EOs. There are many antioxidant studies that were conducted for EOs using ABTS•+ assay which showed promising free radical scavenging, for example the EO of S. macrostema showed 53.10% of DPPH° and 92.12% ABTS•+ scavenging potential which indicate the EO of S. macrostema carry good amount of water soluble antioxidants, most probably of elite class antioxidants. The ABTS•+ assay is performed following the steps given as follow [33];

508  Essential Oils

Table 22.1  Antioxidant activities EOs of various plants. Common name Clove

Alecost, costmary

Scientific name

Most abundant compounds

Abundance (%)

Syzygium aromaticum

Eugenol Eugenyl acetate β-Caryophyllene Tau muurolol Isoeugenol β-Thujone α-Thujone Eucalyptol Thymol

Tanacetum balsamita L.

β-Eudesmol Golden Buttons

Tanacetum vulgare L.

Antioxidant assay

Results

Reference

72.46 4.18 3.73 2.83 2.12

DPPH° Scavenging Assay,

29.36–77.28% inhibition

[38]

β-carotene linoleic acid bleaching assay

9.56–72.68% inhibitory activity

84.43 4.68 4.07 0.67 0.64

DPPH° Scavenging Assay & FRAP Assay

13.59% inhibition 339.1 mol Trolox/g

TransChrysanthenyl acetate β-Thujone (E)-Dihydrocarvone Artemisia cis-chrysanthenol

18.39

13.86% inhibition

14.28 11.02 9.15 3.93

585.6 µmol Trolox/g

Camphor Borneol 1,8-Cineole Camphene bornyl acetate

30.48 14.8 10.8 7.29 5.53

DPPH° Scavenging Assay, FRAP & ABTSº+ Assay

[39]

IC50 = 51 µg/mL

[39]

[40]

(Continued)

Biological Potential of Essential Oils  509

Table 22.1  Antioxidant activities EOs of various plants. (Continued) Common name Conehead Thyme, Persian-hyssop and Spanish Oregano

Scientific name

Most abundant compounds

Abundance (%)

Thymus capitatus L

Thymol Carvacrol Terpinene Trans-13Octadecenoic acid Linalool

51.22 12.59 10.3 9.04

Antioxidant assay

Results

Reference

DPPH & Ferric reducing power Assay

IC50 (0.619 µg/mL) EC50 (2.13 µg/mL) EC50 (0.78 µg/mL)

[41]

2.29

Avishan Persian name

Thymus daenensis Celak

Thymol p-Cymene Carvacrol Carvone Borneol

70.12 5.12 4.99 3.12 2.96

DPPH° Scavenging Assay

IC50 (0.26 mg/mL)

[42]

Thymus

Thymus kotschyanus Celak

Carvacrol Thymol Carvacrol acetate Phytol Thymoquinone

27.8 16.8 6.87 6.8 5.4

DPPH° Scavenging, Phosphomolybdate Assay

IC50 (0.16 mg/mL)

[42]

2.78 mg of AAE/g of dry weight

510  Essential Oils ØØ Prepared the solution by mixing 7.0 mM ABTS•+ and 4.9 mM potassium persulfate (1:1) and incubate in dark for 12 h. ØØ The ABTS•+ was diluted to obtain absorbance 0.701 at 734 nm with DMSO ØØ Mix 1 mL EO solution of different concentrations with equal volume of ABTS•+ solution and incubate for 7 min. ØØ Measure the absorbance at 760 nm by using spectrophotometer. The free radical scavenging activity of EOs was expressed in percent inhibition of ABTS•+ [34]. The antioxidant activities of most commonly used EOs determined by ABTS•+ is shown in Table 22.1.

22.4.7 Bleachability of β-Carotene in Linoleic Acid System Bleachability of beta carotene in linoleic acid assay based on oxidation of carotene via linoleic acid [35]. The general protocol of bleachability of β-carotene in linoleic assay is as follow; • • • •

Easy and simple to conduct Efficient in performance Cost effective Reproducible assay

However, care must be taken for the preparation of emulsified carotene in linoleic acid system. Further, due to different colloidal properties of fatty acid, it affects antioxidants and oxidation initiators. Linoleic acid is excellent in a sense that it is polyunsaturated fatty acid and easily oxidized due to formation of lipid substrates by micelle in aqueous systems and this increased the hydrophilicity of medium and polar antioxidants. Bleaching Betacarotene in linoleic acid system assay is used in many studies from different researchers for evaluating the antioxidant potential of EOs [36]. Following are the basic steps to conduct the bleachability of β-carotene assay; ØØ Prepare the stock solution by mixing 0.5 mg carotene, 1mL chloroform, 25 µL linoleic acid, and 200 mg Tween 40. ØØ Evaporate all the chloroform at reduced pressure using rotary evaporator. ØØ Then add 100 mL oxygenated distilled water in stock solution and the reaction mixture emulsified for 1 min. ØØ Take 2.5 mL emulsion in test tube and mix it with 350 µL of diluted EOs of different concentrations. and BHT (positive control). ØØ Incubate all the reaction mixture for 48 h and record the absorbance at 490 nm. ØØ Repeat the assay using BHT as positive control. The antioxidant potential of EOs was assessed as percent scavenging of β-carotene bleaching, was calculated by following equation [37].

Biological Potential of Essential Oils  511



% Scavenging =

A S (2H) − A C (2H) × 100 A C (0H) − A C (2H)

Where, AS(2H) is absorbance of tested sample at t = 2h; AC(2H) is absorbance of control at t = 2h. AC(0H) is absorbance of the control exactly after 48 h incubation while AC(2H) is absorbance of the control after 2h. The antioxidant activities of most commonly used EOs determined by different antioxidant assays are shown in Table 22.1.

22.5 Antimicrobial Activities of Essential Oils Chemotherapeutic agents, either synthesized chemically or obtain from microbes are used in the treatment of infectious diseases [43]. A hybrid substance is a semisynthetic antibiotic in which the chemist modifies a molecular form produced by the microorganism to attain desired qualities. From the perspective of the host, the most significant quality of an antimicrobial agent is its selective toxicity, which means that the molecule is target specified and only hit its specific target and kills the specified bacteria while having no or less side effects on the host. The antibiotic can be classified on the basis of its action [44]. It may be cidal (killer) and it may be static (inhibitor) for the microorganisms. The ability of the antibiotic to kill or inhibit a particular range of bacteria is called its spectrum which mainly categories into a narrow or broad spectrum antimicrobial agents. Various in vitro and in vivo tests are in practice for the evaluation of antimicrobial activity of natural products, including EOs. Thousands of published research reports showing antimicrobial potential of EOs. However, the only difference between in vitro and in vivo study is the use of living system for particular assay and its detection of effectiveness [45]. Generally, the result of EO’s antimicrobial activities depends on various factors including; the incubation time, media concentration and amount, type of media used, growth phase, ionic strength of media in addition to EO extraction process conditions. However, comparison of published data on reaction conditions and antimicrobial potential is quite complicated [46]. Evaluation of antibacterial activity of EOs at preliminary stage, typically carried out by the disk diffusion method (DDM), in which the zone of inhibition (ZOI) is measured. Various factors may effect on EO’s antibacterial potential including the thickness and size of the paper disc, the volume and the concentration of EOs, the concentration of the bacteria or bacteria account, the composition of the media as well as the incubation temperature and incubation time. The term minimum inhibitory concentration (MIC) is commonly reported to express the antimicrobial potential. The definition of the MIC, is the concentration that inhibit the bacterial growth. Many researchers are involved in improving the existing methods and developing new methods for the evaluation of EOs for their antimicrobial potential. Similarly, development in micro broth dilution method to find the MIC is modified and the absorbance was

512  Essential Oils measured to access the bacterial growth in the media. When using spectrophotometer for optical density (OD) (turbidity) measurement, are critical to count colonies by viable count. The variation in the way to report antibacterial activity of EO in various publications limits the comparisons among researches. The antimicrobial activity of EOs using dilution of EO in nutrient agar or nutrient broth media is best method with least matrix effects. With this development, redox indicator resazurin is used for measuring the antibacterial activity of oil soluble compounds and therefore for measuring MIC without using spectrophotometer. This method lessened the use of absorbance and optical density. The resazurin micro titter plate method is more efficient, accurate and sensitive as compared to trivial antibacterial agar-well diffusion method [47]. One another feature of OD methods is that it varies significantly by varying the solvent or emulsifier amount to dilute and stabilize EOs in water-based culture media or dissolve the EOs. Several substances have been used for this purpose: n-hexane, dimethyl sulfoxide, methanol/ethanol, Tween 80/Tween-40/Tween-20, and agar. However, few reports conclude that use of Tween-40 is unnecessary as an additive. The agar (0.2%) solution was mostly prepared in ethanol to make a homogenous dispersion. It can be concluded from some report that detergent like tween 80, 40 etc. and solvents could decrease the antibacterial activity of EOs [30]. Another drawback of the utilization of detergent to dissolve EOs is the formation of turbidity that decrease the visualization of the solution and difficult to record the OD [47]. The general protocol of disk diffusion assay and agar well diffusion method is as follow;

22.5.1 Disk Diffusion Assay Antibacterial/anti-fungal activity of EOs was preferably determined by disk diffusion assay using following protocol [48]; ØØ Inoculate tryptic soy agar with the microorganisms of ~104 colony forming units/mL. ØØ Place a 6 mm filter paper disk impregnated with 20µL of EOs diluted in DMSO on TS agar and allow for 30 min to diffuse EO in to medium at room temperature. ØØ The plates, then incubate for 24 h at 37°C. ØØ Measure the ZOI in mm by using ruler. ØØ Repeat the experiment in triplicate. ØØ Typically, broad-spectrum or any antibiotic use as positive control and DMSO was used as negative control.

22.5.2 Agar Well Diffusion Method The antibacterial/anti-fungal activity of EOs were also reported using agar well diffusion method described by different research groups [49]. The procedure of this method is mention below.

Biological Potential of Essential Oils  513 ØØ Culture the bacterial strain 24h before starting the assay in nutrient broth and mixed with 9% NaCl solution and then match with Mac Farland turbidity standard of 0.5×106 (colony forming units) CUF/mL. ØØ Prepare the petri plates by pouring 70 mL of seeded Mueller Hinton (MH) Agar and media and allow to solidify. ØØ Engrave five fine wells (8 mm) in each petri plate with sterilized cork borer. ØØ Seal the wells with MH agar and mark followed by addition of 40 µL of EO sample in their respective wells. Use ciprofloxacin (5 µg/mL) as positive control in each plate as well. ØØ Incubate all tested palate at 37°C for overnight. ØØ Measure the ZOI in mm using ruler.

22.5.3 Determination of Minimal Inhibitory Concentration (MIC) Antibacterial potential of EOs was also determined by microtiter broth microdilution method. It is also called the minimum inhibitory concentration (MIC) [50]. Many researchers are involved in improving the existing methods and developing new methods for the evaluation of EOs for their antimicrobial potential. Similarly, development in micro broth dilution method to find the MIC is modified and the absorbance was measured to access the bacterial growth in the media The procedure of MIC measurement is mentioned below [51]; ØØ Dilute the EO in DMSO and subjected to a serial dilution in 96-well plate bearing TS broth for bacteria. ØØ Suspend the bacterial strain in liquid medium and maintain the concentration at 108 CFU/mL. ØØ Incubate the 96-well plate at 37°C for 24 h and measure the OD at 520 nm using Elisa reader/Spectrophotometer. ØØ Calculate the MIC of each sample. Many researchers are involved in improving the existing methods and developing new methods for the evaluation of EOs for their antimicrobial potential [52]. Similarly, development in micro broth dilution method to find the MIC is modified and the absorbance was measured to access the bacterial growth in the media [53]. When using spectrophotometer for optical density (OD) (turbidity) measurement, are critical to count colonies by viable count. The variation in the way to report antibacterial activity of EO in various publications limits the comparisons among researches [54]. On the other hand, bacteria exist in two forms gram-positive and gram-negative bacteria and their sensitively against EOs is different. The antibacterial activities of various EOs of plants have been extensively reported in research reports against both gram-negative [Escherichia coli (E. coli), Salmonella typhimurium (S. typhimurium), Pseudomonas aeruginosa (P. aeruginosa), Camplyobacter spp, Pseudomanas putida (P. putida)] and gram-­positive [Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes)] bacteria: for instant Elettaria cardamomum (cardamom) EO showed MIC 3.75–7.50 µL/mL against E. coli and S. aureus [11] Eugenia caryophyllus EO showed

514  Essential Oils MIC in the range of 1.25–10 µL/mL against B. subtilis, E. coli, P. aeruginosa, P. putida, and S. aureus [55, 56]; Lavandula angustifolia EO showed MIC in the range of 1.33 to 42.67 µL/ mL against E. coli, P. aeruginosa and S. aureus [5]. However in another study it showed MIC in the range of 125–250 µL/mL against E. coli and S. aureus which was quite high as compared to above study. Mentha piperita EO showed 62.5 µL/mL MIC value against E. coli, and S. aureus. All these studies reflect the potential of EOs as an antibacterial agents which render them to use in functional foods and cosmetics for preventing to grow infection in body i.e. a best agents for pre-infection therapy.

22.6 Essential Oils as Natural Antimicrobial Agents Naturally occurring antimicrobials are gaining interest for food preservatives and pharmaceutical applications for many reasons; (1) due to broad spectrum activity broader application in various areas including food and pharmaceutical application, (2) Food manufacturers marked the label “GREEN” on their product to attract the customers, (3) it is not acceptable that the new synthetic preservatives can be approved without proper testing and a through testing not only take time but also increase the cost and also seems to be toxic, (4) the natural antimicrobial are more acceptable also due to the fact that they come from natural sources and seems to be less toxic and generally recognized as safe (GRAS). The food antimicrobials arbitrary was classified into two categories: a naturally occurring and traditional or “regulatory approved”. Some natural antimicrobials including lysozyme and lactoferrin are generally used in various products. The microorganisms develop resistance against synthetic compounds and antibiotics do not completely cure these diseases. The natural compound and isolated natural products such as vinegar, herbs, natural ores, salt, or sugar, and spices have long been used to preserve foods and slow the onset of spoilage. However, recently polar and nonpolar extracts of certain plants and EOs have been gaining interest due to their antimicrobial potential, as well as these are imparting flavor and colors to foods and food products [51]. Some of the bioactive components isolated from these medicinal plants and their metabolites e.g. polyphenols used due to their preservative potentials. The chemical compositions of the active ingredients in these plant EOs and extracts are diverse. The phytochemical analysis revealed that one class of compounds isolated from these is hydrophobic (oil-soluble) and other is hydrophilic (water-soluble). The utilization of polyphenols from plants is increasing day by day due to the potential application as natural antioxidants and antimicrobials and antiseptic properties. Following extracts and EOs from dietary herb, spices and medicinal plants of different plant families like Lamiaceae (mint family), myertaceae, citeraceae, etc. showed the antimicrobial activities; thyme, have been used as antibacterial and antiseptic in foods, rosmarinic acid (from Lamiaceae plant including mint, oregano, thyme, sage etc.) and thymol (from thymus and oregano species) have showed antimicrobial and anti-­ inflammatory properties. Microbiologist reported that food-borne pathogens are serious risk to human health and thyme EOs contained thymol and carvacrol, have potential application against the growth of these kind of bacteria.

Biological Potential of Essential Oils  515

22.7 Anticancer Activity of Essential Oils Essential oils are highly concentrated liquid and have ability to damaging the cells and lipid and protein layers by coagulate the cytoplasm and show the anticancer potential. Moreover, EOs affect calcium and other ionic channels and reduce pH and results in decreasing the membrane potential that stimulate depolarization in mitochondrial membranes of eukaryotic cells. Following assays used to evaluate the anticancer and cytotoxic activities of EOs; resazurin test, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay [57], Trypan Blue exclusion test. MTT assay has been used exclusively due to of its efficiency, sensitivity, accuracy, and simplicity. Various EOs have been reported for their anticancer activity including mint EOs; rosemary EOs; origanum EOs; thymus EOs.

22.8 Cell Culture and Treatment The human cancer cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin and maintained at 37°C with 5% CO2 in humidified atmosphere. Cells were treated with EOs dissolved in DMSO with a final DMSO concentration of 0.05% [58].

22.9 Determination of Cell Viability The anticancer activity of EOs of natural products was determined by cell viability MTT assay as described by us previously [59, 60]. The protocol is mention below. ØØ Culture the cancer cell lines according to the protocol devised for particular cell line 48h prior to assay ØØ Following treatment, add 10µl MTT reagent in EOs sample of concentration 5 mg/mL ØØ Incubate the cancer cell line plate for 4 h at 37°C ØØ Subsequently, add 150 μL DMSO to dissolve the formazan crystals ØØ Note the absorbance at 490 nm in a microplate reader

22.10 Conclusion The in vitro assays for the assessment of EO’s biological potential is a key area of natural product research and it is best needed to develop more reliable and easy to conduct assay for recording antioxidant, antimicrobial and anticancer activities.

Acknowledgement The authors are thankful to Higher Education Commission (HEC), Islamabad, Pakistan and Government College University Faisalabad for providing resources to complete this project.

516  Essential Oils The authors also thankful to Ms. Neelum Iftikhar and Mr. Ali Abbas for partial collection and arranging of material related to in vitro studies.

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Biological Potential of Essential Oils  519 50. Rabib, H., Elagdi, C., Hsaine, M., Fougrach, H., Koussa, T., Badri, W., Antioxidant and antibacterial activities of the essential oil of moroccan Tetraclinis articulata (Vahl) masters. Biochem. Res. Int., 2020, 9638548, 2020. 51. Wiegand, I., Hilpert, K., Hancock, R.E., Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc., 3, 163, 2008. 52. Ahmed, M., Phul, A.R., Bibi, G., Mazhar, K., Ur-Rehman, T., Zia, M., Mirza, B., Antioxidant, anticancer and antibacterial potential of Zakhm-e-hayat rhizomes crude extract and fractions. Pak. J. Pharm. Sci., 29, 895, 2016. 53. Al-Hajj, N.Q.M., Wang, H.X., Ma, C., Lou, Z., Bashari, M., Thabit, R., Antimicrobial and antioxidant activities of the essential oils of some aromatic medicinal plants (Pulicaria inuloides-­ Asteraceae and Ocimum forskolei-Lamiaceae). Trop. J. Pharm. Res., 13, 1287, 2014. 54. Bakht, J., Noor, N., Iqbal, A., Shafi, M., Antimicrobial activity of different solvent extracted samples from the leaves and fruits of Capsicum annuum. Pak. J. Pharm. Sci., 33, 2020. 55. Sanae, L., Evrendilek, G.A., Mouhcine, F., Hicham, K., Rabia, B., Abdelhakim, E.O.L., Combined antibacterial effect of Origanum compactum and Mentha piperita (Lamiaceae) essential oils against ATCC Escherichia coli and Staphylococcus aureus. Vegetos, 35, 74, 2021. 56. Moussii, I.M., Nayme, K., Timinouni, M., Jamaleddine, J., Filali, H., Hakkou, F., Synergistic antibacterial effects of Moroccan Artemisia herba alba, Lavandula angustifolia and Rosmarinus officinalis essential oils. Synergy, 10, 100057, 2020. 57. Caputo, L., Souza, L.F., Alloisio, S., Cornara, L., De Feo, V., Coriandrum sativum and Lavandula angustifolia essential oils: Chemical composition and activity on central nervous system. Int. J. Mol. Sci., 17, 1999, 2016. 58. Jurenka, J.S., Anti-inflammatory properties of curcumin, a major constituent of Curcuma longa: A review of preclinical and clinical research. Altern. Med. Rev., 14, 141, 2009. 59. Mwale, C., Makunike, K.N., Mangoyi, R., Antibacterial activity of Melia azedarach leaves against Salmonella typhi and Streptococcus pneumoniae. Int. Ann. Sci., 8, 47, 2020. 60. Cragg, G.M. and Newman, D.J., Plants as a source of anti-cancer agents. J. Ethnopharmacol., 100, 72, 2005.

23 Biological Potential of Essential Oils: Evaluation Strategies Santanu Chakraborty1, Manami Dhibar1, Aliviya Das2, Kalpana Swain3 and Satyanarayan Pattnaik3* Dr. B. C. Roy College of Pharmacy and AHS, Durgapur, India Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, India 3 Division of Advanced Drug Delivery, Talla Padmavathi College of Pharmacy, Warangal, India 1

2

Abstract

Among the different bioactive phytochemical constituents, essential oils are widely accepted, recognized, and used in food, cosmetic, chemical industries, pharmaceutical industries as well as even in our daily life. They are the complex mixtures of volatile organic compounds which are characterized by a strong odor produced in the form of secondary metabolites in plants. Since essential oils have exhibited diverse biological activities, there is an increasing trend of exploitation of these natural products in the cosmetic and pharmaceutical industries. Characterization and in-vitro, and in-vivo evaluation of these essential oils are very important owing to the regulatory requirements for use in drug and cosmetic product development. This chapter aims at bringing up a summary and critical appraisal of the reported methods, both in-vitro and in-vivo, for assessment of the biological activities of essential oils. Keywords:  Essential oil, biological activity, antibacterial, antimicrobial, antifungal, ­ anti-inflammatory, antidiabetic, anticancer

23.1 Introduction In the present era, natural products have gained much more interest as compared to their synthetic counterparts because of their safety profile, low adverse effects, eco-friendly nature, and easy availability [1]. From ancient times, plants or their specific parts are used for nutritional as well as for medicinal values [2–4]. Plant sources providing various classes of chemical compounds and they are termed phytochemicals. Some of the phytochemicals have medicinal values and are known as bioactive phytochemical constituents. The most important bioactive phytochemical constituents obtained from plant sources are tannins, terpenoids, alkaloids, phenolic compounds, flavonoids, saponins, essential oils, etc. [5]. Among the different bioactive phytochemical constituents, essential oils are widely accepted, recognized, and used in food, cosmetic, chemical industries, pharmaceutical *Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (521–550) © 2023 Scrivener Publishing LLC

521

522  Essential Oils industries as well as even in our daily life. In the late 19th and early 20th centuries, most researches were performed on extraction and structure elucidation of aromatic molecules of essential oils. Essential oils (EOs) are oily, hydrophobic, aromatic, and volatile principles isolated from different parts of the plants [6, 7]. They are also called as ethereal oils and are the complex blends of volatile organic compounds or fragrant constituents which are characterized by a strong odor. These secondary metabolites are mainly involved in defensive mechanisms in plants and contain around 20 to 60 constituents in different concentrations. They mainly consist of terpenes (monoterpenes, sesquiterpenes, and diterpenes), polyunsaturated omega-­6-fatty acids, phenylpropenes, flavonoids, etc. [8]. These constituents of essential oils and their concentration within the same botanical species are significantly influenced by geographical position, climate condition, harvesting time, nature of the soil, extraction process, etc. [9]. Inhalation, absorption through the skin, and ingestion are the different ways by which essential oils can enter the body. Essential oils have a number of therapeutic activities in the body such as antibacterial [10], antifungal [11], antiviral [12], antimalarial [13], anti-­ inflammatories [14], analgesics, antipyretics [15], anticonvulsants [16], antimutagenic [17], hepatoprotective [18], anticancer [19], etc. For mankind, the dengue virus is a major threat and every year so many people have affected by the virus. So many researchers have proved that essential oils have anti-viral potential against the dengue virus [20–23]. Essential oils also have anti-viral properties against Flavivirus. Essential oils from Lippia alba, Artemisia vulgaris, Oreganum vulgare show antiviral activity against the yellow fever virus [24]. Not only in the pharmaceutical and health care field but also in the agriculture sector essential oils have significant applications. Essential oils are widely used as repellent [25], fumigant [26], insecticide [27, 28], etc. The diverse therapeutic applications of essential oils have been established and reported by many researchers across the world (Table 23.1) [12, 13, 29–37]. Table 23.1  Therapeutic applications of essential oils obtain from various plants. Therapeutic applications

Essential oils obtain from plants sources

Anti-inflammatory

Clove, German chamomile, Lavender, Yarrow

Antiseptic

Lavender, Tea tree

Antifungal

Lemongrass

Acaricidal

Lavender

Antiviral

Lavender, Lemon, Melissa

Analgesic

Rosemary, Peppermint, Clove

Aphrodisiac

Jasmine, Black pepper, Sandalwood, Rose

Antibiotic

Tea tree, Lavender (Continued)

Biological Potential of Essential Oils  523 Table 23.1  Therapeutic applications of essential oils obtain from various plants. (Continued) Therapeutic applications

Essential oils obtain from plants sources

Astringent

Frankincense, Cypress, Sandalwood

Antispasmodic

Peppermint, Cedarwood, Marjoram, Rosemary

Antiphlogistic

German chamomile

Antinociceptive

Polygermander, Summer savory

Cytophylactic

Cedarwood, Lavender, Frankincense

Carminative

Melissa, Fennel, Chamomile, Peppermint

Anticancer

Sage

Diuretic

Grapefruit, Juniper, Lemon

Expectorant

Thyme, Eucalyptus, Clary sage, Rosemary, Pine, Cypress, Sandalwood, Fennel, Cedarwood

Emmenagogue

Rosemary, Geranium, Juniper, Clary, sage, Basil, Marjoram, Chamomile, Rose, Ginger

Euphoric

Orange, Neroli, Jasmine, Rose, Grapefruit, Roman chamomile

Hormonal (generally)

Fennel, Jasmine, Rose, Neroli, Clary sage, Geranium

Immunomodulatory

Clove, Ginger, Sage

Laxative

Pine, Marjoram, Peppermint, Fennel, Black pepper, Orange

Psychotropic

Parsley

Rubefacient

Lemongrass, Peppermint, Rosemary, Black pepper

Sudorific

Ginger, Cardamom, Black pepper, Rosemary

Sedative

German chamomile, Carrot seed, Roman chamomile, Lavender, Valerian

Vulnerary

Lavender, Frankincense

Vasodilator

Eucalyptus, Black pepper, Marjoram, Rosemary

Vasoconstrictor

Cypress

Spasmolytic

Coriander, Carway

23.2 Biological Activities of Essential Oils The literature reveals diverse biological activities of essential oils which may be translated for therapeutic application (Figure 23.1). The reported biological activities include antibacterial, antifungal, anti-inflammatory, antioxidant, anticancer, anti-diabetic, antispasmodic, etc. The key biological activities are discussed as follows.

524  Essential Oils

Antibacterial

Antispasmodic

Antifungal

Essential Oils Antiinflammatory

Antidiabetic

Anticancer

Antioxidant

Figure 23.1  Biological activities of essential oils.

23.2.1 EOs as Antibacterial Agents Essential oils have excellent antibacterial applications and are widely used against a broad range of pathogenic bacterial strains. As per the literature, essential oils show antibacterial properties against Helicobacter pylori, Bacillus cereus, Shigella dysentery, Staphylococcus aureus, Salmonella typhimurium, etc. [38, 39]. The following are few examples of essential oils inhibiting different microorganisms: Clove oil [40] inhibits S. typhimurium, A. hydrophila, L. monocytogenes, E. coli, Streptococcus thermophilus, Lactobacillus bulgaricus, S. aureus; Linalool [41] inhibits Botrytis cinerea; Sage oil [39] inhibits S. aureus, B. cereus, S. typhimurium; Coriander oil [42, 43] can act against Bacillus subtilis, Salmonella choleraesuis, Staphylococcus aureus, Corynebacterium diphtheria, Shigella sonnei, Eugenol [44] inhibits A. hydrophila, L. monocytogenes, Microsporum gypseum, Thymol [39] inhibits B. cereus, Shigella sonnei, S. typhimurium, L. monocytogenes, S. aureus; Cinnamon oil [45] can act against Lactobacillus bulgaricus, Streptococcus thermophilus, E. coli; Thyme oil [46] inhibits Pseudomonas putida, E. coli, L. monocytogenes; Cinnamaldehyde [47] active against Pseudomonas putida; Carvacrol can act against L. monocytogenes, S. typhimurium, B. cereus, E. coli. Akor & Anjorin [48] studied the antimicrobial activities of Commiphora africana root extracts against various pathogenic bacterial strains and found excellent antimicrobial

Biological Potential of Essential Oils  525 properties against Candida albicans, Staphylococcus aureus, Escherichia coli. Helicobacter pylori is a gram-negative, highly motile, and gastrointestinal pathogen responsible for chronic superficial gastritis and duodenal ulcer disease in humans. In the present scenario multi-drug therapy such as combinations of proton pump inhibitors, antibiotics, bismuth subsalicylate, H2-blockers is used to treat H. pylori. Sometimes application of this multidrug therapy may cause different side effects. Traditional herbal medicines may be an alternative to overcome these problems and treat H. pylori. Epifano et al. in 2006 [49] reported that essential oil extracted from Commiphora africana is very potent against H. pylori with 1 μL/mL concentration.

23.2.2 EOs as Antifungal Agents Many EOs possess excellent antifungal properties and may be used as a substitute for synthetically developed antifungal therapy. Different researchers have tried to establish naturally extracted essential oils as antifungal additives. The EOs isolated from various plants e.g., thyme, lavender, fennel, rosemary, etc., showed significant antifungal properties against Phytophthora infestans and Botrytis cinerea [50, 51]. Essential oils decrease the cell wall integrity as well as plasma membrane permeability of the fungi. Some researchers also suggested that essential oils disrupt the cytoplasmic membranes. There are so many essential oils obtained from the plant sources that were investigated by various researchers to observe the mechanism of action against different fungi (Table 23.2). Table 23.2  List of essential oils and their mechanism of action against specific fungi. Essential oil

Fungi

Mechanism of action

References

Lavandula multifida L.

Candida albicans

Inhibited the filamentation, cytoplasmic membrane disruption, and propidium iodide staining

[52]

Curcuma longa L.

Aspergillus flavus

Inhibited the aflatoxin production

[53]

Matricaria chamomilla L.

Aspergillus niger

Growth inhibitor

[54]

Cinnamomum jensenianum

A. flavus

Altered the plasma membrane, fibrillar layer, and cytoplasm

[55]

Ocimum sanctum L.

C. albicans

Cytotoxic

[56]

Mentha piperita L.

C. albicans, C. tropicalis, C. glabrata

Decreased the proteolytic activity

[57]

Anethum graveolens L.

C. albicans

Decrease in ATPase activity, and chromatin condensation

[58]

Chenopodium ambrosioides L.

A. flavus

Cytotoxic and inhibited the aflatoxin B1 production

[59]

526  Essential Oils

23.2.3 EOs as Anti-Inflammatory Agents Essential oils isolated from various medicinal plants have excellent anti-inflammatory properties. These oils also help to protect against prolonged inflammation and hence have a significant impact on drug discovery. Essential oils extracted from Cinnamonmum zeylanicum bark are extremely useful on different inflammatory biomarkers such as intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, interferon-inducible T-cell alpha chemo attractant, monocyte chemo attractant protein-1, interferon-gamma induced protein 10, monokine induced by gamma interferon, etc. Essential oils from Cinnamonmum zeylanicum bark can also alter the signaling pathways of inflammation, tissue remodeling, and cancer biology [60]. Another example of essential oil extracted from lavender is also very much effective against acute inflammation [61, 62]. The essential oil extracted from cinnamon leaf contain linalool and cinnamaldehyde also have significant anti-­inflammatory properties [63]. Essential oil from Lindera erythrocarpa also proved as an anti-­inflammatory agent and effective in RAW264 cells as well as helps to decrease LPSinduced pro-­inflammatory mediator’s production [64]. Other examples of essential oils extracted from Jatropha curcas [65] and Eugenia caryophyllata [66] were also reported to have excellent anti-inflammatory activities.

23.2.4 EOs as Antioxidants Proteins, unsaturated lipids, and amino acids are susceptible to oxidation in presence of free radicals and other reactive oxygen species. These free radicals, as well as reactive oxygen species, are responsible for molecular alterations which are an important phenomenon for cancer [67], diabetes, asthma [68], Parkinson’s disease, arteriosclerosis, Alzheimer’s disease [69] as well as aging. Our body has its defense mechanisms to fight against these free radicals and to remove them from the body [70]. But sometimes there may be an imbalance between this free radical synthesis and their removal from the body. In this case, there may be a need to supply antioxidants from outside sources. Essential oils consist of a high number of phenolic compounds which may act as antioxidants or scavengers for free radicals. Essential oils from Origanum tyttanthum, Thymus serpyllus, Mentha longifolia contain a high proportion of phenolic compounds such as thymol and carvacrol which are mainly responsible for antioxidant properties. As per the literature, essential oils extracted from various plant sources such as oregano, clove, nutmeg, thyme, basil has excellent antioxidant properties [71]. For example, the essential oil of Thymus serpyllum L. acts as a scavenger for free radicals [72]. Essential oil from Citrus limonum helps to control the free radicals and thereby prevent lipid peroxidation as well as skin tissue damage [73]. The application of lemon essential oil could also prevent the human skin from oxidative damage [74].

23.2.5 EOs as Anticancer Agents Nowadays cancer is one of the major threats for us and more than six million lives were affected every year [75]. There are so many synthetic anti-cancer drugs are available in the market to treat cancer but they have several side effects. With this respect, nowadays herbal medicines gain much more interest in cancer therapy. In recent times, herbal plants

Biological Potential of Essential Oils  527 and their essential oils are widely used as a phytomedicine to treat cancer [76]. In cancer, the synthesis of reactive oxygen species as well as inflammatory states is very crucial which may propagate the generation of cancerous cells (Figure 23.2) [77, 78]. The excessive production of reactive oxygen species carries the probabilities of oncogenic transformation of the cells [79]. This reactive oxygen species also lead the tumor formation by modulating the redox-mediated signaling pathways. So to treat cancer we need such type of compound which has antioxidant properties, initiates apoptosis as well as can arrest the cell cycle of cancerous cells. In this context, essential oils are extremely useful which have anti-oxidant properties as well as they can initiate the apoptosis process. They can also activate the detoxification process, DNA repair systems, and inhibit metastasis as well as angiogenesis [80]. There are numerous researches that have been done with essential oils to explore their anticancer activities e.g., Abies balsamea [81], Zanthoxylum schinifolium [82], Aniba rosaeodora [83],

Figure 23.2  The arm-in-cage (AIC) test for measuring the efficacy of topical mosquito repellents under laboratory conditions. Hungry female mosquitoes are contained in a test cage and the repellent is applied to the forearm between the wrist and elbow, while the hand is protected by a latex glove through which the mosquitoes cannot bite. Adapted from Ref. [204].

528  Essential Oils Pinus densiflora [84], Melissa officinalis [85], C. bergamia [86], Boswellia sacra [87, 88, 97], Curcuma zedoaria [89], Pinus koraiensis [90], etc. Artemisia capillaris found to exhibit cytotoxic properties and used for human oral epidermoid carcinoma [91]. Cytotoxic properties were exhibited by Thymus vulgaris and explored its possible application in the treatment of head and neck squamous cell carcinoma [92]. Intriguingly, Pogostemon cablin found to induce apoptosis, and decrease cell growth offering an option to treat human colorectal cancer [93]. Similarly, Curcuma wenyujin inhibited tumor growth and proposed for treatment of human cervical cancer [94]. Studies have explored Cryptomeria japonica for treatment of human oral epidermoid carcinoma [95]. In another study, Cephalotaxus griffithii induced mitochondria-initiated apoptosis and found useful for human cervical cancer treatment [96].

23.2.6 EOs as Anti-Diabetic Agents At present time diabetes mellitus is one of the very common diseases and is available almost in every family. It is a hyperglycemia condition that mainly occurs due to less production of insulin by the pancreas. Among its different types, type-2 diabetes mellitus is very common which is depending on insulin secretion [97]. Long-term hyperglycemia also propagates the generation of free radicals and damages the endogenous antioxidants leading to diabetic complications [98]. The researchers are continuously focusing to find a long-term solution for diabetes and its associated complications. The application of synthetic medicines for a long time may also lead to several complications. So the finding of alternative therapy (such as plant products or extracts) is very crucial in this context. Plant extracts such as essential oils are investigated widely for the management of diabetes. Ademiluyi et al. investigated that the essential oils extracted from Ocimum basilicum leaves have excellent anti-diabetic as well as antioxidant properties [99]. Oboh et al. in 2014 examined the anti-diabetic as well as antihypertensive properties of essential oil extracted from black pepper. They stated that the constituents of extracted essential oil from black pepper have significant inhibitory properties of α amylase, α glucosidase, and angiotensin-I converting enzyme and could be used in the management of diabetes and hypertension [100, 101].

23.2.7 EOs as Antispasmodics EOs and their constituents help to give relief from spasm and are thereby widely used as antispasmodic agents [102]. The constituents of some essential oils such as terpineol, ­α-terpinene, tetrahydrocarvone, 4-carvomenthenol, cymene, borneol, aromadendrene, etc. are extremely useful in these conditions. These constituents help to relax the isolated ileum and inhibit receptor-dependent and independent mechanisms and thus prevent the contraction [103]. As per Criddle et al. 1998 essential oil isolated from Croton nepetaefolius have a significant antispasmodic effect and is a potent modulator for intestinal smooth muscle [104]. As per the literature, several researchers have proved the antispasmodic activities of extracted essential oils from different plant sources. Such as Chamomile nobile: Direct smooth muscle relaxation [105]; Elettaria cardamomum: Calcium channels inhibitor [106]; Xylopia frutescens: Calcium channels inhibitor and histaminergic receptors antagonist [107]; Origanum majorana: Calcium channels inhibitor [108]; Salvia officinalis: Potassium

Biological Potential of Essential Oils  529 channels activator [109]; Acorus calamus: Calcium channels inhibitor [110]; Cymbopogon citratus: Calcium channels inhibitor [111].

23.3 In Vitro Assessment of Biological Activities 23.3.1 Antimicrobial Assay To evaluate the antimicrobial properties of EOs, conventional as well as some non-­ conventional methods have been applied [112]. To assess the antibacterial susceptibility of Essential oil, the NCCL method is applied, which is further modified [113, 114]. The two basic conventional techniques for in-vitro antibacterial as well as antifungal activity of essential oils include (i) agar Diffusion method, and (ii) dilution method [112]. The outcome of the in vitro tests depends upon some factors like–the extraction method to extract the essential oil, growth phase, inoculum volume, type of culture media, pH, temperature, incubation time of media, etc. [39, 115]. In the Agar diffusion method, the efficacy of essential oil is measured in terms of the size of zone of inhibition and the morphology of the microorganism [116]. But this method has some limitations in the case of the use of essential oil, as there is a chance of evaporation of volatile components present in the essential oil during the incubation period. Still, it is used as a pre-screening method of a wide variety of essential oil as it can be easily performed with a very less quantity of essential oil [112]. The strength of the antibacterial activity of essential oil can also be determined by Agar or Broth Dilution method, [112, 117, 118]. Besides these above-mentioned conventional techniques, some non-conventional techniques are also there e.g. Bioautographic method, Turbidimetry, Bioimpedimetric method, etc. [112]. For the estimation of vapor-phase antimicrobial activity, some techniques like the Microatmosphere method [112], disk volatilization method [116, 117], etc. are used. The result is expressed in terms of minimum inhibitory concentration (MIC) or minimum inhibitory quantity (MIQ).

23.3.2 Antibacterial Assay Murbach Teles Andrade et al. 2014 used twenty-seven essential oils to investigate in vitro antimicrobial activity by using the agar dilution method against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa strains and minimal inhibitory concentration (MIC50% and MIC90% values) were calculated. Black paper and tea tree oil were highly effective against S. aureus strain, whereas E. coli was highly sensitive to clove and cinnamon Essential oil [119]. In another interesting study, 32 EOs were assessed for the antibacterial activity using Agar diffusion, Microatmosphere, and Microbroth assay, and checkerboard method [120]. Among all the Essential oil, Red bergamot, Red thyme, Chinese cinnamon, clove, Cinnamon bark, Wild bergamot exhibit higher antimicrobial activity in the entire assay due to the presence of carvacrol, cinnamaldehyde, and thymol. Two major components, Trans-anethole and estragole present in Fennel seeds have shown antibacterial activity against several food-borne pathogens [121]. Similar activity

530  Essential Oils was exhibited by Omani basil [122], Thymus daenensis [123], Rosmarinus officinalis L. [124], finger citron essential oil [125], etc.

23.3.3 Antifungal Assay The most commonly used method for antifungal assay is the disc diffusion method. The indication of antifungal activity is expressed by measuring the inhibition zone diameter in mm [126, 127]. Minimum inhibitory concentration (MIC) [128, 129] and minimum fungicidal concentration [130] are measured by another assay i.e., broth microdilution method as based on the Clinical and Laboratory Standards Institute – CLSI reference (formerly NCCLS; M27-A2) [114], tube dilution method [127] and gradient plate technique [126]. Moghtader, 2013 [131] assesses the anti-fungal potential of the EOs of Mentha piperita L. against Aspergillus nigar using the disc diffusion method. The EO has shown antifungal activity owing to Menthol and menthone in it. A similar study was carried out by [132] with Eucalyptus camaldulensis Dehnh. essential oil against five Fusarium spp, [133] with Thymbra spicata L. essential oil, etc. Another study was carried out by [134] by testing eleven essential oils against twelve plant pathogenic fungi. Here also disc diffusion method was followed. Among all the Essential oil, thyme, cinnamon leaf, clove, and aniseed oils had shown the best antifungal activity. Nakahara et al. [135] carried out the in-vitro antifungal assay of citronella oil against Aspergillus, Penicillium, Eurotium by using the vapor-agar contact method. The result was expressed in terms of Minimum inhibitory dose (MID) in mg/L or ppm. Among all the constituents, linalool and citronellal showed the highest antifungal activity.

23.3.4 Antioxidant Assay Antioxidants are the compound capable of protecting cells by slowing or retarding oxidation by free radicals [136] in a very less amount (less than 1%, usually 1-1000mg/L) [137]. Reactive oxygen species are toxic compounds that induce oxidation of biological molecules present in our body and led to aging, cancer, Parkinson’s disease, diabetes, etc. [138]. Over years, researches based on the antioxidant activity of essential oil has been increased as natural antioxidants are relatively nontoxic and less harmful than some synthetic antioxidants like butyl hydroxyl toluene (BHT), Butylated hydroxyl anisole (BHA), etc. [139]. Some of the important assay methods used to test the antioxidant activity of essential oil are summarized in Table 23.3. Other than this above-mentioned methods, ORAC assay, Aldehyde/carboxylic acid assay, FIC assay, Photochemiluminescence (PCL) assay, lipoxygenase inhibitor-screening assay, CUPRAC (cupric reducing antioxidant capacity) assay, malonaldehyde/gas chromatography (MA/GC) assay, β-carotene-linoleic acid (linoleate) assay, etc. are also used [140]. The DPPH assay at room temperature concluded the antioxidant potential of studied EOs in the order-clove >> cinnamon > nutmeg > basil ≥ oregano >> thyme [71]. To assess the antioxidant activity of EOs obtained from oregano, clove, thyme, rosemary, sage, five different antioxidant tests (DPPH, FRAP, TBARS, FIC, and Rancimat) were carried out by [141]. In the DPPH assay, clove showed the highest antioxidant activity stronger

Biological Potential of Essential Oils  531 Table 23.3  Antioxidant assay methods. Tests

Theory

References

DPPH Assay

Widely used. It measures the antioxidant ability to reduce the stable 1,1-diphenyl-2picrylhydrazylradical (DPPH) (λmax-520 nm). Ascorbic acid and BHT is used as standard.

[137, 140–142]

ABTS Assay

It measures the capacity of antioxidants to scavenge the free-radical cation,2,2-azinobis(3-eth-ylbenzothiazoline-6-sulfonate(ABTS•+) (λmax-734nm).

[140, 143]

FRAP Assay

Ferric Reducing Antioxidant Power assay follows the potassium ferricyanide–ferric chloride method. It involves the reduction of Fe3+ to Fe2 by antioxidants. (λmax-590nm).

[137, 140, 141]

TBARS Assay

The assay used to assess the degree of lipid peroxidation, which involves spectrophotometric measurement (λmax - 532 nm) of malondialdehyde-thiobarbituric acid (MDA) 2-TBA pink colored complex.

[137, 141, 144, 145]

Rancimat Assay

It involves The Rancimat apparatus which measures The antioxidant lipid activity of Essential oil by the conductometric method.

[137, 141]

Conjugated Diene Assay

This assay involves the formation of conjugated diene at the lipid peroxidation stage.

[142]

than BHT and Ascorbic acid. The order of IC50 value observed-clove < ascorbic acid < BHT < thyme < oregano < sage < rosemary. In the TBARS assay, the highest inhibition was shown by thyme essential oil. Clove essential oil has shown the strongest antioxidant activity in FRAP assay. Whereas in the Racimat test, oregano essential oil shown better antioxidant activity. A wide application of antioxidant test (conjugated diene assay, the DPPH assay, the aldehyde/­carboxylic acid assay, lipoxygenase inhibitor-screening assay, and the malonaldehyde/gas chromatography (MA/GC) assay) of around twenty-five essential oils was reported [142]. For every antioxidant assay, essential oils behaved differently. Like in the aldehyde/carboxylic acid assay, Cinnamon leaf oil and Bergamot oil exhibit strong antioxidant activity followed by Basil oil in DPPH assay, Eucalyptus, and chamomile oil in the conjugated diene assay, Aloe vera oil in lipoxygenase inhibitor-screening assay, etc. In all the assays, clove oil and Thyme oil exhibited strong antioxidant activity because of the presence of high levels of eugenol and thymol. The EOs obtained from needles of six species of Pinus taxa also shown good antioxidant activity by DPPH assay, FRAP, and ABTS assay [146] because of the presence of α-pinene bornyl acetal, β-caryophellene, α-guaiene, and germacrene D as main constituents.

532  Essential Oils

23.3.5 Anticancer Assay Cancer is the most complex multifactorial disease and worldwide, the second leading cause of death [147] which results from an uncontrollable growth of abnormal cells resulting in malignant tumor formation [148, 149]. Over years, the use of compounds of natural origin in the prevention of cancer has been increased. Among phytochemicals, essential oil possesses anticarcinogenic/antimutagenic/antiproliferation activities [149, 150–154]. Among all the thymus species studied, T. kotschya essential oil has shown the most cytotoxicity against Hela cells (revealed by MTT assay) due to the presence of large amounts of carvacrol [154]. The effect of Zataria multiflora essential oil was comparable. The main constituents of Nectandra leucantha essential oil shown significant cytotoxic activity on B16F10-Nex2, HeLa, U-87, HCT, Siha, HFF, MCF7 cell lines [155]. The sulforhodamine B (SRB) assay was used for the screening of cytotoxicity in different essential oils on C32, ACHN, LNCaP, and MCF-7 cell lines [156]. Among all the essential oils studied, L. nobilis exhibited the most effective effect on C32 and ACHN cell lines.

23.3.6 Anti-Diabetic Assay Essential oils are also found to possess anti-diabetic activity. There is an investigation reporting the in vitro hypoglycemic effect against amylase and glucosidase activities [157]. To assess the in-vitro antidiabetic activity, researchers have used the α amylase inhibitory assay, the α-glucosidase enzyme in the α-glucosidase inhibition test and pancreatic lipase assay [158, 159]. Al-Hajj et al. (2016) [160] carried out α amylase inhibition assay and α glucosidase inhibitory assay to find out in-vitro anti-diabetic activity of the essential oil extracted from leaves of Pulicaria includes. In all the assays, % inhibition and IC50 value were calculated. Eugenol and α-pinene of S. aromaticum and C. cyminum possess in-vitro anti-diabetic activity by α amylase inhibition assay in a dose-dependent mode [161].

23.4 In Vivo Assessment of Biological Activities 23.4.1 Antimicrobial Assay Naveed et al. investigated the antimicrobial properties of few EOs using rabbits as experimental model [162]. EOs treatment reduced the bacterial count on the rabbit skin in the treatment group compared to the control and standard group after 24 and 48 hours. Furthermore, it was reported that EOs-treatment was more effective than conventional antibiotics in treating the subcutaneous staphylococcal infection at the affected location in rabbits. Lu et al. 2012 examined the antimicrobial properties of cassia, basil, geranium, lemongrass, cumin, and thyme essential oils (EOs), as well as their main components, against Phytophthora parasitica var. nicotianae. In this study, they chose cinnamaldehyde for greenhouse studies to test its efficiency against tobacco black shank in-vivo because it had the most powerful effects on the pathogen in-vitro [163].

Biological Potential of Essential Oils  533 Ferrazzano et al. determined the antimicrobial effects of Plantago lanceolata herbal tea on cariogenic bacteria in-vitro and in-vivo, as well as identified the main components of the Plantago lanceolata plant. In this investigation, only the short-term impact of Plantago lanceolata against oral streptococci and lactobacilli was tested in-vivo [164].

23.4.2 Antidermatophytic Assay Dermatophytoses are a serious zoonotic danger in medical and veterinary medicine, affecting various body parts including skin, hair, wool, feathers, nails, claws, and hooves, whether naturally or artificially generated [165]. Essential oils have recently been utilized as an option in the treatment of dermatomycosis [166]. In-vivo methods for assessing the biological activity of active compounds, particularly natural materials, are useful tools for determining their antifungal efficacy for topical administration [167–169]. Selected studies are mentioned below for the readers to refer. Polyscias fulva bark extraction (1:1, v/v) in dichloromethane-methanol: The extract’s preclinical findings were obtained, and concentrations of 1.25, 2.5, and 5%, w/w, were employed. The extract’s action was dependent on the concentration. Moreover, the therapy was effective in elimination of the infection in 14 days [170]. Chenopodium ambrosoides EOs: T. mentagrophytes infection was healed after thirteen days of twice-daily use of the essential oil ointment (conc-50 ppm) [171]. Acute dermal toxicity by Ageratum houstonianum essential oil: This finding suggests that large dosages of Ageratum houstonianum essential oil have a depressive or sedative impact on the CNS. On the motor fibers or nerve centers, this oil may function as a myorelaxant or tranquilizer [172].

23.4.3 Antifungal Assay To prevent fungus invasion, several essential oils found applications in food processing or food packaging [173–176]. A study has been done by Hua et al. 2019 [173] for the in-vivo antifungal efficacy of essential oil. In this study, EOs like cinnamon oil or clove were used to assess antifungal activity against fungi grown on wheat-bread. Intriguingly, the studies EOs arrested the growth of the fungi grown on bread and subsequently extended the shelf-life of the bread samples. Similarly, Sumalan et al. 2020 extended the use of natural volatile chemicals in the preservation of newly cut vegetables and fruit in a modified environment [175].

23.4.4 Anti-Inflammatory Assay Many methods have been followed to assess the anti-inflammatory potential of EOs [177– 184]. The most commonly used method is Carrageenan-induced paw edema in mice [6]. Although this approach may be used to test materials for anti-inflammatory properties, nothing is known about how it works. Trauma-Induced Rat Paw Edema study was carried out by Bounihi et al. 2013 [177]. It has been observed that at all DOE levels, Melissa officinalis essential oil substantially reduced inflammation caused by experimental trauma. A summary of tests used by investigators to assess the activity of EOs against inflammatory conditions are represented in Table 23.4 and the experimental models are presented in Table 23.5.

534  Essential Oils Table 23.4  Tests to evaluate anti-inflammatory activity. Experimental protocol

Anti-inflammatory activity

References

Histological Analyses

Decreasing the thickness of the ear

[178]

In-vivo topical anti-inflammatory activity

Enhanced inflammatory cell recruitment to the wound site

[178]

Carrageenan-induced paw edema in mice

Inhibition of paw edema

[179]

Croton oil-induced ear edema

Inhibition of edema

[180]

Effects on arachidonic acid (AA) and platelet-activating factor induced shock

Prevented the lethal effects of intravenous PAF or AA

[181]

Kidney medulla

Inhibition of PG and leukotriene production

[182]

Antinociceptive activity

The number of acetic acid-induced writhes was considerably reduced.

[183]

Table 23.5  Experimental models to evaluate anti-inflammatory activity. Experimental model

Methods

Carrageenan-induced Acute Inflammatory Model

The thickness of the paws was measured using Vernier calipers every hour until the 6th hour, and then at the 24th hour.

Dextran-induced Acute Inflammatory Model

The thickness of the paws was measured using vernier calipers every hour up to the sixth hour, and then every 24 hours in the presence and absence of turmeric oil.

Formalin-induced Chronic Inflammatory Model

After injecting formalin into the paws, the thickness of the paws was measured using vernier calipers for 6 days.

23.4.5 Antioxidant Assay Oxidative damage is largely a consequence of exposure to ionizing radiation and a variety of chemical agents and as byproducts of normal cellular metabolism. The damage to the biomolecules leads to many chronic diseases like atherosclerosis, cancer, diabetics, rheumatoid arthritis, post-ischemic perfusion injury, myocardial infarction, etc. This has triggered researchers to develop antioxidants from natural or synthetic sources [70, 185]. The quest for obtaining antioxidants from natural resources has led researchers to explore EOs for promising activities [186]. Antioxidant action is attributed to the natural phenolic chemicals found in plants. Numerous in-vivo and in-vitro investigations have verified its action [140].

Biological Potential of Essential Oils  535 Table 23.6  In vivo antioxidant activity assessment. Test

Observation

References

In-vivo acute drug toxicity study

The toxicological effects were measured in terms of mortality and given an LD50 value.

[187]

Teratogenicity test

The treated group remained active and showed no symptoms of toxicity. Also, the anti-diabetic and triglyceride-lowering benefits of Satureja Khuzestanica essential oil may be due to its antioxidant characteristics.

[188]

Antioxidant capacity in-vivo is determined by several parameters that should be considered while evaluating it. Bioavailability is one of these variables. In biological fluids, cells, and tissues, antioxidants must be effectively absorbed, transported, distributed, and maintained. Catalase, superoxide dismutase, glutathione peroxidase, lipid peroxidation, nitric oxide, and reduced glutathione levels were measured in cell line implanted cancer-bearing mice in in-vivo research. Finally, the lungs were removed to examine metastatic histology [187]. A summary of tests used to evaluate the anti-oxidant activity of essential oil is represented in Table 23.6.

23.4.6 Anticancer Assay Essential oils (EOs) from aromatic plants have proven anticancer effects among the wellknown phyto-complexes [189, 190]. Plant volatile oils, or EOs, have long been utilized in traditional medicine, and those derived from aromatic plants (and their components) have been proven to have anticancer effects [190]. Even though research on the use of EOs as anticancer therapeutic agents is still in its infancy, most of the currently deployed anticancer agents are from botanical origin [191]. A summary of tests used to evaluate the anti-cancer activity of essential oil is represented in Table 23.7.

23.4.7 Anti-Diabetic Assay When compared to synthetic drugs, drugs obtained from plants are supposed to be safer and harmless. Furthermore, therapeutic plants are more readily available and less expensive than manufactured medicines. The World Health Organization recommends the use of therapeutic herbs in this regard. Especially for people in remote areas of underdeveloped nations who cannot afford synthetic medicines [196]. As a result, there has been a lot of study into using medicinal plants to treat diabetes and its consequences. Data derived from a variety of aromatic medicinal plant extracts revealed numerous therapeutic benefits for a variety of metabolic diseases, including diabetes mellitus [39, 197–199]. There are different methods involved for the study of in-vivo evaluation of the anti-­ diabetic activity of essential oil stable. Table 23.8 shows some of the important assay methods for assessment of antidiabetic potential of EOs.

536  Essential Oils Table 23.7  Anticancer activity assessment of essential oils. Assay

Applications

References

Human breast cell lines

Essential oil therapy reduced cell viability and increased cell death in human breast cancer cell lines, but the immortalized normal human breast cell line was more resistant to essential oil treatment.

[87]

Cell cytotoxicity assay

Released LDH activity in culture medium from injured cells was used to measure Boswellia sacra essential oil-induced breast cell death.

[87]

Cell growth and viability assay

In the growth media of immortalized MCF10-2A cells and breast cancer cell lines, cell proliferation was measured.

[192]

Genomic DNA fragmentation

Chromosome DNA fragmentation, a biological marker of apoptosis, was used to test if Boswellia sacra essential oil caused apoptosis in breast cancer cells.

[193]

Matrigel invasion assay

A modified in vitro Matrigel outgrowth test was used to assess the potential of Boswellia sacra essential oil to inhibit breast cancer cell invasion.

[194]

MTT assay

Mentha longifolia essential oil and Mentha pulegium essential oil (as bulk samples) as well as ML-SLNs and MP-SLNs were studied for their anticancer properties (as nanoformulations).

[195]

Table 23.8  Assessment of antidiabetic potential of EOs. Test

Observation

References

Acute Oral Toxicity Study

To observe the toxic effect or mortality.

[160]

Oral Glucose Tolerance Test with Essential Oil in Diabetic Rats

At 60, 120, and 180 minutes after glucose injection, blood samples were taken from the retro-orbital location.

[160]

Induction of Diabetes Mellitus

Intraperitoneal injections were used to cause diabetes. Diabetic rats were defined as those with a fasting blood glucose level of 200 mg/dL.

[200]

Determination of LD50

Using varying dosages of A. sieberi oil extract, an acute toxicity investigation was undertaken to assess the LD50.

[201]

Blood collection and glucose determination

Blood samples were obtained using a spinning tail and the glucose level in the blood was analyzed right away.

[201]

Biological Potential of Essential Oils  537

23.4.8 Mosquito Repellent Assay Depending on the species, several approaches are employed to measure repellant activity. The arm-in-cage essay is the most often used method for evaluating EOs [202]. In this study, a solution of EO was applied over the exposed body surface (hand) of a study participant and allowed to place the hand in the cage containing mosquitoes. The number of mosquito bites are recorded and reported against a control (Figure 23.2) [203, 204]. In another study by Giatropoulos et al. 2012, the number of mosquito landings on human skin at a dosage corresponding to 0.2 µL/cm2 diluted with dichloromethane was used to determine the repellant activity of the chosen EOs, isolated compounds, and combinations in vivo [205]. Pavela in the year 2015 found the majority of essential oils with LC50 values less than 100 ppm against mosquito larvae were from five botanical families: Myrtaceae, Apiaceae, Rutaceae, Cupressaceae, and Lamiaceae in a recent review. The Lamiaceae family’s Mentha genus is widely grown, and its oils have been proven to have larvicidal properties against Aedes aegypti larvae [206].

23.5 Conclusion Since ancient times, aromatic plants have been utilized to treat, prevent, and cure illnesses. However, the usage of medicinal plants has grown significantly, prompting research into the characterization, identification, and isolation of new natural compounds having medicinal characteristics. Essential oils (EOs) have exhibited significant therapeutic characteristics and relevance in the cosmetics as well as food industries. Essential oils contain a wide variety of pharmacological characteristics that have been documented in several pieces of research over the years. Plant essential oil was commonly used as one of the most essential and effective components in pharmaceuticals and other medicinal applications. Recently, synthetic additives which were utilized in various fields such as pharmaceuticals, cosmeceuticals, food, and agriculture purposes have raised significant health concerns due to their adverse effects. This has greatly triggered researchers across the world to explore the potentials of aromatic plant materials for diverse application to cater the needs of pharmaceutical, cosmetic and food processing industries apart from other allied sectors.

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548  Essential Oils 187. Manjamalai, A. and Grace, V.M.B., Antioxidant activity of essential oils from Wedelia chinensis (Osbeck) in vitro and in-vivo lung cancer bearing C57BL/6 mice. Asian Pac. J. Cancer Prev., 13, 3065, 2012. 188. Abdollahi, M., Salehnia, A., Mortazavi, S.H.R., Ebrahimi, M., Shafiee, A., Fouladian, F., Keshavarz, K., Sorouri, S., Khorasani, R., Kazemi, A., Antioxidant, antidiabetic, antihyperlipidemic, reproduction stimulatory properties and safety of essential oil of Satureja Khuzestanica in rat in vivo: A oxicopharmacological study. Med. Sci. Monit., 9, 331, 2003. 189. Fitsiou, E. and Pappa, A., Anticancer activity of essential oils and other extracts from aromatic plants grown in Greece. Antioxidants, 8, 1, 2019. 190. Privitera, G., Luca, T., Castorina, S., Passanisi, R., Ruberto, G., Napoli, E., Anticancer activity of Salvia officinalis essential oil and its principal constituents against hormone-dependent tumour cells. Asian Pac. J. Trop. Biomed., 9, 24, 2019. 191. Blowman, K., Magalhães, M., Lemos, M.F.L., Cabral, C., Pires, I.M., Anticancer properties of essential oils and other natural products. Evid. Based Complement. Alternat. Med., 2018, 1, 2018, Article ID 3149362. 192. Yang, Q., Titus, M.A., Fung, K.M., Lin, H.K., 5α-androstane-3α,17β-diol supports human prostate cancer cell survival and proliferation through androgen receptor-independent signaling pathways: Implication of androgenindependent prostate cancer progression. J. Cell. Biochem., 104, 1612, 2008. 193. Mondalek, F.G., Lawrence, B.J., Kropp, B.P., Grady, B.P., Fung, K.M., Madihally, S.V., Lin, H.K., The incorporation of poly(lactic-co-glycolic) acid nanoparticles into porcine small intestinal submucosa biomaterials. Biomaterials, 29, 1159, 2008. 194. Sasaki, C.Y. and Passaniti, A., Identification of anti-invasive but noncytotoxic chemotherapeutic agents using the tetrazolium dye MTT to quantitate viable cells in Matrigel. Biotechniques, 24, 1038, 1998. 195. Kelidari, H.R., Alipanah, H., Roozitalab, G., Ebrahimi, M., Osanloo, M., Anticancer effect of solid-lipid nanoparticles containing Mentha longifolia and Mentha pulegium essential oils: In vitro study on human melanoma and breast cancer cell lines. Biointerface Res. Appl. Chem., 12, 2128, 2021. 196. WHO, Second report of the WHO expert committee on diabetes mellitus, World Health Organization, Technical report series, 646, 1998, Accessed at- http://whqlibdoc.who.int/trs/ WHO_TRS_646.pdf. 197. Vernin, G., Merad, O., Vernin, G.M.F., Zamkotsian, R.M., Parkanyi, C., GC-MS analysis of Artemisia herba alba asso essential oils from Algeria. Dev. Food Sci., 37, 147, 1995. 198. Sabu, M.C. and Kuttan, R., Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property. J. Ethanopharmacol., 81, 155, 2002. 199. Bakkali, F., Averbeck, S., Averbeck, D., Idaomar, M., Biological effects of essential oils-A review. Food Chem. Toxicol., 46, 446, 2008. 200. Cannas, S., Molicotti, P., Ruggeri, M., Cubeddu, M., Sanguinetti, M., Marongiu, B., Antimycotic activity of Myrtus communis L. towards Candidaspp. from isolates. J. Infect. Dev. Ctries., 7, 295, 2013. 201. Irshaid, F., Mansi, K., Aburjai, T., Antidiabetic effect of essential oil from Artemisia sieberi growing in Jordan in normal and alloxan induced diabetic rats. Pak. J. Biol. Sci., 13, 423, 2010. 202. Schreck, C.E., Techniques for the evaluation of insect repellents: A critical review. Annu. Rev. Entomol., 22, 101, 1977. 203. Gillij, Y.G., Gleiser, R.M., Zygadlo, J.A., Mosquito repellent activity of essential oils of aromatic plants growing in Argentina. Bioresour. Technol., 99, 2507, 2008.

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24 Algal Essential Oils and Their Importance in the Ecosystem S.Z.Z. Cobongela

*

Nanotechnology Innovation Centre, Advanced Materials Division, Mintek, Randburg, South Africa

Abstract

Algae produce a wide variety of volatile organic compounds which are components of essential oils. These volatile organic compounds are produced and secreted in the aquatic ecosystem where they play different ecological roles. Essential oils’ major compounds are terpenes, especially monoterpenes and sesquiterpenes. Volatile hydrocarbons, aldehydes, sulfur-containing compounds, and alcohols are also constituents of essential oils. Their production is affected by a number of environmental factors such as temperature, light, chemical stress, etc. Abiotic stress enhances the production of volatile organic compounds to prepare and transfer information between homogeneous and heterogeneous algae. Essential oils principally enhance resistance to abiotic stress and protection against aquatic herbivores. They possess antimicrobial, antifungal, antitumor, and antioxidant activities. Keywords:  Essential oils, volatile organic compounds, algae, terpenes, ecosystem, pheromones, beach-odor, hydrocarbons

24.1 Introduction Algae is often described as a eukaryotic plant-like organism often found floating in the water. It ranges from a simple floating single-cell phytoplankton to macroalgae attached to the ocean floor, commonly known as seaweed. Seaweed is a thallophyte, composing of leaflike structures. They are classified into three distinct groups: brown, green, and red algae. Algae are abundant in oceans and can also be found in other water bodies such as lakes, rivers, ponds, etc. The marine environment is considered more important than terrestrial. This is based on the fact that about 71% of the earth’s surface is covered by water. Planet earth is called the “Blue Planet” due to the abundance of water on its surface. It is, therefore, no surprise that the marine environment plays an important role in the ecosystem between land, sea, and atmosphere. The marine environment has vital ecological functions and enormous biodiversity. Algae have been deemed the “world’s most important plant” due to their significant role in the environment which includes the production of about 50% oxygen [1]. Email: [email protected]

*

Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (551–564) © 2023 Scrivener Publishing LLC

551

552  Essential Oils Their oxygen-producing ability is directly proportional to their carbon dioxide consumption ability. This is vital for the planet’s atmosphere and environment especially animals who depend on oxygen for survival. Most aquatic ecosystems depend on algae as the primary producer. Algae are now a major focus for the possible production of sustainable biofuel. It has produced a number of biopharmaceutical active compounds used in anticancer, antibiotics, antiviral drugs development, etc. [2–4]. Also, microalgae have been used in biomass production and the production of biotechnological important products. The biological activities of marine plants are linked to their bioactive secondary metabolites [5]. Algae produce and secrete secondary metabolites into the water to modify the environment for their benefit. One of the secondary metabolites produced by algae is essential oils.

24.2 Algal Essential Oils Essential oils are commonly present in different parts of terrestrial plants such as leaves, flowers, buds, fruits, peel, grass, seeds, roots, wood, bark, and resin. Considering their intrinsic functions in plants, they have been exploited as bioactive agents in many households. In pharmaceutics and food industries, essential oils are used as food preservatives and antibacterial additives. Essential oils have been found to extend food shelf life while showing inhibition against several pathogenic bacteria, fungi, and yeast [6]. They have also been discovered in different species of algae. Marine essential oils are less familiar than terrestrial odoriferous plants. Due to the complexity of accessing the marine environment, only a few marine plant species are reported to possess essential oils. Essential oils are volatile secondary metabolites produced by plants to protect themselves against pests and predators [7]. Therefore, they form part of the defense system in most plants. They are non-lipid-based odorous/aromatic volatile oils that are commonly used in the pharmaceutical and food industries. For decades, essential oils have been used worldwide by many cultures for different purposes cosmetics, and medicinal use. The earliest documented use of essential oils dates back to 4500 BC where ancient Egyptians used them in cosmetics and ointments [8]. Between 3000 and 2000 BC, India and China also recorded the use of essential oils in traditional medicine [9, 10]. They have since gained momentum in commercial and medical use as they are natural and considered safe in comparison to their synthetic counterparts [11–13]. These volatile compounds have found use in several products such as herbicides/repellents, perfumes, food flavors, and more [11, 14]. Essential oils are extracted using several different extraction methods. These methods are directed by the type of oil extracted and the plant material being used. These methods include water/solvent and steam distillation, expression, solvent-free extraction, etc. [15, 16]. The most common extraction method for essential oils is hydrodistillation using the Clevenger apparatus. These volatile compounds are often analyzed by gas chromatography-mass spectrometry (GC-MS). Algae produce a wide variety of volatile organic compounds that are released into the aquatic ecosystem. The main volatile organic compounds in algal essential oils are terpenes which are by-products of mevalonate and methylerythritol-4-phosphate pathways [17, 18]. Their biosynthesis is linked to fatty acids. The most common terpenes identified in a number of essential oils are monoterpenes and sesquiterpenes. These terpenes were first discovered on a sea hare (Aplysia californica) digestive gland. It was later discovered that the source of these terpenes is the red alga Plocamium pacificum [19]. Essential oils also contain other

Algal Essential Oils  553 Table 24.1  Common compounds present in algal essential oils. Volatile organic compounds

Algae species

Reference

Terpenoid compounds e.g. monoterpenes and sesquiterpenes

Plocamium violaceum Plocamium pacificum Caulerpa taxifolia   Caulerpa racemose Dictyopteris divaricate Dictyopteris dichotoma Capsosiphon fulvescens

[19, 25–31]

Hydrocarbons e.g. alkanes and alkenes

Ulva pertusa Ulva prolifera Ulva linza Chara vulgaris Jania rubens Undaria pinnatifida

[30, 32–35]

Oxygenated hydrocarbons e.g. aldehydes and ketones

Ulva pertusa Ulva prolifera Ulva linza Nizamuddinia zanardinii Palmaria palmata

[30, 33, 36–39]

Amine compounds

Chlorophyceae sp Phaeophyceae sp Rhodophyceae sp Dictyopteris divaricata

[40, 41]

Halogen compounds

Saccharina latissimi Ulva lactuca Asparagopsis sp

[42–45]

chemical classes such as phenols, alkanes, saturated and unsaturated fatty acids, etc. [20]. Other major compounds are simple volatile hydrocarbons, aldehydes, sulfur-­containing compounds, and alcohols [21]. Table 24.1 contains a list of common compounds present in algal essential oils. The secretion of volatile organic compounds is affected by a number of environmental factors such as abiotic stress, temperature, oxygen, light, and nutrition [17, 22–24]. Perturbations in the algae environmental conditions that indirectly affect the chloroplast forces algae to physiologically acclimatize. The acclimatization includes the production of essential oil especially monoterpenes.

24.3 Factors Affecting Algae Essential Oil Production 24.3.1 Temperature An increase in light intensity increases the production of essential oils. This has been tested and observed in different algae species such as Thalassiosira weissflogii, T. pseudonana, Pleurochrysis carterae, and Rhodomonas salina [46]. A similar scenario was observed

554  Essential Oils in marine algae, Hypnea spinella, and Falkenbergia hillebrandii, producing more hydrocarbons in the presence of light and decline in the darkness [47]. The maximum production depends on the light when the photosynthesis rate is maximal.

24.3.2 Light Green alga Dyctiochloropsis were observed to produces essential oil alcohols and aldehydes at a maximum rate during a heat shock exposure [48]. These volatile compounds are quickly secreted into the environment to signal the heat stress to the other species. Amongst the produced compounds in response to heat, β-cyclocitral is the most abundant. β-Cyclocitral is a component of algae essential oils that signals the neighboring species to undergo physiological changes in preparation and response to heat. High temperature increases reactive oxygen species conversely increasing the production of halogenated hydrocarbons [49].

24.3.3 Nutrients Phosphorus and nitrogen are typically the main sources of nutrition in water bodies. In water bodies, phosphorus exists as polyphosphate and orthophosphate and it is a limiting nutrient for algal growth [50]. In the case of limited phosphorus, algae have been observed to produce a vast amount of volatile compounds [51]. The study was conducted on Chlamydomonas reinhardtii, a single-celled green alga. The same phenomenon was observed with nitrogen as a limiting factor. Nitrogen deprivation induces genes that are involved in the formation of essential oil compounds [52]. Under nutrient deprivation, algae produced volatile compounds to inhibit the growth of other algae species. The volatile compounds help the emitters to better compete for the depleting nutrients.

24.3.4 Chemical Stress Algae also increase the production of essential oils in the presence of external chemical stress. Foreign chemicals such as acetic acid in water bodies increase abiotic stress and induce programmed cell death in algae. In the presence of increased concentrations of acetic acid or sodium chloride, C. reinhardtii conversely increases the production of essential oils [23, 53]. The same phenomenon was observed when the same algae species was stressed with sodium carbonate [54]. Abiotic stress causes an increase in oxidative oxygen species. However, the oxidative oxygen species decrease with an increase in volatile compounds production. Furthermore, the production of volatile compounds decreases the density of algae [53]. Decreased density assists the algae for better mobility and floating away from the stressful environment. These observations prove that volatile compounds are produced as a survival strategy for the emitters and thus play an important ecological function. Figure 24.1 presents the ecological importance of algal essential oils.

24.4 Ecological Importance of Algal Essential Oils As the primary producer of aquatic herbivores, algae are preyed on by predators for food. In pursuit to protect themselves, algae produce volatile compounds that are toxic to the

Algal Essential Oils  555

Induces Defense in Homogenous Algae Protects Algae Against Predators

Pheromones

Antifungal Agents

Ecological Functions of Algal Essential oils

Beach-Odor Lowers Oxidative Stress

Antibacterial Agents Allelopathic Effects on Other Heterogenous Algae

Figure 24.1  Ecological importance of algal essential oils.

predators [55]. Damaged algal cells produce toxin volatile compounds while producing predator repellents. The repelling effect is closely associated with aldehydes [56]. β­cyclocitral and other C11-sulfocompounds have been tested for their repellent effect on the predators. These compounds repel the predators such as Daphnia magna while increasing their swimming velocity. Sesquiterpene produced by Caulerpa taxifolia and Caulerpa racemose is the most abundant cytotoxic and repellents [25]. The hydrocarbon compounds also act as a chemical defense system in marine plants. They are deterrents against herbivores such as reef fishes, urchins, etc. [57]. The volatile compounds also inhibit the sea urchins and D. magna from laying eggs [58, 59]. They, therefore, play a role as hormones because they interfere with the sexual reproduction of predators. The main problem encountered by the algae during hostile changes in the ecological system and elevated abiotic stress is the inevitable production of reactive oxygen species. High intracellular reactive species concentration damages essential systems such as photosynthesis apparatus, deoxyribonucleic acids (DNA), proteins, and induced cell death [60, 61]. Volatile compounds, especially monoterpenes act as reactive oxygen species scavengers to protect the cells during abiotic stresses [62]. Ketones, esters, and aldehydes are also associated with decreasing and normalizing reactive oxygen species levels in algal cells [54]. Therefore, other essential oil compounds are directly involved in protecting algae from high temperatures, pH changes, salinity, drought, abiotic stress, etc. Essential oils also help in sending a signal to the neighboring homogeneous algae to prepare for specific abiotic stress. For example, when a normal healthy alga is exposed to volatile compounds released by homogeneous algae undergoing programmed cell death, there is a noticeable decline in normal cells growth while increasing antioxidant enzymes [23]. These compounds are information agents that transfer alerting messages to prepare

556  Essential Oils the neighboring homogeneous algae for foreseeable abiotic stress. This helps in the preservation and sustains the growth of the species population. Algae can also send or receive messages from heterogeneous species in the same environment. In water bodies, especially lakes and ponds, algae and cyanobacteria co-exist with a close relationship. Under abiotic stress, cyanobacteria also produce volatile compounds and toxins against algae [63]. The cyanobacterial compounds are important allelopathic communication between these two heterogeneous species [64]. Volatile compounds from cyanobacteria produced under nutrient deprivation decrease algal cell growth while diminishing the photosynthetic pathway [17, 51]. The same phenomenon is observed between heterogeneous algae species. Volatile compounds from Microcystic algae inhibit the growth of Chlorella vulgaris and C. reinhardtii [65, 66]. β-Cyclocitral, α-ionone, β-ionone, and geranylacetone are some of the volatile compounds that were actively inhibiting the growth and photosynthetic abilities in the heterogeneous algae [67]. The allelopathic communication of volatile compounds observed in aquatic heterogeneous species is mainly for the competition of depleting nutrients.

24.5 Pheromone Properties of Algal Essential Oils Chemical communication is vital for plants, animals, and microorganisms. Animals use chemical communication to mark territory, attract mates, find prey, identify other animals, etc. In plants, chemical communication is important for plant reproduction as their aroma invites pollinators for the dispersion of pollens and seeds in terrestrial plants. Marine plants also employ the same technique for their reproduction. Algae reproductive system produces both motile males and females. Algal essential oil plays a vital role in marine algae intra- and interspecies chemical communication. Chemicals that are responsible for communication and coordinating activities within a specific species are known as pheromones [68]. In algae, these chemicals are responsible for communication and interaction with homogeneous algae and also play an allelopathic role in heterogeneous algae. Pheromones are common in the animal kingdom, however, they have also been observed to play a vital role in plants and algae. Algae female gametes, especially brown algae, naturally produce sexual pheromones to enhance reproduction [69]. Algae female pheromones attract the male gametes for anisogamy. Sperm attraction in brown algae has been observed more than 100 years before the identification and discovery of pheromones. Pheromones have been observed to be effective at a concentration between 1-1000pmol. They are characterized by unsaturated and non-functionalized acyclic and/or alicyclic C11-hydrocarbons [70]. Parenthetically, ectocarpene from Ectocarpus siliculosus algae was the first algae pheromone isolated in 1971 [71]. Scytosiphon lomentaria, Colpomenia bullosa, and Analipus japonicas, commonly known as the sea fir, are examples of brown algae that secrete these pheromones [72]. This sea fir secretes pheromones which have been identified to be C11-hydrocarbons, comprising approximately 98% of ectocarpene and about 2% of both hormosirene and dictyopterene. However, hormosirene has a higher effect on attracting the male gametes compared to the other compounds [69]. Hormosirene, dictyopterene, cystophorene, and ectocarpene have also been identified in a number of brown algae and seaweeds [73, 74]. Dictyota dichotoma and some of Dictyopteris species are primary producers of dictyopterene pheromone [75]. A 2004

Algal Essential Oils  557 study reported the presence of various volatile organic compounds in the natural biofilm of polyethylene pipes used to supply raw lake water [76]. Dictyopterenes were amongst the volatile compounds identified in the natural biofilm. Nevertheless, some Dictyopteris pheromones are patented for use in cosmetics components in fragrances, deodorants, and antiperspirants [77]. Pheromones, especially dictyopterenes, are also associated with the “ocean-odor” [78].

24.6 Algal Essential Oils in “Beach-Odor” Most of the terrestrial essential oils are used in perfumes for their special odor. Few of the marine species also produce odorous essential oils. The odorous effect of essential oils is mostly associated with terpenes and other compounds such as phenols and simple aliphatic esters. A typical sea-breeze has attracted the attention of man since ancient times. It was only in the mid-1900s where the sea-breeze or beach-like odor was associated with algal essential oils containing different secondary metabolites [79]. There are two natural organic compounds mainly associated with beach-like odor. These are C11-hydrocarbons (cyclic and alicyclic) which also act as pheromones and polyunsaturated aldehydes [80, 81]. Several brown algae genera have been found to produce C11-hydrocarbons. These genera include Zonaria, Desmarestia, Ectocarpus, Dictyota, etc. [70]. However, the genus Dictyopteris produces these C11-hydrocarbon compounds in abundance in almost all the species [77]. Dictyopteris divaricata, brown algae, is an example of species that produces an oil with a “beach-odor” [26]. This oil is a mixture of cadinene-type sesquiterpenes (α-­ copaene, γ-cadinene, cadalene, β-elemene, and δ-cadinol) selinene-type sesquiterpene (dictyopterone and α-dictyoptero) and sesquiterpene alcohol [26, 27]. Dictyopteris dichotoma species closely related to D. divaricate also presents the “beach-odor”. It produces similar sesquiterpenes and other chemical compounds such as palmitic acid, hexadecenoic acid, and n-parafines [28]. Aldehydes and alcohol have been the major volatile compounds associated with the aromatic nature of Ulva pertusa, a microscopic green algae species [82]. Similar compounds were identified in another green algae species, Capsosiphon fulvescens [29]. In this species, more compounds associated with the pleasant aroma were detected. These include esters, especially methyl esters from hexadecanoic and 9-octadecenoic acids, which generally give sweet and fruity flavors [29, 83]. The sesquiterpenes alcohols known as cubenols are however the main components of flavor and odor in most aromatic algae [29, 84, 85]. They have been determined in many algal species including C. fulvescens and Dictyopteris divaricate. A sniffing test conducted on cubenol gives out kelp, mint, hey, and ocean odor. In more recent studies, researchers are ocean scent is generally considered as being pleasant and stimulates a sense of peace, lightness, and overall wellbeing [77].

24.7 Algal Essential Oils in “Off-Odor” The presence of these organic volatile compounds from microalgae and cyanobacteria may contribute to the off-odor and taste episodes in drinking water. These compounds do not affect the drinking water quality indicators such as turbidity, suspended matter, etc.,

558  Essential Oils therefore they are often present in drinking water. It is currently not clear if these volatile compounds affect human health, they however increase the cost of water treatment. Other studies associate the earthy-musty odor of lake and pond waters to algal essential oil terpenes such as geosmin, 2-methylisoborneol, β-cyclocitral, and β-ionone [18, 86, 87]. Geosmin in particular is likely to give off-flavors in fish causing major problems in the fish industry [88]. Fishes can easily absorb these compounds through the grill and other tissues exposed to water. The rate of absorption increases with an increase in water temperature and oxygen content [89]. Bioaccumulation of geosmin and other volatile compounds in catfish and tilapia has affected the fish market causing severe economic losses [90, 91]. These compounds are also found in abundance during the algal bloom caused eutrophication in lakes and ponds [92]. Although these essential oil compounds are associated with off-odor, products obtained from the fermentation of these compounds especially hydroxyketones and β-ionone, are important for odor quality in microbial-processed dairy food [90]. β-cyclocitral is a signaling molecule that modulates the activity of many key physiological processes while giving out a pleasant odor together with β-ionone.

24.8 Antibacterial Activities of Algal Essential Oils Marine algae extracts from different genera possess a broad range of antimicrobial, antifungal, and antioxidant activity [93–95]. They also act as an antimicrobial agent to protect the plants from opportunistic microorganisms. Dioscorea membranacea Pierre essential oils possess antimicrobial activity against Staphylococcus aureus and Agrobacterium tumefaciens [96]. Antimicrobial activity of essential oils from a brown algae Padina pavonia against Staphylococcus aureus and Candida albicans [97]. Essential oils extracted from Fucus vesiculosus showed potency against methicillin-resistant Staphylococcus aureus [98]. These can be useful as an alternative to the current antibiotics that are susceptible to bacterial resistance. In recent years, there have been efforts to discover new and effective antimicrobial agents from algae species [99–101]. Algal essential oils extracted from Colpomenia sinuosa, Dictyota dichotoma, Dictyota dichotoma var. implexa, Petalonia fascia, and Scytosiphon lomentaria species have activity against both Gram-positive and Gram-negative bacteria. However, the essential oils show higher activity against Gram-positive especially Bacillus subtilis and Staphylococcus aureus [95]. This might be due to the thin yet complicated structure of the Gram-negative bacterial cell wall [102]. These essential oil extracts are composed of hydrocarbons, terpenes, acids, phenols, sulfur-containing compounds, aldehydes, naphthalene skeleton, and alcohols.

24.9 Antifungal Activities of Algal Essential Oils Animal and plant-fungal infections are indisputable affection public health and crop production. There are several pathogenic fungi that contribute to diseases in both animals and plants. With the vast amount of available antifungal remedies, algae also contribute antifungal active compounds. Seaweed Hormophysa cuneiformis extract mixture possesses antifungal activity against eight pathogenic fungi (Aspergillus flavus, A. fumigatus, Candida

Algal Essential Oils  559 albicans, Trichosporonas ahii, Alternaria alternata, Cladosporium herbarum, Fusarium oxysporum, and Penicillium digitatum [103]. One of the active compounds that contributed to the antifungal activity is essential oils. Again, P. pavonia essential oils show antifungal activity against Macrophomina phaseolina,  Rhizoctonia solani, and  Fusarium solani [97, 104]. The volatile compounds from P. pavonia also have antitumor activity against lung carcinoma cell lines.

24.10 Conclusion Terrestrial essential oils have been used worldwide by many cultures for different purposes. Researchers are slowly focusing on new compounds produced by marine organisms and are currently undergoing detailed investigations to isolating biologically active molecules. Algal essential oils play a vital role in the ecosystem. They are associated with a number of ecological important roles. They contribute to the beach-odor and aroma. However, they are mainly produced for the sole benefit of the algae such as protection against predators, competing for nutrients, etc. Changes in environmental factors such as limited light, hot water temperatures, nutrient depletion, and abiotic stress inversely increase the production of algae essential oil. It can be concluded that the essential oils extracted from algae contain bioactive compounds present in their constitution with interesting bioactivity that can offer significant benefits and biotechnological relevance.

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25 Classical Methods for Obtaining Essential Oils Syed Raza Ali Naqvi1*, Hiba Shahid1, Ameer Fawad Zahoor1, Muhammad Saeed1, Muhammad Usman1, Ali Abbas1, Mamoon Ur Rasheed1 and Tanvir Hussain2 Department of Chemistry, Government College University Faisalabad, Faisalabad, Pakistan 2 Department of Chemistry, The College of Art and Science, Sialkot, Pakistan

1

Abstract

An extraction technique is a separation process, used to separate bioactive components from different parts of plant. Mainly, plant extracts are of two types, volatile and non-volatile hydrocarbons. Volatile hydrocarbon that might be saturated or unsaturated, are commonly known as essential oil which bear specific odor. Their extraction from plant sources commonly conducted with classical methods and rarely using modern techniques on large scale. Classical methods own to its simplicity and handling, and cost-effectiveness mostly preferred in all sectors of extracting essential oils (laboratories to industrial scales). These include mechanical treatment, hydro-distillation (water distillation, and steam distillation), cold pressing, solvent extraction, and soxhlet extraction. Choice of the method depends upon the nature of essential oil, solvent used and sensitivity of the process. However, none of the process is ideal; the researcher or industrialist has to compromise one or other parameter such as yield, time, or quality. The aim of this book chapter is to describe different classic methods of extracting essential oil and their efficiency. Keywords:  Essential oil, hydro-distillation, cold pressing, solvent extraction, soxhlet extraction

25.1 Introduction Extraction of natural component remained a top priority work in scientific studies for the development of health care/pharmaceutical products to manage and treat the diseases with ignorable side effects. Phytochemicals extracted from different parts of plants are of two types, volatile and non-volatile hydrocarbons. Volatile hydrocarbon that might be saturated or unsaturated, are commonly known as essential oil (EO); it carries specific fragrance. Their extraction from plant sources commonly conducted with classical methods and rarely using modern techniques on large scale. The EOs due its fragrance and volatility are best known for its therapeutic values, commonly known as aromatherapy. In general, the EOs are obtained by steaming or pressing various parts of the plants. However, classical techniques are being practiced commonly with the aim to obtain maximum yield of EOs under certain economic conditions [1–3]. Unprocessed material of juicy fruit or edible items can be the major raw material to obtain EOs by steam distillation or mechanical treatment [4]. *Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (565–582) © 2023 Scrivener Publishing LLC

565

566  Essential Oils The smell, yield, and aesthetic quality of EOs define the market value. Old classical methods of extraction were associated with many disadvantages that have been resolved through their modified classical and modern extraction methods [5]. Supercritical fluid extraction has become more beneficial than solvent extraction method to obtain maximum yield of EOs in short time and it does not require further treatment like solvent extraction [6, 7]. Essential oils, chemically consist of hydrocarbons which also include oxygen, nitrogen, or sulfur containing compounds [8–11]. Mainly they consist of terpenes, aldehydes, fatty acids, phenols, ketones, esters, alcohols, oxides and phenols [4, 12]. Regarding the physical characteristics, EOs are soluble in non-polar solvents and insoluble in H2O. They have specific gravity (97% repellency) against Ae. Albopictus [150].

42.3 Conclusion Essential oils stand out among natural plant products due to their widespread use in traditional medical systems around the world. Their constituents have typically effective antimicrobial characteristics that have been employed as preventatives against a variety of microbiological illnesses since Ayurvedic times. The findings discussed in this article are meant to refocus the scientific community’s attention on the wide range of applications of these aromatic oils.  Antimicrobial, antioxidant, anti-inflammatory, antiviral and anticancer properties, in particular, have been reported in a variety of cell and animal models. However, the scarcity of clinical trials in human research needs more attention to achieve a high degree of scientific evidence. EOs can assist in the development of novel medications based on natural ingredients.

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Therapeutic Benefits of EOs  1007 114. Mondal, M., Quispe, C., Sarkar, C., Bepari, T.C., Alam, M.J., Saha, S., Ray, P., Rahim, M.A., Islam, M.T., Setzer, W.N., Analgesic and anti-inflammatory potential of essential oil of Eucalyptus camaldulensis Leaf: In vivo and in silico studies. Nat. Prod. Commun., 16, 1, 2021. 1934578X211007634. 115. Hansen, D., Haraguchi, M., Alonso, A., Pharmaceutical properties of ’sucupira’ (Pterodon spp.). Braz. J. Pharm. Sci., 46, 607, 2010. 116. Spindola, H.M., Servat, L., Rodrigues, R.A.F., Sousa, I.M.O., Carvalho, J.E., Foglio, M.A., Geranylgeraniol and 6α, 7β-dihydroxyvouacapan-17β-oate methyl ester isolated from Pterodon pubescens Benth.: Further investigation on the antinociceptive mechanisms of action. Eur. J. Pharmacol., 656, 45, 2011. 117. Grace, M.H., Esposito, D., Timmers, M.A., Xiong, J., Yousef, G., Komarnytsky, S., Lila, M.A., Chemical composition, antioxidant and anti-inflammatory properties of pistachio hull extracts. Food Chem., 210, 85, 2016. 118. Paterniti, I., Impellizzeri, D., Cordaro, M., Siracusa, R., Bisignano, C., Gugliandolo, E., Carughi, A., Esposito, E., Mandalari, G., Cuzzocrea, S., The anti-inflammatory and antioxidant potential of pistachios (Pistacia vera L.) in vitro and in vivo. Nutrients, 9, 915, 2017. 119. Mogosan, C., Vostinaru, O., Oprean, R., Heghes, C., Filip, L., Balica, G., Moldovan, R.I., A comparative analysis of the chemical composition, anti-inflammatory, and antinociceptive effects of the essential oils from three species of Mentha cultivated in Romania. Molecules, 22, 263, 2017. 120. Han, X. and Parker, T.L., Antiinflammatory activity of cinnamon (Cinnamomum zeylanicum) bark essential oil in a human skin disease model. Phytother. Res., 31, 1034, 2017. 121. Kim, D.H., Kim, C.H., Kim, M.-S., Kim, J.Y., Jung, K.J., Chung, J.H., An, W.G., Lee, J.W., Yu, B.P., Chung, H.Y., Suppression of age-related inflammatory NF-κB activation by cinnamaldehyde. Biogerontology, 8, 545, 2007. 122. Mondal, S. and Pahan, K., Cinnamon ameliorates experimental allergic encephalomyelitis in mice via regulatory T cells: Implications for multiple sclerosis therapy. PLoS One, 10, e0116566, 2015. 123. Hassan, S.B., Gali-Muhtasib, H., Göransson, H., Larsson, R., Alpha terpineol: A potential anticancer agent which acts through suppressing NF-κB signalling. Anticancer Res., 30, 1911, 2010. 124. Torck, M. and Pinkas, M., Camptothecin and derivatives: A new class of antitumor agents. J. Pharm. Belg., 51, 200, 1996. 125. Avemann, K., Knippers, R., Koller, T., Sogo, J.M., Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks. Mol. Cell. Biol., 8, 3026, 1988. 126. Sung, H., Ferlay, J., Siegel, R.L., Laversanne, M., Soerjomataram, I., Jemal, A., Bray, F., Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA. Cancer J. Clin., 71, 209, 2021. 127. Weaver, B.A., How Taxol/paclitaxel kills cancer cells. Mol. Biol. Cell, 25, 2677, 2014. 128. Zhong, Z., Yu, H., Wang, S., Wang, Y., Cui, L., Anti-cancer effects of Rhizoma Curcumae against doxorubicin-resistant breast cancer cells. Chin. Med., 13, 1, 2018. 129. de Araújo-Filho, H.G., Dos Santos, J.F., Carvalho, M.T.B., Picot, L., Fruitier-Arnaudin, I., Groult, H., Quintans-Júnior, L.J., Quintans, J.S.S., Anticancer activity of limonene: A systematic review of target signaling pathways. Phytother. Res., 35, 4957, 2021. 130. Ye, Z., Liang, Z., Mi, Q., Guo, Y., Limonene terpenoid obstructs human bladder cancer cell (T24 cell line) growth by inducing cellular apoptosis, caspase activation, G2/M phase cell cycle arrest and stops cancer metastasis. J. BUON Off. J. Balk. Union Oncol., 25, 280, 2020. 131. Miller, J.A., Pappan, K., Thompson, P.A., Want, E.J., Siskos, A.P., Keun, H.C., Wulff, J., Hu, C., Lang, J.E., Chow, H.-H.S., Plasma metabolomic profiles of breast cancer patients after shortterm limonene intervention. Cancer Prev. Res., 8, 86, 2015.

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43 Essential Oils Used in Packaging: Perspectives and Limitations Khadija El Bourakadi, Abou El Kacem Qaiss and Rachid Bouhfid* Moroccan Foundation for Advanced Science, Innovation and Research (MAScIR), Composites et Nanocomposites Center, Rabat Design Center, Rue Mohamed El Jazouli, Madinat El Irfane, Rabat, Morocco

Abstract

Packaging has been around for hundreds of years, its main role being to contain and transport products safely. Numerous components with various shapes, functions, and materials are frequently used in packaging to satisfy the complementary requirements of a specific product. Food packaging (sensitive and perishable goods) must not endanger human health and must be consistent with the product’s design, physical structure, safety, and deterioration caused by biological or chemical issues. Antibacterial activities of essential oils can help extend the shelf life of both raw and processed foods. Despite this, the food industry has only used a few preservation techniques focused on the use of essential oils. This chapter’s goals are to afford a summary of current knowledge about essential oils’ chemical structure, therapeutic, and biological activities, to define their functional applications, and to evaluate the possibilities and limitations of their use in the food industry. Keywords:  Essential oils, medicinal properties, biological function, active packaging, advantages, future perspectives, limitations

43.1 Introduction Food packaging is essential and ubiquitous by way of practically entirely nutriment is packaged in some way is packaged in some way or another. Food safety and quality would be jeopardized without packaging [1]. Packaging is a product-specific structure designed to prevent food from deteriorating chemically, biologically, or physically, prolong shelf life, and preserve, improve, and ensure product quality and protection [2]. Food packaging may also be described as an additional layer on the outside of a product that helps protect it from the elements, promote movement, and storage during sale and use. Food packaging has continuously been thought of as an inactive barrier that protects food from the influences of the environment [3, 4]. Current research patterns, however,

*Corresponding author: [email protected] Inamuddin (ed.) Essential Oils: Extraction Methods and Applications, (1009–1024) © 2023 Scrivener Publishing LLC

1009

1010  Essential Oils include the creation of packaging materials that communicate favorably with the environment and food, thus aiding in the preservation of both [5]. Food safety, security, comfort, and communication are the four key functions of passive packaging, active packaging, on the other hand, provides active protection by releasing active substances such as antioxidants, antibacterial agents, enzymes, flavorings, and nutraceuticals on a continuous basis [6]. Active packaging extends the shelf life of foods, retains spices, and while retaining product consistency, decreases the usage of chemicals and preservatives in food formulas, thanks to its creative concept and engagement with the packaging, the food, and the climate [7, 8]. Thus, most of the plastic active packaging found in the literature allows for the incorporation of synthetic and artificial active substances in their systems [9]. However, these substances are associated with various adverse human health effects and sometimes carcinogenicity as a result of their migration process through the food packaging to the packaged food and then to the consumer’s body [10]. As a result, new natural alternatives such as essential oils, herbs, and extracts from medicinal plants are being investigated [11]. The US Food and Drug Administration considers essential oils and extracts from aromatic and medicinal plants to be generally healthy [12]. They have been extensively researched not only for being natural products, but also because they have been shown to have biological, nutritive, and beneficial properties for food and human health. They have been widely studied and exploited because of their biological properties [13]. In general, the main bioactive components of essential oils and extracts are the ones responsible for their biological activity, but smaller components can also contribute and show a synergistic effect [14]. Essential oils and extracts from a variety of aromatic plants are used in the production of food packaging [15]. These compounds, on the other hand, have been used to make lowcost non-biodegradable polymers and biopolymers that are difficult to process. The foremost aim of this chapter is to offer a summary of the published data on the biological and medicinal activities of those essential oils and their components that could be measured appropriate for use in functional applications, namely those related to food industry, and to describe their conceivable use in manufacturing of package materials. The current state of knowledge on the potential of these substances is presented; limitation and safety issues are discussed; and future research directions are suggested. This chapter focuses on the biological impacts of essence on infections acquired from food, particularly those for which food packaging systems serve as the primary reservoir.

43.2 Essential Oils: Definition, Preparation, and Composition Essential oil is an odorous substance with a complex composition obtained by steam distillation or other methods from a botanically specified raw material. It is a mixture of various molecules, including terpenes and oxygenated compounds (alcohols, aldehydes, ketones), obtaining essential oils by cold expression (zests). In this last case, there is some ambiguity about the term essential oil. For this type of extract the term essence is also used [16]. Essential oils can be extracted from a variety of plant parts [13]: • Flowers (rose petals), • Fruit peels (lemon, bergamot, orange),

Essential Oils Perspectives and Limitations  1011 • • • • • •

Seeds (aniseed), Leaves (eucalyptus), Berries (juniper), Flower buds (clove), Fruits (parsley), Wood (sandalwood, cinchona bark).

There are several methods of extracting essential oils, such as distillation, hydrodistillation, percolation, and expression, which can change the composition of essential oils. In terms of the quantity needed, the complexity of the procedure, and the application area, Figure 43.1 depicts prepared extraction techniques [17]. Two processes are mainly used and are the subject of a monograph in the Pharmacopoeia: • Cold expression also called cold pressing is used to obtain the essences and is reserved for Citrus fruits (lemon, mandarin, orange...). This process consists of mechanically breaking up fresh citrus peel by subjecting the vegetable substance to a strong pressure using a hydraulic press. • The most common method for extracting essential oils is steam distillation [18]. A heat source heats a still containing water and plants placed on a tray. The heat causes steam to form, which passes through the plants and carries the aromatic molecules with it. A liquid composed of water and essential oil is recovered. The essential oil is lighter and separates from the water [19]. The essential oil content of plants is low, in the order of 1–3%, except for cloves (14– 19%), mace (10–13%), nutmeg (8–9%) and cardamom (4–10%). Essential oils (EOs) are usually classified according to the chemical nature of the major active ingredients, more rarely according to the method of extraction, or the biological effects [20]. Generally, there are eight main classes: • • • •

Sesquiterpene carbides Terpene carbides Alcohols Esters and alcohols

• Supercritical fluid extraction, Subcritical liquid extraction, Ultrasound assisted extraction, Microwave assisted extraction, Microwave hydrodiffusion, Microwave steam distillation, Solvent free microwave extraction

Innovative methods

• Hydrodistillation, Steam-distillation, Hydrodiffusion, Organic solvent extraction, Cold pressing, Dry distillation

Essential oils extraction methods Classical methods

Figure 43.1  Essential oils extraction techniques.

Laboratory scale

• Clevenger distillation, Microdistillation, Headspeace, Solid-phase microextraction, current

1012  Essential Oils • • • •

Aldehydes Ketones Phenols Ethers and peroxides.

Essential oils can be classified into several biochemical families. The therapeutic activity of an essential oil is linked to its biochemical structure, the functional groups of its main compounds (alcohols, phenols, terpenic compounds, etc.) and their synergistic actions.

43.3 Essential Oils: Medicinal and Biological Functions The employment of synthetic chemicals to monitor insects and arthropods, according to some data, raises some clear concerns regarding the environment and human health. As a result, there is an increasing market for natural or alternative repellents. These products are both effective and safe for the community. Aromatic plants and spices have been used for centuries in food preparations not only for the flavor they provide but also for their antibacterial and antifungal properties. Oregano, thyme, sage, rosemary, and cloves are all aromatic plants frequently used as food ingredients. The essential oils of these plants all have a common feature: they are rich in phenolic compounds such as eugenol, thymol and carvacrol [21]. These compounds have a strong antibacterial activity [22]. The most active compound is carvacrol. Since it is non-toxic, it is employed as a protective and food flavoring in beverages, sweets, and other meals [23]. Thymol is the active ingredient in mouthwashes and eugenol is used in cosmetics, food and dental products [24]. These compounds have antimicrobial properties against a wide range of bacteria: Escherichia coli, Bacillus cereus, Listeria monocytogenes, Salmonella enterica, Clostridium jejuni, Lactobacillus sake, Staphylococcus aureus and Helicobacter pyroli [25]. Antibacterial effects are also found in other families of compounds, such as alcohols, aldehydes, and monoterpene ketones (geraniol, linalool, menthol, terpineol, thujanol, myrcenol, thujone, camphor, carvone, etc.), phenylpropanes (cinnamaldehyde) and monoterpenes (yterpinene, p-cymene) [26]. The food, cosmetic and pharmaceutical industries are very interested in the properties of these compounds, especially as they are natural flavorings. As a result, many researchers around the world are studying their potential as preservatives [27]. Most of these compounds are also very good antifungal agents. Thymol, carvacrol, and eugenol are still the most active compounds here [28]. A large number of essential oils compounds have been tested against a wide range of fungi: Candida (C. albicans), Aspergillus (A. niger, A. flavus, A. fumigatus), Penicillium chrysogenum, and many others [29]. Essential oils have biological properties due to many small terpenoids and phenolic compounds, which have also shown high antioxidant activity in pure form. The antioxidant activities of essential oils have recently been broadly studied. The formation of disorders like Alzheimer’s is linked to oxidative stress, which happens when there is an imbalance between the generation of free radicals and antioxidant enzymes [30]. Cinnamon, nutmeg, clove, basil, parsley, oregano and thyme essential oils have powerful antioxidant compounds [31]. The most active compounds are thymol and carvacrol once again. Their behavior is linked to their phenolic composition, as these compounds have oxidative-reductive properties and thus aid in the neutralization of free radicals and the breakdown of organic compounds [32, 33]. Certain alcohols, ethers, ketones, and monoterpene

Essential Oils Perspectives and Limitations  1013 aldehydes, as well as monoterpene aldehydes, are responsible for the antioxidant function of essential oils: tinalool, 1,8-cineoIe, geranial/neral, citronellal, isomenthone, menthone and some monoterpenes: α-terpinene, γ-terpinene and terpinolene [31]. In clinical settings, essential oils are similarly used to treat inflammatory illnesses such as rheumatism, asthma, and arthritis [34]. Essential oils’ broad therapeutic potential has drawn the attention of researchers in recent years to their potential anti-cancer activity. As a result, essential oils and their volatile constituents are now being studied in the search for new natural anti-cancer products [35]. Essential oils can both prevent and suppress cancer. It’s common knowledge that some ingredients, such as garlic or turmeric, are healthy sources of anti-cancer agents useful in cancer prevention [36].

43.4 Functional Application of Essential Oils Due to their numerous and diverse properties, aromatic plants and their essences are used in many fields such as: food, pharmacy, perfumery, aromatherapy and others [37]. Many essential oils are found in the formula of a very large number of pharmaceutical products: syrups, drops, capsules. They are also used in the preparation of infusions such as: verbena, thyme, mint and others [31]. Essential oils’ aromas stimulate the nervous system, triggering a self-regulatory command [38]. Aromatherapy, more specifically, helps the body combat illness by activating the self-healing reflex and altering the chemical composition of body fluids (saliva, blood, lymph). The essential oils moreover have an impact on hormonal secretions, on the endocrine balance and on the body’s neurovegetative reactions [38]. Essential oils are widely used to treat certain internal and external diseases (bacterial or viral infections, humoral or nervous disorders). In dentistry, several essential oils have given very satisfactory clinical results in the disinfection of the dental pulp, as well as in the treatment and prevention of caries [39]. In this sense, thyme and rosemary essential oils have been used to relieve fatigue, headaches, muscle pain and some respiratory problems. Unfortunately, these prescriptions do not have a rigorous scientific basis as they are often derived from empirical practices and trial and error [40]. Another fascinating area is the use of essential oils in creams and gels, which allows these cosmetics to be preserved while maintaining their pleasant scent thanks to their antiseptic and antioxidant function [41]. For example, due to its antibacterial activity, lavender is already used a lot in perfumery for its odoriferous properties, it is found in many perfumes or toilet waters as well as in soaps, detergents, etc. It is also used in cosmetics for its healing properties (floral waters, body care creams) [42]. Essential oils, on the other hand, are mostly employed in the food industry for food preservation due to their scent, flavor, and inherent antimicrobial properties [43]. In this, citrus essential oils such as monoterpenes, sesquiterpenes, and oxygenated derivatives have strong inhibitory activities against pathogenic bacteria, implying that they may be used as flavoring and antioxidant agents [44, 45]. In addition, several food segments use, to varying degrees, essential oils which offer them a formidable potential of their aromatic notes in an infinitely varied register. They are found in almost all food sectors: non-alcoholic drinks, confectionery, dairy products, soups, sauces, bakery products, meat products, etc. [46]. However, it is only recently that the potential for essential oils to be utilized as preservatives

1014  Essential Oils Cosmetics

Food/Beverages

Cleaning products

Essential oils

Care products

Pharmaceuticals

Spa/Salon products

Figure 43.2  Functional/industrial application of essential oils.

has received a lot of consideration, owing to the existence of molecules with antibacterial and antioxidant capabilities in them [32]. The share of essential oils in flavoring continues to grow at the expense of synthetic flavoring compounds, alongside derivatives of fruit processing, essential oils are still likely to have a growing market. Figure 43.2 below summarizes the functional application of essential oils.

43.5 Active Packaging Material Based on Essential Oils There has been a significant rise in research into the manufacture of antimicrobial and antioxidant materials in these days, with the goal of preserving food against oxidative reactions and microbial growth [47]. Furthermore, rather of synthetic compounds, natural additives, such as natural preservatives derived from natural sources, are a current trend in the food sector. The incorporation of essential oils and extracts which are considered as an examples of natural substances and medicinal plants is a new approach in active food packaging [48, 49]. Since the US FDA and European regulations classify natural additives such as essential oils, plant extracts and their constituents as GRAS (Generally Recognized as Safe), healthier and much less toxic than synthetic substances [50]. Packaging manufacturers and discerning consumers consider their incorporation into food packaging films to be an attractive way to prevent microbial spoilage of food [51].

43.5.1 Composite and Nanocomposite Materials Based on Essential Oils According to Regulation (EU) No 450/2009 (European Commission, 2009), “active materials which are designed to contain active components that will release into the packaged food or into the surrounding food environment to prolong the shelf life or preserve or enhance the condition of packaged foods. Once the active compounds are incorporated into the food packaging, they can be released in a regulated manner to keep foods’ organoleptic properties, consistency, and microbiological integrity [52].

Essential Oils Perspectives and Limitations  1015 Currently, a huge interest has been given to the incorporation of essential oils as a bioactive substance into the packaging systems. Several factors, including the properties of the essential oil, the volume incorporated, the use of plasticizer, and the polymer matrix, influence the mechanical properties of active packaging incorporating essential oils for this purpose [53, 54]. Interestingly, Lopez et al. were observed that cinnamaldehyde-enriched polyethylene film was effective at 4% (wt./wt.) in inhibiting the growth of fungi (Penicillium islandicum, Penicillium roqueforti, Penicillium nalgiovense, Eurotium repens, Aspergillus flavus, Candida albicans, Debaryomyces hansenii and zigosaccharomyces rouxii). Bacteria (Listeria monocytogenes and S. aureus) were inhibited at an active agent concentration of 8% (wt./wt.). E. coli, Yersinia enterocolitica, Salmonella choleraesuise and P. aeruginosa were inhibited with a highest concentration of 10% (wt./wt.) (Table 43.1) [55]. Table 43.1  Active food packaging films based on plant extracts/essential oils. Natural extracts/essential oil

Polymeric matrix

Reference

Green tea extracts

Ethylene vinyl alcohol (EVOH) Chitosan

[73] [3]

Bergamot essential oil

Chitosan

[74]

Clove essential oil

Polylactide/graphene oxide nanosheets

[75]

Savory essential oil

Agar-Cellulose

[58]

Grape-seed extract

PA/LDPE Soy protein isolate Whey protein isolate

[76] [77] [78]

Rosemary

PP Gelatin

[51] [79]

Grape seed extract, nisin, and EDTA

Soy protein

[4]

Lemon essential oil

Grass carp collagen-chitosan

[80]

Peppermint/chamomile essential oils

Gelatin nanofibers

[81]

Origanum vulgare L. essential oil

Poly(lactic acid)

[61]

Thymol

Poly(lactic acid)

[60]

Murta fruit extract

Methyl cellulose

[47]

Cinnamon bark oil and soybean oil

Alginate films

[64]

Rosemary, Myrtle and Thyme essential oil

Poly(lactic acid)

[65]

Ginger essential oil

Gelatin

[82]

Cinnamon essential oil

Electrospun polyvinyl alcohol

[83]

Cinnamon oil

Polyvinyl alcohol

[62]

1016  Essential Oils The integration of bioactive natural extracts into food polymeric materials is one of the most recent research trends. Plant extracts, in particular, have been found to provide some physicochemical modifications to films/materials, potentially extending their overall applications [56, 57]. Cinnamon, clove, and star anise extracts were added to a partly hydrolyzed gelatin film to enhance its tensile strength and water vapor permeability [5]. In another hand, many studies have examined the use of various essential oils in food packaging and their effects on the packaging’s and food’s properties. The addition of essential oils to different types of polymers improves the film’s physical and chemical properties in several ways. When essential oils are added to a biocomposite film during the casting process, the mechanical strength is reduced [58, 59]. However, a biocomposite film designed definitely from polylactic (PLA) matrix demonstrates the opposite [60, 61]. Incorporating essential oils into synthetic polymers such as low density polyethylene (LDPE) and polyvinyl alcohol (PVA) results in no significant improvements in tensile strength or elongation at breaks, although it does reduce young’s modulus due to increased crystallinity in the film [62, 63]. While, the water vapor permeability (WVP) of biocomposite film does not change significantly when essential oils are added or a marginal rise [64]. Despite this, the physical properties of most food packaging do not change significantly after adding essential oils. Citronella oil, cinnamon oil, and thyme oil were among the most commonly used essential oils in insect repellent tests [65]. Attributable to their aromatic properties, essential oils are frequently used as insecticidal agents to prevent insects, protecting stored goods in various ways (Figure 43.3). The mixture excellently replaces conventional insecticides thus causing no harm to the environment. In spite of the fact that it is environmentally friendly and non-toxic, their oily and volatile properties make them unsuitable for use in food packaging [66] and complex chemical compositions that can modify the constituent in food packaging, only certain essential oils may be integrated into food packaging. In another interesting work, polylactic acid-based films incorporated with different natural substances to improve the mechanical properties such as thymol [67], Marigold flower extract [68], α-tocopherol resveratrol [69] have been developed to increase the antimicrobial and/or antioxidant properties of food packaging for perishable products.

Power

Sticker

Gel Wood

Forms of Insects Repellent

Food package

Fabric

Lotion

Figure 43.3  Functional application forms of essential oils.

Fume

Essential Oils Perspectives and Limitations  1017 Researchers have developed starch-based films containing extracts of red cabbage [70], oregano [71], rosemary [72], etc., aiming of getting better the antioxidant and/or antimicrobial properties of food packaging. The research cited above reports that natural extracts of bioactive plants may be a promising ingredient for improving the physicochemical properties and/or bioactivity of films for a variety of application.

43.5.2 Advantages Because of their medicinal properties, natural-source drugs have seen a significant increase in demand throughout time, organoleptic and fragrance properties. Essences and extracts prepared from plants can be used in fields as diverse as perfumery, food processing, aromatherapy, pharmacy, phytotherapy and natural cosmetics. During storage, many foods must be safeguarded from microbial deterioration and lipid oxidation. Research is underway to improve product quality and protection to meet the needs of consumers for nutritious natural products that do not use chemical additives, but on the other hand, they have excellent nutritional value. It is required to maintain sensory properties and, most importantly, to control the pathogens that cause food poisoning [15]. Essential oils, which are used as additives, arose from an increasing trend towards using natural preservatives instead of synthetic ones. Essential oil research is particularly interesting in this regard because of their diverse bioactive properties [84]. Generally speaking, natural substances such as essential oils and its derivatives with no substantial medical or environmental effects may potentially be useful antibacterial or antifungal alternatives [85]. The action of many essential oils against both significant human pathogenic microorganisms is discussed in this context [86, 87], as well as microorganisms that source of food spoilage, have also been investigated [20, 88]. The antibacterial effect of essential oils is typically attributed to the tiny terpenoids and phenolic chemicals found in them [89], Despite evidence that tiny components have a significant impact, owing to synergy [90]. Interestingly, owing to its remarkable properties including, antibacterial, antioxidant, antifungal, aside from its ability to repel insects, most essential oils derived from various natural aromatic plants have been discovered to be able to be integrated into packaging, allowing for multifunction’s known as “active or smart packaging”. Essential oils have the potential to change the matrix of packaging materials, resulting in better plastic properties. Another benefit of essential oils is that they have a pleasant scent. Herbs are used in cooking because of their taste and fragrance. Many botanical insecticides, such as thyme, cinnamon, and peppermint oil, are derived from the same plants that we use in our favorite dishes. Their friendly fragrance is all natural and won’t leave you with a chemically scented aftertaste. Worms, on the other hand, would not like the smell. This is ideal for keeping pests out of your home and yard [91]. Residual repellency is provided by some essential oils, such as botanical insecticides; these residues are not harmful to humans or pets. When compared to synthetic chemicals, plant essential oils have a low toxicity. Despite the fact that they are highly effective against bugs, you won’t have to worry about coming into contact with them [92]. Correspondingly, most of essential oils are better for the environment because they don’t leave any toxic residues. These unique products contain natural ingredients that will not

1018  Essential Oils pollute the atmosphere. Unlike synthetic chemicals, they won’t remain in the climate. When long-term security is required, products can be reapplied when needed [93]. In addition, antioxidant properties are found in many essential oils. Antioxidants help to protect cells from free radical damage. This harm can lead to serious illnesses like cancer. Researchers are looking into how adding essential oils to food will help us consume more antioxidants while also extending the shelf life of our food [94]. To resume, essential oils come in a wide range of varieties. Some plants are prized for their pleasant scent. Others claim that they have potent healing powers. However, their efficacy can have negative side effects that you should be aware of. Figure 43.4 below summarizes the most advantages of these natural products on human health.

43.5.3 Limitations As mentioned earlier, the use and application of smart packaging technologies has many advantages. However, there are some drawbacks. One of them is that the effects of migration, protection and quality of nanomaterials, and the theory of migration are unknown.

Stress reduction

Fungal infections Sleep aid

Essential oils benefits

Health benefits

Disease Prevent

Lower toxicity

Figure 43.4  Advantages of essential oils.

Essential Oils Perspectives and Limitations  1019 There are both advantages and disadvantages to using essential oils in the manufacture of packages. Essential oils can be used as an alternative to standard chemicals that are not environmentally friendly, as well as the costly and time-consuming analytical methods that are commonly used to track a packaged food product’s environment in the supply chain. These packaging methods, conversely, face challenges such as system hardness and high manufacturing costs, low robustness and sensitivity, compliance with strict legislation and food safety [95]. Additionally, the toxicity of essential oils might also affect their use in food packaging industry and increase its limitation in this field. The toxicity of essential oil varies according to their composition, which itself varies according to the source plant, which itself may vary according to the land where it is grown. For example, the leaf essential oil of Silvia officinalis L is richer in toxic thujone in Estonia than in the rest of Europe [17]. Cinnamon, citrus, cloves, lemongrass, coriander, oregano, sage, chili, thyme, and rosemary are the most commonly used spices in the food business, with a tiny amount of essential oils allowed in meals [96]. Consumption many natural compounds can cause severe toxicity issues. It’s crucial to strike a balance between the essential oil’s actual amount and the danger of toxicity [74]. Carvacrol, carvone, cinnamaldehyde, citral, p-cymene, eugenol, limonene, menthol, and thymol are chemical components present in essential oils that are registered and recognized as healthy agents in the European Union (EU) countries. On the other hand, estragole and methyl eugenol have been removed from the safety list [91]. Essential oils are not without danger, even though they come from nature and have been used for centuries [52]. Since improper use can result in severe side effects or even poisoning, they must be used and stored in accordance with the manufacturer’s recommendations. Children and pets should not be exposed to essential oils since their bodies cannot absorb the same dosages as adults. Furthermore, someone who is pregnant should seek medical advice before using essential oils.

43.6 Conclusion and Future Perspectives Because of the consumer demand for natural goods and safe food, as well as the introduction of increasingly stringent requirements to avoid foodborne infectious disease, food security, quality maintenance, and protection are becoming increasingly important issues in the food industry. A requirement for the future is the implementation of studies and the application of active packaging with the potential to increase food safety. More research is required on the inclusion and preservation of natural additives in food packaging films, as well as assessments of all food safety implications. For example, essential oils are GRAS compounds that have been discovered to have antibacterial and antioxidant properties, making it an excellent candidate for use in the food industry. Additionally, essential oils can replace or remove synthetic additives. Thus, salad, beef, chicken, fish, and vegetables all displayed positive protection when essential oils were used as additives. Based on the findings of this chapter, it is important to develop an interdisciplinary strategy that brings together professionals from a range of biotechnology sectors, including microbiology, food technology, engineering, and materials science, to provide the use of essential oils and natural plant extracts in food packaging a bright future.

1020  Essential Oils

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Essential Oils Perspectives and Limitations  1023 64. Zhang, Y., Ma, Q., Critzer, F., Davidson, P.M., Zhong, Q., Physical and antibacterial properties of alginate films containing cinnamon bark oil and soybean oil. LWT - Food Sci. Technol., 64, 423, 2015. 65. Yahyaoui, M., Gordobil, O., Herrera Díaz, R., Abderrabba, M., Labidi, J., Development of novel antimicrobial films based on poly(lactic acid) and essential oils. REACT, 109, 1, 2016. 66. Anese, M., Bot, F., Panozzo, A., Mirolo, G., Lippe, G., Effect of ultrasound treatment, oil addition and storage time on lycopene stability and in vitro bioaccessibility of tomato pulp. Food Chem., 172, 685, 2015. 67. Ramos, M., Jiménez, A., Peltzer, M., Garrigós, M.C., Development of novel nano-biocomposite antioxidant films based on poly (lactic acid) and thymol for active packaging. Food Chem., 162, 149, 2014. 68. Samsudin, H., Soto-Valdez, H., Auras, R., Poly(lactic acid) film incorporated with marigold flower extract (Tagetes erecta) intended for fatty-food application. Food Control, 46, 55, 2014. 69. Hwang, S.W., Shim, K., Selke, E.M., Soto-valdez, H., Matuana, L., Auras, R., Poly (L-lactic acid) with added α-tocopherol and resveratrol: Optical, physical, thermal and mechanical properties. Polym. Int., 61, 418, 2012. 70. Chang-bravo, L., López-córdoba, A., Martino, M., Biopolymeric matrices made of carrageenan and corn starch for the antioxidant extracts delivery of Cuban red propolis and yerba mate. React. Funct. Polym., 85, 11, 2014. 71. Goyanes, S. and Bernal, C., Biofilms based on cassava starch containing extract of yerba mate as antioxidant and plasticizer. Starch/Stärke, 67, 780, 2015. 72. Piñeros-Hernandez, D., Medina-Jaramillo, C., López-Córdoba, A., Goyanes, S., Edible cassava starch films carrying rosemary antioxidant extracts for potential use as active food packaging. Food Hydrocolloids, 63, 488, 2016. 73. Muriel-Galet, V., Cran, M.J., Bigger, S.W., Hernández-Muñoz, P., Gavara, R., Antioxidant and antimicrobial properties of ethylene vinyl alcohol copolymer films based on the release of oregano essential oil and green tea extract components. J. Food Eng., 149, 9, 2014. 74. Sánchez-González, L., Cháfer, M., Chiralt, A., González-Martínez, C., Physical properties of edible chitosan films containing bergamot essential oil and their inhibitory action on Penicillium italicum. Carbohydr. Polym., 82, 277, 2010. 75. Arfat, Y.A., Ahmed, J., Ejaz, M., Mullah, M., Polylactide/graphene oxide nanosheets/clove essential oil composite films for potential food packaging applications. Int. J. Biol. Macromol., 107, 194, 2017. 76. Wang, L. and Rhim, J., Grapefruit seed extract incorporated antimicrobial LDPE and PLA films: Effect of type of polymer matrix. LWT - Food Sci. Technol., 74, 338, 2016. 77. Ha, J.-U., Kim, Y.-M., Lee, D.-S., Multilayered antimicrobial polyethylene films applied to the packaging of ground beef. Packag. Technol. Sci., 14, 55, 2001. 78. Lim, G., Jang, S., Song, K.B., Physical and antimicrobial properties of Gelidium corneum/ nano-clay composite film containing grapefruit seed extract or thymol. J. Food Eng., 98, 415, 2010. 79. Bravo, L., Alemán, A., Montero, P., Antioxidant properties of tuna-skin and bovine-hide gelatin films induced by the addition of oregano and rosemary extracts. Food Chem., 112, 18, 2009. 80. Jiang, Y., Lan, W., Sameen, D.E., Ahmed, S., Qin, W., Zhang, Q., Chen, H., Dai, J., He, L., Liu, Y., Preparation and characterization of grass carp collagen-chitosan-lemon essential oil composite films for application as food packaging. Int. J. Biol. Macromol., 160, 340, 2020. 81. Tang, Y., Zhou, Y., Lan, X., Huang, D., Luo, T., Ji, J., Wang, W., Electrospungelatin nanofibers encapsulated with peppermint and chamomile essential oils as potential edible packaging. Biofuels Biobased Mater., 67, 2227, 2019.

1024  Essential Oils 82. Maria, E., Alexandre, C., Lourenço, R.V., Mônica, A., Barbosa, Q., Cristina, I., Moraes, F., José, P., Gelatin-based films reinforced with montmorillonite and activated with nanoemulsion of ginger essential oil for food packaging applications. Food Packag. Shelf Life, 10, 87, 2016. 83. Wen, P., Zhu, D., Wu, H., Zong, M., Jing, Y., Encapsulation of cinnamon essential oil in electrospun nanofibrous film for active food packaging. Food Control, 59, 366, 2016. 84. Sanuja, S., Agalya, A., Umapathy, M.J., Synthesis and characterization of zinc oxide – neem oil – chitosan bionanocomposite for food packaging application. Int. J. Biol. Macromol., 74, 76, 2015. 85. Rodr, A., Batlle, R., Ner, C., The use of natural essential oils as antimicrobial solutions in paper packaging. Part II. Prog. Org. Coat., 60, 33, 2007. 86. Hammer, K.A., Carson, C.F., Riley, T.V., Antimicrobial activity of essential oils and other plant extracts. J. Appl. Microbiol., 86, 985, 1999. 87. Cox, S.D., Mann, C.M., Markham, J.L., Bell, H.C., Gustafson, J.E., Warmington, J.R., Wyllie, S.G., The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). J. Appl. Microbiol., 88, 170, 2000. 88. Benkeblia, N., Antimicrobial activity of essential oil extracts of various onions (Allium cepa) and garlic (Allium sativum). Lebensm. Wiss. Technol., 37, 263, 2004. 89. Helander, I.M., Alakomi, H.-L., Latva-Kala, K., Mattila-Sandholm, T., Pol, I., Smid, E.J., von Wright, A., Characterization of the action of selected essential oil components on gram-­ negative bacteria. J. Agric. Food Chem., 8561, 3590, 1998. 90. Nchez, Ä., Amo, R., Atlle, Ä.N.B., Vapor-phase activities of cinnamon , thyme , and oregano essential oils and key constituents against foodborne microorganisms. J. Agric. Food Chem., 55, 4348, 2007. 91. Demyttenaere, J.C., Recent EU legislation on flavors and fragrances and its impact on essential oils, J.C. Demyttenaere, (Ed.), CRC Prress Taylor and Francis Group, Boca Raton, 2010. 92. Isman, M.B., A renaissance forbotanical insecticides? Pest Manage. Sci., 71, 1587, 2015. 93. Alizadeh-sani, M., Mohammadian, E., Julian, D., Eco-friendly active packaging consisting of nanostructured biopolymer matrix reinforced with TiO 2 and essential oil: Application for preservation of refrigerated meat. Food Chem., 322, 126782, 2020. 94. Blowman, K., Magalhães, M., Lemos, M.F.L., Anticancer properties of essential oils and other natural products. Evidence-Based Complementary and Alternative Medicine, 2018, 1, 2018. 95. Speranza, B. and Corbo, M.R., Essential oils for preserving perishable foods: Possibilities and limitations, in: Application of Alternative Food-Preservation Technologies to Enhance Food Safety and Stability, A. Bevilacqua, M.R. Corbo, S. Mi (Eds.), pp. 35–37, Bentham Books, Singapore, 2010. 96. Tajkarimi, M.M., Ibrahim, S.A., Cliver, D.O., Antimicrobial herb and spice compounds in food. Food Control, 21, 1199, 2010.

Index α-humulene, 241 α-pinene, 232, 242, 445 α-terpinene, 445, 935 α-terpineol, 241 β- pinene, 8, 13 β-bisabolene, 233 β-Caryophyllene, 13, 16–18, 23, 241 β-Cedrene, 241 β-elemene, 237 β-Eudesmol, 13 β-myrcene, 232 β-phellandrene, 248 β-pinenes, 242 β-Santalol, 14–15 γ-aminobutyric acid, 448 γ-eudesmol, 241 Γ-tepinyl acetate, 345 ρ-Cymene, 3, 13, 15 1,8-cineol, 9, 13, 16, 19, 23, 242, 247, 445, 447, 1039 3-β-friedelanol, 245 A. millefolium, 451 A. tumefaciens, 451 A. vitis, 451 Abdominal, 215, 216 Acetoxylation, 177 Acetylcholine, 448 Acetyl-Co-A, 239 Achillea biebersteinii, 451, 453 Achillea gypsicola, 453 Acid, 211, 214 Acinetobacter, 211 Action mechanisms, 318 Active packaging, 322, 1036, 1040 Active packaging system, 54 Acyclovir-resistant, 14, 17, 21

Adenovirus-3, 12 Adriamycin resistant cells, 238 Adsorbent porous materials, 759 Adsorption, 209, 221 Adsorption separation, 753 Advantages, 455 Aedes aegypti, 428, 449 Aerogels, 759 Aerotheleums, 977 Aflatoxin, 212 Agar dilution, 251 Aging, 208, 209, 213, 214 Agriculture, 207–209, 219, 220, 223 Alcohol, 32, 345, 346, 349 Aldehyde, 34, 345, 346 Algae essential oil production, 553 Algal essential oils, 552, 553 Algal essential oils in beach-odor, 557 Algal essential oils in off-odor, 557 Alkaloids, 230 Alkanes, alkenes and benzenoids, 188 Alkoxylation, 177 Allergic, 213, 214, 222 Alloxan, 253 Almond oil, 966 Alpha-bisabolol, 877 Ambrosia artemisiifolia, 452 American bollworm, 428 Amides, 346 Amines, 346 Amomum, 349 Amphene, 345 Analgesic, 126, 127, 130, 131, 133–135, 138, 207, 209, 217, 346 Ancient bollworm, 438 Anesthetic, 127, 138, 147, 207, 346 Anethole, 432 Angelica EO, 453, 455 Animals, 210, 211, 213, 219, 220

1025

1026  Index Anti-aging potential, 566 Anti-avian influenza A, (H9N2) 21 Anti-cancer, 996 Anti-cancerous, 430 Anti-dandruff, 59 Anti-diabetic agents, 528 Anti-diabetic assay, 532, 535 Anti-fungal, 430 Anti-inflammatory, 3, 32, 59, 127, 131, 139–141, 147, 207, 213, 217, 346, 1008, 1020–1022, 1026 Anti-inflammatory activity, 368 Anti-inflammatory agents, 526 Anti-inflammatory assay, 533 Anti-lice, 59 Anti-melanogenic, 208, 213, 214 Anti-proliferative, 346 Anti-quorum sensing, 349 Anti-tumorigenic effect, 198 Anti-viral mechanism, 3, 21 Antibacterial, 254, 340, 343, 346, 349, 350, 352, 1042 Antibacterial activities of algal essential oils, 558 Antibacterial activity, 370, 429 Antibacterial agent, 57 Antibacterial agents, 524 Antibacterial assay, 529 Antibacterial effects, 34 Antibacterial mechanism, 349 Antibiofilm, 207, 211, 339, 340, 346–353 Antibiotic, 339, 340, 342–344, 350, 351, 353 Antibiotic resistance, 339, 343, 344, 429 Antibiotics, 211, 217 Anticancer 3, 235 Anticancer agent, 57 Anticancer agents, 526 Anticancer assay, 532, 535 Antidermatophytic assay, 533 Antidiabetic, 253 Antifeedant activity, 432 Antifungal, 139, 141, 142, 248, 276, 283, 340, 347, 348, 1014, 1042 Antifungal activities, 371, 558 Antifungal agents, 525 Antifungal and anti-oomycete properties, 450 Antifungal assay, 530, 533 Antihistaminic, 434 Antiinflammatory, 239 Antimicrobial, 3, 139, 141–143, 147, 148, 207–211, 213, 218, 219, 221, 339, 340, 342–346, 349–353, 420, 698, 1003, 1010–1011

Antimicrobial action, 291 Antimicrobial activity, 275, 317 Antimicrobial assay, 529, 532 Antimicrobial efficacy, 984 Antimicrobial purpose, 291 Antioxidant, 59, 139, 141, 144, 145, 147, 207–222, 277, 346, 435, 699, 1039 Antioxidant activity, 369 Antioxidant assay, 530, 534 Antioxidant effects, 32 Antioxidant potential, 469, 504 Antioxidants, 526 Antiparasitic ,127, 128, 133, 135 Antiproliferative, 141, 143, 144 Antiseizure and anticonvulsant agent, 195 Antisolvent separation, 754 Antispasmodic, 126, 139 Antispasmodics, 528 Antiviral, 34, 243, 340, 1015–1017 Antiviral activity, 103, 371 Antiviral agent, 58 Antiviral, antimicrobial, antifungal activities, 186 Anxiety, 215–217 Apiaceae family, 434 Apigenin, 194 Apiol, 345 Apoptosis, 235 Apparent solubility, 730 Arbovirus, 434 Aroma, 207–211 Aromatherapy, 56, 116, 190, 207–210, 214–217, 223, 565, 770, 919, 960, 1039 Aromatherapy, 933 Aromatic, 502 Aromatic compounds, 186 Aromatic oils, 229 Aromatic plants, 316 Aromatic shrub, 437 Artemisia multiflora, 10 Artificial neural network, 700 Ascaridol, 345 Aspergillus, 212 Aspergillus flavus, 429 Aspergillus sp., 452 Asthma, 130, 132, 134, 135, 139, 141, 144, 191 Atropine, 230 Attention, 428 Avian, 989 Avocado oil, 967

Index  1027 Ayapana triplinervis, 12 Ayurvedic, 979 B. nigrifluens, 450–451 Baccharis dracinculifolia, 494 Bacillus thuringiensis, 382, 407 Bacterial resistance, 339, 340, 342–344 Bactericidal properties, 450 Bakuchiol, 875 Basil, 345, 347 Bay, 346 Benzene, 434 Benzoic acid, 876 Benzyl alcohol, 345 Bergamot, 214, 221, 345, 348, 940 Bergamot EO, 455 Beta carotene, 510 Betulinic acid, 244 Beverages, 207–211 Bioactive, 208–210, 212 Bioactive compounds, 298, 604, 614 Bioactive constituents, 32 Bioactive films added EO, 304 Bioactivities, 280 Biochemical and physiological dysfunction, 449 Biodegradable, 208, 212, 219 Biodegradable packaging, 197 Biofertilizer, 166 Biofilm, 339–353, 1012 Biofilm formation, 340, 341, 344 Biological, 207, 215 Biological activities, 466, 635, 636, 637 Biological activities of essential oils, 523 Biological activity, 302, 588 Biological applications, 364 Biological effects, 982 Biological potential, 503 Biopesticide, 60, 166, 210, 219, 443, 444, 447, 458 Biotechnologic, 218, 219 Biotic stress, 428 Bisabolol, 345 Bisabolol oxide, 345 Bisabolone oxide, 345 Body, 213, 215, 222 Borneol, 345 Borneol and eucalyptol, 193 Bornyl acetate, 345 Bovine, 987

Bovine herpes, 14 Brain, 215 Broad spectrum, 429 Brochothrix, 211 Bunium persicum, 434 Butylhydroxytoluene, 991 C. aurantium L, 451 C. michiganensis, 450–451 C. scolymus, 451 Cadalene, 241 Cadinene, 345 Caffeine, 230 Calamenene, 433 Camphor, 3, 14, 222, 345, 346, 448–449, 903, 1038 Camphor oil, 970 Camphor tree, 433 Campylobacter, 211 Cancer, 143, 145, 146, 148, 210, 213, 215, 216, 365 Candida, 348 Candida albicans, 212, 219, 221 Canola oil, 968 Capsaicin, 875 Capsid disintegration, 6–7 Captothecin, 235 Caraway, 210 Carbon dioxide (CO2), 628 Carbon units, 188 Cardiovascular diseases, 367 Cardiovascular disorders, 924 Carom seeds, 431 Carotenoids, 34 Carrageenan, 995 Carvacrol, 3, 13–17, 210, 221, 235, 322, 345, 346, 348–351, 1011–1013, 1016, 1019–1021 Carveol, 345 Carvone, 242, 345, 349, 1038 Caryophyllene, 432 Caryophyllene oxide, 241 Cedral, 248 Cedrene, 345 Cedrol, 345 Cedryl acetate, 345 Cells, 215, 220 Cellulose, 212, 219 Chamazulene, 345 Chamomile, 345, 938

1028  Index Chavicol, 231 Chemical composition, 51, 317 Chemical constituents, 429 Chemical nature, 796 Chemical setup, 980 Chemotherapeutic agents, 511 Chikungunya, 219, 431 Chitosan, 197, 212, 218, 219, 220 Cineol, 345 Cinnamaldehyde, 210, 214, 231, 322, 346, 349, 350 Cinnamic Acid, 875 Cinnamic aldehyde, 345 Cinnamomum, 214 Cinnamomum zeylanicum, 11, 13, 15 Cinnamon, 210–212, 214, 221, 222, 345, 346, 348, 1005, 1008, 1012, 1015, 1018, 1021–1022, 1024, 1043 Cinnamon EO, 323, 445, 452 Cinnamon essential oil, 197 Cinnamon oil, 231, 922 Cinnamyl alcohol, 234 Cinnamylacetate, 240 Citral, 345, 904 Citronella, 210, 212, 221, 223, 902, 931, 1015, 1025–1026 Citronella, EO 445, 447, 454 Citronella oil, 348 Citronellal, 234, 345, 432, 1039 Citronellol, 211, 345, 347 Citronellyl acetate, 345 Citrus, 345, 348, 487, 1011, 1015, 1023, 1025 Citrus bergamia, 8, 10, 15 Clary sage, 345 Classical methods, 579 Clinical, 208, 210, 215–219 Clinical practice, 960 Clove, 210–212, 214, 222, 223, 345–347, 349, 431 Clove EO, 445, 452, 454 Clove essential, 323 Clove oil/eugenol oil, 895 Coacervation process, 623 Coating materials, 620 Coconut oil, 968 Cohobation, 847 Cold pressing, 231, 576, 772, 887, 906 Cold pressing method/expression, 844 Color, 211–213, 219 Complexation, 605

Composition, 583, 585, 589, 591 Concentration of oil, 302 Conclusion, 559 Contamination, 208–210, 212, 213, 219 Controlled release, 610 Conventional, 583, 592, 593, 595 Conventional extraction, 52 Core element, 980 Coriander, 211 Coriandrum, 492 Coronavirus, 243, 1016–1017 Cosmetic, 207–209, 213, 214, 223, 438 Cosmetic aromatherapy, 917 Cosmetic industry application, 190 Cosmetics, 957 Cosolvent, 746 Cosolvent addition, 713 Cost of manufacture, 678 Cotton, 220, 221, 439 Coxsackie virus, 11, 15 Crystal violet, 352 Cumin, 230 Cuminaldehyde, 434 Cuminic aldehyde, 345 Curcuma oil, 348 Curing illness, 979 Current constraints, 455 Cyclodextrin complexes, 276 Cyclooxygenase, 240 Cymbopogon flexuosus, 11 Cymbopogon nardus, 8, 11, 16 Cytokine, 239 Cytopathogenic reduction assay, 9 Cytotoxicity, 140, 145, 147, 148 D-Limonene, 445, 455 D. solani, 451 Dairy products, 324 Damage, 210, 220 Damascus, 216 Dandruff, 371 Degradation, 212 Dengue virus, 8, 12, 14, 16 Dental, 209, 217, 218, 917, 924, 925 Dental applications of essential oils, 895 Dentistry, 892 Depression, 209, 215, 216 Dermatological problems, 947 Diabetes, 208, 209, 217, 368

Index  1029 Dillapiole, 240 Dimethyl allyl diphosphate (DMAPP), 239 Dipentene, 345 Disc diffusion, 251 Diseases, 208, 213, 215, 217 Disk-diffusion method, 295 Disorder, 208, 209, 214, 215, 217 Disruption, 985 Distillation method, 710 Diterpenes, 346 DNA, 220 Dose dependent, 437 Doxorubicin, 238 DPPH scavenging assay, 506 Drought, 69, 71, 72, 81, 82, 91 Drug, 209, 211, 215, 217, 218 Drug delivery, 22 Dysmenorrhea, 216 Dysphania ambrosioides, 11, 15 E. africanus L, 451 E. amylovora, 451 E. carotovora, 451 Ecological importance, 470 Ecological importance of algal essential oils, 554 Ecological-niches, 2 Economic, 212, 214 Economic analysis, 733 Economical crops, 428 Ecosystem, 551 Edible essential oil, 964 Edible films with EO, 303 Efficacy of essential oils to insects, 447 Egg hatching, 436 Electrospinning material, 198 Emotional, 209, 215 Emulsification, 606, 626, 643, 644, 648 Encapsulation, 277, 281, 351, 636, 637 Endocrine, 214, 222 Enfleurage, 803 Enfleurage method, 879 Enhance, 996 Enterobacter, 211, 344, 350, 351 Enterococcus faecalis (E. Faecalis), 344, 348, 351 Entrainer, 746 Enveloped, 989 Environment, 210, 215, 216, 219 Enzyme, 213, 214, 217 Enzyme-assisted SCFE, 786

Epi-taraxerol, 244 Epirubucin, 238 EPS synthesis, 341 Eradication concentration, 352 Escherichia coli, 211, 212, 220, 221, 344, 346–350, 513 Essenal oils odorants, 486 Essential oil, 270, 272, 276, 427, 637, 645, 1036 Essential oil algae, 467 Essential oil biopesticides, 447 Essential oil extraction, 847 Essential oil safety issue, 372 Essential oil-based therapies, 942 Essential oil-based therapy, 933 Essential oil(s) – EO(s), 69–91 Essential oils, 50, 99, 185, 466, 565, 586, 708, 742, 768, 917, 929 Essential oils in packaging, 302 Esters, 345, 899 Eucalyptol, 211, 345, 347 Eucalyptol, α-pinene, 196 Eucalyptus, 210, 214, 217, 221, 222, 345, 347, 349, 383, 387, 389, 396, 397, 401, 402, 404, 408, 409, 410, 411, 429, 902 Eucalyptus bicostata, 11, 15 Eucalyptus caesia, 10, 13 Eucalyptus EO, 445, 454–455 Eucalyptus globulus, 8, 11, 16 Eugenol, 3, 13–16, 18, 210, 222, 231, 345–347, 349, 350, 445–448, 452 Eugenol acetate, 345 Eugenol components, 431 Euphoric, 215 Expectorant, 127, 130, 131, 133 Extracellular polymeric substances (EPS), 339, 340, 341, 342, 343, 352 Extract, 207, 210, 211, 213, 222 Extraction, 978 Extraction kinetic curve, 749 Extraction methods, 52 Extraction of emulsions (SFEE), 629 Extraction of essential oil, 363 Extraction pressure, 749 Extraction process, 747 Extraction temperature, 749 Extraction time, 698 Extraction yield, 293, 715, 724, 749 Extrusion technology, 624

1030  Index Farnesene, 345 Farnesol, 8, 13, 0345 Fatty acids, 34 Fenchone, 345 Fennel, 345 Fir, 345 Flame ionization detector, 578 Flavonoid, 991 Flaxseed oil, 969 Flow rate, 751 Foeniculum, 491 Food, 207–213, 215, 220–223 Food and drug administration (FDA), 32, 211, 212, 215 Food contact surface, 302 Food industry, 37 Food packaging, 319, 1035 Food physicochemical and sensory characteristics, 302 Food preservation, 54 Food preservative, 957 Food products, 322 Food safety, 316, 1036 Food-borne illnesses, 31 Formulations, 208, 209, 213, 214, 219 Fractioning, 754 Frankincense, 345 Free radicals, 213, 214 Friedelane, 244 Fungi, 71, 81, 86, 87, 89, 90, 91 Fungicidal, 209, 219, 220 G. citri-aurantii, 452 GABA neurotransmitter, 448 Garlic, 211, 345, 430 Gas chromatography olfactometry, 478 Gastrointestinal, 208, 213, 215 Gastrointestinal nematode, 437 GC-O data processing methodology, 481 Generally recognized as safe, 32, 672 Genotoxicity, 140, 145–148 Geraniaceae, 348 Geranial, 345, 348, 432 Geraniol, 236, 322, 345, 902, 1038 Geraniol acetate, 345 Geranium, 213, 214, 223, 345, 347 Germacrene, 241 Ginger, 345, 348 Gingerdione, 236

Globulol, 248 Glycoprotein, 7 Glycyrrhizin, 244 Gram negative, 39 Gram-positive, 36 GRAS, 212 GRAS (generally recognized as safe), 330 Green strategy, 32 Groundnut oil, 969 Guaiol, 241 Harmful, 210, 220, 221 Headache, 209, 215 Health, 207, 210, 211, 213–215, 217, 219, 221, 223 Heavy metals, 69, 71, 73, 85, 86, 91 Helicoverpa armigera hubner, 438 Helminthes, 437 Hemagglutinin, 7, 16 Hemiterpenes, 230 Hemodialysis, 217, 972 Hepatitis-B-Virus, 245 HePG2 cells, 235 Herbicidal, 209, 219 Herbicidal/Weedicide properties, 452 Herbs, 427 Herpes simplex virus, 18, 245 Heteroatomic metabolites, 875 High fever, 434 Hinokitiol, 878 Hippocrates, 979 Home care, 957 Horticulture, 61 HSV-1, 7, 9–10, 12–13, 17, 21–22 HSV-2, 10, 12–13, 22 Human, 213, 214, 217, 219, 220 Human head lice, 428 Human health, 439 Human rotavirus, 14 Humulene, 431 Hydration, 177 Hydro distillation, 231, 570, 903, 905, 689, 710, 772, 804, 805, 806, 807, 865, 873, 875, 889 Hydro-diffusion, 566, 772, 803, 865 Hydrocarbons, 34 Hydrophobicity, 984 Hydrostatic pressure, 429 Hyperpigmentation, 208, 209, 214 Hyphal, 987 Hypoglycemia, 217

Index  1031 Illicium verum, 10, 13 Immortelle, 191 Immune, 209, 211, 215, 222, 223 Implant, 209, 217, 218 Indolizine, 236 Indomethacin, 240 Industrial, 207–210, 223 Industrial applications, 54, 756 Infection, 208, 211, 213, 218, 219, 428 Infectious diseases, 369 Inflammation, 209, 213, 222 Influenza A virus, 245 Influenza virus, 7, 9–11, 14–19 Inhalation, 215, 216, 222 Inhibitory, 210, 218 Inhibitory activity, 295 Inhibitory concentrations, 352 Inhibitory effect, 297 Innovative extraction, 53 Insecticidal, 207, 209, 219, 220 Insects, 209, 210, 219, 220 Insect repellents, 449 Insomnia, 209, 215, 216 Integrated pest management (IPM), 450, 456 Interventions, 428 Irritation, 209, 213, 222 Isoborneol, 436, 903 Isobornyl acetate, 345 Isomenthone, 1013 Isopentyl diphosphate (IPP), 239 J. regia L. (shells), 451 Japanese encephalitis, 12 Jasmine, 345 Jaundice, 437 Juniper, 345 Ketoconazole, 251 Ketone, 345, 346 L-bornyl acetate, 241 Lactones, 874 Lamiaceae, 347 Lanadulol, 345 Lapachol, 872 Larvae, 429 Larval growth, 431 Larvicidal, 141, 144, 253, 430 Lauraceae, 347

Laurel oil, 347 Laurus, 211 Lavandula, 346 Lavandula officinalis, 10 Lavender, 212, 214–217, 221, 345, 347, 436, 605 Lavendulyl acetate, 345 Legal aspects, 330 Legumes maize, 427 Lemon, 211, 212, 221, 345 Lemongrass, 345, 432, 902 Lemongrass EO, 447, 452 Leuconostoc, 211 Life cycle assessment, 679, 680 Life cycle of mosquito, 431 Light, 69, 71, 72, 76, 83, 84, 91 Lignins, 230 Limonene, 13, 232, 345, 348, 429 Linalol, 345 Linalol oxide, 345 Linalool, 239, 347, 459, 351, 902, 1038 Linalool or citral, 322 Linalyl acetate, 345 Linolenic acid, 878 Lipid, 208, 210, 212, 219 Lipid peroxide, 239 Lipid-based nanocarriers, 22 Lipopolysaccharide, 241 Lipopoxygenase, 241 Liposomes, 218, 270 Lippia alba, 12 Liquid absorption separation, 754 Listeria, 211 Litopenaeus vanname, 23 Litsea cubeba, 437 Long-term prospects, 455 Lupulone, 875 Luteol, 239 M2 ion channels inhibition, 21 Maceration, 568, 803, 804, 875, 876, 902 Macrodilution, 251 Macrodilution and microdilution methods, 295 Malaria, 427 Malathion, 430 Managing menopause symptoms, 947 Mandarin, 345 Mankind, 427 Maricariarecutta and anthemisnobilis, 911 Marjoram, 230

1032  Index Massage, 215, 216 Massage aromatherapy, 917 Mathematical modeling, 714, 751 Mechanism, 215, 217 Mechanism of action of essential oils, 893 Mechanisms of resistance, 342 Medical, 207–209, 217–219, 223 Medical aromatherapy, 917 Medicinal application, 57 Melaleuca, 346, 347 Melanin, 213, 214 Melissa, 217, 220, 345, 346, 348 Melissa officinalis, 21–22 Melvalonic acid, 239 Membrane permeability, 349 Memory, 209, 215 Menstrual cramps, 193 Menta, 347 Mentha, 126, 127, 129, 134, 135, 139–141, 143–145, 147, 212, 217, 218, 488, 1007, 1012, 1014 Mentha aquatica L., 128, 130 Mentha arvensis, 129–135, 142–146 Mentha canadensis, 132 Mentha citrata, 135 Mentha gentilis, 128 Mentha longifolia Huds, 145 Mentha longifolia L, 145, 146 Mentha mozaffarianii, 145, 146 Mentha piperita L, 126, 128–131, 133–139, 141, 142, 144–148 Mentha piperita var. citrata, 131 Mentha pulegium L, 126, 128, 129, 131–134, 136–139, 143, 145, 147 Mentha rotundifolia, 141, 143 Mentha sativa, 133 Mentha spicata L, 126, 128–133, 135–139, 141, 143–146 Mentha suaveolens, 9–10, 13, 21, 126, 129, 130, 135–139 Mentha sylvestris, 130 Mentha villosa, 128, 129, 131, 135 Mentha viridis, 128, 132 Mentha x villosa Huds., 130, 131, 135 Menthol, 3, 13, 195, 233, 345, 347, 1038 Menthone, 345, 1039 Metabolic anti-viral mechanisms, 9 Metabolites, 316 Metformin, 253

Methicillin-resistant, 986 Methods, 583, 590, 591, 592 Methoprene, 430 Methyl chavicol anethole, 345 Methyl erythritol 4-phosphate (MEP), 239 Methyl nonyl ketone, 345 Mexican oregano, 10, 12, 15–17 Microbes, 60, 429 Microbial contamination, 31 Microbial interactions, 162 Microbicidal action, 299 Microcapsules, 218, 220, 221 Microcrustaceans, 220 Microemulsion, 281 Microencapsulation, 191, 220, 221 Microparticles, 218 Microwave assisted extraction, 758, 773, 908 Migraine, 217 Mint, 126, 127, 138, 144, 211 Mint EO, 445, 450 Miscellaneous, 428 Mode of action, 934 Molecules, 212, 215, 221 Monoterpenes, 3, 7, 17–19, 23, 34, 317, 346, 447–449, 454–455 Morphine, 230 Morphological study, 6 Mosquito antennae hairs, 449 Mosquito bite, 434 Mosquito repellent, 958 Mosquito repellent assay, 537 Mosquitoes, 428 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide), 515 Multi drug resistance, 238 Murine norovirus (MNV), 7, 23 Muscle, 215, 219 Muurolene, 254 Myrcene, 345, 432, 445 Myrcenol, 1012 Myrcenone, 248 Myristicin, 345 Myrrh citronella, 345 Myrtaceae, 347 Myrtle family, 445 Nano-formulated essential oils, 117 Nanocapsules with EO, 303

Index  1033 Nanocarriers, 22 Nanoemulsification, 434 Nanoemulsion, 351, 456–457, 281 Nanogels, 22 Nanoparticle, 351, 212, 218–220, 606, 614 Nanoprecipitation method, 625 Nanosystems, 22 Narcissus, jasmine, 191 Natural, 207, 208, 210, 212–215, 219–223 Natural plants products, 584 Natural source, 300 Natural-based essential oils, 467 Nausea and vomiting, 940 Neem, 383, 392, 398, 413 Nepeta cataria, 433 Neral, 345, 445 Nerol, 345, 902 Neroli, 194 Neuraminidase, 7–8, 16 Nitric oxide, 239 Nitrous oxide synthase, 246 Non-enveloped viruses, 6, 15–16, 23 Non-thermal methods, 196 Non-volatile, 874 Nontherapeutic uses, 957 Nutrients, 71, 77, 82, 84, 85, 87 O. ciliatum, 451 O. heracleoticum, 451 Obesity, 368 Ocimum, 347 Ocimum basilicum, 12 Octopamine, 448, 455 Odor, 208, 210, 214, 215, 221, 477 Odor acvity value (OAV), 481 Odor detecon threshold, 481 Oil pulling, 893 Olfactory aromatherapy, 917 Olfactory aromatherapy and psychoaromatherapy, 938 Ombellulone, 345 Opioids, 215, 216 Optimization, 699 Oraganophosphates, 430 Oral, 209, 214, 218 Oral conditions, 894 Orange, 211, 216, 217, 223, 432 Orange EO, 445 Oregano, 210, 211, 220, 221, 345, 346, 350

Organic compounds, 34 Origanum, 211, 346 Origanum heracleoticum, 451 Origanum majorana, 433 Osmophores, 589 Osmunda regalis, 11, 15 Ovicidal effect, 429 Oxidation, 208–213, 222 Oxidative, 208, 212, 213, 217 Oxidative stress, 501 Oxides, 345, 346 Oxygen, 211–213 Oxygenated compounds, 189 p-cimeno, 179 p-cymene, 241, 350 P-vetivone, 345 Packaging, 207–210, 212, 223, 279 Pain, 208–210, 215–217, 219 Pain and inflammation, 933 Palliative, 208, 215, 216 Palmarosa, 345 Particle diameter, 604 Particles, 606, 607, 610, 611 Particles from gas saturated solutions (PGSS), 628 Parts of plants, 298 Patchouli, 3, 11, 16–19 Patent, 678 Pathogens, 209–211, 219, 221, 300 Pathophysiology, 208, 210 Pathways of EOs’ pesticide action, 455 Patients, 208, 210, 215–218 Pediculus humanus capitis, 435 Pelargonium graveolens, 10 Penetration, 984 Penicillium species, 452 Peppermint, 126, 135, 138, 139, 146, 195, 212, 221, 230, 345, 347, 351 Peppermint EO, 445, 450 Peppermint oil, 896 Perfume, 191 Perfumery, 208–210, 213, 223 Perillaldehyde, 345 Perpetual plant, 434 Persister cell, 342–344 Perspectives, 760 Pest management, 62 Pesticide, 957 PGPF, 164

1034  Index PGPR, 164 Pharmaceutical application, 57, 806, 809–864, 866–872 Pharmacological properties, 198 Pharmacopoeia, 570 Phase diagram, 774 Phase equilibria, 745 Phellandrene, 345 Phenolic compounds, 34, 584 Phenolics, 230 Phenols, 345, 346 Phenylpropane, 231 Phenylpropanoids, 34, 239, 900 Pheromone, 470, 556 Photothermal, 212 Phyto-stimulators, 166 Phytochemical compounds, 23 Phytochemistry, 32, 363 Phytochemistry and sources of EOs, 445 Phytopathogenic bacteria, 451 Phytopathogenic fungi, 450 Pigments, 584 Pimpinella, 222 Pine, 345 Pinene, 188, 345 Pinocamphone, 345 Pinocarvone, 345 Pinus, 493 Piperitenone oxide, 248 Piperitone, 345 Placebo, 216, 217 Planktonic bacteria, 339–343, 352, 353 Plant, 207, 210, 213–217, 219, 220, 222, 223 Plant biomass production, 165 Plant derived, 981 Plant essential oil, 445 Plant extracts, 32 Plant growth promoters, 164 Plasmodium falciparum, 433 Plasmodium vivax, 433 Plastic, 212 Poaceae, 348 Polar modifiers/co-solvants, 697 Polarity of bioactive components, 17 Polyethylene, 1016 Polylactic acid, 1016 Polymerization, 603, 605, 606 Postoperative, 215, 216, 219 Pregnant, 215

Preservation, 208, 212 Pressure, 723, 726 Pressure and temperature assisted separation, 753 Process temperature, 695 Proofreading, 2 Protease, 237 Protectants, 62 Protection, 209, 212, 213, 219 Protein inhibition, 7 Proteus, 211 Pseudomonas, 211 Pseudomonas aeruginosa (P. Aeruginosa), 340, 341, 343, 344, 347, 348, 350 Pseudomonas spp., 451 Psycho-aromatherapy, 917 Psychological disorders, 933 Pulegone, 345 Pulmonary edema, 246 Pyripoxyfen, 430 Quinine, 230 Quorum sensing, 341, 342, 349 R. fascians, 450–451 R. stolonifer, 452 Rapid expansion of supercritical solutions (RESS) technique, 628 Ravensara, 345 Reactive oxygen, 991 Reactive oxygen species (ROS), 213, 214, 220 Ready-to-eat, 199 Reference tool, 977 Release mechanism, 636, 637, 643 Repelent, 141, 144 Replication cycle of virus, 3, 4, 6, 9, 15 Resins, 230 Resistance, 211, 217 Respiratory, 208, 209, 215 Respiratory diseases, 134, 135, 139, 147 Respiratory syncytial virus, 14 Respiratory tract diseases, 367 Response surface methodology, 781 Rhizome, 435 Rose, 345 Rosemary, 211, 221, 223, 345, 346 Rosemary EO, 445, 447, 450 Rosemary oil, 196 Rosewood, 345

Index  1035 Rosmarinus, 211, 219 Rosmarinus officinalis, 8, 10 Rutaceae, 348 S. aromaticum, 451 S. hortensis, 451 S. macrostema, 507 S. scandens, 451 Sabinene, 345, 432 Safe, 208, 210–212, 214, 221, 222 Safe insecticide, 439 Safety, 208, 211, 221 Safety issues, 897 Safety precautions, 972 Saffron, 230 Safrole, 240, 345 Sage, 210, 211 Salinity, 71, 79, 82, 83, 90, 91 Salmonella, 211, 221 Salvia desoleana, 10, 17 SARSCoV-2, 23 Satureja, 210, 220, 348 Satureja hotensis, 10 Savory, 210, 221, 345 Scale-up, 676, 752 Scar, 209, 213 Sclareol, 875 Second-order model, 719 Secondary metabolites, 157–159, 345, 346 Secondary plant metabolisms, 466 Sedative, 127, 133 Self-defense, 978 Semiochemicals, 62 Sensorial characteristics, 299 Sensory, 210, 211 Separation, 753 Sesame oil, 970 Sesquiterpene, 3, 18, 23, 34, 230, 345, 346, 446–447 Sessile cell, 340–343, 352, 353 Shelf life, 278, 291, 1036 Shikonin, 875 Shrubs, 427 Side effects of EOs, 971 Simplified equilibrium model., 721 Skin, 208, 209, 212–214, 217, 219, 222 Skin penetration enhancers, 59 Sleep, 208–210, 215–217 Smart packaging, 1017

Sodium sulphate, 576 Solubility, 745 Solvent extraction, 584, 710, 772, 804, 805, 865–869, 875, 890, 902 Solvent flow, 732 Solvent recycling, 748 Solvent regeneration, 755 Soothing, 126, 131, 132 Sorghum, 427 Sources, 51 Sources of essential oil, 10–12, 362 Sovová model, 716 Soxhlet extraction, 577, 688, 803–805, 869, 873, 903 Spectrophotometer, 512 Spodoptera, 384, 386, 391, 392, 393, 394 Spray drying process, 626 Spruce, 345 Stability, 269 Staphylococcus aureus (S. aureus), 211, 213, 219–221, 344, 346–351 Star anise, 431 Steam and hydro-distillation, 584 Steam distillation, 231, 574, 672, 710, 772, 803, 804, 806–865, 869, 873, 888, 903, 907 Steroids, 230 Sterols, 34 Stimulating skin coolness mediator receptors, 907 Stimulodeterrent diversionary strategy (SDDS), 450 Streptococcus mutans (S. Mutans), 344, 347, 348 Stressors, 2 Subtropical, 434 Sun gold, 192 Sunflower oil, 970 Supercritical anti-solvent (SAS), 629 Supercritical CO2, 671, 672 Supercritical encapsulation techniques, 628 Supercritical fluid, 575 Supercritical fluid extraction, 687, 711, 729, 734, 773, 904, 908 Superoxide dismutase, 239 Suppressing, 987 Swartziadione, 878 Sweet marjoram, 345 Symbiosis, 162 Symptoms, 208, 214, 216, 222 Synergistic effects, 21

1036  Index Synergistic formulations, 454 Synthetic, 207, 210, 212–214, 221, 223, 1005–1006, 1010, 1016–1017 Synthetic insecticide, 435 Synthetic pesticides, 428 Syzygium, 347, 349 Syzygium aromaticum, 434 T-cadinol, 241, 242 T. fallax, 451 Tanacetum aucheranum, 451 Tannins, 230 Tansy, 345 Taste, 208, 211, 213, 215, 218 Tea tree, 212, 217, 218, 221, 222, 436 Tea tree essential oil, 908 Tea tree oil, 347, 349, 900–901 Tegetone, 345 Temperature, 69, 71, 72, 80, 83, 723, 728 Terpene, 2, 7, 15, 17, 34, 230, 345, 346, 348, 349, 446–448, 587–588 Terpinen-4-ol, 347, 445 Terpinene, 345 Terpineol, 345, 1038 Terpinolene, 248, 1039 Teucrium pseudochamaepitys, 11 Textile, 207–209, 220, 221 Therapeutic applications, 522 Therapeutic guidelines, 972 Therapy, 216 Thermal shifts assays, 6 Thermodynamic properties, 744 Thimus, 490 Thin film hydration method, 627 Thujanol, 1012 Thujone, 345, 1038 Thyme, 210–212, 219–221, 223, 230, 345, 346, 1007, 1012, 1014, 1018–1021, 1025 Thyme EO, 445, 450, 452 Thyme, mint, mustard, 191 Thymohydroquinone dimethyl ether, 14 Thymol, 3, 9, 13–14, 16–17, 233, 322, 345, 346, 347, 349, 350, 432 Thymoquinones, 235 Thymus, 346 Thymus capitatus, 10–12 Time of addition assay, 4 Time-of-addition, 989 Tinalool, 1013

Tomato, 427 Topical, 215, 217, 222 Toxic, 208, 210, 212, 218, 220–223 Toxic effects of essential oils, 454 Toxicity, 141, 144, 145, 147, 148 Trachyspermum ammi, 12 Trans-anethole, 13 Transdermal therapeutics, 59 Treatment, 209, 210, 213, 215, 217–219 Treatment of dementia, 921 Trichomes, 589 Turmeric, 211, 212, 220, 610, 672 Turmerone, 348, 676 Turpentine, 874 Ultrasonication-assisted extraction, 883 Ultrasound extraction, 878 Ultrasound-assisted extraction, 578, 759, 773 Ultrasound-assisted SCFE, 786 Ultraviolet radiation, 429 Unprocessed, 978 US FDA, 196 Vanilla, 211, 214 Vetiver, 345 Vetiverol, 345 Vincristine, 235 Viral attachment assay, 6 Viral disease, 431 Viral fusion assay, 6 Viral inhibitory activity, 105 Viral replication, 102 Viral-RNA, 6 Viridiflorene,   Virus, 219, 221 Virus infects and their forms, 1–2 Volatile chemical compounds, 467 Volatile oil, 989 Wall material, 642 Water vapor permeability, 1016 Waxes, 34 Weeds, 452, 453, 455, 457 West nile virus, 20 White spot syndrome virus, 23 Wintergreen oil, 971 Worldwide, 427 Wound, 218, 219, 221

Index  1037 Xanthomonas spp., 450–451 Yarrow, 345 Yellow fever virus, 12, 16, 19 Ylang–ylang, 345 Z-ligustilide, 254 Zataria multiflora, 10, 20

Zika infection, 431 Zika virus, 12, 14, 16 Zingiberaceae, 348, 435 Zingiberene, 348

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