Nutraceuticals and Health Care 9780323897792, 0323897797

Table of contents :
Front Cover
Nutraceuticals and Health Care
Nutraceuticals and Health Care
Copyright
Dedication
Contents
Contributors
Preface
1 - Nutraceutical-A deep and profound concept
1.1 Introduction
1.2 Facts of nutraceuticals
1.2.1 Growth
1.2.2 Health maintenance
1.2.2.1 Immune function
1.2.2.1.1 Gastral well-being
1.2.2.2 Mental health
1.2.2.3 Aging
1.2.2.4 Physical performance
1.2.3 Effect on chronic ailments (e.g., Heart disease, diabetes and metabolic disease, musculoskeletal disease)
1.2.4 Musculoskeletal disease
1.3 Fiction of nutraceuticals
1.3.1 Supplements are meant for men and women
1.3.1.1 No need of supplements with balanced diet intake
1.3.1.2 Protein burden on kidneys
1.3.1.3 Undeclared nutraceutical ingredients are not safe
1.3.1.4 Antioxidants prevent cancer
1.3.1.5 Zn for curing cold symptoms
1.3.1.6 Prevention of bone fractures in older age by calcium supplements
1.3.1.7 Nutraceuticals are as good as diet
1.4 Developmental strategy for nutraceuticals
1.5 Disease prevention claims of nutraceuticals
1.6 Sources of nutraceuticals and chemical nature
1.7 Plant food by-products as source of nutraceuticals
1.8 Nutraceutical profile of prominent nutraceuticals
1.9 Mechanism of action of nutraceuticals
1.10 Nutraceuticals in ayurveda
1.11 Recent trends in nutraceuticals
1.12 Recent developments in nutraceuticals
1.13 Patents on nutraceuticals
1.13.1 Berberine
1.13.2 Beta glucan
1.13.3 Gamma oryzanol
1.13.4 Lignans
1.13.5 Curcumin
1.14 Regulatory aspect of nutraceuticals
1.15 Current status
1.16 Indian and global nutraceutical market size
1.17 Establishment of nutraceuticals in market
1.17.1 Demands of nutraceuticals in market
1.17.2 Factors affecting future market
1.17.3 Steps for market development of nutraceuticals
1.18 Marketing barriers of nutraceuticals
1.19 Status of nutraceuticals and future prospects
References
Further readings
2 - Cereal proteins
2.1 Introduction
2.2 Cereal grains
2.2.1 Rice
2.2.2 Wheat
2.2.3 Barley
2.2.4 Oats
2.2.5 Sorghum
2.2.6 Rye
2.2.7 Maize
2.2.8 Millet
2.2.9 Other cereals
2.3 Nutritional profile of cereals
2.3.1 Macronutrients
2.3.1.1 Carbohydrate
2.3.1.2 Proteins
2.3.1.3 Lipids
2.3.2 Micronutrients
2.3.2.1 Minerals
2.3.2.2 Vitamins
2.3.3 Non-starch polysaccharides
2.4 Phytochemicals
2.5 Antinutrients
2.6 Classification and chemistry of cereal proteins
2.6.1 Cereal storage proteins
2.6.1.1 Storage prolamins
2.6.1.1.1 Prolamins from Triticeae (wheat, barley, and rye)
2.6.1.1.2 Prolamins from maize
2.6.1.1.3 Prolamins from oats, rice, and sorghum
2.6.1.2 Storage globulins and glutelins
2.6.1.3 Other proteins
2.6.1.3.1 Hydrolytic enzymes and their inhibitors
2.6.1.3.2 Starch granule proteins
2.6.1.3.3 Antimicrobial and cysteine-rich proteins
2.7 Extraction and characterization techniques
2.7.1 Extraction techniques
2.7.1.1 General method for whole seed protein extraction
2.7.1.1.1 Solubilization after precipitation
2.7.1.1.2 Direct resolubilization
2.7.1.2 Extraction of specific proteins
2.7.1.2.1 Albumins and globulins
2.7.1.2.2 Amphiphilic proteins
2.7.1.2.3 Starch granule proteins
2.7.2 Characterization techniques
2.7.2.1 Analytical ultracentrifugation
2.7.2.2 Electrophoresis
2.7.2.2.1 SDS-PAGE
2.7.2.2.2 Capillary zone electrophoresis
2.7.2.2.3 Isoelectric focusing
2.7.2.3 Chromatography
2.7.2.3.1 Anion and cation exchange chromatography
2.7.2.3.2 Gel filtration chromatography
2.7.2.3.3 Size-exclusion chromatography
2.7.2.3.4 High-performance liquid chromatography
2.7.3 Detection techniques
2.7.3.1 Immunological methods
2.7.3.2 Enzymatic method
2.7.3.3 Differential scanning calorimetry
2.7.3.4 FTIR and Raman spectroscopy
2.7.3.5 Western and northern blotting
2.7.3.6 Light scattering
2.7.3.7 Microscopy
2.8 Cereal proteins; toxicity, and safety
2.8.1 α-Amylase/trypsin inhibitors and lectins
2.8.2 Nonceliac gluten sensitivity
2.8.3 Immunoglobulin-mediated allergic response
2.8.3.1 Baker's asthma
2.8.3.2 Wheat-dependent exercise-induced anaphylaxis
2.8.4 Other allergenic responses
2.8.5 Celiac disorder
2.9 Bioavailability
2.9.1 Digestion of proteins
2.9.1.1 Internal factors
2.9.1.2 External factors
2.9.1.3 Effect of processing
2.9.1.3.1 Size reduction or milling
2.9.1.3.2 Heat and pressure treatment
2.9.1.3.3 Extrusion and explosion puffing
2.9.1.3.4 Fermentation and germination
2.9.1.3.5 Protein hydrolysis
2.9.1.3.6 Bread-making process
2.9.1.3.7 Indigestible proteins
2.10 Applications of cereal proteins
2.10.1 Cereal proteins in food industries
2.10.2 Cereal enzymes in food industries
2.10.3 Specific application of cereal proteins
2.10.4 Nutraceutical applications
2.10.4.1 Cereal protein–based bioactive peptides
2.10.4.1.1 Antihypertensive peptides
2.10.4.1.2 Anticancer cereals peptides
2.10.4.1.3 Antioxidant peptides
2.10.4.1.4 Antidiabetic peptides
2.10.4.1.5 Antiinflammatory peptides
2.11 Conclusion
References
3 - Lignans
3.1 Introduction
3.2 Sources/derivatives
3.2.1 Lignan content of various regional diets
3.3 Extraction and characterization techniques
3.4 Chemistry
3.5 Mechanism of action
3.6 Bioavailability
3.7 Stability, safety, and toxicology
3.8 Applications (clinical and pharmacological)/health benefits
3.8.1 Cancer prevention
3.8.2 Diabetes
3.8.3 Dyslipidemia
3.8.4 Hypertension
3.8.5 Management of hot flashes
3.8.6 Diet and sexual health
3.9 Conclusion
References
4 - Betalain
4.1 Introduction
4.2 Sources/derivatives
4.2.1 Betalain yield improvement in beet
4.2.2 Alternative plant source for betalains
4.2.3 Betalain production through metabolic engineering
4.3 Extraction and characterization techniques
4.3.1 Tools of betalain characterization
4.3.1.1 UV-Vis spectra absorption
4.3.1.2 Fourier transform infrared spectroscopy data of betanin
4.3.1.3 NMR data of betanin
4.3.1.4 LC-MS spectrum of betanin
4.3.1.5 Thermogravimetric analysis
4.4 Chemistry
4.5 Mechanism of action
4.5.1 Regulation of cardiovascular disease
4.5.2 Regulation of hyperglycemia
4.5.3 Regulation of microbe's activity
4.5.4 Regulation of cell apoptosis
4.6 Bioavailability of betalains
4.6.1 Stability of betalain related to structure and degradation pathways
4.6.2 Effect of isomerization
4.6.3 Effect of deglycosylation
4.6.4 Effect of hydrolysis
4.6.5 Effect of decarboxylation
4.6.6 Effect of dehydrogenation
4.6.7 Betalain stability affected by factors
4.7 Health benefits of betalains
4.7.1 Free radical chelating character
4.7.2 Anticancer activity
4.7.3 Antilipidemic effects
4.7.4 Antimicrobial activity
4.8 Conclusion
References
5 - Flavonoids
5.1 Introduction
5.2 Sources/derivatives
5.3 Extraction and characterization techniques
5.4 Chemistry
5.5 Mechanism of action
5.6 Bioavailability
5.7 Flavonoids stability and safety and toxicology
5.7.1 Safety and stability
5.8 Clinical and pharmacological applications
5.9 Health benefits
5.10 Conclusion
References
6 - Lycopene
6.1 Introduction
6.2 Sources of lycopene
6.3 Extraction and characterization techniques
6.4 Chemistry/structure of lycopene
6.5 Pharmacokinetics, bioavailability, and pharmacodynamics of lycopene
6.6 Mechanism of action of lycopene
6.7 Isomerization and stability of lycopene
6.8 Safety and toxicity studies of lycopene
6.9 Therapeutic properties of lycopene
6.9.1 Antioxidative effects
6.9.2 Antiinflammatory activity
6.9.3 Anticancer effects
6.9.4 Cardioprotective effects
6.9.5 Antidiabetic effects
6.9.6 Osteoprotective effects
6.9.7 Hepatoprotective effects
6.9.8 Skin protective effects
6.9.9 Additional health benefits of lycopene
6.10 Conclusion
References
7 - Carotenoids
7.1 Introduction
7.2 Sources of carotenoids
7.3 Extraction and characterization techniques
7.3.1 Soxhlet extraction
7.3.2 Ionic liquids as a solvent for extraction
7.3.3 Microwave-assisted extraction
7.3.4 Ultrasonic-assisted extraction
7.3.5 Enzyme-assisted extraction
7.3.6 Supercritical fluid extraction
7.3.7 Characterization of carotenoids
7.4 Chemistry of carotenoids
7.4.1 Chemical properties
7.4.2 Physical properties
7.4.3 Electrochemical properties
7.5 Mechanism of action of carotenoids
7.5.1 Mechanism of action of carotenoids as antioxidants
7.5.2 Mechanism of action of carotenoids in cancer
7.5.3 Mechanism of action of carotenoids in cardiovascular diseases
7.5.4 Mechanism of action of carotenoids in diabetes and associated complications
7.5.5 Mechanism of action of carotenoids in neurodegenerative diseases
7.5.6 Mechanism of action of carotenoids in ophthalmic disorders
7.5.7 Mechanism of action of carotenoids as immunity booster
7.6 Bioavailability of carotenoids
7.6.1 Factors affecting bioavailability of carotenoids
7.7 Stability, safety, and toxicity
7.8 Health benefits of carotenoids
7.8.1 Carotenoids as antioxidant and its health benefits
7.8.2 Carotenoids in cancer
7.8.3 Carotenoids in cardiovascular diseases
7.8.4 Carotenoids in diabetes mellitus and associated complications
7.8.5 Carotenoids in neurodegenerative diseases
7.8.6 Carotenoids in age-related eye disorders
7.9 Conclusion
References
8 - Curcumin
8.1 Introduction
8.2 Sources/derivatives
8.3 Extraction techniques
8.4 Chemistry
8.5 Mechanism of action
8.5.1 Curcumin and transcription factors
8.5.2 Curcumin and adhesion molecules
8.5.3 Curcumin and autophagy
8.6 Bioavailability
8.6.1 Solubility
8.6.2 Permeability
8.6.3 Novel strategies for enhancing the bioavailability of curcumin
8.7 Safety and toxicology
8.8 Applications (clinical and pharmacological)/health benefits
8.8.1 Anticancer
8.8.2 Hepatoprotective
8.8.3 Antidiabetic role
8.9 Conclusion
References
9 - Eugenol
9.1 Introduction
9.2 Chemistry (physical and chemical properties)
9.3 Sources of eugenol
9.4 Extraction and characterization techniques
9.4.1 Solvent extraction
9.4.2 Steam distillation
9.4.3 Hydrodistillation
9.4.4 Supercritical fluid extraction
9.4.5 Microwave-assisted (MWA) extraction
9.4.6 Ultrasound-assisted extraction
9.5 Derivative of eugenol
9.6 Mechanism of action
9.7 Bioavailability
9.8 Applications (clinical and pharmacological)/health benefits
9.8.1 Other bioapplications of eugenol and its derivatives
9.8.1.1 Enzyme inhibitor
9.8.1.2 Antibiotics
9.8.1.3 Bio-based packaging materials
9.8.1.4 Green synthesis of metal nanoparticles
9.8.1.5 Nanoparticles or nanodiamonds
9.8.1.6 Synthesis of biocopolymers
9.8.1.7 Synthesis of composite resins/gums
9.8.1.8 Preparation of dendrimers
9.8.1.9 Protective biofilm
9.8.1.10 Corrosion inhibitors
9.9 Safety and toxicology
9.9.1 Acute and short-term toxicity
9.9.2 Chronic toxicity
9.9.3 Immunotoxicity
9.9.4 Reproductive toxicity
9.9.5 Genotoxicity
9.9.6 Carcinogenicity
9.9.7 Clinical management
9.9.8 Ecotoxicology
9.9.9 Exposure standards and guidelines
9.10 Conclusion
References
Further reading
10 - PUFA and MUFA
10.1 Introduction
10.1.1 Monounsaturated fatty acids
10.1.2 Polyunsaturated fatty acids
10.2 Sources/derivatives
10.3 Extraction and characterization techniques
10.3.1 Chromatographic methods
10.3.2 Distillation methods
10.3.3 The low-temperature fraction crystallization method
10.3.4 Supercritical CO2 extraction method
10.3.5 Enrichment by enzymatic methods
10.3.6 Urea fractionation method
10.4 Chemistry
10.5 Mechanism of action
10.6 Bioavailability
10.7 Stability, safety, and toxicology
10.8 Applications (clinical and pharmacological)/health benefits
10.8.1 Effect of MUFA and PUFA on cardiovascular diseases
10.8.2 Effects on eye health, brain, and nervous system
10.8.3 MUFA–PUFA and neurodegenerative diseases
10.8.4 Antidepression effects of EPA and DHA
10.8.5 The health effect of omega-3 fatty acids on the skin and inflammatory diseases
10.8.6 The health effect of omega-3 fatty acids on diabetes
10.8.7 The health effect of omega-3 fatty acids on diabetes
10.8.8 The health effect of omega-3 fatty acids on asthma
10.9 Conclusion
References
11 - Resveratrol
11.1 Introduction
11.2 Sources
11.3 Extraction and characterization techniques
11.4 Chemistry
11.5 Mechanism of action
11.6 Bioavailability of resveratrol
11.7 Stability, safety, and toxicology of resveratrol
11.8 Pharmacological and clinical applications of resveratrol
11.8.1 Role of resveratrol in cardiovascular health
11.8.2 Therapeutic effects of resveratrol on liver disorders
11.8.3 Anticancer properties of resveratrol
11.8.4 Antimicrobial properties of resveratrol
11.8.5 Neuroprotective effects of resveratrol
11.8.6 Antiinflammatory activity of resveratrol
11.8.7 Antioxidant properties of resveratrol
11.9 Conclusion
References
12 - Glucosinolates
12.1 Introduction
12.2 Chemical structure and hydrolysis
12.3 Sources
12.4 Bioaccessibility and bioavailability
12.5 Mechanism
12.5.1 Regulation of xenobiotic metabolism
12.5.2 Regulation of oxidative stress
12.5.3 Cell cycle arrest
12.5.4 Induction of apoptosis
12.5.5 Inhibition of angiogenesis
12.5.6 Other biological activities
12.6 Processing and cooking effects
12.7 Toxic and antinutritional effect
12.8 Conclusion
References
13 - Gamma oryzanol
13.1 Introduction
13.2 Sources
13.3 Extraction and characterization techniques
13.3.1 Extraction and purification of γ-OZ
13.3.2 Characterization methods
13.4 Chemistry
13.5 Mechanism of action
13.6 Bioavailability
13.7 Stability, safety, and toxicology
13.8 Application
13.8.1 Antioxidant effect
13.8.1.1 Free radical scavenger
13.8.1.2 Antioxidant enzyme activator
13.8.1.3 Nrf2 inducer
13.8.2 Antiulcer effect
13.8.3 Antiinflammatory effect
13.8.4 Antidiabetic property
13.8.5 Antihyperlipidemic property
13.8.6 Anticancer property
13.8.7 Improvement of menopausal symptoms
13.8.8 Antiallergic property
13.8.9 Application of γ-OZ
13.9 Conclusion
References
14 - Tocopherol
14.1 Introduction
14.2 Sources
14.3 Extraction and characterization techniques
14.3.1 Esterification and transesterification
14.3.2 Direct solvent extraction
14.3.3 Saponification
14.3.4 Distillation
14.3.5 Chromatographic methods
14.3.6 Liquid–liquid extraction
14.3.7 Crystallization
14.3.8 Enzymatic methods
14.3.9 Supercritical fluid extraction
14.3.10 Soxhlet extraction
14.3.11 Cold pressing
14.3.12 Deep eutectic solvent extraction
14.3.13 Ultrasound-assisted extraction
14.3.14 Partition of tocopherol homologues for separate use
14.4 Chemistry and biosynthesis of tocopherol
14.5 Mechanism of action: antioxidant properties and degradation
14.5.1 Antioxidant properties
14.5.2 Degradation of alpha tocopherol
14.6 Bioavailability
14.7 Stability, safety, and toxicology
14.8 Applications (clinical and pathological): health benefits
14.8.1 Antioxidant activity
14.8.2 Antiinflammation
14.8.3 Immunity
14.8.4 Cancer
14.8.5 Metabolic disorders
14.8.6 Skincare
14.8.7 Eye health
14.8.8 Liver health
14.9 Conclusion
References
Further readings
15 - Alpha-linolenic acid
15.1 Introduction
15.2 Sources
15.3 Extraction and characterization techniques
15.4 Chemistry
15.5 Mechanism of action
15.6 Bioavailability
15.7 Stability, safety, and toxicology
15.8 Applications
15.9 Conclusion
References
16 - Ascorbic acid
16.1 Introduction
16.2 Sources/derivatives
16.3 Methods of extraction and characterization
16.4 Chemistry
16.5 Mechanism of action
16.6 Bioavailability of ascorbic acid
16.7 Stability, safety, and toxicology
16.7.1 Toxicity
16.8 Applications of l-Ascorbic acid and its health benefits
16.8.1 Clinical and pharmacological relevance
16.8.1.1 Role in cardiovascular disease
16.8.1.2 Role in biosynthesis
16.8.1.3 Role as antioxidants
16.8.1.4 Role as an antiaging agent
16.8.1.5 Role as an anticancer
16.8.1.6 Role in immunity
16.8.1.7 Role in lipid metabolism
16.8.1.8 Role in protection from heavy metals toxicity
16.8.1.9 Role in endocrinology system
16.8.1.10 Role in preventing health disorders
16.8.1.11 Deficiency disorders of ascorbic acid
16.8.2 Other commercially viable applications
16.8.2.1 Role in bakery
16.8.2.2 Role in water treatment and purification
16.8.2.3 Role as a beverage and health drink
16.8.2.4 Role in the meat industry as a preservative
16.8.2.5 Role in fruit preservation
16.8.2.6 Role in personal care and cosmetics
16.8.2.7 Role in the manufacturing industry
16.9 Conclusion
References
17 - Phenolic acids
17.1 Introduction
17.2 Classification and chemical structure of phenolic acids
17.3 Biosynthesis and extraction of phenolic acids
17.4 Sources
17.4.1 Cereals
17.5 Health benefits
17.5.1 Antidiabetic properties
17.5.2 Antioxidant properties
17.5.3 Anticancer properties
17.5.4 Nephroprotective and hepatoprotective
17.5.5 Antiallergic
17.5.6 Neuroprotective
17.5.7 Antimelanogenic
17.5.8 Lipid peroxidation prevention
17.5.9 Antimicrobial
17.5.10 Antiinflammatory properties
17.5.11 Antiviral properties
17.6 Storage and processing stability
17.7 Absorption and metabolism in human
17.8 Impact on food quality and sensory properties
17.9 Conclusion
References
18 - Anthocyanins
18.1 Sources/derivatives/chemistry
18.2 Extraction and characterization techniques of anthocyanins
18.2.1 Preextraction treatments
18.2.2 Solute–solvent interaction
18.2.3 Solvent extraction
18.2.4 Ultrasound-assisted extraction
18.2.5 Microwave-assisted extraction
18.2.6 High-pressure processing
18.2.7 Enzymatic-assisted extraction
18.2.8 Supercritical fluid extraction
18.3 Health and pharmacological benefits of anthocyanins
18.3.1 Antioxidant activity
18.3.2 Cardiovascular effects
18.3.3 Anticancerous activity
18.3.4 Antiinflammatory properties
18.3.5 Antiobesity and antidiabetic activity
18.3.6 Neuroprotective properties
18.4 Bioavailability
18.5 Stability
18.5.1 Factors affecting anthocyanins stability
18.5.1.1 pH
18.5.1.2 Temperature
18.5.1.3 Oxygen
18.5.1.4 Light
18.5.1.5 Enzyme
18.5.1.6 Effect of concentration
18.5.1.7 Impact of water activity (aw)
18.6 Safety and toxicology
18.7 Conclusion
References
19 - Genistein and daidzein
19.1 Introduction
19.2 Sources
19.2.1 Genistein sources
19.2.2 Daidzein sources
19.3 Extraction and characterization techniques
19.3.1 Analytical methods for determination and characterization of genistein and daidzein
19.3.1.1 UV–visible spectroscopy
19.3.1.2 Mass spectrometry
19.3.1.3 Liquid chromatography/tandem mass spectrometry
19.3.1.4 Ultra performance liquid chromatography/mass spectroscopy
19.3.1.5 Reverse-phase high-performance liquid chromatography
19.3.1.6 Capillary zone electrophoresis
19.4 Chemistry of genistein
19.4.1 Chemical nature
19.4.2 Chemical structure
19.4.3 Active principles
19.4.3.1 Phytoestrogens and mammalian estrogens
19.4.3.2 Anticancer effects
19.4.3.3 Stability
19.4.4 Biological properties
19.5 Metabolic pathway and mechanism of action
19.6 Bioavailability of genistein
19.7 Stability, biosafety, and toxicology of genistein
19.8 Applications/health benefits
19.9 Conclusion
References
20 - Beta-glucan
20.1 Introduction
20.2 Sources/Derivatives
20.3 Extraction and characterization techniques
20.3.1 Extraction of beta-glucan from cereals
20.3.2 Extraction of beta-glucan from yeast
20.3.3 Extraction of beta-glucan from fungi
20.4 Chemistry
20.5 Mechanism of action
20.6 Bioavailability
20.7 Stability, safety, and toxicology
20.7.1 Stability
20.7.1.1 Effect of milling and germination
20.7.1.2 Effect of cooking and baking
20.7.1.3 Effect of extrusion
20.7.1.4 Effect of roasting
20.7.1.5 Effect of freezing and storage
20.7.2 Safety and toxicity
20.8 Health benefits
20.8.1 Regulation of blood glucose and insulin level
20.8.2 Reduction of cholesterol level and prevention of coronary heart diseases
20.8.3 Lowering of fat for obesity
20.8.4 Reduction of blood pressure
20.8.5 Improving gut flora and laxative effect
20.8.6 Antitumor effect
20.8.7 Antiinflammatory action
20.9 Conclusion
References
21 - Berberine
21.1 Introduction
21.2 Chemistry, source, and uses
21.2.1 Chemistry of berberine
21.2.2 Source and uses of berberine
21.3 Extraction and characterization techniques
21.3.1 Extraction methods
21.3.2 Characterization techniques
21.4 Mechanism of action
21.4.1 Diabetes
21.4.2 Cardiovascular disorders
21.4.2.1 Cholesterol metabolism
21.4.2.2 Triacylglycerol metabolism
21.4.3 Cancer
21.4.4 Severe acute respiratory syndrome coronavirus 2
21.4.5 Other health benefits
21.5 Stability, safety, and toxicology
21.5.1 Solution stability
21.5.2 Safety of berberine
21.5.3 Toxicity of berberine
21.6 Health benefits
21.6.1 Antiparasitic potential
21.6.2 Antifungal effect
21.6.3 Antibacterial activity
21.6.4 Antiviral and immune modulatory function
21.6.5 Neuroprotective activity
21.6.6 Canker sores
21.6.7 Ulcerative Colitis
21.6.8 Diabetes
21.6.9 Diabetic complications
21.6.10 Gut issues
21.6.11 High blood pressure
21.6.12 High cholesterol levels
21.6.13 Antiinflammatory and antioxidant activities of berberine
21.6.14 Memory and learning
21.6.15 Liver health
21.6.16 Mitochondrial function
21.6.17 Anxiety
21.6.18 Polycystic ovary syndrome
21.6.19 Weight loss
21.7 Conclusion
References
Index
A
B
C
D
E
F
G
H
I
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
Back Cover

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Nutraceuticals and Health Care

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Nutraceuticals and Health Care

Edited by

Jasmeet Kour Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, Sangrur, Punjab, India

Gulzar Ahmad Nayik Department of Food Science & Technology, Government Degree College Shopian, Jammu & Kashmir, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2022 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www. elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-323-89779-2 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

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This book is a dedication to my beloved family. Without their unending support this work wouldn’t have been accomplished.

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Contents Contributors Preface

xv xix

1. Nutraceutical-A deep and profound concept Jasmeet Kour, Hitesh Chopra, Saba Bukhari, Renu Sharma, Rosy Bansal, Monika Hans and Dharmesh Chandra Saxena 1.1 Introduction 1.2 Facts of nutraceuticals 1.2.1 Growth 1.2.2 Health maintenance 1.2.3 Effect on chronic ailments (e.g., Heart disease, diabetes and metabolic disease, musculoskeletal disease) 1.2.4 Musculoskeletal disease 1.3 Fiction of nutraceuticals 1.3.1 Supplements are meant for men and women 1.4 Developmental strategy for nutraceuticals 1.5 Disease prevention claims of nutraceuticals 1.6 Sources of nutraceuticals and chemical nature 1.7 Plant food by-products as source of nutraceuticals 1.8 Nutraceutical profile of prominent nutraceuticals 1.9 Mechanism of action of nutraceuticals 1.10 Nutraceuticals in ayurveda 1.11 Recent trends in nutraceuticals 1.12 Recent developments in nutraceuticals 1.13 Patents on nutraceuticals 1.13.1 Berberine 1.13.2 Beta glucan 1.13.3 Gamma oryzanol 1.13.4 Lignans 1.13.5 Curcumin 1.14 Regulatory aspect of nutraceuticals 1.15 Current status

1 2 2 2

1.16 Indian and global nutraceutical market size 1.17 Establishment of nutraceuticals in market 1.17.1 Demands of nutraceuticals in market 1.17.2 Factors affecting future market 1.17.3 Steps for market development of nutraceuticals 1.18 Marketing barriers of nutraceuticals 1.19 Status of nutraceuticals and future prospects References Further readings

19 20 20 20 21 21 21 22 28

2. Cereal proteins 3 3 3 3 4 5 6 6 7 7 8 9 10 11 11 11 15 16 16 16 19

Cherakkathodi Sudheesh, Zahid Rafiq Bhat, Basheer Aaliya and Kappat Valiyapeediyekkal Sunooj 2.1 Introduction 2.2 Cereal grains 2.2.1 Rice 2.2.2 Wheat 2.2.3 Barley 2.2.4 Oats 2.2.5 Sorghum 2.2.6 Rye 2.2.7 Maize 2.2.8 Millet 2.2.9 Other cereals 2.3 Nutritional profile of cereals 2.3.1 Macronutrients 2.3.2 Micronutrients 2.3.3 Non-starch polysaccharides 2.4 Phytochemicals 2.5 Antinutrients 2.6 Classification and chemistry of cereal proteins 2.6.1 Cereal storage proteins 2.7 Extraction and characterization techniques 2.7.1 Extraction techniques

29 29 29 29 30 30 30 30 30 30 31 31 31 33 33 33 33 33 34 40 40

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2.7.2 Characterization techniques 2.7.3 Detection techniques 2.8 Cereal proteins; toxicity, and safety 2.8.1 a-Amylase/trypsin inhibitors and lectins 2.8.2 Nonceliac gluten sensitivity 2.8.3 Immunoglobulin-mediated allergic response 2.8.4 Other allergenic responses 2.8.5 Celiac disorder 2.9 Bioavailability 2.9.1 Digestion of proteins 2.10 Applications of cereal proteins 2.10.1 Cereal proteins in food industries 2.10.2 Cereal enzymes in food industries 2.10.3 Specific application of cereal proteins 2.10.4 Nutraceutical applications 2.11 Conclusion References

45 47 48 48 49 49 50 50 50 50 53 53 54 54 54 56 57

3. Lignans Syeda Saniya Zahra, Ihsan-ul Haq and Omer Farooq 3.1 Introduction 3.2 Sources/derivatives 3.2.1 Lignan content of various regional diets 3.3 Extraction and characterization techniques 3.4 Chemistry 3.5 Mechanism of action 3.6 Bioavailability 3.7 Stability, safety, and toxicology 3.8 Applications (clinical and pharmacological)/health benefits 3.8.1 Cancer prevention 3.8.2 Diabetes 3.8.3 Dyslipidemia 3.8.4 Hypertension 3.8.5 Management of hot flashes 3.8.6 Diet and sexual health 3.9 Conclusion References

61 61 65 66 69 71 76 78 79 79 79 79 79 80 80 82 82

4. Betalain Varun Kumar and Amarjeet Kumar 4.1 Introduction 4.2 Sources/derivatives 4.2.1 Betalain yield improvement in beet 4.2.2 Alternative plant source for betalains

4.2.3 Betalain production through 89 metabolic engineering 4.3 Extraction and characterization techniques 89 4.3.1 Tools of betalain characterization 90 4.4 Chemistry 91 4.5 Mechanism of action 93 4.5.1 Regulation of cardiovascular disease 93 4.5.2 Regulation of hyperglycemia 94 4.5.3 Regulation of microbe's activity 95 4.5.4 Regulation of cell apoptosis 95 4.6 Bioavailability of betalains 96 4.6.1 Stability of betalain related to 97 structure and degradation pathways 4.6.2 Effect of isomerization 97 4.6.3 Effect of deglycosylation 97 4.6.4 Effect of hydrolysis 97 4.6.5 Effect of decarboxylation 97 4.6.6 Effect of dehydrogenation 98 4.6.7 Betalain stability affected by 98 factors 4.7 Health benefits of betalains 98 4.7.1 Free radical chelating character 98 4.7.2 Anticancer activity 99 4.7.3 Antilipidemic effects 99 4.7.4 Antimicrobial activity 99 4.8 Conclusion 99 References 100

5. Flavonoids Prerna Gupta, Jasmeet Kour, Manish Bakshi and Rhythm Kalsi 5.1 Introduction 5.2 Sources/derivatives 5.3 Extraction and characterization techniques 5.4 Chemistry 5.5 Mechanism of action 5.6 Bioavailability 5.7 Flavonoids stability and safety and toxicology 5.7.1 Safety and stability 5.8 Clinical and pharmacological applications 5.9 Health benefits 5.10 Conclusion References

105 106 106 106 107 107 107 107 110 110 111 111

6. Lycopene 87 87 88 89

Nusrath Yasmeen, Aga Syed Sameer and Saniya Nissar 6.1 Introduction

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6.2 Sources of lycopene 6.3 Extraction and characterization techniques 6.4 Chemistry/structure of lycopene 6.5 Pharmacokinetics, bioavailability, and pharmacodynamics of lycopene 6.6 Mechanism of action of lycopene 6.7 Isomerization and stability of lycopene 6.8 Safety and toxicity studies of lycopene 6.9 Therapeutic properties of lycopene 6.9.1 Antioxidative effects 6.9.2 Antiinflammatory activity 6.9.3 Anticancer effects 6.9.4 Cardioprotective effects 6.9.5 Antidiabetic effects 6.9.6 Osteoprotective effects 6.9.7 Hepatoprotective effects 6.9.8 Skin protective effects 6.9.9 Additional health benefits of lycopene 6.10 Conclusion References

115 116 116 118 119 120 120 121 121 121 123 125 126 127 127 127 128 128 128

7. Carotenoids Sweta Priyadarshini Pradhan, Santwana Padhi, Monalisa Dash, Heena, Bharti Mittu and Anindita Behera 7.1 Introduction 7.2 Sources of carotenoids 7.3 Extraction and characterization techniques 7.3.1 Soxhlet extraction 7.3.2 Ionic liquids as a solvent for extraction 7.3.3 Microwave-assisted extraction 7.3.4 Ultrasonic-assisted extraction 7.3.5 Enzyme-assisted extraction 7.3.6 Supercritical fluid extraction 7.3.7 Characterization of carotenoids 7.4 Chemistry of carotenoids 7.4.1 Chemical properties 7.4.2 Physical properties 7.4.3 Electrochemical properties 7.5 Mechanism of action of carotenoids 7.5.1 Mechanism of action of carotenoids as antioxidants 7.5.2 Mechanism of action of carotenoids in cancer 7.5.3 Mechanism of action of carotenoids in cardiovascular diseases

7.5.4 Mechanism of action of carotenoids in diabetes and associated complications 7.5.5 Mechanism of action of carotenoids in neurodegenerative diseases 7.5.6 Mechanism of action of carotenoids in ophthalmic disorders 7.5.7 Mechanism of action of carotenoids as immunity booster 7.6 Bioavailability of carotenoids 7.6.1 Factors affecting bioavailability of carotenoids 7.7 Stability, safety, and toxicity 7.8 Health benefits of carotenoids 7.8.1 Carotenoids as antioxidant and its health benefits 7.8.2 Carotenoids in cancer 7.8.3 Carotenoids in cardiovascular diseases 7.8.4 Carotenoids in diabetes mellitus and associated complications 7.8.5 Carotenoids in neurodegenerative diseases 7.8.6 Carotenoids in age-related eye disorders 7.9 Conclusion References

ix

144 144 145 145 145 146 146 147 147 147 148 148 149 150 150 150

8. Curcumin 135 135 136 138 138 138 138 139 139 139 141 141 142 142 143 143 143 144

Srinivasan Krishnamoorthy, R. Paranthaman, J.A. Moses and C. Anandharamakrishnan 8.1 8.2 8.3 8.4 8.5

Introduction Sources/derivatives Extraction techniques Chemistry Mechanism of action 8.5.1 Curcumin and transcription factors 8.5.2 Curcumin and adhesion molecules 8.5.3 Curcumin and autophagy 8.6 Bioavailability 8.6.1 Solubility 8.6.2 Permeability 8.6.3 Novel strategies for enhancing the bioavailability of curcumin 8.7 Safety and toxicology 8.8 Applications (clinical and pharmacological)/health benefits 8.8.1 Anticancer 8.8.2 Hepatoprotective 8.8.3 Antidiabetic role

159 159 162 163 164 164 165 165 165 166 166 167 168 168 169 169 170

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8.9 Conclusion References

170 171

9. Eugenol Ajay Sharma, Garima Bhardwaj, Harvinder Singh Sohal and Apurba Gohain 9.1 Introduction 9.2 Chemistry (physical and chemical properties) 9.3 Sources of eugenol 9.4 Extraction and characterization techniques 9.4.1 Solvent extraction 9.4.2 Steam distillation 9.4.3 Hydrodistillation 9.4.4 Supercritical fluid extraction 9.4.5 Microwave-assisted (MWA) extraction 9.4.6 Ultrasound-assisted extraction 9.5 Derivative of eugenol 9.6 Mechanism of action 9.7 Bioavailability 9.8 Applications (clinical and pharmacological)/health benefits 9.8.1 Other bioapplications of eugenol and its derivatives 9.9 Safety and toxicology 9.9.1 Acute and short-term toxicity 9.9.2 Chronic toxicity 9.9.3 Immunotoxicity 9.9.4 Reproductive toxicity 9.9.5 Genotoxicity 9.9.6 Carcinogenicity 9.9.7 Clinical management 9.9.8 Ecotoxicology 9.9.9 Exposure standards and guidelines 9.10 Conclusion References Further reading

177 177 179 180 180 180 181 181 181 181 182 184 187 187 190 191 191 191 191 191 191 192 192 192 192 192 192 198

10. PUFA and MUFA Mustafa Öz, İlknur Ucak and Gulzar Ahmad Nayik 10.1 Introduction 10.1.1 Monounsaturated fatty acids 10.1.2 Polyunsaturated fatty acids 10.2 Sources/derivatives 10.3 Extraction and characterization techniques 10.3.1 Chromatographic methods 10.3.2 Distillation methods

199 199 200 202 203 203 203

10.3.3 The low-temperature fraction crystallization method 10.3.4 Supercritical CO2 extraction method 10.3.5 Enrichment by enzymatic methods 10.3.6 Urea fractionation method 10.4 Chemistry 10.5 Mechanism of action 10.6 Bioavailability 10.7 Stability, safety, and toxicology 10.8 Applications (clinical and pharmacological)/health benefits 10.8.1 Effect of MUFA and PUFA on cardiovascular diseases 10.8.2 Effects on eye health, brain, and nervous system 10.8.3 MUFAePUFA and neurodegenerative diseases 10.8.4 Antidepression effects of EPA and DHA 10.8.5 The health effect of omega-3 fatty acids on the skin and inflammatory diseases 10.8.6 The health effect of omega-3 fatty acids on diabetes 10.8.7 The health effect of omega-3 fatty acids on diabetes 10.8.8 The health effect of omega-3 fatty acids on asthma 10.9 Conclusion References

203 203 203 204 204 204 206 207 207 207 208 209 209

210 210 210 211 211 211

11. Resveratrol Zahid Rafiq Bhat, Abida Bhat, Bharti Mittu, Kappat Valiyapeediyekkal Sunooj and Rasiya Ul Zaman 11.1 Introduction 11.2 Sources 11.3 Extraction and characterization techniques 11.4 Chemistry 11.5 Mechanism of action 11.6 Bioavailability of resveratrol 11.7 Stability, safety, and toxicology of resveratrol 11.8 Pharmacological and clinical applications of resveratrol 11.8.1 Role of resveratrol in cardiovascular health 11.8.2 Therapeutic effects of resveratrol on liver disorders

217 217 218 219 223 225 226 226 226

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Contents

11.8.3 Anticancer properties of resveratrol 11.8.4 Antimicrobial properties of resveratrol 11.8.5 Neuroprotective effects of resveratrol 11.8.6 Antiinflammatory activity of resveratrol 11.8.7 Antioxidant properties of resveratrol 11.9 Conclusion References

227 227 227

233 233 234 234 236 236 237 237 237 238 238 238 239 239 239

13. Gamma oryzanol Sangeeta Saikia and Himjyoti Dutta 13.1 Introduction 13.2 Sources 13.3 Extraction and characterization techniques 13.3.1 Extraction and purification of g-OZ 13.3.2 Characterization methods 13.4 Chemistry 13.5 Mechanism of action 13.6 Bioavailability 13.7 Stability, safety, and toxicology 13.8 Application 13.8.1 Antioxidant effect 13.8.2 Antiulcer effect 13.8.3 Antiinflammatory effect 13.8.4 Antidiabetic property 13.8.5 Antihyperlipidemic property

254 254 254 255 255

14. Tocopherol 227 227 228

Rohini Bhat Introduction Chemical structure and hydrolysis Sources Bioaccessibility and bioavailability Mechanism 12.5.1 Regulation of xenobiotic metabolism 12.5.2 Regulation of oxidative stress 12.5.3 Cell cycle arrest 12.5.4 Induction of apoptosis 12.5.5 Inhibition of angiogenesis 12.5.6 Other biological activities 12.6 Processing and cooking effects 12.7 Toxic and antinutritional effect 12.8 Conclusion References

253

227

12. Glucosinolates 12.1 12.2 12.3 12.4 12.5

13.8.6 Anticancer property 13.8.7 Improvement of menopausal symptoms 13.8.8 Antiallergic property 13.8.9 Application of g-OZ 13.9 Conclusion References

xi

245 245 245 246 247 249 249 250 250 251 251 252 252 252 253

Jinku Bora, Thoithoi Tongbram, Nikhil Mahnot, Charu Lata Mahanta and Laxmikant Shivnath Badwaik 14.1 Introduction 14.2 Sources 14.3 Extraction and characterization techniques 14.3.1 Esterification and transesterification 14.3.2 Direct solvent extraction 14.3.3 Saponification 14.3.4 Distillation 14.3.5 Chromatographic methods 14.3.6 Liquideliquid extraction 14.3.7 Crystallization 14.3.8 Enzymatic methods 14.3.9 Supercritical fluid extraction 14.3.10 Soxhlet extraction 14.3.11 Cold pressing 14.3.12 Deep eutectic solvent extraction 14.3.13 Ultrasound-assisted extraction 14.3.14 Partition of tocopherol homologues for separate use 14.4 Chemistry and biosynthesis of tocopherol 14.5 Mechanism of action: antioxidant properties and degradation 14.5.1 Antioxidant properties 14.5.2 Degradation of alpha tocopherol 14.6 Bioavailability 14.7 Stability, safety, and toxicology 14.8 Applications (clinical and pathological): health benefits 14.8.1 Antioxidant activity 14.8.2 Antiinflammation 14.8.3 Immunity 14.8.4 Cancer 14.8.5 Metabolic disorders 14.8.6 Skincare 14.8.7 Eye health 14.8.8 Liver health 14.9 Conclusion

259 259 260 261 262 262 262 263 263 264 264 264 265 265 265 265 265 266 267 267 268 268 270 271 271 272 272 272 273 273 273 273 274

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References Further readings

274 278

15. Alpha-linolenic acid Jessica Pandohee 15.1 Introduction 15.2 Sources 15.3 Extraction and characterization techniques 15.4 Chemistry 15.5 Mechanism of action 15.6 Bioavailability 15.7 Stability, safety, and toxicology 15.8 Applications 15.9 Conclusion References

279 280 281 282 282 282 284 284 285 285

16. Ascorbic acid Bharti Mittu, Zahid Rafiq Bhat, Ashish Chauhan, Jasmeet Kour, Anindita Behera and Mahaldeep Kaur 16.1 Introduction 16.2 Sources/derivatives 16.3 Methods of extraction and characterization 16.4 Chemistry 16.5 Mechanism of action 16.6 Bioavailability of ascorbic acid 16.7 Stability, safety, and toxicology 16.7.1 Toxicity 16.8 Applications of L-Ascorbic acid and its health benefits 16.8.1 Clinical and pharmacological relevance 16.8.2 Other commercially viable applications 16.9 Conclusion References

17.4 Sources 17.4.1 Cereals 17.5 Health benefits 17.5.1 Antidiabetic properties 17.5.2 Antioxidant properties 17.5.3 Anticancer properties 17.5.4 Nephroprotective and hepatoprotective 17.5.5 Antiallergic 17.5.6 Neuroprotective 17.5.7 Antimelanogenic 17.5.8 Lipid peroxidation prevention 17.5.9 Antimicrobial 17.5.10 Antiinflammatory properties 17.5.11 Antiviral properties 17.6 Storage and processing stability 17.7 Absorption and metabolism in human 17.8 Impact on food quality and sensory properties 17.9 Conclusion References

304 309 309 309 309 310 310 310 310 310 310 310 311 311 311 311 312 313 313

18. Anthocyanins 289 289 290 292 293 294 295 296 297 297 299 299 300

17. Phenolic acids Md Nazmus Saqib and Md Ramim Tanver Rahman 17.1 Introduction 303 17.2 Classification and chemical structure of 303 phenolic acids 17.3 Biosynthesis and extraction of phenolic 304 acids

Amir Gull, Mohd Aaqib Sheikh, Jasmeet Kour, Beenish Zehra, Imtiyaz Ahmad Zargar, Altaf Ahmad Wani, Surekha Bhatia and Mushtaq Ahmad Lone 18.1 Sources/derivatives/chemistry 317 18.2 Extraction and characterization 317 techniques of anthocyanins 18.2.1 Preextraction treatments 318 18.2.2 Soluteesolvent interaction 318 18.2.3 Solvent extraction 318 18.2.4 Ultrasound-assisted extraction 318 18.2.5 Microwave-assisted extraction 319 18.2.6 High-pressure processing 319 18.2.7 Enzymatic-assisted extraction 319 18.2.8 Supercritical fluid extraction 320 18.3 Health and pharmacological benefits of 320 anthocyanins 18.3.1 Antioxidant activity 321 18.3.2 Cardiovascular effects 321 18.3.3 Anticancerous activity 322 18.3.4 Antiinflammatory properties 322 18.3.5 Antiobesity and antidiabetic 322 activity 18.3.6 Neuroprotective properties 323 18.4 Bioavailability 323 18.5 Stability 324

18.5.1 Factors affecting anthocyanins stability 18.6 Safety and toxicology 18.7 Conclusion References

324 326 326 326

19. Genistein and daidzein Loveleen Sarao, Sandeep Kaur, Tanu Malik and Ajay Singh 19.1 Introduction 19.2 Sources 19.2.1 Genistein sources 19.2.2 Daidzein sources 19.3 Extraction and characterization techniques 19.3.1 Analytical methods for determination and characterization of genistein and daidzein 19.4 Chemistry of genistein 19.4.1 Chemical nature 19.4.2 Chemical structure 19.4.3 Active principles 19.4.4 Biological properties 19.5 Metabolic pathway and mechanism of action 19.6 Bioavailability of genistein 19.7 Stability, biosafety, and toxicology of genistein 19.8 Applications/health benefits 19.9 Conclusion References

331 331 331 332

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20.7.2 Safety and toxicity 20.8 Health benefits 20.8.1 Regulation of blood glucose and insulin level 20.8.2 Reduction of cholesterol level and prevention of coronary heart diseases 20.8.3 Lowering of fat for obesity 20.8.4 Reduction of blood pressure 20.8.5 Improving gut flora and laxative effect 20.8.6 Antitumor effect 20.8.7 Antiinflammatory action 20.9 Conclusion References

351 351 351

352 352 352 352 353 353 354 354

332

21. Berberine 333 334 334 334 334 336 336 336 337 338 338 338

20. Beta-glucan Hanuman Bobade, Antima Gupta and Savita Sharma 20.1 Introduction 20.2 Sources/Derivatives 20.3 Extraction and characterization techniques 20.3.1 Extraction of beta-glucan from cereals 20.3.2 Extraction of beta-glucan from yeast 20.3.3 Extraction of beta-glucan from fungi 20.4 Chemistry 20.5 Mechanism of action 20.6 Bioavailability 20.7 Stability, safety, and toxicology 20.7.1 Stability

Contents

343 343 344 344 345 345 347 348 349 349 349

Santwana Palai 21.1 Introduction 21.2 Chemistry, source, and uses 21.2.1 Chemistry of berberine 21.2.2 Source and uses of berberine 21.3 Extraction and characterization techniques 21.3.1 Extraction methods 21.3.2 Characterization techniques 21.4 Mechanism of action 21.4.1 Diabetes 21.4.2 Cardiovascular disorders 21.4.3 Cancer 21.4.4 Severe acute respiratory syndrome coronavirus 2 21.4.5 Other health benefits 21.5 Stability, safety, and toxicology 21.5.1 Solution stability 21.5.2 Safety of berberine 21.5.3 Toxicity of berberine 21.6 Health benefits 21.6.1 Antiparasitic potential 21.6.2 Antifungal effect 21.6.3 Antibacterial activity 21.6.4 Antiviral and immune modulatory function 21.6.5 Neuroprotective activity 21.6.6 Canker sores 21.6.7 Ulcerative Colitis 21.6.8 Diabetes 21.6.9 Diabetic complications 21.6.10 Gut issues 21.6.11 High blood pressure

359 359 359 360 360 360 362 362 362 363 363 363 363 364 364 364 364 364 364 365 365 365 365 365 365 366 366 366 366

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Contents

21.6.12 High cholesterol levels 21.6.13 Antiinflammatory and antioxidant activities of berberine 21.6.14 Memory and learning 21.6.15 Liver health 21.6.16 Mitochondrial function 21.6.17 Anxiety

366 366 367 367 367 367

21.6.18 Polycystic ovary syndrome 21.6.19 Weight loss 21.7 Conclusion References Glossary Index

367 367 368 368 369 371

Contributors Basheer Aaliya, Department of Food Science and Technology, Pondicherry University, Puducherry, India

Saba Bukhari, Department of Animal Genetics and Breeding, Skuast, Kashmir, India

C. Anandharamakrishnan, Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology Entrepreneurship and Management Thanjavur (NIFTEM - T), Ministry of Food Processing Industries, Government of India, Thanjavur, Tamil Nadu, India

Ashish Chauhan, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur, Punjab, India

Laxmikant Shivnath Badwaik, Department of Food Engineering and Technology, School of Engineering, Tezpur University, Napaam, Assam, India Manish Bakshi, School of Agriculture, Lovely Professional University, Jalandhar, Punjab, India Rosy Bansal, GSSDGS Khalsa College, Patiala, Punjab, India Anindita Behera, School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan Deemed to be University, Bhubaneswar, Odisha, India Garima Bhardwaj, Department of Chemistry, Sant Longowal Institute of Engineering and Technology, Sangrur, Punjab, India Abida Bhat, Department of Immunology and Molecular Medicine, Sher-e-Kashmir Institute of Medical Sciences, Srinagar, Jammu Kashmir, India Rohini Bhat, Interactions Lab, National Centre for Biological Sciences, Bangalore, Karnataka, India Zahid Rafiq Bhat, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur, Punjab, India

Hitesh Chopra, Chitkara College of Pharmacy, Chitkara Univeristy, Rajpura, Punjab, India Monalisa Dash, Institute of Pharmaceutical Technology, Cuttack, Odisha, India Himjyoti Dutta, Department of Food Technology, Mizoram University, Aizawl, Mizoram, India Omer Farooq, Department of Pharmacognosy, Shifa College of Pharmaceutical Sciences, Shifa Tameer-eMillat University, Islamabad, Punjab, Pakistan Apurba Gohain, Department of Chemistry, Assam University, Silchar, Assam, India Amir Gull, Department of Food Science and Technology, University of Kashmir, Srinagar, Jammu and Kashmir, India Antima Gupta, Department of Food Science and Technology, Punjab Agricultural University, Ludhiana, Punjab, India Prerna Gupta, School of Agriculture, Lovely Professional University, Jalandhar, Punjab, India Monika Hans, Government PG College for Women, Jammu, Jammu and Kashmir, India Ihsan-ul Haq, Department of Pharmacy, Quaid-i-Azam University, Islamabad, Punjab, Pakistan

Surekha Bhatia, Department of Processing and Food Engineering, Punjab Agricultural University, Ludhiana, Punjab, India

Heena, GSSDGS Khalsa College, Patiala, Punjab, India

Hanuman Bobade, Department of Food Science and Technology, Punjab Agricultural University, Ludhiana, Punjab, India

Mahaldeep Kaur, Department of Microbial Biotechnology, Panjab University, Chandigarh, India

Jinku Bora, Department of Food Technology, Jamia Hamdard, New Delhi, Delhi, India

Rhythm Kalsi, School of Agriculture, Lovely Professional University, Jalandhar, Punjab, India

Sandeep Kaur, Department of Agriculture, MM University, Ambala, Haryana, India

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Contributors

Jasmeet Kour, Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Sangrur, Punjab, India Srinivasan Krishnamoorthy, Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology Entrepreneurship and Management Thanjavur (NIFTEM - T), Ministry of Food Processing Industries, Government of India, Thanjavur, Tamil Nadu, India Amarjeet Kumar, Department of Home Science, Rohtas Mahila College, Sasaram, Veer Kunwar University, Ara, Bihar, India Varun Kumar, Department of Home Science, Ramesh Jha Mahila College, Saharsa, B.N. Mandal University, Madhepura, Bihar, India Mushtaq Ahmad Lone, Directorate of Planning and Monitoring, Sher-e- Kashmir University of Agriculture Sciences and Technology, Shalimar, Srinagar, India Charu Lata Mahanta, Department of Food Engineering and Technology, School of Engineering, Tezpur University, Napaam, Assam, India Nikhil Mahnot, Department of Food Technology, Rajiv Gandhi University, Rono Hills, Doimukh, Arunachal Pradesh, India Tanu Malik, Centre of Food Science and Technology, CCSHAU, Hisar, Haryana, India Bharti Mittu, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur, Punjab, India

Santwana Palai, Department of Veterinary Pharmacology & Toxicology, College of Veterinary Science and Animal Husbandry, Odisha University of Agriculture & Technology, Bhubaneswar, Odisha, India Jessica Pandohee, Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, WA, Australia R. Paranthaman, Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology Entrepreneurship and Management Thanjavur (NIFTEM - T), Ministry of Food Processing Industries, Government of India, Thanjavur, Tamil Nadu, India Sweta Priyadarshini Pradhan, School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan Deemed to be University, Bhubaneswar, Odisha, India Md Ramim Tanver Rahman, Medicinal Chemistry Laboratory, Centre Hospitalier Universitaire (CHU) de Québec Research Center, Université Laval, Quebec, QC, Canada Sangeeta Saikia, Food Researcher, Guwahati, Assam, India Aga Syed Sameer, Department of Basic Medical Sciences & Quality Unit, College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia; King Abdullah International Medical Research Centre (KAIMRC), National Guard Health Affairs, Jeddah, Saudi Arabia; Molecular Diseases & Diagnostics Division, Infinity Biochemistry Pvt. Ltd., Srinagar, Kashmir, India

J.A. Moses, Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology Entrepreneurship and Management - Thanjavur (NIFTEM - T), Ministry of Food Processing Industries, Government of India, Thanjavur, Tamil Nadu, India

Loveleen Sarao, Department of Microbiology, Punjab Agricultural University, Ludhiana, Punjab, India

Gulzar Ahmad Nayik, Department of Food Science & Technology, Government Degree College Shopian, Jammu and Kashmir, India

Dharmesh Chandra Saxena, Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Sangrur, Punjab, India

Saniya Nissar, Department of Biochemistry, Government Medical College, Shri Maharaja Hari Singh Hospital, Srinagar, Kashmir, India; Molecular Diseases & Diagnostics Division, Infinity Biochemistry Pvt. Ltd., Srinagar, Kashmir, India

Ajay Sharma, Department of Chemistry, Chandigarh University, Mohali, Punjab, India

Md Nazmus Saqib, School of Food Science & Technology, Jiangnan University, Wuxi, Jiangsu, China

Renu Sharma, Department of Applied Sciences, Bhai Gurdas Degree College, Sangrur, Punjab, India

Mustafa Öz, Aksaray University, Faculty of Veterinary Medicine, Aksaray, Turkey

Savita Sharma, Department of Food Science and Technology, Punjab Agricultural University, Ludhiana, Punjab, India

Santwana Padhi, KIIT Technology Business Incubator, KIIT Deemed to be University, Bhubaneswar, Odisha, India

Mohd Aaqib Sheikh, Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Sangrur, Punjab, India

Contributors

Ajay Singh, Department of Food Technology, Mata Gujri College, Fatehgarh Sahib, Punjab, India Harvinder Singh Sohal, Department of Chemistry, Chandigarh University, Mohali, Punjab, India Cherakkathodi Sudheesh, Department of Food Science and Technology, Pondicherry University, Puducherry, India Kappat Valiyapeediyekkal Sunooj, Department of Food Science and Technology, Pondicherry University, Puducherry, India Thoithoi Tongbram, Department of Food Engineering and Technology, School of Engineering, Tezpur University, Napaam, Assam, India _Ilknur Ucak, Ni gde Ömer Halisdemir University, Faculty of Agricultural Sciences and Technologies, Ni gde, Turkey Altaf Ahmad Wani, FAO, Wadura, Sher-e- Kashmir University of Agriculture Sciences and Technology, Shalimar, Jammu and Kashmir, India

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Nusrath Yasmeen, Faculty of Pharmacology, College of Nursing, King Saud Bin Abdul Aziz University for Health Sciences, Jeddah, Saudi Arabia; King Abdullah International Medical Research Centre (KAIMRC), National Guard Health Affairs, Jeddah, Saudi Arabia Syeda Saniya Zahra, Department of Pharmacognosy, Shifa College of Pharmaceutical Sciences, Shifa Tameer-eMillat University, Islamabad, Punjab, Pakistan Rasiya Ul Zaman, Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu Kashmir, India Imtiyaz Ahmad Zargar, Division of Food Science and Technology, Sher-e- Kashmir University of Agriculture Sciences and Technology, Shalimar, Srinagar, India Beenish Zehra, Department of Nutrition and Dietetics, Sharda University, Greater Noida, Uttar Pradesh, India

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Preface Our earth comprises of a plethora of plants enriched with certain ingredients which have tremendous nutraceutical potential. These plant-based nutraceuticals have been able to carve a niche in the global market in the form of medicinal and health-related food products as well. These nutraceuticals play a dual role as a food ingredient as well as a therapeutic agent preventing various diseases. This industry has been able to acquire tremendous attention not only from consumers but also from scientific communities and various food manufacturing organizations. The nutraceutical market has been evolving since past few years due to growing attention of researchers and modernistic and advanced techniques for the analysis of several qualitative and quantitative characteristics. Across the globe, a wide variety of chronic diseases such as cardiovascular diseases, cancers, diabetes, and obesity are rising tremendously and these diseases have been reported to contribute a very high percentage of 56.5 million deaths and high fraction of the diseases. Amid this scenario, consumers are drifting toward alternative beneficial products instead of expensive and high-tech disease treatment approach in the modern medicines. Consumers are aware of diet-related health problems and need to increase consumption of fruits and vegetables; however, this is not so feasible in our everyday life. Hence the incorporation of dietary supplements seems to be an only alternative for the procurement of these health friendly ingredients. The proposal of this book is designed in this way to highlight the inevitable and impeccable association between nutraceuticals of plant origin and health. The aim of this book is to undergo the elucidation of several pivotal nutraceuticals on the basis of several significant aspects such as derivatives, extraction, chemistry, mechanism of action, pharmacology, bioavailability, safety, and manifold applications. Nevertheless, majority of the plant-based nutraceuticals have been mentioned and studied in literature; more in depth and comprehensive knowledge about the selected nutraceuticals such as gamma oryzanol, lignans, beta-glucans, betalain, etc., is required. In this regard, keeping the future benefits of the researchers, academicians, health professionals, and lastly but not the least, public health in view, this proposed book is designed. Apart from this, toxicity and safety of these selected nutraceuticals will be also thoroughly discussed along with their bioavailability for the safe utilization into various foods. The various chapters in the proposed book based on different selected nutraceuticals will be an exploration of those ingredients which are worthy of attention on many aspects and need to be incorporated into our diet on daily basis. Hence, this book would lay a very solid foundation for analyzing the efficacy and validity of various plantderived nutraceuticals which can be exploited as a quintessential therapeutic tool in the prevention of chronic and degenerative diseases such as cardiovascular diseases, cancer, obesity, and diabetes as well as to promote sound human health. Dr. Jasmeet Kour Dr. Gulzar Ahmad Nayik

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Chapter 1

Nutraceutical-A deep and profound concept Jasmeet Kour1, Hitesh Chopra2, Saba Bukhari3, Renu Sharma4, Rosy Bansal5, Monika Hans6 and Dharmesh Chandra Saxena1 1

Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Sangrur, Punjab, India; 2Chitkara

College of Pharmacy, Chitkara Univeristy, Rajpura, Punjab, India; 3Department of Animal Genetics and Breeding, Skuast, Kashmir, India; 4

Department of Applied Sciences, Bhai Gurdas Degree College, Sangrur, Punjab, India; 5GSSDGS Khalsa College, Patiala, Punjab, India;

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Government PG College for Women, Jammu, Jammu and Kashmir, India

1.1 Introduction Consumer awareness regarding role of nutraceuticals is the important key factor which generates demand for nutraceutical sector. Consumers have wistful longing for specialty nutrition which leads to digestive health, beauty enhancement, specific chronic health problems, and so on. The major bone problems prevailing in society like osteoporosis and arthritis have also propelled the nutritionists to work in this direction. The protein progression has also led to design specialty nutraceuticals for children. Nutraceuticals working as pharma foods help in inhibition of cardiopathy, high blood pressure, osteoporosis, high blood glucose, and for lowering saturated fatty acids (Salmeron et al., 1997). This demand has revolutionized the food world to be offering a notable benefaction to good health as well as well-being of human beings. Nutraceutical acting as preventive foods also helps in improving the gastric and stomach problems as they work as probiotics and prebiotics as well. Overall nutraceuticals improve the immune system thus helping in fighting with harmful extraneous microorganisms. Many lifestyle-related diseases like cancer can be treated with the sensible intake of nutraceuticals. Although nutraceuticals are not the magic bullets which are directly targeting the cancer cells but they can prevent the further infections and inflammations and in turn will boost the immune system (Kessler et al., 2001). Wrong food intake habits can lead to colon cancer because of the conversion of precarcinogens to carcinogens by intestinal microflora. The enzymes like glycosides, azoreductases, and nitroreductases present in intestinal microflora convert these precarcinogens to carcinogens. The use of probiotic strains like Lactobacillus acidophilus and Lactobacillus casei helps to reduce the levels of these enzymes and so the generation of these enzymes will be reduced by imparting them anticancer effects. Many of the natural foods like fish, tomato, and green leafy vegetables have bioactive compounds which enable the oxidation of LDL. Some neurogenerative diseases like Parkinson’s disease which are known to be triggered by wrong foods and on the other hand can be reduced by nutraceuticals. Nutraceuticals are associated with following properties: l l l l l l l l l

Antioxidant properties Antiinflammatory properties Insulin sensitivity Anticancerous properties Affecting cell differentiation Increasing enzyme activity which helps in detoxification Upkeep of DNA mending Upsurge the programmed cell death of cancer Diminution in cell propagation

Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00021-1 Copyright © 2022 Elsevier Inc. All rights reserved.

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Nutraceutical mainly includes natural food and supplemented foods. Natural foods contain naturally bioactive nonnutritive components in high concentration that provides health benefits. Supplemented foods are the formulated foods which include the foods especially designed with increased biologically active compounds. So to summarize nutraceuticals potentially promote health beyond basic nutrition (Faisal & Varma, 2009). The research and development in therapeutic diets has come up with the concept of nutraceutical, nutraceuticals, designer foods, phyto foods, pharma foods, etc. These foods have great nutritional significance and all have one thing in common that is the presence of specific food ingredients which are targeted toward a specific physiological function of the body. Functional food components are beneficial components which are naturally a part of foods or they can be fortified in foods as per the requirements. These include, for example, polyphenols, carotenoids, isoflavones, dietary fiber, fatty acids, vitamins, minerals, phytoestrogens, isoprenoids, soya protein, prebiotics, and probiotics (Shinde et al., 2014). The nutritional components have a major role in health improvement as well as prevention of disease. All the foods which have positive effects on health and provide medical and health benefits are also known as nutraceuticals (Subbiah, 2007). The only difference between nutraceutical and pharmaceutical is that nutraceuticals target general state of health and work on disease prevention principle while pharmaceuticals influence particular state of health thus curing a specific disease with synthetic inputs (Das et al., 2012).

1.2 Facts of nutraceuticals There are various facts which state that nutraceuticals are beneficial for mankind. These are as follows.

1.2.1 Growth The nourishment of child starts in mother’s womb so mother diet influences the growth and development of child. The nutrition course during prenatal period and childbearing period and the composition of breast milk provides good quality proteins along with nutrients primarily including n-3 and n-6 polyunsaturated fatty acids, amino acids, micronutrients in the form of zinc, folic acid, iron, and iodine. These nutrient components are beneficial to health in cell and tissue growth as well. The bones development during adolescence requires calcium, Vitamin D and K, fluorides, etc., which are functional food components. The gastrointestinal growth during early years is dependent on probiotics and prebiotics (oligosaccharides and inulin).

1.2.2 Health maintenance It further includes following parameters to be considered (Pandey et al., 2010).

1.2.2.1 Immune function The nutraceutical components that help in boosting immune system are antioxidants, vitamins, trace minerals (zinc, copper, manganese), PUFA, arginine along with nucleotides, nucleosides, prebiotics, probiotics, and synbiotics. 1.2.2.1.1 Gastral well-being The gastrointestinal health is subjected to a proper equilibrium of good bacteria thus inhibiting the entry of harmful bacteria in intestinal tract. The intake of probiotics, prebiotics, and synbiotics can enhance the metabolic functionality of GI tract microflora. The probiotics are beneficial in reducing the GI infections and so improve the overall gut functioning along with reduction in constipation and diarrhea. The prebiotics are indigestible food components which stimulate the metabolic functioning of specific bacteria present in gut. They are proven in reducing colon cancer. The absorption of minerals like calcium and magnesium is being supported by beneficial bacteria. The dairy products, table spreads, baked foods, cereals, salad dressings, meat products, and some confectionary foods may contain prebiotics.

1.2.2.2 Mental health The nutraceuticals are helpful in promoting optimum mental state and mental performance. These foods may influence person’s intellect, temperament and liveliness, reaction to anxiety, short-term remembrance, attentiveness, and commitment. Glucose is known to influence mental performance, sucrose helps in reducing discomfort and pain, caffeine improves cognitive performance, and B vitamins improve mental health of old people. Many potential functional components like S-adenosyl methionine and folic acid are known to improve depression.

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1.2.2.3 Aging Aging leads to oxidative stress on the body. By delaying aging many diseases can be prevented. The defense against oxidative stress is present in our body itself. These are antioxidants, mineral and trace elements like selenium (Se), manganese (Mn), and copper (Cu), Vitamins C and E, carotenoids which are all free radical scavengers. Natural antioxidants like flavonoids, certain vitamins such as C and E, carotenoids, and other polyphenolic compounds existing in foods are possibly useful applicants for nutraceuticals (Berger & Shenkin, 2006). Herbal diets containing fruits like berries, grapes, pomegranate, mangosteen, and tomato are known to be explored by the nutraceutical sector as budding antioxidants.

1.2.2.4 Physical performance Nutraceuticals help in providing physical activity. Micronutrient supplementation during preparing nutraceutical potentially improves physical performance during physical training and the main ingredients include caffeine, precise amino acids, creatine, and carnitine. 1. Reduced risk of Obesity There are a lot of food components such as chitosan, medium-chain triglycerides, conjugated linoleic acid, green tea, calcium, and capsaicin that help in reducing appetite and enhance satiety or reduce fat absorption. This nutritional approach of weight management helps in reducing the risk of obesity (Kasbia, 2005).

1.2.3 Effect on chronic ailments (e.g., Heart disease, diabetes and metabolic disease, musculoskeletal disease) Nutraceuticals are protective against cardiovascular diseases which are mostly induced by dietary fats. Hence foods low in saturated fats and trans fats reduce LDL (low-density lipoprotein) levels. MUFA like olive oil and PUFA like linoleic and linolenic acids help in reducing LDL without leaving any effect on HDL cholesterol. Long-chain fatty acids like eicosatetraenoic acid and docosahexaenoic acid present in fish oil reduce plasma triglycerols and promote blood vessel integrity. The foods containing these functional unsaturated fatty acids help in reducing cardiovascular diseases (Hollman & Katan, 1999). Soluble fibers (psyllium and dietary fructans like inulin, oligofructose) as dietary intakes are also known to reduce LDL (Baljit, 2007). Phytosterols are found in fruits and vegetables, seeds and nuts, cereals and legumes, oils obtained from vegetables and plants. Diets containing herbal foods rich in polyphenolic compounds are associated with less chances of CVD and other long-lasting illnesses (Ramaa et al., 2006). The compounds flavonoids having varied array of polyphenolic composites including flavanols, flavones, flavanones, anthocyanins, and anthocyanidins existing unsurprisingly in foods obtained from plants are potential nutraceutical ingredients. Foods rich in fiber like whole foods, fruits, and vegetables, diets low in saturated fats, and foods having small glycemic index (GI) are recommended for obese and overweight people who are more at the menace of heart diseases and type 2 diabetes. Spices like cinnamon, garlic, coriander, turmeric, and ginger are beneficial for diabetes (Mechanick, 2005).

1.2.4 Musculoskeletal disease The most prevailing condition affecting older people is osteoarthritis. The diet therapy can be used with intake of substances like glucosamine and chondroitin as food supplements. Bone health can be maintained by nutrients like vitamins D, K, and C along with minerals like manganese (Mn), copper (Cu), and zinc (Zn). Vitamin D has an important share in bone mineral mass and bone development. Plant bioactive ingredients are useful ingredients for bone health.

1.3 Fiction of nutraceuticals There are many myths related to supplemented foods and nutraceuticals that does not support scientific evidences (Espin et al., 2007). These are as follows.

1.3.1 Supplements are meant for men and women It is believed that supplements should be taken by men and not women as this intake can lead to muscular look in females and take away their feminine look.

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1.3.1.1 No need of supplements with balanced diet intake Food is the great source of nutrients which are present in supplements but appropriate proportions are required to maintain the healthy body regime. The body needs vital nutrients to meet all the nutritional requirements and this can be made possible by supplements to cope up the nutritional deficiency disorders.

1.3.1.2 Protein burden on kidneys Sometimes it is believed that high proteins are a stress on human kidneys but this is not true. Kidneys efficiently remove the extraneous material and are meant for filtration. It is suggested to increase the water intake in case of high protein diets.

1.3.1.3 Undeclared nutraceutical ingredients are not safe It is misconception that the ingredients undeclared are disaster but the fact is that although these products are labeled as supplements but they are categorized as unapproved drugs legally. Many supplements like weight loss diets, sporty nutrition, and products related to sexual disorders are covered under adulterated category.

1.3.1.4 Antioxidants prevent cancer Research suggests that antioxidants like vitamins C and E can damage the free radicals thus preventing cancers. According to some findings b carotene can intensify the menace of pulmonary cancer in chain smokers (Thanopolou et al., 2006).

1.3.1.5 Zn for curing cold symptoms Research studies suggest that syrups containing zinc can reduce the cold duration if zinc is taken within 24 h of the initiation of cold but high amount of zinc intake can also hinder the copper absorption thus weakening the immune system. It has been advised to take 75 mg of zinc per day and to refrain from nasal sprays of zinc which can lead to smell loss.

1.3.1.6 Prevention of bone fractures in older age by calcium supplements It is suggested to supplement calcium in diet as deficiency of calcium can cause bone-related problems but it is a misconception that it will prevent the bone fractures in older age. The calcium required by body is to be provided in the form of supplements as normal diet rich in calcium cannot fulfill the adequate calcium requirements of approximately 1200 mg by human body in old age.

1.3.1.7 Nutraceuticals are as good as diet In research studies it has been indicated that nutrients isolated in pill form are having little advantages over dietary intake because in whole food, the nutrients work synergistically thus benefitting the body. The single nutrients contained in pills are not effective against chronic diseases and sometimes can even lead to harmful effects on body, e.g., excess vitamin A can cause osteoporosis leading to bone fractures. Similarly, antioxidants as nutraceuticals can aggravate cell growth leading to cancer (Rajasekaran et al., 2008). It is usually believed that nutraceuticals are not quality tested like other drugs as the nutraceutical companies do not require to provide the safety and effectiveness of their products but in contrast to this even the nutraceuticals are to be controlled by regulations and they are not allowed to be sold in the drugstores without appropriate approvals (Hathcock, 2001). There are many examples like scientists from Canada who tested the nutraceuticals from 12 companies and found that only 2 companies were having 100% purity and rest were adulterated with other plants having misleading components. Similarly, Attorney General Officials from New York performed DNA testing on herbs in 2015 and concluded that they were not containing actual herbs.

1.4 Developmental strategy for nutraceuticals Nutraceuticals are developed according to consumer preferences as follows: 1. Specialty nutrition for digestive health and these foods can be designed by food fortification with functional food components like vitamins, more minerals, fish oils, omega-3 fatty acids, and probiotics. 2. Natural forms which state that such foods should be devoid of any artificial additives like sweeteners. The foods should be in natural form and should be minimally processed or unprocessed. 3. Beauty enhancing foods which includes natural drinks, energy drinks, sports beverages, and cent percent juices.

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4. Protein-rich foods that are foods help in maintaining healthy bones, joints, and strengthen the immune system of muscle buildup. 5. Children-specific foods which include specific nutrients and calories specially required for kids’ growth and development. 6. Pharma foods which are helpful in preventing heart disease, hypertension, osteoporosis, type 2 diabetes, and also for lowering cholesterol. 7. Special food for allergic people like gluten free, lactose free, meatless substitutes like lentils and legumes, dairy free (can include soya milk, rice milk, or coconut milk in place of animal milk). 8. Sports foods that are given as supplements to sports persons like energy bars, nutrition bars, or energy drinks. 9. Weight management foods which are more in demand in today’s lifestyle and include whole grains, fiber-rich foods.

1.5 Disease prevention claims of nutraceuticals The major health claims related to specific diseases and their prevention by nutraceuticals are as follows: 1. Osteoporosis: Calcium intake and healthy diet accompanied by consistent exercise help children and adults to acquire good health of bones thus reducing severe diseases like osteoporosis in later stages of life. 2. Cancer: Cancer development is based on many factors. The foods low in dietary fat may reduce cancer risk. The body growth depends not only on the equilibrium between production of cells and demise of specialized cells but also on the imbalance resulting from unlimited cell proliferation inducing tumors. Apart from this, the increase in free radicals also leads to oxidative DNA damage which increases the onset of cancer. Amid this, the supplementation of diet with antioxidants has proven to be a boon. Low-fat diets that are rich in fiber having grains, fruits, and vegetables help in reducing the onset of cancer. 3. Hypertension: Diets with low sodium content diminish high blood pressure. 4. Coronary heart disease: Fruits, vegetables, and cereal products rich in fiber, mainly soluble fiber, help in reducing the risk of heart disease. Certain foods such as garlic, black cumin, ginseng, onion, guava leaves, and grape extracts have outshined in reducing cardiovascular ailments. 5. Immune dysfunction: Nutraceuticals and their bioactive components containing omega-3 fatty acids, dietary fibers, antioxidative compounds, plant sterols, and flavonoids play a pivotal beneficial role for human health. Phytochemicals act as immunomodulators along with possessing potent antiinflammatory properties. 6. Diabetes mellitus: This is usually associated with heart-related ailments and immune dysfunction in addition to insufficient liver enzymes primarily glutathione peroxidase, catalase, glucose-6-phosphate dehydrogenase, and glutathione S-transferase activity leading to ingress of oxidative stress damaging liver pancreatic b-cells. Functional foods inhibit diabetes apart from reducing its complications by virtue of antioxidant-rich foods like garlic, green tea, fenugreek, black cumin, Bauhinia forficata, Cissus sicyoides L., etc. Fiber-rich foods, e.g., barley, may aid in curbing high levels of cholesterol, triglycerides, and free fatty acid. A modern concept of self-care states that diet is correlated with health advantages that coexist with conventional therapeutic solutions to illness management. Scientific studies have clearly shown the potential dietary advantages of lowering the incidence of illness, and customers are already conscious that food has a larger effect on well-being. At the other side, consumers understand the costly, time-consuming, and impersonal issues of the new health-care program. Functional foods fit into a system that ranges from maintenance/promotion of health to treatment of disease. The primary goal in public health is to reduce the incidence in illness in a wide portion of the population through improving their lifestyle. In future, functional foods are going to be essential components of the established health programs to minimize the risk of specific diseases thus increasing consumer control and minimizing cost. The insights gained from biotechnological advances in genetic research give opportunities to prevent diseases and improving health with functional foods. This has made it possible to consider the role of nutrients in different systems at the cellular stage of the human body. Dependent influences of dietary components on each organism may also be extensively analyzed. Understanding the relationship between diet and genes is also a new discovery that will create new opportunities for potential generations to improve their fitness and well-being. Nutrigenomics, proteomics, as well as metabolomics are three emerging fields which will lead to the accelerated production of functional foods (Martirosyan and Singharaj, 2016, pp. 410e424). Nutrigenomics is known as the relationship between diet and genes. Dietary components of concern can include important nutrients such as pivotal vitamins, fats, minerals, certain bioactive compounds (e.g., phytochemicals), or metabolites in food additives (e.g., retinoic acid, eicosanoids). There is no proper description of a good or bad food but diets can be good or bad and overall lifestyle

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including smoking, physical activity, stress, etc., can impose effect on the overall health. Functional foods are more in demand by health-conscious sector and are still in nascent stage. The claims related to health benefits should be based on some particular scientific criteria (Clydesdale, 1997). There are a number of factors which weaken certain scientific foundation. These factors may include complexity of food components, effects on foods, metabolic changes occurring due to diet, and insufficient surrogate markers developing diseases. So additional research based on potential health benefits is required which will advance the public health. Functional food growth is expanding because of consumer awareness regarding natural foods providing protection against physiological threats. Although there are a series of products established in the market with unidentified health claims which create chaos to the consumers in terms of their real effectiveness. So, there is a need of uniform international policy for functional foods for their role in preventing physiological threats.

1.6 Sources of nutraceuticals and chemical nature Our earth is an abundant source of wide variety of plant sources having medicinal properties. Presently the two major concerns of developing countries are hunger and malnutrition. Several plants, herbs, fruits, and vegetables are blessed with important nutritive components such as carbohydrates, protein, vitamins, minerals, and essential amino acids (Murphy et al., 2012). Among the various fruits and vegetables, the prominent ones including bananas, grapes, watermelon, oranges, lemon, tomato, carrot, pomegranate, and ginger possess significant nutraceutical (Mahima et al., 2013). Plants and vegetables produce a wide range of phytochemicals which possess significant antioxidant potential. The various antioxidant pigments include anthocyanidins, catechins, carotenoids, flavonoids, isothiocynates, phytonutrients, and polyphenols. It is established that around 4000 phytochemicals are reported in organic products, green vegetables, and grains (González-Sarrías et al., 2013). Flavonoids comprising hydroxylated phenolic components are acknowledged for performing various pharmacological activities (Dixon et al., 1983). These are further categorized into several other subgroups such as flavonol, flavanone, flavone, and isoflavones. Citrus flavonoids namely naringin and naringenin were recognized for lowering of expression of vascular cell adhesion molecule-1 and monocyte chemotactic protein-1, with expected application in anticipation of atherosclerosis (Lee et al., 2001). Bioflavonoids such as naringin, isolated from grape fruit peel, are found to fix capillary permeability along with brittle nature of vascular tissues (Kosseva, 2013). Wine is an abundant source of essential flavanols such as catechins, proanthocyanidins that constitutes an important class of phenolic compounds and exhibits numerous beneficial effects (Shrikhande, 2000). In plants, flavanoid (apigenin) occurs as flavones in chamomile, flavanones (hesperidins) in citrus fruits, quercetin, kaempferol in tea, and ginkgo flavonglycosides in ginkgo (Majoa et al., 2005). Among vegetables, onion bulbs (Allium cepa L.) are reported to have significant amounts of dietary flavonoids which exert considerable effects on cancer, inflammation, and cardiovascular diseases (Okamoto, 2005). Lycopene, a principal carotenoid found abundantly in tomatoes, is known for imparting red color apart from protection from degenerative diseases such as cardiovascular diseases and cancer (Kosseva, 2013). Lutein, another naturally occurring carotenoid, contained in significant amounts in green leafy vegetables like broccoli, spinach, kale, yellow carrots, etc., is known for providing immense nourishment to our eyes and skin. Numerous antioxidant vitamins like vitamins C and E as well as carotenoids contained in considerable amounts in fruits and vegetables are well known for providing protection by scavenging free radicals (Singh & Devi, 2015).

1.7 Plant food by-products as source of nutraceuticals Fruits and vegetables produce about 25%e30% of nonconsumable waste products (Ajila et al., 2010). The failure to recover such materials results in generation of unnecessary waste as well as exhaustion of natural resources (Bhalerao et al., 1989). Several regions of vegetables and fruits are embedded with significant concentrations of bioactive components such as phenolics, carotenoids, flavonoids, and anthocyanins (Ayala-Zavala et al., 2004). Grape pomace, a by-product in most of the cases, is observed to have elevated level of antioxidants and antimicrobial substances obtained during wine-making process and is well recognized for its antioxidative, antiviral, and antiinflammatory activities. These health beneficial characteristics might be credited to higher concentration of phenolic substances. These polyphenolic substances may include anthocyanins, quercetin, and proanthocyanidins (Brazinha et al., 2014). Grape seeds are considered to be adequately rich in proanthocyanidins which act as nutraceuticals in numerous products. The proanthocyanidin extract of grape seeds is observed to neutralize free radicals and thus has the ability to impart protection against the oxidative stress induced by free-radical mechanism (Feng & Chen, 2003; Spranger et al., 2008).

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Biomass generated in fruit-processing industries prominently apple pomace including peels, core, calyx, seeds, and stem tissues is a storehouse of polyphenols (Vendruscolo et al., 2008). It is more prone to microbial disintegration due to its higher moisture content and the occurrence of carbohydrates prone to fermentation (Bhushan et al., 2008). Moreover, its dumping in open zones prompts serious ecological issues. It is generally utilized for extraction of various products such as pectin (Canteri-Schemin et al., 2005), organic acids (Shojaosadati & Babaeipour, 2002), enzymes (Zheng & Shetty, 2000), natural antioxidants (Lu & Foo, 2000), and edible fibers (Masoodi et al., 2002). Apple seed extracts contain prominent compounds such as phloretin-20-xyloglucoside, 5-caffeoylquinic acid, p-coumaroylquinic acid, and epicatechin (Fromm et al., 2012).

1.8 Nutraceutical profile of prominent nutraceuticals Lignans comprise diphenolic compounds which contain 2,3-dibenzylbutane skeleton (Setchell, 1995). These are formed by combining two coniferyl alcoholebased residues which are prevalent in the cell wall of plants (Westcott & Muir, 2003, pp. 55e73). In spite of having widespread occurrence in plant kingdom such as flaxseed, sesame seed, pumpkin seed, soybean, berries, and huge consumption in Western countries, these have received comparatively little attention (Touré & Xueming, 2010). Lignans are of immense interest owing to their capability of showing a broad range of health promoting effects which prominently include antioxidative, antiviral, and antitumorigenic (Ayres & Loike, 1990). Apart from this, lignans are also known to be antidiabetic and anti-obesity agents (Bhathena & Velasquez, 2002) and may even impart protection against cardiovascular diseases (Vanharanta et al., 1999). Several in vitro and animal studies have proven that lignans impart immense protection in Graves’ diseases like breast cancer, prostate cancer, colon cancer (Bowen, 2001; Denmark-Wahnefried et al., 2001; Yang et al., 2001), as well as cardiovascular diseases (Prasad, 1999, 2001). A considerable research attention has been attained by beta-glucans due to their significant health benefits as well as various physicochemical properties. Beta-glucans have been known to lower plasma cholesterol, glycemic index, as well as improving lipid metabolism (Delaney et al., 2003; Keenan et al., 2007). These may also contribute in minimizing the incidence rate of coronary as well as ischemic heart diseases (Maki, Galant et al., 2007) in addition to lower down total, LDL cholesterol and ratio of total to HDL cholesterol (Behall, 1997). Another major aspect related to the nutraceutical profile of b-glucans is that they provide humoral as well as cell-mediated immunity and enhance the immunological activity when consumed on daily basis (Estrada et al., 1997). Gamma oryzanol (GO), one of the most significant constituents of rice bran oil, has been reported to reveal numerous health-promoting effects such as decline in plasma cholesterol and improvement in plasma lipid profile and HDL cholesterol levels (Cicero & Gaddi, 2001). Various biological activities like antioxidative (Ismail et al., 2010), antiinflammatory (Akihisa et al., 2000), and antitumor activities (Yasukawa et al., 1998) have been reported by GO. Owing to the significant nutraceutical properties of beta-glucans, lignins, and GO an isolation process was derived for the extraction of beta-glucans from barley flour, lignin concentrate derived from flaxseed powder, and GO concentrate from rice bran oil in fairly good yield (Kour et al., 2019).

1.9 Mechanism of action of nutraceuticals There is a wealth of hidden food ingredients and nutrients that possess significant biological activities. In the recent years, nutraceutical industry has been growing rapidly in the present food market. It has been significantly proven that food ingredients can be helpful to prevent several disorders. Numerous research studies have highlighted the role of fruits and vegetables in the inhibition of cardiovascular diseases (Hu & Willett, 2002). The protective action of plants may be credited to the existence of various phytochemical components. German and Walzem (2000) evaluated that polyphenolic compounds contained in grapes and wine can prevent arterial disease by interfering with cellular metabolism and signaling. Antioxidant substances comprise an essential part of nutraceuticals. A wide range of compounds possess significant direct apart from indirect antioxidative properties. These antioxidants are known for prevention of deleterious effects owing to oxidation process. Oxidative stress might be due to generation of highly reactive oxygen and nitrogen related to body. Free radicals are the major cause of various degenerative diseases (Cornelli, 2009). Antioxidant substances can provide protection against various degenerative diseases as they can curb the production of free radicals by lowering the energy of these highly reactive species. For example, consumption of vitamin E is highly beneficial for preventing Parkinson’s disease (De Rijk et al., 1997). Terpenes, one of the prominent categories of phytonutrients prominently seen in green foods, cereals, and soy plants, exhibit exceptional antioxidant activity. These substances react with radical species by disassociation into fatty membranes

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with their long carbon side chain and facilitate in treatment of various diseases (Jain & Ramawat, 2013). Antioxidants are also recognized for inhibiting mutation and preventing dreadful diseases due to their ability to scavenge superoxides, hydroxyl, as well as peroxy radicals (Hochstein & Atallah, 1988). Apart from scavenging of free radicals, these antioxidants also participate in the formation of complexes of redox-catalytic metal ions (Varzakas et al., 2016). Phenolic compounds hail from that class of secondary plant metabolites which contain either one or several aromatic rings having hydroxyl groups in their basic structural composition (Balasundram et al., 2006). This heterogenous group of chemical compounds is recognized for varied activities of biological origin (Ignat et al., 2011; Popa et al., 2008). These play a significant role in maintaining the nutritional profile of fruits and vegetables (Lapornik et al., 2005). Phenolic antioxidant substances comprising tocopherols, phytoestrogens, and green teaederived polyphenols aid in decreasing oxidative cell injuries, inflammatory reactions leading to an efficient brain health (Bates et al., 2002; Youdim & Joseph, 2001). Flavonoids which form another prominent class of naturally obtained ingredients consisting of a benzo-g-pyrone structure are also recognized for performing various biological activities. Their chemical properties are based on their structure, extent of hydroxylation, class, conjugation, substituents, and degree of polymerization (Heim et al., 2002). The protective mechanism of flavonoids is to block enzymes which convert angiotensin that raises blood pressure and blocks cyclo-oxygenase known as “suicide” enzyme which is responsible for breaking down prostaglandins thus preventing platelet aggregation (Chintale et al., 2013). Flavonoids exhibit a significant role in inhibition of dreadful and degenerative diseases like cardiovascular diseases and cancer (Okamoto, 2005). These major active nutraceutical ingredients in plants are highlighted to exhibit antiallergic, antiinflammatory, hepatoprotective, antiviral, anticarcinogenic, and antioxidative characteristics (Singh & Devi, 2015).

1.10 Nutraceuticals in ayurveda Hippocrates, father of medicine, well said ‘Let food be thy medicine and medicine be thy food.’ Nutraceuticals refer to those nontoxic foods that are not considered as essential nutrients but have biological significance such as disease resistance, antioxidants, and regulating the immune systems. It refers to herbal products, prebiotics, and probiotics that act as medicine for the health of humans by inhibiting chronic diseases and performing far above than basic nutritional function. Due to sedentary lifestyle their has been increase in the occurrence of the disease. Nutraceuticals hold impeccable importance because of their potential nutritional status, safety, and therapeutic effects. Quality of life has sparked the ‘Nutraceutical Revolution’ and has led to seek complementary beneficial products. There has been an elevating interest in nutraceuticals which have great health benefits and work better than modern medicine and have their roots deep embedded into the traditional system of Indian medicine, ‘‘Ayurveda.’‘ The Ayurvedic came into light when it was developed between 2500 and 500 BCE in India (Subhose & Narayana, 2005). The literal meaning of Ayurveda is “Science of life or Science of Longevity” because it offers to live a long and healthy life. It rejuvenates the body by means of diet therapy. From ancient times, Ayurveda along with other Indian sciences significantly contribute to the field of medicine and the art of living inclusive of culture in all areas of human needs. “Annam bramha” is a firm belief in ancient Indian culture, which translates to mean the presence of divinity is perceived in all that is consumed. Food has been priced the most important aspect in life as it nourishes mind and body. “Nutraceuticals,” “functional foods,” “wellness foods,” “medicinal foods,” “pharma foods” are the terms which have been able to grab significant attention today which is worth appreciation among common masses. A nutraceutical is demonstrated to unleash physiological role by imparting protection against chronic disease. Ayurveda has also stated the same principle as “Swasthurrjaskar Chikitsa” that means the different ways by which one can achieve health. Primary principal of Ayurveda is also to keep the body healthy. But Ayurveda has a broader vision because “Rasayana Chikitsa” means rejuvenation therapy. Rejuvenation means to return to the normal from a diseased state, it also talks about “Achar Rasayana” that proves mental stability, which is equally important for physical stability. Nutraceuticals coupled with traditional medicine are given whole-hearted acceptance by the consumers backed by modern medical research. As health-related awareness increases in the population, people become more conscious about their future health. Ayurveda, the 5000-year-old health science, has mentioned tremendous benefits of food for medicinal purposes which are aimed at providing basic nutrition and at the same time reducing disease risk. The use of herbal products as nutraceuticals has been successful in unleashing therapeutic properties having negligible side effects along with great monetary gains. The concept of ‘Ajasrika Rasayana’ relates to those foods which can be consumed to improve the quality of life and it protects from stress induced by both external and internal sources. Nutraceuticals in common usage belonging to

Nutraceutical-A deep and profound concept Chapter | 1

9

Ayurveda include Chyavanprash (for general health and prevention of respiratory disorders); Phala Ghrita (for reproductive health); Arjuna Ksheerpaka (for cardioprotection); Brahma Rasayana (for protection from mental stress); and Rasona Ksheerpaka (for cardioprotection) and Shatavari Ghrita (for general health of women during various physiological states). Dietary supplements and herbal remedies are popular healthy alternatives for people. These supplements complete the nutritive value of the diet and some of them are: Asparagus racemosus (Wild Shatavari) is a potent Ayurvedic rejuvenative and strengthens the immune system; Commiphora mukul is a major ingredient in joint and possesses strong immunomodulating properties; Cyperus scariosus maintains healthy genitourinary system along with hepatoprotective action. Garcinia cambogia is a fruit having hydroxycitric acid which hampers lipids and fatty acids. Hydroxycitric acid is responsible for the inhibition of the enzyme ATP-citrate leading to decrease in the production of acetyl-CoA which plays a crucial role in metabolizing fat and carbohydrates. Glycyrrhiza glabra (licorice) is a versatile medicine for gastrointestinal health. Being a laxative, it helps in the healing of mucous membranes apart from relaxing the muscle spasms. It shows antioxidative action in cancer protection and has key role in interferon production. Gymnema sylvestre has glycolytic action and Melia azadirachta (neem) is used as astringent that promotes healing. Momordica charantia (L. Karela, bitter melon) finding its widespread usage in Ayurveda contains gurmarin, which is similar to bovine insulin that suppresses responses of neurons in brain to sweet taste. Moringa pterygosperma is abundantly used in numerous health requirements and contains physiologically active principles. It contains “pterygospermin,” an antibiotic-like substance. Nardostachys jatamansi (DC. Jatamansi, musk root) is a plant effective for mental health. Piper longum (L. pippali, Indian long pepper) is a powerful stimulant for both the digestive and the respiratory systems and has a rejuvenating effect on lungs. Pippali is a typical Ayurvedic complementary component whose benefit is to increase the bioavailability and enhance absorption of the other active ingredients. Piper nigrum (L. Maricha, black pepper) is one of the most important spices which is widely used to amplify the body’s ability to absorb nutrients contained in the food and aid the digestive process. Bergenia ligulata (Wall Pasanavheda) is diuretic in action. Terminalia chebula (Retz. Haritaki) is effectively used as a purgative, expectorant and is a strong antimutagenic agent. Tinospora cordifolia (Miers Guduchi) is a potent immunity booster having high content of vitamin C. Withania somnifera (L.) Dunal Ashwagandha holds a place similar to Ginseng in traditional Chinese medicinal therapies. The “Indian Ginseng” Zingiber officinale Rosc Sunth enhances absorption and prevents gastrointestinal side effects. The nutraceutical market is growing by leaps and bounds in functional foods and herbal supplements. Consumer’s awareness and market strategies need to be developed to increase the consumption of these nutritional foods.

1.11 Recent trends in nutraceuticals Nutrition is a need. Many illnesses related to health result from an improper nutrition. Nutraceuticals is an emerging alternative approach which is a fast-growing food industry with millions of people in the world using natural products. There has been a tremendous growth in global nutraceutical market in last decade. In India, functional foods are expected to witness much higher growth rates when compared to dietary supplement over the next 5 years. Nutraceutical food comes into medicinal format such as tablet, capsule, or powder while modern nutraceuticals are available as forms of food or as whole food itself or included in foods. With the billions of rupees being spent on costly drugs and surgical procedures, nutraceutical-based approach seems a magical and logical intervention which is relatively less invasive and inexpensive. In the modern era of fast foods, sedentary lifestyles, and increasing rates of preventable disease, physicians and clinical nutritionists should be encouraged to further knowledge with regard to safe alternatives available to treat the diseases. The majority of the nutraceuticals do possess multiple therapeutic benefits and have been claimed to have physiological benefits or provide protection against various diseases such as the cardiovascular, obesity, diabetic, cancer, and chronic inflammatory disorders. The raised demands for health care have dramatically increased the cost of medical care. Therefore, people have tried to achieve a better quality of life by eating more plant foods or taking dietary supplements. Development of nutraceuticals with distinctive traits has the potential to deliver healthy and cheaper products to the world. The current demand for novel and healthy foods together with the increasing lifestyle diseases has created a new market for healthy cereals and legumes as a replacer to fast foods. The current focus has now been shifted toward millets and pulses for enhancing nutritional and functional quality for high-end consumer. The current trends in use of extruded flours as novel green ingredients as phenolic rich hydrocolloids, gluten free, low glycemic fat replacers are used nowadays as functional foods. Recent work on nutraceuticals highlights the potential of extruded flours as healthy snacks, breakfast cereals, or as additions to breads and mayonnaises (Martinez et al., 2014; Román, Santos et al., 2015). Modified functionality of extruded flours offers wide applications in food industry as thickening, gelling agents, functional ingredients, and fat replacers (Mason & Thompson, 2014; Román, Martínez, & Gómez 2015).

10

Nutraceuticals and Health Care

The novel trend in nutraceuticals is now the introduction of extrusion technology which involves extensive processing of cereals for development of healthy snacks. Extrusion is a novel technology with art of making quality foods beyond the traditional foods. Extrusion is a process to improve stability and texture of food by the processing conditions. Improved functionality of processed flours can be used in development of gluten-free, high fiber, high phenolic, high mineral, lowfat, and low-glycemic foods. Processed flours mimic the properties of hydrocolloids and thus can be used as alternatives to synthetic hydrocolloids with “green label.” This technology can supplement baking industry to develop products to the current changing needs and taste of consumers. From a nutritional viewpoint, they are high in starch and fats and with low dietary fiber, thus with high glycemic load, known to cause childhood obesity and trigger type 2 diabetes (Brennan et al., 2013; Omwamba & Mahungu, 2014). Present day snacks are deficient in essential amino acids and hence have low protein and biological value (Devi et al., 2013). This calls for enrichment of snacks with high protein, dietary fiber, mineral content so to qualify them as functional foods. Use of whole grains, millets, pulses, pseudocereals, and other naturally derived ingredients is a new open window in development of functional healthy snacks. The nutraceutical industry is rapidly growing (7%e12% per year). With extensive anecdotal data on exciting health results, nutraceuticals promise significant contributions to disease prevention. The global nutraceuticals market is estimated at 117 billion US dollar of which India’s share is a meager 0.9%. The United States and Japan are key markets for nutraceutical intake. Indian nutraceuticals market is about 1 billion USD which is increasing day by day. Globally the nutraceuticals are the fastest growing industry (http://www.ficcinutraceuticals.com/). Various nutraceutical combinations have entered the global market through introduction of ethnopharmacological claims made by different traditional practices. To live a long and healthy life, the diet must be nutritious. Nutraceuticals play a large role in providing optimum nutrients. These nutraceuticals construct a protective barrier against large number of infections and allergies.

1.12 Recent developments in nutraceuticals In developing countries where under nutrition, awareness and increased exposure to infectious diseases are common due to poverty and inadequate sanitation thereby increasing the need for healthier, nutritious, and less costly alternative. During the pandemic of COVID-19, there is an increased demand for nutraceutical ingredients such as herbal supplements, dietary nutrition products for boosting immunity, and thus market growth of immunity booster product grows on steady-state. The worldwide nutraceutical market is growing day by day in the form of dietary supplements and functional foods and beverages. But dietary supplements are not as effective as their natural sources. They need to be enriched further with minerals and vitamins. Therefore, there is a need for the development of methods to increase the nutritional value of the foods and this can be achieved by the use of molecular technology. Biotechnological methods can be used for studying and cloning of various genes which can help to induce the expression of bioactive compounds. Development of nutraceuticals for novel health benefits, elucidating mechanisms of action of these products, and development of study systems such as in vitro coculture cell models can help in the elimination of various health issues. Modeling new eating habits using the existing knowledge is needed for the eventual ideal of ‘Nutrition for all’ vision. WHO estimates that 60% of the cardiac patients in the world will be Indians by 2030. Asia is expected to have 190 million diabetes cases, more than half of them are in India and China (Jasinski et al., 2013). Researchers say that the percentage of overweight people in India is on track to rise from 9% in 1995 to 24% in 2025. Use of nutraceuticals in a sports industry is a new future development in the nutraceutical industry which is exciting and opens a way for greater research and efficacy in nutraceutical world. Some recent developments in nutraceuticals industry include launching by Kemon Industry (US) in May 2020, the new immunity-boosting nutraceutical ingredients made of algae source beta-glucan. In December 2017, Arla Foods and PT Indofood CBP Sukses Makmur Tbk (Indonesia) launched a joint venture to expand Arla Foods’ presence in the Indonesian market. In August 2018, ADM announced that it would add Onavita algae DHA powder to its line of omega-3 product solutions. The new omega-3 powder is part of ADM’s portfolio of health and wellness ingredients, including probiotics, vitamins, plant extracts, and nourishing oils. In October 2018, Cargill launched Latitude, a plant-based, sustainable alternative omega-3 source for fish feeding applications. In 2019, the Asia-Pacific region became a crucial region in the global nutraceutical market as consumer health issues increased. The main key players in the market are Archer Daniels Midland Company, Cargill, Incorporated, Royal DSM N.V, BASF SE, Nestle S.A., Groupe Danone S.A, E. I. du Pont de Nemours and Company, PepsiCo Inc., Aland (Jiangsu) Nutraceutical Co., Ltd., General Mills, Inc., Blytheco, Gamajet, Pharmachem Laboratories, Balchem Corp, Alpha Packaging, Aker BioMarine, Barrington Nutritionals Premier Nutraceutical Pvt Ltd., Sydler India Marlyn Nutraceuticals. Nutritional therapy has emerged as new healing concepts which has quickly and widely spread in recent years. Strong recommendations for consumption of nutraceuticals, use of nutritional therapy, and phytotherapy have become

Nutraceutical-A deep and profound concept Chapter | 1

11

progressively popular to improve health, and to prevent and treat diseases. There is need of hour to improve awareness about conventional nutraceutical ingredients to the Indian consumers which is severely limited which will unravel an opportunistic window to the growing Indian nutraceutical market.

1.13 Patents on nutraceuticals The nutraceuticals are the products that cover broad term of food sources that provides extra benefit apart from basic nutritional value. The number of patents has been assigned for various nutraceutical products starting from extraction method to formulation for various products. Some of them are available at commercialization level, and some of them got expired. The section provides bird eye view about the various nutraceuticals patented for various uses and method of preparation.

1.13.1 Berberine The berberine (BBR) is a main active ingredient of Rhizoma coptidis, which is traditional Chinese medicinal herb that found its use in inflammatory disorders and diabetes mellitus (Pang et al., 2015; Tan et al., 2016). Chemically, it is ammonium salt in quaternary form derived from the isoquinoline class of alkaloids, having molecular weight of 336.36 g/ mol (Battu et al., 2010; Wang et al., 2017). It has sparing solubility in water, and possesses characteristic bitterness. Nevertheless, BBR has also been used as antibacterial drug for human use (Peng et al., 2015) but it has shown some serious side effects, leading to ban on use as injections. Then it was formulated as tablet dosage form, but it has poor oral absorption, solubility being a limiting factor (Zhu et al., 2013). The BBR as salt form has higher solubility (Miyazaki et al., 1981). Kaimin (2003) filed the WO/2003/090749A1 patent for high solubility of BBR and its applications in various ailments. The highly soluble BBR was able to dissolve in 500 parts of solvent, while to qualify for solubility it must be able to dissolve in 100 parts, showing very promising results. The BBR thus prepared was able to treat blood glucose levels and treating complications of diabetes. The BBR has also been utilized as the oral administered formulation showing eulipidemic, hypocholesterolemia and hypotriglyceridemic, antioxidant, and protective agent of the vascular endothelium (Luigi et al., 2012). As per patent no. US8673864B2, the mangiferin-berberine salt was synthesized as AMPK activator (Teng & Wei, 2014). The magniferin as its own is acidic while berberine is alkaline in nature. Under certain conditions they form salt, but the complex is not so strong and held by weak Van der Waals forces. It gets dissociated under physiological conditions, causing both moieties to act separately and play their independent role. Table 1.1 shows various patents related to berberine.

1.13.2 Beta glucan From the past two decades, the beta-glucan has been investigated as the potent immunological activator. They have also known to have anticancerous (Aleem, 2013) or antiinflammatory activity (Bacha, Nasir, Iqbal, & Anjum, 2017), insulin resistance (Zheng et al., 2013), antihypertension (Maki, Davidson et al., 2007; Maki, Galant et al., 2007), and anti-obesity effects (Li et al., 2019). Also it has been reported to cause activation of immune system (Chan et al., 2009), and modulate the humoral and cellular immunity. Gordon worked on cancer therapy and used neutral soluble glucan and monoclonal antibodies (Gordon, 2016). Betaglucan is supposed to enhance the tumoricidal activity via binding to the C3 compliment protein receptor CR3. The method further described the use of neutral soluble glucan along with rituximab for the eradication of non-Hodgkin’s lymphoma. The glucans has also been investigated as the hematopoietic agents, for example. In US patent No. 5532223, the researchers revealed the use of glucan that is neutral and soluble that stimulates the hematopoietic and immunological effects without stimulating the production of unwanted cytokines (Spiros et al., 1996). The beta glucan exists as globular glucans having particle size of 5e50 microns, though the size can be reduced when subjected to external force for particle size reduction; however, when it comes in contact with the rehydration process it gets reaggregated to form clumps. The US patent No. 6,476,003B1 investigated method for production of small particles size nonaggregated microparticulate beta 1,3/1,6 Glucan. The 1/3 and 1/6 glucan obtained after the patent process was able to normalize, potentiate, and modulate the immune response from macrophages (Frank et al., 2002). Ikewaki et al. (2007) compared the immunological activity of Sophy-Beta-glucan US Patent 6956120 and Japan Patent 2004e329077, obtained from the black yeast Aureobasidium pullulans strain AFO-202. The glucan was able to inhibit the DNA synthesis and IFN-alpha production. Moreover the presence of immunoglobulin G (IgG) was detected in sera derived from normal adults. Table 1.2 has depicted various patents related to beta-glucans.

12

TABLE 1.1 List of patents related to berberine.

Patent No.

Description of patent

Inventor

Patent publishing year

1

WO2003090749A1

Prepared medicament with highly soluble berberine

Kaimin Wu

2003

Wu (2003)

2

WO2004093876A2

Described use of berberine and antimicrobial agent for the treatment of oral pathogens

Douglas Kinghorn, Sara Kate Roberts, Christine D Wu

2004

Kinghorn et al. (2004)

3

CN102989017B

Described method for use of berberine in tumor diagnosis

Fan Chengzhong, Wu Xiaoai, Mei Xiaoli, Liang Zhenglu, Li Yunchun, He Ling, Xiao Hengyi

2014

Chengzhong et al. (2014)

4

US9358261B2

Additional artemisinin and berberine compositions and methods of making

Bob Rosen

2016

Rosen (2016)

5

CN105560233A

Berberine and fenofibrate pharmaceutical composition and use thereof

Su Xianying, Zhao Limei, Xing Dan, He Xin, Sun Jukui, Song Lin, Na Xinzhu

2016

Xianying et al. (2016)

6

CN102151260B

Andrographolide and berberine combination and application thereof

Wang Yuqiang, Du Enming, Xu Lipeng, Cheng Qian, Ren Junlan

2012

Yuqiang et al. (2012)

7

US6280768B1

Berberine alkaloids in the treatment of diarrhea induced by protozoa

Joseph T. McDevitt

2001

McDevitt (2001)

8

WO2004032924A1

Described the use of berberine as insulin sensitizer

Jiandong Jiang, Jing Wei, Zizheng Wang, Huaining Pan

2004

Jiang et al. (2004)

9

JP2020073546A

Method of preparing and application of berberine salts, ursodeoxycholic acid and its salt

Liping Liu

2020

Liu (2020)

10

CN102949375B

Berberine hydrochloride solid lipid nano-preparation and preparation method thereof

Yang Shuyu, Xue Mei, Li Xuejun

2015

Shuyu et al. (2015)

11

EP2448577B1

Disclosed preparation containing berberine for its use in treating rosacea and red face related diseases

Shuen-LuHung, Wen-Hung Chung, Tse-Wen Chang

2016

Hung et al. (2016)

12

CN105920018A

Use of tripterine and berberine for metabolic syndrome treatment.

Ning Guang, Zhang Yifei, Zhang Zhiguo, Wang Shujie

2019

Guang et al. (2019)

13

CN1279946A

Berberine hydrochloride suppository and its producing method

Li Zhongkun, Wang Chongjing, Wang Yan

2004

Zhongkun et al. (2004)

14

WO2017027971A1

Delivery of berberine and its compounds across epidermis

Joseph Gabriele, David Baranowski, Beth Buchanan, Jonathan Zuccolo, Mikaela Teris

2017

Gabriele et al. (2017)

15

CN101461949B

Berberine cyclodextrin inclusion compound, preparation thereof, and preparation method

Cui Yuanlu, Zhang Ye, Tian Junsheng, Zhang Yanbin

2011

Yuanlu et al. (2011)

17

CN101940229A

Berberine plant source insecticide and preparation method thereof

Zhang Rong, He Jia, Zhang Yi, Chen Honghao, Gao Liyuan, Ma Jianhua, Zhu Mengmeng, Wang Fang

2011

Rong et al. (201)1

S.No.

References

Nutraceuticals and Health Care

Berberine

18

CN103372210A

Application of berberine combined chemotherapeutic medicament in antitumor therapy

Gu Wenwen, Wang Yun, Cheng Tao

2013

Wenmen et al. (2013)

20

US20110281852A1

Use of berberine composition for weight gain therapy and obesity related to antipsychotic drugs

Gareth Davies, Yueshan Hu

2011

Davies and Hu (2011)

21

CN104069066A

Berberine-sodium caprate solid dispersion and application of same in treating diabetes and complications thereof

Chen Li, Zhang Ming, Li Jing, Meng Zhaojie

2017

Li et al. (2017)

22

CN104997735A

Berberine hydrochloride taste-masked pellet, and preparation thereof

Han Zhiqiang

2015

Zhiqiang (2015)

23

CN102813928A

Method for modifying bitter taste of berberine, berberine hydrochloride and related compound preparations

Wang Youjie, Xu Desheng, Liang Shuang, Feng Yi, Ruan Kefeng

2012

Youjie et al. (2012)

24

CN1589793A

Application of tannic acid berberine in preparation of medicine for treating ulcerocolonitis

Jiang Wanglin, Fu Fenghua, Tian Jingwei, Wang Chaoyun, Sun Fang

2005

Wanglin et al. (2005)

25

US20120321726A1

Berberine-containing pharmaceutical composition for inhibiting cancer stem cell growth or carcinoma metastasis and application thereof

Hsiu-Mei Hsieh, Chen-Yu Lee, Chih-Chien Shen, Tien-Chun Wang

2012

Hsieh et al. (2012)

26

CN102349520B

Application of berberine as herbicide

Zhou Lijuan, Huang Jiguang, Xu Hanhong

2013

Lijuan et al. (2013)

27

EP3243823A1

Mangiferin-6-o-berberine salt and preparation method and use thereof

Houlei Teng, Wei Wu, Jingzhuo Zhang, Zhe Lin

2014

Teng and Wu (2014)

Nutraceutical-A deep and profound concept Chapter | 1 13

TABLE 1.2 List of patents related to beta-glucans. Beta-glucan

14

Description of patent

Inventor

Country

WO2016073763A3

Beta-glucan methods and compositions that affect the tumor microenvironment

Nandita Bose, Keith Gorden, Anissa Sh. Chan, Steven Leonardo, Jeremy Graff, Xiaohong Qiu, Takashi Kangas, Kathryn A. Fraser, Adria Bykowski Jonas, Nadine Ottoson

France

2016

Bose et al. (2016)

US8753668B2

Production of beta-glucans and mannans

Joseph James Sedmak

United States

2014

Sedmak (2014)

US6323338B1

Method for concentrating b-glucan

Richard C. Potter, Philip A. Fisher, Kirk R. Hash, Sr. John D. Neidt

United States

2001

Potter et al. (2001)

US20070042930A1

Described the effect of beta-glucan on tissue repair

Gordon Ross, Trunetta Jo Ross

United States

2007

Ross et al. (2007)

US7550584B2

Methods of purifying beta-glucans

Arun K. Bahl, Sharon V. Vercellotti, John R. Vercellotti, Elias Klein

United States

2009

Bahl et al. (2009)

US7786094B2

Described the use of glucan against the biological warfare weapons

Gary R. Ostroff

United States

2010

Ostroff (2010)

US20040023923A1

Defined the process of preparation of cold water esoluble beta-glucan

Keith Morgan

United States

2004

Morgan (2004)

US20050245480A1

Methods of using beta-glucan as a radioprotective agent

Gary Ostroff, Gordon Ross, Trunetta Ross

United States

2005

Ostroff et al. (2005)

CN100467583C

Disclosed novel microorganisms capable of producing beta glucan

Tsubaki Kazufumi, Sugiyama Hiroshi, Tokai Lin Yoshiwa

China

2005

Kazufumi et al. (2005)

US20170056432A1

Cancer therapy using beta-glucan and antibodies

Gordon D. Ross, JR.

United States

2017

Ross (2017)

CN100540010C

The application of beta-glucan in the medicine of preparation opposing biological war weapon and the pathogen that comprises anthrax

Gary R. Ostrov

China

2009

Ostrov (2009)

US7777027B2

Purified beta-glucan composition

Arun K. Bahl, Sharon V. Vercellotti, John R. Vercellotti, Elias Klein

United States

2010

Bahl et al. (2010)

KR20090009513A

Defined the method of producing the low-molecularweight beta-glucan by irradiation

Jae-Hoon Kim, Jae-Hoon, Hong Sung Nak Yoon, BeomSeok Song, Joo-Woon Lee

Korea

2009

Kim et al. (2009)

EP1453909B1

Cereal beta-glucan compositions, methods of preparation, and uses thereof

Mark J. Redmond, David A. Fielder

Europe

2007

Redmond and Fielder (2007)

KR20120117011A

Dietary fiber composition containing beta-glucan

Rueddy Durs

South Korea

2012

Durs (2012)

KR100862096B1

Formulation containing beta glucan and bitter melon extract for antidiabetic activity

KiKim Rin-soo, Park Bok-ryeon, Seobu Il, Ahn Hyo-chan, Yang Gun-ju, Lee Hyung-sik, Jang Hee-jung, Jo Hyeongrae

South Korea

2008

Rin-soo et al. (2008)

WO2001078534A2

Beta-glucan compositions for reducing hypercholesterolemia and controlling of postprandial blood glucose and insulin levels

Jeffrey John Kester, Robert Lawrence Prosise, Kevin Patrick Christmas, Joseph James Elsen, Ralph Lawrence Helmers, Jr. Thomas Joseph Wehmeier

World patent

2002

Kester et al. (2002)

References

Nutraceuticals and Health Care

Patent No.

Patent publishing year

Nutraceutical-A deep and profound concept Chapter | 1

15

1.13.3 Gamma oryzanol GO is the product obtained as the unsaponifiable matter of crude rice bran oil, obtained after milling processes of rice (Lakkakula et al., 2004). It has been found that it is mixture of variable quantity of ferulic acids esters of phytosterol and triterpens alcohols. About 10 varieties of sterylferulates have been identified in the GO, e.g., cycloartenylferulate, 24-methylenecycloartanyl ferulate, campestenylferulate, campesterylferulate, stigmastenylferulate, sitosterylferulate, D7-stigmastenyl ferulate, stigmasterylferulate, campestanylferulate, and sitostanylferulate (Xu, 1999). The components of GO have been purified via use of HPLC and isomeric units have been identified by crystallization, NMR, and MS (Bao et al., 2013; Feng & Chen, 2003; Norton, 1995). It has been proven that GO is one of the scavenger of 2,2-diphenyl-1picrylhydrazyl (DPPH), hydroxyl, and superoxide radicals (Juliano et al., 2005). Also it has been evidenced that GO reduces the lipoprotein such as very-low- and low-density content and cholesterol level and increase the high-density lipoprotein in hypercholesterolemic hamsters (Wilson et al., 2007). Kour et al. (2019) evaluated effect of GO concentrate on the nutritional, textural, pasting, thermal, structural, and morphological properties of corn and rice flour blend based RTE extrudates. The Chinese patent No. CN101292942A discloses dermatological formulation of GO and sodium bisulfate (Saii et al., 2008). The GO has fat-soluble properties for dermatological application; it is dissolved in oil phase as emulsion which during preparation is of clear color while it gets discolored subsequently. The discoloration issue was resolved in patent by use of sodium bisulfate. In case of transdermal system, when GO is applied it causes to increase the stability of active material entrapped inside it due to its inherent antioxidizing property hence increasing the physicochemical stability of active material. The Korean patent No. KR20120114730A discloses method for preparation of microemulsion using lecithin and GO (Kim, 2012). When the microemulsion is applied on the skin, the interfacial membrane is destroyed during applying and the active material enclosed is released and penetrated into epithelial tissue. The various prominent patents related to GO are depicted in Table 1.3.

TABLE 1.3 List of patents related to gamma oryzanol. Gamma oryzanol

Patent No.

Description of patent

Inventor

Country

Patent publishing year

CN103200826B

Method for producing oil or fat that contains gamma oryzanol

Takuo Tsuno, Takashi Yamanaka, Hiroaki Segoshi

China

2014

Tsuno et al. (2014)

CN104394864B

Composition containing sesamin-class compounds, gamma oryzanol, and rice germ oil

Yu Motomatsu, Yu Ipponmatsu, Yoshiko Ono, Namino Tomimori, Daitsu Takemoto, Toshiaki Sueyasu

China

2016

Motomatsu et al. (2016)

CN101292942A

Dermatologic preparation composition containing gamma oryzanol

Isamu Saii, Yuichiro Kano, Hiroyuki Kawashima

China

2008

Saii et al. (2008)

KR20120114730A

Microemulsion as a transdermal delivery system comprising gamma oryzanol

Byung-cheol Kim

Korea

2012

Kim (2012)

JP2008120748A

Gamma oryzanolesolubilized liquid composition

Yoshiko Honma, Emi Ishida

Japan

2015

Honma and Ishida (2015)

WO2012110303A2

Cosmetic preparation of material particles and gamma oryzanol

Ce´cile Grare, Catherine Marion, Ce´line Philippon

World patent

2013

Grare et al. (2013)

EP1475073A2

Use of hair cleansing cosmetic preparation containing gamma oryzanol and low metal ion concentration

Horst Argembeaux, Andreas Koller

Europe

2004

Argembeaux and Koller (2004)

References

16

Nutraceuticals and Health Care

1.13.4 Lignans Lignans are compounds obtained from higher plants formed by coupling of two coniferyl alcohol residues to form diphenolic compounds (Kour et al., 2019). Flaxseeds are the richest source of principal lignin compounds known as secoisolariciresinol diglycoside (SDG) that is converted to mammalian lignans as enterodiol and enterolactone in human colon. Flaxseed lignans have proven as a boon to exhibit therapeutic effects including antioxidative, antiinflammatory, antiatherosclerogenic, and antiestrogenic ones, thus suggesting ability to reduce risk and protect against cancer (Puukila et al., 2017). Various patents related to lignans are depicted in Table 1.4.

1.13.5 Curcumin Curcumin is obtained from Curcuma longa which is the main constituent of Indian spices. Turmeric is the rhizomatous plant from ginger family. Chemically curcumin is polyphenolic in nature. It has been used for its antioxidant, antiinflammatory (Lestari & Indrayanto, 2014) and antimutagenic, antimicrobial (Reddy et al., 2005), and anticancer properties (Vera-Ramirez et al., 2013). Table 1.5 has depicted various patents related to curcumin.

1.14 Regulatory aspect of nutraceuticals Currently there has been new trend in nutraceuticals market, as people are using them as preventive therapies and selfmedication rather than based on prescriptions. So there is strong need to regulate it. As there is no such strict laws for its potency and nomenclature so the patient remains as confused. There is strong need to regulate the nomenclature and claims for nutraceuticals. They are not regulated as various drug categories; rather they are further divided into different food categories. The legal definition defines them based on two categories such as one is origin and other is pharmacological benefit it is providing. In the United States, they are called as dietary supplements, while in Canada they are known as natural health products, and Japan lists them as Foods for Special Health Use (FOSHU). In the United States, the Food and Drug Administration (FDA) regulates the approval of foods, additives, drugs, and cosmetics. However, the nutraceuticals fall outside the scope of FDA and Dietary Supplement, Health and Education Act (DSHEA) of 1994 that controls them as dietary supplements (Wollschlaeger, 2003). The dietary supplements are defined as the means a product (other than tobacco) intended to supplement the diet that bears or contains one or more of the following dietary ingredients: l l l l l l

a vitamin; a mineral; an herb or other botanical; an amino acid; a dietary substance for use by man to supplement the diet by increasing the total dietary intake; or a concentrate, metabolite, constituent, extract, or combination of any ingredient described in above clause.

There is also new term called new dietary supplements which can be defined as supplements which were not sold in the United States before October 15, 1994 (Starr, 2015). There are certain set of labels regulations in DSHEA that needs to be followed including use of full name of product stating it as supplement, name and place of business of manufacturer, packer or distributor, along with complete list of ingredients and net contents of package. Along with it, also the nutritional labeling in the form of supplement facts should be present. Federal Register Final Rule-62 FR 49826 defines the labeling requirements for other constituents present in the container. In European Union, the nutraceuticals if are to be administered for any medical claim or pharmacological action, then it must be fulfilling the requirements for European Union legislation in the pharmaceutical sector for medicinal products for veterinary use according to Commission Directive 2001/82/EC (Directive, 2001). Nutraceuticals can be administered as feed or feed additives when they are found to have no medicinal claim or pharmacological action. If they found to have medicinal action they must comply with the Commission Regulation (EU) No 68/2013 (EC, 2013). When they are used as feed additives for animal nutrition, nutraceuticals as herbs or their extract, prebiotics and probiotics shall comply with No. 1831/2003 regulation. The European Food Safety Authority (EFSA) regulates the nutraceuticals in European countries and has devised two separate regulations as follows:

TABLE 1.4 List of patents related to lignans. Lignans S. No.

Patent publishing year

References

Description of patent

Inventor

US9545119B2

Specific vanillyl lignans and use thereof as taste improvers

Michael Backes, Jakob Peter Ley, Katharina Reichelt, Susanne Paetz

2017

Backes et al. (2017)

2

US20080057140A1

Application of Lignans for the prevention and elevation of symptoms related to estrogen deficiency

Mikko Unkila

2008

Unkira (2008)

3

US9629869B2

Application of fructus schisandrae total polysaccharides in preparation of medicine or nutraceuticals used for treating coughing

Kefang, L. A. I., Zhong, S., Gan, Z., Liu, X., Yichu, N. I. E., and Zhong, N.

2017

Kefang et al. (2017)

4

US8350066B2

Disclosed method of extraction of bioactive lignans resulting in high yield and purity from sesame oil

Chami, Arumughan, Chandrasekharan Pillai Balachandran, Mullan Velandy Reshma, Andikannu Sundaresan, Shiny Thomas, Divya Sukumar, and Syamala Kumari Sathyanandan Saritha

2013

Arumughan et al. (2013)

5

CZ2014870A3

Extraction method of lignans from coniferous tree branch knots and use of the extract for food purposes

Pavel Hic, Josef Balik, Jana Kulichova, Jan Trı´ska, Jan Strohalm, Nadezda Vrchotova, Milan Houska

2016

Hic et al. (2016)

6

CZ30250U1

Chocolate with an increased content of natural lignans

Jana Kulichova, Josef Balik, Pavel Hic, Miroslav Horak, Jan Triska, Nadezda Vrchotova, Milan Houska, Jan Strohalm, Pavla Novotna

2017

Kulichova et al. (2016)

7

CN100496751C

Method and device for dehulling separation of flaxseed

Li Qun, Hu Xiaojun, Liang Xia, Xu Guangying

2006

Qun et al. (2006)

8

CA1302254C

Disclosed the method of preparing the therapeutic seed composition

Paul A. Stitt

1992

Stitt (1992)

9

US5069903A

Disclosed the method of preparing the therapeutic seed composition

Paul A. Stitt

1991

Stitt (1991)

10

CN100365005C

Method for extracting and purifying secoisolariciresinol diglucoside from flaxseed

Xu Shiying, Wang Zhang, Zhang Wenbin, Zhang Xiaoming, Zhong Fang

2008

Shiying et al. (2008)

11

KR101908201B1

Seasoned oil composition containing an ingredient of a flaxseed and preparing process thereof

Insuk Kim

2018

Kim (2018)

12

KR101040206B1

Aqueous power of flaxseed oil comprising omega-3 fatty acid

Shin Bong-seok, Ahn Nam-soon

2011

Bong-seok and Nam-soon (2011)

13

US6777591B1

Legume-like storage protein promoter isolated from flax and methods of expressing proteins in plant seeds using the promoter

Sarita Chaudhary, Gijs van Rooijen, Maurico Moloney, Surindor Singh

2004

Chaudhary et al. (2004)

Nutraceutical-A deep and profound concept Chapter | 1

Patent No.

1

17

18

Curcumin Patent No.

Description of patent

Inventor

Patent publishing year

References

1

US10512616B2

Composition to enhance the bioavailability of curcumin

Benny Antony

2018

Antony (2018)

2

US20170224637A1

Intranasal delivery of curcumin

Thomas M. DiMauro

2017

DiMauro (2017)

3

US7745670B2

Curcumineresveratrol hybrid molecule

Thomas M. DiMauro

2010

DiMauro (2010)

4

US9447023B2

Curcumin and tetrahydrocurcumin derivatives

Krishnaswami Raja, Probal Banerjee, Andrew Auerbach, Wei Shi, William L’Amoreaux

2013

Raja et al. (2013)

5

US20180318217A1

Liposomal curcumin for treatment of diseases

Razelle Kurzrock, Lan Li, Kapil Mehta, Bharat Bhushan Aggarwal, Lawrence Helson

2018

Kurzrock et al. (2018)

6

US10159654B2

Prepared curcumin formulation with enhanced bioavailability

Benny Antony

2018

Antony (2018)

7

US9012411B2

Prepared formulation containing the derivatives of curcumin, paclitaxel, and aspirin

James N. Jacob

2015

Jacob (2015)

8

US20190175628A1

Phospholipid complexes of curcumin having improved bioavailability

Andrea Giori, Federico Franceschi

2019

Giori and Franceschi (2019)

9

EP1299406B1

Prepared derivatives of curcumin with the enhancement in solubility

Barbara Bertram, Manfred Hergenhahn, Bernd L. Sorg, Manfred Wiessler

2003

Barbara et al. (2003)

10

US20080076821A1

For the treatment of Alzheimer’s disease, intranasal administration of prodrugs of curcumin

Thomas M. DiMauro

2008

DiMauro (2008)

11

US20080103213A1

Liposomal curcumin for treatment of neurofibromatosis

Razelle Kurzrock, Lan Li, Kapil Mehta, Bharat Aggarwal

2008

Kurzrock et al. (2008)

12

US9138411B2

Prepared liposomal preparation based on curcumin for sustained release of nanocurcumin

Amalendu Prakash Ranjan, Anindita Mukerjee, Jamboor K. Vishwanatha, Lawrence Helson

2015

Ranjan et al. (2015)

13

US20200108148A1

Enhancement in bioavailability of curcumin

Benny Antony, Moni Abraham Kuriakose

2020

Antony and Kuriakose et al. (2020)

14

US20100179103A1

Curcumin cyclodextrin combination for preventing or treating various diseases

Ketan Desai

2010

Desai (2010)

15

US8288444B2

Delivery of curcumin using iontophoresis for the treatment of Alzheimer’s disease

Sean Lilienfeld, Thomas M DiMauro

2011

Lilienfeld and DiMauro (2011)

S.No.

Nutraceuticals and Health Care

TABLE 1.5 List of patents related to curcumin.

Nutraceutical-A deep and profound concept Chapter | 1

l

l

19

Food supplementsdThe Food Supplements Directive (FSD) 2002/46/EC establishes a list of allowable vitamins and minerals (others not covered under this directive), and sets labeling requirements for these food supplements along with the dosage limits (Vlietinck et al., 2009). Novel foodsdFood supplements also need to comply with the EU Novel Foods Regulation which lays down rules for novel foods that were not used before 1997 (Vettorazzi et al., 2020).

While in Japan, the term Health food is generally used, which accommodates the foods, drinks, or dietary supplements, that carry nutrient functions and health claims, and are designed such that they are more effective in comparison to common foods in helping the people to maintain and promote their health (Shimizu, 2003). In India, in the year 2006, food safety and security act was passed, and in 2008 FSSAI came into picture (Singh et al., 2016). The FSSAI consists of 21 chapters. And in 4th chapter means 22 of act states that nutraceuticals, dietary supplements, and various functional foods and they can be produced/manufactured, marketed, or sold by any company. While the articles 23 and 24 states about the packaging and labeling. In the year 2011, the FSSAI issued guidelines about the process of licensing and registration of food business, packaging and labeling, food product standards and food additives, prohibition of sale, contaminants, and residues, as well as laboratory sample analysis (Keservani et al., 2014). In order to qualify for the Nutraceuticals as per FSSAI, it should have any of the following constituents: Schedule Schedule Schedule Schedule Schedule Schedule

I: Vitamins and minerals II: Essential amino acids and other nutrients IV: List of plants and botanical ingredients VI: List of ingredients as nutraceuticals VII: List of strains as probiotics VIII: List of prebiotic compounds

1.15 Current status Nutraceuticals market size was worth of about US $382.51 billion in the year 2020. It is estimated to rise beyond US $722.49 billion, by the year 2025. Moreover due to increase in diseases related to lifestyle, people are moving from chemically derived products to healthier items like nutraceuticals. As due to COVID-19, the people became more aware about the principles and role of immunity, there has been steady rise in the market trend for nutraceuticals in year 2020. With increase in awareness level of calorie reduction and weight loss issues in the United States, China, and India, it is assumed that it will surely increase the demand over there and will impact the industry in positive growth. More over the digestive system area along with immune system is enhancing the cognitive behavior of consumers. The personalized health-care facilities are also used by manufacturers to formulate better suited end-products for customers. Companies are also spending money on evaluating the proper purchasing behaviors of consumers that is helping them to produce the tailored products. The role of epigenetics and nutrigenomics has also been highlighted in the identification of nutritional requirements and providing better clarity to the consumers that each one behave to food in different ways. This not only results in boost of one’s health but also helps to lessen the chances of adverse drug reactions.

1.16 Indian and global nutraceutical market size Functional foods are introduced to the market with the help of researchers, scientists, nutritionists, and government authorities. The development of functional foods will help masses of people in treatment of specific disease and chronic illness. The nutraceuticals available in Indian markets are available in natural as well as processed form. The foods are available in altered forms (the foods experienced manual interference) or pure forms (natural food forms). The alteration in nutraceuticals can also be done by changing its chemical composition. Some countries consider pills and capsules in the category of nutraceuticals while others include only traditional foods in this category. There are many regulatory bodies that regulate the health claim parts and labeling part. Consumers today being health conscious must be aware of right number of doses of phytochemicals and nutrients that claim health benefits. Safety issues of nutraceuticals are becoming popular day by day throughout the world. There is a very fine line between the safety scale of functional foods and nutraceuticals. There is some restrictive specific limit for the consumption of nutraceuticals. Although nutraceuticals are fast acting than functional foods. Functional foods can be consumed as basic food stuff along with the main meals, e.g., the probiotic curd can be consumed in daily diet without any prescription. But in case of nutraceuticals proper consultation of doses is necessary as they are in concentrated form (Espín et al., 2007). The functional foods and nutraceuticals provide targeted nutritional disease preventing health benefits to consumers and so they have opened a scope of new opportunities

20

Nutraceuticals and Health Care

for health promotion. The nutrition claims are any claim which states, suggests, or implies that the food has a particular beneficial property due to energy value and also provide at a reduced or increased rate so different nutrition claims like low energy value, energy free, sodium free, salt free, or very low sodium. According to regulatory bodies all nutrition claims should be mentioned on the label. There are no well-defined regulations guiding the labeling of these products but usually the labeling requirements under FSS Act are to be followed. India has been targeted as a nutraceutical hub with lots of research material and studies along with the magazines and newspapers flooded with information of nutraceuticals. The calculation of global market size of nutraceuticals is cumbersome because of ambiguity in their origin. The current calculated value is 40 billion dollars which is progressively increasing in terms of sales and variations in ingredients benefitting health.

1.17 Establishment of nutraceuticals in market Nutraceutical market is currently hotter than internet in the United States as stated by Rolf Bjerndell CEO of Skånemejerierne in Sweden. Although it is in nascent stage in Europe. The new food product development, innovations in food by researchers, and consumer awareness are an added bonus to establish nutraceuticals in current market. There are large number of consumers that are switching over to disease prevention through nutrient components instead of going in for synthetic drugs. People have started believing in preventive measures of a disease through diet instead of going to doctors for treatment. Nutraceuticals are anticipated to counter nutritional disorders and help in serious ailments like excessive weight, cardiovascular diseases, osteoporosis, cancer, digestive disorders, skin problems, and much more. The pharma industry has explored all these parameters and is thus including nutraceuticals in wide range of products. The consumers can be benefitted by nutraceuticals in three manners: (i) Perception of healthy living by consumer (ii) Perception of cutting down the expenses on health (iii) Perception of food industry to incorporate value addition to their products Consumers believe that if something is added in food from outside as additive, it makes the food impure and only natural food is a pure food. The enriched foods are perceived as unhealthy and nutraceuticals are considered as synthetic food items. Consumers believe that nutraceuticals are having lesser nutritional value and they are genetically engineered foods and thus are not that healthy as their conventional foods as shown in Fig. 1.1.

1.17.1 Demands of nutraceuticals in market The major key factors generating the demand of nutraceuticals are as follows: (a) Consumer awareness in health maintenance (b) Sociocultural factors like availability of local cuisine, religion, rituals (c) Presence of biologically active components acting in harmony with health

1.17.2 Factors affecting future market There are many factors which are vital for the development of future market in nutraceuticals. (a) Consumer acceptance of nutraceuticals (b) Synergy between academicians and industrialists (c) Regulations and labeling laws for health claims

Natural Food

Unnatural Food

Convenonal

Nutraceucal

Ecological

Genecally Engineered

FIGURE 1.1 Consumer perception of natural food and nutraceuticals.

Nutraceutical-A deep and profound concept Chapter | 1

21

1.17.3 Steps for market development of nutraceuticals All the steps include relationship of foods in curing diseases for which in vitro and in vivo studies are being conducted on humans by administering suitable quantities of nutraceuticals and checking the antagonistic effects if any, and finally the carrier is developed (Fig. 1.2). Afterward marketing is done to make public aware of the health benefits of nutraceuticals. Lastly the research is done for sustainability of products and attitude of individuals is measured (Martirosyan & Singh, 2016). Although many significant factors like metabolites, dose dependency, toxic components, and bioavailability of nutraceuticals require lot of scientific evidence and so nutraceutical market is not well-established market in most countries (Valls et al., 2013) but the nutraceutical terminology has been used by industries since a decade. There are many terms like functional foods, nutraceuticals, pharmaconutrients, and dietary supplements which are used by industry for nutrientenriched foods that help in disease prevention. Nutraceuticals are being sold in the form of pills, tablets, or nutrient extract but functional foods are considered as ordinary foods and there are no separate rules and regulations framed to distinguish functional foods from nutraceuticals. The pharmaceutical market is growing immensely on the grounds of highend research and development on nutraceuticals and thus this sector is having tall profits. The industry has to epitomize the growth by protecting the lucid practice of these drug designs based on appropriate clinical trials and possible drug interactions along with cost-effectiveness (Mechanick, 2005). 1. Market Challenges for Nutraceuticals: The recognition of ingredients present in a food acting as nutraceutical is a key factor to design a pill or drug and that also depends on the consumer acceptance (Mechanick, 2005). The research on such foods includes: (a) Identification of key components present in food which provides the potential health benefits (b) Development of biomarkers (c) Possible utilization of nontechniques to develop nutraceuticals (d) Anticipation of demands of nutritional requirements (e) Stabilization of nutritional components during the processing as well as reaching the target organs (f) Establishing dietary references for a wide range of nutrient component

1.18 Marketing barriers of nutraceuticals There are several hinderances in marketing of nutraceuticals; the legislations at national and global level in terms of health claims are one of the biggest hindrances. Apart from legislations, the record keeping of scientific evidences, consumer perceptions, limited knowledge regarding nutraceuticals, convincing consumers toward nutraceutical benefits, and grave voice of investors are some of the barriers in marketing of nutraceuticals. The ignorance and hard rules of patenting pull the food manufacturers in competitive market where they end up with little profits because of copying off their food components having medicinal properties by other manufacturers so the investment on such projects is not safe instead of uniqueness in the product. According to research studies, it is depicted that 70% of patients (Kessler et al., 2001) refer to their physicians beforehand the conventional treatments which directs the condemnation of nutraceutical benefits.

1.19 Status of nutraceuticals and future prospects Pharma sector is having a silver lining in India as large number of therapies have emerged and have shown greater results. There is subsequent increase in variety of products which are commercially viable boosting Indian economy leading to improved earnings of people. Besides challenges and perseverance, lot of support vision and business insights are contributing advantageous market hub nutraceutical sector in India. The country is blessed with traditional herbs, good climatic conditions, qualified quacks, research and development facilities. The nutraceutical market in India is estimated to be 1 billion US dollars. The global market size is growing at a rate of 7% CAGR while Indian CAGR is 18% which is much faster (Verhagen et al., 2010). The dormant market size in India is 2e4 times the recent market scenario and is having approximately 150 million budding customers. The US market is 1 billion dollars which is having 54% share of therapeutic foods and 32% share contributed by supplemented foods and 14% share is captured by functional beverages.

Phytonutrient Analysis

Preclinical Screening

Clinical Trials

product Development

Markeng

FIGURE 1.2 Steps for market development of nutraceuticals.

Epidemiological studies

Market Survey

22

Nutraceuticals and Health Care

The fast moving consumer goods companies are providing a greater advantage to nutraceutical industry in India (FICCI, 2009). The nutraceutical industry in countries like India, Brazil, and China is burgeoning and many suppliers have established themselves locally and globally which has bloom the nutraceutical glory across globe (Shimizu, 2003). According to Hippocrates, father of medicine, “thy food thy medicine.” He always said that let the food be your medicine and your medicine should be your food. If the consumers will follow this idea only then the nutraceutical market can come to its full bloom. However, the nutraceutical market has bigger challenges of disseminating information regarding health claims and inclining them toward nutraceuticals choice instead of conventional accompaniments. It is not easy to force a consumer to feed on the products provided by industry regardless of their nutritional and health benefits. The food manufacturer can move the things in right direction and fortunately can understand the consumer perception of tastes but cannot create the consumer needs. Development and marketing of nutraceuticals is tedious task. Consumers are not having the clarity regarding nutraceuticals and for them anything rich in fiber, having herbs or yoghurts acting as probiotics that are beneficial for health come under nutraceuticals (Espin et al., 2007). The consumer is showing deep interest in nutraceuticals because of its therapeutic value and no side effects as being posed by other kind of treatments. The Indian and global market of nutraceuticals is increasing because of deviation of people’s mindset toward preventive measures and having increased disposable income (Siro et al., 2008). Moreover, consumer expenditure for health and more promising hike in pharma retail chain creating better prospects for nutraceuticals sector. The major challenge faced in India is the standardization flaws, lack of appropriate health knowledge, paying capacity, marketing although the market of nutraceuticals is immensely growing in India along with the United States and Europe (Shinde and Namdeo, 2014). Proper business models and active rules and regulations can lead to a quicker admittance to nutraceutical market. Nutraceutical market is an innovative approach for treating several diseases and pharma sector should be encouraged to overcome all the hindrances and government and health centers must support them to establish market of nutraceuticals (Shashank, 2006).

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Wanglin, J., Fenghua, F., Jingwei, T., Chaoyun, W., & Fang, S. (2005). Application of tannic acid berberine in preparation of medicine for treating ulcerocolonitis. CN1589793A. Wenwen, G., Yun, W., & Tao, C. (2013). Application of berberine combined chemotherapeutic medicament in antitumor therapy, CN103372210A. Westcott, N. D., & Muir, A. D. (2003). Chemical studies on the constituents of Linum spp. Flax the genus Linum. New York (USA): Taylor and Francis. Wilson, T. A., Nicolosi, R. J., Woolfrey, B., & Kritchevsky, D. (2007). Rice bran oil and oryzanol reduce plasma lipid and lipoprotein cholesterol concentrations and aortic cholesterol ester accumulation to a greater extent than ferulic acid in hypercholesterolemic hamsters. The Journal of Nutritional Biochemistry, 18(2), 105e112. Wollschlaeger, B. (2003). The Dietary Supplement and Health Education Act and supplements: dietary and nutritional supplements need no more regulations. International Journal of Toxicology, 22(5), 387e390. Wu, K. (2003). Use of berberine with high solubility in preparation of medicament .World . Patent No. WO2003090749A1 Washington, DC: U.S. Patent and Trademark Office. Xianying, S., Limei, Z., Dan, X., Xin, H., Jukui, S., Lin, S., & Na, Xinzhu (2016). Berberine and fenofibrate pharmaceutical composition and use thereof, CN105560233A. Xu, Z., & Godber, J. S. (1999). Purification and identification of components of g-oryzanol in rice bran oil. Journal of Agricultural and Food Chemistry, 47(7), 2724e2728. Yang, C. S., Landau, J. M., Huang, M. T., & Newmark, H. L. (2001). Inhibition of carcinogenesis by dietary polyphenolic compounds. Annual Review of Nutrition, 21(1), 381e406. Yasukawa, K., Akihisa, T., Kimura, Y., Tamura, T., & Takido, M. (1998). Inhibitory effect of cycloartenol ferulate, a component of rice bran, on tumor promotion in two-stage carcinogenesis in mouse skin. Biological and Pharmaceutical Bulletin, 21(10), 1072e1076. Youdim, K. A., & Joseph, J. A. (2001). A possible emerging role of phytochemicals in improving age-related neurological dysfunctions: A multiplicity of effects. Free Radical Biology and Medicine, 30(6), 583e594. Youjie, W., Desheng, X., Shuang, L., Yi, F., & Kefeng, R. (2012). Method for modifying bitter taste of berberine, berberine hydrochloride and related compound preparations. CN102813928A. Yuanlu, C., Ye, Z., Junsheng, T., & Yanbin, Z. (2011). Berberine cyclodextrin inclusion compound, preparation thereof and preparation method, CN101461949A. Yuqiang, W., Enming, D., Lipeng, X., Qian, C., & Junlan, R. (2012). Andrographolide and berberine combination and application thereof, CN102151260B. Zheng, J., Shen, N., Wang, S., & Zhao, G. (2013). Oat beta-glucan ameliorates insulin resistance in mice fed on high-fat and high-fructose diet. Food and Nutrition Research, 57. https://doi.org/10.3402/fnr.v57i0.22754 Zheng, Z., & Shetty, K. (2000). Enhancement of pea (Pisum sativum) seedling vigour and associated phenolic content by extracts of apple pomace fermented with Trichoderma spp. Process Biochemistry, 36(1e2), 79e84. Zhiqiang, H. (2015). Berberine hydrochloride taste-masked pellet, and preparation thereof, CN104997735A. Zhu, J. X., Tang, D., Feng, L., Zheng, Z. G., Wang, R. S., Wu, A. G., Duan, T. T., He, B., & Zhu, Q. (2013). Development of self-microemulsifying drug delivery system for oral bioavailability enhancement of berberine hydrochloride. Drug Development and Industrial Pharmacy, 39(3), 499e506. Zhongkun, L., Chongjing, W., & Yan, W. (2004). Berberine hydrochloride suppository and its producing method, CN1279946A.

Further readings Fang, N., Yu, S., & Badger, T. M. (2003). Characterization of triterpene alcohol and sterol ferulates in rice bran using LC-MS/MS. Journal of Agricultural and Food Chemistry, 51(11), 3260e3267. Kajla, P., Sharma, A., & Sood, D. R. (2015). Flaxseedda potential functional food source. Journal of Food Science and Technology, 52(4), 1857e1871. Mason, W. R. (2009). Starch use in foods. In J. BeMiller, & R. Whistler (Eds.), Starch chemistry and technology (pp. 745e795). New York: Academic Press. North Carolina Association for Biomedical Research. (July 2007). Nutraceuticals. WWW. About bioscience. Org. Redmond, M. J., & Fielder, D. A. (2006). Extraction and purification method for cereal beta-glucan. US patent 012149 A1.

Chapter 2

Cereal proteins Cherakkathodi Sudheesh1, Zahid Rafiq Bhat2, Basheer Aaliya1 and Kappat Valiyapeediyekkal Sunooj1 1

Department of Food Science and Technology, Pondicherry University, Puducherry, India; 2Department of Pharmacology and Toxicology, National

Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur, Punjab, India

2.1 Introduction Cereals are defined as the edible seed or grain of the grass family Gramineae and are the major source of food. Wheat, barley, and rye belong to the genus Triticale, rice to Oryzae, oats to Aveneae, millet to Paniceae, corn and sorghum to Andropogoneae. Current knowledge on the sources of cereal proteins: rice and wheat are the major cereals in human nutrition which account for 55% of total cereals production. The major cereals consumed by the world population are in the order of sorghum < millet < rye < oats < barley < maize < rice < wheat. Moreover, these cereals act as a major source of primary metabolites such as carbohydrates, proteins, vitamins (B vitamins), and minerals, and secondary metabolites (bioactive compounds) for the human diet. Among these metabolites, protein consists of approximately 6%e15%. However, various production methods and agricultural practices are attributed to change the protein content among the cereals (Esfandi et al., 2019). The storage protein present in the rice is oryzenin (glutelin), wheat has glutenin (glutelin) and gliadin (prolamin), maize has zein (prolamin), and oats have albumin, globulin, and avenin (prolamin), while barley has glutelin and hordeins (prolamin). Commonly, the quality of proteins depends on their amino acid composition; cereal proteins are limited in the essential amino acids such as tryptophan, lysine, and threonine. However, cereal proteins contain a higher amount of sulfur-containing amino acids such as methionine and cysteine. Hence, the combination of cereal proteins with lysine-rich and sulfur-deficient legume proteins has higher significance from the nutrition and health point of view. The nutritional significance of cereal proteins also depends on factors such as protein digestibility and bioactive peptides. The physicochemical and functional properties of cereal proteins are widely used in the food processing, pharmaceutical, and nutraceutical industries. In this chapter, the current knowledge on the cereal protein sources, the extraction process, characterization techniques, bioavailability, allergenicity, health benefits, and pharmaceutical/nutraceutical applications are summarized.

2.2 Cereal grains 2.2.1 Rice Rice (Oryza sativa) is an important cereal crop and acts as the major staple food for the world population, especially Asians. Rice is one of the oldest domesticated cereals in the world, and it was first domesticated 8200e13,500 years ago. Rice has a higher affinity to absorb the trace element arsenic present in the soil and water. Hence, the Food and Drug Administration (FDA) permitted only 100 ppm inorganic arsenic in the cereals used for infant food formulations. Asian regions are the best suited for rice cultivation due to the high rainfall. Rice is consumed mainly in the form of breakfast cereals and is also used for the production of brew sake in Japan. Rice grains contain husk or hull as the outer layer and edible caryopsis. Brown rice consists of the pericarp (pigmented layer), seed coat, embryo, and endosperm. Wild rice is not related to rice, and it is the grain of the North American plant, Zizania aquatica. It is more expensive as compared to other grains and contains a high amount of proteins than rice (Bleakley, 2018; McKevith, 2004).

2.2.2 Wheat Wheat belongs to Triticum family, and its commercially important subspecies are Triticum aestivum, Triticum durum, and Triticum compactum. It originated about 9200 years ago and is grown in both winter and spring seasons. Wheat is mainly Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00010-7 Copyright © 2022 Elsevier Inc. All rights reserved.

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cultivated in the western part of the world and is used for different kinds of food such as baked products, pasta, and breakfast cereals. It is also used for the production of alcoholic beverages such as beer and vodka. Raw wheat used for food production is either in the form of whole-grain or dehulled; the ground wheat flour is widely used in food sectors. The ear of the wheat is also called spikelet, and whole wheat grains are enclosed between lemma and palea. The wide acceptability of wheat in the food industries is attributed to the presence of gluten proteins such as glutelin and gliadin. These proteins give viscoelastic properties to the dough, and they improve the texture of the finished products (Bleakley, 2018; McKevith, 2004).

2.2.3 Barley Barley (Hordeum vulgare) has been cultivated since 15,000 BCE. It has good resilient properties and exists in a wide range of conditions. Barley is mainly used for animal feed, manufacturing of beer, and distilled spirit (whisky). A small amount of barley has been used for food purposes. Barley shows poor sensory quality. Hence, it has less significance in food production as compared with wheat and other cereals. Barley, nowadays, is a part of human diet in countries such as India, China, Morocco, and Ethiopia (Bleakley, 2018). The barley (spike) is made of spikelets and is connected to the rachis in an alternating pattern. The outer layer of barley consists of the husk, which completely covers the grain, followed by the pericarp, seed coat, and aleurone layer (McKevith, 2004).

2.2.4 Oats Oats (Avena sativa) have a higher tolerance to wet and dry conditions as compared to wheat, rye, and barley. It is an annual crop and mainly cultivated for animal feed, but a small portion of oats is used for human consumption in the form of porridge, oatcake, breakfast cereals, and infant foods. However, whole grain oats contain a higher amount of fiber; it decreases their digestibility and nutritional values. Oats is also used for nonfood purposes such as cosmetics and adhesives. An oat kernel consists of a spikelet, and the hull surrounds each kernel with two layers (lemma and palea). The hull is loosely connected to the groat and the bran layers (pericarp, seed coat, and aleurone cells) envelope the groat which consists of about 65%e85% of oat kernel (McKevith, 2004).

2.2.5 Sorghum Sorghum (Sorghum bicolor L. Moench) is a heat-tolerant plant and used as a staple food in the parts of Asia, Africa, and the Middle East regions. The major portion of sorghum cultivated in North, South, and Central America is used for animal feed. The caryopsis of sorghum grains consists of pericarp, endosperm, and germ. Grain sorghum, forage sorghum, grass sorghum, Sudan sorghums, and broomcorn are the major classes of sorghum. This classification is based on factors such as color and thickness of pericarp, pigmented testa, endosperm thickness, and type (McKevith, 2004).

2.2.6 Rye Rye is a winter crop and mainly grown in the cold temperature zone. It is a major crop in Russia, Poland, Germany, and Scandinavian regions. It is used for the production of crispbread and alcohol and used as animal feed. The husk of the rye grain is called a glume, and the grains are arranged in an alternating form across the rachis. Rye grain consists of endosperm, pericarp, testa, and germ; its color is greyish yellow (McKevith, 2004).

2.2.7 Maize Maize is the major source of starch and is cultivated mainly in the Western Hemisphere; it is widely used as animal feed. Dent maize, flint maize, sweet corn, and popcorn are the major commercially important maize varieties. Germ, endosperm, pericarp, and tip cap are the major parts of maize grain. Maize is also used as the source of starch, cooking oil, and sweeteners (McKevith, 2004).

2.2.8 Millet Millets are small-grained annual cereals. The pearl millet is the most important type among the millets. Other minor millets such as finger millet (ragi), proso millet, and foxtail millet are used for human consumption, and those account for less than 1% of their production. Nevertheless, these crops have played an important role in the food security of certain regions like Asia and Africa (McKevith, 2004).

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2.2.9 Other cereals Buckwheat and quinoa are the other cereals which are important in certain regions of the world. Buckwheat (Saracen corn) is produced from the plant Fagopyrum esculentum. It is consumed in the form of porridge, baked pancake, and cooked cereals. Quinoa is produced from Chenopodium album and is consumed mainly in the form of bread in countries such as Chile and Peru.

2.3 Nutritional profile of cereals Cereals are the staple food and are the major sources of the carbohydrate, proteins, minerals, and B vitamins for the global population. It contains a number of secondary metabolites having excellent health benefits. Cereals also contain antinutritional factors in specific amounts.

2.3.1 Macronutrients 2.3.1.1 Carbohydrate Cereals contain about 75% carbohydrate. Starch is the primary carbohydrate present in the endosperm of cereal grains (Fig. 2.1). Starch exists as granular form, and their size varies from 5 to 50 mm. Cereal starch granules exist in different shapes such as polygonal, polyhedral, round, oval, or spherical. Amylose and amylopectin are the two fractions present in the starch granules and vary in different cereals. In general, cereals contain 25%e27% of amylose, but in waxy corn and rice most of the starch is amylopectin. Cereals contain a certain amount of starch that is not digested by the enzymes present in the human body. These starch are called resistant starch and function like dietary fibers. Resistant starch is categorized as: RS1 implies physically inaccessible starch for digestion which are present in the whole or partially milled grains; RS2 implies native resistant starch granules found in raw banana, potato, and high amylose maize starch; RS3 refers to retrograded starch which is mainly found in the cooked and cooled potato, pasta, corn flakes, and bread; RS4 denotes chemically modified starches (McKevith, 2004).

2.3.1.2 Proteins Cereals contain about 6%e15% proteins (Fig. 2.2). Cereal proteins are the richest source of amino acids. However, some of the essential amino acids are limited in the cereals that are lysine, threonine, and tryptophan. Hence, this limitation can be overcome by combining a diet with legumes (it contains high amount of lysine) (McKevith, 2004). Oats proteins mainly contain globulin, so it has a more balanced amino acid profile than other cereals (Mäkinen et al., 2016). Fig. 2.3 presents the essential amino acid profile of various cereals. Glutenin and gliadin proteins present in the wheat are accredited to their higher industrial values; these proteins give wheat dough viscoelastic properties and result in the improved texture of the wheat-based products. The proteins present in the cereals contain different kind of enzymes involved in the synthesis of

FIGURE 2.1 Chemical composition of whole cereal grain. Adapted from Bekes and Wrigley (2004).

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FIGURE 2.2 Protein content of cereal grains. Adapted from Bekes and Wrigley (2004).

FIGURE 2.3 Amino acid composition of various cereals. Adapted from McKevith, B. (2004). Nutritional aspects of cereals. Nutrition Bulletin, 29(2), 111e142. https://doi.org/10.1111/j.1467-3010.2004.00418.x.

other grains’ components. It also accounts for the breakdown of the chemical components present in the cereal grains. The proteins present in the matured grains act as a major storage reservoir for the amino acids involved in the growth of plant after germination. Generally, a major portion of proteins is present in the endosperm of the grains. However, in grains like barley, the proteins are higher in certain non-endosperm part, like germ (Bekes & Wrigley, 2004). The protein content of grains improves their baking quality (especially in wheat grain). However, malting process prefers grains with a lower amount of proteins. The desirable level of proteins in the barley used for malting process is w8%. Here the protein helps to control the supply of hydrolytic enzymes during the malting process (Bekes & Wrigley, 2004).

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2.3.1.3 Lipids Cereals contain a high amount of lipids. Rice, barley, wheat, and rye contain about 1%e3% lipids. Maize and oats contain 5%e9% and 5%e10% of lipids. Cereal lipids are the richest source of essential fatty acids like linoleic acid.

2.3.2 Micronutrients 2.3.2.1 Minerals Most cereals are the richest source of potassium. Cereals are a poor source of sodium, and also contain iron, zinc, magnesium, and trace elements like selenium. Rice grain has higher selenium content (10e13 mg/100 g of cereals) than other cereals. The important factor that affects the selenium content of cereals is the selenium content of the soil. Wheat grown in North America generally has a higher amount of selenium than that grown in the European countries.

2.3.2.2 Vitamins Cereals are the richest source of B1, B2, and B3. It also contains a considerable amount of vitamin E. b-carotene present in the yellow corn is the richest source of vitamin A. Cereals are a poor source of Vitamin C and B12 (McKevith, 2004).

2.3.3 Non-starch polysaccharides Cereals grains are a good source of non-starch polysaccharides, viz soluble and insoluble non-starch polysaccharides. They play an important role in weight control by extending the food retention time in the stomach. The content of the insoluble non-starch polysaccharides in most of the cereals is similar, but the composition of soluble non-starch polysaccharides is varied. Arabinoxylan and beta-glucans are major water-soluble non-starch polysaccharides. Arabinoxylan is present in wheat, rye, and barley, while beta-glucan is found in oats. Barley (3%e11%), oats (3%e7%), and rye (1%e2%) contain a higher amount of arabinoxylan and beta-glucans as compared with wheat (10 kDa) obtained from barley glutelins by the alcalase exhibited strong activity against DPPH. Smaller peptide (molecular weight baicalein greater than baicalin then galuteolin after that daidzein followed by rutin which is found to be greater than luteolin and at the end daidzein. Further for better knowing the solubilization characterization of quercetin and baicalein of the mechanism of action of the polysaccharides phasesolubility method was used. Baicalin and baicalein have shown to have antibacterial, antiinflammatory, antioxidant, and neuroprotective effects as extracted from Scutellaria baicalensis Georgi (SBG) (Cruz et al., 2017). Barbosa et al. (2007) have observed that rutin, hesperidin, and D-catechin reduce capillary fragility and unusual permeability that promote hemostasis in preventing capillary hemorrhage to treat hypertension and act as an auxiliary drug for treating arteriosclerosis. In another study the stability of quercetin, epigallocatechin gallate (EGCG), and cyanidin-3-glucoside (C3G) with respect to bloodebrain barrier cytotoxicity and neuroprotection was checked (Pogacnik et al., 2016) with respect to different incubation time in neurobasal media and ringer-HEPES solution. The solution was used for the neuron and

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HBMEC cultures at 37
C. All the three selected flavonoids, i.e., quercetin, EGCG, and C3G, are said to be formidable antioxidants and thus easily oxidized in biological media. Among the three EGCG has the tendency to outreach brain at the desired target cells where they can wield neuroprotective properties. Flavonoids are known to exert anticancer, antiaging, hepatoprotective, antiinflammatory, cardiovascular function enhancement, treating chronic prostatitis, enhancing immunity, and regulating the endocrine system (Fuchs-Tarlovsky et al., 2013). In contrast to safety and toxicity, flavonoids are considered as safe with low toxicity and varied structures as they can chelate the metal ions forming different complexes whose pharmacokinetic attribute increases as compared to other free flavonoids (Kumar & Pandey, 2013).

5.8 Clinical and pharmacological applications Researchers have concluded that fruits and vegetables are majorly suited for pharmacological, nutraceutical, pharmaceutical, medicinal, and cosmetic applications due to their antioxidative, free radical scavenging capacity, hepatoprotective nature, antiinflammatory, anticancer, and antiviral activities (Grassi et al., 2016; Panche et al., 2016). Flavonoids, in plants, help in combating oxidative stress (allows attenuation of reactive free radicals to become less reactive aroxyl radicals) and act as growth regulators and have potency to be utilized in treatment of cancer, e.g., the protective ability of rutin, a flavonoid, is due to its interfering activity on the enzyme XO (Tapas et al., 2008). Flavonoids are generally considered as nutritional and medicinal and are absorbed via the GI tract and/or by directly administering to the infected tissue (Havsteen, 2002). They generally get modified upon absorption by small intestine especially via conjugation or metabolism thereby in the large intestine by the colon microflora and further by hepatic microflora. They act by affecting the duplication and infectivity of certain RNA and DNA of the viruses. The pivotal enzymes which erase toxins from the body are quinone reductase, glutathione s-transferase, and uridine 5-diphospho-glucuronyl transferase (Rodriguez et al., 2015). Wu et al. (2008) have discussed that quercetin, naringin, hesperetin, and catechin are responsible for antiviral activity. Quercetin extracted from lotus leaves also acts as antibacterial agent for periodontitis (inflammation of tissue around the teeth) according to Li and Xu (2008) and Basu et al. (2016). Flavonoids like tannins, stilbenes, curcuminoids, coumarins, lignans, quinines, 2,3-dihydroxyflavone, tangeritin, 3-hydroxyflavone, 3,4-dihydroxyflavone, fisetin, apigenin, luteolin, daidzein, and genistein are reported to show chemoprotective properties and induce apoptosis by locking the cell cycle, managing carcinogen metabolism (Kang et al., 2011) by exerting its likely effect on cytochrome P450 pathway to inhibit the activities of certain P450 isozymes and ontogenesis expression. Clinical trials of genistein and daidzein have shown prevention against postmenopausal bone loss in women and estrogenic activity (Srivastava & Bezwada, 2015). Another flavonoid, baicalin has the capability to be used in industries as anti-HIV factor by intervening with the association of the HIV-1 envelope proteins with chemokine coreceptors and preventing the HIV-1 entry to specific target cells (Grassi et al., 2016; Kang et al., 2011; Rodriguez et al., 2015).

5.9 Health benefits Based on its bioactive composition flavonoids are said to be complex groups with unknown biological functions. They do possess specific and nonspecific activity based on some characteristics (Fraga et al., 2010; Ravishankar et al., 2013). One of the most important features of flavonoids is that they generally occur in the glycosidic form in the gastrointestinal pathway. Though, they are majorly known for their antioxidant activity preventing radical damage in both in vivo and in vitro system (Arct & Pytkowska, 2008). They also behave as secondary antioxidant defense mechanism in plant tissues open to different abiotic and biotic stresses (Kozlowska & Szostak, 2014). Supplementation with flavonoids reduces the risk of cardiovascular (improving coronary vasodilatation) and neurological diseases like Alzheimer, Parkinson, and dementia, stimulating neuronal regeneration and prevention of oxidative neuronal damages (Grassi et al., 2016), cancer, antibacterial, antiapoptotic, antinecrotic, and antigiardial activities, inhibition of cell damage, reducing allergic reactions, etc. (Benavente-García & Castillo, 2008). A recent study has proven to have positive effects of flavonoids to rise blood flow toward brain and sensory systems and show beneficial effects on the peripheral and central nervous system (Sokolov et al., 2013). Flavonoids are also known for other salient biological activities such as protection of skin from UV exposure, protecting DNA from rupturing, building of capillaries, antiinflammatory effect, and defensive action against radiation, moistening, softening, soothing, antiseptic, etc. (Herrera et al., 2009). These properties highlight the use of flavonoids as a beneficial ingredient in the fabrication of cosmetics and pharmaceutical products. They also impede with nucleic acid or proteins and show antimicrobial and pharmacological effects (pathological disorders of gastric and duodenal ulcers) (Patel et al., 2018) and act as natural inhibitors against inflammation (Hoensch & Oertel, 2015). Various examples of food

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containing flavonoids are green vegetables, fruits, olive and soybean oils, red wine, chocolate, and tea (Yao et al., 2012). Although several flavonoids are found in the plant, flavanol, flavonol, flavanone, anthocyanin, hydroxycinnamate, and stilbene are the major flavonoids showing beneficial health effects (Pandey & Rizvi, 2009).

5.10 Conclusion Flavonoids are massive group of phytochemicals, largely occurring in plants. They reveal many biological effects, such as anticancer, antiaging, hepatoprotective, antiinflammatory, cardiovascular function, curing chronic prostatitis, improving immunity, and modulating the endocrine system. The only thing with these is they are not bioavailable or have low bioavailability hence required in the form of dietary supplement daily in the diet.

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The International Journal of Biochemistry & Cell Biology, 45(12), 2821e2831. https://doi.org/10.1016/j.biocel.2013.10.004 Rodriguez, E. E., Reyes, M. A., Rivera, G., Espinosa, L. G., & Palos, V. (2015). Alimentos funcionales y compuestos bioactivos. Editores P y V México, 63. Samanta, A., Das, G., & Das, S. K. (2011). Roles of flavonoids in plants. Carbon, 100(6), 12e35. Sambandam, B., Thiyagarajan, D., Ayyaswamy, A., & Raman, P. (2016). Extraction and isolation of flavonoid quercetin from the leaves of Trigonella foenum-graecum and their anti-oxidant activity. International Journal of Pharmacy and Pharmaceutical Sciences, 8(6), 120e124. http:// innovareacademics.in/journals/index.php/ijpps/article/download/10943/4982. San, Sithisarn, P., & Gritsanapan, W. (2002). Appropriate extraction method for high contents of total phenolics, total flavonoids and free radical scavenging properties of Ziziphus mauritiana seed extracts. Biomedicine and Pharmacotherapy, 79(13), 276e282. 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Chapter 6

Lycopene Nusrath Yasmeen1, 3, Aga Syed Sameer2, 3, 5 and Saniya Nissar4, 5 1

Faculty of Pharmacology, College of Nursing, King Saud Bin Abdul Aziz University for Health Sciences, Jeddah, Saudi Arabia; 2Department of

Basic Medical Sciences & Quality Unit, College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, Saudi Arabia; 3King Abdullah International Medical Research Centre (KAIMRC), National Guard Health Affairs, Jeddah, Saudi Arabia; 4Department of Biochemistry, Government Medical College, Shri Maharaja Hari Singh Hospital, Srinagar, Kashmir, India; 5Molecular Diseases & Diagnostics Division, Infinity Biochemistry Pvt. Ltd., Srinagar, Kashmir, India

6.1 Introduction Phytochemicals, phycobiliproteins, and phytopigments are considered to be the best sources from which the modern therapeutic agents are obtained since time immemorial. Currently because of the rise in the number of infectious diseases, pandemics like coronavirus disease-19 (COVID-19), and several existing epidemics like human immunodeficiency virus (HIV), cancer, etc., the research arena around the globe has had a drastic transformation. Scientists are more prone toward extracting the active constituents from natural products that can be developed as first-line therapeutic agents or as therapeutic adjuvants, leading to a boom in complementary alternative medicine therapies. One such phytopigment that is exhaustively studied due to its immense therapeutic properties is “Lycopene” regarded as The Miracle nutrient. Lycopene is one of the 600 naturally occurring pigments that belong to the tetraterpene carotenoid family (Petyaev, 2016). It is a bioactive, lipophilic carotenoid that imparts red color to fruits and vegetables. It exhibits a variety of roles in both plants and humans. In plants it is known to play a role in the photosynthetic process, and is also found to be abundantly present in the chromoplasts which are responsible for imparting color to the fruits, ripening, enhance attraction thereby improving consumption and seed disposal by herbivores (Collins et al., 2006). It is the most powerful singlet oxygen quencher known for its excellent antioxidant properties. Consequently, lycopene is used to treat several chronic ailments in humans (Ferreira & Corre, 2013). Lycopene (C40H56) is a dietary phytochemical with numerous potential benefits in humans. The deep red phytopigment with a crystalline nature was first isolated from berries of Tamus communis L. in 1873. Later Millard et al. (1875) isolated solanorubin a crude mixture from tomatoes (Singh and Goyal, 2008). The name Lycopene was coined by Schunck to the red phytopigment of tomatoes, when he reported difference in the absorption spectra of pigments from tomatoes and carrots (Schunck, 1904). Duggar (1913) used the term Lycopersicon to the lycopene in his experimental work observing the effect of growth conditions on the content of lycopene (Kun et al., 2006; Duggar, 1913).

6.2 Sources of lycopene Lycopene is a natural phytopigment synthesized by plants and certain microorganisms. Humans and animals cannot synthesize this pigment, hence they are mostly dependent upon diet for its supply. Lycopene is available abundantly from natural sources such as ripe tomatoes (Lycopersicon esculentum) lodging highest percent of lycopene, also in processed tomatoes (Bhat et al., 2020). Additionally, it is present in papaya (Carica papaya), red grapefruits (Citrus paradisi), watermelon (Citrullus lanatus), passion flower fruit (Passiflora edulis), guava (Psidium guajava), and carrot (Daucus carota) (Estevez-Santiago et al., 2016). Apart from these lycopene is present in good amounts in pureed rose hips and dried apricots (Mourvaki et al., 2005) (Fig. 6.1).

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FIGURE 6.1 Sources of Lycopene.

Furthermore, nutraceutical lycopene is obtained from red carrots, bitter melon, autumn olive, and Gac fruit (Momordica cochinchinensis) (Nanta et al., 2020). Nearly 80% of the dietary lycopene is available to humans from tomato and tomatobased products such as tomato soups, tomato paste/processed tomato, tomato ketchup, and tomato sauces (Imran et al., 2020).

6.3 Extraction and characterization techniques Lycopene is a lipophilic red pigment practically insoluble in water, slightly soluble in methanol, ethanol, and with enhanced solubility in organic solvents such as hexane, benzene, ethyl acetate, chloroform, petroleum ether, methylene chloride, and acetone (Grabowska et al., 2019). Hence it is evident that the extraction of lycopene is possible using the abovementioned organic solvents. However, the extraction process using organic solvents has disadvantages of residual toxicity. Subsequently, scientists have shifted the extraction of lycopene toward green solvent approaches with the technological advancements. The various novel approaches of lycopene extraction currently being utilized are supercritical carbon dioxide SC-CO2, microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), enzyme-assisted extraction (EAE), etc. Solvents such as dimethyl ether with GRAS (generally regarded as safe) status are being used for the extraction of lycopene. The novel extraction methods are used to increase the yield under optimized experimental conditions. The different procedures with their principles for lycopene extraction are described in Table 6.1.

6.4 Chemistry/structure of lycopene Lycopene is carotenoid belonging to C40 terpenoids; basically it is an isomer of b-carotene (Palozza et al., 2012). It is a highly unsaturated, symmetrical, and acyclic hydrocarbon. The molecular formula of lycopene is C40H56, and its chemical name is 2,6,10,14,19,23,27,31-octamethyl-2,6,8,10,12,14,16,18,20,22,24,26,30-dotriacontatridecaene (Gupta et al., 2015; Mozos et al., 2018). The molecular weight of lycopene is 536.85 Daltons, with absorption maximum detected at 470 nm (Ferreira & Corre, 2013). Lycopene is a carotenoid representative that lacks provitamin A activity due to the absence of beta-ionone ring (Story et al., 2010). The tetraterpene symmetric structure of lycopene is due to eight isoprene (octaprene) units having head to tail binding processes except in the middle where the tail to tail bindings exist (Ciriminna et al., 2016) (Fig. 6.2). The acyclic hydrocarbon units of lycopene have 13 double bonds that are linearly aligned of which 11 are conjugated and 2 nonconjugated (Naviglio et al., 2019). The antioxidant power of lycopene is attributed to its linearly aligned polyene conjugated double-bond system (Papaioannou & Karabelas, 2012). Additionally, this system of lycopene is regarded to be the crucial feature that constitutes the key to its biological activity; also it is regarded as a chromophore responsible for imparting the orange-red color of the pigment (Bhuvaneswari & Nagini, 2005).

TABLE 6.1 Novel technologies used in the extraction of Lycopene, with a description of their principles, procedures and the extraction conditions. Principle

Sample used

Extraction conditions maintained

Yield reported

References

Enzyme-assisted extraction (EAE)

The principle behind EAE method is the plant cell wall by utilizing various hydrolyzing enzymes that work as a catalyst, and promote release of the intracellular components under optimized experimental conditions.

Whole tomatoes Tomato peel Fruit pulper wastes Industrial tomato wastes

Cellulose pectinase enzymatic reaction temperature ¼ 40 C, enzymatic reaction time ¼ 5 h, enzyme: substrate ratio ¼ 0.2 mL/g, solvent: substrate ratio ¼ 5 mL/g, extraction time ¼ 1 h, enzyme: enzyme ratio ¼ 1

132 mg/g (198%) 108 mg/g (224%) 429 mg/g (107%) 1104 mg/g (206%) 119 mg/g (23%) 190 mg/g (52%) 202 mg/g (61%) 156 mg/g (45%) 11.5 mg lycopene/g

(Choudhari & Ananthanarayan, 2007) (Catalkaya & Kahveci, 2019)

Microwave-assisted extraction (MAE)

In this process microwaves interact with the water and organic components of the sample plant material; thereby heat is generated following dipole moments and ionic conduction mechanisms. The unidirectional transfer of heat and mass in MAE creates a synergistic effect to improve the yield by accelerating the extraction process.

Tomato peels

ethyl acetate: acetone 0:10 solvent ratio at 400 W power of microwave with a 24 kJ equivalent (1 min)

13.592 mg/100 g

(Ho et al., 2015)

Ultrasound-assisted extraction (UAE)

The principle behind UAE is utilization of ultrasonic energy to improve the yield by accelerating the extraction process, by creating cavitation through high pressures and high temperatures.

Tomato paste processing wastes

35:1 (v/w) solvent solid ratio, 90 W ultrasonic power for 30 min run

89.9 (mg/kg)

(Kumcuoglu et al., 2014)

Supercritical fluid extraction (SCFE)

SCFE is the process of extracting the desired component from a matrix using supercritical fluids such as CO2 (extracting solvent). The other optimal conditions for this process are the critical temperature of 31  C and critical pressure of 74 bar.

Watermelon Tomato peel Tomato peel byproduct þ tomato seed oil

extraction temperature (70e90 C), pressure (20.7e41.4 MPa), and cosolvent ethanol addition (10%e15%) 86  C, 34.47 MPa, and 500 mL of CO2 at a flow rate of 2.5 mL/min temperatures of 70e90 C, pressures of 20e40 MPa, a particle size of 1.05  0.10 mm, and flow rates of 2e4 mL/min of CO2 for 180 min extraction time

38 mg/g 7.19 mg lycopene/g 56%

(Vaughn Katherine et al., 2008) (Rozzi et al., 2002) (MacHmudah et al., 2012)

Conventional organic solvent extraction (COSE)

Lycopene and carotenes, as lipophiles, are usually extracted with nonpolar lipophilic organic solvents such as dichloromethane, hexane, THF, benzene, and chloroform.

Freeze-dried Lycopersicon esculentum

Hexane, ethyl acetate, and ethanol 1 h using 1 L of organic solvents

3.58, 4.39, and 1.25 mg/g, respectively

(Roh et al., 2013) (Papaioannou et al., 2016)

Selective inclusion complex method

It is a selective enclathration process wherein bile acid derivatives form the channels to entrap organic guest molecules showing hosteguest assemblies.

Tomato paste

Bile acid derivatives such as DCA and cholic acid used

0.119 g/kg

(Seifi et al., 2013) (Fantin et al., 2007)

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FIGURE 6.2 Structure of lycopene: (A) 2D structure and (B) 3D structure of lycopene, respectively.

6.5 Pharmacokinetics, bioavailability, and pharmacodynamics of lycopene Lycopene cannot be synthesized de novo in humans; hence they are completely dependent upon diet for its usage in body (Marques et al., 2015). The studies pertaining to the absorption, metabolism, physicochemical properties, and bioavailability of lycopene are the current arena for research in order to ascertain its beneficial effects. Lycopene is highly lipophilic pigment insoluble in water which is regarded as main factor affecting the efficiency of micellization by bile acids and its subsequent uptake by mucosa cells. Absorption of lycopene is a complex phenomenon involving several processes such as release from food matrix, micellar dissolution, uptake by the intestines, chylomicron incorporation, tissue distribution and transportation from core of VLDL, and transformation to LDL, mechanism of liver uptake (Moran et al., 2018). It is clearly evident that absorption of lycopene from processed foods such as tomato sauce and tomato paste or from tomato juice or soup cooked in oil is greater compared to its absorption from raw tomato (Gärtner et al., 1997). This shows that several processes such as heat processing, presence of plant matrix, dietary fiber, dietary fat incorporation, and homogenization are attributed to enhance absorption of lycopene (Meroni & Raikos, 2018; Singh and Goyal, 2008). Absorption of lycopene is by both passive diffusion and active transport facilitated by scavenger receptor class B type 1 (SR-B1) transporter protein, depending on the site of absorption (Moussa et al., 2008). It is absorbed and distributed with the lipid fraction via circulatory system and predominantly found in adrenal gland, liver, testes, adipose tissue, and prostate tissues. The plasma concentration range of lycopene on an average was found to be between 0.22 and 1.06 mmol/L. Halflife of lycopene was reported to be 2e3 days (Schwedhelm et al., 2003). The elimination half-life of lycopene from a tomato paste formulation was found to vary between 28 and 62 h depending on dosage (10e120 mg) as reported by Gustin et al. (2004) in their Phase I study of lycopene. They also reported that the maximum total lycopene concentration was attained at 16e33 h with maximum levels of 0.075e0.210 mM. Lycopene is metabolized into carbon dioxide partially by b-oxidation, additionally beta, beta-carotene 90 ,100 -oxygenase enzyme (BCO2) catalyzes the eccentric cleavage of lycopene apo-100 -lycopenoids (Wang, 2012). Khachik et al., reported the presence of 5,6-dihydroxy-5,6-dihydrolycopene a lycopene metabolite in vivo in human serum; they also found epimeric 2,6-cyclolycopene-1,5-diols in human serum and milk (Khachik et al., 1995; Lindshield et al., 2007). The primary route of excretion of lycopene is via the fecal route whereas the polar metabolites are excreted through urine (Hwang, 2005). Additional routes for elimination of lycopene are via breast milk and sebaceous glands. Another most important factor effecting lycopene absorption efficiency and bioavailability is isomerization (Faisal et al., 2010). Different isomeric forms (all-E)- and (Z)-isomers of lycopene exhibit varied absorption properties and consequently varied bioavailability in humans. The Cis/Z-isomers are considered to be more bioavailable than all-transisomer (Honda et al., 2019). Nearly 90% of lycopene from natural fruits and vegetables like raw tomatoes exists in alltrans forms in the crystalized state, in spite of this, the Z-isomer accounting to nearly 50% is detected in the human body in plasma and other tissues or organs, such as the liver (Saini et al., 2020). Lycopene isomerization from all-E-isomer to Z-forms is reversible and can occur in vivo during any of the pharmacokinetic phases (Huang & Hui, 2020). The process of isomerization occurs in the gastrointestinal lumen, intestinal absorptive cells called enterocytes (Richelle et al., 2010),

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and liver (Teodoro et al., 2009), also it is known to be enhanced by the acidic condition of the gastric milieu (Re et al., 2001; Ross et al., 2011). Numerous factors are known to affect lycopene bioavailability of the isomeric forms such as the release from the food matrix, thermal processing of lycopene such as cooking, presence of fat content, coadministration with other carotenoids, soluble compounds, and lycopene dosage (Shi & Le Maguer, 2000). Borel et al., reported that a nutritional dose of divalent Ca2þ can impair bioavailability of dietary lycopene in healthy humans (Borel et al., 2016). It is also evident from several studies that the difference in the bioavailabilities of lycopene isomeric forms is attributed to their solubility in mixed micelles facilitating their absorption. Subsequently, cis-isomers have greater solubility in lipophilic solutions along with lower tendency to aggregate and crystallize, and hence more bioavailable. This was proved by the study done by Cooperstone et al., who reported that the lycopene in tangerine tomatoes is rich in cis-lycopene that are more bioavailable than the lycopene in red tomatoes that is all-trans-lycopene. Moreover, they reported that cis-lycopene is present in chromoplasts that are globular lipid structures. In contrast, all-trans lycopene is present in the form of large crystalline aggregates responsible for poor solubilization and lower bioavailability (Cooperstone et al., 2015). Tomato extract or tomato processed foods can be regarded as ideal source of lycopene containing highly bioavailable cis/Z-isomers that are stable and devoid of retroisomerization (Soares et al., 2019).

6.6 Mechanism of action of lycopene Lycopene is regarded as a potent nutraceutical successfully used in the treatment of chronic diseases such as cancer, hypertension, cardiovascular disease (CVD), neurodegenerative disease, and osteoporosis (Giovannetti et al., 2012). One of the most important characteristic of lycopene is its antioxidant nature that is attributed by singlet oxygen (O2) quenching effect, its power to deactivate an array of free radicals (e.g., hydroxyl radical, hydrogen peroxide, nitrogen dioxide, and thiol and sulfonyl radicals and lipid peroxyl radicals) (Shixian et al., 2005). The following reaction processes are considered to be responsible for the antioxidative effect of lycopene, firstly adduct formation, transfer of electrons to the radical species, and also allylic hydrogen abstraction (Kong et al., 2010). Furthermore, it is important to note that the reactivity of lycopene is highly dependent on its molecular structure, site of cellular action, interaction with other antioxidants, partial oxygen pressure, and the concentration at the site (Sharifi-Rad et al., 2020). Also, lycopene exhibits antiinflammatory effects, immune enhancement, lipid modulation properties, neuroprotective activity by enhancing gapjunctional intercellular communication, and antineoplastic effects by varying mechanism of action. The various therapeutic properties of lycopene with their respective mechanism of action are depicted in Fig. 6.3.

FIGURE 6.3 Therapeutic properties of lycopene with their respective mechanism of action.

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6.7 Isomerization and stability of lycopene Lycopene exists in cis- and trans-isomeric configurations due to the presence of double bonds in its structure. However, lycopene with a few exceptions, naturally, is present (almost 90%) in an all-trans-isoform, referred to as all-E-lycopene and the remaining 10% is found in its Cis/Z-isomeric forms of lycopene. Largely existing cis forms are 5Z, 9Z, and 13Z (Ashraf et al., 2020; Petyaev, 2016). The all-trans-isoform of lycopene can undergo mono- or poly-isomerization. Cis-isomerization can be initiated when lycopene is exposed to light, high thermal energy, chemical reactions (using oxygen, catalysts, acids, and metal ions), during processing and storage, upon light irradiation, cooking (Shi, 2000; Urbonaviciene et al., 2017). This isomerization is important for increasing the bioavailability of lycopene. Many geometrical isomers can occur from all-E-lycopene, but the trans-cis isomerization is practically possible for specific ethylenic groups; this is due to 1,4 interactions around the double bonds leading to steric hindrance (Zhang et al., 2012). Lycopene can exist in numerous geometric configurations of which more than 72 are favorable structurally. The most important lycopene isomeric forms are all-E-lycopene, 5Z-lycopene, neolycopene A (6-cis-lycopene), prolycopene (1-, 3-, 5-, 7-, 9-, 11-cis-lycopene), and cis-lycopene (1-, 3-, 5-, 6-, 7-, 9-, 11-cis-lycopene). The cis-isomers are Z-isomers of lycopene that are thermodynamically most stable. It is reported that the stability of 13Z-lycopene was much less than either the 5Z- or the 9Z-, or the all-trans-isomer. Moreover, of all the isomeric forms of lycopene the most stable form with high antioxidant activity is 5-cis-lycopene (Müller et al., 2011). The order of stability of lycopene isomeric forms is: 5-cis > alltrans>9-cis>13-cis>15-cis>7-cis>11-cis which is in accordance to the ab initio calculations (Lambelet et al., 2009). The various trans-cis isomeric forms are depicted in Fig. 6.4.

6.8 Safety and toxicity studies of lycopene The safety of natural and synthetic lycopene consumption at dietary levels was evaluated in animals, various populations, and subpopulations of humans including children and women of childbearing potential. Moreover, several in vitro studies were performed to assess the toxicity, reproductive side effects, liver uptake, and genotoxicity of lycopene (Trumbo, 2005). In 90-d oral toxicity study done in Wistar albino rats, it was observed that lycopene has a large margin of safety. There were no adverse effects observed upon administration of lycopene up to a dose of 586 mg/kg body weight/d in male rats and up to 616 mg/kg body weight/d females in rats (Jonker et al., 2003). Based on the evidence from several toxicology studies, no-observed-adverse-effect level of 3 g/kg/day consumption of natural and synthetic lycopene is considered to be safe and free from unwarranted side effects (Mellert et al., 2002). Hence, the Institute of Medicine has not determined

FIGURE 6.4 Various trans-cis isomeric forms of lycopene.

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upper limit for lycopene consumption due to lack of substantial data related to lycopene adverse effects (IOM, 2000). Lycopene treatment also showed no evidence for reproductive toxicities. Albeit, the crystalline form of natural lycopene if stored improperly might upon exposure to light and air get converted to a mutagenic compound as a result of degradation. However, this is not the case with formulated lycopene due to incorporation of antioxidants in its composition, degradation is bypassed (McClain & Bausch, 2003). Consequently, the average daily uptake range for lycopene is 0.5e5 mg/day, but 20 mg/day or more can be obtained from consumption of vegetables and tomato juice. Excessive consumption of lycopene can lead to yellow-deep orange discoloration of skin a condition known as lycopenodermia/lycopenemia only side effect observed, which is reversible (Gupta et al., 2015; Reich et al., 1960).

6.9 Therapeutic properties of lycopene 6.9.1 Antioxidative effects Oxidative stress is considered to be the most significant factor leading to several chronic diseases. There exists a balance between oxidant and antioxidant system in the body. The generation of free radicals and reactive oxygen species (ROS) tend to disturb the balance and curb the endogenous antioxidant defense system. Several strategies to boost the antioxidant system are under spotlight (Niki, 2010). Dietary antioxidants that can ensure protection from oxidative damage by reducing the ROS generation were being considered the most efficient preventive strategic method; however, due to lack of substantial evidence and clinical trials their efficacy still remains dubious (Hajhashemi et al., 2010). Lycopene exhibits antioxidant property attributed to its highly lipophilic nature and the presence of conjugated double bonds (Tvrdá et al., 2016). The prooxidant/antioxidant activity of dietary antioxidants depends mainly on oxygen tension, redox hemostatic status in the cell, and their dosage. It is evident from the previous studies that the in vivo prooxidant/antioxidant activity of lycopene is not only dependent upon the factors mentioned above but also varies depending on the presence of other antioxidant substances like vitamin C or E. Its antioxidant activity is mostly at cellular levels causing interaction with biological membranes and lipid components (Kurutas, 2016). Therapeutic potential of lycopene may be due to its involvement in the array of chemical reactions that ensure the protection of biomolecules against oxidation such as DNA, lipids, and proteins. Lycopene administration at a dose of 100 ng/mL showed increase in the Nrf2, pNrf2, and HO-1 levels; expression of PCNA was increased, decreased Bax expression, and increased Bcl-xL expression levels in D-gal-induced aged ovarian tissues of hens. These results proved that lycopene attenuated oxidative stress via activation of Nrf2/HO-1 pathway in Dgal-induced aged ovarian tissues of hens (Liu et al., 2018). Similarly, it was observed that addition of lycopene supplement to cryopreservation medium holding bovine sperm resulted in an increased sperm motility. It also led to increased sperm mitochondrial activity, exhibited sperm and acrosomal membrane protective effects, and most importantly showed decrease in levels of ROS and intracellular superoxide. This concludes that lycopene exhibited antioxidant properties and increased the survival and efficiency of cryopreserved bovine sperm (Tvrda et al., 2017). Lycopene treatment was found to regulate various biochemical pathways involving sirtuins (SIRT1), nuclear factor-erythroid 2erelated factor 2 (Nrf2), and proprotein convertase subtilisin/kexin type 9 (PCSK9) in order to combat oxidative stress. The several mechanisms through which lycopene exerts antioxidant effects are schematically depicted in Fig. 6.5.

6.9.2 Antiinflammatory activity Chronic inflammation is identified as the major contributing factor toward the causation and progression of several pathological diseases. Lycopene obtained from tomato products or lycopene supplementation has been reported to exhibit protective effect against pathological processes by downregulating the inflammatory cascade. Antiinflammatory activity of lycopene is associated with several cellular mechanisms such as modifications of eicosanoid synthesis or eicosanoid generating enzymes and the downregulation of pro-inflammatory cytokines synthesis and release, inhibition of ROS that are pivotal pro-inflammatory mediators, changes in the expression of cyclooxygenase and lipoxygenase, and modulation of signal transduction pathways that include mitogen-activated protein kinase (MAPK), activator protein-1 (AP-1), and nuclear Factor-kB (NF-kB) signaling. It is also reported that lycopene can exhibit antiinflammatory effects via activation of transcription factor and reduction of PAF biosynthesis. Moreover, some studies suggest it may be via induction of apoptosis in activated immune cells. Enzymes inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) have been implicated in disturbance of redox hemostasis by ROS production in TNF-a, IL-1, lipopolysaccharide (LPS), or 4-O-tetradecanoylphorbol-13-acetate (TPA) stimulated cells (Palozza et al., 2010). Lycopene has been reported to counteract the effects of iNOS and COX-2 by

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FIGURE 6.5 Schematic representation of antioxidant effects of lycopene. (A) Major therapeutic benefits of lycopene attributed to its radical scavenging or antioxidant effects. Hence it is regarded as singlet oxygen scavenger. (B) The several pathways through which lycopene exerts its antioxidant effects majorly it activated Nrf2-HO-1 and Bcl2 pathway inhibit apoptosis; on the other hand it inhibits Bax pathway to inhibit apoptosis.

inhibiting nitric oxide production and/or by downregulating NF-kB, signal transducer and activator of transcription-1-a (STAT-1-a), and interferon regulatory factor-1 (IRF-1) at protein and mRNA levels (Kim et al., 2014). Lycopene inhibits iNOS levels stimulated by LPS induction in mouse macrophage cell (Rafi et al., 2007). Lycopene was shown to increase NRF2 levels, nearly doubling nicotinamide adenine dinucleotide phospho (NAD(P)H) quinone oxidoreductase 1 (NQO1) protein and mRNA levels. Kawata et al., in their study reported that lycopene exerted potent antiinflammatory activity in RAW264.7 cells by suppressing LPS-induced expression of Cox2, Nos2, and Tnfa gene expression and also by upregulating the gene expression of Hmox1 (Kawata et al., 2018). In another study done by Bignotto et al., it was reported that acute and repeated administration of lycopene at two doses (25 and 50 mg/kg) inhibited carrageenan-induced paw edema and also reverted the abnormal liver injury markers (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and g-glutamyl transferase) and malondialdehyde back to normal (Bignotto et al., 2009). Cytokines can be pro-inflammatory or antiinflammatory. Pro-inflammatory cytokines are TNFa, IL-1, IFN-g, IL-12, IL-18, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and antiinflammatory cytokines are IL-4, IL10, IL-13, IFN-a, and transforming growth factor-b (TGF-b) (Opal & Depalo, 2000). C-reactive protein (CRP) is regarded

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as clinical marker of inflammation (Sproston & Ashworth, 2018). Goncu et al., in their study demonstrated that lycopene exhibits significant antiinflammatory efficacy in the LPS-induced rat endotoxin-induced uveitis (EIU) model. They reported that pretreatment with lycopene 10 mg/kg for 3 consecutive days significantly decreased aqueous humor concentrations of inflammatory markers such as NO, IL-6, TNF-a thereby reducing uveal inflammation (Goncu et al., 2016). Very few interventional studies in humans exist that serve as basis demonstrating the antiinflammatory effects of lycopene (Fig. 6.6). The results from these studies are inconsistent. Hence, further critical research needs to be undertaken especially in humans considering several variables and factors to identify the potential of lycopene/its supplements as antiinflammatory agent.

6.9.3 Anticancer effects Cancer is a multifactorial disease that must be targeted in several directions in order to avoid cancer development and progression. The existing treatment for cancer is both a boon and bane to the society as it can curtail the disease to some extent; but, at the same time the chemotherapeutic agents used are themselves associated with several deleterious effects. Hence, cancer research is always top priority among the scientists who are trying to develop ideal cancer drugs from a variety of sources. Phytochemicals, in this regard, always occupy the first place of drug discovery. Several studies suggest that a diet enriched with fruits and vegetables with higher antioxidant compounds has potential anticancer properties. Numerous epidemiological studies have proven that there exists an inverse relationship between consumption of lycopenerich tomatoes and cancer risk (Barber & Barber, 2002). Lycopene is regarded to be an excellent phytonutrient with proven antioxidant properties. It is found to retard cancer progression in different types of cancers such as prostate cancer, gastric cancer; breast, stomach, ovary, and colon cancer; etc. The various possible mechanisms by which lycopene may prevent cancer may include-modulation of growth factor signaling pathways, cell cycle progression, and alteration of cell survival via changes in intracellular signaling pathways. Lycopene also exhibits antineoplastic effects by enhancing its antiinflammatory, antiangiogenic, antimetastasis, antiinvasive, and immunomodulatory actions. It might halt carcinogenic progression by modulating cellular processes through several molecular targets such as AP-1, NF-kB, STAT (signal transducers and activator of transcription), HIF (hypoxia-inducible factor), Nrf2 (nuclear factor-erythroid 2erelated factor 2), PI3K/AKT (phosphoinositide 3-kinase/AKT), MAPKs (mitogen-activated protein kinases), and VEGF (vascular endothelial growth factor) (Trejo-Solís et al., 2013).

FIGURE 6.6 Antiinflammatory effects of lycopene. Lycopene decreases the pro-inflammatory and inflammatory mediators by downregulating several inflammatory signaling cascades such as those involving NF-kB, MAPK, JNK, ERK, p53, IL-1b. It also inhibits the ROS-related oxidative stress. Consequently, it shows its antiinflammatory effects.

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Data from existing preclinical and clinical research studies prove the anticancer properties of lycopene. In addition, there is an immense research indicating the mechanisms by which these properties are exhibited. For instance, Zhou et al., demonstrated that treatment with lycopene inhibits growth of HGC-27 cells via the upregulation of p-ERK proteins expression, showing its antitumor effect on HGC-27 cells and gastric carcinogenesis (Zhou et al., 2016). In another study administration of lycopene to the gastric carcinomaeinduced rats led to the upregulation of antioxidants and immunity activities thereby reducing the risk of gastric cancer. Lycopene exhibited its anticancer effects by increasing levels of IL-2, IL-4, IL-10, TNF-a levels and decreasing the IL-6 level in carcinoma induced rats (Luo & Wu, 2011). Kim and Kim in a review suggested that oxidative stressemediated gastric carcinogenesis can be prevented by appropriate consumption of raw lycopene or lycopene supplements. They also proposed mechanism by which lycopene can regulate gastric carcinoma. Accordingly, lycopene scavenges free radicals generated due to stress and subsequently the antioxidant enzymes system is activated that protects gastric mucosa against oxidative stress via ERK activation, p53 induction, modulation of cell cycle, and restoration of immune function (Kim & Kim, 2015). Mekuria et al., performed meta-analysis evaluating the anticancer activity of lycopene in animal models of hepatocellular carcinoma (HCC), which showed that lycopene interferes with the initiation and progression steps of carcinogenesis in animal models (Mekuria et al., 2020). Evidences from in vitro and in vivo experimental data suggest that administration of lycopene can exhibit anticancer effects against liver cancer by inhibiting NF-kB. Jhuo et al., confirmed that lycopene treatment at dose of 0.1e5 mM exhibited antimetastatic effect on liver adenocarcinoma (SK-Hep-1) cells via downregulation of NADPH oxidase 4 (NOX4). The treatment eventually exhibited decrease in the intracellular ROS levels, causing attenuation of TGF-b-induced signaling along with the cancer cell metastasis (Jhou et al., 2017). Also, it is influenced by presence or absence of BCO2 and lycopene supplementation. Bhatia et al., demonstrated that lycopene enriched tomato extract could possibly delay the initiation of HCC (Bhatia et al., 2018). Effect of lycopene treatment on human breast cancer cell lines of type estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 was studied by Wang and Zhang who concluded that lycopene inhibited cell cycle progression halting the growth of ER-positive MCF-7 cells. Whereas, ER-negative MDA-MB-231 cell growth was inhibited by G1 phase cell cycle arrest and apoptosis induction (Wang & Zhang, 2007). Koh et al., reported that lycopene exhibits antiproliferative and/or antiinvasive/migratory effects in H-Ras MCF10A and MDA-MB-231 breast carcinoma cell lines by inhibiting ERKs and Akt signaling pathways (Koh et al., 2010). Nahum et al., proposed that lycopene might inhibit cell cycle progression in human breast (MCF-7 and T-47D) and endometrial (ECC-1) cancer cells via reduction of the cyclin D levels causing retention of p27 in cyclin E  cdk2 complex, thus leading to inhibition of G1-CDK activities (Nahum et al., 2010). Holzapfel et al., in their study evaluated the antitumor potential of lycopene treatment on ovarian cancer using intraperitoneal animal model. They reported that lycopene consumption significantly reduced the metastatic load of cancer in mice induced with ovarian cancer, and also diminished tumor burden. It exhibited synergistically enhanced antitumorigenic effects of paclitaxel and carboplatin. Not only this, lycopene also reduced the number of cancer proliferating cells. It reduced the overtly expressed ovarian cancer biomarker, CA125. Lycopene treatment also is associated with decreased expression of several markers like ITGA5, ITGB1, FAK, MMP9, EMT, and ILK. Along with decreased expression of the integrin a5 protein and finally with downregulation of MAPK pathway all these attributed to the antimetastatic and antiproliferative effects (Holzapfel et al., 2017). In another study it was revealed that LPS-stimulated SW 480 human colorectal cancer (CRC) cells when treated with lycopene at concentrations of 0, 10, 20, and 30 mM suppressed the expression of TNF-a, IL-1b, IL-6, PGE2, COX-2, NO, and iNOS in a dose-dependent manner (P < .05). This shows that lycopene could curtail the MAP kinases (ERK1/2, p38, and JNK) subsequently inhibiting NF-kB signaling pathways which might help in the prevention and treatment of CRC (Cha et al., 2017). Siler et al., studied the effects of lycopene and vitamin E in MatLyLu Dunning prostate cancer model. They reported that lycopene led to downregulation of 5-a-reductase by interfering with local testosterone and hence decreased expression of steroid target genes like cystatin-related protein 1 and 2, probasin, prostatic spermineebinding protein, and prostatic steroidebinding protein C1, C2, and C3 chain). Additionally, lycopene caused downregulation of prostatic IGF-I and IL-6 expression. Also, it was observed that androgen signaling was reduced by Vitamin E (Siler et al., 2004). Kim et al., reported that lycopene at concentrations of 10e6 and 10-5 M showed crucial reduction in the growth of LNCaP human prostate cancer cells in a dose-dependent manner (Kim et al., 2002). Graff et al., in a prospective cohort of 46,719 men revealed that lycopene consumption in the form of tomato sauce could help in reducing common gene fusion transmembrane protease, serine 2 (TMPRSS2):v-ets avian erythroblastosis virus E26 oncogene homolog (ERG), i.e., TMPRSS2:ERG-positive prostate cancer disease (Graff et al., 2016). Several epidemiological studies exist which were done to evaluate association between prostate cancer and lycopene consumption. However, they remain inconclusive due to mixed results obtained (Wei & Giovannucci, 2012) (Fig. 6.7).

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FIGURE 6.7 Anticancer effects of lycopene. Lycopene exhibits its anticancer properties by downregulating several signaling pathways such as NF-Kb, MAPK. It downregulates VEGFR, IGFR, FASL, PGDFR receptor modulated pathways.

6.9.4 Cardioprotective effects Antioxidant property of lycopene in conjunction with its antiinflammatory effects attributes lycopene with cardioprotective effects. Several animals, cell line, and epidemiological studies were done to find out the association between lycopene treatment and cardiovascular disorders. In a study done Bansal et al. (2006) evaluated cardioprotective effects of lycopene. Adult male albino Wistar rats were used as experimental models to study the myocardial ischemiaereperfusion injury (MIRI). It was observed that treatment with lycopene 1 mg/kg dissolved in olive oil normalized MAP and HR, reduced lipid peroxidation levels as demonstrated by reduction in MDA levels. It also showed increase in the levels of GSH content (P < .05) and antioxidant enzyme GSHPx. However, the activities of other antioxidant enzymes like SOD and CAT were restored. Furthermore CK-MB isoenzyme which is a specific cardiac marker levels were restored by lycopene treatment (Bansal et al., 2006). In another experimental study done by Upaganlawar et al., myocardial infarction was induced by isoproterenol in rats. Here they reported that pre-coadministration of lycopene (10 mg/kg/day, p.o.) for a period of 30 days reversed all the abnormal parameters such as Naþ, Kþ, and Ca2þ electrolytes, CRP, Caspase-3 protease along with myeloperoxidase, and nitrite levels. Furthermore, it reverted back the vitamin E, uric acid, serum protein, hemodynamic, and apoptotic parameters to normal (Upaganlawar et al., 2012). In another study Zeng et al., observed that lycopene exhibits cardioprotective effects by modulating ROS-dependent MAPK and Akt/GSK3b signaling, activating the antioxidant system, enhancing the mitochondria function. Hence, it provided protection against cardiac hypertrophy in both in vivo and in vitro models (Zeng et al., 2019). Moreover, Duan et al., demonstrated that coadministration of lycopene with ischemic postconditioning (IPoC) in hypercholesterolemic rats exhibits cardioprotective effects and prevents MIRI via inhibition of endoplasmic reticulum stress markers, and upregulation of the reperfusion injury salvage kinase (RISK) (Duan et al., 2019). In a study done by He et al., it was reported that lycopene (10 mg/kg/day) decreased the expression of collagen I and collagen III along with decrease in TGF-b1, TNF-a, IL-1b, also the apoptotic proteins like caspase-3, caspase-8, and caspase-9 were reduced. In this study it was suggested that lycopene treatment attenuated cardiac remodeling post-MI via downregulating the NF-kB signaling pathway thereby reducing inflammatory responses and cardiomyocyte apoptosis (He et al., 2015). Pretreatment with lycopene (1.5 mg/kg/ day) provides protection against ischemia/reperfusion (I/R) injury as seen in in vitro and in vivo studies done by Yue et al. They reported that lycopene protects cardiomyocytes from mitochondrial dysfunction and oxidative damage induced by I/R injury. This was possible via inhibition of mitochondrial ROS production and mitochondrial transcription factor A

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(Tfam) stabilization as observed by reduction in mt8-hydroxyguanine (8-OHdG) and mtDNA content along with decreased mtDNA transcription levels (Yue et al., 2015). In a study done by Li et al., it was reported that lycopene can be used to treat cardiac injury induced by atrazine owing to its antiinflammatory effect, via downregulation of the NF-kappa B pathway and inhibition of NO production thus exhibiting its cardioprotective effects (Li et al., 2017). Furthermore, lycopene treatment might regulate cholesterol levels by a mechanism similar to statins, i.e., inhibition of enzyme HMG-CoA reductase via downregulation of PCSK9 mRNA synthesis in cholesterol biosynthesis (Alvi et al., 2017). Subsequently, lycopene was known to enhance activity of LDL receptors in macrophages (Cheng et al., 2019), improve the LDL/HDL ratio, and reduce synthesis of dysfunctional HDL (Thies et al., 2016). In a meta-analysis done by Ried and Fakler it was suggested that consumption of lycopene at doses 25 mg daily could reduce LDL cholesterol by 10% similar to consumption of low doses of statins in patients with moderate hypercholesterolemia (Ried & Fakler, 2011). However, some research studies also exhibited no effect of lycopene treatment on CVD (Mozos et al., 2018). As per the existing literature it is documented that lycopene may reduce expression of cell surface adhesion molecules along with reduction in the thickness of intima-media suggesting it to be useful in the prevention of atherosclerosis (Martin et al., 2000). In conclusion treatment with lycopene or its supplementation can provide cardioprotective and lipid-lowering effects. However, further clinical trials are warranted to know the exact mechanism and dosage required.

6.9.5 Antidiabetic effects Diabetes is an array of metabolic diseases characterized by hyperglycemia primarily due to dysfunctional insulin signaling. Hyperglycemia can cause oxidative stress due to glucose auto-oxidation and formation of AGEs (advanced glycation end products) which are considered to be the main reason for an end organ damage. Yin et al., reported that lycopene oil solution at a dose of 10 mg/kg or 20 mg/kg body weight upon oral administration once a day for 10 weeks reverted the abnormal parameters like increased fasting blood glucose (FBG) levels, lipid levels in blood and liver, glycosylated hemoglobin, homeostasis model assessment ratio (HOMA-IR) in streptozotocin (STZ)-induced diabetic rats to normal also normalized circulating insulin levels. They concluded that lycopene and tomato products upon consumption activate the antioxidant enzyme and regulate glycolipid metabolism in STZ-induced diabetic rats (Yin et al., 2019). In an experimental study utilizing HK-2 cells and Wistar rats done by Tabrez et al., it was found that AGEs formed from D-ribose can lead to the upregulation of RAGE expression both in HK-2 cells and Wistar rats’ kidneys causing complications of diabetic nephropathy. Subsequently, administration of lycopene altered AGE-RAGE axis by its indirect antiglycated effect. It also inhibited the inflammatory proteins (NF-kB, TNF-a, MMP-2) in HK-2 cells providing evidence that the process of inflammation is either delayed or halted (Tabrez et al., 2015). It is hypothesized that hyperglycemia can cause neuronal cell death. Several studies carried out in animal models of type-1 diabetes reported that hippocampal neuronal death via apoptosis is the main reason for cognitive disorders in diabetic rats (Li et al., 2002). Soleymaninejad et al., from their experimental study concluded that treatment of diabetic rats with lycopene alone or its coadministration with insulin for 8 weeks caused downregulation of proapoptotic Bax gene and upregulation of antiapoptotic Bcl-2 and Bcl-xL genes along with restoration of hippocampal areas (Soleymaninejad et al., 2017). Sharma et al., studied the antidiabetic effects of lycopene niosomes and reported that the formulation significantly reduced blood glucose level along with the other biochemical parameters in diabetic-induced rats on treatment with lycopene niosomes at doses of 100 and 200 mg/kg for 14 days (Sharma et al., 2017). Figueiredo et al., demonstrated that treatment of STZ-induced diabetic rats with combination of lycopene þ metformin showed synergistic effects and significantly decreased the postprandial glycemia, activated the antioxidant defenses leading to damage of the ROS and AGEs formed, attenuated dyslipidemia, and upregulated PON1 activity (Figueiredo et al., 2020). Gao et al., from their large cross-sectional study reported that an inverse association exists between lycopene intake and FBG (Pfor trend< 0$001) and GDM risk particularly among prim gravid Chinese women; they concluded that intake of dietary lycopene decreased risk of GDM effectively among primigravid women mainly by lowering FBG levels (OR 0.20;95% CI 0.07, 0.55 in the highest vs. the lowest quartile of intake; Pfor interaction ¼ 0$036) (Gao et al., 2019). Neyestani et al., conducted a clinical trial utilizing 35 patients in Iran. In this study they reported the beneficial effects of physiological dose of lycopene in patients with type 2 diabetes mellitus. It was observed that lycopene is an immune enhancer, can enhance the total antioxidant capacity (TAC), inhibit formation of MDA-LDL adducts, and may enhance the innate arm of the immune response (antiatherogenic) while diminishing T-cell-dependent adaptive (proatherogenic) immune response. Additionally, it might prevent macrophage uptake of ox-LDL, and also prevent foam cell formation. All these events might prove to be effective in modulating oxidative stress and serum levels of immunoglobulin M in patients with type 2 diabetes mellitus and help in the long-term prevention of diabetic complications, especially CVD (Neyestani et al., 2007).

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6.9.6 Osteoprotective effects Bone is a dynamic connective tissue comprising of osteoblasts, osteocytes, and osteoclasts. The risk of osteoporosis is mostly evaluated through bone mineral density (BMD). Bone loss may be enhanced by increased levels of tumor necrosis factor-a (TNF-a) a pro-inflammatory cytokinin. Osteoporosis is the major disorder that decreased levels of estrogen in postmenopausal women, might be attributed to suppression of osteoprotegerin (OPG), and increase in activity of receptor activator of nuclear factor kB ligand (RANKL), thereby increasing osteoclastogenesis (Florencio-silva et al., 2015). Evidence from several in vitro and in vivo studies using animal models, cell lines, and epidemiological studies exists that show the beneficial effects of lycopene on bone health. Investigation done by Liang et al., showed that lycopene treatment with three doses for 8 weeks was able to revert the increased blood Ca and P back to normal that was induced by the ovariectomy (OVX), showing that lycopene treatment downregulated the rate of bone loss. Furthermore, in an interventional study done on postmenopausal women by Mackinnon et al., it was reported that lycopene supplementation for 4 months significantly decreased the bone resorption marker N-telopeptide (NTx) levels. This was achieved by reducing the oxidative stress parameters that might consequently reduce the risk of osteoporosis (MacKinnon et al., 2011). Iimura et al., showed that lycopene intake significantly inhibits bone loss by suppressing bone resorption in ovariectomized 6-week-old female SpragueeDawley. They observed an increase in the BMD of the lumbar spine and the tibial proximal metaphysis due to lycopene treatment in a dose-dependent manner. This increase in BMD is due to enhanced bone formation and decreased bone resorption (Iimura et al., 2014). The epidemiological and clinical studies done by several researchers demonstrated that there can be significant reduction in the bone resorption markers by incorporation of dietary lycopene up to approx. 30 mg/day in postmenopausal women. Russo et al., in their pilot controlled clinical study reported that treatment with lycopene-rich tomato sauce at the dose of 10 mM halted the bone loss in postmenopausal women through the activation of the WNT/b-catenin and ERK1/2 pathways, upregulation of RUNX2, alkaline phosphatase, COL1A, and downregulation of RANKL Saos-2 (Russo et al., 2020).

6.9.7 Hepatoprotective effects Liver is the most prominent site for metabolism and detoxification. It helps in maintaining a balance between oxidante antioxidant system. Imbalance in this system is attributed to hepatic damage characterized by oxidative stress. AbdelRahman et al., studied the effects of lycopene against Bisphenol A (BPA)-induced hepatotoxicity in cyclic female rat model. They reported that hepatic oxidative injury caused by BPA along with apoptotic effect were reverted to normal by MDA suppression and via amelioration of SOD and GPx activities. Lycopene also caused overregulation of CYPR450 metabolic enzymes which helped in subsequent removal of BPA metabolites and decreasing further exposure of liver to harmful effects of BPA. All these hepatoprotective effects of lycopene were attributed to downregulated hepatic caspase-3, thereby apoptosis was reduced and hepatic integrity was maintained (Abdel-Rahman et al., 2018). Study done by Jiang et al., suggests that pretreatment with lycopene (5, 10, and 20 mg/kg) exhibits a hepatoprotective role on nonalcoholic fatty liver disease in rat. They reported that treatment with lycopene significantly reduced MDA levels, levels of lipid products LDL-C, FFA, and improved HDL-C levels. Similar study was done by Pinto et al. They studied effects of pretreatment of lycopene in CCl4-intoxicated rats. They reported that pretreatment with lycopene significantly increased antioxidant enzymes (SOD, CAT, GPx, GST, and GSH) levels. Moreover, the study showed an increased lipoxygenase (LOX) activity in CCl4-intoxicated rats pretreated with lycopene (Pinto et al., 2013). Furthermore, its hepatoprotective effect might be associated with downregulation of the TNF-a and CYP2E1 expression, also observed reduction in the infiltration of liver fats (Jiang et al., 2016). Shimzu et al., studied the effects of lycopene against concanavalin A (Con A)-induced hepatic damage in BALB/c male mice. They reported that pretreatment with lycopene (25 mg/kg) significantly reduced the serum hepatic marker (ALT and AST) levels. It also significantly decreased the abnormally altered levels of IL-6, IFN-g, and TNF-a in serum. The data from these experimental studies indicate that lycopene has antioxidant and cell protective effects, which were attributed to lycopenic modulation of oxidative stress. This study confirmed the hepatoprotective effects of lycopene (Shimzu et al., 2018).

6.9.8 Skin protective effects Lycopene is a carotenoid attributed with antiaging effects. It is also known for its photoprotective effect on skin against UV-induced damage. Chronic UVR exposure is associated with photodamage, sunburn, and premature skin aging characterized by loss of skin elasticity, pigmentation, and appearance of wrinkles. Earlier research studies reported that

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enhanced dietary intake of lycopene is associated with its increased concentration in human skin, and hence can be used as a protective agent from UVR-induced erythema (Stahl et al., 2001). Rizwan et al., demonstrated that Lycopene is a potent nutraceutical that provides protection against photodamage by the UVR possibly through induction of mtDNA deletion and reduction in procollagen (pC) I, fibrillin-1, and matrix metalloproteinase (MMP)-1 levels by negating the damage caused by ROS and oxidative stress. Finally, they concluded that consumption of lycopene from tomato paste provides significant protection from acute and chronic photodamage (Rizwan et al., 2011). Ascenso and colleagues have suggested that the lycopene formulated as ethosomes or transferosomes exhibited enhanced skin retention properties due to efficient vesicular dermal delivery systems, hence can be used for the treatment of skin inflammatory disorders (Ascenso et al., 2013). In a randomized, crossover study done by Beck et al., it was revealed that the intake of lycopenerich tomato nutrient complex (TNC) completely hindered UVA1- and UVA/B-stimulated upregulation of hemeoxygenase 1 (HO-1), intercellular adhesion molecule 1 (ICAM-1), and matrix metallopeptidase 1 (MMP-1) mRNA expression. Hence providing molecular evidence for photoprotective effects of lycopene against ultraviolet damage of human skin (Grether-Beck et al., 2017).

6.9.9 Additional health benefits of lycopene Lycopene exhibits neuroprotective effects against neurodegenerative diseases like Alzheimer’s diseases and modulates amyloidogenesis, by decreasing Ab accumulation, downregulating the expression of neuronal b-secretase beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), APP, and upregulating the a-secretase A disintegrin and metalloproteinase 10 (ADAM10) levels (Prakash & Kumar, 2014). Lycopene consumption downregulated the expression of IBA-1, a marker of microgliosis, hence considered to be useful in the prevention of age linked neuroinflammatory diseases (Zhao et al., 2018). Lycopene also exhibits neuroprotective effects against Parkinson’s disease (Prema et al., 2015). Several experimental models provide evidence about neuroprotective effects of lycopene; however, there are no clinical trials present. Lycopene treatment can be used to overcome fertility-related issues. It was demonstrated by various studies that lycopene prevents sperm DNA fragmentation, and improved the sperm count and motility (Durairajanayagam et al., 2014; Zini et al., 2010). Additionally, lycopene treatment increased glutathione peroxidase (GPx) activity (Hekimoglu et al., 2009) and decreased MDA levels (Kaya et al., 2019). Clinical trials reported that upon consumption of lycopene twice daily at dose of 2 mg for approximately 3e12 months showed significant alteration in sperm characteristics and pregnancy rates (Gupta & Kumar, 2003; Majzoub & Agarwal, 2018).

6.10 Conclusion In this chapter the authors tried to shed a light on the chemical, biological, and pharmacological aspects on the nutraceutical phytopigment - lycopene. The data gathered from several epidemiological studies reveal that lycopene is an effective free radical scavenger and singlet oxygen quencher possessing astonishing anti-oxidant and anti-inflammatory properties. These characteristic properties have attributed lycopene with anticancer, antidiabetic, and hepato- and neuroprotective activities. However, the bioavailability of the isomeric forms and their appropriate delivery methods is still naïve. Moreover, the evidence existing also is contradictory in several instances due to pro/antioxidant nature of lycopene. Consequently, substantial research in variety of experimental models with human clinical trials is further warranted to conclude the exact therapeutic potential, its underlying mechanism, and to determine the dose of lycopene and its supplements.

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Journal of Nutrition, 138(8), 1432e1436. https://doi.org/10.1093/jn/138.8.1432 Mozos, I., Stoian, D., Caraba, A., Malainer, C., Horbanczuk, J. O., & Atanasov, A. G. (2018). Lycopene and vascular health. Frontiers in Pharmacology, 9(MAY), 1e16. https://doi.org/10.3389/fphar.2018.00521 Müller, L., Goupy, P., Fröhlich, K., Dangles, O., Caris-Veyrat, C., & Böhm, V. (2011). Comparative study on antioxidant activity of lycopene (Z)-isomers in different assays. Journal of Agricultural and Food Chemistry, 59(9), 4504e4511. https://doi.org/10.1021/jf1045969

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Nahum, A., Hirsch, K., Danilenko, M., Watts, C. K. W., Prall, O. W. J., Levy, J., & Sharoni, Y. (2010). Lycopene inhibition of cell cycle progression in breast and endometrial cancer cells is associated with reduction in cyclin D levels and retention of p27 Kip1 in the cyclin E  cdk2 complexes. Oncogene, 2001, 3428e3436. Nanta, N., Songsri, P., Suriharn, B., Lertrat, K., Lomthaisong, K., & Patanothai, A. (2020). Seasonal variation in lycopene and b-carotene content in Momordica cochinchinensis (Lour.) Spreng. (Gac fruit) genotypes. Pakistan Journal of Botany, 52(1), 235e241. https://doi.org/10.30848/ PJB2020-1(39) Naviglio, D., Sapio, L., Langella, C., Ragone, A., Illiano, M., Naviglio, S., & Gallo, M. (2019). Beneficial effects and perspective strategies for lycopene food enrichment: A systematic review. Systematic Reviews in Pharmacy, 10(2), 383e392. https://doi.org/10.5530/srp.2019.2.49 Neyestani, T. R., Shariatzadeh, N., Gharavi, A., Kalayi, A., & Khalaji, N. (2007). 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British Journal of Dermatology, 164, 154e162. https://doi.org/10.1111/j.1365-2133.2010.10057.x Roh, M. K., Jeon, M. H., Moon, J. N., Moon, W. S., Park, S. M., & Choi, J. S. (2013). A simple method for the isolation of lycopene from Lycopersicon esculentum. Botanical Sciences, 91(2), 187e192. https://doi.org/10.17129/botsci.413 Ross, A. B., Vuong, L. T., Ruckle, J., Synal, H. A., Schulze-König, T., Wertz, K., Rümbeli, R., Liberman, R. G., Skipper, P. L., Tannenbaum, S. R., Bourgeois, A., Guy, P. A., Enslen, M., Nielsen, I. L. F., Kochhar, S., Richelle, M., Fay, L. B., & Williamson, G. (2011). Lycopene bioavailability and metabolism in humans: An accelerator mass spectrometry study. American Journal of Clinical Nutrition, 93(6), 1263e1273. https://doi.org/10.3945/ ajcn.110.008375 Rozzi, N. L., Singh, R. K., Vierling, R. A., & Watkins, B. A. (2002). Supercritical fluid extraction of lycopene from tomato processing byproducts. Journal of Agricultural and Food Chemistry, 50(9), 2638e2643. https://doi.org/10.1021/jf011001t Russo, C., Ferro, Y., Maurotti, S., Salvati, M. A., Mazza, E., Pujia, R., Terracciano, R., Maggisano, G., Mare, R., Giannini, S., Romeo, S., & Pujia, A. (2020). Lycopene and bone: An in vitro investigation and a pilot prospective clinical study. Journal of Translational Medicine, 18, 1e11. https:// doi.org/10.1186/s12967-020-02238-7 Saini, R. K., Alaa, A. E. D., Roohinejad, S., Rengasamy, K. R. R., & Keum, Y. S. (2020). Chemical stability of lycopene in processed products: A review of the effects of processing methods and modern preservation strategies. Journal of Agricultural and Food Chemistry, 68(3), 712e726. https:// doi.org/10.1021/acs.jafc.9b06669

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Chapter 7

Carotenoids Sweta Priyadarshini Pradhan1, Santwana Padhi2, Monalisa Dash3, Heena4, Bharti Mittu5 and Anindita Behera1 School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan Deemed to be University, Bhubaneswar, Odisha, India; 2KIIT Technology Business

1

Incubator, KIIT Deemed to be University, Bhubaneswar, Odisha, India; 3Institute of Pharmaceutical Technology, Cuttack, Odisha, India; 4GSSDGS Khalsa College, Patiala, Punjab, India; 5National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur, Punjab, India

7.1 Introduction Nutraceuticals also known as bioceuticals are the food products which can also be considered as alternative to pharmaceutical drugs due to its various health benefit properties (Banach et al., 2018). It has been established by FDA as “food additives” and “dietary supplements,” under the authority of Federal Food, Drug and Cosmetic Act (Singh et al., 2018). Nutraceuticals are available in many food derivative forms like amino acids, vitamins, minerals, herbs, etc. From a nutritive perspective, nutraceuticals are sources of both nutrients (carbohydrates, proteins, fats) and nonnutrients (probiotics, prebiotics, phytochemicals, fibers, and enzymatic regulators) (Bergamin et al., 2019). It is responsible for promoting health, prevention of diseases, and available as an adjunctive therapy of treating different ailments like oxidative stress, Alzheimer’s disease (AD), Parkinson’s disease (PD), diabetes, cancer, inflammatory diseases, osteoarthritis, and cardiovascular diseases (CVDs) (Dutta et al., 2018). Nutraceuticals in today’s world create a new era of research as it endorses the eminence of life, diminishes the risk of diseases, and lifts the immune system of body significantly (Rajasekaran et al., 2008). In this present era, the usage of nutraceuticals is continuing to rise, with no feasible limit given in the recurrent development of the nutraceuticals product range. Carotenoids or tetraterpenoids are red, yellow, and orange organic pigments that are mainly produced from plants, algae, bacteria, and fungi. They are responsible for providing characteristic colors to different food products and living organisms such as pumpkins, carrots, tomatoes, cranberries, corn, flamingo, lobster, salmon, daffodils, and shrimp. Carotenoids are mainly obtained from fats and other basic organic metabolic building blocks of several creatures. It is also produced by aphides, spider mites, and endosymbiotic bacteria in whiteflies (Novakava et al., 2012). There are about 1100 carotenoids present till now which can be majorly divided into two main groups, i.e., carotene and xanthophylls (Yabuzaki et al., 2017). All the derivatives of carotenoids are tetraterpenes, i.e., they are generally derived from 8-isoprene molecule and consist of 40 carbon atoms. About 40 types of carotenoids can be obtained by typical human diet and 20 types of carotenoids have been detected in human blood cells and tissues. Carotenoids can also absorb wavelength ranging from 400 to 900 nm (violet to green color) that’s why the compounds of carotenoids show the complementary colors like deep red, orange, and yellow. Epidemiological studies reported that the diet enriched with carotenoids reduces the incidences of heart diseases (CVD), osteoporosis, diabetes, cancer, age-related macular degeneration (AMD), cataract, and also HIV infections (Milani et al., 2017). In this chapter, carotenoids as important nutraceuticals are being discussed with its sources/derivatives, mechanism of action, extraction processes, techniques of characterization, and its therapeutic applications.

7.2 Sources of carotenoids Carotenoids are the group of phytonutrients (“plants chemicals”) which are mainly found in plant, algae, and bacteria (Meléndez-Martínez et al., 2019). Carotenoids are mainly classified into two groups, i.e., carotene (contain only carbon (C) and hydrogen (H) atoms, e.g., lycopene, aecarotene, and b-carotene) and xanthophylls (contain also oxygen along with carbon and hydrogen atoms, e.g., astaxanthin and cryptoxanthin) (Milani et al., 2017). Carotenoid-containing foods are generally red, yellow, or orange in color but not always as they are also present in green leafy vegetables Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00006-5 Copyright © 2022 Elsevier Inc. All rights reserved.

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(Abe-Matsumoto et al., 2018). Carrots, yams, watermelon, papaya, tomatoes, bell pepper, cantaloupe, mangoes, sweet potato, papaya, spinach, kale, orange, and avocado are among fruits and vegetables which possess greater sources of carotenoids. Tomatoes are mainly rich with lycopene and the green vegetables such as broccoli, spinach, and green beans are the rich sources of a- and b-carotene. Common sources of b-cryptoxanthin are mandarin, peas, egg yolk, corns, and oranges. Green leafy vegetables like broccoli, spinach, collard, and kale are rich in lutein and zeaxanthin. The edible flower called Tropaeolum majus are enriched with lutein. Lutein is also present in Spondias lutea (caja) and Myrciaria dubia (Camu camu). Some of the microalgae like Chlorella sorokiniana MB 1, Chlorella vulgaris, and native microalga called Scenedesmus obliquus CNW-N are enriched with lutein. Zeaxanthin is abundantly found in Caryocar villosum (pequi) and the Indigenous aquatic microalgae Chlorella saccharophila (Singh, Barrow et al., 2015). Astaxanthin and canthaxanthin are obtained from salmon and crustaceans. Astaxanthin is reddish-pink in color and richly available in marine animals such as crabs, lobster, shrimp, and microalgae such as Haematococcus pluvialis, Phaffia rhodozyma, and C. vulgaris (Kim et al., 2016). However, carotenoids cannot be obtained from animal sources so it can be obtained from the daily diet. Mainly the carotenoids are present in the chromoplast of plant cells and isolated as caroteneeprotein complex (Chunhua et al., 2013). Xanthophylls are highly present in vegetables than in fruits, but fruits like papaya and persimmon are virtuous sources of xanthophyll. b-Carotene exists in chlorophylls which are converted to chlorophyllecarotenoid complex in fruits and vegetables (Natália & Sandra, 2016). Some carotenoids can also be obtained from industries by chemical synthesis like astaxanthin, canthaxanthin, becarotene, etc. The industrial production of b-carotene from microalga source of Dunaliella is the largest one in India and probably used for various pharmaceutical use. Likewise, astaxanthin is also obtained from Haematococcus, which have high commercial use for its pigment and high market growth for aquaculture such as salmonids and crustacean’s pigmentation (Torregrosa et al., 2018). Stigmas of saffron contain apocarotenoids like crocetin, crocin, picrocrocin, and safranal. These are water-soluble yellow pigments responsible for the color, taste, and smell (Frusciante et al., 2014). Various types of carotenoids are also found from marine sources and enlisted in Table 7.1.

7.3 Extraction and characterization techniques There have been intensive exertions for the advancement of enhanced extraction procedures of carotenoids over last few decades. Nevertheless, due to the existence of several chemical and physical barriers in complex matrix it leads to low recovery rate. The low recovery rate arises generally because of prevention of mass transfer of carotenoids and diverse levels of polarity of carotenoids during extraction (Saini & Keum, 2018). As like dissolves like, for the extraction of polar carotenoids, the use of more polar solvents like acetone, ethanol, and ethyl acetate etc., is preferred. On the other hand, nonpolar solvents such as tetrahydrofuran, hexane, and petroleum ether are more appropriate for the extraction process of nonpolar carotenoids. The extraction of carotenoids can be classified into five categories: i) ultrasound-assisted extraction (UAE) or microwave-assisted extraction (MAE), ii) accelerated solvent extraction (ASE) technique, iii) supercritical fluid extraction (SCFE), iv) pulse electric fieldeassisted extraction (PEFAE), and v) enzyme-assisted extraction (EAE) (Adadi et al., 2018). All the above-listed extraction methods offer different modes of disintegration, pressure, and temperature for efficient extraction of carotenoids (Li et al., 2017). Traditionally, carotenoids are mainly extracted by low-pressure extraction processes such as soxhlation, agitation, centrifugal, and shaking extraction techniques. Though these methods are simple but they have major limitations. It includes mainly excessive use of organic solvent for the extraction, less recovery rate, poor automation, and more time to process the reaction rate (Strati et al., 2015). The abovementioned drawbacks have mainly fortified scientific inventions to meet the requirements of society and industry. The other emerging technologies include pressurized liquid extraction (PLE), subcritical water extraction (SWE), EAE, and supercritical fluid extraction (SCFE) (Poojary et al., 2016). Earlier most of the extraction processes are conducted by the use of petroleum ether, hexane, methylene chloride, but all these solvents impose adverse health effects due to their toxic nature. The advanced extraction methods include use of green solvents for an efficient extraction of carotenoids (Saini & Keum, 2018). But some limitations of green solvents like high viscosity, high boiling point, and requirement of monitoring during extraction to check lipid oxidation and acidification failed to justify its use for extraction. As a result more potential green solvents commonly used for extraction of carotenoids have been taken under consideration involving ionic liquids, supercritical fluid, bio-based solvents, and hydrotropic solvents. Bio-based solvents like ethanol exhibit low selectivity and sensitivity for hydrophobic compounds due to its polar nature (Singh, Ahmad et al., 2015). The detailed description of all the other emerging solvents used for an efficient and reliable extraction process is discussed in the following sections.

TABLE 7.1 List of different carotenoids found in marine microorganisms with their pharmacological applications with potential benefits for human health. Name of the carotenoid

Marine microorganisms from which carotenoids obtained

b-Carotene Lycopene

Pharmacological applications

References

Microalga, bacteria, and cyanobacteria

Antioxidant, antiinflammatory, antidiabetic, antitumor, benefits for CNS function, and atherosclerosis

Marshall et al. (2007); Misawa et al. (2011); Vilchez et al. (2011)

Microalgae and bacteria

Immune modulation, prevention of atherosclerosis, antioxidant, antiulcer activity, antitumor activity, gene regulation, and coronary heart disease

Igielska-Kalwat et al. (2015); Rao & Rao (2007); Rodrigo-Ban˜os et al. (2015); Viuda-Martos et al. (2014);

Bacteria, microalgae, and haloarchaea

Antitumor effect

Rodrigo-Ban˜os et al. (2015)

Microalgae and haloarchaea

Antioxidant, inhibition of retinitis and cataract, atherosclerosis, antiapoptotic effect and prevention of age-related macular degeneration (AMD), coronary heart disease, and heart stroke

Huang et al. (2015); Nataraj et al. (2016)

Astaxanthin

Microalga, bacteria, cyanobacteria, and haloarchaea

Antioxidant, antitumor, antihyperglycemic, rheumatoid arthritis, diabetic nephropathy, asthma, immune modulation, ocular protective, cardiovascular and neurodegenerative disorders

Relevy et al. (2015); Sluijs et al. (2015)

Zeaxanthin

Microalga, bacteria, and cyanobacteria

Reduction of cardiovascular risk factors, antioxidative, antiinflammatory, breast cancer, maintains visual function

Bernstein et al. (2016); Gammone et al. (2015)

Canthaxanthin

Microalga, haloarchaea, bacteria, and cyanobacteria

Antioxidant, antitumor activity, and provitamin A activity

Mordi et al. (2016); Rodrigo- Ban˜os et al. (2015)

b-Cryptoxanthin

Cyanobacteria

Antioxidant properties, stimulates bone formation, enhances respiratory function, depresses lung cancer tariffs, antiinflammatory, prevents neurodegenerative diseases, and modulates response to phytosterols in postmenopausal women

Min et al. (2017); Liu et al. (2016)

Fucoxanthin

Microalgae

Anticancer, antiobese, antidiabetic activity, hepatoprotective, antimalarial, antiangiogenic effect, reduction of cardiovascular disease, ocular and bone protective effect

Gammone et al. (2015); (Peng et al., 2011)

Salinixanthin

Microalga and haloarchaea

Anticancer activity

Rodrigo- Ban˜os et al. (2015)

Echinenone

Microalga and cyanobacteria

Antioxidant effect

Kent et al. (2015)

Sioxanthin

Actinomycetes

Antioxidant effect

De Jesus Raposo et al. (2015)

Bacterioruberin

Haloarchaea

Antioxidant and anticancer activity

Rodrigo- Ban˜os et al. (2015)

Saproxanthin

Bacteria

Antioxidant and apoptosis-inducing effect

Gammone et al. (2015)

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Phytoene Lutein

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7.3.1 Soxhlet extraction Soxhlet extraction is one of the most used conventional techniques giving the highest recovery of carotenoids. Though, it uses significant amount of solvent, costly, and time-consuming, in most of the cases it acts as a promising method for evaluating the other processes used for extraction of carotenoids. Sometimes higher temperature causes the cisetrans isomerization and thermal degradation of carotenoids. Cardenas-Toro et al. compared the soxhlet extraction, PLE, and percolation in terms of yield, profile, and economic value for carotenoids extracted from pressed palm fibers. The study concluded that the extraction efficiency of carotenoids was highest by soxhlet extraction as compared to pressurized and percolation extraction methods but the selectivity of extraction of carotenoids (a- and b-carotene) was higher and production cost was lower for ASE than soxhlet extraction (Cardenas-Toro et al., 2015).

7.3.2 Ionic liquids as a solvent for extraction To enhance the sustainability, more environment friendly ionic liquids and solvents have been discovered for the extraction process of carotenoids. These include nonvolatile and nonflammable solvents which are made up of loosely held positive and negative ions such as bromide, chloride. Therefore these solvents are indicated as green solvents for the extraction purpose (Grosso et al., 2015). Currently, Leonardo et al. have exploited ionic liquids as a promising solvent for pretreatment stage at mild temperature with an efficient extraction of carotenoids (de Souza Mesquita et al., 2020). These solvents can enter the cell wall by damaging the H-bond network of cellulose. Among numerous classes of ionic liquids, imidazolium-based ionic liquids were found to be the most eficient solvent for extraction of carotenoids, compared to ammonium- and phosphonium-based ionic liquid. The research group evaluated ethanolic and aqueous solution of ionic liquids for the purification and extraction of carotenoids from Bactris gasipaes fruits. The investigation of ionic liquids has improved the selectivity and yield for extraction of carotenoids. However, a limited report is available regarding these features. Thus the properties, cytotoxicity, and environmental impact of ionic liquids can be examined for extraction of different carotenoids from varied sources in future. Murador et al. developed a sensitive and selective supercritical fluid extraction method of high-performance liquid chromatography with diode array detection method hyphenated with atmospheric pressure chemical ionization and triple quadruple mass spectrometry detection for the simultaneous extraction and identification of carotenoids and esters (Murador et al., 2019).

7.3.3 Microwave-assisted extraction To ensure recovery of extra pure carotenoids form food matrix without any interference adequate extraction methodology is needed to be developed with more selectivity and efficiency. This method is quite simple, straight-forward, and economic for the extraction of carotenoids due to less solvent and time consumption (Ho et al., 2015). It is subcategorized into two parts, mainly microwave-assisted solvent extraction and microwave-assisted solvent-free extraction. In case of solventassisted extraction, the solvents and material mixed together and exposed to microwave energy where samples containing carotenoids are heated to boiling point where solvent enters into the material matrix, solubilize and leach out the carotenoids for the further characterization and analysis. On the other hand, microwave hydrodiffusion and gravity (MHG) are a type of solvent-free extraction developed for the extraction of carotenoids (Vian et al., 2008). Due to intense heating system prompt bursting of secondary metabolites like carotenoids occur but in the same time cooling system outside the reactor to decrease the temperature of extract before harvesting is required. The key aspect in improving the yield of extraction of carotenoids includes pretreatment of plant material (biological, chemical, mechanical). However, the major demerit that occurs in microwave-assisted procedure involves intense heating, which decreases the bioavailability of nutraceuticals due to degradation (Rahul et al., 2018).

7.3.4 Ultrasonic-assisted extraction UAE is an important technique of food processing including intracellular and extracellular metabolite extraction. The ultrasonic waves cause acoustic cavitation which leads to rupture of cells which makes bulk extraction of material. The factors need to be optimized during this process of extraction include temperature, intensity, density, and power of ultrasonic waves. Singh et al. have optimized the pulse length to 19.7 s, solvent to CDW ratio of 68.38 mg per liter, and 13.48 min of extraction time in order to acquire the maximum yield of zeaxanthin and carotene from algae C. saccharophila (Singh et al., 2015b). In another experiment, Yan et al. have compared five different solvents like acetone, chloroform, petroleum ether, methanol, and acetone: petroleum ether (1:1) and found the highest carotenoids content from acetone: petroleum ether extract (Yan et al., 2015).

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7.3.5 Enzyme-assisted extraction It is usually being followed as a pretreatment before the solvent extraction procedure in order to enhance the yield of target molecule. Generally, pectinase and cellulose hydrolytic enzymes are used for extraction of carotenoids. Recently the extraction of carotenoids is rising progressively with improved techniques and solvents focused on economic and efficient extraction. Ricarte et al. (2020) investigated the EAE of carotenoids from waste of sunflower by green solvents. Lombhardelli et al. (2020) have used tailored synergetic method for an extraction of carotenoids from tomatoes by using enzymes cellulase, polygalacturonase, xylanase, and pectin lyase at variable temperatures, enzyme dosage, and process time with pH ranging from 3 to 8. They optimized the treatment for 180 min with 25 U/g for the maximum recovery and efficiency. Strati et al. have also reported EAE along with high pressure in order to increase the yield of extracted carotenoids. They reported that high pressure enhanced the extraction yield as compared to ambient temperature by 80% e85% (Strati et al., 2015).

7.3.6 Supercritical fluid extraction Supercritical fluid extraction (SCFE) was an effective method for the extraction process of carotenoids from liquid or solid samples using supercritical carbon dioxide (Sc-CO2) as a solvent for extraction. The lower viscosity and higher diffusion coefficients of Sc-CO2 allow the deeper diffusion inside the complex matrix which enables the higher efficiency as compared to other conventional methods available for an extraction. The final product obtained is highly concentrated. Macías-Sánchez reported an efficient extraction method for carotenoids from Scenedesmus almeriensis using Sc-CO2 as extraction solvent (Macías-Sánchez et al., 2010). Guedes et al. (2013) have reported more selective, sensitive, and faster extraction method to separate carotenoid pigments and antioxidants from microalgae. Furthermore, the supercritical fluid can be recycled so it is an eco-friendly process. In general, extraction pressure (300e400 bar), temperature (40e60
C), flow rate (1e5/mlmin), time (30e120 min), entrainer’s concentration (5%e25% v/v), and CO2 density (solvent power) are the five utmost vital parameters for SFE of carotenoids (Khalil et al., 2016). Recently, Andrade Lima et al. conveyed the method of extraction of carotenoids from vegetable waste matrices including peels of potato, tomato, peach, apricot wastes of yellow, green and red pepper by SCFE (de Andrade Lima et al., 2019). The optimized conditions of extraction in the study have been claimed to give maximum yield of carotene. Other potential green solvents and methods used for a selective and efficient extraction of carotenoids include bio-based solvents, pulse field extraction, hydrotropic solvents, SWE, instant control dropeassisted extraction (Lu, Feng, Han, & Xue, 2013; Martínez et al., 2020). The brief outlook of all the emerging techniques used for an efficient extraction of carotenoids is summarized in Table 7.2.

7.3.7 Characterization of carotenoids Carotenoids are colored organic compounds containing extensive conjugated double bond system. Therefore, for the part of their characterization UVevisible spectroscopy plays an imperative role in which the bathochromic and hypsochromic shift illustrate the properties too. Moreover, due to constrained polyene structure it also possesses cis and trans isomers which can easily be distinguished via infrared spectroscopy. When carotenoids are heated at higher temperatures, they become unstable for which they can’t be characterized by gas chromatography. So, HPLC, MS, UVevisible, or even their combinations are used for characterization of carotenoids in a variety of samples (Giuffrida et al., 2018). The common methods used for identification and quantification of carotenoids are HPLC with UV detection in reverse as well as normalphase HPLC. Generally normal-phase HPLC is not appropriate method for an efficient separation of carotenoids as they are nonpolar in nature. In spite of these techniques, liquid chromatographyemass spectroscopy offers numerous advantages like high selectivity and sensitivity for analysis of carotenoids. In food samples, HPLC coupled with MALDI-TOF is also reported to be a dominant tool for detection of carotenoids (Gupta et al., 2015). Ambati et al. (2017) have studied different analytical techniques for identification of carotenoids composition in algae. Due to extreme health benefits of carotenoids, extensive research has been done worldwide. Therefore, in order to provide quantitative and qualitative data of analysis, it has been a great challenge to develop improved methods for characterization of carotenoids (Mercadante et al., 2017). Recent reports for characterization of carotenoids are described in Table 7.3.

140

TABLE 7.2 Emerging techniques for extraction of carotenoids with advantages and disadvantages. Principle

Advantages

Disadvantages

Reference

Ionic liquid Extraction

These have been prepared by tertiary ammonium salts in order to increase the extraction yield and efficiency.

ILs have been studied as solvents, cosolvents, cosurfactants, electrolytes, as well as used in the creation of IL-supported materials for separation purposes

ILs have proved to be excellent solvent for extraction, they are still expensive in comparison to the conventional solvents commonly used in the extraction of natural products. Moreover, the recycling of ionic liquids is a major concern.

de Souza Mesquita et al. (2020)

Microwaveassisted extraction

Moisture of target material is evaporated by heat from microwaves. The pressure applied induces migration of the solvent into the matrix.

Simple, time efficient, high extraction yield

Sometimes required additional separation method to eliminate solid particles. Thermal degradation is a major issue

Ho et al. (2015)

Ultrasonic extraction

Sound waves at a frequency greater than the human hearing range disrupt the target matrix. The solvent penetrates and releases the extract.

Less solvent consumption, time efficient, higher extraction yield

Ultrasonic waves for the longer time if passed produce heat effect which might degrade the sample

Singh, Ahmad et al. (2015)

Supercritical fluid extraction

CO2 in supercritical state kept in contact with matrix. Penetrates the solid matrix and dissolves mainly lycopene.

Nonflammable, higher selectivity, extraction time less, green solvent usage

Expensive setup, multistage extraction procedure, difficult to handle in laboratories

de Andrade Lima et al. (2019)

Subcritical water extraction

This extraction takes place in hot water under critical temperature, i.e., 100e375
C and at high pressure of about 10e60 bar.

Usage of noxious solvent is prevented for higher extraction yield

Depends on dielectric constant variations of different types of carotenoids

Joana GilCha´vez et al. (2013)

Pressurized liquid extraction

High pressure enhances the diffusion of material inside the porous cell to increase the extraction yield.

Faster extraction, less solvent consumption, higher extraction yield

Thermal degradation

Singh, Ahmad et al. (2015)

Pulsed electric field

The cell wall is ruptured due to high intensity electric field and external field diverges the sheath of plant samples to release carotenoid.

High extraction yield

Parameters have to be optimized before operating the system

Buckow et al. (2013)

Instant drop eassisted extraction

Work based on thermomechanical effect by revealing that the material will be under saturated vapor followed by rapid drip in pressure to vacuum.

No thermal degradation, high extraction yield, no toxic solvent

Expensive setup, multistage extraction procedure

Joana GilCha´vez et al. (2013)

Enzymeassisted extraction

Enzyme causes degradation under minor process condition and hence affluences carotenoid extraction.

Higher extraction yield

Cost of enzyme is expensive

Lombardelli et al. (2020)

Nutraceuticals and Health Care

Extraction method

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TABLE 7.3 Different analytical techniques used for analysis of carotenoids. Analytical technique

Research findings

Reference

Spectrophotometric method (UVevisible)

On the basis of adsorption coefficients quantification of carotenoids has been carried out.

Zaghdoudi et al., 2017

High-performance liquid chromatography (HPLC)

Maximizing carotenoid extraction from algae as a food additive. In pepper carotenoid characterization has been done in profiling 10 carotenoids.

Cero´n-Garcı´a et al. (2018); Li et al. (2017)

Ultrahigh-performance liquid chromatography ephotodiode arrayemass spectroscope (UHPLCPDA-MS)

Comparison of different analytical techniques for the analysis of carotenoids.

Giuffrida et al. (2018)

Ultrahigh-performance liquid chromatography eatmospheric pressure chemical ionization equadruple time-of-flight mass spectroscope (UHPLC-APCI-QTOFMS)

Spectrophotometry coupled to identification of plastoglobules as a site of cleavage of carotenoid.

Rottet et al. (2016)

Rapid separation liquid chromatography (RSLC)

Improved fast and selective method for carotenoid determination.

Abate-Pella et al. (2017)

7.4 Chemistry of carotenoids Carotenoids belong to pigments having characteristic double bond system. These are 40-C isoprenoid components biosynthesized from the five-carbon isoprene units (Saini et al., 2015). Carotenoids are mainly composed of eight isoprenoid units connected in a specific manner so that the nonterminal methyl groups remain in 1,5-position and the two central methyl groups get organized in 1,6-position (Takashi, 2019). Naturally, these are biosynthesized via stepwise addition of three isopentenyl pyrophosphate (IPP) units to one dimethyl allyl diphosphate (DMAPP) to form the 20-carbon compound geranyl geranyl diphosphate (GGPP), the precursor. Two molecules of the formed precursor are further united by phytoene synthase forming the first carotenoid in the biosynthetic pathway, called phytoene. Phytoene is then unsaturated to lycopene with 11 conjugated double bonds from which nearly all the carotenoids are being derived. a- and b-carotene can be formed by cyclization of lycopene on either or both the ends which further can be oxygenated to form xanthophylls like lutein, zeaxanthin, etc. They share a common formula of C40H56O2 and illustrated in Fig. 7.1 (Sun, Tadmor, & Li, 2020). Carotenoids with carbon less than 40 due to the loss of carbon either inside the chain, called as norcarotenoids, or loss of carbon from the end of the chain, called as apocarotenoids. Whereas carotenoids with carbon atoms greater then 40, i.e., 45 to 60, are called as the homocarotenoids (Harrison & Bugg, 2014). Each carotenoid has several isomers due to the presence of altering single and double bonds (Maoka, 2019). Isomers are mostly in all-trans form, although light can also lead to transformation of cis form into trans form depending upon the surrounding environment. Trans forms are more rigid with a greater propensity to crystallize than the cis forms. The shape of the molecule is determined by its isomeric form and can even affect the solubility and absorptivity (Sun, Tadmor, & Li, 2020).

7.4.1 Chemical properties Carotenoids are hydrophobic in nature and so get solubilized in organic solvents. If they are integrated within liposomes or cyclic oligosaccharides like cyclodextrins, they can get solubilized in aqueous solvents. The solubility of the carotenoids can be altered by affecting its polarity which can be done by adding hydroxyl groups to the end groups of the carotenoid. When in solution form, these are sensitive toward elevated temperature, presence of oxygen, light, and acid, thus subjected to oxidative degradation (Saini et al., 2015). The conjugated polyene chromophore in carotenoid molecule is responsible for absorption of light, thus responsible for the color and photoprotective actions. The chromophore absorbs certain wavelengths of visible light and the complementary color arises (Kiokias et al., 2015). Carotenes and xanthophylls are found in different fruits and vegetables with their characteristic color due to difference in their chemical structure, no. of carbon atoms, and no. of conjugations. b-Carotene is the dominating isomer in comparison to a-carotene but high content of a-carotene is found in some of the vegetables and fruits, such as spinach, broccoli, green beans, sweet potatoes, pumpkin, and carrots (Khoo et al., 2011).

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FIGURE 7.1 Different types of Carotenoidsdcarotenes, xanthophylls, and apocarotenoid.

7.4.2 Physical properties Carotenoids being hydrophobic in nature are associated with the bilayer membranes. The hydrophobicity of the molecule gets altered with the addition of the polar group such as the hydroxyl group, thus altering the orientation with respect to the membranes. The physical nature and the function of the membrane can be affected by the differences in orientation of the molecules to the membrane surface to maintain the hydrophobic environment. Lycopene and b-carotene are positioned parallel to the surface of the membrane whereas xanthophylls such as lutein being polar are aligned at right angles to the membrane surface keeping their hydroxyl groups in hydrophobic environment (Khoo et al., 2011). In nature, carotenoids are found as fine dispersions in aqueous media (b-carotene in oranges) and as crystalline aggregates (lycopene in chromoplasts) (Saini et al., 2015).

7.4.3 Electrochemical properties The conjugated double bond systems of carotenoids lead to their electrochemical properties. Delocalization of p-electrons occurs over the entire polyene chain due to the altering p and s bond system. These delocalized electrons acquire energy to transit from ground state to the excited state. The polyene chain even permits the transfer of singlet or triplet energy. The energy needed to do so depends upon the chromophores attached and the length of the conjugated polyene chain (Saini et al., 2015).

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7.5 Mechanism of action of carotenoids 7.5.1 Mechanism of action of carotenoids as antioxidants Carotenoids are the rich source of antioxidants and produce beneficial effects in oxidative stress by interfering with the free radicals produced during oxidative stress. Free oxygen radicals play a vital role in the pathogenesis process of inflammation and induce stress-related disease such as cancer, cardiovascular disorders, neurodegenerative disorders, and ophthalmic diseases (Zaid et al., 2019). Carotenoids inhibit the active free radicals by replacing the electrons and donating hydrogen (H) atoms to radicals or attaching to radicals. Carotenoids scavenge the free radicals by 3 steps in the electron transfer method, i.e., Oxidation and reduction CAR þ ROO$ / CA Rþ þ RO O

(7.1)

CAR þ RO O$ / ROOCAR

(7.2)

ðCAR þ RO O$ / CAR þ ROOHÞ

(7.3)

Addition

Abstraction

The conjugated double bonds present in the carotenoids accept electrons from reactive species and neutralize the free radicals (Cicero & Colletti, 2017). The combination of two lipophilic vitamins like vitamins C, E and carotenoids mainly b-carotene leads to synergistic effect which significantly scavenges reactive nitrogen species (RNS) and inhibits lipid peroxidation levels. Reactive oxygen species (ROS) and RNS are the cytotoxic chemicals produced as a result of inflammatory response. ROS and RNS not only damage parasites and pathogens but also destroy the cells and tissues of host cells which lead to decrease in immunity and cause several chronic diseases due to destruction of lipids, proteins, and nucleic acids (Young & Lowe, 2018). Antioxidant enzymes play vital role in controlling the destructive factors such as ROS and RNS. There are three major antioxidant enzymes called as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). SOD helps in conversion of superoxide into hydrogen peroxide and further detoxification to water and oxygen by GPx and CAT. Additionally, carotenoids can decrease the vulnerability of breaking of single-stranded nucleic acid in cell lines. The oxidative stress is reduced by scavenging free radicals responsible for DNA damage or by controlling the repair mechanisms of DNA (Meléndez-Martínez, 2019). Moreover, overexpression of certain antioxidant genes is stimulated by carotenoids like Nrf2 (Nuclear factor erythroid 2erelated factor 2) transcriptional factors which mainly aid in declining diabetes and neurological disorders (Kaulmann & Bohn, 2014).

7.5.2 Mechanism of action of carotenoids in cancer Carotenoids as candidate for antitumor activity face some limitations like low bioavailability, bioaccessibility, assimilation, and transformation into a lipophilic compound (Fernández-García et al., 2012). As the primary sources of carotenoids in food are from plants, the compounds are very less soluble causing low bioavailability. Similarly the less bioaccessibility is due to interaction of carotenoids with other dietary fibers and phytosterols. Another leading cause of less bioavailability is the transformation or degradation of carotenoids in the food matrix or after ingestion in the gastric environment (McClements et al., 2015). The carotenoids attenuate the cancer signaling pathways by dysregulation of cellular signaling systems like transcription of genes associated with phosphatidylinositide 3-kinase (PI3K), protein kinase B (PKB or Akt), and mammalian target of rapamycin (mTOR). This controls the proliferation, survival, invasion, and metastasis of cancer cells (Sever & Brugge, 2015). Carotenoids trigger the apoptosis or programmed cell death of cancer cells by affecting both intrinsic and extrinsic pathways of apoptosis with activation of caspases (Saini et al., 2020). Unregulated cell cycle causes proliferation, invasion, and metastasis of cancer cells and carotenoids block progression of cell cycle by blocking the key transition points. Mainly the suppression of phosphorylation of cancer suppressor proteins which actively participate in different stages of cell cycle like retinoblastoma (Rb) in S-phase transcription. Phosphorylation of Rb is achieved by activated cyclin D1/cyclin-dependent kinase 4 and 6 complex which causes S-phase transcription and G1eS phase transition. Mostly carotenoids arrest cell cycle by downregulating cyclin D1, cyclin D2, CDK4, and CDK6 expression resulting in upregulation of GADD45 a which restricts the access of the cell into S phase (Rowles & Erdman, 2020). ROS is another leading cause for cancer and the carotenoids maintain the balance of the oxidative stress by acting as dynamic antioxidant for normal cells and as prooxidant in cancer cells, thereby selectively kill the cancer cells (Vijay et al., 2018).

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Growth and metastasis of tumor cells are also influenced by angiogenesis and regulated by growth factors like vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and epidermal growth factor (S¸ahin et al., 2012).

7.5.3 Mechanism of action of carotenoids in cardiovascular diseases One of the major causes for CVDs is oxidative stress. Carotenoids as antioxidants are one of the most preferred agents to prevent the change in structure of plasma lipoprotein (Fiedor & Burda, 2014). Oxidized low-density lipoproteins (OxLDLs) which act as a proatherogenic agent, their formation is inhibited by carotenoids (Bryk et al., 2017). Transendothelial migration, adhesion of leukocytes, aggregation of platelets, and increased expression of growth factors are stimulated by carotenoids which facilitate the regeneration of new blood vessel walls and reduce the bioavailability of nitrous oxide. Physiologically the antioxidant enzymes suppress the overproduction of ROS (Bartekova et al., 2019). Oxidative stress decreases the levels of blood vessels relaxant prostacyclin by arresting their synthesis. Similarly increase in free radicals in the body increases the concentration of F2-isoprostanes, which contract the blood vessels resulting in hypertension. So carotenoids scavenge the free radicals and reduce the blood pressure (Piwowar, 2014). CVD is very common in T2DM patients due to hypertensive vascular damage (Strain & Paldánius, 2018). Carotenoids reduce thrombosis and atherosclerosis in dose-dependent manner by balancing the redox reactions (Pietro et al., 2016). It is responsible as a limiting factor of low-density lipoprotein (LDL) and regulates ROS-induced vasoconstriction which modulates the mean arterial pressure and fluidity in hypertensive animals. Moreover, it reduces inflammatory cytokines pathways and hemostatic disorders without affecting renal functions and in this way, it prevents CVS disorders (El-Baz et al., 2020).

7.5.4 Mechanism of action of carotenoids in diabetes and associated complications Carotenoids act with multiple mechanisms in diabetes mellitus (DM) and the complications associated with it. The microvascular and macrovascular complications in diabetes cause retinopathy, nephropathy, neuropathy, and cardiovascular risks (Padhi et al., 2020). The carotenoids act as the most efficacious antioxidants for reducing the glucose concentration in blood and also activate enzyme systems which protect the body from the diabetes-associated complications (Roohbakhsh et al., 2017). High serum content of carotenoids lowers the risk of development of type II DM (Minoru, Mieko, Kazunori, Yoshinori, & Masamichi, 2015). The mechanisms of diabetic retinopathy mainly lead to retinal oxidative stress and chronic inflammation, hemodynamic changes in retinal pathways, overexpression of growth factors, impairment in signaling pathway of neurotrophic factor and its receptors which further damage to retinal neurons, glial cells, and retinal vessels (Sayon et al., 2017). In diabetes, the superoxide scavenging systems reduce the properties of antioxidant enzymes SOD, GSH, glutathione peroxidase, glutathione reductase, and manganese superoxide dismutase (MnSOD). They also alter the enzymes causing modifications of DNA and histone (Dehdashtian et al., 2018). Diabetic nephropathy and neuropathy also include the same pathophysiology as retinopathy but in case of diabetic nephropathy there is also increase in the ROS which is linked with TGF-b and excessive extracellular matrix (ECM) causing renal impairment and fibrosis (Jha et al., 2016). Carotenoids are the natural substitutes which contain many therapeutic active constituents like lutein, zeaxanthin, astaxanthin, carotene, etc., which can be useful in prevention of diabetic retinopathy, nephropathy, and neuropathy. Carotenoids play a significant protective action in all types of diabetes by reducing the increased intracellular level of AGEs, lipid peroxidation, ROS, overexpression of VEGF and metalloproteinase-2 induced by higher glucose concentration, and cell proliferation in cultured human retinal epithelial cells (Benlarbi-Ben Khedher et al., 2019). Carotenoids also improve oscillatory potentials and decrease the biomarkers of oxidative stress like MDA, superoxide ion, 8-hydroxy-2deoxyguanosine, and MnSOD in the retinal tissue db/db mice, thus suggesting antioxidant activity against diabetes, and help in regulation of pathological cellular functions (Bohn, 2019). Carotenoids also decrease nuclear factor-kB (NF-kB) p65 subunit in parts of brain like cerebral cortex and hippocampus which further declines the concentrations of inflammatory mediators such as IL-1b, IL-6, and TNF-a (Ying et al., 2015). Carotenoids trigger the P13K/Akt pathway suppressing caspases 3 and 9 activity protecting brain cells from apoptosis and advanced hippocampalebased cognitive functions (Xu et al., 2015).

7.5.5 Mechanism of action of carotenoids in neurodegenerative diseases In CNS the activation of oxidative stress results in different neurodegenerative disorders such as PD, AD, Huntington’s disease (HD), multiple sclerosis, and amyotrophic lateral sclerosis (ALS). Several diseases occur due to Ca2þ instability for signaling of molecules. Neurodegenerative disorders are the result of progressive degeneration of neurons associated with protein aggregates (Cho et al., 2018). AD occurs by senile plaques with b-amyloid (Ab) aggregations and tangling of fibrils

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with hyperphosphorylated tau (Hardy & Selkoe, 2002). Similarly PD, HD, and ALS are associated with accumulation of asynuclein, huntingtin, and TAR DNA-binding protein 43, respectively (Cho et al., 2018). In neurodegenerative diseases, the ROS level is elevated by many cellular activities like mitochondrial insults and production of redox metals which interact with oxygen, ultimately causing neuronal cell death (Uttara et al., 2009). Neuroinflammation is also found in CNS as a result of neurodegeneration, trauma, and different autoimmune disorders. In chronic conditions of neuroinflammation, cytokines and chemokines are liberated by microglia and the oxidative stress is elevated causing damages to the neurons (Frank-Cannon et al., 2009).

7.5.6 Mechanism of action of carotenoids in ophthalmic disorders The most prevalent eye problems with aging are cataract, glaucoma, AMD, and diabetic retinopathy which may lead to partial or complete loss of vision (London & Beezhold, 2015). Oxidative stress is the main reason behind these abnormalities of eye so carotenoids are considered to be the potent antioxidant and antiinflammatory effect (Martínez-Fernández de la Cámara et al., 2013). Oxidative stress in eyes is the result of contact to light, higher concentration of mitochondria, and increased metabolic rate of photoreceptors. This leads to inequilibrium between production and neutralization of ROS. This leads to oxidation of components of cell and malfunction and degradation of retinal tissues (Mitra et al., 2016). Cataract occurs due to change in activity of Naþ/Kþ adenosine triphosphatase, oxidative stress, accumulation of lens protein, activation of polyol pathway, and advanced glycation end-products formation (Erdurmus et al., 2014). Glaucoma is a type of optical neuropathy with successive degeneration of the ganglion cells of retina. The degeneration of these cells results in successive optical atrophy and permanent loss of vision (Weinreb et al., 2014). High intraocular pressure is also associated with glaucoma with ganglion cell loss (Saccà et al., 2014). Diabetic retinopathy is the major complication of DM (Fathalipour et al., 2020). Hyperglycemia activates number of abnormalities in metabolic processes in the retina and subsequently produces ROS leading to oxidative stress (Cheng et al., 2017). Oxidative stress induces lesions in retina inducing endothelial cell dysfunction, angiogenesis, and periocitary apoptosis of Rouget cells and/or mural cells (Cheng et al., 2017). AMD is a continuous degeneration of retinal pigment epithelium (RPE) and the retinal macula due to increase in age (Datta et al., 2017). Pathogenesis of AMD is dependent on excess production and accumulation of ROS. Increase in ROS level in retina and antioxidant cell defense systems cause injury and apoptosis of RPE cells and photoreceptors due to oxidative stress (Zarbin, 2004). Dry eye disease is an ailment of exterior of eye and the tears that influence disturbances in vision, discomfort of eye, and instability of tear film (Seen & Tong, 2017). Zeaxanthin and lutein are the macular carotenoids which protect the photoreceptor cells by neutralization of the oxidation reactions (Bernstein et al., 2016). Macula is protected by these carotenoids as eye-protective nutrients, through oxidation process and a sequence of transformations. ‘3-Hydroxy-b, ε-caroten-30 -one’ is one of the metabolic products of lutein found in the retinas by oxidation (Bernstein et al., 2016).

7.5.7 Mechanism of action of carotenoids as immunity booster Carotenoids also help in differentiation of cell and growth of tissue by converting into provitamin A and boost immune functions by upregulation of communication between the cells by elevating the exchange of signals for regulation of growth leading to apoptosis in damaged cells (Bhatt & Patel, 2020). It is also investigated that supplementation of astaxanthin is associated with increased total hemocyte count and survival rate of different species. They are also responsible in regulating expression of several immunomodulatory genes like peptidoglycan recognition receptor proteins (PGRPs) gene, prophenoloxidase (ProPO) gene (Babin et al., 2010), thioredoxin-like protein (TRX) gene (Zhang et al., 2018), vitellogenin (Vg) gene, ferritin gene (Zhang et al., 2019), toll-like receptor (TLR) gene (Dai et al., 2016), heat shock protein (HSP 70 and HSP 90) (Cheng et al., 2020), and CuZnSOD gene (Han et al., 2016). They also regulate major nonenzymatic antioxidant enzymes such as polyunsaturated fatty acids (PUFAs), uric acids, vitamins C and E, GSH, and tripeptide (L-g-glutamyl-L-cysteinyl-L-glycine) and possess antioxidant property which increases different immune functions by aiding endogenous enzymes such as CAT, SOD, and GPx and free radicals generated during immune activity get detoxified (Karsoon et al., 2020).

7.6 Bioavailability of carotenoids The portion of the carotenoids being assimilated in the human body, entering the systemic circulation, thus becoming accessible for either use in various physiological activities or for storage in the body is referred as bioavailability (Kopec & Failla, 2018). Several steps are involved in the absorption of carotenoids. These include the following (Donhowe & Kong, 2014):

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Discharge of carotenoids from the food matrix Dispersion in the liquid emulsion Solubilization of carotenoids into the enzymes of pancreas and bile salts Development of mixed micelles and movement through microvilli Uptake of carotenoids by mucosal cells of intestine Incorporation of carotenoids into chylomicrons Finally enter the lymph system and blood circulation b-Carotenes found in plants are low in bioavailability, i.e., around 10%e65%; due to achievement of enough release of carotenoids, they show confrontation of complexes of carotene with protein, fibers, and cell walls to metabolize (Rein et al., 2013). b-Carotene is integrated into bile salts and gastric emulsions which are found to be inhibited by the soluble proteins leading to the indication of determination of the extent of carotenoid assimilation in the intestine by the interfacial characteristics of gastric emulsions (Lemmens et al., 2014). The transport of carotenoids found in vegetables and fruits requires mass transfer, but the limitation of movement from the matrices from the aqueous phase to the lipid phase affects the bioavailability. This is considered as the major factor restricting the bioavailability of carotenoids (Palafox-Carlos et al., 2011). The release of carotenoids depends upon digestion and degradation level of the food matrices and is supported by the mechanical processing that occurs prior to digestion (Palafox-Carlos et al., 2011). Carotenoids get absorbed if mixed with micelles. Addition of fats via diet increases the bioavailability of carotenoids (Lemmens et al., 2014). Naturally lipid content in vegetables and fruits is low so addition of extra lipids in the diet plays an important role in digestion. Carotenoids present in vegetables are somewhat less bioavailable than fruits (Schweiggert et al., 2014). Carotenoids from vegetable sources hugely contribute in diets (Bowen et al., 2015, pp. 31e67). Lipid addition in the diet, mainly long-chain fatty acid like oleic acid, is always beneficial in case of nonpolar carotenoids such as carotenes in comparison to polar carotenoids such as xanthophylls (Victoria-Campos et al., 2013).

7.6.1 Factors affecting bioavailability of carotenoids Assimilation of carotenoids depends on various factors such as (Desmarchelier & Borel, 2017): Type of food matrices Intracellular intactness of the carotenoids within the matrix Type and amount of dietary fat present in the meal Actions of enzymes of digestive system Efficiency in transport through the electrolytes A mnemotechnic term “SLAMENGHI” is suggested to enlist every factor affecting the bioavailability of the carotenoid. Each letter of the proposed term represents a factor, where (Desmarchelier & Borel, 2017). “S” stands for “species of the carotenoids,” where these are categorized according to their physicochemical properties. “L” stands for “linkage between molecules,” where it mainly states the functional groups like esters, aldehydes, etc., are linked to the carotenoids. “A” stands for “amount of carotenoids consumed in the diet.” “M” stands for “matrix where carotenoid is incorporated,” mainly indicating the matrices where carotenoids are present. “E” stands for “effectors of absorption,” where outcome of nutrients and drugs is referred for carotenoid assimilation. “N” stands for “nutrient status of the host,” which focuses mainly on the status of vitamin A. “G” stands for “Genetic factors,” that represent effect of polymorphisms or epigenetic modifications of genes. “H” stands for “Host-related factors,” where distinctiveness of the individual is considered such as age, gender, etc. “I” stands for “mathematical Interactions,” where differences in the observed effects are referred when any two factors mentioned above act jointly with the sum of their individual effects.

7.7 Stability, safety, and toxicity Carotenoids are more stable in trans isomeric form. Factors such as light, heat, acids, and presence of cis forms promote minute loss of color and its provitamin activity (Lara et al., 2020). Carotenoids are more vulnerable to enzymatic or nonenzymatic oxidation due to their structure. Oxygen availability, enzymes, high temperature, metals, prooxidants and

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antioxidants, and exposure to light cause loss of stability of carotenoids. Carotenoids are mainly lipophilic and hydrophobic in nature and mainly water insoluble and soluble in different organic solvents like chloroform, acetone, and isopropyl alcohol (Ligia Focsan et al., 2019). The instability characteristics of carotenoids can be overcome by nanoforms by evaluating different properties like size and size distribution, zeta potential, polydispersity index (PDI), morphology, pH, drug loading capacity, and encapsulation efficiency to improve stability, safety, solubility, bioavailability, therapeutic index, and pharmacokinetic profile of carotenoids (dos Santos et al., 2018). The US Food and Drug Administration has also continually accepted the familiar supplements of different carotenoid-rich products such as zeaxanthin, lycopene, lutein, etc., including palm oil carotenoids (FDA, 2010, 2014, 2016). However, higher dose of b-carotene cannot be recommended during pregnancy and breastfeeding and is unsafe when used for long term and can change the skin color to yellow and orange and this condition is called as carotenoderma which can be prevented by discontinuing the supplements of bcarotene and dietary carotene. It also increases the danger of death and can cause cancer and other serious adverse effect. Combination of antioxidants, vitamins, and b-carotene can cause serious problem in angioplasty; persons who exposed to asbestos and smoking can increase the risk of colon, prostate, and lung cancer (Meléndez-Martínez, 2019). Generally, bcarotene is typically linked with favorable effect in animal models and healthy volunteers and nonsmokers (MeléndezMartínez, 2019).

7.8 Health benefits of carotenoids The benefits and availability of wide varieties of carotenoids add values for nutrition and health benefits for number of lifestyle-related and age-related problems of human. The multiple ways of mechanism of action of carotenoids open its application in treatment and amelioration of many diseases like cancers, CVDs, diabetes, neurodegenerative diseases, and eye and immunity boosting effects.

7.8.1 Carotenoids as antioxidant and its health benefits However, it is reported that supplementation of high-diet carotenoids can lead to decrease in activity of SOD and increases the content of GPx which produces potent antioxidant effect (do Nascimento et al., 2020). It was also observed that food supplement containing carotenoids mainly astaxanthin can avert immunity through immunosuppression (MeléndezMartínez, 2019). It is also reported from one study that carotenoids found in Micrococcus luteus and Micrococcus roseus possess antioxidant activity through DPPH free radical scavenging property (Pérez-Gálvez et al., 2020). The utilization of carotenoids as antioxidant is the primary mechanism on which various ailments are treated. In the chemical structure of carotenoids like astaxanthin and fucoxanthin presence of oxygen atom represses the expression of cytokines such as IL-6, IL-1b, and TNF-a and acts as pro- and antiinflammatory compounds (Jaswir & Monsur, 2011). It scavenges free radicals of oxygen so no longer can intermingle with NF-kB resulting in formation of macrophage foam cells and reduce the activity of TNF-a (Di Pietro, Di Tomo, & Pandolfi, 2016).

7.8.2 Carotenoids in cancer A vast research has been reported for use of different carotenoids in the treatment of different types of carcinoma. Carotenoids like fucoxanthin, lycopene, and b-carotene control the cell cycle in different phases like G2/M phase and G0/G1 phase (Haddad et al., 2013; Yu et al., 2011). Carotenoids like lycopene and siphonoxanthin act as antiangiogenic agent retarding the development and spreading of tumor cells (Huang et al., 2013). Moreover, carotenoids like lycopene and bcryptoxanthin initiate suppression of signaling pathway of NF-kB producing beneficial effect against prostate and lung cancer (Lim & Wang, 2020). b-Carotene reported to possess antiangiogenic activity which assists to stop the progress of generating new vasculature which is generally observed in tumors. Carotenoids like lycopene, b-carotene, and astaxanthin are proved as prooxidants and can inhibit triggered ROS-mediated apoptosis of tumor cells (Koklesova et al., 2020). Besides, it produces synergistic action with cytotoxic drugs and minimizes the harmful effect of the cytotoxic drugs to noncancerous cells (by acting as an antioxidant) without interfering the cytotoxicity of drugs to cancer cells (by acting as a prooxidant). Lycopene downregulated the androgen metabolism and altered the prostatic gene expression of Srd5a1 (encoding steroid 5a-reductase) by reducing up to 2.5-fold (Chen et al., 2015). The reduction in growth factor signaling helps in reduction of cell proliferation and cell cycle progression (Soares et al., 2013). Lycopene, a-carotene, and bcarotene are being studied widely for prostate cancer. The effect of dietary lycopene was found to be linear with doseeresponse in decreasing the risk by 1% for prostate cancer for each milligram of lycopene administered. Similarly the

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risk for prostate cancer was reduced by 4% for each 10mg/Dl circulating lycopene (Rowles et al., 2017). b-Carotene decreases the activation of growth protein signaling and similarly lycopene decreases proliferation and increased apoptosis in cell lines of breast cancer (MCF-7 cells) (Sowmya Shree et al., 2017). Prevention of inflammation can arrest the growth of breast cancer (Ramel et al., 2012). Lutein was reported to inhibit the augmentation of breast cancer cells by arresting the cell cycle and caspase-independent cell death (Gong et al., 2018). Astaxanthin also inhibited the proliferation and breast cancer cell movement so controls the growth and metastasis of breast cancer (McCall et al., 2018). In case of lung cancer, lycopene and b-carotene were found to reduce the risk significantly up to 30% (Abar et al., 2016). b-Carotene, b-cryptoxanthin, lycopene, lutein plus zeaxanthin also reduced the oral and pharyngeal cancer (Emanuele et al., 2016). Crocetin is also reported as an active anticancer agent and it acts by multiple mechanisms like inhibition of nucleic acid synthesis, enhanced antioxidative effect, initiation of apoptosis, and impairing different signaling pathways of growth factors (Gutheil et al., 2012). Crocetin was reported to be efficacious in cancers of breast, cervix, colorectal region, liver, lungs, pancreas, skin, and leukemia (Gutheil et al., 2012).

7.8.3 Carotenoids in cardiovascular diseases CVDs like atherosclerosis, hypertension associated with DM, and disturbances in lipid profile can be treated by carotenoids. Kim et al. (2011) published the outcome of lycopene on markers of endothelial function and oxidative stress. Eightweek intake of lycopene of 15 mg/day increased the levels of serum SOD and reduced the concentration of cardiovascular risk factors like C-reactive protein, vascular cell adhesion molecule-1 (VCAM-1), and intercellular cell adhesion molecule (ICAM). The study observed the decrease in systolic pressure by 3.2 mmHg (Kim et al., 2011). Jiang et al. reported the hypolipidemic effect of lycopene in sheep. The decrease was observed for total cholesterol, LDL, and triglyceride levels but no change in HDL level (Jiang et al., 2015). Kulczyn’ski et al. also reported hypolipidemic results of lycopene by studying the enzymes participating in the metabolism of cholesterol. Lycopene reduced the action of ACAT, HMG-CoA reductase enzymes and augmented the activity of LDL receptors. Lycopene showed antioxidant activity with decreasing the levels of total cholesterol, LDL, triglyceride level, and Apo-B level but improved HDL and Apo-A1 level. Lycopene lowered the lipid peroxidation in human and animals (Kulczynski et al., 2017). Yang, Jin et al. (2011) and Yang, Seo et al. (2011) investigated the hypolipidemic effect of astaxanthin in atherosclerosis animal model. Monroy-Ruiz et al. published the outcomes of astaxanthin in reduction of not only the systolic pressure but also decrease of left ventricular hypertrophy (LVH). Astaxanthin decreased the oxidative stress and increased the bioavailability of nitric oxide (Monroy-Ruiz et al., 2011). El-Din et al. (2015) experimented on the lipid lowering effect of b-carotene. b-Carotene reduced the concentration of triglycerides, total cholesterol, LDL, and VLDL with enhancement in HDL. Bechor et al. reported the action of isomeric form of b-carotene on mice. The b-carotene repressed the intensification of atherosclerosis by enhanced removal of cholesterol from macrophages (Bechor et al., 2016).

7.8.4 Carotenoids in diabetes mellitus and associated complications Carotenoids like astaxanthin, bixin, crocin, lutein, cryptoxanthin, zeaxanthin, and lycopene are the most studied molecules against DM and associated complications. These molecules also trigger the sensitivity of insulin and increase the immunity in patients of DM (Roohbakhsh et al., 2017). Utmost antioxidant activity was found in astaxanthin as comparison to other carotenoids like lutein, b-carotene, and zeaxanthin (Yeh et al., 2016). Astaxanthin was reported for protective antioxidant activity on beta cells of pancreas thereby decreasing the glucose tolerance, increasing the insulin level in db/db mice with type II DM (Uchiyama et al., 2002). Astaxanthin binds to PPARg receptors and affects the mRNA expression, which metabolizes the carbohydrates and regulates the glucose level in blood (Inoue et al., 2012). Kianbakht et al. reported the carotenoids found in saffron, crocin showing the antidiabetic activity by increasing the serum insulin content (Kianbakht & Hajiaghaee, 2011). Rajaei et al. (2013) also reported the antihyperglycemic activity of crocin in liver and kidney of diabetic rats which decreased the lipid peroxidation. Fucoxanthin was reported to show the antidiabetic activity by upregulating the mRNA expression of GLUT4 in skeletal muscles by activating the PPARg coactivator-1a. The mRNA of insulin receptors is upregulated with augmentation in phosphorylation of Akt which regulates the translocation of GLUT4 (Nishikawa et al., 2012). Fucoxanthin inhibited the aldose reductase, advanced glycation end-product formation, protein tyrosine phosphatase 1B and a-glucosidase, and increased PPARg and GLUT4 gene expression which contributed for its antidiabetic activity (Jung et al., 2012). Insulin resistance is the result of phosphorylation of insulin receptor substrate 1 (IRS-1). Phosphorylation of IRS-1 is regulated by two endogenous regulators, i.e., JNK and IKKb. So the inhibition of these endogenous regulators can decrease the insulin resistance. Crocetin selectively inhibited the PKCq, activator of both JNK and IIKb signaling pathway thereby blocked the palmitate-induced insulin resistance (Yang et al., 2010). Astaxanthin

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showed the antidiabetic effect by modulating the actions of the enzymes involved in metabolism of carbohydrate like hexokinase, fructose-1,6-bisphosphatase, glucose-6-phosphatase, glycogen phosphorylase, and pyruvate kinase. Hyperglycemia causes activation of neutrophils and macrophages which results in generation of excess ROS. Increased levels of ROS cause damage to different cell components like proteins, lipids, and nucleic acids and also activate inflammation (Otton et al., 2010). Excessive ROS affects the secretion of insulin and increases in insulin resistance (Yang, Jin et al., 2011, Yang, Seo et al., 2011). The oxidative stress is mainly responsible for precipitation of most of the diabetic complications. As carotenoids are the super-antioxidants so mostly adopted for reduction of diabetic complications. Bixin was reported to increase the levels of glutathione reductase, thioredoxin reductase, SOD, and serum nitric oxide level decreasing the complications of DM (Roehrs et al., 2014). Astaxanthin reduced the hyperglycemia-induced hepatic disorders by decreasing the inflammation-related proteins (Park et al., 2015). Similarly lycopene showed antidiabetic effect by lowering the levels of hydrogen peroxide and lipid peroxidation with increase in concentration and upregulation of mRNA transcription of SOD, CAT, and GPx in diabetic rats (Aydin & Celik, 2012). In streptozotocin-induced diabetic rats, astaxanthin protected the functions of retina and prevented diabetic retinopathy significantly (Yeh et al., 2016). Astaxanthin also inhibited mediators of oxidative stress like nitrotyrosine, acrolein, and 8-hydroxy-2-deoxyguanosine thereby improving the antioxidant enzymes and reducing inflammatory mediators like monocyte chemoattractant protein-1, intracellular adhesion molecule-1, and fractalkine. This is mainly associated with downregulating NF-kB transcription factor which can be used for the treatment of diabetic nephropathy (Sun et al., 2011). Astaxanthin also produced inhibitory effect against animal models of T2DM by blocking aldose reductase enzyme, which played a key role of diabetic complications in the polyol pathway including diabetic retinopathy in gerbil (Psammomys obesus sp.) (Benlarbi-Ben Khedher et al., 2019). In diabetic nephropathy, astaxanthin reduced overproduction of TGF-b and ECM as it controls excess production of fibronectin and TGF-b1 and returned to normal physiological levels of creatinine and uric acid which prevented glomerular hypertrophy and renal fibrosis (Xiaoyu et al., 2018). Astaxanthin is also responsible in upregulation of the frontal cortex which decreases expression of glial fibrillary acidic protein (GFAP) in brain stem, reduces ROS and inflammatory mediators, and finally increases the synthesis of antioxidant enzymes, which can be used in the treatment and prevention of diabetic neuropathy (Li et al., 2018).

7.8.5 Carotenoids in neurodegenerative diseases Neurodegenerative diseases can be controlled up to some extent by carotenoids as the neurodegeneration is an irreversible process; carotenoids prevent it by controlling different gene expression, oxidation processes, and signaling pathways. Lutein decreases the neuroinflammation and reduced lipid peroxidation and proinflammatory cytokines discharge by deactivating NF-kB pathway due to oxidative stress (Liu et al., 2017). The carotenoids of saffron, crocin and crocetin also reduced the proinflammatory cytokines and nitric oxide with lipopolysaccharide, interferon-g, and b-amyloid (Ab) by activation in microglial cells (Nam et al., 2010). Astaxanthin decreased the neuroinflammation in hippocampus and retina and reduced deficits in cognition, oxidative stress in retina (Yeh et al., 2016). Similarly fucoxanthin reduced inflammation against a variety of stimulus through Akt, NF-kB, and mitogen-activated protein kinase pathways (Zhao et al., 2017). Lycopene reduced the neuroinflammation, depression, and inflammation-induced cognitive dysfunction (Zhao et al., 2017). Another approach of treatment of neurodegenerative diseases is autophagy, which is a catabolic process for cleaning up of damaged cells, proteins and even causes recycling of nutritional building blocks. In many neurodegenerative diseases, autophagy clears the misfolded or accumulated protein clusters like tau fibrils in AD and Lewy bodies in PD (Rahman & Rhim, 2017). Lutein was reported to decrease the autophagy induced by cobalt chloride via the mTOR pathway whereas it induced autophagy in retinal epithelial cell by upregulating Beclin-1 (Chang et al., 2017). Crocin causes autophagy in hypoxia and inhibits autophagy during reperfusion (Zeng et al., 2016). Fucoxanthin protected neuronal cell death by activating autophagy and the nuclear factor erythroid 2erelated factor pathway (Zhang et al., 2017). Dietary lycopene improved the cognitive function in AD patients (Yu et al., 2017). Carotenoids like lycopene, astaxanthin, and b-carotene aid in transportation process in brain leading to reduced improper signaling events (Rzajew et al., 2020). Astaxanthin can significantly reduce the risk of diseases by various pharmacological mechanisms such as decrease in cerebral infraction in tissues of brain, antiapoptosis, decrease in incidences of ischemia due to induced apoptosis, and decrease in the release of glutamate and reduce cellular damage by free radicals (Kowsalya et al., 2019; Rzajew et al., 2020). It has been also investigated that lycopene increases bioavailability in bloodebrain barrier (BBB) and decreases certain diseases. In AD, ROS is produced with activation of caspase along with Akt/GSK-3-b signaling (Aziz et al., 2020). Carotenoids normalize this signaling and inhibit activation of caspase. ROS decreases Nrf2/HO-1 and HO-1 with cytotoxicity of Ab and results in

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reperfusion of injury and ischemia in cerebral region with chronic neurodegeneration which significantly halts by intake of carotenoids in the daily diet (Bhatt and Patel, 2020).

7.8.6 Carotenoids in age-related eye disorders Carotenoids protect the eye by reducing formation of lipofuscin with aging. The RPE accumulates lipofuscin, a fluorescent colored mixture of protein with lipid produced from metabolism of vitamin A with products of lipid peroxidation (Bhosale et al., 2009). Lipofuscin is poisonous to the mitochondria and induced apoptosis of RPE cells upon exposure to blue light (Ben-Shabat et al., 2001). Lutein and zeaxanthin reduced the level of lipofuscin formed in vivo and in cultured RPE cells (Bhosale et al., 2009). Lutein and zeaxanthin can protect the eye against photooxidative damage and filter the blue light which generates ROS causing photodamage of retinal cells (Alves-Rodrigues & Shao, 2004). Zeaxanthin was confirmed to decrease retinal oxidative lesions due to diabetes and increased concentration of VEGF and ICAM-1 in retina (Kowluru et al., 2008) whereas lutein could reduce oxidative stress and lipid peroxidation in retinal tissues of diabetic patients (Arnal et al., 2009). Carotenoids are the rich source of vitamin A and also called as provitamin A and the deficiency of vitamin A causes night blindness. Appropriate intake of carotenoids in daily diet can improve vision quality and leads to good vision (Berendschot & Plat, 2014). Moreover, zeaxanthin and lutein are found in the macular region of retina and serve for detailed and sharp vision. They also act as a filter for blue light from screens and scavenge free radical oxygen from retina. They also prevent AMD process from retina and cataract in eyes (Bhatt and Patel, 2020). Bungau et al. reported the effective prevention and therapeutic activity of lutein, zeaxanthin, and mesoxanthin in age-related eye disorders. These carotenoids prevent the macular degeneration due to antioxidant and antiinflammatory property (Bungau et al., 2019). Along with lutein and zeaxanthin, b-carotene also shows mechanistic protection for various eye disorders as it is a precursor of vitamin A (Tuj Johra et al., 2020).

7.9 Conclusion Carotenoids are colorful lipid-soluble pigments and epidemiological findings have found that elevated dietary consumption of carotenoids is associated with decrease in the risk of breast, cervical, vaginal, colorectal cancers, and cardiovascular and eye diseases. Antioxidant functions of carotenoids are based on different mechanisms like quenching free radicals, mitigating damage from reactive oxidant species, and hindering lipid peroxidation. In spite of the numerous therapeutic benefits conferred by carotenoids, certain issues are related to its usage which requires to be taken care of. The major ones being are the carotenoids extremely susceptible to oxidation, chemical or enzymatic transformations and may therefore be transferred to other products whose implications are not completely established and various genetic expressions can contribute to change in the response of people with identical dietary intakes to carotenoid therapy. Further research should explain the efficacy, metabolism, and biological attributes of carotenoids prior they are considered to prevent carcinogenesis.

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Chapter 8

Curcumin Srinivasan Krishnamoorthy, R. Paranthaman, J.A. Moses and C. Anandharamakrishnan Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology Entrepreneurship and Management - Thanjavur (NIFTEM - T), Ministry of Food Processing Industries, Government of India, Thanjavur, Tamil Nadu, India

8.1 Introduction Curcumin, one of the principle curcuminoid compounds of the turmeric (Curcuma longa) rhizome, is a yellow-orange crystalline material. Turmeric, which is used since 4000 years ago especially in South Asia, is recognized as “Golden spice” or “Indian saffron” for its medicinal applications (Akbik et al., 2014). The discovery of curcumin dates back to over 200 years as a pure compound in 1842. However, the last decade has witnessed substantial progress in curcumin research and received great moments for its multiple therapeutic effects on the human body. The yellowish polyphenol compound curcumin received greater attention after the antibacterial property of the curcumin molecules was revealed and reported by Schraufstätter and Bernt (1949). A literature search in “PubMed” with the topic “curcumin” revealed that there is an increasing trend (Fig. 8.1) in the publications of curcumin-based research and its applications. A 40-fold increase in the curcumin-based publications from 2001 to 2019 implies the compound’s growing importance due to the anticancerous and immune-boosting properties. Though most of the researchers investigated the biological effects of curcumin, some biological and chemical researchers also exploited the chemistry behind the biological activity. Curcumin studies in all the chemistry branches, including physical, organic, inorganic, and analytical chemistry, explained various mechanisms in the formulation and development of curcumin-based drugs and nutraceuticals(Ganapathy, Preethi, Moses, & Anandharamakrishnan, 2019). While the extraction and synthesis of curcumin were dealt with in organic chemistry, the inorganic chemistry studies explained the metal-chelating abilities to form novel structural entities through the b-diketo groups. Likewise, while the physical chemist exploited the spectroscopic properties of curcumin and its interactions with other biological matrices, the analytical chemist utilized the unique absorption pattern of curcumin to identify and quantify trace elements like boron. Similarly, the chemical reactivity with free radicals, formation, and degradation of nonconjugated and other addition reactions was mostly studied in curcumin-based food and pharmacological research (Santos-Sánchez et al., 2019). The curcumin research spanning more than four decades has revealed diverse pharmacological and chemopreventive effects against several chronic and lifestyle diseases (Adams et al., 2019). The popular Asian spice has been used widely as an indigenous medicine to treat various diseases such as ulcers, cough, hepatic disease, sinusitis, biliary disorders, etc. In the Ayurvedic practice, turmeric powder is used to expulse gas, strengthen the body’s energy, dissolve gall stones, alleviate arthritis, and improve digestion. The modern-day applications include antiaging, anticancer, wound-healing, and immunostimulant activities.

8.2 Sources/derivatives C. longa L., the tuberous rhizome of the perennial herbaceous plant commonly referred to as turmeric, is the source of the diarylheptanoid compound “curcumin.” The yellow-colored curcuminoids are of three main categories: curcumin, bisdemethoxycurcumin, and demethoxycurcumin (Omosa et al., 2017). Many sesquiterpene compounds have been isolated and characterized from turmeric, including turmerone, germacrone, zingiberene, b-sesquiphellandene, a-curcumene, b-bisabolene, dehydrocurdinone, curcumenol, procurcumadiol, bisacumol, isoprocurcumenol, zedoaronediol, curlone, epiprocurecumenol, and turmeronols (Mishra et al., 2018).

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FIGURE 8.1 The number of publications during the last three decades indexed by “PubMed” with the topic “curcumin”.

The curcumin derivatives are synthesized through chemical structure modification and have different receptor binding and pharmacological activities due to their varied pharmacokinetics and physiochemical properties (Amalraj et al., 2017). The structural advantage of curcumin and the essential pharmacophores of the molecule facilitate derivatization of multiple natural and synthetic analogs (Fig. 8.2). The functional moieties, as can be observed, consist of two phenyl rings substituted with methoxyl and hydroxyl groups linked through 7 carbon keto-enol linker molecules. However, the derivatives are generally produced through chemical reactions between acetylacetone and aryl-aldehydes. This assembly yields multiple chemical analogs and

FIGURE 8.2 Reactive functional groups in curcumin structure.

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functional side groups to facilitate further derivatization reactions (Tomeh et al., 2019). A structureeactivity relationship of curcuminoid compounds and their derivatives confirms the presence of coplanar hydrogen donor groups and b-diketone moieties, which are critical for curing diseases like prostate cancer (Cheng et al., 2020). Dimethylcurcumin is a novel curcumin analog associated with androgen receptor degradation and has a prominent role in cancer treatment (in particular prostate cancer) (Tomeh et al., 2019). Further, the dimethyl compound of curcumin also exhibits remarkable antiproliferative activity against breast cancer cells. The addition of a methyl group enhanced the targetability and biological activity of the molecules. Moreover, the hydrophobicity of the dimethylcurcumin is higher than the curcumin molecules and enhances the administrable dose in cancer therapies (Perrone et al., 2015). Table 8.1 summarizes the list of curcumin derivatives, its chemical structural modifications, and its specific therapeutic properties. Tetrahydrocurcumin (THC) is one of the curcumin derivatives with nearly similar structural and pharmacological characters of curcumin molecules. However, several studies reported the strong antioxidant property of THC, among other curcuminoids, and are widely considered in food industries for their nontoxic nature (Aggarwal et al., 2015). Owing to the polymorphic molecular targets, cellular responses, and signaling pathways, THC is considered superior to curcumin and has demonstrated reports against inflammation, hepatotoxicity, cancer, and nephrotoxicity (Amalraj et al., 2017). Hydrazinocurcumin is another synthetic derivative of curcumin which has superior pharmacological and biological activity comparable to curcumin. Interestingly, the hydrazine compound has a beneficial role in breast cancer, although the molecular mechanism is largely unknown (Zhou et al., 2020). Metallo-curcumins are proven to diminish the cytotoxic potential and enhance pharmacodynamic effects due to their ability to bind with DNA through groove binding, intercalation, and electrostatic interactions. The modulating property at the molecular level is key in designing new therapeutic drug candidates with antitumor properties (Vellampatti et al., 2018).

TABLE 8.1 Curcumin derivatives, its chemical modifications, and its therapeutic properties. Curcumin derivatives

Chemical modification

Therapeutic properties

References

Glycosylated curcumin derivative

Substitution of glycol groups on the chemical moiety (aromatic rings)

Aqueous solubility, chelating properties, and enhanced biological properties

Gurung et al. (2017)

Aromatic analogues of curcumin

Structural modifications with cyclohexane bridges

During lymphoma therapy, improved mitochondrial membrane permeability

Stanic (2017)

Tetrahydrocurcumin (THC)

Hydrogenated curcumin molecules with diketone moiety

Loss of STAT3 inhibition and DNA binding properties and moderate antioxidant activity

Tomeh et al. (2019)

Dimethylcurcumin

Substitution of methyl groups on R2 and R4 sites

Anticancer activity against prostate and breast cancer

Shi et al. (2012)

Vanadium, indium, and gallium complexes

Metal complexation at b-diketones sites

Improved cytotoxic activity

Zhang et al. (2019)

Metallo-curcumin (Cu2þ/Ni2þ/Zn2þ)

Metal complexation by the b-diketones

Improved DNA binding ability and enhanced water solubility

Vellampatti et al. (2018)

Copper conjugate of curcumin analogues

Conjugation through keto-enol moiety

Induced activation of NF-kB cells in KBM-5 leukemic cells

Mukherjee et al. (2016)

Curcumin carbocyclic analogues

Addition of carboxyl group at the diketone moiety

Stronger inhibition of HIV protease and enhanced antioxidant activity

Tomeh et al. (2019)

Semicarbazone

Introducing formaldehyde semicarbazone at the keto and enol moieties

Enhanced antiradical, antioxidant, and antiproliferative activity

Malik et al. (2018)

Hydrazinocurcumin

Substituting hydrazine derivative with diketone moiety

Inhibition of colon cancer via antagonism of calcium or calcium module function

Tomeh et al. (2019)

Cyclic curcumin derivatives

Aldol condensation mediated through boron trioxide

Enhanced antioxidant activity and cytostatic, antitumor

Amalraj et al. (2017)

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A growing number of curcumin derivatives are engineered with a focus to resolve limitations associated with the clinical usage of curcumin. The main problems associated with curcumin application are poor bioavailability, drug formulation issues, and lower biological activities. Despite these, the novel technological approaches such as nanotechnology successfully improved the formulation approaches with enhanced pharmaceutical activity.

8.3 Extraction techniques Even though the common extraction technique for curcumin is the solvent extraction process, there are still many advanced methods reported to protect the bioactive compounds effectively from light and heat. Further, several methods reported to use steam, percolation, and hydrodistillation methods subsequent to the solvent extraction process to recover the entire active chemicals, pigments, and other curcuminoid compounds (Zielinska et al., 2020). The commonly used curcumin extraction processes in practice are listed in Table 8.2. As a supercritical solvent, carbon dioxide has been in commercial use for extracting curcuminoids in recent times (Santana et al., 2017). The increasing popularity of carbon dioxideebased extraction is due to its inert, nontoxic, nonflammable, and recyclable conditions. Further, the supercritical technology is superior in preserving the heat-labile compounds (Chang et al., 2006). The curcumin extraction can be accelerated through supercritical carbon dioxide in addition to ethanol and enzyme treatments (Nagavekar & Singhal, 2019). Deep eutectic solvent, an ionic fluid with selective properties, is gaining importance for its eco-friendly properties and thermal protection. These deep solvents are prepared using quaternary ammonium salt and hydrogen-bond donors such as sugars, amino acids, and polyalcohols at different rates. Aydin and his team used deep eutectic solvents and vortex techniques for microextraction of curcumin and other related phenols from turmeric-based food products (Aydin et al., 2018). Moreover, the recovery rate of curcumin through the ionic fluid method is around 93%. Ultrasonic-assisted solvent extraction is another emerging green technology popularly employed in phenolic compound extraction in recent times. Menghwar and research team used the ultrasonic-assisted supramolecular solvent method to extract curcumin. In this method, the chemical groups (eOH and eO) on the curcumin structure form hydrogen bonds in the dissolution solvent, where the van der Waals and other dispersion forces of the solvents attract the apolar portion of curcumin (Menghwar et al., 2018). This method further reduces the extraction time to 20 min using the ultrasonic waves. Microwave-assisted extraction is another rapid and high-efficiency method that employs ionic conduction and rotation dipole principles for the separation and concentration of bioactive compounds from food products (Zielinska et al., 2020). Likewise, Mandal et al. extracted curcumin from dried rhizome through microwave process and showed time reduction and less solvent usage (Mandal et al., 2008). The extraction rate was 27% more than the conventional Soxhlet process, which might be due to the dual heating process of the Soxhlet method. Other popular curcumin extraction methods include steam distillation, polar, and nonpolar solvent extractions (Zieli nska et al., 2020). In most studies, turmeric oil and oleoresin were the major compound of interest from C. longa. In

TABLE 8.2 Common extraction methods and their conditions for curcumin. Methods

Source of extraction

Conditions

References

Vortex-assisted deep eutectic solvent (DES)

Turmeric liquid extract

Emulsification liquideliquid microextraction

Aydin et al. (2018)

Ultrasound-assisted ionic liquid-dispersive

Dried rhizomes

Liquid microextraction

Altunay et al. (2020)

Microwave-assisted extraction

Dried rhizomes

Microwave energy for analyte partition

Mandal et al. (2008)

Supercritical antisolvent solution (SAS)

Dried rhizomes

Supercritical carbon dioxide

Lv et al. (2018)

Liquideliquid microextraction

Mixture of curcuminoids

Two-phase aqueous extraction using ultrasound and imidazolium

Shu et al. (2016)

Hydrodistillation

Dried rhizomes

Vaporization-condensation cycle

Raina et al. (2002)

Steam distillation

Dried rhizomes

Fractional distillation based on boiling point

Raina et al. (2002)

Soxhlet extraction

Curcumin oleoresin

Percolation (boiler and reflux)

Negi et al. (1999)

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the steam distillation method, an autoclave machine with controlled pressure and temperature extracts essential oils from turmeric rhizome. Similarly, the hydrodistillation process is used to extract turmeric oil through a hydrodistillation machine meant for extraction of essential oils (Manzan et al., 2003). In the conventional Soxhlet extraction process, the best solvent composition was 5% ethyl acetate in hexane, and major compounds like turmerone, curlone, and ar-turmerone were identified (Zieli nska et al., 2020). Table 8.2 summarizes the most common curcumin extraction methods and the ideal conditions employed by various other researchers.

8.4 Chemistry Chemically, the IUPAC name of curcumin is 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadien-3,5-dione and known as diferuloylmethane (C21H20O6). The polyphenol compound’s melting point is 183 C, and the molecular mass is 368.37 g/mol. The b-diketone moiety links the two aryl rings of the compound, and ortho-methoxy phenolic groups are coupled to the ring structure. Further, the predominant form of curcumin is keto in an acidic or neutral medium, and it is enol form in an alkaline medium. Similarly, the color of the compound also changes with the change in pH, whereas the curcumin forms bright yellow color in acidic pH range 2.5e7.0 and changes to dark red in pH above 7 (Esatbeyoglu et al., 2012). Despite the chemical advantage of the curcumin molecules (structure and form), the prominent character is its ability to undergo metabolic conjugation and reduction activities. However, this functional potential also leads to poor bioavailability of the compounds in new product formulations. The principal mechanism of curcumin metabolism is shown in Figs. 8.3 and 8.4. In the phase I metabolism of curcumin, the principal reactions are reduction and glucuronidation, which yield four heptadiene-3,5-dione molecules of curcumin. The metabolites were dihydrocurcumin, THC, as well as hexahydrocurcumin. Further, the reduction process was catalyzed by curcumin reductase present in the liver tissues (Hoehle et al., 2007). Interestingly, the intestinally adapted Escherichia coli have genes like curA, producing curcumin-metabolizing enzymes (Hassaninasab et al., 2011). Meanwhile, in phase II metabolic process, enzymes like UDPglucuronosyltransferases and sulfotransferase catalyze curcumin’s conjugation reactions through glucuronidation and sulfation (Ireson et al., 2002). The common metabolites in this conjugation reaction of curcumin include curcumin glucuronide, glucuronide sulfate, curcumin sulfate, and hydrocurcumin derivatives. In line with this report, Hoehle et al. declared that they observed curcumin glucuronide and its conjugates in tissues like plasma and jejunum in an in vivo study. Esatbeyoglu et al. revealed a similar trend of the report, which supports the fact that the curcumin metabolites are found abundant in the tissues before it starts circulating in plasma fluids (Esatbeyoglu et al., 2012). The metabolic reactions like

FIGURE 8.3 Keto-Enol tautomerism forms of curcumin. From Lee, W. H., Loo, C. Y., Bebawy, M., Luk, F., Mason, R. S. & Rohanizadeh, R. (2013). Curcumin and its derivatives: their application in neuropharmacology and neuroscience in the 21st century. Current Neuropharmacology, 11(4), 338e378. https://doi.org/10.2174/1570159x11311040002.

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FIGURE 8.4 Glucuronidation and reduction mechanism of curcumin. From (2013). Curcumin and its derivatives: Their application in neuropharmacology and neuroscience in the 21st century. Current Neuropharmacology, 11(4), 338e378. https://doi.org/10.2174/1570159x11311040002.

the glucuronidation process occur more extensively in intestinal tissues than liver in humans. However, in the case of rats, this metabolic process occurs less extensive in intestinal tissues than the liver. These metabolic differences might result from differential conjugation-hydrolyzing enzymes in the liver and intestine of humans and rats (Zeng, Shen, et al., 2017).

8.5 Mechanism of action The biological mechanism behind curcumin’s therapeutic activities is well explained in various disease and pathological states of cells and organisms. Though numerous scientific phenomena are identified for their superior medicinal value, the apoptosis of cancerous cells, nuclear factor triggered mitochondrial stress, and free radicals regulation are key mechanisms described in various clinical studies. Moreover, the capacity to interfere with the cell cycle (G2/M phase) facilitates apoptosis of cancer cells (Liczbi nski et al., 2020).

8.5.1 Curcumin and transcription factors The transcription factors such as nuclear factor-kappa light chain of B cells (NF-kB) comprise a group of proteins that regulates cellular processes in pathophysiological conditions such as inflammation. Several cancer cell line studies have

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shown that NF-kB is constitutively triggered during cancer cell growth due to the oncogenic mutations and other inflammatory microenvironments (Gupta et al., 2010). The receptors like interleukin-6 mediate tyrosine phosphorylation, which activates signal transducer and activator of transcription 3 (STAT3) factors (Tadashi, 2015). Interestingly, it was noted that the STAT molecules have prominent roles in oncogenic parameters such as immune system evasion, angiogenesis, apoptosis, and cell proliferation (Ashrafizadeh et al., 2020). The role of curcuminoids in cancer treatment through the regulation of transcription factors and genes involved in apoptosis has been well described in many cancer cell line studies. Curcumin has a potential role in suppressing anagenesis through downregulation of genes for tumor proliferation, apoptosis, and invasion mechanisms (Lee et al., 2019). It is reported that curcumin affects STAT3 molecules and NF-kB signaling pathways involved in cancer cell progression (Wang et al., 2019). Moreover, curcumin can work against metastasis and lung cell invasion through the regulation of HLJ1 genes in tumorigenesis (Tajuddin et al., 2019). The speciality protein (Sp-1) belongs to a group of transcriptional factors that usually express high in case of cancerous cell growth in organs like breast, gastric organs, and thyroid glands. The effect of Sp-1 factors is dominant in cells containing GC-rich Sp-1 binding sites and interacts with multiple corepressors and activators, especially during carcinogenesis (Shi & Zhang, 2019). The role of Sp-1 is well implicated in multiple cellular reactions like nuclear factors (O’Connor et al., 2016). Moreover, the Sp-1 is known for constitutive transactivation of housekeeping and TATA genes related to cancer and other cell differentiation processes (Wing et al., 2018). Considering the significance of Sp-1 factors in cancer cell progression and its migration, suppressing those factors will be a key tumor prevention strategy (Ganesan et al., 2016). The bioactive compound curcumin has superior control over Sp-1 factors, and its gene expression may be targeted due to its inhibitory action against calmodulin and SEPP1 involved in tumorigenesis (Golonko et al., 2019). Many other authors also reveal these observations about the curcumin role on Sp-1 factors. The curcumin is also involved in inhibiting Sp-1 binding onto the lung carcinoma cells (Chen et al., 2019). Moreover, curcumin has the potential capacity to reduce colony formation in colorectal cancer cell lines (Vallianou et al., 2015).

8.5.2 Curcumin and adhesion molecules The pleiotropic effects of curcumin are well documented, which reveal that the bioactive compound interacts and regulates multiple molecular targets such as focal adhesion kinase to modulate the expression of extracellular components. Further, the curcumin metabolites improve cell adhesion capabilities of numerous extracellular components like laminin in line with the concentration. It is evident that the curcumin has a suppressing role over the FAK activity through modification and inhibition of phosphorylation sites and induces certain adhesion-based molecular components to prevent detachment of cancer cells (Liu & Khalil, 2017). Thus, this ultimately leads to the inhibition of FAK genes and supports the antiinvasive curcumin property (Fan et al., 2015). Likewise, in the colorectal cancer cell line study, the curcumin possesses an inhibiting role in CD24 expression and supports tumorigenesis treatment. Curcumin has also been proven to upregulate E-cadherin and inhibit epithelialemesenchymal transition (Wang et al., 2019).

8.5.3 Curcumin and autophagy Autophagy, a biological process, is essential for the survival of the cell during stressful conditions through protein degradation pathways. Likewise, the endoplasmic reticulum (ER) is a major cellular component in protein synthesis and maturation. The proteasome degrades ubiquitinated proteins through the ER-linked degradation pathway through autophagy (Beese et al., 2020). Further, various pathological conditions are known to affect ER-based protein homeostasis and disrupt protein folding and cause imbalances in ER protein loading and folding capacities, resulting in ER stress (Adams et al., 2019). In this scenario, the unfolded protein response signaling is reported to induce a cancer microenvironment (Shen et al., 2018). Hence, the ER stress, apoptosis, and unfolded protein response are documented for regulating the cancer cell’s future (Sisinni et al., 2019). The recent studies advocate that curcumin induces apoptosis through ER stress and autophagy mechanisms in hepatocellular carcinoma and epithelial ovarian cancer cell lines (Liu & Khalil, 2017). The curcumin treatment suppresses malignant glioma cells’ growth through the autophagy process (Vallianou et al., 2015).

8.6 Bioavailability The biopharmaceutical system has classified curcumin as a class IV substance. This classification indicates the poor permeability across the tissue membranes (Paolino et al., 2016). Moreover, the compound undergoes rapid metabolism and exhibits low serum concentration, resulting in poor bioavailability of curcumin (Suresh & Nangia, 2018).

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8.6.1 Solubility The extremely lower solubility of curcumin in water is mainly because of the molecule’s hydrophobic nature and pHdependent wettability (Iqbal et al., 2020). In a recent curcumin solubility study in water, the maximum value at pH 5 was 11 ng/mL. Meanwhile, when the pH was altered to 6.8, the solubility has increased to 0.1 mg/mL, which further dropped to 0.06 mg/mL at pH 7.4 (Song et al., 2016). The solubility in different water and buffer solutions at various pH is provided in Fig. 8.5. A successful oral drug’s fundamental property should be its ability to get dissolved in the gastrointestinal fluids, followed by higher absorption. The gastrointestinal solubility of curcumin in comparison with the water at different pH (0.01 and 0.1 at pH 5.0 and 6.8, respectively) revealed that the solubility is much higher in simulated gastric fluids though it is low and insignificant (3 mg/mL) (Song et al., 2016). The presence of pepsin in the simulated gastric fluid might be why the slightly higher solubility in human gastrointestinal fluids. In general, the pepsin improvises lipophilic drugs’ solubility, although the mechanism is not revealed (Pinnamaneni et al., 2016). In line with these in vitro studies, a clinical trial conducted on human beings has shown a low plasma concentration of curcumin with an oral dose of 12 g (Klickovic et al., 2014). The results of curcumin bioabsorption studies through animal models widely differ based on the feed material and concentration, but still, the plasma curcumin level was less than 1% (Liu et al., 2020). In a recent research work on human and rats (Hassanzadeh et al., 2020), the plasma concentration of curcumin was found to be 1.35  0.23 mg/mL in rats and 0.006  0.005 mg/mL in human. It is clear that even at a high dose of curcumin, the solubility in a bioabsorption is poor, which requires technological interventions.

8.6.2 Permeability The permeability of curcumin compounds is another prime factor affecting oral administration. Moreover, a bioactive compound’s bioabsorption relies on its permeability across the gastrointestinal tract membrane (Parikh et al., 2016). This fact is evident from the animal and human studies, and interestingly, the absorption of curcumin was better at the duodenal tissues of rat small intestine, followed by jejunum and colon, but it was poorest in the ileum (Righeschi et al., 2016). The Caco-2 cell line has been commonly used to evaluate the permeability of novel drug compounds due to its similarity with human intestinal epithelial cells, especially in polarity and compact cell membrane (Parikh et al., 2018). The in vitro cell model grown at permeable filter support for studying the curcumin permeability is depicted in Fig. 8.6. In this cell line study, curcumin’s permeability was decreased from the apical side to the basolateral side (A to B) of the model through increasing the dose of curcumin. However, in the opposite direction (i.e., B to A), the permeability found increasing, which indicates the trends in the absorption of curcumin (Zeng, Cai, et al., 2017). The permeability coefficient (Papp) value was used to express the absorption of nutrients in the cell line model (Wang, Ke, Zhang, & Yang, 2017b). Furthermore, curcumin at a concentration of 5 mg/mL, increased absorption of curcumin was seen in PappA-B

FIGURE 8.5 The solubility of curcumin in aqueous and buffer medium.

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FIGURE 8.6 Caco-2 cell monolayer grown at a permeable filter support. From (2017). Transport of curcumin derivatives in Caco-2 cell monolayers. European Journal of Pharmaceutics and Biopharmaceutics, 117, 123e131. https://doi.org/10.1016/j.ejpb.2017.04.004.

than PappB-A. Likewise, it was observed that when the curcumin concentration was raised to 20 mg/mL, the PappB-A value was greater than the PappA-B. The inference from these results demonstrates that the curcumin absorbed by the intestinal cells is lower than the compound being pumped out of the cell when the concentration rises above 10 mg/mL. These research findings elucidate the dogma behind the curcumin absorption in varied concentrations of curcumin bioactive compounds. Another study on curcumin bioabsorption using Caco-2 study hypothesized that the efflux pathway has an influencing role in its poor bioavailability. This inference is made on the fact that the curcumin was poorly permeable with the Papp (A to B) value 2.93  0.94  106 cm/s, which was higher than the Papp (B to A) value of 2.55  0.02  106 cm/s at a concentration of 62.6 mg/mL curcumin.

8.6.3 Novel strategies for enhancing the bioavailability of curcumin One of the significant barriers to curcumin’s clinical efficacy is its poor bioavailability, as discussed in Sections 8.5.1 and 8.5.2. Several formulations were developed in the last decade to enhance the bioavailability of the natural phenol compound “curcumin.” Researchers also envisage improving the metabolic potential of curcumin, such as circulation time, resistance to metabolic damage of cometabolites, and absorption. Nanoparticles, micelles, phospholipid complexes, and liposomes were some of the trending formulations found effective in increasing the bioavailability of curcumin compounds. The following text from the recent literature indicates the increased efficacy of curcumin through novel encapsulation techniques. Anandharamakrishnan and his research group exploited nanotechnology for encapsulation of curcumin to increase its bioavailability and targeted delivery (Aadinath et al., 2016). The curcumin-in-b-cyclodextrin-in-nano magneto liposomes were created with iron oxide nanoparticles engineered within the liposome molecules. The encapsulation efficiency was 71%, and the encapsulated nanosized particles exhibited a low IC50 value, i.e., 64.78 mg/mL, signifying an enhanced radical scavenging activity as a result of synergistic coencapsulation with b-cyclodextrin. In another coencapsulation study, Leena et al. encapsulated curcumin over resveratrol using a modified spray drying approach. The nozzle was modified to two-fluid and three-fluid nozzles, and the encapsulation materials used were sodium alginate and carboxymethylcellulose. The encapsulation efficiency was significantly improved through this novel approach, and it was 82.91% for resveratrol and 59.64% for curcumin. Further, the novel three-fluid nozzle approach found promising to coencapsulate curcumin into polysaccharides with superior scale-up potential (Leena et al., 2020). Many other researchers have also explored the encapsulation approach for increasing the bioabsorption and availability in the target organs through a nanotechnological approach. One method employed by Li et al. has applied starch nanoparticles for fabricating the curcumin-loaded starch solution. It was reported that the 3% weight ratio of curcumin in the starch molecule had shown the maximum encapsulation efficiency and bioabsorption as well (Li et al., 2017; Liu et al., 2019). The starch nanocomposites with soluble curcumin have better antioxidant activity and stability against UV irradiation, beneficial for the industrial food application. Another research team proposed cationic surfactants of doublechained molecules and found that the system improved the antioxidant properties solubility of curcumin molecules (Kumar et al., 2016). However, with the emergence of emulsions, the curcumin bioavailability research got a turning point, which paved the way for enhanced bioactive compound delivery (Shah et al., 2016). The recent research suggests that curcumin in pellets, powder, tablets, and solution forms rather than the matrix form having increased solubility and absorption inside the living cells (Shah et al., 2016). The solid lipid nanoparticles of curcumin have the advantage that the formulation has prolonged stability and enhanced digestibility in the gastrointestinal tracts. For example, Pinheiro et al. demonstrated that the curcumin nanoemulsions could be further stabilized through different emulsifiers such as lactoferrin for regulating its bioactivity, safety, and protection for the human consumption

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purpose. In another sense, the low-temperature emulsification methods are most commonly used over high-temperature processes because high temperature is undesirable for thermolabile curcumin compounds (Pinheiro et al., 2016). The novel investigations target the delivery system suitable for the human purpose to enhance the bioavailability of curcumin. Polysaccharides, proteinepolysaccharides, and proteinelipid combinations are frequently used coating materials to protect the bioactive compounds from degradation due to the enzymatic action and better release during the gastrointestinal tract passage. These delivery systemebased measures have considerably enhanced the bioabsorption, bioavailability, and bioactivity in the targeted tissues. Owing to chitosan’s excellent compatibility characteristics, the chitosan hydrogel was also applied for the extended-release of hydrophobic curcumin compounds (Delmar & BiancoPeled, 2016). Grafting is another new technique in creating an encapsulating material like polysaccharide with the potential to design delivery materials with tailored properties and is proven to overcome problems like low bioavailability and limited solubility. Mutalik et al. used pH-sensitive polyacrylamide-grafted-xanthum gum nanoparticles for loading curcumin for colon targeted drug delivery (Mutalik et al., 2016). These nanoparticle conjugates were effective in the treatment of colonic inflammation in rats. Other novel and recent investigations on curcumin bioavailability studies include excipient nanoemulsion and acetatelinked polymer micellar nanoparticles. The excipient nanoemulsion comprised of lipid droplets dispersed within different aqueous mediums was found to increase the oral bioavailability by increasing absorption (Zou et al., 2015). Meanwhile, the pH-responsive acetal-linked polymeredrug micellar nanocarriers with curcumin molecules are proven to initiate a faster drug release and create new hopes for its enhanced pharmacological actions (Li, Shin, et al., 2016).

8.7 Safety and toxicology Curcumin with multiple pharmacological activities and antimicrobial and antioxidant properties is proven to be quite safe in humans and animals with a long-established safety record even at a dose of 8 g/day. In the light of the established safety and functional significance of the bioactive compound “curcumin,” the USFDA declared curcumin as generally regarded as a safe (GRAS) compound (Sharifi-Rad et al., 2020). Despite the well-established safety studies of curcumin, a few studies on curcumin toxicity conducted in and out of the human body have highlighted some of the deleterious side effects in certain conditions. The safety of curcumin consumption at different dosage was studied through several in vitro experiments which also revealed some adverse side effects. The Kawanishi research team showed the damaging property of curcumin on DNA molecules in the presence of cytochrome p450 isoenzymes and copper (Sakano & Kawanishi, 2002). Likewise, the toxic property of curcumin is also experimented by Frank research team (Frank et al., 2003). In the study on liver cancer through rat model, curcumin combines with copper and enhanced oxidative stress and toxicity instead of inhibiting hepatic tumor formation. In terms of acute toxicity develop after curcumin dosage, there is no established proof-of-concept in animals. In the curcumin toxicity study conducted in human volunteers, nonserious (Grade 1 of the WHO classification) side effects were recorded, and they declared curcumin safe for consumption (Vareed et al., 2008). Some authors also suggested that turmeric powder intake of around 1.5 g did not show any clinical side effects, and the human volunteers have acquired the beneficial biological and pharmacological effects of this super spice (Sharma et al., 2007). Chainani-Wu (2012) conducted a human clinical trial on the antiinflammatory activity and safety of curcumin from 1966 to 2002 with 25 subjects to 8 g of curcumin/day for 3 months. The study confirmed that no toxic effects are associated with curcumin at a daily dose of 12 g (Goel et al., 2008). Overall, curcumin at a safe dosage improves human health and cures numerous ailments. Further, the safe dosage varies among the individuals based on the physiology and metabolism rate. There exists a considerable body of literature for the safe daily intake dosage of curcumin (0e3 mg/kg body weight) (Kocaadam & S¸anlier, 2017). International organizations for food control like WHO expert committee and European food safety authority report also suggest that the curcumin at the safe dosage has superior health-promoting properties.

8.8 Applications (clinical and pharmacological)/health benefits Numerous scientific studies revealed curcumin’s pharmacological properties through in vivo and in vitro model systems over the decades. The pharmacological activities include anticancer, antihyperlipidemic, antipsoriasis, antithrombotic, antiangiogenic, antimicrobial, antihypertensive, wound healing, antioxidant, antihepatotoxic, and antithrombotic properties (Leena, Anukiruthika, Moses, & Anandharamakrishnan, 2022). Meanwhile, the neuroprotective property is due to a combination of properties like immunomodulatory, antiapoptotic, antiamyloidogenic, antiexcitotoxic, antiinflammatory,

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and antioxidant activity. Considering the growing interest in curcumin’s biological activity in the prevention of cancer, hepatic damage, and diabetes, this section selectively elaborates the recent advances in research works exploiting the inhibitory role of curcumin specific to those diseases (Farooqui & Farooqui, 2017).

8.8.1 Anticancer The anticarcinogenic property of curcumin has been extensively studied over the decades. The presence of functional groupdhydroxyphenyl compounddin the second position of the curcumin compound and its derivatives is a supportive factor in the cancer treatment. In a case study in urothelial cells of human being, curcumin inhibited the proliferation of cancer cells through alteration of ERK5/AP-1 protein pathway (Liu et al., 2017). Yang et al. (2017) reported that the autophagy process was induced in castration-resistant cancer cells of prostate organs through the administration of curcumin. The bioactive curcumin combined with methotrexate and paclitaxel inhibited adenocarcinoma cells and increased the cytotoxicity of the cancerous cells (Ali et al., 2017). Moztarzadeh and his research team successfully established this observation that curcumin has cytotoxicity activity against the tumorous cells by blocking the cell cycle (Baghbani & Moztarzadeh, 2017). This study demonstrated the scientific phenomena behind this process as curcumin upregulated p53 proteins and promoted high phosphorylation of CDC cells in the MCF-7 cancer cell line. Moreover, curcumin is also reported for its interference with the G2/M phase of the cell cycle and its role in increasing the reactive oxygen species in killing the cancerous cells. It activates caspase 9 and cytochrome c, which are key molecular agents in the cancer prognosis (Rauf et al., 2018). Upon activation, the MAPK (including the active ERK5/AP-1 protein pathway) phosphorylates nuclear transcription factors and through the construction of microRNA transcription factors. The popular gene networks such as miR-34a-5p were found to be involved signaling pathway of multiple gene networks, including WNT1, through the supportive role of LEF1 transcription factors (Jiao et al., 2017). The study conducted by Kumar et al. on breast cancer cell lines (MC-7) also revealed a sequence of molecular events (Kumar et al., 2016). A complete reversal of promoter molecules like glutathione S-transferase and hypermethylation further leads to the expression of genes involved in the transferase protein even after 72 h period (Kumar et al., 2017). Instead, curcumin-induced modulations in the expression pathway of microRNAs, such as Akt, strongly inhibit the cancer cell progression and are a proven method for targeted tumor therapy (Zhou et al., 2020). Besides, curcumin acts on the sites of cancer cell prognosis and decreases colony formation. For example, Feng et al. (2017) demonstrated the inhibitory role of curcumin against cancer cell colonization through the common curcumin analog L45H37 compound. Further, the author revealed many facts behind the molecular events in this antitumor activity. The novel analog of curcumin L45H37 induced the production of reactive oxygen species, ER stresserelated protein, and autophagy-related cellular factors in a sequential manner. However, the N-acetyl cysteine could fully reverse ER-related stress due to L45H37 by blocking reactive oxygen species while inhibiting the growth of lung cancer cells. Combining pharmaceutical drugs with curcumin against various types of cancer is evident in emerging clinical studies. For example, Banerjee research team combined the docetaxel compounds with curcumin biomolecules and evaluated its potency against prostate cancer (Banerjee et al., 2017). These compounds have proven clinical records and are widely in practice for metastatic prostate cancer cells. The molecular targets for these compounds in the prevention of cancer cells are RTKs, p53, P13K, and other CSC markers (Banerjee et al., 2017). The authors further reported that the curcumin compound has also proven to suppress the activation of sonic Hedgehog pathways and WNT/b-catenin. Likewise, another common phenomenon during tumorigenesis is the proliferation of hepatic stellate cells and extracellular compounds’ secretion. Chen research group revealed the role of curcumin in promoting stellate cells’ senescence through histocompatibility genes, and its expressed proteins lie UL16 (Cheng et al., 2020).

8.8.2 Hepatoprotective Curcumin, the bioflavonoid compound, possesses many beneficial properties, including protection against liver inflammation. The bioactive compound reduces serum enzymes, MDA content and has the potential to attenuate pathological damage due to lipopolysaccharide-induced acute liver injury. The sole mechanism lies in the expression of genes related to the antioxidant defense genes like Nrf-2 and suppression of TNF-a levels induced by galactosamine (Xie et al., 2017). The toxicants of microbial origin like aflatoxin cause tumor in association with cytochrome P450 like enzymes. Curcumin at a dosage of 450 mg/kg has an attenuating role over the liver damage caused by the aflatoxin and cytochrome enzymes (Muhammad et al., 2017). Curcumin molecules monitored the enzymes related to liver injury to protect against the biliary ducterelated disorders.

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Other popular mechanisms of the suppressive role of curcumin in liver inflammation and injury are inhibition of lipid peroxidation, amelioration of cellular factors in the management of oxidative stress such as CAT, SOD, etc., and the potential free radical scavenging activity on reactive oxygen species (Ghoreshi et al., 2017). The oxidative species developed in liver tissues create stressful conditions in hepatic cells and disrupt cellular calcium ions’ homeostasis. This process ultimately results in irreversible cellular damage. The curcumin treatment at a dosage of 5 mM has preventive potential against any free radicals and hydrogen peroxide ions (Kheradpezhouh et al., 2016). Emerging evidence signifies that Hedgehog pathways become activated during liver injury, which is also accompanied by a sequence of extracellular molecular events that leads to entire cellular damage. Intake of curcumin at a concentration of 20 mM halted glycolysis-related proteins and their mRNA expression. The effect of curcumin also decreased the phosphorylation process in the liver cells and prevented lactate production (Lian et al., 2016). In another study, curcumin downregulated the key genes for hepatic stellate cell damage induced by chloroform in an albino rat study (Delmar & Bianco-Peled, 2016).

8.8.3 Antidiabetic role The antidiabetic activity of curcumin is another major research area which is widely investigated by researchers across the globe. Though curcumin’s hypoglycemic activity was revealed for its potency to protect the pancreatic cells against inflammation and oxidative damage, its role against vasculopathy and neuropathy was subsequently investigated. The protective role of curcumin against glycation and collagen cross-linking mitigates complications developed through diabetes. Regulation of polyol pathways is another key pathway through which curcumin reduces blood glucose levels. In an in vivo study conducted on db/db mice model developed through recessive diabetes genes (db), curcumin on renal manifestation and its related pathological complications were reported. The study exemplified several renal tissueerelated changes with the curcumin intake, such as suppressed mesangial matrix expansion and decreased albuminuria content in the blood. The curcumin also blocked the expression of proteins related to renal failure such as cleaved caspase-1, fibronectin, etc. (Liu et al., 2017). The effect of curcumin was examined in double transgenic mice with diabetes. The curcumin supplementation significantly ameliorated the defective insulin pathway and other insulin-regulating factors like IR (Wang et al., 2017a). The glucagon-like peptide 1 (GLP-1) is a type of incretin protein which has hypoglycemic activity. Kato et al. revealed that curcumin treatment improves the secretion of GLP-1 factors and hence controls the diabetic complication (Kato et al., 2017). In line with these reports, Al-Ali research team established that curcumin possesses hypolipidemic activity and controls adiponectin synthesis, Interleukin-6, and C-reactive proteins which create hypersensitivity in the pancreatic glands (Al-Ali et al., 2016). The curcumin complexation with metals like zinc is proven antidiabetic in several in vivo and in vitro studies. In streptozotocin-induced diabetic rats, the effect of administration of curcuminezinc complex has lowered the hemoglobin and lipid content at a significant level while improved the plasma insulin levels (Al-Ali et al., 2016; Dehghan et al., 2016). Other remarkable antidiabetic roles exhibited by curcumin include its ability to inhibit the inactivation of calmodulindependent kinases, lower nitric oxide synthase, lipoprotein-linked cholesterol, and systolic blood pressure (Bulboaca et al., 2016). Similarly, curcumin dominates insulin resistance and has a prominent role in regulating hyperglycemiarelated factors like phosphorylated AKT and kinase enzymes (Jiménez-Osorio et al., 2016). In this series of the antidiabetic role of curcumin, curcumin’s ability to promote the multiplication of hepatic stellate cells into healthy active insulin producers is also remarkable. In a streptozotocin-induced diabetic rat, curcumin at a dosage of 50 mg/kg decreased the transition of stellate cells into cancerous myofibroblast cells (Mustafa, 2016). Sun et al. cracked the links between reactive oxygen species and the podocyte apoptosis after curcumin administration and revealed its association with caveolin-1 phosphorylation (Sun et al., 2016).

8.9 Conclusion The yellow crystalline “curcumin” has a long history of culinary applications and its potential therapeutic benefits in Indian Ayurveda and many global ancient medicine systems. In the modern scientific context, the golden spice is mostly exploited in food, cosmetics, pharmacological, and other health systems. Over the decades, curcumin’s safety is well established and recognized for its GRAS status by many standard food organizations. Though bioavailability is a major limitation in the therapeutic mechanism, the micro- and nanoencapsulation approaches and novel drug delivery systems have proven to solve this problem. The pleiotropic therapeutic effects of curcumin comprise various lifestyle diseases such as cardiovascular diseases, diabetes, gastric disease, and many others. The molecular mechanism behind the curcumin

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pharmacological activities includes suppressing Sp-1 and STAT transcription factors, downregulation of gene expressions of ADEM10, SEPP1, and calmodulin. Other key mechanisms well established for its antitumor property are autophagy mechanism, oxidative stress, and ER stresseassociated apoptosis. The recent nanoemulsion and novel Pickering curcumin emulsions support various clinical ailments, including the medication which needs to cross the bloodebrain barrier, such as b-amyloid plaque. Despite all this, there is still a lot more research essential to unravel the promising properties of curcumin.

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Chapter 9

Eugenol Ajay Sharma1, Garima Bhardwaj2, Harvinder Singh Sohal1 and Apurba Gohain3 1

Department of Chemistry, Chandigarh University, Mohali, Punjab, India; 2Department of Chemistry, Sant Longowal Institute of Engineering and

Technology, Sangrur, Punjab, India; 3Department of Chemistry, Assam University, Silchar, Assam, India

9.1 Introduction Eugenol is a volatile phytoconstituent present in various essential oils isolated from different aromatic medicinal plant. Eugenol acts as a natural antioxidant and has been extensively employed for the treatment of different lifestyle-related ailments. Conventionally, it has been endorsed to cure innumerable problems such as diarrhea, bronchitis, hyperlipidemia, hyperglycemia, arthritis, inflammation liver ailments, cancer, cardiovascular, and skin diseases. Eugenol has been acknowledged as GRAS (generally recognized as safe) by WHO (World Health Organization) and has been recognized as nonmutagenic (Khalil et al., 2017; R, 2015). It has been commonly used in the Ayurveda and Chinese primeval medicines systems. In the modern world, main source of eugenol, i.e., clove is mainly cultivated in Indonesia, Zanzibar, India, Pakistan, and Sri Lanka. Owing to wide range of therapeutic potential and practical applications in day-to-day life, eugenol has drawn the attention of numerous scholars and unlocked the doorway of research about its employment as a medication to cure innumerable diseases. Therefore, it is very substantial to represent the research outcomes associated to the beneficial properties of eugenol in order to explicate its mechanisms of action involved in prevention of several lifestyle-related illnesses and its significance for human health.

9.2 Chemistry (physical and chemical properties) IUPAC Name: 4-allyl-2-methoxyphenol. Nonproprietary and common names: Eugenol, eugenic acid, 4-allylguaiacol, caryophyllic acid, allylguaiacol, 4-allyl-1hydroxy-2-methoxybenzene, 4-allylcatechol-2-methyl ether, 4-allyl-2-methoxyphenol, 1-allyl-4-hydroxy-3-methoxybenzene, 1-hydroxy-2-methoxy-4-allylbenzene, 2-methoxy-4-allylphenol, 1-hydroxy-4-allyl-2-methoxybenzene, 2-methoxy-1hydroxy-4-allylbenzene, and 1-hydroxy-2-methoxy-4-propenylbenzene. Chemical formula: C10H12O2 Molecular mass: 164.2 g/mol. Elemental Analysis: Carbond73.15%, hydrogend7.37%, oxygend19.49% m/z: 164.08 (100.0%), 165.09 (11.0%) Color: Clear, colorless to pale yellow liquid. Odor and taste: Strongly aromatic and pungent odor of clove and spicy taste. Solubility (organic solvents): Extremely soluble. Solubility (water): Partially soluble. Body metabolism: Absorption via small intestine. Elimination and excretion: Through urination and as pass away CO2 Structural Formula: Fig. 9.1. Refractive Index: 1.540e1.542 at 20
C. Density: 1.064e1.068 g/mL. Specific Gravity: 1.0652 at 20
C. Melting Point: 9.2 to 9.1
C. Boiling Point: 254e255
C at 760 torr and 93e95
C at 10 torr. Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00007-7 Copyright © 2022 Elsevier Inc. All rights reserved.

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FIGURE 9.1 Eugenol.

Biosynthesis: Amino acid tyrosine is the starting point in the biosynthesis of eugenol. In first step, the tyrosine ammonia lyase enzyme converts L-tyrosine to p-coumaric acid. Then p-coumarate 3-hydroxylase enzyme in the presence of oxygen and NADPH (nicotinamide adenine dinucleotide phosphate) converts p-coumaric acid to caffeic acid. In next step, methylation of caffeic acid is carried out in the presence of S-adenosyl methionine to form ferulic acid, which is in turn by reacting with 4-hydroxycinnamoyl-CoA ligase enzyme converted to feruloyl-CoA. Further, feruloyl-CoA upon reduction by cinnamoyl-CoA reductase enzyme produces coniferaldehyde. Then, coniferaldehyde upon reduction with sinapylalcohol dehydrogenase or cinnamyl-alcohol dehydrogenase enzyme harvests coniferyl alcohol. Later on, coniferyl alcohol undergoes esterification with substrate CH3COSCoA to produce an ester, namely, coniferyl acetate. Finally, coniferyl acetate reacts with eugenol synthase 1 enzyme in the presence of NADPH to produce eugenol (Fig. 9.2) (Harakava, 2005). Chemical Synthesis: Chemically, eugenol can be produced via the allylation of guaiacol by allyl bromide. Initially, guaiacol reacts with allyl bromide in presence of potassium carbonate under reflux condition to produce guaiacol allyl ether, which finally under reflux condition at high temperature undergoes rearrangement to produce eugenol and o-eugenol (Fig. 9.3) (Moraes et al., 2020). Eugenol can also be produced through biotechnological approaches like biotransformation by means of different microorganisms’ participation, for instance, Amycolatopsis spp., Corynebacterium spp., Pseudomonas ssp., Escherichia coli, Streptomyces spp., and Bacillus cereus (Mishra et al., 2013; Molina et al., 2013).

FIGURE 9.2 Biosynthesis of eugenol.

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FIGURE 9.3 Chemical synthesis of eugenol.

Stability: Different strong oxidizing agents like potassium permanganate, ferric chloride, zinc, and iron are compatible with eugenol. Eugenol also reacts with various strong alkalis. On exposure to air, eugenol gets thicken and darken. It also gets blacken with age and may decay on exposure to light. Stability analysis with NMR (nuclear magnetic resonance) shows that the eugenol solutions prepared in dimethyl sulfoxide are stable up to 24 h (Yuwono et al., 2002). Eugenol is rapidly decomposed by different microbes living in the soil, particularly by bacterial action. Laboratory studies verified Pseudomonas fluorescensemediated decomposition of eugenol. Eugenol is not hydrolyzed under anaerobic environment in water. It is rapidly decomposed from the application spot by volatilization and decayed upon reaction with hydroxyl radicals and ozone existing in atmosphere (Sellamuthu, 2014).

9.3 Sources of eugenol For time immemorial living beings always have had the pleasure of taking what nature has provided them in the form of plants, be it food, medicine, shelter, weapons, etc. Human beings as a species are very hardy and resilient to many harsh conditions and diseases, but there are many more which are impervious to the human immune defense. It was the early men, experimenting with one particular plant that gave him the consciousness of its magical healing components and thus started the revolution of medicine derived from plants. One such early plant which gave men a medicinal touch was clove (Syzygium aromaticum) and tulsi (Ocimum tenuiflorum). These have been first used by the Chinese at the start of the Chinese civilization and the Indus Valley civilization, respectively, both of which are considered by many scholars to be the oldest civilizations (Kamatou et al., 2012; R, 2015). The plants with a high content of eugenol are generally used by traditional medicine experts as curative agents for countless diseases in their regular practice. These experts use different parts of the therapeutic plants, for example, flower, seeds, stem, leaves, root, and even whole plant (Kamatou et al., 2012; Khalil et al., 2017; R, 2015; Yadav et al., 2015). Traditionally, the extracts produced from different parts of these therapeutic plants have been endorsed to cure innumerable ailments including cancer, bronchitis, arthritis, hyperlipidemia, liver ailments, diarrhea, hyperglycemia, inflammatory disturbances, cardiovascular, and skin diseases. Clove was the first plant from which eugenol was initially extracted. Currently, eugenol has been produced in substantial amount from the extracts and essential oils of several herbal medicinal plants like clove leaves and buds, tulsi leaves, cinnamon leaves and bark, oregano, thyme, pepper, ginger, and turmeric (Khalil et al., 2017; R, 2015). Apart from these, many other aromatic herbs such as nutmeg, marjoram, bay, basil, and mace are also known to act as significant source of eugenol. Species like Illicium anisatum, Melissa officinalis L., Myristica fragrans Houtt. (Jaganathan & Supriyanto, 2012), Pogostemon cablin (Chen et al., 2013), Aphanamixis polystachya (Shaikh et al., 2012), Aegle marmelos (L.) Correa (Baliga et al., 2013), Abutilon indicum L. (Sharma et al., 2013), Acorus calamus L. (Avadhani, 2013), Piper betel (Gundala & Aneja, 2014), Pimenta pseudocaryophyllus (Gomes) Landrum (D’angelis & Negrelle, 2014), and Rabdosia japonica var. glaucocalyx (Xiang et al., 2015) have been also known to possess eugenol in considerable amount. Eugenol is generally isolated from the aerial parts of herbal plants (e.g., flowers, leaves, stem, and bark) as these parts are known to have a considerable volume of essential oils (Kamatou et al., 2012; Khalil et al., 2017). The amount of eugenol in these plant parts may fluctuate with season. However, various scientific reports revealed that highest concentration of eugenol can be attained in the fall time of year in comparison to the summer varieties (Khalil et al., 2017; Yadav et al., 2015). Table 9.1 represents the concentration of eugenol in some of its natural sources.

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TABLE 9.1 Natural sources of eugenol. Common name

Botanical name

Family/Genus

Eugenol content

References

Clove

Syzygium aromaticum

Myrtaceae

44%e55% in bud oil and 60.0%e 72.4% in the oil from pedicel

Bullerman et al. (1977), Lee and Shibamoto (2001), Mbaveng and Kuete (2017)

Nutmeg

Myristica fragrans

Myristicaceae

45%e90%

Bennett et al. (1988)

Sweet basil

Ocimum basilicum L.

Lamiaceae

6.6%

Johnson et al. (1999)

African basil

Ocimum gratissimum

Labiatae

68.8%

Gupta et al. (2014)

Tulsi or holy basil

Ocimum tenuiflorum

Lamiaceae

52.60%

Gupta et al. (2014)

Tezpat

Cinnamomum tamala

Lauraceae

91.4%

Dighe et al. (2005)

Cinnamon

Cinnamomum verum

Cinnamomum

65%e92%

Bullerman et al. (1977)

Japanese star anise

Illicium anisatum L.

Schisandraceae

0.4%e1%

Ize-Ludlow et al. (2004)

Balm mint

Melissa officinalis

Lamiaceae

3%e40%

Pavithra (2014)

9.4 Extraction and characterization techniques Nowadays, various conventional (hydrodistillation or steam distillation) and modern extraction (supercritical fluid extraction, ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE)) techniques have been employed for the isolation of eugenol. The characterization of the isolated eugenol can be done using advanced spectroscopic techniques like MS (mass spectroscopy), NMR, FTNIR (Fourier transform near-infrared spectroscopy), and FT-IR (Fourier transform infrared spectroscopy) whereas the purity of isolated eugenol can be verified using GCMS (gas chromatography and mass spectroscopy), LCMS (liquid chromatographyemass spectroscopy), HPLC (high-pressure liquid chromatography), and HPTLC (high-performance thin-layer chromatography) (Yuwono et al., 2002; Mahapatra et al., 2009; Khalil et al., 2017). Some significant techniques employed for the extraction and isolation of eugenol from different natural sources are discussed herein.

9.4.1 Solvent extraction It is a simple and economic method used for the isolation and extraction of vital essential oils from the medicinal plants. It is the one of the most usual and widely employed method for the separation of essential oils from their natural sources. Eugenol can be isolated by employing different organic solvents such as methanol, ethanol, n-hexane, petroleum ether, etc. The major drawback of solvent extraction is the isolation of some additional soluble classes of secondary metabolites along with eugenol. In this process, the fine powder of plant material is enfolded in filter paper and then the filter paper is placed inside the thimble. Finally, the thimble is inserted on the reflux flask of 500 mL capacity. The isolation is carried out by employing an appropriate solvent in Soxhlet apparatus (Quan et al., 2004). Temperature is set according to the solvent used. At the end, dried extract is obtained by evaporating the solvent from isolated extract by mean of rotary vacuum evaporator. Presently, numerous amendments have been done in the primeval solvent extraction procedures, which demonstrate higher proficiency in comparison to the conventional process. Apart from Soxhlet extraction method, maceration method can also be used for the isolation of eugenol (Kar Mahapatra et al., 2009; Khalil et al., 2017; Quan et al., 2004).

9.4.2 Steam distillation Usually the isolation of eugenol has been carried out by steam distillation (Khalil et al., 2017). During the isolation and extraction process of eugenol, firstly the plant material is air-dried and converted to a fine powder. This fine powder is used

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for the isolation of essential oil using different extraction methods. Once the essential oil is isolated then it is mixed with 3% solution of potassium or sodium hydroxide in order to extract the eugenol. This process resulted in the formations of alkali salt of eugenol. The unsolvable share of essential oil or the extract is then further subjected to steam distillation or solvent extraction. The residual basic solution is then acidulated at low temperature which resulted in the release of free eugenol. The free eugenol is then isolated from residual mixture via various techniques, for instance, fractional distillation, CC (column chromatography), TLC (thin-layer chromatography), and HPLC (Kar Mahapatra et al., 2009; Antic, 2014; Khalil et al., 2017; Yazdani et al., 2005).

9.4.3 Hydrodistillation Hydrodistillation is another commonly used method for extraction of essential oils (Jeyaratnam et al., 2016). In this process, 100 g dry and powdered clove buds are soaked in water. To execute the process dry cloves are taken in a volumetric flask (500 mL) and exposed to hydrodistillation for 5e6 h. Afterward, the volatile concentrate is collected and saturated with NaCl followed by the addition of suitable organic solvent. After some time, organic and aqueous layer get separated out. Organic layer is dehydrated using anhydrous Na2SO4. Finally, for the recovery of pure essential oil organic layer is heated at 60
C on water bath. The average yield of oil obtained comes out to be 11.50% although the concentration of eugenol obtained is 50.6%e53.6% (Khalil et al., 2017).

9.4.4 Supercritical fluid extraction Supercritical fluid (carbon dioxide) (SC-CO2) extraction is also an effective way for the isolation of clove oil from clove buds (Chatterjee & Bhattacharjee, 2013). Guan et al. (2007) analyzed the composition of clove oil using supercritical carbon dioxide extraction and other conventional methods (steam distillation, solvent extraction, molecular distillation). The results revealed that the composition of clove oil is alike in both the modern and conventional method. The quantity of CO2 used during experiment should be minimum up to 10 times excessive than sample and the whole process should be carried out in stainless steel container (Yazdani et al., 2005). Gopalakrishnan et al. (1990) evaluated the effectiveness of SC-CO2 and liquid extraction and found that former has better extraction frequency for clove oil. Through many experiment it has been observed that by increasing the flowing rate of CO2, the yield of eugenol in essential oil can be raised in case of SC-CO2 extraction (Reverchon & Marrone, 1997). Even though numerous investigations have shown the efficacy of SC-CO2 extraction as competent tool for the isolation of clove oil still analysis on eugenol yield optimization using SC-CO2 method is scanty (Khalil et al., 2017). Therefore, further investigations are required to highlight the effectiveness of SC-CO2 extraction technique for the isolation of eugenol from the clove bud.

9.4.5 Microwave-assisted (MWA) extraction Primeval techniques employed for the isolation of eugenol have many disadvantages, for example, thermal decomposition, leaching of fragrance, and hydrolysis of the products. To overcome such issues, variety of economical and efficient modern techniques have been introduced for the production of high yield of essential oil or extract and MWA extraction is one of them. This technique provides rapid extraction rate in low cost expenditure than conventional methods. Several arrays of methods have been developed by exploitation of microwave extraction process for the efficient isolation of essential oils. Some of these methods are MWHD (microwave-assisted hydrodistillation) (Golmakani & Rezaei, 2008), MWSD (microwave-assisted steam distillation) (Chemat et al., 2006), coaxial MWDH (coaxial microwave-assisted hydrodistillation), and MWHG (microwave-assisted hydrodiffusion and gravity) (Vian et al., 2008). Among all of these green extraction techniques, microwave-assisted hydrodistillation is found to be most efficient as it saves time of heating by 400% and energy consumption by 30% as well as this method is cost-efficient. Latterly on comparing the conventional hydrodistillation method and coaxial MWHD method for extraction of eugenol, it has been found that the capability of coaxial MWHD method for extraction of essential oil or eugenol from various herbs including clove is better. The eugenol extracted by coaxial MWHD exhibits higher thermal stability and extraction time is also less as compared to hydrodistillation method (González-Rivera et al., 2016). Hence, it has been found that MAE techniques are very beneficial to use at industrial scale as compared to other traditional methods used for eugenol extraction.

9.4.6 Ultrasound-assisted extraction UAE is another green extraction method, which remarkably speeds up the whole process of extraction of essential oil by reducing the demand of energy. Some more advantages of this technique are that one can easily handle the instrument,

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have high efficacy, no residue, eco-friendly, and degradation of extract can be prevented (Chemat & Khan, 2011). The analysis of effectiveness of UAE and conventional methods for extraction of clove oil revealed that the former is better in the isolation of clove oil as compared to latter with the extraction rate of 1350 mL/min (Alexandru et al., 2013). Tekin et al. (2015) implemented ultrasound extraction of eugenol from clove by using central composition design. The independent variables which were evaluated in the study include concentration of plant (3%e7%), time of extraction (30e60 min), and temperature (32e52
C), whereas clove extract was the dependent variable. The frequency of ultrasound waves was fixed at 53 kHz. The results revealed that the yield of extract was greatly affected by temperature (Tekin et al., 2015). The UAE is known to have various advantages over conventional methods as it is easy to handle, moderate use of solvent, reproducible, sustainable, and eco-friendly nature. The only drawback associated with this method is the degradation of phytocompounds at high frequency of ultrasound waves (Medina-Torres et al., 2017). It has been concluded that there are many methods that can be employed for the extraction and isolation of eugenol, e.g., steam distillation, hydrodistillation, solvent extraction, supercritical carbon dioxide extraction, MAE, UAE, etc. Extraction by all of these methods is very effective but microwave- and ultrasound-assisted extraction of eugenol has been recently developed techniques which are easy to implement, eco-friendly, low cost and provide high yield as compared to other methods.

9.5 Derivative of eugenol Eugenol is eco-friendly, inexpensive, present in abundance, and its chemical modifications are simple with a high yield; therefore eugenol can be utilized as biological lead molecule to synthesize variety of derivatives with promising healthpromoting potential (Lee & Shibamoto, 2001). The recent studies revealed that the eugenol derivatives possess diverse medicinal and biological characteristics like antibacterial, antityrosinase, antifungal, antiacne, antiseptic, antipyretic inhibition, antioxidant, anticancer, antiarthritis, antiinflammatory, etc. (Bullerman et al., 1977; Bennett et al., 1988; Dighe et al., 2005; Gupta et al., 2014; Johnson et al., 1999; Lee & Shibamoto, 2001; Mahapatra & Roy, 2014; R, 2015). Eugenol derivatives also possess high mechanical and thermal properties which lead to increase in flexibility of conetworks. These properties of eugenol derivatives provide immense applications in biofilm, bio-emulations, and biopolymer for the preparation. The different groups attached to eugenol molecule can change its chemistry and hence bioactivity, for instance, if phenol group is attached to eugenol it will enhance its free radical, reactive oxygen (ROS) and reactive nitrogen species (RNS) scavenging property (Ize-Ludlow et al., 2004). Antibacterial property of any molecule is mainly attributed to the presence of amine, ammonium, phenolic group; hence the introduction of these functional groups may enhance the biological potential of eugenol (Pavithra, 2014). Eugenol and its derivatives have vast applications in pharmaceutical, cosmetic, medicinal, and food industries. Owing to their antifungal, algacidal, and antifouling properties, eugenol and its derivatives are added to paint base in order to provide effective coating to many materials which help to inhibit the growth of algae and fungi. Although eugenol shows many medical and biological applications, its derivatives are more effective (Gupta et al., 2014; Johnson et al., 1999; R, 2015). Some of the derivatives of eugenol are mentioned below with their preparation and properties. Isoeugenol and methyl eugenol are the two naturally occurring isomers/derivatives of eugenol present in nutmeg, which play significant role in advancement of organic, carbohydrate, natural products, and medicinal chemistry. Isoeugenol has been extensively used as a vital constituent in organic synthesis whereas methyl eugenol is employed as a flavor and fragrance ingredient in variety of food products, perfumes, toiletries, and detergents (Ratnamala & Jamatsing, 2016) (Fig. 9.4). Ester and nitro (1e4) derivatives of eugenol can be synthesized through etherification and nitration reaction of eugenol and acetyl chloride/acetic anhydride and sodium nitrate (Fig. 9.5) (Kaur et al., 2019). These derivatives were evaluated for their in vitro anticancer potential against cancer cells like oral squamous carcinoma (KB) and androgen-insensitive prostate (DU-145) cancer cells. Both the derivatives showed potent cell growth inhibitory potential. The nitro derivative showed even better anticancer activity (P < 0.01) than that of eugenol with IC50 values 19.02  106 mol/L against DU-145 cells

FIGURE 9.4 Natural derivative of eugenol.

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FIGURE 9.5 Nitro and acylated derivatives of eugenol.

and 18.11  106 mol/L whereas against KB cells. Hence, signifying the occurrence of acetyl, nitro, and hydroxyl group plays significant role in displaying the anticancer potential of these constituents (Carrasco et al., 2008). Carrasco et al. again synthesized different nitro and acylated derivatives of eugenol (1e8; Fig. 9.5) and evaluated their antifungal potential against human pathogenic fungi. The results revealed that all derivatives showed remarkable antifungal potential. The structureeactivity relationships revealed that the influence of derivatives on biological potential is mainly associated with hydroxy group (at C-1), an allyl substituent (at C-4) and methoxy group (at C-2) or the existence of one or two nitro groups at diverse positions on benzene ring (Carrasco et al., 2012) (Fig. 9.5). 1,3-benzoxazine (Fig. 9.6) derivatives were derived from basic component eugenol by Mannich reaction (reaction in the presence of aldehyde and secondary amine), and then this 1,3-benzoxazine further undergoes hydrolysis to give aminomethyl derivatives. These 1,3-benzoxazine derivatives of eugenol showed potent antidepressant and antimalarial activities (Rudyanto et al., 2014). The eugenol undergoes two types of reactions, i.e., first with potassium carbonate along with benzyl halide and another with acyl chloride in dichloromethane. These reactions lead to formation of derivatives 1A-4A and 1B-9B (Fig. 9.7). These derivatives possess antibacterial activities in different ratios. Moreover, among all 13 derivatives only compound number 7B showed highest antibacterial potential (Abdul Rahim et al., 2017) (Fig. 9.7). Biphenyls derivatives (1e3) (Fig. 9.8) of eugenol show interesting biological activities. These derivatives have potent antiinflammatory and antinociceptive potential (Kurokawa et al., 1998) (Fig. 9.8). The benzoyl and acetyl derivatives (Fig. 9.9) of eugenol show potent antileishmanial activity with more inhibition and less toxicity. Among these two, benzoyl derivatives show more inhibition (Morais et al., 2014) (Fig. 9.9).

FIGURE 9.6 Scheme for the synthesis of 1,3-benzoxazine and aminomethyl derivatives.

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FIGURE 9.7 Scheme for the synthesis of benzyl derivatives of eugenol.

FIGURE 9.8 Biphenyls derivatives of eugenol.

FIGURE 9.9 Benzoyl and acetyl derivatives of eugenol.

9.6 Mechanism of action Several studies have demonstrated the therapeutic power of eugenol that enlightened its significance as one of the key bioactive phytoconstituent with numerous health-endorsing properties (Yogalakshmi et al., 2010). Till now, innumerable investigations have been done in order to understand the mechanism associated with the therapeutic potential of eugenol. Broadly, the mechanism of action involved in the functional and biological activeness of eugenol is mainly attributed to its free radical, RNS and ROS neutralizing power, microbial protein and DNA damaging potential, cellular and cytoantioxidant boosting influence (Singh & Panwar, 2014) (Fig. 9.10). The various mechanisms associated with the health-promoting, biological, and functional characteristics of eugenol are represented in Table 9.2. The mechanism of eugenol activity on bacterial cell has been described clearly in Fig. 9.11. Primarily it disrupts the cytoplasmic membrane which causes an increase in the membrane nonspecific permeability due to this reason: the transport of ATPs and ions gets affected. Eugenol modifies membrane permeability, due to which there is a loss of ions and extensive other cellular contents, and results in cell damaging and death. Eugenol alters the cell membrane of different bacteria as it affects and alters the cytotoxicity of cell owing to the generation of intracellular ROS and RNS. This leads to the inhibition of cell growth and damaging of the cell membrane and DNA which leads to the cell death (Marchese et al., 2017) (Fig. 9.11).

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FIGURE 9.10 Mechanism involved in antibacterial and antifungal potential of eugenol.

TABLE 9.2 Mechanism of efficacy of eugenol associated with different bioactivities. Bioactivities

Mechanisms

References

Antioxidant activity

Shows inhibitory effect on lipid peroxidation Helps to inhibit RNS and ROS formations

Gu¨lc¸in, (2011), Ito et al. (2005), Lee and Shibamoto (2001)

Antimicrobial activity

Helps in induction of cell lysis in gram-positive and gram-negative bacteria Helps in inhibition of ERK, IKK/NF-kB, and p38MAPK signaling pathways

Benencia and Courrges (2000), Charan Raja (2015), Marchese et al. (2017)

Antiinflammatory potential

Helps in prevention of inflammatory cytokine expression Shows inhibitory potential on prostaglandin synthesis Also retarding cyclooxygenase activity Helps in inactivation of tumor necrosis factors Helps in inhibiting nuclear factor-kappa B pathways

Huang et al. (2015), Koh et al. (2013), Markowitz et al. (1992)

Anticancer activity

Helps in triggering cell apoptosis Helps in targeting E2/E1 surviving pathways Helps in apoptosis of breast cancer cells (MCF-7) in human Helps to overpower COX-2 gene in HT-29 colon cells in human (Fig. 9.12) Helps in inhibiting prostaglandin E2 production Helps in reducing DNA oxidation Helps in inhibiting matrix metalloproteinase action (MMP-9) Helps in inactivation of extracellular-signal-regulated kinase (ERK) proteins/pathways

Behbahani (2014), Majeed et al. (2014), Vidhya and Devaraj (2011), Yin et al. (2018)

Continued

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TABLE 9.2 Mechanism of efficacy of eugenol associated with different bioactivities.dcont’d Bioactivities

Mechanisms

References

Neuroprotective and antistress-related perspectives

Helps in inhibition in lipid peroxidation Helps in preventing reduction in dopamine contents Shows inhibitory activity on 5-lipoxygenase activity Helps in reducing ROS, NO, and malondialdehyde concentrations Augmenting glutathione contents in cerebellum and cortex regions Attenuating cytosolic calcium content and acetylcholinesterase action in brain regions Helps in regulating serotonergic system in amygdala region Helps in diminishing reactive oxygen speciese induced upsurge in plasma corticosterone levels Augmenting changes in serotonin (5-HT) concentration Helps in decreasing concentration of norepinephrine in brain Helps in reducing ulcer index Modulates brain monoaminergic systems and hypothalamicepituitaryeadrenal

Garabadu et al. (2011, 2015), Kabuto et al. (2007), Khalil et al. (2017), Prasad and Muralidhara (2013)

Antidiabetic activity

Helps in inhibiting a-glucosidases activity Helps in inhibiting formation of advance glycation endproduct (AGE) Helps in preventing attachment of glucose to serum albumin Helps in upregulating the concentration of antioxidative enzymes

Genc¸ Bilgic¸li et al. (2019), Karthikesan et al. (2010), Srinivasan et al. (2014), Tahir et al. (2016)

Hypocholesterolemic perspectives

Helps in scavenging free radicals Helps in maintaining antioxidant level of body Helps in reducing cellular oxidative damage Helps in increasing the level of superoxide dismutase enzymes Helps in decreasing the concentration of serum malondialdehyde Shows inhibitory action on 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase)

Qi et al. (2018), Venkadeswaran et al. (2014)

FIGURE 9.11 Broad mechanism of efficacy of eugenol.

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FIGURE 9.12 Mechanism involved in anticancer potential of eugenol.

The activity of eugenol toward bacteria and fungi may be credited to the existence of a free eOH group (Nazzaro et al., 2013). In recent years, it has been hypothesized by many of the researchers that the hydroxyl group present on eugenol prevents the enzyme action of any bacteria or fungi by binding with the protein group (Burt, 2004).

9.7 Bioavailability Bioavailability refers to the availability of a drug or metabolite according to its dosage to produce its therapeutic action in body. In simple words, it tells how quickly a substance or metabolite gets absorbed by the body and enters the target area where it can take its action. When we talk about bioavailability of any bioactive compound, we discuss about how much medicine will be absorbed by our body and rest of it is deactivated by liver and thrown out from the body through kidneys (Chow, 2014). Thus, bioavailability of any bioactive compound describes as its activity toward the target site and its amount of consumption. Activity of eugenol is completely defined by its mechanism in disruption of cell wall membranes in microorganisms like bacteria and fungi. Therefore, the activity and bioavailability of eugenol is completely dependent upon its dose of consumption and different studies proved that it shows different effect toward target site at different doses, i.e., eugenol at low dose shows good bioavailability while at high dose it has proved to be harmful and may result in necrosis and inflammation of organs (Barboza et al., 2018). Various research studies have proved that oral consumption of eugenol has provided a very good bioavailability. Eugenol possesses a very insubstantial solubility and volatility due to which it has a good effectiveness when taken through mouth (Michiels et al., 2008). Eugenol has been absorbed and metabolized at faster rate in the liver when taken orally, whereas 95% of it expelled out from the body within 24 h. Further, various essential oils containing eugenol are absorbed rapidly in the proximal small intestine and stomach after pulmonary, oral, and dermal intake (Majeed et al., 2016). Therefore, eugenol should be encapsulated in order to inhibit its absorption before its action and also to enhance its ability and water solubility (Arana-Sánchez et al., 2010; Marchese et al., 2017). The bioavailability and stability of eugenol can be enhanced by various methods such as preparation of nanoemulsions of eugenol with different carrier oils (Majeed et al., 2016; Zhao et al., 2008), by encapsulation of eugenol with chitosan, 2-hydroxypropyl-b-cyclodextrin and b-cyclodextrin, by emulsionediffusion techniques through polycaprolactone, and by making nanofibers using electrospinning method (Choi et al., 2009; Hill et al., 2013). All these techniques provide a better solution and method to overcome the various difficulties associated with the handling and storage of eugenol. These techniques help to enhance the shelf life and stability of bioactive metabolite by providing protection against oxidation, reduction, light, and heat. Further, these techniques also help to improve the solubility (toward water) and to reduce the various side effects associated with bioactive metabolites, i.e., eugenol (Marchese et al., 2017; Woranuch & Yoksan, 2013a, 2013b). Furthermore, all of these techniques have no effect on the bioactivity of the active metabolite (eugenol) used to prepare all these nanoformulations.

9.8 Applications (clinical and pharmacological)/health benefits Clove oil is a major source of eugenol and has been used as a fragrance and spice from ancient times in Asian countries. An analgesic (dental) potential of clove oil was also stated in European countries in the 17th century. During last three centuries, the use of clove oil has been increased extensively owing to its immense health benefits and pharmacological potential. Nowadays, clove oil has been used in various consumer products such as detergents, soaps, food flavoring agent,

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mouthwashes, filler for dental cavities (zinc oxideeeugenol), analgesic (for toothache), food preservatives, bioinsecticides, biofungicide, cosmetics, clove cigarettes, mosquito repellent, and as an intermediate compound in vanillin synthesis since the 19th century in the United States (Ratnamala & Jamatsing, 2016; Sellamuthu, 2014). Clove oil has been widely employed in Ayurveda, ancient Chinese medicine, and other ancient systems of medicine all around the world as an antispasmodic for the cure of gastrointestinal disorders and as carminative to eliminate excessive gas present in the intestine. Apart from this, clove oil and eugenol have been well known for their pharmacological potential such as anticonvulsant, anesthetic, analgesic, antiparasitic, antiinflammatory, anticancer, antioxidant, cardioprotective, antibacterial, antifungal, antiviral, and insect repellent. Broadly speaking, eugenol is natural phytoconstituent with malleable pharmacological potential on virtually all systems (Maurya et al., 2020; Ratnamala & Jamatsing, 2016; Sellamuthu, 2014). Eugenol has been used for variety of health benefits including indigestion, blood impurities, generalized stress, cough, parasitic infestations, toothaches, and headache. It is well known for relieving verbosity and can in fact help to endorse good digestion as well as metabolism. The expert panel of German commission lately approved the use of eugenol oil as a tropical anesthetic and antiseptic. Another research has stated the antinociceptive and local anesthetic potential of eugenol in the periphery of orofacial region (Maghbool et al., 2020). Eugenol has been also employed for the cure of acne, rheumatoid arthritis, chronic diarrhea, asthma, and against various allergies (Joardar et al., 2020). Table 9.3 represents the diverse clinical and pharmacological properties of eugenol and clove oil along with effective dose.

TABLE 9.3 Pharmacological potential of eugenol and clove oil. Activity studied

Experimental

Effect (dose/concentration)

Analgesic (Daniel et al., 2009; Ferland et al., 2012; Park et al., 2011)

(a) Mice, (b) male SpragueeDawley rats

Extraordinary pain modulatory effect (100 mg/kg) Extended reaction time (40 mg/kg)

Antioxidant (Bondet et al., 1997; Bortolomeazzi et al., 2010; Mahapatra & Roy, 2014; Nagababu & Lakshmaiah, 1994; Oroojan et al., 2020; Pulla Reddy & Lokesh, 1994; Sueishi & Nii, 2019; Yogalakshmi et al., 2010)

(a) Microsomal mixed function oxidase facilitated peroxidation, (b) low-density lipoprotein oxidation, (c) lipid peroxidation, (d) reactive oxygen scavenging activity, (e) free radical reaction, (f) rat intestine, (g) lipid peroxidation, (h) DPPH scavenging activity, (i) hydroxyl radical scavenging

Controlled thiobarbituric acid reactive substances Hinders oxygen uptake Monooxygenase action inhibition (4e15 mM) Suppressed oxidation (1.5 mM) Exceptional suppression (0.05e0.15 mM) Significant activity (18.50 mU/100 mM sol) Hinders the lipid peroxidation at proliferation step (62.5e250 mM) Caused glutathione-S-transferases (1000 mg/kg) Hindered (0.1e1.0 mM/kg) Similar responses as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (20 mg/mL) Activity is larger in comparison to quercetin (0.6 mg/mL)

Protective (Aboubakr et al., 2019; Kar Mahapatra et al., 2009; Kumar et al., 2016; Nagababu et al., 1995)

(a) N-methyl-D-aspartate neurotoxicity, (b) carbon tetrachloride intoxicated rat liver, (c) liver monooxygenase activity and carbon tetrachlorideeinduced lipid peroxidation, (d) induced gastric lesions in rat, (e) induced ulcer in male Spraguee Dawley rats, (f) adult male Wistar rats, (g) nicotine-induced oxidative damage

Prevents acute neuronal swelling as well as reduced neuronal death (100e300 mM) Countered metabolic enzyme reduction (10.7 mg/kg/day) Inhibition (5 and 25 mg/kg) Reduced number and sternness of ulcers reported (10e100 mg/kg) Gastroprotective activity (100 mg/kg) Curtailed thioacetamide-induced hepatic injury (10.7 mg/kg/day) Dose-dependent protection (1e20 mg/mL)

Anesthetic (Guenette et al., 2006; Gue´nette et al., 2007; Park et al., 2009; Sell & Carlini, 1976)

(a) Randomized, single-blind study, (b) male SpragueeDawley rats

Activity equivalent to benzocaine Reversible and dose-dependent action (5e60 mg/kg)

Eugenol

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TABLE 9.3 Pharmacological potential of eugenol and clove oil.dcont’d Activity studied

Experimental

Effect (dose/concentration)

Antibacterial, antifungal, and antiviral (Campaniello et al., 2010; Chami et al., 2004; Gavanji et al., 2015; Hamzah et al., 2018; Jafri et al., 2020; Khan et al., 2005; Schnitzler, 2019)

(a) Candida albicans biofilms, (b) C. albicans, (c) oral candidiasis in immune spread rats, (d) vaginal candidiasis in immune spread rats, (e) Penicillium species, (f) Aspergillus species, (g) Fusarium species, (h) anti-herpes simplex virus

Active against sessile cells (20e2000 mg/L) Antifungal action through envelope damage (0.2%) Incomparable reduction in the colony counts (0.5 mL, 24 mM) Decrease in the colony counts of C. albicans (20 mg/kg/day) Growth retardation (100 mg/L) Growth retardation (100e140 mg/L) Growth retardation (140e150 mg/L) Active against herpes simplex viruses 1 and 2 (16.2 and 25.6 mg/mL)

Anticonvulsant (Dallmeier & Carlini, 1981; Dallmeier Zelger et al., 1983)

Maximal electroshock seizure test

Synthetic or artificial eugenol derivatives presented substantial action

Antiinflammatory (Grespan et al., 2012; Ito et al., 2005; Ma et al., 2017; Magalha˜es et al., 2019; Mateen et al., 2019; ¨ ztu¨rk & O ¨ zbek, 2005) O

(a) Prostaglandin H synthase inhibition, (b) human macrophages, (c) male SpragueeDawley rats, (d) mice

Restricts cycloxygenase (100e200 mM) Inhibited the release of proinflammatory mediators and restricts the cyclooxygenase-2 Reduced both paw as well as joint inflammation in induced arthritis (33 mg/kg) Reduced lipopolysaccharide-induced lung swelling (160 mg/kg, ip)

Penetration enhancer (Ahad et al., 2016; Mutalik & Udupa, 2003)

(a) In vitro evaluation in mouse skin, (b) in vitro penetration through porcine epidermis

Increased drug flux (5%) Considerable improvement of penetrability coefficient of drug (5%)

Cardiovascular (Choudhary et al., 2006; Lahlou et al., 2004; Mnafgui et al., 2016; Sensch et al., 2000)

(a) Rat mesenteric vascular bed, (b) Guinea-pig heart muscle, (c) hypertensive rats, (d) smooth muscle relaxant effect, (e) isoproterenol-induced cardiac hypertrophy

Dose-dependent, reversible vasodilatation (0.2e20 mmol) Potassium current inhibition (60 e600 mmol/L) Vascular relaxation (0.006e6 mM) Action directed via endothelial-generated nitric oxide (300 mM) Oxidative stress along with apoptosis reduction (1 mg/kg twice daily)

Anticancer (Bezerra et al., 2017; Kaur et al., 2010; Manikandan et al., 2011)

(a) Mice, (b) Swiss mice

Resistance toward chemically tempted skin cancer (30 mL) Restraint of skin carcinogenesis at dysplastic phase (1.25 mg/kg)

Antioxidant (Bamdad et al., 2006; Gu¨lc¸in et al., 2012; Jirovetz et al., 2006; Radu¨nz et al., 2019)

(a) DPPH scavenging activity, (b) hydroxyl radical scavenging, (c) lipid peroxidation

Action comparable to that of BHA and BHT (0.5 mg/mL) Shows higher activity than quercetin (0.2 mg/mL) More potential than BHT

Antiinflammatory (Banerjee et al., 2020; Han & Parker, 2017; Mektrirat et al., 2016)

Mice

Caused cytokine inhibition (200 mg/kg)

Anticancer (Bhalla et al., 2013; Miyazawa & Hisama, 2003; Periasamy et al., 2016)

Antimutagenic activity against MNNG, 4NQO, AfB1, and Trp-P-1 cell lines

Suppressive effect on mutagens (ethyl acetate extract)

Aphrodisiac (Colombi Cansian et al., 2014)

Male Swiss mice

Enhanced sexual function (500 mg/kg of 50% ethanolic extract)

Penetration enhancer (Herman & Herman, 2015; Shen et al., 2007)

Excised rabbit abdominal skin In vivo percutaneous absorption in rabbit

Extraordinary rise in drug flux (1%e3%) Increase in drug flux (1%e3%)

Clove oil

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9.8.1 Other bioapplications of eugenol and its derivatives 9.8.1.1 Enzyme inhibitor 15-lipoxygenase enzyme causes cancer by reacting with fatty acids. Eugenol esters work better against this enzyme and possess anticancer properties (Orafaie et al., 2018; Sadeghian et al., 2008).

9.8.1.2 Antibiotics Eugenol has potential to break the cell wall of bacterial strains and to inhibit their growth owing to which it shows antimicrobial action against variety of microorganisms. Due to which, it can act as antibiotic agent (Felgueiras et al., 2020).

9.8.1.3 Bio-based packaging materials The bio-based packaging materials formed from organic compounds like eugenol are more renewable, efficient, cheap, and abundant. Eugenol nanoparticles developed using emulsions, due to their antioxidant and antimicrobial activity, are used as bio-packaging materials that help to increase shelf life of materials (Woranuch & Yoksan, 2013a, 2013b).

9.8.1.4 Green synthesis of metal nanoparticles Green synthesis always dominates in the preparation of any compound or nanoparticles. Eugenol and acetyleugenol due to their phenolic and antioxidant nature behave as natural reducing agents in the synthesis of metal nanoparticles, i.e., silver nanoparticles. Apart from natural reducing agents these also help to stabilize the synthesized nanoparticles by forming case like structure around them (Sangar et al., 2019).

9.8.1.5 Nanoparticles or nanodiamonds Plant-based nanoparticles or nanodiamonds are cheaper, less toxicity, more surface area, and easy to synthesize, and these have vast number of applications in pharmaceutical, medicinal, cosmetic, and other industries. The eugenol-based nanodiamonds are easy to penetrate in skin and show minimum toxicity with high stability, so these are used in skin treatment in drug delivery (Namdar & Nafisi, 2018).

9.8.1.6 Synthesis of biocopolymers Eugenol also has high hydrolytic stability due to the formation of covalent bonds with the other molecules; this property of eugenol makes it more soluble in variety of solvents. The emulsion materials developed from eugenol are known to show antimicrobial properties and also inhibit in vitro human herpes. Due to more thermal stability and wettability, the eugenol derivatives are used in the preparation of diverse nature of copolymers, which are used in root canal therapy, ophthalmology, and also in manufacturing tiny intraocular lenses for eyes (Rojo et al., 2008).

9.8.1.7 Synthesis of composite resins/gums Composite resins/gums are usually used by dentists for cavity filling or root canal therapy. These resins/gums are also known as “white filling.” The eugenol in combination with ZnO and methacrylate is used as composites that provide temporary cementing to the tooth. The methacrylic derivatives of eugenol also provide antimicrobial potential to these resins and also help to provide orthopedic relief (Almaroof et al., 2016; Bayindir et al., 2003; Rojo & Deb, 2015; Rojo et al., 2009).

9.8.1.8 Preparation of dendrimers The dendrimers are the polymers used in gene therapy and act as diagnosing agents. Eugenol-based dendrimers contain aromatic ring that is responsible for the fluorescent properties of these dendrimers. The dendrimers of carbosinane with amino functional group attached to eugenol ligand interact with drugs like penicillin and form hosteguest interaction, i.e., anionic guest and cationic dendrimer. So, these dendrimers work best for anionic drugs.

9.8.1.9 Protective biofilm Polymeric form of eugenol acts as nonconducting electropolymerized film for detecting oxygen and this is very useful in the field of neuroscience (Paul et al., 2013).

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9.8.1.10 Corrosion inhibitors The phytoconstituents which possess potent antioxidant potential are very effective against corrosion of iron and steel in acidic condition. The essential oils containing eugenol and its derivative are effective inhibitors of corrosion (due to their antioxidant properties) and their efficiency is directly proportional to concentration and temperature (Chaieb et al., 2005).

9.9 Safety and toxicology As a food additive, eugenol is considered healthy, but because of the wide array of applications as well as the extensive use of clove oil and eugenol and its accessibility, there is a great deal of doubt about its toxicity. Liver, lung, and the nervous system are primarily affected by the toxicity of clove oil or eugenol. Several reports have been presented on accidentally intake of 5e10 mL clove oil or eugenol especially by kids and young children, which causes several health problems such as nervous system depression, lever failure, coma, severe toxicity causing seizures, acidosis, and paralysis (Sellamuthu, 2014). Clove cigarette smokers mainly suffer from acute respiratory signs like edema of lung, aspiration pneumonia, hemoptysis, and bronchospasm. In rats’ model, similar respiratory problems were also detected (Sellamuthu, 2014).

9.9.1 Acute and short-term toxicity Phenol is connected with the eugenol chemical structure. But the toxicity of eugenol does not contain corrosive activities of phenol; gastroenteritis, secretion of mucin, and vomiting and to some extent toxicity of eugenol are similar to the phenol. No analysis has demonstrated acute toxic effects of eugenol. In general eugenol toxicity is low in mammals, and in the United States, eugenol is listed as Category III by the Environment Protection Agency; in rodents the LD50 value is > 1930 mg/kg (Sellamuthu, 2014).

9.9.2 Chronic toxicity The systemic toxicity of eugenol is discussed below in humans. Chronic tests in animals that were diagnosed with 87 mg/ kg or up to 1% eugenol were not harmful to rats; deaths and enlargement of liver and adrenal gland were recorded at higher doses [4000 mg/kg BW (body weight)] (Sellamuthu, 2014). The 10% eugenol doses in diet for 14 days bring about the death of all female and one male rat; and 1.25% eugenol doses in diet for 90 days bring about the decrease in weight gain in rats. Alike findings were also noted in mice where 10% doses of eugenol were toxic and all the mice died, while a low dose (0.6%) did not bring any toxicity in mice. The 14-day dietary intake of 10% eugenol led to the deaths of one male rat and all female rats, while the 90-day dietary intake of 1, 2, and 5% eugenol contributed to a decrease of rats’ weight gain. Similar effects were recorded in mice; 10% of eugenol was toxic and causes all mice dead and hence no toxicity in mice was caused at a low dose (0.6%) (Sellamuthu, 2014).

9.9.3 Immunotoxicity Results are controversial for clove oil and eugenol causing hypersensitivity and allergy. After the use of eugenolcontaining dental products, various adverse effects were observed. In recent studies, various ill effects were reported, i.e., ulcers, skin irritation, dermatitis, and tissue necrosis. Clove or eugenol is a slow sensitizing agent which exhibited systemic reactions like stomatitis and urticaria. Only a single case is reported of eugenol-containing hypersensitivity reactions when exposed to eugenol products (Sellamuthu, 2014).

9.9.4 Reproductive toxicity Reproductive toxicity is not as such reported. But the spermicidal action of clove oil and eugenol has been reported in vitro studies (Sellamuthu, 2014).

9.9.5 Genotoxicity There are no human data on genotoxic effects. In vitro trials have shown some effect on chromosome aberrations and mutagenesis. In Syriac hamster embryo cells, eugenol mediated chromosomal aberrations was found to be 3.5% aberrant cells in case of V79 cells and for S9 liver fractions, it was 15% aberrant cells. The DNA helix untwisting enzyme topoisomerase II was inhibited by eugenol (Huang et al., 2015; Sellamuthu, 2014).

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9.9.6 Carcinogenicity The carcinogenesis of eugenol in human beings is not known, and there are also minimal animal studies. Eugenol has not induced cancer in rodents, but it has developed mice’s liver tumor. Moreover, mixed experiments for mutagenesis were conducted. On the basis of these findings, eugenol is listed as a Category III (not classifiable) compound for carcinogenicity by the International Research Agency for Cancer, and eugenol is not used in the US National Toxicology Program as fairly anticipated to cause cancer (Sellamuthu, 2014; Smith et al., 2002).

9.9.7 Clinical management No eugenol toxicity antidotes are available. Patients are also treated with hepatological and pulmonary toxicity and are treated based on symptoms such as coma or seizures. Furthermore, N-acetyl cysteine should be prescribed to avoid hepatotoxicity (Sellamuthu, 2014; Yadav et al., 2015).

9.9.8 Ecotoxicology There are no bird toxicity trials of eugenol or clove oil. Its effects on insects are extremely complex, and it is highly poisonous to bacteria. The low dosage of clove is employed as an anesthetic for fishes and high dosage or longer time of exposure can cause fishes death. Lice and caterpillars are not known to be eugenol sensitive. Values of LD50/LC50 of clove oil and eugenol for snail is LC50 22e28 mg/L, for aquatic invertebrates is LC50 30 mg/L (24 h), for mosquitos is LC50 33 mg/L (unknown duration), for tiger prawn is LC50 130 mg/L (1 h) and 30 mg/L (24h), for fishes is LC50 9 mg/L (12h) and 81 mg/L (10 min), for lice is LD50 0.25 mg cm2, and for dust mites is LD50 3.7e12 mg/cm2 (Sellamuthu, 2014).

9.9.9 Exposure standards and guidelines As such, there are no well-defined time-weighted average, working exposure limits, or other guidelines for clove oil or eugenol. However, based on a study in rats, WHO suggested suitable dietetic consumption for humans is 2.5 mg kg1 day1 (Sellamuthu, 2014).

9.10 Conclusion Presently, scientists have keen interest in small therapeutic molecules obtained from medicinal plants which serve as a potential source for the cure of variety of infections or diseases. Eugenol is an easily available, inexpensive, small, and structurally simple natural product, that offers a widespread application and prospects in different fields such as bio/ chemical synthesis, pharmaceutical, cosmetic, food, and flavor. It is a highly versatile molecule that serves as a starting material for the synthesis of high-valued low-molecular-weight variety of natural products and their derivatives. It also acts as a building block for the formation of highly complex functionalized bioactive constituents. This chapter explicates the usefulness of eugenol as a potent therapeutic tool that can be used in combination with various foods and herbal medications for curing variety of metabolic disorders. Eugenol and its derivatives have been well known for their pharmacological potential like antibacterial, antifungal, antiviral, antioxidant, anticancer, anticonvulsant, antiinflammatory, penetration enhancement, antigenotoxic, and cardioprotective. Recently, owing to their exceptional medicinal and biological potential, eugenol and its derivatives have also attracted a lot of consideration of the scholars from all around the world. The fascinating numbers on therapeutic supremacy of eugenol and its derivatives are reassuring. However, further more investigations are needed to fully explore the stipulate dosage amount, stability, safety, toxicology, overall human health promotion potential, and various other hidden abilities of eugenol and its derivatives for innumerable practical applications related to the betterment and advancement of mankind.

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Further reading Das Mahapatra, K., & Kumar, B. (2012). A review on therapeutic uses of Ocimum sanctum Linn (tulsi) with its pharmacological actions. International Journal of Research in Ayurveda and Pharmacy, 3(5), 645e647. https://doi.org/10.7897/2277-4343.03512

Chapter 10

PUFA and MUFA _ ¨ z1, Ilknur Mustafa O Ucak2 and Gulzar Ahmad Nayik3 1

Aksaray University, Faculty of Veterinary Medicine, Aksaray, Turkey; 2Nigde Ömer Halisdemir University, Faculty of Agricultural Sciences and

Technologies, Ni gde, Turkey; 3Department of Food Science & Technology, Government Degree College Shopian, Jammu and Kashmir, India

10.1 Introduction Fats are among the most important organic substances for human and consist of hydrogen, carbon, and oxygen atoms. Besides being a considerable source of energy, these organic substances contain fat-soluble vitamins, form lipoproteins, and involve in metabolic activities (Mol, 2008). Fatty acids are important structural components that have critical roles in metabolic events like energy storage and transport and regulation of gene functions (Robertson et al., 2013, pp. 45e99). Moreover, they are also required for cell membrane structures (Abedi & Sahari, 2014). Fatty acids can be found intracellularly both as free form and in lipid complex (Rustan & Drevon, 2005). Today, more than 1000 fatty acids are now distinguished in microorganisms, plants, and animals. By the way, around 20 of them are generally found in nature and palmitic, linoleic, and oleic acids are the most well-known fatty acids (Gunstone et al., 2007). Fatty acids which are formed by carbon chains are organic acids that have one methyl group (CH3) at one end and a water-dissolvable carboxyl gathering (-COOH) at the opposite end (Schubert, 1973). All fatty acids are grouped based on their carbon chain lengths, single or double bonds in their structures, and the degree of unsaturation (Serpek & ve Kalaycõoglu, 2000, p. 457). In natural oils, fatty acids which are straight-chain products are divided into two types. They are saturated and unsaturated fatty acids (Karaca & Aytaç, 2007). Fats are the subject of investigations of the relationship between certain diseases and nutrition. In particular, the research has focused on the saturated or unsaturated structure of fatty acids, their cis/trans structure, as well as the cholesterol and essential fatty acid content of fats and their oxidative stability. As a crucial part of human nutrition, fats are a very important source of energy, contain functional compounds such as fat-soluble vitamins, regulate blood lipid levels, and have functional groups like omega-3 fatty acids (Çakmakçõ & Kahyaoglu, 2012). Unsaturated fatty acids consist of molecules which have at least a single double bond on the chain. Since a fatty acid molecule has two ends, one of which containing carboxyl and the other methyl carbon, methyl carbon is called omega “un” carbon and unsaturated fatty acids are also named such as n-3, n-6, n-7, or n-9 according to the “n” carbon in which methyl carbon end is by the first double bond (Öztecik, 2019, pp. 21e22).

10.1.1 Monounsaturated fatty acids Monounsaturated fatty acids (MUFAs) are acids having a single double bond, they have two hydrogen atoms deficit, and their two carbon atoms in their structure are connected by a double bond. The human body makes these fatty acids by using saturated fatty acids and uses them in various physiological events (National Research Council, 1989). Almonds, nuts, walnuts, avocados, and especially olive oil are the main sources of MUFA (Peou et al., 2016; Wang et al., 2015). MUFAs which can be found in olive oil and canola oil are resistant to oxidant stresses. Mediterranean-style diets with plenty of carbohydrates and rich in unsaturated fatty acids are considered indicative of a healthy diet (olive oil, fresh fruit, and vegetables) (Esposito, 2004). In excess of 100 unsaturated fatty acids have been found to the day and the most common MUFAs are given in Fig. 10.1. The human body can synthesize MUFAs similarly to fatty acids which are saturated (Fig. 10.2). MUFAs are known as n-9 fatty acids. While the effects of MUFAs on LDL (low-density lipoprotein) cholesterol are neutral, it is known that they raise HDL (high-density lipoprotein) levels. The blood HDL is generally labeled as good cholesterol (Gogus & Smith, 2010). Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00004-1 Copyright © 2022 Elsevier Inc. All rights reserved.

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FIGURE 10.1 The most common MUFAs (Gunstone, 1996).

FIGURE 10.2 Monounsaturated fatty acid: oleic acid (Öztecik, 2019, pp. 21e22).

10.1.2 Polyunsaturated fatty acids Omega (n-3) and omega (n-6) fatty acids, containing more than one double bonds in the cis configuration in their chemical structure, can be given example for polyunsaturated fatty acids (PUFAs). Carbon atoms in fatty acids’ numbers come from the carboxyl group. Next to the carboxyl group is the carbon atom number 2 and it is also known a-carbon atom. Carbon atom number 3 is the b-carbon. The carbon atom by the methyl group is known as u- or n-carbon atom. In every n-3 fatty acids, the primary double bond is placed in the third to fourth carbon atoms from the u-carbon atom. Similarly, in every fatty acid n-6, the first double bond is found in the sixth to seventh carbon atoms adjacent to the u-carbon atom (Dubois et al., 2007). The PUFAs contain multiple double bonds in their form (Fig. 10.3). These fatty acids are sorted into two subgroups as fatty acids n-3 or n-6 based on the first carbon from the methyl group in which the double bond is located. Alpha-linolenic acid is the source for fatty acids of omega-3 (Öztecik, 2019, pp. 21e22). While fatty acideintegrated lipids provide thermal and mechanical insulation, PUFAs are vital for different mechanisms such as the production of eicosanoids, interaction with enzymes, maintenance of membrane fluidity, lipid peroxidation, protein acylation, and gene interactions (Liu et al., 2014; Rustan & Drevon, 2005). Humans and other mammals have the desaturase enzyme, which can form a double bond up to the ninth carbons from the carboxyl group. Therefore, only oleic acid (18:1n-9), a single MUFA of class n-9, can be synthesized in humans and animals. On the other hand, fatty acids which are n-3 and n-6 cannot be synthesized, and they must be taken externally

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FIGURE 10.3 Metabolic pathways of n-3 and n-6 polyunsaturated fatty acids (Marangoni et al., 2020).

through diet. 18-carbon linoleic (LA, 18:2, u-6, cis, cis, 9, 12-octadecadienoic acid) and linolenic (ALA, 18:3, u-3, cis, cis, 9,12,15-octadecatrienoic acid) cannot be synthesized in the organism, have to be taken through nutrients, and it makes them named as essential fatty acids (Waruda et al., 2006). Mainly, essential fatty acids of n-3 and n-6 are found in the body. The n-3 fatty acids contain alpha-linolenic acid (ALA, 18:3) having 18 carbons and 3 double bonds, and the n-6 fatty acids contain cis-linoleic acid (LA, 18:2), and it includes 18 carbons and 2 double bonds. Oleic acid (OA, 18:1) among the n-9 fatty acids and palmitoleic acid (PA, 16:1) among the n-7 fatty acids are those generally used in the organism; however, it does not make them essential. Essential fatty acids are the precursor to eicosanoids and their products (prostaglandin: PG, thromboxane: TX, and leukotrienes: LT) in the organism. Eicosanoids exert important roles regulating gastrointestinal, reproductive, and immune systems (Harris et al., 2007). PUFAs are subjected to further processes such as the addition of carbon atoms and desaturation after they are produced in fatty acid metabolism. Besides, PUFAs are metabolized through b-oxidation in mitochondria or peroxisomes (Rustan & Drevon, 2005). PUFAs have two groups based on their chain lengths. For instance, LA and ALA are short-chain PUFAs (SC-PUFAs), while EPA, DHA, and AA are classified as long-chain (LC-PUFAs). ALA and linoleic acid are two fatty acids which are essential and should be taken with diets since humans cannot synthesize these essential fatty acids repeatedly due to the lack of D12 and D15 desaturases enzymes that form double bonds in the fatty acid backbone (Russo, 2009). Therefore, daily diets should contain appropriate fatty acids (Collinius, 2016). Linoleic acid and ALA are essential precursors for LC-PUFAs, i.e., AA, DHA, and EPA. EPA and DHA are made of linoleic acid and ALA through elongation and denaturation steps. The LC-PUFA biosynthesis from LA and ALA is

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completed by the b-oxidation step in peroxisomes (Robertson et al., 2013, pp. 45e99). Enzymes in the human body can synthesize LC-PUFA from LA and ALA, but not to the extent of the required amounts (Wiktorowska-owczarek et al., 2015).

10.2 Sources/derivatives PUFAs which belong to n-3 series are found in fish such as mackerel, herring, sardines, trout, and salmon and a small amount in eggs as an animal source (Fig. 10.4). ALA is rich in some vegetable sources such as green leafy vegetables.

FIGURE 10.4 Sources of omega-3 essential fatty acids (Schwalfenberg, 2006).

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Soybean oil, canola oil, hemp seed oil, flaxseed, pumpkin seeds, walnuts, purslane, legumes, and rapeseed may be given as examples for these vegetables. Also, n-3 fatty acids are vastly found in human milk. DHA and EPA are mainly found in marine fish. The main sources of n-9 sources are unrefined raw olives, olive oil, hazelnut oil, almonds, avocados, cashew walnuts, peanuts, sesame oil, pecan nuts, pistachios, nuts, canola oil, and flaxseed oil (Konukoglu, 2008).

10.3 Extraction and characterization techniques For the extraction of fatty acids, various methods such as chromatography, distillation, low-temperature fraction crystallization, supercritical CO2 extraction, enzymatic hydrolysis, and urea fractionation have been used.

10.3.1 Chromatographic methods In chromatographic methods of separation, fatty acids are first adsorbed on substances with a high adsorbing ability such as silica and alumina. Usually, adsorbents are filled into columns (column chromatography). Adsorbed fatty acids on the adsorbent are then eluted from the system with aqueous acetone or methanol. Depending on different adsorption properties, fatty acids are separated from each other. In silver ion chromatography, silica impregnated with silver nitrate in the column is used as an adsorbent. Unsaturated fatty acids form complexes with silver ions. Based on the variations of complex formations according to the double bonds’ number and the molecule’s length of chain, the separation and therefore purification of fatty acids can be performed (Anonymous, 2020).

10.3.2 Distillation methods In these methods, fatty acids are generally fractioned after converting them into methyl esters and subjecting to vacuum distillation at 250
C under a pressure of 0.2e0.5 mmHg. Under these conditions, thermal polymerization of highly unsaturated fatty acids is difficult to prevent. Oxidation of free fatty acids can also occur (Anonymous, 2020).

10.3.3 The low-temperature fraction crystallization method Depending on the molecular structures of triacylglycerols and fatty acids, their solubilities in solvents are different. In this method, oil or fatty acids are generally subjected to fractional crystallization in acetone 25 and 40
C. In a solvent-free environment also, it is possible to fractionate triacylglycerol at low temperatures. In industry, palm stearin rich in palmitic acid and palm olein rich in oleic acid are produced from palm oil with this method. The method requires special cooling units and the crystals need to be filtered quickly, especially at low temperatures (Timms, 2005).

10.3.4 Supercritical CO2 extraction method Since the CO2 critical temperature is 31
C, the extraction process can be performed at very low temperatures such as 35
C. This method is then preferred for temperature-sensitive substances. At a pressure of 200 bar, the density of CO2 is also very close to the density of the hexane. Therefore, it displays nonpolar solvent properties similar to hexane. However, under critical conditions, the solubility of triacylglycerols in CO2 is very low, so it is not suitable for the enrichment of fats by PUFAs. In the supercritical CO2 extraction method, since the fatty acids can only be fractionated according to the number of carbons, regardless of the degree of unsaturation, working directly with fatty acids is almost impossible. As methyl esters of fatty acids can be fractionated with this method, it is possible to produce PUFA-rich products. Although this method is still considered an expensive method, the effort has been made to increase its industrial applicability (Anonymous, 2020).

10.3.5 Enrichment by enzymatic methods Lipases naturally hydrolyze triacylglycerols into diacylglycerols, monoacylglycerols, glycerin, and free fatty acids at the inner surface between the water and oil phases. In organic solvents that do not mix with water, they catalyze the esterification and transesterification reactions of lipid substrates.

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Lipases can be produced from various tissues of plants and animals by fermentation processes that employ microorganisms such as mold, yeast, and bacteria. Lipases can be manufactured easily and cost-effectively at industrial quantities with fermentation method and have a great applicability in the industry (Kuo et al., 2002; Sharma et al., 2001).

10.3.6 Urea fractionation method Urea fractionation is a classic method developed and used since 1940 to separate ester, alcohol, and other derivatives of fatty acids from other fatty acids, fats, and nonparticipating substances. The length of carbon chain and the presence and molecules’ unsaturation degree are of importance. Saturated fatty acids and MUFAs are easily eliminated by the urea participation with the appropriate application of this method. This simple, quick, and inexpensive method is based on the fact that urea combines with linear chain compounds to give crystalline end-products, which are easily separated (Kent, 2009).

10.4 Chemistry Fatty acids are nearly totally aliphatic carboxylic acids of unbranched chain. Although the most frequently seen length of natural fatty acids are between 4 and 22 carbon length (C4 to C22) and the most common is C18, the most extensive definition contains every chain length. Natural fatty acids have biosynthesis of a common pathway. The synthesis of chain starts with a couple of carbon molecules and the double bonds of cis are added by desaturase enzymes at definite positions coming from the carboxyl group which yields fatty acids of equal chain length having a characteristic methylene-interrupted cis double bond model. Many fatty acids having degree of unsaturation and variable chain lengths are formed in this way. In general use, systematic names of fatty acids are very laborious, and shorter alternative names are commonly used. Two numbers which are divided by two dots in a row show the chain length and the double bonds’ numbers which are, respectively, 18 carbon octadecenoic acid and 1 double bond are given as 18:1. The position of double bonds is determined in several ways: it defines the configuration and location, or the location of a double bond from the carboxyl or methyl ends. The location of double bond by the methyl end is given as n-x or o-x. Here, x is given to the carbons number coming from the methyl end. Two types are commonly used, while n-system is generally preferred. The names and structures of fatty acids in commercial oils and fats are given in Figs. 10.5 and 10.6 (Scrimgeour, 2005). Omega-3 fatty acids are those of heterogeneous group which has a double bond in the third to fourth carbon atoms from the methyl end (from the u1 carbon atom). Generally, they are distinguished as MUFAs (single double bond in the carbon chain) and PUFAs (multiple double bonds in the carbon chain) (Cholewski et al., 2018). Due to the length of hydrocarbon chain, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) are referred as very long chain of n-3 fatty acids. EPA, DHA, and DPA are present in cod liver oil, fish oils, krill oil, some algae oils, and oily fish as well as some preparations of pharmaceutical grade. EPA, DHA, and DPA are bonded metabolically. There is a way to simply synthesize EPA from plant-derived n-3 fatty acids (Calder, 2014). Fatty acids of omega-3 (n-3) are a family of PUFAs beneficial to the health of human. EPA and DHA are the most important among n-3 fatty acids in a functional manner; however, n-3 DPA also emerges as a critical omega-3 fatty acid (Calder, 2014; Cockbain et al., 2012).

10.5 Mechanism of action LC-PUFAs are the main constitutional phospholipids component in the cell membranes of all tissues in the body and affect the fluidity and ion transfer of the membranes. n-3 PUFA is especially abundant in retina, myocardial, brain, and spermatozoa, and they are indispensable for the development, physiological functions of these tissues, and many other physiological events. In general, n-3 fatty acids (ALA, DHA, and EPA) exert active roles in preventing and treating diseases such as rheumatoid arthritis, cardiovascular, cancer, Alzheimer’s (AD), and asthma as well as retinal and brain development in infants (Brown, 2000, pp. 299e318; Lauritzen et al., 2001). DHA, one of the most important compounds of the brain and retinal membrane, cannot occur in sufficient amounts in ALA deficiency. Furthermore, linoleic acid inhibits the synthesis of EPA and DHA when taken at very high amounts in the diet. Therefore, diets that include linoleic aciderich corn and sunflower oil and low ALA cause EPA and DHA deficiency. In these cases, EPA and DHA must be taken through diet (Calabrase, 1999; Stoll, 1999). The significance of nutrition for the healthy development and functioning as well as protection for the human body from diseases has recently become an

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FIGURE 10.5 Fatty acids in commodity oils and fats, nomenclature, and structure (Scrimgeour, 2005).

important issue. Animal-origin foods are the first in line meeting the demand for energy, protein, vitamin, and mineral by people in proper and balanced nutrition. Eating habits are important in human health. Essential nutrients such as fatty acids, amino acids, and some sources of minerals and vitamins need to be adequate and balanced in the diets. Since fatty acids are triglycerides consisting of variable length carbon chains with different degrees of saturation, they are both an important part of complex lipids and a great source of energy. Fatty acids are either saturated or unsaturated. Unsaturated fatty acids are also sorted into two groups which are MUFA and PUFA. Fatty acids of n-6 and n-3 are defined as PUFAs; they are liquid at ambient temperature and are also significant for human health. The main n-6 sources are corn and soybean oil, which contain important amounts of linoleic acid while n-3 is abundant in flaxseeds, walnuts, and especially in oily fish and plankton. DHA and EPA are vastly found in seafood in the food range. Firstly, marine algae synthesize such fatty acids and then planktons and other small sea animals take them. Thus, they join the food range. EPA, DPA, and DHA (respectively, C20:5, u-3; C22:5, u-3; C22:6, u-3) are n-3 series of fatty acids, which are vastly found in fish (Gordon & Ratliff, 1992). The most important fatty acids are ALA in flaxseed and walnuts and EPA and DHA in fish oil. DHA and EPA are not synthesized in sufficient levels by the body of human; they are called indispensable fatty acids and should be taken through diet (Calabrase, 1999; Stoll, 1999).

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FIGURE 10.6 Fatty acids in commodity oils and fats, occurrence (Scrimgeour, 2005).

Besides, the kind of fish consumed and the way it is prepared can also be important in its effectiveness against cardiovascular diseases (Mozaffarian et al., 2003). In many studies, it has been reported that the environment, diet, and gender of fish change their fatty acid content (Oz, 2016, 2017, 2019; Oz et al., 2018; Öz & Dikel, 2015). A Danish study also found that fish oils protect the heart by regulating the heart rate (Christensen, 2001). The main purpose of changes in the diet to be protected from and to prevent cardiovascular diseases is to maintain the blood lipid in the normal levels. In order to do this, nutrients having saturated fatty acids of low levels and PUFA of high levels are added to the diet, therefore the recommendation is to reduce animal-derived foods in which saturated fatty acids are rich such as red meat, pork, whole milk, and instead increase fish consumption (Rondeau et al., 2003) because it is now very well known that the consumption of fish decreases serum triglyceride levels and increases HDL cholesterol concentrations (Dewailly et al., 2003; Undeland et al., 2004).

10.6 Bioavailability The nutritional benefits are due to the relevant substance amount in food and its bioavailability. Bioavailability means the absorption of nutrients from the intestines, passage to the circulation, and transport to where it is needed. Likewise, the efficiency of pharmaceuticals varies by the bioavailability of the active substances in them (Schuchardt & Hahn, 2013). PUFAs of omega-3 type are commonly found as triacylglycerides and free fatty acids in fish and fish oil (Maki et al., 2009; Schuchardt et al., 2011).

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The omega-3 fatty acids bioavailability depends on their chemical forms. In a study, three common forms of EPA þ DHA, esterified triglycerides, ethyl esters, and phospholipids, were compared in terms of bioavailability. Participants were given three chemical forms in equal doses and their absorptions were investigated. The highest absorption was found to be in the group given phospholipid form and the lowest absorption was with ethyl ester form (Schuchardt et al., 2011). Omega-3 fatty acids which have the form of phospholipid are much more efficient than triglyceride form in the cellular function and structure (Chandrasekar et al., 1996). Compared to other forms, phospholipid form is absorbed better in the intestines, therefore bioavailability is greater (Jacobsen et al., 2013; Maki et al., 2009). It was reported in a previous study that the bioavailability of omega-3 fatty acids from krill oil could be greater than that of fish oil since krill oil has mostly the omega-3 phospholipids (Nash, et al., 2014; Sanlõer & Bolukbasõ, 2016). Omega-3 fatty acids concentration can be measured in serum, plasma, lymph, and blood cells. Although plasma fatty acids profile reflects the supply of fatty acids which are between short and medium-long in length in the diet, the amount of fatty acids found in blood cells is generally a good indicator for long-term bioavailability (Miura et al., 2018; Schuchardt & Hahn, 2013).

10.7 Stability, safety, and toxicology It was reported that the US Food and Drug Administration (FDA) certified the overall fish oil safety which contains both EPA and DHA and that it is considered safe to take up to 3 g/day n-3 LC-PUFAs (Lien, 2009). In the studies that evaluated the DHA toxicity, no stable toxicity indicators were reported in animals fed by high doses of DHA, and the genotoxicity was not observed. In the study, it was suggested that DHA levels in breastfeeding milk were about 315 mg/day for infants of younger than 6 months of age. When evaluating the clinical safety in adults, lipid metabolism, platelet function, glycemic control, oxidation potential, and immune function were considered. No important side effects were recorded in people who consumed DHA from 1 to 7.5 g/day and most studies used a 2e6 g of DHA per day (Lien, 2009). Since the daily n-3 fatty acids intake with diet and the daily need (0.2e1 g/day for children; 1e1.5 g/day for adults) are low, no serious side effects due to their consumption were considered. Taking ALA in flaxseed oil in very high doses (30 g/day) can cause diarrhea and gas, but there were no side effects regarding EPA and DHA consumption. Also, people at risk of bleeding are recommended to be careful as n-3 fatty acids reduce blood viscosity. Moreover, since these fatty acids have many double bonds, it is recommended to add antioxidants to diets when n-3 fatty acids are consumed at high levels (Ruxton et al., 2005). It is suggested that the blood pressureelowering potential of fish consumption may have a beneficial effect against cardiovascular diseases, but it should be noted that the amount of fish oil that is needed to normalize blood pressure is quite high. Their use at this level may have some negative effects on the blood rheology and can also attenuate or potentiate the effects of some drugs used routinely (Undeland et al., 2004). The most common problem for PUFA of omega-3 intake is its potential to increase bleeding risk through the antiplatelet effect. In some cases, gastrointestinal discomfort with dietary fish oil has also been demonstrated. Caution needs be taken when optimizing their benefits to balance between toxicity and activity. Even though fish oil products and PUFA have been utilized for in vivo and in vitro studies, clinical trials for topical application are limited today (Huang et al., 2018).

10.8 Applications (clinical and pharmacological)/health benefits PUFAs of n-3 series have protective and beneficial effects for human development and health. Earlier experimental studies have shown the evidence that PUFAs of n-3 series have ability to suppress most cancer developments such as breast, prostate, colon, liver, and pancreas (Ucak et al., 2019). Since omega-3 fatty acids are beneficial against diseases of wide range from cancer to heart diseases and from brain-related disorders to AIDS, it is suggested that the consumption of fatty aciderich oily fish or their fats at all stage of life, from the stage of fetus development to old ages (Mol, 2008). Omega-3 fatty acids, taken especially through fish consumption, are proven to reduce the risk of cardiovascular diseases and death from cardiovascular diseases, are effective in cancer prevention and treatment, decrease AD risk, relieve clinical symptoms in skin diseases, asthma, allergic diseases, rheumatoid arthritis, eczema, seborrhoeic dermatitis, psoriasis, and reduce the risk of diabetes (Tilami & Sampel, 2018; Calder, 2017).

10.8.1 Effect of MUFA and PUFA on cardiovascular diseases Regular fish or the fish oil consumption has been known to be as a supplement attenuates the coronary heart disease risk and prevents sudden cardiac death. The potential protective effects of fish and fish oil against cardiovascular diseases

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were first mentioned in the 1940s by British physiologist Hugh Sinclair’s description of Eskimo diets, with the hypothesis that insufficiency of some fatty acids can lead to cardiovascular diseases (Sinclair, 1982). Studies later showed that Eskimos living on the island of Greenland interestingly consumed very high levels of fat (40% of total calorie intake) with diet, but the mortality rate from cardiovascular diseases was very low (Lee & Lip, 2003; Çelebi et al., 2017). Bucher et al. (2002) recorded that n-3 unsaturated fatty acid intake from the dietary as well as nondietary sources helped to reduce general mortality rates because of sudden death and myocardial infarction in patients carrying coronary heart disease (Bucher et al., 2002). Marchioli et al. (2001) also demonstrated that adding n-3 fatty acid of 1 g/day to the diet of patients with myocardial infarction decreased the risk of mortality and relapse of myocardial infarction. It has been reported that fish or fish oils consumption that contain EPA and DHA reduces cardiovascular mortality and consuming 1 g of EPA and DHA every day were beneficial (Breslow, 2006). In men consuming more than 30 g of fish daily, it was found that the mortality rate due to cardiovascular diseases was 40%e50% less than in men who never consumed fish and that when fish or fish oil was added to diets of individuals with cardiovascular disease, total and cardiovascular mortality rates, especially sudden deaths, decreased significantly (Zhang et al., 1999). Daviglus et al. (1997) stated that the rate of developing cardiovascular diseases and myocardial infarction in individuals was 38% lower in individuals who consumed fish for 35 g/day compared to those of nonconsumer. Results of a study which lasted 16 years and was conducted with 84,664 women demonstrated that the rate of deaths due to cardiovascular disease in women who consumed fish one time in a week was 29%e34% less than those who consumed fish once a month (Hu et al., 2003). Many health experts recommend 2 or 3 servings of oily fish for every week or taking EPA þ DHA of 250e500 mg daily to contribute to health and decrease the risk of cardiovascular disease (Richter et al., 2016). Omega-3 fatty acids of fish oils are protective against coronary heart disease. In these days of many pharmacological options for cardiovascular disease, many people think that basic dietary changes or nutritional supplements may be a natural and more acceptable way for the benefit (Din et al., 2004). PUFAs of omega-3 are available in nature and are well tolerated and vastly utilized for the prevention of cardiovascular diseases (Cockbain et al., 2012).

10.8.2 Effects on eye health, brain, and nervous system Fatty acids of omega-3 are vital for the brain. Brain’s dry weight is formed by fats in the amount of 50%e60% and omega fatty acids account for 30%e35% of this. With an adequate DHA in the last trimester of pregnancy, 15%e20% of brain cells and 30%e60% of the retina develop. This development continues during the lactation period (Gow et al., 2018; Lauritzen et al., 2001). During the gestational period, omega-3 fatty acids intake at recommended amounts lead to an extension of the gestation period (2e8 days), prevention of preterm births, increase in birth weight (200 g) and head circumference, increase visual sharpness in the first 1 year after birth, acceleration in cognitive development. It also decreases the frequency and severity of autoimmune diseases, atopic dermatitis, eczema, and postpartum depression (Arita & Miyata, 2015; Kar et al., 2016). PUFAs of n-3 series are known to increase problem-solving ability in infants between and 10 months old (Lauritzen et al., 2001). Low DHA levels cause low levels of brain serotonin, which increases the tendency for depression, suicide, and violent behavior (Tanskanen et al., 2001). In a study, hyperactivity, defined as emotional imbalance, being unable to concentrate on a task, attention deficiency, excessive physical mobility, and learning disabilities, was reported to be common in 30%e40% of school-age children. Fish oils, vitamins, and minerals have been reported to be much more effective on hyperactivity. Research conducted among children aged 6e12 with predetermined behavioral disorders found that approximately 40% of 53 children with low levels of n-3 fatty acids had attention deficiency due to hyperactivity disorder (Arnold, 2001). DHA is a significant structural component for the eye and brain and supplementation of DHA during the development of these tissues is valuable in optimizing visual and neurological development. Therefore, diet of pregnant and lactating women must contain adequate amounts of DHA. Recent studies emphasize EPA and DHA have potential to promote improving mental development, learning, and behavior in childhood and reducing the negative effects of psychiatric diseases in adults (Calder, 2014). For optimal vision and neural development, omega-3 PUFA is required. Furthermore, many evidences show that high intake of PUFA may benefit in various neurological and psychiatric disorders, and especially in neurodegenerative conditions. It is shown by the evidence that omega-3 PUFAs provide significant neuroprotective potential in acute neurological damage. Therefore, these fatty acids offer potential therapeutic approaches in both chronic and acute cases (Dyall & Michael-Titus, 2008). In a recent study, infants born on time were supplemented with DHA starting immediately after birth until they were 12 months old. The visual sharpness of the infants given DHA at a ratio of 0.32%, 0.64%, or 0.96%

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in their food was compared with the control group not supplemented with DHA. In all groups supplemented with DHA, the visual sharpness at the 12th month was significantly better when we compare it to the control group. However, this difference was not dose dependent (Birch et al., 2010). Unsaturated fatty acids with long-chain length, especially DHA, are known to be structurally and functionally effective on vision, motor nervous system development, cognitive-emotional and behavioral development. Recent research showed that DHA is important in retinal function in infants and vision performance (Lauritzen et al., 2001). Studies have demonstrated that DHA deficiency decreases the retinal response to light, decreases vision sharpness, and delays the time of adaptation to darkness. Dietary DHA is known to accelerate the development of visual sharpness in premature babies (SanGiovanni et al., 2000).

10.8.3 MUFAePUFA and neurodegenerative diseases Neurodegenerative diseases can be defined as diseases caused by progressive and irreversible damage to the cellular pathways of the brain. Motor disorders, speech impairments, and/or cognitive impairments are major clinic observations. The most common of these diseases are AD, Parkinson’s disease (PD), motor neuron disease, and Huntington’s disease. AD is the most famous disease of cognitive impairment. The first and main symptom is memory impairment and forgetfulness and is a result of cortex damage, which is the residential area of the hippocampus and neurons. Parkinson’s and Huntington are basal ganglia disorders. As for motor neuron (ALS) disease, it is characterized by muscle weakness caused by spinal, bulbar, and cortical neuron loss (Adapinar, 2019). Alzheimer’s is a dementia disease because of structural degeneration of the brain’s transmission system. Amyloid accumulation between brain cells is known to be the main cause of the disease. Studies have shown that fish oil PUFA prevents Alzheimer’s-causing amyloid accumulation in animal models by increasing the release of an amyloid-carrier protein (Morris et al., 2003). It was reported in a previous study that omega-3 fatty acids improved inflammatory status by reducing the production of inflammatory lipid end products with their competitive arachidonic acid (AA) inhibitor activity. Therefore, omega-3 fatty acids can promote the brain function as well as attenuate pathological conditions of Alzheimer’s (Desale & Chinnathambi, 2020). Studies which are limited in number have investigated the positive or negative effects of essential fatty acids in diet and levels of cholesterol in Parkinson patients and the relationship has not yet been clarified. However, it has been reported that neurotransmitter systems are affected due to the peroxidation of PUFA and cholesterol existing in high ratios within the brain and that insufficient consumption of a-linolenic acid affects the brain fatty acid profile, and that maintaining brain DHA levels, which has antiinflammatory effects, has also gained importance in Parkinson patients (Mermer & Yõldõran, 2020). Another study reported that n-3 PUFAs can cure PD by suppressing the release of proinflammatory cytokines, increasing neurotrophic factor expression and restoring membrane fluidity and mitochondrial function as well as reducing oxidized production levels (Li & Song, 2020). All the nutritional studies regarding the protection and prevention of neurodegenerative diseases could indicate that humans should consume fish in their diet. The balance for omega-3 and other fatty acids should be protected every week. Marine sources with rich omega-3 fatty acid sources, such as sardines, mackerel, salmon, and tuna, must also be consumed (Adapinar, 2019). In daily nutrition, LC-PUFA supplements have been reported to prevent major depression and AD because they have significant positive effects on cognitive functions (Collinius, 2016).

10.8.4 Antidepression effects of EPA and DHA Depression is a problem that develops due to stress, hormonal changes, biochemical abnormalities, and other causes and is characterized by a feeling of unhappiness and despair. Epidemiological studies report a higher risk of depression in people with low blood EPA and DHA levels (Edwards, 1998; Tiemeier & Tuijl, 2003). The decrease in DHA levels in brain cells has led to the emergence of diseases such as depression, Alzheimer’s, memory lapse, schizophrenia, and visual impairments. Moreover, it is also stated that concentration problems, hyperactivity, and low IQ levels are associated with low amounts of DHA (Canbulat & Özcan, 2008). Studies have demonstrated that any changes in dietary ratio of n-6/n-3 can accordingly change composition of the brain in terms of the fatty acid, and thereby the functions of the brain cell membrane. This change is among the important causative factors of the development of depression. Health experts emphasize that n-3 PUFAs are protective against depression and point to the lower frequency of depression in countries with high fish consumption (Hibbeln, 1998; Small, 2002). Omega-3 fatty acids of DHA and EPA help regenerating cells in the brain and they allow the brain and retinal cells to multiply. It has been shown in the studies that there is a negative relationship between the consumption of n-3 fatty acids

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and problems such as schizophrenia, Alzheimer’s, depression, memory lapse, and visual impairments (Canbulat & Özcan, 2008). Fatty acids of n-3 series also showed positive results in the treatment of individuals with antisocial behaviors, learning disabilities, and mental imbalances that lead to schizophrenia and depression (Marangell et al., 2003). Although the clinical trials recommend adding fish oil (DHA and EPA) in the treatment of depression, the function of this positive effect is unknown (Peet, 2003; Small, 2002). Schizophrenia is a mental disease caused by structural disorders in red blood cells and blood plasma. Unsaturated fatty acids of low levels in the blood, such as AA, DHA, and EPA, could increase schizophrenic symptoms. In the related studies, it was observed that these symptoms disappeared when these fatty acids, especially, EPA, were taken at normal doses. For 6 months, hallucinating people were administered EPA 2 g/day and their schizophrenic symptoms were found to be decreased by 85% (Conquer, 2000).

10.8.5 The health effect of omega-3 fatty acids on the skin and inflammatory diseases The epidermis layer of the skin plays a significant role in protection of the skin from external factors such as harmful bacteria and ultraviolet (UV) radiation. The dermis layer is formed by collagen and elastin tissue, while subcutaneous adipose tissue (hypodermis) is formed by the elastin tissue of the fat cells. All of these layers protect the integrity and health of the skin (Sies & Stahl, 2004). It has been reported in the studies that the harmful effects of UV light on skin aging can be reduced by the fatty acids in a certain amount in the diet. DHA of fish oil positively affects the thickness of the dermis and subcutaneous tissue (Rhodes, 2004; Sies & Stahl, 2004). Fish oil fatty acids are reported to promote the function of skin barrier, suppress inflammation and hyperpigmentation caused by UV, relieve dry skin and itching caused by dermatitis, facilitate the healing of the wound on the skin, and prevent the development of skin cancer (Huang et al., 2018). All benefits of these fatty acids can be obtained through different means of applications. These means include oral nutritional supplement, topical application, and intravenous injection. According to the clinical findings, epidemiological, biochemical, and animal studies, these fatty acids have positive effects such as reducing inflammation in the rheumatoid arthritis. It has been found that EPA and DHA suppress the production of inflammatory agents from AA, reduce bone pain in these patients, and have disease retardant effects (Cleland et al., 2003). The working mechanism of fish oil for the psoriasis treatment is mostly because of a change in the composition blood cell membrane and epidermal lipids. In psoriatic skin lesions, AA was reported to exist at high levels and psoriasis inflammation is mainly caused by metabolite leukotriene B4. In case lipoxygenase or cyclooxygenase enzymes metabolize EPA instead of AA in cell membranes, the end products of EPA metabolization will help alleviate inflammation (Mayser et al., 1998; Ricketts et al., 2010). Intravenous and oral applications of PUFAs of n-3 series derived from the sea have been stated to have positive effects and can be used as an auxiliary treatment in the treatment of psoriasis (Mari et al., 2017). On the preventing, reducing, and treating rheumatoid arthritis symptoms such as swelling, pain, difficulty in moving, fever fatigue, and weakness in the joints, omega-3 fatty acids have very important effects. Although the cause of the disease is not yet fully known, links between genetic factors and autoimmune processes are considered. Omega-3 fatty acids reduce inflammation and alleviate the symptoms of arthritis and autoimmune diseases (Eseceli et al., 2006). Eicosanoids sourced from EPA and DHA have also very considerable functions in cell signaling and inflammatory processes (Mozaffarian & Wu, 2011).

10.8.6 The health effect of omega-3 fatty acids on diabetes A recently conducted meta-analysis that examined the effect of LC n-3 PUFAs on type 2 diabetes patients showed that these fatty acids, especially EPA and DHA, lower serum triglyceride levels (Chen and Shao, 2015). Epidemiological studies have determined that the occurrence of diabetes mellitus is lower in societies where consumption of fish is high. In clinical trials, it was shown that the reason of it is that by n-3 PUFAs in fish are able to reduce insulin resistance by reducing blood pressure and plasma triglyceride levels (Sidhu, 2003). Besides, the ability of n-3 PUFAs to reduce platelet aggregation and their antiarrhythmic effects could decrease the incidence of cardiovascular disease in diabetic individuals. Conclusively, it can be stated that the cardiovascular illness occurrence is decreased in diabetic persons who consume higher amounts of fish and fish oil (Hu et al., 2003).

10.8.7 The health effect of omega-3 fatty acids on diabetes Today, cancer is one of the most important and rapidly increasing health problems of developed and developing countries. Cancer causes more than 9.6 million deaths each year, accounting for 18.4% of deaths across the world. Every year, 18.1

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million people are diagnosed with cancer. According to the estimates of the world health institutions, an increase of 22 million in cancer cases is predicted by 2030. When we evaluate the relationship between cancer and nutrition according to the analysis of the studies conducted in the last 30 years; the role of foods and nutrition in the prevention of cancer is important. It has been shown that more than 60%e70% of cancer cases can be prevented by nutrition and lifestyle (WCRF/AICR, 2018). Epidemiological, ecological, and experimental studies in recent years have demonstrated that DHA and EPA may suppress the development of most types of cancer involving colon, breast, liver, prostate, and pancreatic cancer (Gomez-Candela et al., 2011). Fish oils also have beneficial effects on cancer diseases. In a study with mice with tumors, it was discovered that fats containing n-3 or purified fatty acids of n-3 series can slow the progress of various cancer types such as lung, colon, prostate, and breast. Furthermore, the effectiveness of chemotherapy drugs was amplified by the addition of n-3 fatty acids to the diet. n-3 fatty acids also reduce cachexia related to cancer and promote life quality, and chemotherapy in breast cancer was shown to be succeed more in those who consumed n-3 fatty acids compared to nonor less consumers. Fish oils are also known to slow tumor development (Hardman, 2004). A growing body of experimental, epidemiological, and clinical evidence shows that omega n-3 PUFAs exert antiecolorectal cancer (CRC) activity. It is known that n-3 PUFAs involve in various phases of CRC management, such as preventing CRC and treatment of its metastatic progression (Cockbain et al., 2012). Numerous epidemiological research studies compared the effect of excessive or less consumption of fish on various types of cancer. Nonetheless, it has not been found that fish consumption leads increase in risk of any type of cancer. However, for some types of cancer, the risk-reducing effects of having cancer have been reported. Excessive fish consumption is reported to promote protection against gastrointestinal, thyroid, and multiple myeloma cancers (WCRF/AICR, 2018).

10.8.8 The health effect of omega-3 fatty acids on asthma A study demonstrated that consuming fish weekly decreased asthma risk among children. A prospective study conducted by the same team also showed a significantly reduced risk of developing asthma in 574 children aged between 8 and 11 who consumed fresh and oily fish (Hodge et al., 1996). The low incidence of asthma in the Eskimo population with high fish consumption rate could indicate that the fatty acids of omega-3 have beneficial effects in asthma (Reisman, 2011). In countries having high fish consumption like Japan, the prevalence of asthma is also lower than in countries where consumption is low. This condition is considered to be associated with omega-3 fatty acids that reduce inflammatory cytokine levels (Pu et al., 2015). A study with 1673 asthmatic children stated that as the frequency of fish consumption increased, the prevalence of asthma decreased and omega-3 intake had protective effects on asthma (Takemura et al., 2002).

10.9 Conclusion PUFAs and MUFAs have beneficial and protective impacts on human health and development. Numerous experimental evidences show that fatty acids mentioned above exert a strong role in suppressing most cancer development and reducing risk of cardiovascular diseases. The main source of PUFAs of n-3 series, with DHA and EPA being in the lead, usually exists in fish, while MUFAs’ was mostly found in olive oil and canola oil. In meeting the increasing demand from the consumers who have high awareness on health, the purification and extraction of PUFAs and MUFAs are very crucial and efficient extraction techniques with the proper combinations need to be developed with the high yield.

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Meta-analysis of dietary essential fatty acids and long chain polyunsaturated fatty acids as they relate visual resolution acuity in healthy preterm infants. Pediatrics, 105, 1292e1298. _ ¸ kisi. In M. Aslan (Ed.), Omega Yag Asitleri ve Saglõk Üzerine Etkileri. Ankara: Eczacõ Sanlõer, N., & Bolukbasõ, H. (2016). Krill Yagõ ve Saglõk Ilis Odasõ. Schubert, K. (1973). MI Gurr and AT James, Lipid Biochemistry: An Introduction. 231 S., zahlreiche Abb. und Tab. London 1971: Chapman and Hall LTD,£ 2.50. Zeitschrift für allgemeine Mikrobiologie, 13(5), 454. Schuchardt, J. P., & Hahn, A. (2013). Bioavailability of long-chain omega-3 fatty acids. Prostaglandins, Leukotrienes and Essential Fatty Acids, 89(1), 1e8. Schuchardt, J. P., Schneider, I., Meyer, H., Neubronner, J., Schacky, C. V., & Hahn, A. (2011). Incorporation of EPA and DHA into plasma phospholipids in response to different omega-3 fatty acid formulations-a comparative bioavailability study of fish oil vs. krill oil. Lipids in Health and Disease, 10, 145e151. Schwalfenberg, G. (2006). Omega-3 fatty acids: their beneficial role in cardiovascular health. Canadian Family Physician, 52(6), 734e740. Scrimgeour, C. (2005). Chemistry of fatty acids. In F. Shahidi (Ed.), Bailey’s industrial oil and fat products (pp. 1e43). New York, USA: John Wiley & Sons, Inc. Serpek, B., & ve Kalaycõoglu, L. (2000). Biyokimya. Ankara: Nobel Yayõn Dagõtõm. Sharma, R., Christi, Y., & Banerjee, C. (2001). Production, purification, characterization, and application of lipases. Biotechnology Advances, 19, 627e662. Sidhu, K. (2003). Health benefits and potential risks related to consumption of fish or fish oil. Regulatory Toxicology and Pharmacology, 38, 336e344. Sies, H., & Stahl, W. (2004). Nutritional protection against skin damage from sunlight. Annual Review of Nutrition, 24, 173e200. Sinclair, H. M. (1982). The relative importance of essential fatty acids of the linoleic and linolenic families: studies with an eskimo diet. Progress of Lipid Research, 20, 897e899. Small, M. F. (2002). The happy fat. New Scientist, 24, 34e37. Stoll, A. L. (1999). Omega-3 fatty acids in bipolar disorder. Archives of General Psychicatry, 56, 407e412. Tanskanen, A., Hibbeln, J. R., Hintikka, J., Haatainen, K., Honkalampi, K., & Viinanaki, H. (2001). Fish consumption, depression, and suicidality in a general population. Archives of General Psychiatry, 58, 512e513. Takemura, Y., Sakurai, Y., Honjo, S., Tokimatsu, A., Gibo, M., Hara, T., … Kugai, N. (2002). The relationship between fish intake and the prevalence of asthma: the Tokorozawa childhood asthma and pollinosis study. Preventive Medicine, 34(2), 221e225. https://doi.org/10.1006/pmed.2001.0978 Tiemeier, H., & Tuijl, H. R. (2003). Plasma fatty acid composition and depression are associated in the elderly: the Rotterdam study. American Journal Clinical Nutrition, 78, 40e46. Tilami, S. K., & Sampel, S. (2018). Nutritional value of fish: lipids, proteins, vitamins, and minerals. Journal of Reviews in Fisheries Science and Aquaculture, 26(2), 243e253. Timms, R. E. (2005). Fractional crystallisation-the fat modification process for the 21st century. European Journal of Lipid Science and Technology, 107, 48e57.

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Ucak, I., Oz, M., & Maqsood, S. (2019). Products based on omega-3 polyunsaturated fatty acids and health effects. In C. M. Galanakis (Ed.), The role of alternative and innovative food ingredients and products in consumer wellness (pp. 197e212). Academic Press. Undeland, I., Ellegard, L., & Sandberg, A. S. (2004). Fish and cardiovascular health. Scandinavian Journal of Nutrition, 48, 119e130. Wang, L., Bordi, P. L., Fleming, J. A., Hill, A. M., & Kris-Etherton, P. M. (2015). Effect of a moderate fat diet with and without avocados on lipoprotein particle number, size and subclasses in overweight and obese adults: a randomized, controlled trial. Journal of the American Heart Association, 4(1). e001355. Waruda, D., Joshi, K., & Harsulkar, A. (2006). Polyunsaturated fatty acids: biotechnology. Critical Reviews in Biotechnology, 26, 83e93. Wiktorowska-Owczarek, A., Berezinska, M., & Nowak, J. Z. (2015). PUFAs: structures, metabolism and functions. Advances in Clinical and Experimental Medicine, 24, 931e941. World Cancer Research Fund International. (2018). Diet, nutrition, physical activity and cancer: a global perspective: a summary of the third expert report. World Cancer Research Fund International. Zhang, J., Sasaki, S., Amano, K., & Kesteloot, H. (1999). Fish consumption and mortality from all causes, ischemic heart disease, and stroke: an ecological study. American Journal of Preventive Medicine, 28, 520e529.

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Chapter 11

Resveratrol Zahid Rafiq Bhat1, Abida Bhat2, Bharti Mittu5, Kappat Valiyapeediyekkal Sunooj3 and Rasiya Ul Zaman4 1

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur,

Punjab, India; 2Department of Immunology and Molecular Medicine, Sher-e-Kashmir Institute of Medical Sciences, Srinagar, Jammu Kashmir, India; 3Department of Food Science and Technology, Pondicherry University, Puducherry, India; 4Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Jammu Kashmir, India; 5National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur, Punjab, India

11.1 Introduction Resveratrol is the type of phytoalexin compounds produced by plants as an act of mechanical defense by them against fungal and other parasitic attack, presented it a biologically active substance. They also prove to be protective for plants in response to environmental stress (UV radiations, chemical compounds) and mechanical damage. The biological function of resveratrol includes antiinflammatory and antioxidative properties by which they show an increase in superoxide dismutase (SOD) and suppress the activity of cyclooxygenase (COX), hypoxia-inducible factor 1a, and vascular endothelial growth factor (VEGF) inside cells (Diaz-Gerevini et al., 2016). Experimental models in vitro/in vivo certified its role in neurodegenerative diseases by activating AMP-activated protein kinase (AMPK) and HO-1 (Heme Oxygenase1) that reduce ROS production via NF-кB signaling pathway modulation (Bastianetto et al., 2015). Defects in the mitochondrial functioning are linked to various metabolic, cardiovascular, and neurodegenerative diseases. In terms of its health benefits it is shown to be a potent activator of SIRT1/PGC-1a which acts as a mediator of activating the mitochondrial respiration and lipid oxidation (Kulkarni & Cantó, 2015; Lagouge et al., 2006). Autophagy (type II programmed cell death), as one of the biological housekeeping pathway, maintains cell survival during stress or starvation. Resveratrol administration in mouse models augmented autophagic flux by modulation of its key targets inside the cell, i.e., LC3, p62, SIRT1, and S6K1 (Armour et al., 2009; Fernández-Rodríguez et al., 2020). The physiological result of resveratrol in cardiovascular diseases has shown decrease in the oxidative stress via endothelial nitric oxide synthase (eNOS) and the levels of inflammatory cytokines (IL-6, IL-1) (Gal et al., 2020). As a radioprotective mediator it protects the cells by downregulating ROS and NOX4 signaling mechanism toxicity (Agbele et al., 2020). Thus resveratrol acts as the multitarget drug with its target ligands in different signaling pathways that make it potent biomolecule for the study.

11.2 Sources Nine families of plant kingdom which contain resveratrol oligomers are Dipterocarpaceae, Vitaceae, Cyperaceae, Leguminosae, Paeoniaceae, Gnetaceae, Umbellifers, Haemodoraceae, and Musaceae. First resveratrol tetramer (Hopeaphenol) extracted from Dipterocarpaceae was isolated by Coggon et al. Natural resveratrol (3,5,4-trihydroxystilbene) were isolated first from the roots of white hellebore (Veratrum grandiflorum). It was identified and named by Dr. Michio Takaoka (Japanese chemist) (Fremont, 2000; Nakata et al., 2012). From another medicinal plant Polygonum cuspidatum (Kojo-kon in Japanese) belonging to a family of Viniferins was also used for its isolation. Sources of resveratrol include grape skin, blueberry, raspberry, mulberry, strawberry, and cranberry (Nawaz et al., 2017; Pirola & Fröjdö, 2008). Concentration wise berries contain 10% less amount than grapes and heating/cooking them degrade resveratrol in them. Commercially it can be found in white/red wine, Japanese knotweed, peanuts, pistachios, peanut butter, and chocolate (Kiselev, 2011). Red wine consumption is considered protective to health as it contains greater content of polyphenols (e.g., resveratrol, procyanidin B) with antioxidant properties released from grape’s skin (Guerrero et al., 2009).

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11.3 Extraction and characterization techniques As a natural source of resveratrol (trans-3,5,40 -trihydroxystilbene) grape berries contain greater concentration during its green stage and decline steadily with ripening (Liang et al., 2008). As for the natural production in plants it takes place via phenylpropanoid pathway mechanism (Fig. 11.1). Production of resveratrol by fermentation process in Escherichia coli or yeast by the insertion of trans-gene product of 4-CL2 and STS gene into the media containing p-coumaric-acid is seen more productive in yeast than E. coli (Pervaiz & Holme, 2009). Chemical biosynthesis of resveratrol and its derivates is carried out either by the Wittig or Heck methods (Antus et al., 2015; Liu et al., 2015; Niesen et al., 2013; Pezzuto et al., 2013). Wittig reaction as a classical biosynthesis of the stilbene products is the reliable method for the olefination of positional selectivity of resveratrol derivates (Fig. 11.2). Wittig reaction method yields E/Z isomers of resveratrol in a reaction mixture. Heck reaction method is more efficient in a way that it yields only E-isomer of resveratrol in the reaction mixture (Fig. 11.3).

FIGURE 11.1 Concentration of resveratrol at different stages.

FIGURE 11.2 Wittig reaction as a classical biosynthesis of the stilbene products.

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FIGURE 11.3 Heck reaction method to yield only E-isomer of resveratrol.

11.4 Chemistry Resveratrol chemically named as 3,5,40 -trihydroxystilbene and according to IUPAC nomenclature it is named as 5-(E)-2(4-hydroxyphenyl) ethenyl, benzene-1,3-diol. Methods like NMR, chromatography, mass spectrometry (MS), circular dichroism (CD), 2D NMR spectra (COSY, HSQC, HMBC) provided detailed characterization features of resveratrol and derivatives. Structurally Fig 11.4 it consists of two isomeric forms, i.e., cis and trans, both containing the stilbene ring structure. In solution these two isomeric forms of resveratrol are interconverted to each other in presence of light. The stability of trans-isomer depending on pH of solution ranges from hours to days is considered biologically active form of resveratrol (Matsuoka et al., 2002). Further the pKa values of trans-mono-, di-, and triprotonated forms are 9.3, 10, and 10.6. For the detection procedure both trans and cis isomers are detected using technique UV-HPLC at 308 and 288 nm, respectively (Cheng et al., 2006). Structurally resveratrol has >100 analogs present with different biological active roles (Szekeres et al., 2011; Zimmermann Franco et al., 2012). Table 11.1 lists the group of important hydroxyl, methoxy, and halogenated derivates of resveratrol (Beekwilder et al., 2006; Jeandet et al., 1991; Guiso et al., 2002). The addition of extra hydroxyl group or the presence other functional groups, e.g., halogens increased their biological activity, e.g., antioxidant, antiproliferative, antiangiogenic, etc., properties than the parent resveratrol compound.

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FIGURE 11.4 Structural features of resveratrol.

TABLE 11.1 List of a group of important hydroxyl, methoxy, and halogenated derivates of resveratrol. Derivative 3,40 ,5-Trans-trimethoxystilbene (natural analogue of resveratrol)

Structure

Function Proapoptotic, antioxidant, and potent vascular targeting analog than resveratrol

OH

OH OH

3,40 ,5-Trihydroxy-diphenylacetylene

Weak antioxidant property but stronger antitumor and antiinflammatory activity

OH OH OH

0

3,5,3 -Trihydroxy-trans-stilbene

OH

3,5-Dihydroxy-trans-stilbene

Antioxidative properties

OH

OH

OH

OH

Antioxidant property

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TABLE 11.1 List of a group of important hydroxyl, methoxy, and halogenated derivates of resveratrol.dcont’d Derivative 3,40 -Dihydroxy-trans-stilbene

Structure

Function Binds to estrogen alpha and strong antioxidant property

OH

OH

3,30 -Dihydroxy-trans-stilbene

Antiinflammatory and antioxidative properties

OH

OH

3-Hydroxy-trans-stilbene

Antioxidative properties

OH

4-Hydroxy-trans-stilbene OH

Binding to estrogen receptor alpha (ERa) presents its distinct activity in estrogensensitive cancer cells

Trans-3,4,5,40 -tetramethoxystilbene

Shows strong bioactivity compared to resveratrol for suppressing growth of cancer cells by inhibiting signaling components of VEGFR2, Akt, FAK, c-Src, mTOR

Pentamethoxystilbene (PMS), synthetically methoxylated analog of resveratrol

Antiproliferative and strong inducer of apoptosis, major role playing in breast cancer

Continued

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TABLE 11.1 List of a group of important hydroxyl, methoxy, and halogenated derivates of resveratrol.dcont’d Derivative

Structure

Trans-4,40 -dihydroxystilbene (DHS)

Function Increased inhibitory potential in tumor metastasis against lung cancer

OH

OH

Trans-3,30 ,4,50 -tetrahydroxystilbene (THS)

Therapeutic target in cardiovascular diseases such as atherosclerosis and myocardial ischemia by acting as a potent antiinflammatory, antiangiogenic, and antioxidant

OH

OH

OH 0

0

0

3,3 ,4,4 ,5,5 -Hexahydroxy-trans-stilbene (HDS), synthetic resveratrol derivative

OH

Antioxidant, antiproliferative, and induce apoptosis in various malignancies such as colon, breast, and leukemia cancers

OH

OH

OH

OH

OH OH

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TABLE 11.1 List of a group of important hydroxyl, methoxy, and halogenated derivates of resveratrol.dcont’d Derivative

Structure

Function

3,4,5-Trimethoxy-4-bromo-cis-stilbene (BCS)

More potent antiproliferative compared to resveratrol in lung cancer

Br

4-Bromo-resveratrol

OH

Potent inhibitor of sirtuins than resveratrol

OH

Br

Pterostilbene (3,5-dimethoxy-40 hydroxyltrans-stilbene)

OCH3

OH

Antioxidant, antiproliferative, antiinflammatory, and antiapoptotic properties

OCH3

An experimental study showed the presence of ortho-dihydroxyl or 4-hydroxy-3-methoxyl group analogues (3,4,30 ,40 THS, 3,4-DHS, 3,4,40 -THS, and 3,30 -DM-4,40 -DHS) bears high antioxidant potential toward LDL peroxidation than parent resveratrol (Chalal et al., 2012). In vitro study models testing the antitumor result of resveratrol and its derivatives proved that hydroxylated resveratrol derivates are more potent antitumor compounds (Kang et al., 2009). Resveratrol and its derivates being antioxidative in nature have the potential to inhibit tyrosinase enzyme involved in the synthesis of melanin pigment in the skin. An enzymatic reaction procedure showed that the presence of para-hydroxylation to parent resveratrol compound (3D structure in Fig. 11.5) enhanced its tyrosinase inhibitory activity (McNulty & Das, 2009).

11.5 Mechanism of action In vitro/vivo experimental studies on resveratrol and its analogues have demonstrated its wide physiological and biochemical properties on the human health. Resveratrol targets multiple cell signaling ligands that control the function and expression of multiple genes that regulate antiapoptotic genes, protein kinases, antioxidant enzymes, metastatic genes, and inflammatory biomarkers (Das & Das, 2010). Based on its multidisciplinary roles it can be considered as the molecule with multiple targets. The protective role of resveratrol against cardiovascular diseases came from the epidemiological data of “French paradox,” i.e., French population in spite of high-fat diet exhibit low cardiovascular diseases because of their high wine consumption. In vitro study on pulmonary arterial hypertension (PAH) has shown the administration of resveratrol

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FIGURE 11.5 3D structure of resveratrol.

decreased inflammatory process by inhibition of cytokines/chemokines (TGF-b, PDGF-a/b, IL-10, IL-6, IL-18, IL-8) inhibiting the activation of HIF1a, HIF-2a via various signaling pathways (NF-кB, PI3K/AKT, mTOR, RhoA-ROCK, Sirtuin 1/PGC-1a, NOX/VPO1, JAK/STAT) (Mirhadi et al., 2020; Peng & Jiang, 2018; Zhu et al., 2020). Resveratrol and its derivatives also acts as the activators of multiple protein kinases (AMP kinases, MAPK, cyclin-dependent kinase) and inhibitor of cyclic oxygenases (COX-1/2) by which they exert the effects of being antiinflammatory, antiproliferative, and antioxidative in various diseases as described in Fig. 11.6. (Dasgupta & Milbrandt, 2007; Malaguarnera, 2019; Utreras et al., 2011).

FIGURE 11.6 Mechanism of action of the resveratrol and its derivatives with its multiple targets inside the cell modulating the different cellular signaling pathways (Berretta et al., 2020; Chang et al., 2017; Ochiai & Kuroda, 2020).

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11.6 Bioavailability of resveratrol In addition to the absorption, distribution, metabolism, excretion, and toxicology of a drug or an active pharmaceutical or nutrient, bioavailability of an active form is increasingly gaining importance not only in terms of therapeutic efficacy but also in the area of drug development. Bioavailability is simply defined as “the rate and extent to which an active ingredient is absorbed from a dosage form and becomes available in the systemic circulation in an unchanged form.” In pharmacokinetics, the method of measurement of the bioavailability of a drug is based on calculating AUC (area under curve) by plotting blood concentrations sampled at different intervals (Allam et al., 2011). The beneficial effects of resveratrol in multiple diseases are well established including cardiovascular diseases, cancer, and metabolic diseases. However, such effects have been established from cell-based in vitro studies primarily and unable to show the similar level of efficacy from in vivo studies. Reports have suggested that the reason for such change in efficacy is fast drug metabolism and decreased bioavailability. Evidences have proven that with an oral intake of resveratrol (25 mg), a concentration peak of around beta w gamma > alpha) and separation of these esters from free tocopherols in a column. Deacylation of delta homologue acetate undergoes rapidly within 15 min, while acetates of beta and gamma homologues take about 2 h; alpha homologue acetates are unreactive. Since cyclic amines are expensive, they can be replaced with methanol, ethanol, or propanol but the reaction requires 190e210
C temperatures inside a pressurized vessel. To overcome this limitation, a basic catalyst such as potassium carbonate, potassium hydroxide, or sodium hydroxide that enables the deacylation reaction to happen at beta > gamma > alpha (in vitro) and alpha > beta > gamma > delta (in vivo). This highest activity of the alpha homologue within living tissues is due to the action of hepatic alpha-tocopherol transfer protein that occurs in higher levels in plasma and tissues, allowing alpha-tocopherol to be preferentially retained and incorporated into lipoproteins. Additionally, tocopherols also react with singlet oxygen or other reactive species (ROS) as part of their antioxidant functions  (Spika et al., 2016).

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14.5.2 Degradation of alpha tocopherol Tocopherols are oxidized by ROS, mainly by lipid peroxyl radicals; the mechanism can proceed differently (Fig. 14.6). Oxidation of tocopherols to tocopheryl radicals may occur by one-electron transfer, which can be rereduced to tocopherols by an ascorbateeglutathione system (Asc/GSH). In absence of ascorbate or glutathione, tocopheryl radicals can form adducts (quinones) or self-coupling products (dimers and/or trimers). In another oxidation pathway involving a twoelectron transfer and singlet oxygen, tocopherols get converted to hydroperoxide, which irreversibly gets hydrolyzed to tocopherol quinone (TQ). This conversion happens in chloroplast lumen under mildly acidic conditions. TQ can be enzymatically transformed to tocopherol quinol (TQH2), catalyzed by NADPH-dependent reactions. Both TQ and TQH2  formed from alpha-tocopherol elucidate antioxidant properties (Spika et al., 2016).

14.6 Bioavailability Bioavailability of food is the fraction of food ingredients placed at the disposal of tissues after ingestion. The bioavailability of tocopherols, commonly known as Vitamin E, in humans, is assessed using the level of plasma tocopherol. This availability is essential for biological activity, as this fraction will only contribute toward our physiological activities. Out of the four vitamers (homologues) of tocopherol (vitamin E), a-tocopherol is the dominant fraction present in the human body and has the highest biological activity. b- and g-tocopherol have been shown to have reduced vitamin activity (10%e30%), whereas d-tocopherol has no activity (Reboul, 2017). In another definition, bioavailability refers to the ingested component that becomes accessible to absorption in the gastrointestinal (GI) tract, followed by its metabolism and further distribution in the body. Bioavailability constitutes three steps: bioaccessibility, absorption, and transformation of the ingested component (vitamin E in this case). To make referencing easier, we shall use the term “vitamin E” synonymously with “tocopherol” from this point onward.

FIGURE 14.6 Possible pathway for degradation of alpha tocopherol. a-TQ, a-tocopherol quinone; a-TQH2, a-tocopherol quinol; Asc/GSH, ascorbateeglutathione cycle; DH, unknown dehydratase; TC, tocopherol cyclase; TMPBQ, 2,3,5-trimethyl-6-phytyl-1,4-benzoquinone; TMPBQH2, 2,3,5trimethyl-6-phytyl-1,4-benzoquinol. From Lushchak, V. I., & Semchuk, N. M. (2012). Tocopherol biosynthesis: chemistry, regulation and effects of environmental factors. Acta Physiologiae Plantarum, 34(5), 1607e1628. https://doi.org/10.1007/s11738-012-0988-9.

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Firstly, let us go through how vitamin E is metabolized in the body. Vitamin E is fat-soluble, and is shown to be associated with major lipids and absorbed mainly in the upper GI tract. However, its absorption is not very efficient. Metabolism of vitamin E in the upper GI tract includes emulsification; incorporation into the micelles; transportation through the unstirred water layer (glycocalyx); assimilation by an apical membrane of enterocytes (intestinal absorptive cells); solubilization into the intestinal lipoproteins; and secretion out of the intestinal cells into the lymph or into the portal vein. The initial phase is the dissolution of vitamin E in the lipid phase of the meal it is present in, which occurs during mastication of food. This is followed by the action of gastric enzymes (pepsin, amylase, and gastric lipase), which assist in the release of vitamin E from the food matrix. It is well established that a-tocopherol does not undergo any significant degradation or absorption in the stomach (Reboul, 2017). In the duodenum (first part of the small intestine), the digestive enzymes (proteases, amylases, and lipases) continue degrading the food matrix, thereby contributing toward further release. Here, the absorption mechanism of vitamin E is quite similar to that of dietary fats. Vitamin E requires biliary and pancreatic secretions in order to form micelles for subsequent uptake by the intestinal epithelial cells (Traber, 2007). The main site for vitamin E absorption is supposedly the midsection of the GI tract. Intestinal absorption of vitamin E is quite complex (Gagné et al., 2009); it is partly mediated by class-B type-1 (SR-B1) scavenger receptors, which are also involved in cholesterol uptake (Reboul, Richelle, et al., 2006). Other mechanisms include intracellular trafficking proteins; modulation of nuclear receptors; and activity of ATP-binding cassette transporters (Traber, 2004). The efficiency of vitamin E absorption is not similar along the intestine; the major sites are where the concentration of vitamin E in micelles and possibly vesicles is the highest. Also, the repartition of vitamin E transporters and distribution of SR-B1 scavengers are not uniform (Borel et al., 2001; Reboul et al., 2007). The efficiency of vitamin E transportation across the intestinal wall is quite variable ranging from 10% to 95% (Borel et al., 2013; Emmanuelle Reboul, 2017). However, in one study where deuterium-labeled vitamin E was studied for absorption, the range dropped to 10%e33% (Bruno et al., 2006). There are numerous factors that modulate vitamin E bioavailability. A mnemonic term ‘SLAMENGHI” is currently in considerable use to list all factors contributing to vitamin E bioavailability (Desmarchelier et al., 2018, pp. 1181e1196). This term was initially proposed to access carotenoids bioavailability and other fat-soluble micronutrients (West & Castenmiller, 1998). Each term corresponds to one factor: S for “Species of vitamin E” (referring to relative bioavailability of the vitamers); L for “Molecular linkages” (e.g., esterification of vitamin E); A for “Amount of vitamin E consumed in a meal”; M for “Matrix in which vitamin E is incorporated” (e.g., vegetable oil or supplement); E for “Effectors of absorption” (i.e., the effect of other nutrients or drugs); N for “Nutrient status of the host with respect to vitamin E levels”; G for “Genetic factors”; H for “Host related factors” (viz. individual characteristics such as age, sex, pathologies, etc.); and I for “Mathematical interactions” (referring to interacting effects of two or more of the described factors). Species of vitamin EdThere is less number of studies regarding the variation of vitamin E species in humans. Overall, it has been reviewed by Desmarchelier et al. (2018, pp. 1181e1196) that the relative bioavailability of stereoisomers, RRRand SRR-a-tocopherol bioavailability, presented with no significant difference in human studies. Also, a- and g-tocopherol bioavailability carried out with a low number of human subjects did not reveal any significant difference between them as well. Molecular linkagesdMostly, dietary vitamin E is consumed in its free form or as supplements. Supplements are usually esterified to protect the hydroxyl group against oxidation. However, no significant differences in bioavailability in human were observed for the free form or esters of succinates and acetates of tocopherols in healthy individuals (Burton et al., 1988; Cheeseman et al., 1995; Nagy et al., 2013). Amount of vitamin EdThe studies comparing nutritional doses with supplemental or pharmacological doses are currently lacking. It has been assumed that the efficiency of vitamin E absorption decreases with increased dose owing to blood saturation. On the contrary, there is no strict evidence of the same. However, one case study has shown that vitamin E levels in chylomicrons increased on the consumption of meals containing large dosages (432 or 937 IU) of a-tocopherol acetates (Borel et al., 1997). Matrix effectsdThe matrix within which vitamin E is incorporated is a key factor that governs its bioavailability. Vitamin E needs to be bioaccessible, i.e., to become available for absorption. The bioaccessibility is quite variable among food matrices. For instance, in banana, lettuce, and bread, vitamin E is almost completely bioaccessible, whereas in apples and orange it is quite low (Reboul, Klein, et al., 2006). It has also been found that the addition of eggs to durum wheat pasta reduces the bioaccessibility of vitamin E from around 70%e50% (Werner & Böhm, 2011). In the case of juices blended with whole milk, an increase in bioaccessibility was observed (Cilla et al., 2012). In other studies, where oilin-water emulsions fabricated with natural emulsifiers and long-chain triglycerides were used, better bioavailability of a-tocopherol was observed (Yang et al., 2017).

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Effectors of absorptiondDifferent authors (Desmarchelier et al., 2018, pp. 1181e1196; Reboul, 2017) have concluded from various studies that the amount of dietary fat in a food matrix facilitates vitamin E extraction, stimulates biliary secretion, and promotes micelle formation to increase its bioaccessibility. For example, consumption of toasted bread with butter or cereals with whole fat milk or raw vegetables consumed with canola oil and eggs led to better absorption of vitamin E, as compared to consuming the same without the fat components. On the other hand, the presence of certain micronutrients like vitamin C, carotenoids, and polyphenols negatively impacts intestinal absorption of tocopherol (Reboul et al., 2007). Whereas, with respect to dietary fibers intake, no adverse effects on vitamin E absorption was concluded among different studies concerning rats as well as humans (Desmarchelier et al., 2018, pp. 1181e1196). However, further studies are needed to identify the impact of various other micronutrients and draw real conclusions. Nutrient status of the hostdVitamins being essential and large amounts of fat-soluble vitamins can lead to toxicity; it is suggested that vitamin E absorption is mediated by the vitamin E status of the host. Studies have suggested that tocopherols can modulate directly or indirectly several nuclear receptors and can act as transcriptional factors for genes encoding proteins for vitamin E uptake (Borel et al., 2013; Desmarchelier et al., 2018, pp. 1181e1196). Genetic as well as host-related factorsdThe involvement of intestinal proteins/enzyme in vitamin E absorption has stimulated the hypothesis that genetic factors can modulate vitamin E absorption efficiency. Intestinal absorption of vitamin E requires normal digestive functions, and thus people with genetic diseases such as cystic fibrosis and abetalipoproteinemia suffer from impaired vitamin E absorption. Further, the effect of sex on vitamin E absorption is difficult to access in males and females, as female hormones affect lipid and lipoprotein metabolism differently (Borel et al., 2013). However, a nonsignificant difference in vitamin E levels has been observed in certain studies (Desmarchelier et al., 2018, pp. 1181e1196). Considering aging effects, it has been observed that the bioavailability of a-tocopheryl acetate is apparently lower in healthy older individuals than in younger ones (Borel et al., 1997), which were attributed to age-related altered digestive functions. Mathematical interactionsdThis includes synergistic or antagonistic effects on vitamin E absorption when considering the interaction of two or more factors with one another. Overall understanding of various factors relating to vitamin E absorption can ultimately transfer benefits leading to higher bioavailability, and one can suggest a personalized recommendation for individuals to confer potent health benefits.

14.7 Stability, safety, and toxicology Lipid oxidation is the major cause of quality deterioration of food products and the destruction of biological membrane structures. Lipid soluble antioxidants such as tocopherols can prevent the oxidation of lipids by competing with unsaturated fatty acids for the lipid peroxy radicals. The reaction rate of tocopherol is 100,000 times faster than the lipid with the lipid peroxy radical (Niki et al., 1984). However, tocopherols themselves may degrade due to improper storage, presence of free radicals, exposure to molecular oxygen, light and elevated temperature, grossly leading to the loss of antioxidant activity, or their role as prooxidants may become available (Choe & Min, 2006; Pignitter et al., 2014). At higher concentrations, tocopherol loses their antioxidant activity or becomes prooxidants, whereas at lower concentrations they have the highest antioxidant activity. The antioxidant activity of tocopherol is inversely related to the stability of tocopherol in vegetable oil (Jung & Min, 1992). The a-tocopherol of soybean oil due to its higher antioxidant activity is destroyed faster than the g- and d-tocopherol of soybean oil during storage at 50
C (Fig. 14.7). Moisture content, the types of oil, and the concentration of tocopherol homologues significantly influence the stability of tocopherol homologues (Jung & Min, 1992). The decomposition of a-tocopherol can be reduced by forming a proteinenutrient complex of a-tocopherol with b-lactoglobulin (Liang et al., 2011). Vitamin E deficiency is a common phenomenon in humans with fat malabsorption syndromes. Primarily, a-tocopherol is administered in humans to prevent vitamin E deficiency. Tocopherols as food additives have the Generally Recognized as Safe (GRAS) status in the United States. The recommended daily intake of vitamin E is reported as 15 mg (Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Carotenoids: A Report of the Panel on Dietary Antioxidants and Related Compounds, Subcommittees on Upper Reference Levels of Nutrients and Interpretation and Uses of Dietary Reference Intakes, and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine., 2000). However, the recommended daily intake may increase with the increase in the content of unsaturated fatty acids in a diet (Belitz & Grosch, 1999). Apparently, the adequate intake of this vitamin is not defined and may vary among the population of the world depending on the physiological conditions and diet. Vitamin E daily intake can be increased up to 300 mg without any complications (Yap et al., 2001). Even short-term high-doses and supranutritional (more than nutritionally required) doses administration of vitamin E has no reported adverse effects on health (Curtis et al., 2014; Final Report on the Safety Assessment of Tocopherol, Tocopheryl Acetate, Tocopheryl

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FIGURE 14.7 Degradation of a-, g-, and d-tocopherol in soybean oil on a storage time scale of 24 d at 50
C. From Player, M. E., Kim, H. J., Lee, H. O., & Min, D. B. (2006). Stability of a-, g-, or d-Tocopherol during soybean oil oxidation. Journal of Food Science, 71(8), C456eC460. https://doi.org/10. 1111/j.1750-3841.2006.00153.x.

Linoleate, Tocopheryl Linoleate/Oleate, Tocopheryl Nicotinate, Tocopheryl Succinate, Dioleyl Tocopheryl Methylsilanol, Potassium Ascorbyl Tocopheryl Phosphate, and Tocophersolan, 2002). However, the risk of developing side effects in some group of patients at the risk of cardiovascular diseases such as thrombotic risk cannot be ruled out and supplementation must be considered with precautions (Final Report on the Safety Assessment of Tocopherol, Tocopheryl Acetate, Tocopheryl Linoleate, Tocopheryl Linoleate/Oleate, Tocopheryl Nicotinate, Tocopheryl Succinate, Dioleyl Tocopheryl Methylsilanol, Potassium Ascorbyl Tocopheryl Phosphate, and Tocophersolan, 2002). Therefore at present, there is no need for recommendation of higher or supranutritional doses of vitamin E which may lead to health complications in the long term. Taking into consideration with regard to what we know at present, the efficacy and supplementation of vitamin E is worth investigating.

14.8 Applications (clinical and pathological): health benefits 14.8.1 Antioxidant activity Prolonged oxidative stress could lead to the onset of many metabolic and lifestyle-associated disorders. Such stress is caused due to an imbalance of free radical generation. Free radicals are generated as an impact of various metabolic processes in human. Widely known free radicals such as hydroxyl, superoxide anion, peroxide, singlet oxygen, nitric oxide, etc., are very reactive and capable of damaging DNA, proteins, carbohydrates, and lipids in the cell, leading to unwanted biochemical reactions (Saikia & Mahanta, 2016). These biochemical reactions later lead to serious metabolic and nonmetabolic disorders in human. However, innate defense in humans against such radicals associated damage is modulated by enzymes like superoxide dismutase, glutathione peroxidase, and micronutrients that quench or scavenge such radicals (Lobo et al., 2010), acting as antioxidants. The antioxidant activity of vitamin E (a-tocopherol) is attributed to its ability to neutralize or intercept lipid peroxyl radicals (LOO) thereby terminating the lipid peroxidation. However, vitamin E is not much of a potent scavenger of other radicals, viz.,_OH and alkoxyl radicals (RO.) (Nimse & Pal, 2015). A recent study conducted on rats demonstrated that the effectiveness of vitamin E supplementation was effective for decreasing lipid peroxidation and attenuating oxidative stress (Abdulaziz et al., 2020). Studies have also demonstrated improved oxidative stress and antioxidant status in elderly women on intakes of dietary antioxidants, such as carotenoids,

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vitamin E, and vitamin C (Boaventura et al., 2020). A recent review has suggested that vitamin E supplementation may lead to increased exercise performance in athletes (Higgins et al., 2020). The same study suggested that vitamin E tends to block free radicals generated during exercise which act as signaling molecules as protection against physical stress. In action, quenching of such radicals supposedly enhances endurance during exercise or sports performance. Thus, it is simple to deduce that vitamin E intake will potentially lead to better antioxidant status in the body, possibly providing protection against health disorders (Lobo et al., 2010).

14.8.2 Antiinflammation Inflammation is a result of an overreactive immune response to a harmful stimulus (chemical or biological). On such a stimulus, a cascade of reactions is initiated. Inflammation is characterized by the overproduction of reactive oxygen/nitrogen species and pro-inflammatory mediators, including lipid mediators, notably prostaglandins and leukotrienes, and cytokines such as TNF-a and interleukin-6 (IL-6). Chronic inflammation is a major contributor to the pathogenesis of chronic diseases such as cancer, cardiovascular diseases, rheumatoid arthritis, and asthma. Studies on tocopherols dosage on varied animal models induced with burn injury, airway inflammation, and colon inflammation have suggested a significant decrease in inflammatory factors (Jiang, 2014). Vitamin E indirectly reduces inflammation by affecting inflammatory mediators (Lewis et al., 2019). A meta-analysis study carried out by taking into consideration 33 randomized clinical trials suggested that a-tocopherol proved to be more effective in reducing serum levels of C-reactive proteins and IL-6 and overall alleviating subclinical inflammation in adults (Asbaghi et al., 2020). Studies pertaining to role of inflammation in arthritis, vitamin E supposedly retards the progression of osteoarthritis by ameliorating oxidative stress and inflammation of the joints (Chin & Ima-Nirwana, 2018).

14.8.3 Immunity Having a strong immunity is of utmost importance. It is widely being noted owing to a prevailing scenario where outbreaks of known or unknown infections can cause a toll on human health. The healthy immune response is linked to increased immunoglobulin levels, antibody responses, lymphocyte proliferation, and interleukin (IL)-2 productions. Numerous studies on dietary supplementation on varied animal and human models have shown the immunomodulatory effect of vitamin E. Vitamin E functions as an antiinflammatory agent by modulating T cell function by directly impacting T cell membrane integrity, signal transduction, and cell division, and also indirectly by affecting other inflammatory mediators (Lewis et al., 2019). In animal studies with cows, chicken, and rats, vitamin E supplementation led to overall increased immune responses (Lee et al., 1998). However, with human subjects, multiple studies have reported increased immune function, but at levels more than dietary recommendation (Lewis et al., 2019). Still, there are other studies suggesting no significant effects on immune functions. This might possibly be due to variation in dosage, age of subjects, and determination methodologies utilized in different studies (Lee & Han, 2018). For mice model studies on wound infections with methicillin-resistant Staphylococcus aureus, and Streptococcus pneumoniae infection of the respiratory tract, vitamin E therapy resulted in good immune responses and subsequent lower microbial counts (Bou Ghanem et al., 2015; Pierpaoli et al., 2017). In humans as well, lower levels of infection in pneumonia, malaria, and the common cold have been reviewed and reported (Lee & Han, 2018). It would be beneficial to focus on further research leading to the identification of optimal doses specific to age health conditions, nutritional status, and genetic variability.

14.8.4 Cancer Owing to its strong antioxidant nature, tocopherols are linked to reduced cancer risks. Certain studies have shown that deficiency of vitamin E is associated with increased risk in certain cancers (Wilson & Mucci, 2019). Vitamin E vitamers have been reviewed to be effective in inducing growth arrest, apoptosis, autophagy, and endoplasmic reticulum stress in cancer cells (Petronek et al., 2021). However, other studies with human subjects revealed a nonsignificant impact of vitamin E on the prevention or delay of lung cancer and pancreatic carcinoma and urinary tract cancer in humans (Petronek et al., 2021). Nevertheless, it has been suggested that vitamin E can be used as an adjuvant along with other active components such as selenium, doxorubicin for cancer prevention (Fernandes et al., 2018; Fred Gey, 1998; Wilson & Mucci, 2019). Cancer cell line and animal model studies have suggested that tocopherols help in modulating nuclear receptors such as PPARg (by upregulation) and ERa (by downregulation) to induce cell proliferation and apoptosis in breast cancer (Das Gupta & Suh, 2016). Overall, the impact of tocopherols is minimal, and the data pertaining to its effects are rather inconsistent.

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14.8.5 Metabolic disorders Metabolic disorders constitute a cluster of medical conditions majorly including obesity, hyperglycemia, dyslipidemia, and hypertension. Vitamin E is suggested as a promising agent for the treatment of such disorders (Wong et al., 2017). The impact of vitamin E in diabetic patients has been extensively carried out. One study carried out on Finnish men and women revealed that dietary intake of vitamin E was significantly associated with a reduced risk of type II diabetes (Montonen et al., 2004). In one study with 44 women aged between 20 and 50 years, it was assessed that consumption of grape seed oil rich in tocopherols improved insulin resistance in obese women (Irandoost et al., 2013). Next, in a clinical study in type I and type II diabetic patient vitamin E supplementation was found to delay the onset of diabetic and reduce blood pressure (Baburao Jain & Anand Jain, 2012). In a recent study, vitamin E evaluation on healthy subjects from Singapore suggested that vitamin E could play a role in delaying the onset of type II diabetes (Bi et al., 2019). Dyslipidemia is characterized by increased triglycerides and lowering of low-density lipoproteins (LDLs). Studies have suggested that supplementation of tocopherols do not confer any benefits in dyslipidemia as such but supplementing with tocotrienols or tocotrienol-rich fractions resulted in significant benefits (Wong et al., 2017). On the other hand, a contrasting effect of vitamin E intake was reported in recent Mendelian randomization-based observational study, in which vitamin E was linked to elevated levels of LDL and triglycerides (Wang & Xu, 2019). Thus, future research on revaluation of the therapeutic potential of vitamin E along with an emphasis on mechanistic understanding will be necessary to better confirm and elucidate beneficial effects of vitamin E in metabolic disorders.

14.8.6 Skincare Vitamin E has been used in dermatological applications for more than 50 years now as a potent antioxidant. Skin is subjected to damage owing to continuous solar radiations which lead to lipid peroxidation in membranes and age-related collagen cross-linking. Tocopherols are found to protect against both lipid peroxidation and collagen cross-linking. Tocopherols stabilize cell membrane by inhibiting the oxidation of arachidonic acid of membrane phospholipids. Also, the topical application of vitamin E has been reviewed to reduce erythema, sunburned cells, UV-inflicted skin damage, and photocarcinogenesis (Schagen et al., 2012). More recently, a study reported synergistic effects of vitamin E with ascorbic acid to improve skin health and brightening effects in the case of female subjects (Rattanawiwatpong et al., 2020), suggesting combinatorial therapies to be better than monotherapies. In another study, authors reported that topical formulation with phosphorylating a-tocopherol monomers better diffuse into skin epidermis thereby increased potential toward damage against UV radiations (Saleh et al., 2021). Existing studies considering preclinical and clinical studies have suggested the benefits of Vitamin E in the case of atopic dermatitis (Ehterami et al., 2019; Teo et al., 2020). Recent studies are more targeted toward enhanced delivery of a-tocopherol in the skin using nanoemulsions (Harun et al., 2021) to benefit skin health.

14.8.7 Eye health Oxidative stress leads to oxidative damage to the eye lens and is regarded as the major factor leading to the pathogenesis of senile cataract (Nartey, 2017). A meta-analysis evaluation suggested that both dietary and supplemental intake of vitamin E could significantly be associated with reduced age-related cataract development (Zhang et al., 2015). Like other antioxidants, tocopherols are also supposed to minimize oxidative damage. In a recent study, nanomicelles consisting of inulinD-a-tocopherol succinate bioconjugates loaded with curcumin were able to protect the blooderetina barrier against high glucose levels, thus suggesting that tocopherol can prevent diabetes-induced retinopathy (Rassu et al., 2021). The lens contains a-crystallin (a molecular chaperone) whose function is to maintain the correct folding of other protein and is also affected by oxidation. In one study, it was observed that rats injected with selenite-a-tocopherol had better a-crystallin function when supplemented with coffee. The study suggested that targeting such chaperone activity can be useful in the development of anticataract drug (Nakazawa et al., 2017). Overall, synergistic beneficial effects on eye health for tocopherols are observed with other antioxidants.

14.8.8 Liver health Nonalcoholic fatty liver disease (NAFLD) is referred to as the accumulation of excessive fat in the liver, without alcohol consumption. It is also strongly associated with obesity and related metabolic disorders such as insulin resistance, dyslipidemia, and oxidative stress. NAFLD also leads to nonalcoholic steatohepatitis (NASH), characterized histologically

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by the presence of hepatic steatosis, lobular inflammation, and hepatocyte ballooning leading to cirrhosis and hepatocarcinoma (Hadi et al., 2018; Pacana & Sanyal, 2012). A significant improvement in steatosis, inflammation, ballooning, and resolution of steatohepatitis in adult nondiabetic patients with aggressive NASH can be brought about with vitamin E therapy (Pacana & Sanyal, 2012). From numerous studies on animals, it has been concluded that vitamin E therapy could recover depleted hepatic glutathione (depletion is linked with oxidative stress and marked increase in hepatic fibrosis); ameliorate steatosis, necroinflammation, hepatic stellate cell activation, and collagen mRNA expression (triggered by increase in oxidative stress and metabolic abnormality due to NAFLD); and reduce serum transaminase levels (elevated levels are associated with NAFLD). These effects have been associated with suppressed expression of the fibrotic genes TGF-b and MMP-2, inflammatory factor COX-2, and proapoptotic genes (Bax), inhibition of factor kappa B (NFkB), and increased hepatic superoxide dismutase activity. On the contrary, multiple human clinical trials with longterm vitamin E monotherapies have reportedly shown both significant and no significant improvement on liver biochemistry and histopathology. However, long-term (>2 years) combinatorial treatment strategies such a vitamin E þ ursodeoxycholic acid or vitamin E þ vitamin C þ atorvastatin have demonstrated overall modest benefits in liver health and histopathological improvements in majority of adults and pediatric patients (Abdel-Maboud et al., 2020; Hadi et al., 2018). On meta-analysis of a controlled clinical trial carried out, the effect of dosage and formulation variation among various clinical studies makes it difficult to ascertain their effects comparatively (Amanullah et al., 2019). Still, there is a need for further studies to comprehend the physiology of NADH/NASH which would help us to better understand and develop a targeted approach for treatments using vitamin E.

14.9 Conclusion This chapter presents essential and relevant information on the sources, extraction, antioxidant properties, and healthbenefiting properties of tocopherols, which are the most important and active forms of vitamin E. After a century of studies since its discovery, some aspects of tocopherols are still far from being completely compiled in literature, especially the nutritional recommendations, therapeutic applications, and disease prevention. Taking into account all these aspects, the research studies on this compound are gaining interest and hence, reemerged as a topic of intense research for the scientific community.

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Journal of Oleo Science, 68(10), 951e958. https://doi.org/10.5650/jos.ess19146 Lobo, V., Patil, A., Phatak, A., & Chandra, N. (2010). Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy Reviews, 4(8), 118e126. https://doi.org/10.4103/0973-7847.70902 Lushchak, V. I., & Semchuk, N. M. (2012). Tocopherol biosynthesis: Chemistry, regulation and effects of environmental factors. Acta Physiologiae Plantarum, 34(5), 1607e1628. https://doi.org/10.1007/s11738-012-0988-9 Mendes, M. F., Pessoa, F. L. P., & Uller, A. M. C. (2005). Optimization of the process of concentration of vitamin e from DDSO using supercritical CO2. Brazilian Journal of Chemical Engineering, 22, 83e91. Montonen, J., Knekt, P., Järvinen, R., & Reunanen, A. (2004). Dietary antioxidant intake and risk of type 2 diabetes. Diabetes Care, 27(2), 362. https:// doi.org/10.2337/diacare.27.2.362 Nagy, K., Ramos, L., Courtet-Compondu, M.-C., Braga-Lagache, S., Redeuil, K., Lobo, B., Azpiroz, F., Malagelada, J.-R., Beaumont, M., Moulin, J., Acquistapache, S., Sagalowicz, L., Kussmann, M., Santos, J., Holst, B., & Williamson, G. (2013). Double-balloon jejunal perfusion to compare absorption of vitamin E and vitamin E acetate in healthy volunteers under maldigestion conditions. European Journal of Clinical Nutrition, 67(2), 202e206. https://doi.org/10.1038/ejcn.2012.183 Nakazawa, Y., Nagai, N., Ishimori, N., Oguchi, J., & Tamura, H. (2017). Administration of antioxidant compounds affects the lens chaperone activity and prevents the onset of cataracts. Biomedicine & Pharmacotherapy, 95, 137e143. https://doi.org/10.1016/j.biopha.2017.08.055 Nartey, A. (2017). The pathophysiology of cataract and major interventions to retarding its progression: A mini review. Advances in Ophthalmology & Visual System, 6. https://doi.org/10.15406/aovs.2017.06.00178 Niki, E., Saito, T., Kawakami, A., & Kamiya, Y. (1984). Inhibition of oxidation of methyl linoleate in solution by vitamin E and vitamin C. Journal of Biological Chemistry, 259(7), 4177e4182. Nimse, S. B., & Pal, D. (2015). Free radicals, natural antioxidants, and their reaction mechanisms. RSC Advances, 5(35), 27986e28006. https://doi.org/ 10.1039/C4RA13315C Pacana, T., & Sanyal, A. J. (2012). Vitamin E and nonalcoholic fatty liver disease. Current Opinion in Clinical Nutrition and Metabolic Care, 15(6), 641e648. https://doi.org/10.1097/MCO.0b013e328357f747 Petronek, M. S., Stolwijk, J. M., Murray, S. D., Steinbach, E. J., Zakharia, Y., Buettner, G. R., Spitz, D. R., & Allen, B. G. (2021). Utilization of redox modulating small molecules that selectively act as pro-oxidants in cancer cells to open a therapeutic window for improving cancer therapy. Redox Biology, 101864. https://doi.org/10.1016/j.redox.2021.101864 Pierpaoli, E., Orlando, F., Cirioni, O., Simonetti, O., Giacometti, A., & Provinciali, M. (2017). Supplementation with tocotrienols from Bixa orellana improves the in vivo efficacy of daptomycin against methicillin-resistant Staphylococcus aureus in a mouse model of infected wound. Phytomedicine, 36, 50e53. https://doi.org/10.1016/j.phymed.2017.09.011 Pignitter, M., Stolze, K., Gartner, S., Dumhart, B., Stoll, C., Steiger, G., Kraemer, K., & Somoza, V. (2014). Cold fluorescent light as major inducer of lipid oxidation in soybean oil stored at household conditions for eight weeks. Journal of Agricultural and Food Chemistry, 62(10), 2297e2305. https://doi.org/10.1021/jf405736j Quek, S.-Y., Chu, B.-S., & Baharin, B. S. (2007). In the encyclopedia of vitamin E. Trowbridge, UK: Cromwell Press.

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Rassu, G., Pavan, B., Mandracchia, D., Tripodo, G., Botti, G., Dalpiaz, A., Gavini, E., & Giunchedi, P. (2021). Polymeric nanomicelles based on inulin D a-tocopherol succinate for the treatment of diabetic retinopathy. Journal of Drug Delivery Science and Technology, 61, 102286. https://doi.org/ 10.1016/j.jddst.2020.102286 Rattanawiwatpong, P., Wanitphakdeedecha, R., Bumrungpert, A., & Maiprasert, M. (2020). Anti-aging and brightening effects of a topical treatment containing vitamin C, vitamin E, and raspberry leaf cell culture extract: A split-face, randomized controlled trial. Journal of Cosmetic Dermatology, 19(3), 671e676. https://doi.org/10.1111/jocd.13305 Reboul, E. (2017). Vitamin E bioavailability: Mechanisms of intestinal absorption in the spotlight. Antioxidants, 6(4). https://doi.org/10.3390/ antiox6040095 Reboul, E., Klein, A., Bietrix, F., Gleize, B., Malezet-Desmoulins, C., Schneider, M., Margotat, A., Lagrost, L., Collet, X., & Borel, P. (2006). Scavenger receptor class B type I (SR-BI) is involved in vitamin E transport across the enterocyte. Journal of Biological Chemistry, 281(8), 4739e4745. https:// doi.org/10.1074/jbc.M509042200 Reboul, E., Richelle, M., Perrot, E., Desmoulins-Malezet, C., Pirisi, V., & Borel, P. (2006). Bioaccessibility of carotenoids and vitamin E from their main dietary sources. Journal of Agricultural and Food Chemistry, 54(23), 8749e8755. https://doi.org/10.1021/jf061818s Reboul, E., Thap, S., Perrot, E., Amiot, M.-J., Lairon, D., & Borel, P. (2007). Effect of the main dietary antioxidants (carotenoids, g-tocopherol, polyphenols, and vitamin C) on a-tocopherol absorption. European Journal of Clinical Nutrition, 61(10), 1167e1173. https://doi.org/10.1038/ sj.ejcn.1602635 Ribeiro, P. P. C., Silva, D. M. de L., Dantas, M. M., Ribeiro, K. D. da S., Dimenstein, R., & Damasceno, K. S. F. da S. C. (2019). Determination of tocopherols and physicochemical properties of faveleira (Cnidoscolus quercifolius) seed oil extracted using different methods. Food Science and Technology, 39, 280e285. Saikia, S., & Mahanta, C. L. (2016). In vitro physicochemical, phytochemical and functional properties of fiber rich fractions derived from by-products of six fruits. Journal of Food Science and Technology, 53(3), 1496e1504. https://doi.org/10.1007/s13197-015-2120-9 Saleh, M. M., Woods, A., Harvey, R. D., Young, A. R., & Jones, S. A. (2021). Nanomaterials fusing with the skin: Alpha-tocopherol phosphate delivery into the viable epidermis to protect against ultraviolet radiation damage. International Journal of Pharmaceutics, 594, 120000. https://doi.org/ 10.1016/j.ijpharm.2020.120000 Schagen, S. K., Zampeli, V. A., Makrantonaki, E., & Zouboulis, C. C. (2012). Discovering the link between nutrition and skin aging. DermatoEndocrinology, 4(3), 298e307. https://doi.org/10.4161/derm.22876   Spika, M. J., Kraljic, K., & Skevin, D. (2016). Tocopherols: Chemical structure, bioactivity, and variability in Croatian virgin olive oils. In Products from olive tree (p. 317). BoDeBooks on Demand. Sure, B. (1924). Dietary requirements for reproduction: II. The existence OF a specific vitamin for reproduction. Journal of Biological Chemistry, 58(3), 693e709. https://doi.org/10.1016/S0021-9258(18)85329-7 Teo, C. W. L., Tay, S. H. Y., Tey, H. L., Ung, Y. W., & Yap, W. N. (2020). Vitamin E in atopic dermatitis: From preclinical to clinical studies. Dermatology. https://doi.org/10.1159/000510653 Traber, M. G. (2004). The ABCs of vitamin E and b-carotene absorption. The American Journal of Clinical Nutrition, 80(1), 3e4. https://doi.org/10.1093/ ajcn/80.1.3 Traber, M. G. (2007). Vitamin E regulatory mechanisms. Annual Review of Nutrition, 27(1), 347e362. https://doi.org/10.1146/annurev.nutr.27.0614 06.093819 Turkiewicz, I. P., Wojdyło, A., Tkacz, K., & Nowicka, P. (2020). Carotenoids, chlorophylls, vitamin E and amino acid profile in fruits of nineteen Chaenomeles cultivars. Journal of Food Composition and Analysis, 93, 103608. https://doi.org/10.1016/j.jfca.2020.103608 Wang, T., & Xu, L. (2019). Circulating vitamin E levels and risk of coronary artery disease and myocardial infarction: A mendelian randomization study. Nutrients, 11(9). https://doi.org/10.3390/nu11092153 Werner, S., & Böhm, V. (2011). Bioaccessibility of carotenoids and vitamin E from pasta: Evaluation of an in vitro digestion model. Journal of Agricultural and Food Chemistry, 59(4), 1163e1170. https://doi.org/10.1021/jf103892y West, C., & Castenmiller, J. (1998). Quantification of the “SLAMENGHI” factors for carotenoid bioavailability and bioconversion. International Journal for Vitamin and Nutrition Research, 68(6), 371e377. Internationale Zeitschrift Fur Vitamin- Und Ernahrungsforschung. Journal International de Vitaminologie et de Nutrition http://europepmc.org/abstract/MED/9857264. Wilson, K. M., & Mucci, L. A. (2019). Diet and lifestyle in prostate cancer. Advances in Experimental Medicine and Biology, 1210, 1e27. https://doi.org/ 10.1007/978-3-030-32656-2_1 Wong, S. K., Chin, K.-Y., Suhaimi, F. H., Ahmad, F., & Ima-Nirwana, S. (2017). Vitamin E as a potential interventional treatment for metabolic syndrome: Evidence from animal and human studies. Frontiers in Pharmacology, 8, 444. https://www.frontiersin.org/article/10.3389/fphar.2017. 00444. Xu, Z. (2008). Comparison of extraction methods for quantifying vitamin E from animal tissues. Bioresource Technology, 99(18), 8705e8709. https:// doi.org/10.1016/j.biortech.2008.04.065 Yang, Y., Xiao, H., & McClements, D. J. (2017). Impact of lipid phase on the bioavailability of vitamin E in emulsion-based delivery systems: Relative importance of bioaccessibility, absorption, and transformation. Journal of Agricultural and Food Chemistry, 65(19), 3946e3955. https://doi.org/ 10.1021/acs.jafc.7b00955 Yap, S. P., Yuen, K. H., & Wong, J. W. (2001). Pharmacokinetics and bioavailability of a-, g- and d-tocotrienols under different food status. Journal of Pharmacy and Pharmacology, 53(1), 67e71. https://doi.org/10.1211/0022357011775208

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Zhang, L., Wang, S., Yang, R., Mao, J., Jiang, J., Wang, X., … Li, P. (2019). Simultaneous determination of tocopherols, carotenoids and phytosterols in edible vegetable oil by ultrasound-assisted saponification, LLE and LC-MS/MS. Food Chemistry, 289, 313e319. https://doi.org/10.1016/ j.foodchem.2019.03.067 Zhang, Y., Jiang, W., Xie, Z., Wu, W., & Zhang, D. (2015). Vitamin E and risk of age-related cataract: A meta-analysis. Public Health Nutrition, 18(15), 2804e2814. https://doi.org/10.1017/S1368980014003115

Further readings Granado-Lorencio, F., Olmedilla-Alonso, B., Herrero-Barbudo, C., Blanco-Navarro, I., Pérez-Sacristán, B., & Blázquez-García, S. (2007). In vitro bioaccessibility of carotenoids and tocopherols from fruits and vegetables. Food Chemistry, 102(3), 641e648. https://doi.org/10.1016/ j.foodchem.2006.05.043 Jung, J., Gim, S. Y., Lee, C., Kim, M.-J., & Lee, J. (2017). Stability of tocopherol homologs in soybean, corn, canola, and olive oils under different moisture contents at 25
C. European Journal of Lipid Science and Technology, 119(6), 1600157. https://doi.org/10.1002/ejlt.201600157

Chapter 15

Alpha-linolenic acid Jessica Pandohee Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, WA, Australia

15.1 Introduction Alpha-linolenic acid (ALA) is an essential omega-3 polyunsaturated fatty acid that has gained increased interest in research and the general population in the last decades. Although it is widely known as a nutraceutical readily obtainable from plants and oil crops, ALA is an important raw material for the synthesis of larger lipids vital for cell membrane structure, hormone function, insulation, and even energy storage. Lipids are complex biological macromolecules consisting of thousands of organic chemical compounds that are predominantly nonpolar in nature. Lipids can be divided into four groups according to their chemical structures and these are glycerides, nonglyceride lipids, complex lipids, and fatty acids (as shown in Fig. 15.1). Fatty acids are simple molecules with a long linear hydrocarbon chain with a carboxylic group at the end of the chain. They exist in two forms: saturated or unsaturated fatty acids. Saturated fatty acids have only single bonds connecting the carbon atoms and do not have any double bonds; they are usually solid at room temperature (Sanders, 2003). Unsaturated fatty acids on the other hand have at least one double bond between the carbonecarbon atoms and are usually liquid at room temperature, for example, stearic acid. Unsaturated fatty acids can be subdivided into two groups: monounsaturated fatty acids, which as its name suggests have only one carbonecarbon double bond (for example, oleic acid), and polyunsaturated fatty acids (PUFAs), which have more than one carbonecarbon double bonds. PUFAs are an important group of compounds because they consist of the simplest fatty acids that will eventually be used as building blocks for larger molecules indispensable for nerve function, brain health, and heart function (Chen et al., 2018; Schoeler & Caesar, 2019). PUFAs are classified as nonessential PUFAs, which can be synthesized by the body, and essential fatty acids, which cannot be synthesized by the body and therefore must be obtained through diet. Finally, the two essential PUFAs can be classified as omega-3 (u-3 or n-3) and omega-6 (u-6 or n-6) fatty acids (Astorg et al., 2004). All essential PUFAs are manufactured by plants and cannot be synthesized by humans and other mammals due to the absence of 12 and 15 desaturase enzymes. Omega-6 PUFAs include linoleic acid (the precursor molecule), gammalinolenic acid, and araquidonic acid, while omega-3 PUFAs include ALA (the precursor molecule), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Humans obtain linoleic acid and ALA through the consumption of plant foods. These in turn go through a series of desaturation elongation reactions in the human body to make longer PUFAs which have various functions. The function of ALA has long been thought to only be the precursor in the synthesis of longchain PUFAs such as EPA and DHA. Lately it has been shown that ALA has a role to play in inflammation diseases, cardiovascular diseases (Abdelhamid et al., 2020), diabetes (Barre, 2007), brain growth and development (and therefore infant growth) (Baumgartner et al., 2014; Blondeau et al., 2015; Innis, 2007), and various types of cancers (Hanson et al., 2020; Serini & Calviello, 2018). This has caused scientists, dieticians, nutritionists, and the population at large to rethink the role and consumption of food containing ALA. The recent years have seen our nutrition, especially those in Western countries, changed dramatically. In many cases, this has meant an increase in the consumption of saturated fatty acids and a preference for omega-6 fatty acid over omega-3 fatty acids, via fast food or easily obtainable food which uses cheap corn and safflower oils. The higher proportion of linoleic acid compared to ALA in our diet means that there is a lower production of PUFAs such as EPA and DHA as both linoleic acid and ALA use the same enzymes. With u-6 fatty acid competing with u-3 fatty acid enzymes, a decrease in level of EPA and DHA in the body as well as an increase in antiinflammatory and metabolic diseases have been observed.

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FIGURE 15.1 Lipid concept map showing where omega-3 fatty acids such as alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid are situated in the lipid family.

Sioen et al. (2017) reviewed the consumption of u-3 and u-6 PUFAs against the recommendations from the European Food Safety Authority and found that people consume on an average less PUFAs than recommended by the World Health Organization. From the 53 studies across 17 European countries, they found that the mean linoleic acid intake was within the recommendation in 52% of the countries with inadequate intakes more likely in lactating women, adolescents, and elderly. Mean ALA intake within the recommendation in 77% countries and in 26% of the countries mean EPA and/or DHA intake was as recommended. Over 50 years of research support the health benefits of omega-3 fatty acids, especially the importance of EPA and DHA in infant growth, cardiovascular disease, cancer, among others have been acknowledged. While ALA was thought to be a precursor and reagent metabolite in the production of EPA and DHA, it is being associated with new roles in the human body.

15.2 Sources The word SLA is derived from the Greek word “linon” meaning flax and “oleic” meaning related to oleic acid or producing oleic acid. Indeed, flaxseed or linseed (Linum usitatissimum) is the richest food source of ALA and is cultivated for consumption in countries such as India, China, the United States, and Canada (Kajla et al., 2015). Nutritionally, flaxseed has a high level of protein, dietary fiber, B vitamins, minerals, lignans, and phytochemicals (Rubilar et al., 2010). Above all, the main constituent of flaxseed is high-quality lipids. Flaxseed is known to contain between 35% and 45% oil, of which 45%e52% is ALA, which is approximately 22 mg/100 g of flaxseed. Moreover, it has been reported to have a negligible amount of saturated fat such as palmitic acid and stearic acid (Goyal et al., 2014). Due to the high amount of ALA present in flaxseed, the latter has been used in various studies to investigate the effect of ALA (via flaxseed ingestion)

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on noncommunicable diseases such as cardiovascular (Parikh et al., 2018), inflammatory disorders (Galli & Calder, 2009), and cancers (Calado et al., 2018; DeLuca et al., 2018). Originally from Mexico and Guatemala, chia (Salvia hispanica L.) is an oilseed that has been cultivated for its use as a staple crop or paint by the Mayas and Aztecs in the pre-Columbian era (Ullah et al., 2016). Derived from the Spanish word “chian” meaning “oily,” chia is a good source of oils containing between 25% and 40% oil, 60% of which is ALA and 20% is linoleic acid (Peiretti & Gai, 2009). In general, chia seeds are rich in fiber, protein, B vitamins, vitamin C, minerals, and antioxidants (Muñoz et al., 2013). Compared to the ALA content in flaxseed, chia seed would usually not be described as a high source of ALA. However, being a good source of other essential compounds such as dietary fiber and antioxidants, chia seeds have been used in many studies to investigate their effect on the incidence of cardiovascular disease (de Souza Ferreira et al., 2015), nonalcoholic fatty liver disease (Medina-Urrutia et al., 2020) and diabetes (Chicco et al., 2009; Parker et al., 2018). Another source of ALA is rapeseed (Brassica napus) oil, and the latter is known to contain a reasonably high amount of ALA compared to other oil crops (not including flaxseed and chia seed). Authentic rapeseed crops initially contained a high amount of erucic and glucosinolates, attributing a bitter taste and toxicity to the oil. Several years of breeding of the B. napus strains in Canada resulted in the development of seeds low in erucic acid and glucosinolates (Barth, 2009), which was labeled as “Can. O., L. -A” (Canadian oilseed, low acid). From this abbreviation the artificial name “Canola” was created. Canola oil is nowadays the only type of rapeseed oil that is allowed for human nutrition and can often be described as Canola-quality meaning “double low” or “double zero” or “00-quality” which signifies low in erucic acid and in glycosinolates. Rapeseed is cultivated across the world during winter in Europe and Asia and during spring in Canada and Australia. Due to the relatively low content of ALA in canola oil, studies investigating the effect of ALA from canola oil on human health are limited to heart diseases (Harland, 2009), cancer (Hardman, 2007), impaired glucose metabolism (Schwab et al., 2018), and high cholesterol (Stricker et al., 2008). Other sources of ALA include walnut, green leaves, marine algae, soybean (soybean oil), pumpkin seeds, perilla seed, tofu, and peony seed (Chen et al., 2018; Schoeler & Caesar, 2019). Due to the importance of ALA, there has been an increase in enrichment of ALA in food especially dairy food such as eggs (Fraeye et al., 2012) and milk (Gebreyowhans et al., 2019; Moallem, 2018).

15.3 Extraction and characterization techniques The use of flaxseed as a rich source of ALA is common in nutritional intervention to investigate the effect of ALA of various diseases such as cardiovascular, diabetes, and cancers. As discussed in Section 15.2, flaxseed also contains hundreds of other bioactive compounds, making it difficult to confirm if the benefits of flaxseed are arising from its high ALA content only, other bioactive nutraceuticals, or a combination of compounds including ALA. Moreover, causality investigations and mechanism of action experiments work best with purified sources of a bioactive compound. Therefore, highly purified ALA is very attractive for pharmaceutical and pharmacological research and applications. Some considerations to bear in mind with ALA extraction include lipid oxidation and stability. While flaxseed, linseed, and other oilseeds contain high levels of PUFAs and these are susceptible to oxidation. Lipid oxidation causes the formation of hydroperoxides and degradation products that decrease the nutritional qualities, sensory properties and shorten storage life. Moreover, the radical oxygen species can react with DNA, protein, or other lipids causing more damages than benefits. ALA purification usually is done in several steps and includes lipid/oil extraction, PUFA characterization, and finally ALA isolation. Lipid extraction is a common procedure in the food industry. These include distillation, mechanical pressing, and solvent extraction of the oilseeds. Even though it is still widely used, cold pressing of oilseeds has limited recovery of oils from seeds and is nowadays a preparation step to increase crush the oilseeds open and expose as much oil as possible to the solvents used for extraction. Solvent extraction is by far the most efficient way to recover oil. Factors such as polarity of solvent, solubility of oils in solvent, solvent to solid ratio, temperature, and time of extraction have been optimized to minimize cost and increase recovery and efficiency resulting up to 80% ALA recovery (Ishak et al., 2020; Zanqui et al., 2016). The use of ultrasound-assisted enzymatic extraction by Li et al. (2017) has shown similar physicochemical oil characteristic as solvent extract and cold pressing extraction but higher efficiency in recovering ALA. The use of supercritical carbon dioxide extraction has been shown to be a promising technique in regard to the ALA isolation providing up to 60% recovery of oil and being a nontoxic and nonflammable method (Bozan & Temelli, 2002; Zhang et al., 2018). The method of choice to characterize ALA is using gas chromatography (Li et al., 2017). Oilseeds or extracted ALA requires to be derivatized to fatty acid methyl esters via an esterification reaction, most commonly using methanol in boron trifluoride, in order to render the fatty acids volatile and nonpolar for analysis (Pandohee & Jones, 2016). The extracts are

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then vaporized, separated through a capillary column before they are detected with a flame ionization detector (GC-FID) or a mass spectrometer (GC-MS). This method has been used to profile PUFAs in crops (Saini et al., 2014) and in serum (Onozato et al., 2015). While the use of liquid chromatography (HPLC) to characterize ALA is possible, the lack of chromophore on fatty acids makes the use of HPLC-UV challenging (Pandohee et al., 2019). Zhou et al. (2019) developed a rapid and sensitive method for simultaneous determination of ALA, EPA, DHA, and DPA in plasma of hyperlipidemic and normolipidemic subjects using LC-MS/MS, and Wu et al. (2009) applied near-infrared spectroscopy to profile fatty acids.

15.4 Chemistry ALA is an organic compound consisting of a long linear chain of 18 carbon atoms and a carboxylic acid end. It has three cis-double bonds at the carbon positions 9, 12, and 15. The chemical structure of ALA is shown in Fig. 15.2. ALA has a CAS number of 463-40-1 (cis). The chemical formula of ALA is C18H30O2 and its average molecular weight is 278.4296 and monoisotopic molecular weight is 278.224580204. ALA exists as a liquid at room temperature; it has a melting point of
11 C and a boiling point of 232 C. It is also known as all-cis-octadeca-9,12,15-trienoic acid (IUPAC name); (9Z, 12Z, 15Z)-9,12,15-octadecatrienoic acid (CAS name); 9-cis,12-cis,15-cis-octadecatrienoic acid; linolenic acid; cis-D(9,12,15)octadecatrienoic acid; FA(18:3(9Z,12Z,15Z)); FA(18:3n3); or simply abbreviated to ALA. Other unique identifiers for ALA are: SMILES: CC/C]C/C/C]C/C/C]C/CCCCCCCC(O)]O; InChI Identifier: InChI ¼ 1S/C18H30O2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18(19)20/h3-4,6e7,9e10 H,2,5,8,11e17H2,1H3,(H,19,20)/b4-3-,7-6-,10-9InChI Key: DTOSIQBPPRVQHS-PDBXOOCHSA-N.

15.5 Mechanism of action For a very long time, the role of ALA was thought to act as the primary component and precursor to EPA and DHA. Health benefits such as brain development, antiinflammatory, and anticoagulation properties have been linked to lowering the risk to cardiovascular disease, Alzheimer’s disease, diabetes, metabolic diseases, and nonalcoholic fatty liver disease, among others. There has been an increase in the number of studies associating similar benefits to the consumption of ALA. However, it remains unclear whether it is ALA that is directly being involved in preventing noncommunicable diseases or it is the increase in EPA and DHA from the consumption of ALA that is providing the health benefits. Therefore significant research is still required to underpin the role of ALA in preventing diseases and their mechanism of action.

15.6 Bioavailability ALA is not synthesized in the body and is obtained through the consumption of plant-based foods (Lane et al., 2014). The World Health Organization recommends an ALA intake of 0.5 g/day for infants, 0.7 g/day for children aged 1e3 years,

FIGURE 15.2 Structure of omega-3 fatty acids. ALA: alpha-linolenic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid; EPA: eicosapentaenoic acid, (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid; DHA: docosahexaenoic acid, (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid.

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0.9 g/day for children aged 4e8 years, 1.2 g/day for children aged 9e13 years, 1.6 g/day for adolescents and adults. ALA together with other fatty acids are present in food in the form of triacylglycerides, large lipid molecules made of three fatty acids attached to a glycerol backbone, which are hydrolyzed in the gastric and intestinal lumen by pancreatic enzymes and lipases. Bile salts also provide assistance to digestion and absorption by producing micelles. Absorption of ALA into the blood occurs in the small intestines and a healthy average adult normally absorbs 80%e95%. The normal concentration of ALA in blood of a healthy adult is about 0.6 Mol% (Punia et al., 2019). Once absorbed, ALA is transported to tissues for usage or to be converted to longer PUFAs. One of the most important uses of ALA is in the biosynthesis of PUFAs such as EPA and DHA (Burdge & Wootton, 2002). The pathway for the conversion of ALA to EPA and DHA occurs in the endoplasmic reticulum and mitochondria of the liver (Burdge, 2006), as shown in Fig. 15.3. ALA (C18:3 n-3) is converted to stearidonic acid (C18:4 n-3) through the addition of a double bond at the 6 position by the 6 desaturase enzyme. Stearidonic acid (C18:4 n-3) is then converted to cis-8,11,14,17-eicosatetraenoic acid (C20:4 n-3) by the addition of two carbons by elongase activity. cis-8,11,14, 17-eicosatetraenoic acid (C20:4 n-3) is converted to EPA (C20:5 n-3) through the addition of a double bond at the 5 position by the 5 desaturase enzyme. EPA (C20:5 n-3) is then converted to docosapentaenoic acid (C22:5 n-3) by the addition of two carbons by elongase activity. Docosapentaenoic acid (C22:5 n-3) is converted to tetracosapentaenoic acid (C24:5 n-3) by the addition of two carbons by elongase activity. Tetracosapentaenoic acid (C24:5 n-3) is converted to

FIGURE 15.3 A general pathway for the conversion of alpha-linolenic acid to docosahexaenoic acid.

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tetracosahexaenoic acid (C24:6 n-3) through the addition of a double bond at the 4 position by the 4 desaturase enzyme. All the desaturation and elongation reactions converting ALA to tetracosahexaenoic acid (C24:6 n-3) occur in the endoplasmic reticulum. Tetracosahexaenoic acid (C24:6 n-3) is then transported to the mitochondria where it is converted to DHA (C22:5 n-3) (Stark et al., 2016). A study by Yu and Salem (2007) showed that organs such as plasma, stomach, and spleen have the highest amount of ALA 4 h after mice have ingested ALA. Eight hours after ingestion, other organs and blood would contain the most amount of ALA, which coincide with digestion and absorption time since ingestion. Other PUFAs such as DHA were found to be stored in larger organs such as the brain, spinal cord, heart, and eye. The authors (Yu & Salem, 2007) also reported 78% of the ingested ALA and linoleic acid were catabolized or excreted and that about 16%e18% of ALA would eventually be stored in adipose tissue and skin. Unabsorbed ALA is eliminated in urine and stool.

15.7 Stability, safety, and toxicology ALA is a PUFA with three carbonecarbon double bonds that can be easily degraded when exposed to air, heat, or light. Oil containing high amount of ALA or other PUFAs are usually prone to oxidation forming products that render the foods bitter and often toxic (Souza et al., 2017). Lipid oxidation (and ALA oxidation) results in the formation of hydroperoxides and reactive oxygen species which can further damage proteins and DNA in our body. For these reasons, oils containing PUFAs are usually stored in light-resistant containers and/or in a cool and dark cupboard. The use of antioxidants such as tocopherol or tea polyphenol palmitate has been shown to improve oxidative stability and shelf life of flaxseed oil (Lu et al., 2020) and tocopherol and ascorbyl palmitate for chia seed oil (Bodoira et al., 2017). On its own, ALA is considered as a safe nutraceutical; an average healthy adult is recommended to consume about 1.6 g/day from vegetable oils or nuts such as flaxseed, canola oil, soybean oil, or walnuts. However, as ALA is obtained in the form of triglycerides, people with high levels of triglycerides are often advised to not take ALA supplements or consuming large amounts of ALA-containing foods.

15.8 Applications In the last three decades there has been a rise in noncommunicable diseases such as antiinflammatory, metabolic, and cardiovascular diseases. It is believed that malnutrition, the consumption of unbalanced diet and preference for fast food, highly processed food, and readily available food with high u-6 fatty acids are a major factor in the increase occurrence of noncommunicable diseases (Abdelhamid et al., 2020; Sioen et al., 2017). To remedy this, dietary interventions with a focus on increasing the ALA consumption have been carried out in healthy adults as well as patients suffering from noncommunicable diseases (DeLuca et al., 2018; Galli & Calder, 2009) to investigate its health-promoting properties (Hennessy et al., 2011). The need for ALA starts as early as a few weeks old feti. During the development of a fetus a large amount of essential fatty acids such as ALA, EPA, and DHA is necessary for the formation of structural components in the cell membranes, intracellular signaling, and storage for postnatal life. In addition to a rapidly growing brain and neurological system requiring the manufacture of specialized cell membrane lipids in the nerve cells, DHA and arachidonic acid are also stored in the brain and the retina (Blondeau et al., 2015). There has also been accumulating evidence that an adequate amount of u-3 fatty acid plays an important role in fetal programming and reducing the impact on metabolic health (Shrestha et al., 2020). Thus, supplementation of ALA and other PUFAs such as EPA and DHA is quite common in the early stages of pregnancy (Poniedziałek-Czajkowska et al., 2014) and in preterm infants (Moon et al., 2016). It is well known that the diets rich in PUFAs and low in saturated and trans fats are key to prevent heart diseases. Coronary heart disease is one of the most fatal diseases in middle-aged adults nowadays and survivors from heart problems require frequent health monitoring to avoid relapse, therefore adding pressure to the health-care system (Wei et al., 2018). As a result, diets rich in whole grains, green vegetables, fish, olive, and canola oil have been widely popular. ALA is an essential nutraceutical against the prevention of stroke; the consumption of chia seeds and flaxseeds has been shown to have cardiovascular benefits (de Souza Ferreira et al., 2015; Parikh et al., 2018). In their systematic review, Wei et al. (2018) associated a reduced risk of coronary heart disease with a consumption of ALA of less than 1.4 g/day and reduced risk of fatal coronary heart disease with a high ALA intake. The evidence of the benefits of ALA extends to food containing lower amounts of ALA. For example, Marangoni et al. (2007) showed that an intake of four walnuts a day together with their normal diets for 3 weeks significantly increased blood levels of ALA and longer PUFAs. Moreover the consumption of rapeseeds in the form of “optimized oil” has been shown to increase the HDL cholesterol content while not increasing the total to HDL cholesterol ratio and the LDL to HDL

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cholesterol ratio (Gladine et al., 2013). This means that intake of the “optimized rapeseed oil” could potentially have cardioprotective effects and therefore prevent the risk of cardiovascular diseases. Rats fed with flaxseed oil experienced a similar effect and showed a reduced risk of development of atherosclerosis (Ali et al., 2017). Type 2 diabetes is a condition where the body becomes resistant to insulin and/or cannot produce enough insulin or the insulin produced does not work effectively on the cells therefore reducing the intake of glucose which results in high levels of blood glucose. More than 80% of diabetes cases is type 2 diabetes and this is a condition believed to be able to manage through lifestyle and incorporation of regular physical activities, balanced diet, and weight loss. While the link between ALA and type 2 diabetes is unclear, Wu et al. (2009) performed a systematic review and showed that the consumption of fish and seafood was not associated with type 2 diabetes and more interestingly suggested that ALA may be associated with modestly lower risk of diabetes. Fatty liver disease is a complication of diabetes and insulin resistance obesity. It is nowadays a chronic metabolic disorder affecting millions of people across the world and fortunately is possible to reverse through lifestyle changes such as weight loss. Murase et al. (2005) investigated the effect of diglycerides on lipid accumulation in rats and found that hepatic triglyceride accumulation was significantly reduced via consumption of diglycerides containing ALA. More recently Medina-Urrutia et al. (2020) showed that the consumption of chia seeds by nonalcoholic fatty liver disease patients increased the plasma concentration of ALA to 75% and further observed a decrease in total cholesterol, nonehighdensity lipoprotein cholesterol, and free fatty acids.

15.9 Conclusion ALA is a nutraceutical with health benefits that have been so far underestimated. Its importance in the biosynthesis of EPA and DHA has been well studied to date. The consumption of food rich in ALA has proven to be beneficial against noncommunicable diseases such as metabolic diseases, inflammatory diseases, and cardiovascular diseases, among others. However, the mechanism of action of ALA and its specific role is still unclear. Substantial research needs to be completed to further reduce the gap in knowledge in the interaction of ALA with other metabolites and biological molecules in the body to provide a better understanding of the ways ALA is behaving as a nutraceutical.

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Zanqui, A. B., da Silva, C. M., de Morais, D. R., Santos, J. M., Ribeiro, S. A. O., Eberlin, M. N., Cardozo-Filho, L., Visentainer, J. V., Gomes, S. T. M., & Matsushita, M. (2016). Sacha inchi (Plukenetia volubilis L.) oil composition varies with changes in temperature and pressure in subcritical extraction with n-propane. Industrial Crops and Products, 87, 64e70. https://doi.org/10.1016/j.indcrop.2016.04.029 Zhang, Z. S., Liu, Y. L., & Che, L. M. (2018). Optimization of supercritical carbon dioxide extraction of Eucommia ulmoides seed oil and quality evaluation of the oil. Journal of Oleo Science, 67(3), 255e263. https://doi.org/10.5650/jos.ess17153 Zhou, B., Lin, C., Xie, S., Zhou, X., Zhang, F., Ye, X., Lin, F., Hu, L., & Huang, A. (August 2019). Determination of four omega-3 polyunsaturated fatty acids by UPLC-MS/MS in plasma of hyperlipidemic and normolipidemic subjects. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 1126e1127, 121762. https://doi.org/10.1016/j.jchromb.2019.121762

Chapter 16

Ascorbic acid Bharti Mittu1, Zahid Rafiq Bhat5, Ashish Chauhan1, Jasmeet Kour2, Anindita Behera3 and Mahaldeep Kaur4 1

National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur, Punjab, India; 2Department of Food Engineering and

Technology, Sant Longowal Institute of Engineering and Technology, Sangrur, Punjab, India; 3School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan Deemed to be University, Bhubaneswar, Odisha, India; 4Department of Microbial Biotechnology, Panjab University, Chandigarh, India; 5

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, Sangrur,

Punjab, India

16.1 Introduction Ascorbic acid is commonly known as Vitamin C. In 1928 Szent-Gyorgyi isolated ascorbic acid from the adrenal cortex. He also demonstrated the reducing property of ascorbic acid and named this compound “hexuronic acid” (Szent-Gyorgyi, 1928). The chemical structure of vitamin C being hexonic acid aldono-1,4-lactone with an enediol group on C2 and C3 was achieved by Norman Haworth in 1933 (Haworth & Hirst, 1933). It is also called L-ascorbic acid, a powerful antioxidant, and free radical scavenger that protect our tissues, cell membranes, and DNA from oxidative damage. It is found to be stable in its dry condition but it quickly oxidizes in solution form on exposure to heat or light. It is an essential micronutrient and a key element for the metabolism of almost all living organisms. It is abundantly available among animals and plants naturally as they have the capability to biosynthesize ascorbic acid. Vertebrates such as mammals, reptiles, and birds are capable of synthesizing ascorbic acid; only a few species of animals such as guinea pigs, human beings, and primates require ascorbic acid in their diet (Hornig et al., 1975). Humans are unable to synthesize vitamin C endogenously due to the lack of an enzyme, L-gulono-gamma-lactone oxidase (Sheraz et al., 2011). It acts as an essential cofactor and electron donor during collagen hydroxylation that boosts the maturation of intracellular and extracellular collagen production. It also protects the body against environmental stress and is used for repairing and growth of bodily tissues and is also involved in protein metabolism. Its deficiency causes scurvy and capillary fragility, which results in weakness of the tissues and collagens an essential dietary component (Li et al., 2007).

16.2 Sources/derivatives The human body is unable to synthesize and store ascorbic acid. Ascorbic acid must be consumed in the diet. Therefore, it is important to include an adequate amount of ascorbic acid in our daily diet from exogenous supplements or sources. The current daily value (DV) for vitamin C is 90 mg. The bioavailability of ascorbic acid from food is assumed to be very high; Kakadu plums being the richest source contain 5300 mg of ascorbic acid per 100 g. The other sources include red acerola cherries, rose hips, fruits and vegetables, citrus fruits, tomatoes, and potatoes. Other food sources include red and green peppers, kiwifruit, broccoli, strawberries, brussels sprouts, and cantaloupe. It is not naturally present in grains instead it is added to some fortified breakfast cereals. Consuming five varied servings of fruits and vegetables a day can provide more than 200 mg of vitamin C. According to the Food and Drug Administration (FDA), one serving of any of the foods mentioned above contains more than 20% of the recommended DV of vitamin C. Along with this, it is also found in dietary supplements in the form of nutraceutical. Nutraceuticals are oral dietary components naturally found in foods believed to have medical or health benefits. The global vitamin C production is currently estimated at 11,000 tons annually. Supplements typically contain vitamin C which has equivalent bioavailability to that of naturally occurring ascorbic acid in foods, such as orange juice and broccoli (Segall & Mayono, 2008; Carita et al., 2020). Other forms of vitamin C supplements include sodium ascorbate, calcium ascorbate, other mineral ascorbates, ascorbic acid with bioflavonoids, and combination products, such as Ester-C, which contains calcium ascorbate, dehydroascorbate, calcium threonate, Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00015-6 Copyright © 2022 Elsevier Inc. All rights reserved.

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TABLE 16.1 Sources of ascorbic acid and its content. S. No.

Source

Content per 100 gm

1

Kakadu plums

5300 mg (Richest source)

2

Red acerola cherries

1677 mg

3

Rose hips

426 mg

4

Green chili peppers

242 mg

5

Guavas

228 mg

6

Yellow peppers

183 mga

7

Blackcurrants

181 mg

8

Thyme

160 mg

9

Parsley

133 mg

10

Mustard spinach

130 mg

11

Kale

120 mg

12

One medium kiwi

71 mg

13

Broccoli

89 mg

14

Brussels sprouts

85 mg

15

Lemons

77 mg

16

Lychees

72 mg

17

American persimmons

66 mg

18

Papaya

62 mg

19

Strawberries

59 mg

20

Oranges

53 mg

a

The highest vitamin C content.

xylonate, and lyxonate. The vast applications of ascorbic acid in the food industry, pharmaceutical industry, and cosmetic industry have led to quantification, identification, and qualification of ascorbic acid and enhanced its importance among researchers, pharmaceutical, and food industries as well. Content of ascorbic acid per 100 gm of sources has been calculated in Table 16.1 (Nermin et al., 2018). Ascorbic acid cannot be synthesized in the human body due to the absence of an enzyme that converts glucose to ascorbic acid. Ascorbic acid can be easily oxidized to form dehydroascorbic acid (DHAA) while can also be reversed easily (Groff et al., 1995). Thus, it is more important to derivatize the ascorbic acid and few such derivatives of ascorbic acid are mentioned in Table 16.2.

16.3 Methods of extraction and characterization Ascorbic acid is a water-soluble vitamin having the chemical formula C6H8O6. It plays a crucial role in human health and involving several physiological functions of the human body. It is associated with health risks considering its deficiency and excess in the body. Thus it is important to establish the approaches for monitoring the concentrations of vitamins in different matrices. This vitamin is not formed in the human body due to the absence of an enzyme that converts glucose to vitamin C. This vitamin can be easily oxidized to form DHAA while can also be reversed easily (Groff et al., 1995). As vitamin C requirement can’t be fulfilled by the human body, it needs to be supplied from diet or supplements (Nermin et al., 2018; Shrikhande et al., 1974). However, an overdose of vitamins is also not recommended and can be toxic as well. In order to take vitamins in an adequate amount, it is necessary to have some methods in place for the extraction and characterization of different vitamins. Different methods have been developed for the extraction and characterization of vitamins. Sample preparation for analysis of water-soluble vitamins involves extraction using diluted HCl solution or methanol. Ascorbic acid was found to be highly soluble in methanol than in an aqueous solution. Liquid chromatography has been employed for the quantification of total vitamin C in several food samples. The method is employed for ascorbic acid

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TABLE 16.2 Various derivatives of ascorbic acid. S.No.

Derivatives

Functions

References

1

Ascorbic acid (AA)

Strong reducing agent, antioxidant agent, synthesize collagen

Elmore (2005)

2

Sodium ascorbyl phosphate (SAP)

Used in cosmetic products for UV protection, collagen production, antioxidant action, and skin lightning and brightening effects also enhance hepatocyte growth factor production

Tsao et al. (1990)

3

Magnesium ascorbyl phosphate (MAP)

For suppression of skin pigmentation, whitening of dark skin, boost collagen synthesis of skin as antiaging agent, exfoliating effect

Kameyama et al. (1996)

4

Ascorbylpalmitate (AA-PAL)

Antimutagenic, antineoplastic, antioxidant role

Shimpo et al. (1996)

5

3-O-Ethyl ascorbate (EAC)

Antioxidant activity, reducing skin darkening after UV exposure, promotion of growth of nerve cell decreases the damage due to chemotherapy

Sitren (1987)

6

Ascorbylglucoside (AA-2G)

Inhibits the melanin production, helps in production of collagen

Butwong et al. (2020)

extraction using 3% metaphosphoric acideacetic acid and relatively its oxidation to DHAA. Ultrasound-assisted extraction is employed for the extraction of vitamin C. It is a less time-consuming method that gives the highest yield (Hong & Van, 2012; Verma et al., 2020). Moreover, supercritical fluid extraction is yet another beneficial approach for its extraction. It works on the solvent power and density relation of the solute and the solvent. Moreover, it is also beneficial for thermolabile substances (Pellicano et al., 2019). Similarly, dispersive liquideliquid microextraction (DLLME) has also been employed for the extraction of ascorbic acid after oxidationereduction reaction with methylene blue (Zhang et al., 2018) (Fig. 16.1).

FIGURE 16.1 Methods of extraction of ascorbic acid.

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TABLE 16.3 Methods of characterization of ascorbic acid. S.No.

Characterization technique

Features

References

1

Capillary electrophoresis

Analytical and quantitative method, separates sample components according to their sizes and charge, fast, high separation efficiency

Voeten et al. (2018)

2

High-performance liquid chromatography

Analytical method, high selectivity and sensitivity, used for quantification of different vitamins

Noh et al. (2020)

3

Reversed-phase high-performance liquid chromatography

Determination of water-soluble vitamins

Noh et al. (2020)

4

High-performance liquid chromatography etandem mass spectrometry

Detection of vitamins

Noh et al. (2020)

5

Microbiological assays

Perceived by global authority who established this method as the best quality level for a long time, improved precision, and accuracy

Zhang et al. (2018)

6

Biosensors

Excellent tool for detecting vitamins in different matrices is optical biosensors. This technique has been associated with simplicity, low cost, and its application in field analysis

Zhang et al. (2018)

Ascorbic acid is most often characterized by reversed-phase HPLC. The mobile phase system used for watersoluble vitamins involves combinations of methanolewater in various ratios along with triethylamine or ion-pair reagents (sodium heptylsulfonate, sodium octylsulfonate, or sodium dodecyl sulfate). However, it involves several drawbacks in resolution, dead retention volume, and low pH that causes increased degradation and dissolution of the silica-based analytical column. Thus, it is necessary to adjust the pH of the mobile phase below the pKa of L -ascorbic acid, i.e., 4.17, to stop the column degradation (Zhang et al., 2018). Likewise, ultrahighperformance liquid chromatography has also been employed for the quantification of vitamin C in fruits and vegetables which is a rapid, sensitive, and reproducible method (Noh et al., 2020). Moreover, the other analytical and quantitative method like capillary electrophoresis is used to quantify ascorbic acid according to their sizes and charges. This technique is fast, associated with high separation efficiency. It can analyze several samples concurrently in multicapillary systems (Voeten et al., 2018). Moreover, an excellent tool for detecting vitamins in different matrices is optical biosensors. This technique has been associated with simplicity, low cost, and its application in field analysis (Zhang et al., 2018). Electrochemical method and microfluidic device are the two detection methods of vitamins and traces of vitamins B1, B2, and C, respectively. One such example of this method is an electrochemical synthesis of poly(3,4-ethylenedioxythiophene)ezirconia nanocomposite for the examination of vitamins B2, B6, and C (Baghizadeh, 2015). Different characterization techniques used for ascorbic acid are enlisted in Table 16.3.

16.4 Chemistry The generic name of L-ascorbic acid is vitamin C which is a freely water-soluble vitamin (300 g/L at 20
C). It has numerous chemical names such as ascorbate. It is made up of asymmetrical six-carbon atoms (C6H8O6) which is structurally correlated to glucose. The molecular weight of this vitamin is 176.12 g/mol having a melting point of 190e192
C and having a density of 1.65 g/cm3. It shows a density of approximately 1.65 g/cm3. It is difficult to solubilize in alcohol (20 g/L at 20
C) and is not soluble in chloroform, ether, and benzene. It has two pKa values: 4.2 and 11.6. The pH of a 5% (w/v) solution in water is 2.2e2.5. It acts as an antioxidant due to its high reducing power. It is also used as food additives, thereby preventing the deterioration of food, and is also used to improve the color and baking property of flour or dough by acting as an additive (Fig. 16.2). This vitamin is not formed in the human body due to the absence of an enzyme that converts glucose to vitamin C. This vitamin can be easily oxidized to form DHAA while can also be reversed easily (Groff et al., 1995). Thus, it is more important to derivatize the ascorbic acid and few such derivatives of ascorbic acid are mentioned in Fig. 16.3.

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FIGURE 16.2 Conversion of ascorbic acid to dehydroascorbic acid.

FIGURE 16.3 Important derivatives of ascorbic acid.

16.5 Mechanism of action Vitamin C has remarkable actions with a broad spectrum of mechanisms. It holds a unique position among the vitamins. The roles of vitamin C are getting more transparent and clear with advanced research and have delineated its significance in biological systems. Vitamin C facilitates the structural process of tissue repair along with collagen formation, involved in oxidationereduction reactions as well as other metabolic reactions. Vitamin C exerts its reducing action due to the presence of the enediol group (Harris, 1953). Endothelial cells contain a sodium-dependent transport system across the membrane that carries ascorbate ions into these cells while DHAA is transferred through facilitative glucose that is further reduced to ascorbate. However, the efflux of ascorbate could be enhanced by the calcium-dependent mechanisms which are able to retain intracellular concentration at a much higher rate than the extracellular one. Reports suggest that during sepsis inflammatory cytokines resist ascorbate uptake in endothelial cell culture. Moreover, ascorbate deficiency is also caused in endothelial cells due to acute level of hyperglycemia in septic patients and impairs endothelium-based modulation. Ascorbic acid has key roles to play in the brain, immune system, sepsis, and bone (Wilson, 2009). All the concerned organs occupied different modes of action (Wolbach & Howe, 1926). Interestingly, cessation in bone formation and new tooth formation was first reported on guinea pigs (Harris, 1953). Similarly, in human beings, the impact of ascorbic acid is more on the cessation of osteogenesis. Aghajanian et al. (2015) analyzed several epidemiological studies and genetic mouse models regarding the effect of vitamin C show a positive effect on bone health (Aghajanian et al., 2015). There are wellknown functions of an ascorbic acid described in Table 16.2 (Kuiper & Vissers, 2014). Certain kinds of recurrent infections result from the genetic disorder of neutrophil function such as rare autosomal deficiency syndrome, defective leukocyte

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FIGURE 16.4 Mode of action of ascorbic acid.

generation, and ChediakeHigashi syndrome. The dosage of ascorbic acid stabilizes the microtubules via the mechanism of phagocytosis as represented in Fig. 16.4 (Boxer et al., 1979).

16.6 Bioavailability of ascorbic acid Like ADME (absorption, distribution, metabolism, excretion) and toxicology of a drug or an active pharmaceutical or nutrient, the bioavailability of an active constituent is increasingly gaining importance in therapeutic efficacy. This is one of the prime factors in drug development and drug discovery. As per the definition by US FDA, bioavailability is “the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed from a drug product and becomes available at the site of drug action.” Generally, bioavailability is measured by determining the area under a curve, or AUC. AUC is a technique of quantitative measurement of the bioavailability of a drug and determined with a plot of concentrations of the drug in blood analyzed at frequent intervals. AUC and concentration of unaltered drugs in the patient’s blood are directly proportional (Allam et al., 2011; Veber et al., 2002). The bioavailability of vitamin C in humans is highly complex. As vitamin C is a micronutrient, its bioavailability (in drug, dietary forms, or formulations) can be represented by the amount absorbed by the intestines and available for different metabolic processes in the human body. Vitamin C levels in vivo are majorly influenced by its pharmacokinetic processes. Active transport of vitamin C into the body has been found to occur via SVCT1 and SVCT2 which are the two sodium-dependent vitamin C transporters (Łukawski et al., 2020; Savini et al., 2008). Interestingly, SVCT1 and SVCT2 also exhibit different affinity for their distribution in various tissues and expressions which influence the rate of uptake of vitamin C. Moreover, one of the transporters (SVCT1) found in epithelial cells/tissues is accountable for uptake by the intestine and renal reabsorption of vitamin C when ingested orally. It helps in maintaining the homeostasis of body homeostasis by preventing excess urinary loss (Savini et al., 2008; Wohlrab et al., 2017). In addition, another transporter, SVCT2 shows different expression and distribution patterns, i.e., specialized and metabolically active tissues. SVCT2 delivers ascorbic acid at the sites where the requirement of this vitamin is for some enzyme-mediated reactions or to protect the tissues from oxidative stress (Savini et al., 2008). Ascorbic acid acts as a reducing agent and for the antioxidant activity; it undergoes one- and two-electron oxidations and gets converted to ascorbyl radical and DHAA. Accumulating shreds of

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evidence have reported that DHAA can be transported to the small intestine by the facilitative glucose transporters GLUT2 and GLUT8 (Corpe et al., 2013). Whereas, DHAA is internalized into the cells by GLUT1 and GLUT3 followed by reduction to ascorbate intracellularly (Corti et al., 2010). Interestingly, the pharmacokinetics has shown variations in the level of vitamin C in blood and rate of excretion by urine after intake of the vitamin Cecontaining test substance. Maximal plasma levels (Cmax) of vitamin C are attained about 2 h after ingestion. Moreover, an early animal study has reported that Cmax of vitamin C provided in citrus fruit media is delayed as compared to synthetic source but the bioavailability of vitamin C in citrus fruits is more than the synthetic source (Lykkesfeldt & Tveden-Nyborg, 2019). In addition to this, a comparable trend was observed in a clinical trial where the subjects are supplemented with 500 mg of vitamin C with or without a citrus fruit extract (Vinson & Bose, 1988; Padayatty et al., 2004). The citrus fruit extract showed a much similar trend in the fact that it achieved the maximal plasma levels late by 1 h but the bioavailability of vitamin C increased by 35%. The citrus fruit extract increased the urinary excretion of vitamin C by 24 h in the presaturated but decreased in nonsaturated patients than the patients treated with only a synthetic source of vitamin C. So the baseline status of vitamin C affects the bioavailability of vitamin C. In two other studies, the urinary excretion of vitamin C is increased in presaturated patients with a source of fruit juice (Levine et al., 1998; Lykkesfeldt & Tveden-Nyborg, 2019). Another presaturation study showed the effect of bioflavonoids on the plasma levels and 24 h urinary excretion of vitamin C (Johnston & Luo, 1994). The intestinal bioavailability of a dose of 500 mg of vitamin C is lesser as the same quantity cannot be available through a normal daily diet (Padayatty et al., 2004). Pharmacokinetic studies have indicated that the relative availability of vitamin C from synthetic sources or natural form in foods or fruit juices (Kondo et al., 2012; Uchida et al., 2011). An intestinal triple lumen tube perfusion model was chosen by Nelson et al. for a comparative study of absorption of vitamin C from the synthetic origin and a natural source like orange juice solution. The study measured the intraluminal events and no difference was found between the two test solutions (Nelson et al., 1975). Some pharmacokinetic studies have reported the decreased Cmax and urinary excretion of vitamin C in presence of food and fruit juices but the differences are minimal (Kondo et al., 2012; Uchida et al., 2011). A study suggests that the bioavailability (plasma levels) of synthetic versus a natural source of vitamin C from kiwifruit in nine nonsmoking male individuals of 18e35 years had optimized levels of plasma vitamin C (>50 mM) (Carr et al., 2013). The individuals were given either a chewable tablet of vitamin C (200 mg) or an equal dose of crude kiwifruit. After an intervention, fasting blood and urine levels of vitamin C were checked after half an hour, then an hour, and subsequently for 8 h. Ascorbate level in plasma increased after 30 min from the intervention but no significant differences in the AUC were observed between the two interventions. The net increase in the level of vitamin C ensures the indicated complete absorption of the ingested ascorbate tablet and vitamin C derived from kiwifruit. Similarly, there was a proportional increase in excretion of vitamin C by urine as compared to creatinine after 2 h. A significant difference between these two groups was found with respect to the amount of excretion of ascorbate in the urine. About w40% of the ingested dose of the tablet and w50% of the vitamin C derived from kiwifruit were reported. So the pharmacokinetic parameters showed comparable bioavailability of vitamin C from the natural source and synthetic source (Carr et al., 2013; Lykkesfeldt & Tveden-Nyborg, 2019). For general consumption dose of vitamin C up to 2000 mg/day is considered safe (Hathcock et al., 2005). However, smaller doses of vitamin C are better than taking larger doses as the reports from pharmacokinetic studies indicated that the bioavailability of a single dose of ascorbic acid greater than 200 mg has lesser bioavailability (Levine, 1996). So ingestion of the number of smaller doses is more bioavailable than a single larger dose. The bioequivalence study established that the relative bioavailability of vitamin C from different tablet formulations is different as the slow-release formulations provide superior vitamin bioavailability than the immediate release (Padayatty et al., 2004). The sodium and calcium salt forms of vitamin C are also being evaluated. Interestingly, in preclinical studies, the calcium form was absorbed more rapidly and excreted slowly as compared to natural vitamin C (Bush & Verlangieri, 1987). Additionally, the calcium form is better tolerated in individuals sensitive to acidic foods (Gruenwald et al., 2006) (Fig. 16.5).

16.7 Stability, safety, and toxicology The ascorbic acid content in different sources like fruits, vegetables, pharmaceutical products, and cosmetics decreases with an increase in temperature and pH (Emese & Nagymate, 2008). The third most commonly consumed vitamin C has been taken in the form of a supplement by a majority of adults. Thus it is necessary to regulate the stability of vitamin C in foods and supplements. It is added to food in its salt forms. It is majorly used in several formulations like creams, cosmetic products with alpha-tocopherol. A substantial content of vitamin C in food gets diminished while storage and preparation. The stability of this vitamin in different formulations is a critical issue and should be taken care of while manufacturing and while storage of products.

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FIGURE 16.5 Bioavailability of ascorbic acid.

TABLE 16.4 Physical factors influencing stability of ascorbic acid. Physical factors

Influence

References

Moisture

Moisture shows a key role in affecting degradation rates of ascorbic acid, and also causes discoloration of solid form of ascorbic acid

Tsao et al. (1996)

Air and light

Upon exposure to air and light, ascorbic acid converts to dehydroascorbic acid

Sheraz et al. (2015)

pH

Alkaline environment have been rapidly affecting the oxidation of ascorbic acid

Yuan and Chen (1998), Buettner and Jurkiewicz (1996)

Temperature

Temperature fluctuations affected the concentration

Jeney-Nagymate & Fodor (2008)

Major factors influencing the stability of ascorbic acid and degradation patterns are temperature, sunlight, moisture, oxygen, pH, and viscosity. It is also catalyzed by metal ions, particularly Cu2þ, Fe2þ, and Zn2þ. Ascorbic acid breaks down with time in tablets and syrups and even in the pure powder during storage. All the formulations of ascorbic acid including the pure powder under the various storage conditions demonstrate that the concentration of ascorbic acid in the various products reduced with time. Storage under refrigeration gave the highest stability, and minimized breakdown. Physical factors affecting the stability of ascorbic acid have been discussed in Table 16.4.

16.7.1 Toxicity Ascorbic acid has low toxicity and is not associated with any serious adverse effects even at a higher level of consumption. The common adverse effects are due to the osmotic effect of unabsorbed vitamin C in the gastrointestinal tract, thus causing diarrhea, nausea, abdominal cramps, and other gastrointestinal disturbances (Jacob & Sotoudeh, 2002). The highest limit of intake of vitamin C is 2000 mg/day. Vitamin C worsens the state of patients having high iron load (Slivaka et al., 1986; Nermin et al., 2018), thereby resulting in tissue damage (Jacob & Sotoudeh, 2002). The other adverse effects that are associated with the high intake of this vitamin include reduced vitamin B12 and copper levels, metabolism, and allergic responses (Wyngaarded, 1987; Nermin et al., 2018). Enamel erosion resulted in the usage of the unbuffered form of ascorbic acid (Flemming et al., 2002). Vitamin C also acts as a prooxidant, thereby causing oxidative damage under certain conditions. Earlier reports suggested as a prooxidant it results in chromosomal and/or DNA damage, thereby causing cancer development (Lee et al., 2001). An intake of 1 g/day for a period of 3 months resulted in the generation of

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the stones in kidney (Alkhunaizi and Chan, 1996; Baxmann et al., 2003). High vitamin C intakes can also increase urinary oxalate and uric acid excretion, thus causes to the formation of kidney stones in patients with renal disorders (Nermin et al., 2018).

16.8 Applications of L-Ascorbic acid and its health benefits 16.8.1 Clinical and pharmacological relevance 16.8.1.1 Role in cardiovascular disease Ascorbic acid protects the cardiovascular system by decreasing the systolic blood pressure, thus improving heart functioning (Chen et al., 2013; Roberts, 2009; Sesso, 2008). It keeps the arteries flexible and strengthens the blood vessel wall. It prevents damage by a heart attack, peripheral artery disease, and stroke by decreasing the chances of atherosclerosis (Ashor et al., 2014).

16.8.1.2 Role in biosynthesis L-Ascorbic acid is essential for the body to synthesize collagen for the cartilage as reported by Ballaz and Rebec (2019). Ascorbic acid promotes wound healing by stimulating the formation of the epidermal barrier. It increases collagen synthesis, wound closure time, and decreases the inflammatory responses of the wound in a healthy person (Charlesworth, 2019; D’Aniello, 2017; Sato, 2017).

16.8.1.3 Role as antioxidants L-Ascorbic

acid performs an important role in protection against oxidative stress in numerous tissues as discussed (Abraham, 2014). It acts as an antioxidant and resists the damage caused by free radicals. Intake of diets rich in vitamin C reduces the risk of arthritis. It has been observed that higher content of ascorbic acid in joints shows higher protection level against the damage (Carr & Frei, 1999). Ascorbic acid is a strong antioxidant that can increase blood antioxidant levels in the body. This process can lessen the risk of chronic diseases like heart disease (Ljiljana et al., 2015).

16.8.1.4 Role as an antiaging agent The antiaging effect of ascorbic acid pertains to its potent antioxidant effect that stimulates collagen formation and refrains damage and wrinkles (Harrison, 2012). Vitamin C in combination with other antioxidants like beta-carotene, vitamin E, and zinc protects the eyes and vision from degeneration that leads to blindness. Maintaining a healthy level of vitamin C shows a protective function against age-related cognitive dysfunction, Alzheimer’s disease, gout, inflammation, and preand postsurgery pain (Hansen et al., 2014; Marzocchella et al., 2011). Aging of the skin is a very common problem, which can be seen by signs such as wrinkles, sagging of skin, uneven skin tone, and dryness. In a recent evaluation improvements in skin integrity, aging, pigment appearance on the skin have been seen with the synergistic antiaging effects of vitamin C and other ingredients. Topical products vitamins C with E have shown protective effects in the process of aging as human skin needs water-soluble as well as lipid-soluble nutrient components (Pattarawan et al., 2020).

16.8.1.5 Role as an anticancer Population-based studies evaluated that intake of foods rich in vitamin C lowers the rate of cancer associated to the skin, cervical dysplasia, and breast. Vitamin C plays an eminent role in protecting cellular integrity as a scavenger, preventing oxidation of cellular protein, lipid, and DNA. L-Ascorbic acid is one of the nutritive nonenzymatic antioxidants (Cadeau, 2016).

16.8.1.6 Role in immunity Ascorbic acid participates in immune defense by improving the cellular functions of both the innate and adaptive immune systems. It controls the epithelial barrier layer against pathogens. It is accumulated in the phagocytic cell-like neutrophils to enhance chemotaxis, phagocytosis, reactive oxygen species that kill microbes. It is also required for apoptosis and clearance of macrophages. Ascorbic acid deficiency renders impaired immunity and higher susceptibility to infections (Schmidt et al., 2011). In a recent study on COVID-19 patients, the administration of ascorbic acid increased the chances of survival of COVID-19 patients by attenuating increased activation of the immune response. Ascorbic acid enhances antiviral cytokines and free radical formation, suppressing viral yield. It also attenuates maximum inflammatory responses

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and hyperactivation of immune cells. Vitamin C is considered an antiviral agent as it increases immunity. A daily allowance of vitamin C can enhance nutritional defense that can be beneficial in patients at risk of or diagnosed with coronavirus disease 2019 (Minkyung & Hyeyoung, 2020).

16.8.1.7 Role in lipid metabolism L-Ascorbic

acid has a great influence on the lipids existing either in intra- or extracellular form. Vitamin C improves the metabolism of the lipid by restricting the oxidation of the unsaturated lipids and lipoproteins (Alul et al., 2003).

16.8.1.8 Role in protection from heavy metals toxicity L-Ascorbic acid facilitates the heavy metal absorption from the intestine such as iron while lowering levels of lead in blood (Dawson et al., 1999). It plays a significant role in the synthesis of carnitine, an enzyme that acts as a cofactor to increase the absorption of nonheme iron in the gastrointestinal tract. It also increases the formation of reduced iron favorable for absorption (Teucher et al., 2004).

16.8.1.9 Role in endocrinology system L-Ascorbic acid contributes to the development of several hormones like adrenaline, endogenous serotonin, and hydroxylation of aromatic compounds in the liver (Anderson et al., 1980; Schmidt et al., 2011).

16.8.1.10 Role in preventing health disorders L-Ascorbic acid, if taken orally, improves a genetic disorder in newborns in which blood levels of the amino acid tyrosine are too high. Vitamin C supplements also help in managing anemia in people undergoing dialysis. Vitamin C supports reduction in symptoms of exercise-induced asthma, eczema, and allergic rhinitis (Finkelsteinn et al., 2011; Frikke-Schmidt et al., 2011; Michiels et al., 2010).

16.8.1.11 Deficiency disorders of ascorbic acid Ascorbic acid has immense health benefits (Fig. 16.6). Scurvy is a deficiency of ascorbic acid that results in absence of healing of a wound and fractured bones due to collagen deformation. Other symptoms of scurvy are body weakness, legs, and arms edema, nose, skin, and gums hemorrhage, infections, vasculitis, bone and cartilage damage, internal bleeding, anemia (Finkelsteinn et al., 2011; Frikke-Schmidt et al., 2011; Michiels et al., 2010).

FIGURE 16.6 Health benefits of ascorbic acid.

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16.8.2 Other commercially viable applications 16.8.2.1 Role in bakery acid is widely used as an additive to the flour. It enhances a number of desired features such as strengthened gluten, greater loaf volume, finer crumb, tenderness to the crumb, a faster-rising loaf for quicker baking. The utilization of ascorbic acid in bakery contributes to higher profit yields (Sahi, 2014; Zhou et al., 2014).

L-Ascorbic

16.8.2.2 Role in water treatment and purification L-Ascorbic

acid combination with sodium ascorbate serves as a useful means to neutralize chlorinated water than other sulfur-based chemical methods and makes chlorinated water safe for drinking (Urbansky et al., 2000; Gao et al., 2020).

16.8.2.3 Role as a beverage and health drink L-Ascorbic

acid as a natural additive in juice and beverage enhances the nutritional value of the beverage without interference to its taste while abstaining from its spoilage (Maryam et al., 2012).

16.8.2.4 Role in the meat industry as a preservative L-Ascorbic

acid’s primary function in the meat environment is to prevent its oxidation (Kanatt et al., 2018; King et al., 2016; Lee et al., 2018).

16.8.2.5 Role in fruit preservation The use of ascorbic acid slows down the oxidation with its low pH. The ascorbic acid helps in the prevention of microbial growth. Ascorbic acid resists browning, which preserves the freshness of fruits as studied by Nagy and Smoot (1977) and Roig et al. (1995).

16.8.2.6 Role in personal care and cosmetics L-Ascorbic

acid regulates the synthesis of the structural protein collagen and hydroxylation of collagen molecules. It has been shown to stabilize collagen mRNA for collagen protein synthesis of the damaged skin. It plays a vital role in photoprotection, wrinkling, and wound healing so it is preferred in skin care lotions, creams, and cosmetics (Soledad et al., 2019).

16.8.2.7 Role in the manufacturing industry L-Ascorbic

acid is used in a wide range of industrial applications. It helps in masking the taste of iodine in sterilized and potable water. It is used in manufacturing plastic (Oster & Fechtel, 2012; Pizzocaro, 1993; Rahman, 2007).

16.9 Conclusion L-Ascorbic

acid is an abundant multifunctional molecule that serves as an essential nutrient for the growth and development of plants and animals including man. It is a naturally occurring, water-soluble, potent reducing, and antioxidant agent that has numerous commercial, biological, and nutraceutical applications. The role of vitamin C in providing better esthetics exhibits immense significance. Due to its protective role, the supplementation of vitamin C serves as a prerequisite to sustaining life with a significant rise in pollution. It is vital in improving immunity by fighting infections and detoxifying reactions. It renders the formation, maintenance, and repair of collagen in fibrous tissue, teeth, bones, connective tissue, skin, and capillaries. It is a cofactor for enzymes biosynthesis of collagen, carnitine, and neurotransmitters that can quench a variety of reactive oxygen and nitrogen species in aqueous environments. The therapeutic use of ascorbic acid includes prevention of ascorbic acid deficiency in a patient at risk, in infants, treatment of scurvy, anemia, and acidifying the urine in urinary tract infection. It acts as a hypertensive and hypocholesterolemic agent. It helps in the reduction of cold and age-related health problems. Adequate amount intake of vitamin C prevents from breast, cervix, and colon cancers. It is recommended to have an intake of 40 mg per day as per India National Institute of Nutrition, Hyderabad, and 45 mg per day or 300 mg per week as per the World Health Organization. Vitamin C gets destroyed by heat and light. Smokers, patients with kidney disease, heredity iron overload disorder patient should use ascorbic acid wisely. L-Ascorbic acid has immense potential in the biomedical application that still remains unnoticed. It is speculated that young scientists would take up ascorbic acid as challenge for research to explore and expedite remarkable achievements.

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Chapter 17

Phenolic acids Md Nazmus Saqib1 and Md Ramim Tanver Rahman2 School of Food Science & Technology, Jiangnan University, Wuxi, Jiangsu, China; 2Medicinal Chemistry Laboratory, Centre Hospitalier

1

Universitaire (CHU) de Québec Research Center, Université Laval, Quebec, QC, Canada

17.1 Introduction Phenolic compounds, including phenolic acids, are secondary metabolites from plants and fungi produced from the primary metabolites (carbohydrates, amino acids, lipids). These compounds are produced in plants and fungi to protect against UV light, insects, viruses, and bacteria. Even certain plant species are known to develop unique phenolic compounds to inhibit other plant competitors’ growth. It is presumed that phenolic compounds were fundamental for plants to conquer their terrestrial environment, such as lignin which stimulates the vascular system’s development, giving stiffness to the vessels (Mitsunaga et al., 2009). However, the phenolic acids, one of the largest groups of phenolic compounds, are plant phenolics having a carboxylic acid (dCOOH) group. They are available in almost all plant-derived foods and exhibit a substantial antioxidant property. Their structure, biological, and epidemiological activities are described in the latter part of this chapter.

17.2 Classification and chemical structure of phenolic acids Phenolic acids are produced in different plant maturation stages depending on their variety, physiology, and environment. The characteristics, quantity, and type of these acids also altered during different maturation stages, processing, and environmental factors. Phenolic acid can be exemplified with alcohol, where there’s an aromatic ring instead of aliphatic carbon. The hydroxyl group’s presence in the benzene ring makes them proton donators; thus, they are considered weak acids. This proton donation ability and the hydroxyl group’s variable position in the ring create a versatile structure with different functionality. As a result, it draws the attention of researchers all around the globe. Phenolic acids can be divided into two distinct groups based on their structure, namely the hydroxycinnamic and hydroxybenzoic structures (Fig. 17.1). The basic structure is familiar with a benzene ring. It’s the number and position of the eOH group that makes the difference. The aromatic ring makes structural changes by methylation and hydroxylation. Other types of phenolic acids that are not very common can be found only in selective sources. Phenolic acids are distinguishable from flavonoids by their structure. Flavonoids consist of multiple aromatic rings, while phenolic acids only have one. The basic flavonoids structure consists of 15 carbon atoms in three aromatic rings. On the contrary, in phenolic acid, variable groups are attached to a single ring (Macheix, Fleuriet, & Billot, 2018). The earliest medical science in human civilization, irrespective of ethnicity, e.g., plant-based medicines in Mesopotamia, Chinese traditional medicine, Ayurveda in South Asia, are all reported to be mainly based on the functionality of plant phenolics. However, physicians of this era don’t have any knowledge about plant phenolics. They learned it by trials and experiences and preserved it from generation after generation (Alves & Rosa, 2007; Ekor, 2014). Apart from the plant sources, Piazzon et al. (2012) described the ex vivo synthesis of phenolic acid for the first time. The complete metabolic pathway or mood of action or contribution toward phenolic acid’s human physiology is still unknown even though it has already been widely used in pharmaceuticals, cosmetics, and food industries (Kumar & Pruthi, 2014; Kumar & Goel, 2019).

Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00014-4 Copyright © 2022 Elsevier Inc. All rights reserved.

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FIGURE 17.1 Groups of phenolic acids.

17.3 Biosynthesis and extraction of phenolic acids Phenolic acids are formed through the Shikimate pathway from aromatic amino acids (L-phenylalanine, L-tryptophan, and L-tyrosine) in most bacteria, fungi, and plants. The biosynthesis of phenolic acids involves several enzymes, methylation, and hydroxylation in the aromatic ring (Marchiosi et al., 2020). Several authors have summarized the biosynthesis in different publications (Vermerris et al., 2008; Marchiosi et al., 2020; Santos-Sánchez et al., 2018). Different extraction procedures (with distinct advantages and disadvantages) have been introduced to extract phenolic acids, such as Soxhlet extraction, liquideliquid extraction, microwave-assisted extraction, ultrasound-assisted extraction, supercritical fluid extraction, and accelerated solvent extraction. The extracted concentration of phenolic acids varies from plant to plant, which also depends on the extraction method, solvents, and the application of hydrolysis (Valanciene et al., 2020; Jitan et al., 2018). Many protocols have been developed to use in UVevisible spectroscopy, infrared spectroscopy, nuclear magnetic resonance, and high-performance liquid chromatography for the purification and identification of phenolic acids.

17.4 Sources Almost all plant species, including vegetables, legumes, fruits, cereal, and seaweeds, constitute phenolic acids distributed through roots, leaves, stems, seeds, and flowers. The amount and type depend on geography, climatic condition, variety, maturity, etc., factors (Macheix, Fleuriet, & Billot, 2018; Macheix, Fleuriet, Billot, Macheix et al., 2018). Their existence as a free acid is sporadic. Instead, they remain conjugate with other structural components like cellulose, lignin, or protein in a plant (Lam et al., 2001; Brett et al., 1990; Andreasen et al., 2000). Phenolic acids are divergently distributed through the whole plant from root to leaf. The same plant showed different distribution depending on season and geography, and the same fruit showed variation in various stages of maturity (Sikorska, Matławska, Głowniak, & Zgórka, 2000; EllnainWojtaszek, Kruczy nski, & Kasprzak, 2001). Again, the different parts of the same fruit have distinguishable cinnamic and benzoic acid content. For example, in the study (Mattila et al., 2006), strawberry in the year 2005 had vanillic acid while it was not detectable in the previous year the same species. In apple, a different degree of phenolic acid was detected between the peel and whole fruit, while two other breeds showed variable results. So, it is difficult to quantify and finalize the exact phenolic acid contents of any plant species. Here, an attempt to summarize (Tables 17.1e17.4) the phenolic acid content of commonly available species based on the currently available literature has been initiated. Different literature followed different methods from the separate timeline, other units, and a further explanation. To simplify, we tried to find similar measuring units and similar techniques. The same research has variation in the same sample data. For better understanding, we excluded all the statistical analyses and did not consider the fluctuation between breeds. Instead, it is expressed as a range. It is highly recommended to follow the original article for any analyzing or citing purpose.

TABLE 17.1 Phenolic acid content of some commonly consumed fruits. Hydroxybenzoic acid (mg/100 g fresh weight)

Hydroxycinnamic acids (mg/100 g fresh weight)

Fruits

p-Hydroxybenzoic acid

Vanillic acid

Gallic acid

Protocatechuic acid

Syringic acid

Cinnamic acid

p-Coumaric acid

Ferulic acid

Sinapic acid

Caffeic acid mg/100 g

Apple

ND

0.0e0.13

0.3e5.4

0.0e1.4

ND

0.4e4.7

0.27e0.80

0e0.1

5.4e18.3

Banana

0.12

0.445

ND

ND

0.22

Bilberry

ND

6.0e6.9

3.2e1.53

1.4e4.2

13.9e15.2

ND

0.46

5.4

ND

0.2

6.1e8.1

1.1e1.2

0.3e05

9.5e10.6

Blue berry

ND

2.0

2.70

2.0

15.6

0.41

1.65

1.29

0.7

59.1

Cherry

0.88

1.17

ND

3.01

0.88

5.1

0.46

ND

17.1

Cranberry

ND

3.1

ND

2.35

0.99

0.49

1.4

0.605

0.52

2.3

Gooseberry (yellow)

ND

0.256

ND

ND

ND

ND

0.11

0.29

0.269

12.1

Grape

ND

0.0e1.07

2.8e3.1

ND

0.0e6.8

1.17e3.8

0.0e0.43

ND

14.8e15.2

Grapefruit

0.44

1.66

ND

0.66

0.98

1.35e3.8

10.7e11.6

0.99

3.245.5

Kiwi

ND

0.19

ND

ND

ND

0.25

0.19

ND

1.5

Mandarin

ND

0.64

ND

ND

ND

0.88

9.24

1.51

6.6

Orange

0.35e0.54

0.44e1.28

ND

ND

ND

1.71e1.81

9.4e9.9

0.8e2.2

3.3e5.6

Peach

0.22

0.25

ND

ND

0.26

0.52

0.11

ND

5

Pear

ND

0.27

ND

ND

ND

0.7

0.29

0.104

6.5

Plum

ND

1.27

ND

ND

ND

2.1

1.47

0.14

23.4

Raspberry

1.64e2.0

1.04

21e22

ND

ND

0.21e0.33

0.9e1.8

0.76e0.94

0.2e0.35

0.691e1.08

Strawberry

4.5e6.3

0e0.24

2.1e5.3

ND

ND

0.54e2.7

2.9e4.5

0.2e0.32

ND

0.171e0.34

Watermelon

ND

0.23

ND

ND

0.86

0.37

0.35

ND

0.12

Phenolic acids Chapter | 17

Blank cell, Data not available; ND, Not detected. Sorted, simplified, and retabulated from Mattila, P., Hellstro¨m, J., & To¨rro¨nen, R. (2006). Phenolic acids in berries, fruits, and beverages. Journal of Agricultural and Food Chemistry, 54(19), 7193e7199. https://doi.org/ 10.1021/jf0615247.

305

TABLE 17.2 Phenolic acid content of some commonly consumed vegetables. Hydroxybenzoic acid (mg/100 g fresh weight)

Hydroxycinnamic acids (mg/100 g fresh weight)

Vanillic acid

Protocatechuic acid

Syringic acid

p-Coumaric acid

Ferulic acid

Sinapic acid

Caffeic acid

Avocado

0.137

0.217

ND

0.157

0.81

1.17

0.97

0.42

Basil

ND

0.137

ND

0.947

0.6

1.5

ND

23.15

Broccoli

0.417

0.257

0.137

ND

0.85

4.1

8.07

1.5

Button mushroom

ND

ND

ND

ND

ND

ND

ND

0.06

Carrot

4.2e5.07

0.98e1.2

ND

ND

0.17

1.17e1.570

ND

15e22

Cauliflower

0.427

ND

0.117

0.327

1.2

0.35

1.8

0.38

Celery root

ND

0.307

ND

ND

0.2

0.81

ND

ND

Chinese cabbage

ND

0.137

ND

ND

0.42

1.4

5.2

0.54

Cucumber

ND

ND

ND

ND

0.08

0.057

ND

ND

Garlic

0.27

ND

ND

ND

0.09

0.63

0.66

ND

Green bean/fresh

0.117

0.497

ND

0.087

1.2

1.2

ND

0.46

Iceberg lettuce

ND

ND

ND

ND

0.09

0.08

ND

4.9

Lettuce

ND

0.0e0.097

ND

ND

0.17e0.25

0.33e1.4

0.0e0.227

20.08e49.1

Onion

0.147

0.067

0.207

ND

0.21

0.08

0.1

ND

Parsley

0.217

ND

ND

ND

5.8

0.23

ND

ND

Parsnip

0.227

0.167

ND

0.767

0.34

2.2

0.2

1.8

Pea/frozen

ND

ND

ND

ND

ND

0.26

0.15

0.13

Peanut

0.98

2.97

ND

ND

77.31

8.770.21

14.03

2.47

Radish

0.937

0.077

ND

ND

5.6

4.6

0.12

1

Red beet

0.057

0.347

0.357

0.517

0.65

25.2

ND

ND

Red cabbage

0.277

0.21

1.07

ND

9.3

6.47

22.18

1.6

Soya bean

1.57

10.7

ND

25.19

12.49

12.002

12

0.33

Spinach/frozen

0.067

0.17

ND

ND

3.1

7.4

ND

ND

Sweet pepper/green

0.157

0.177

ND

ND

2.2

0.37

0.18

1.3

Sweet pepper/red

0.317

0.317

ND

ND

2.4

0.55

0.38

1.2

Sweet pepper/ yellow

0.25

0.267

ND

ND

1.9

0.53

0.32

1.2

Tomato

0.067

ND

0.147

ND

1

0.29

ND

2.07

Turnip

ND

ND

0.097

ND

0.09

0.23

0.84

0.2

White cabbage

0.197

ND

ND

0.117

0.18

0.27

2.8

0.29

Zucchini

ND

0.167

ND

ND

0.32

0.28

ND

0.11

ND, Not detected. Sorted, simplified, and retabulated from Mattila and Hellstro¨m (2007).

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p-Hydroxybenzoic acid

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Vegetables

TABLE 17.3 Phenolic acid content of some common oils seed, oils, and drinks (Mattila et al., 2006). Hydroxybenzoic acid (mg/100 g fresh weight) Drinks

Vanillic acid

Gallic acid

Protocatechuic acid

Syringic acid

Hydroxycinnamic acids (mg/100 g fresh weight) Cinnamic acid

p-Coumaric acid

Ferulic acid

Sinapic acid

Caffeic acid mg/100 g

Black tea

ND

26
2.0

ND

ND

ND

2.0
0.13

0.16

ND

1.42
0.052

Green tea

ND

34
2.7

ND

ND

ND

1.00
0.028

ND

ND

1.340
0.0028

Coffee

ND

ND

ND

ND

ND

1.27
0.053

9.1
0.36

ND

87
2.2

Cocoa powder

3.7
0.2

ND

40
1.6

4.1
0.21

ND

ND

ND

ND

ND

Apple juice

ND

10
1.4

0.756
0.017

ND

0.12
0.0085

1.20
0

0.10
0.013

ND

3.6
0.14

Orange juice

0.52
0.020

ND

ND

ND

ND

1.0
0.13

4.7
0.42

0.47
0.042

0.25
0.015

Apple cider

ND

ND

ND

0.12
0.012

ND

0.39
0.019

0.076
0.0067

ND

1.16
0.035

Red wine

0.9
0.13

ND

ND

5.6
0.21

ND

5.0
0.28

ND

ND

3.2
0.14

Soybean

10.7

25.19

12.002

12.49

12

0.33

Peanut

2.97

14.03

77.31

8.770.21

2.47

Blank cell, Data not available; ND, Not detected.

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Cereals

Hydroxybenzoic Acid (mg/1000g dry matter) Vanillic acid

p-Hydroxybenzoic acid

Syringic acid

Wheat (whole meal) Wheat (refined)

Hydroxycinnamic Acids (mg/1000g dry matter) Gallic acid

Caffeic acid

Ferulic acid

Sinapic acid

p-Coumaric acid

Caffeic acid mg/100g

1e37

2e90

890

63

37

37

120

8

3.8

3000

200

90

120

17

38

240

20

76

250

55

330

90

4

Wheat (bran) Rice (white)

13

Rice (brown)

17

1e27

Oat (whole meal flake)

18

20

Oat (bran)

24

28

Millet

11

Buckwheat flour Rye (flour)

1e05

3 110

260 120e175

85

22

Rye (bran) Barley flour

2e47

77 7

Corn flour blank cell¼ Data not available (Source: Stuper-Szablewska & Perkowski, 2019)

38

18 21

15

860

120

41

2800

480

140

250

11

40

380

57

31

26

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TABLE 17.4 Phenolic acid content of some popular cereal grain.

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17.4.1 Cereals Cereal grains have abounded with phenolic acids. The most common ones are caffeic acid, ferulic acid, p-coumaric acid, and sinapic acids (hydroxycinnamic); gallic acid, p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, and syringic acids (hydroxybenzoic). Most of the phenolic acids are found in the outer layer of grains, and the amount is becoming less toward starchy endosperm (Deng et al., 2012). Among the popularly consumed cereal, oat has shown the highest antioxidant properties. It has characterized by a significant amount of caffeic acid, ferulic acid, protocatechuic, p-hydroxybenzoic, or vanillic acids. Wheat, barley, rice, and rye are also rich in phenolic acids; caffeic acid and ferulic acid are the most abundant (Zielinski & Kozłowska, 2000; Slavin et al., 2001; Kähkönen et al., 1999; Baublis et al., 2000). Like other cereals, ferulic acid and p-coumaric acid are the dominant phenolic acids in rice. Among other phenolic acids, gallic acid, vanillic acid, caffeic acid, and syringic acid can be mentioned. Brown rice has more phenolic acid than milled or polished one as the bran itself consists of 70%e90% of the total phenolic acids. However, there’s no evidence that suggests a significant difference between rice variety in terms of total phenolic acid content but storage and milling condition make the difference. Phenolic acids in rice are comparatively higher than barley and wheat (Zhou et al., 2004).

17.5 Health benefits 17.5.1 Antidiabetic properties Diabetes mellitus has become the single most public health concern that every nation worldwide is struggling to cope with it. In simple words, it’s the disruption of insulin secretion or action or both causing chronic hyperglycemia that hampers regular physiological metabolism. It can also be described as the imbalance between free radical formation and oxidization in an individual (Vinayagam et al., 2016). It has been proved by many researchers that the phenolic compound can regulate the key pathway of carbohydrate metabolism. Experimental studies on ferulic and sinapic acid show their ability to increase insulin levels in diabetic rats (Eun et al., 2007; Cherng et al., 2013). Phenolic acids as an antidiabetic agent have shown different functionality with different mechanisms. The most interesting and well-documented feature is its ability to inhibit the two key enzymes (a-glucosidase and a-amylase) responsible for carbohydrate breakdown (Hanhineva et al., 2010; Manzanaro et al., 2006). Phenolic acid is found effective against postprandial glycemia. It can affect glucose intolerances by facilitating insulin response (Meng et al., 2013). Another mechanism as an antidiabetic agent is, it can influence the functions of glucose and insulin receptor (Ong et al., 2013; Cherng et al., 2013; Hanhineva et al., 2010; Eun et al., 2007; Ong et al., 2012; Prabhakar & Doble, 2011a,b). Fruits and vegetables are commonly available sources of phenolic acids, and researchers suggest that consuming 1e2 g/Day have a potential antidiabetic effect (Gandhi et al., 2014; Choi et al., 2011). All the available drugs for diabetic have limitations or side effects; scientists are searching for a natural compound to treat or prevent diabetic, and phenolic acids are the most potential solution so far. There are several kinds of literary publishing from different aspects regarding plant phenolics as a natural remedy.

17.5.2 Antioxidant properties Phenolic acid is hydrophilic, so it’s become less effective in the fat/oil system. But as the carboxylic group is easily ionized, it becomes more effective in the aqueous system. And the number of the carboxyl group of a phenolic content determines their performance as an antioxidant (Siquet et al., 2006; Zhang & Ji, 2003). Our body produces highly reactive molecules during different metabolism, commonly known as reactive oxygen species (ROS) ($O2, $OH, $RO2, H2O2, HOCl). Inability to neutralize these ROS cause lots of metabolic disorder that gradually leads to organ failure or even death (Furukawa et al., 2004). The role of antioxidants is to inhibit the activity of these free radicals by scavenging, damaging, or binding with another component to prohibit the production. Phenolic compounds, including phenolic acids, are well known to be one of the most abundantly and widely available in dietary sources that have free radical scavenging properties. It also has the ability to inhibit the enzymes that are responsible for the production of ROD. Like vitamin C and vitamin E, phenolic acid can be classified as a nonenzymatic antioxidant (Thyagaraju & Muralidhara, 2008). But all the phenolic acids don’t show the same effectivity; they all have a different mood of action with variable output. Most of the phenolic compounds can act by donating an electron or donating a proton. This phenomenon is termed as a dual age sword behavior.

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17.5.3 Anticancer properties There are several research endeavors for investigating the anticancer properties of phenolic acid. The variety of cancer cells (e.g., prostate, colon, breast, leukemia, etc.) and phenolic acid functionality make it to draw a general conclusion: it is evident that phenolic acid possesses anticancer properties. Gallic acid is one of the most investigated phenolic acids for anticancer properties (Badhani et al., 2015). There are several pathways for anticancer mechanisms mediated in different literature. The common mechanism in simple understanding is that phenolic acid can induce cell death or cell arrest associated with mitochondrial dysfunction, oxidative stress, or intracellular calcium level (Isuzugawa et al., 2001; Dalla Pellegrina et al., 2005).

17.5.4 Nephroprotective and hepatoprotective Plant phenols have shown effective nephroprotective activity in different animal model studies (Nitin, Ifthekar, & Mumtaz, 2013; Shehab et al., 2015). Nicotine, through the smoking cigarette, is responsible for an oxidative cellular injury that initiates the smoking-related disease. Ferulic acid has the ability to reverse this lipid peroxidation process induced by nicotine and increase the amount of antioxidant released by ferulic acid that prevents oxidative damage (Sudheer et al., 2005).

17.5.5 Antiallergic Gallic acid has been shown antiallergic agent by blocking the release of histamine, which would otherwise result in immediate hypersensitivity (Kim et al., 2006).

17.5.6 Neuroprotective Oxidation of dopamine can produce excess ROS that are often responsible for Alzheimer’s and Parkinson’s diseases (Qi et al., 2005; Liu et al., 2008; Crispo et al., 2010). Phenolic acid as an antioxidant can also function as an antineurodegenerative agent. Kasture et al. (2009) evaluated the effectiveness of gallic acid and its derivative as an anti-Parkinson agent.

17.5.7 Antimelanogenic Hyperpigmentation is caused by excess accumulation of melanin in the skin caused by increased melanocytes or melanogenic enzymes. The triggering factors may be abnormal a-MSH hormone release, UV light, chronic inflammation, etc. (Im et al., 2002; Ortonne & Nordlund, 2006). UV radiation from the sun can cause sunburn, skin irritation, tissue degradation that can lead to skin cancer or premature aging. Children are more vulnerable to these phenomena. Although synthetic sunscreen is quite available, in quest of a natural alternate scientist found phenolic compound as a good replacement. Phenolic acid shows an indirect activity by activating endogenous protective enzymes (Stevenson & Hurst, 2007). Gallic acid exhibits extraordinary antityrosinase properties. It has stronger inhibition than standard antityrosinase. It can also protect against UV-mediated melanogenesis (Kim, 2007; Panich, Onkoksoong, Limsaengurai, Akarasereenont, & Wongkajornsilp, 2012).

17.5.8 Lipid peroxidation prevention Lipid oxidation is an uncontrolled reaction that is responsible for the deteriorating shelf-life of processed food commonly known as rancidity. Phenolic acids are used as an antioxidant utilizing free radical scavenging properties. At the same time by adding as a food preserver can also attribute to functional and health benefits. As there is growing concern about synthetic preservatives, there is more demand for their natural replacement for food manufacturers. Phenolic acids have great potentiality in this respect.

17.5.9 Antimicrobial Phenolic acids have antimicrobial properties. The structure, the chain length, and functional groups attached to the aromatic ring determine the magnitude of antimicrobial activity (Cueva et al., 2010). As a result, the two major groups the hydroxybenzoic and the hydroxycinnamic acids showed different antimicrobial properties based on their hydroxyl and

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311

methoxy groups. Hydroxybenzoic acids have an inverse relationship with hydroxyl group where hydroxycinnamic acids didn’t show significant changes in terms of antimicrobial activity. Similarly, replacement of the hydroxyl group by methoxy group increases the antimicrobial activity in hydroxybenzoic acids but a minor change in hydroxycinnamic acids. This antimicrobial variation is also bacterial strain dependent (Sánchez-Maldonado et al., 2011). Phenolic acids are weak organic acids so like other acids they kill bacteria by acidification of the cytoplasm. Low pH increases the antimicrobial activity. So, pKa, lipophilicity, a saturation of chain, and pH are the key factors to determine the antimicrobial activity of a specific phenolic acid (Almajano et al., 2007).

17.5.10 Antiinflammatory properties Phenolic acid can function as an antiinflammatory agent. It can either work directly against the myeloperoxidase enzyme, enzyme that is responsible for inflammation, or it can destroy the harmful metabolites produced by myeloperoxidase enzyme by scavenging (Rosso et al., 2006).

17.5.11 Antiviral properties Phenolic acids showed antiviral properties as a defense mechanism of plant. It protects plant from different virus, bacteria, or fungus (Cowan, 1999; Hamauzu et al., 2005). As a consumable product through food or medicine some phenolic acids have proven as an antiviral component in human. Gallic acid has shown antiviral activity against HIV-1, HSP-1, HSP-2, and human rhinovirus (Nishide et al., 2019; Choi et al., 2010; Kratz et al., 2008; Uozaki et al., 2007; Ghasemzadeh & Ghasemzadeh, 2011).

17.6 Storage and processing stability There are different corelation patterns that exist between phenolic acids on thermal treatment. The heat treatment method also impacts native phenolic acid degradation. The source of phenolic acid also plays a very important role in degradation. It is almost impossible to conclude or generalize anything about the pathway of thermal degradation of phenolic acids, the scientist has discovered different mechanism and behavior of phenolic acids. For example, soybean steaming found less sensitive than conventional cooking methods for the survival of phenolic acids (Xu & Chang, 2008). Sometimes one phenolic acid breaks down to another one or it turns into another antioxidant. The phenolic acids of garlic found increased after thermal treatment in the study of Kim et al. (2013). This phenomenon can be explained by their stability or they become more biologically active after heat treatment. For cereal grains, milling significantly reduces the phenolic acid content by roughly 70%e80%, depending upon the raw material, of the phenolic acid constitute in the bran. Phenolic acids’ survival to hydrothermal processing depends on the nature of cereal grain. Extrusion cooking found more effective for retaining phenolic acids in grains particularly ferulic acid. A study has shown fivefold of increment of phenolic acids in grain by extrusion processing (Zielinski et al., 2001). So, for a cereal grain, whole grain and processed grain have different sorts of phenolic attribution. It is also the amount of free phenolic acid and bound phenolic acid that determines the final phenolic acid content of processed food. The study of Challacombe et al. (2012) showed bread and cracker have higher free phenolic acids than original wheat flour that indicates thermal process initiates the breakdown of bound phenolics and that contributes to increase free phenolic acid. The process of chemical or enzymatic change due to long-term storage of cereal grains also has a great impact on the phenolic acid content. The degree of conversion basically depends on the storage condition (temperature, water activity, humidity, oxygen availability, pH, etc.) and raw material. Chemical oxidation found less destructive than enzymatic oxidation. Partial oxidation or degradation sometimes found to increase the antioxidant properties of some grains. During germination and fermentation phenolic compounds also changed (Van der Sluis et al., 2002). In a nutshell, the process of degradation or conversion during storage or thermal processing is a very complex process depending upon a lot of external and internal factors.

17.7 Absorption and metabolism in human It’s the human body where phenolic acids entered into their last stage of activity and metabolized thereafter. The exact metabolic pathway and their function in human body has yet to be discovered. The variation of chemical structure of phenolic acids and variation of human metabolism according to age, race, gender, and physiology make it burdensome to investigate the metabolic pathway. As an acid or an antioxidant, it also has the ability to take part in different physiological

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FIGURE 17.2 Possible metabolic pathway for phenolic acid.

metabolic reaction. So, it’s inexpedient to sum up the metabolic pathway of phenolic acids. Instead a synopsized idea has been provided here in this section. A probable outline of phenolic acid absorption in body has been shown in Fig. 17.2 (Heleno et al., 2015). Phenolic acids transformed during digestion processes into readily accessible forms are transported in the bloodstream to target sites in human cells. Caffeic acid and ferulic acid are the most abundantly occurred phenolic acid and can be the representative of others. In human GI tract they are metabolized extensively by methylation, glucuronidation, and sulfation. During this process their structure undergoes changing and becomes available for biological utilization. Bourne and Rice-Evans (1998) found the total absorption of ferulic acid is 11%e25% of total ingestion while the rest of the phenolic acids excrete from body.

17.8 Impact on food quality and sensory properties In spite of innumerous health benefits, phenolic acids are often blamed to be responsible for unacceptable flavor and taste. In general, phenolic acids are known to be a bitter flavor attributor to foods (Drewnowski, 1997). Hydroxycinnamic acids can contribute to sensory properties by different mechanisms. Hydroxycinnamic derivatives have also been reported as being bitter (Macheix, Fleuriet, & Billot, 2018; Macheix, Fleuriet, Billot, Macheix et al., 2018). It can interact with Maillard reaction and impact final flavor development. Thermal processing can lead to the breakdown (decarboxylation and oxidative reaction) of phenolic acids that may change the taste in a combination with other sensory contributors. Hydroxycinnamic acids can be converted to amides that give an astringent and mouth-drying sensation (Jiang & Peterson, 2010). Not all the conversion or reaction of phenolic acid is undesirable. Popcorn (Schieberle, 1991), sweet corn (Buttery et al., 1994), tortillas (Buttery & Ling, 1995), and rice (Zeng et al., 2008) are reported with positive flavoring impact of phenolic acids. Phenolic acids are responsible for fruit maturity, flavor development, and at the same time, they contribute toward astringency and bitterness. The mechanism depends on fruits, geography, and interaction with other phenolic acids and flavonoids. It is not possible to summarize one single compound or phenolic acid to be the single factor for either good or bad organoleptic properties. For example, astringency is generally occurred because of tannin and protein interaction. Phenols and phenolic acids contribute to this interaction or they can interact with protein by themselves but this individual impact is negligible (Macheix et al., 2018). Caffeic acid is the contributor of bitter taste of espresso coffee (Jiang & Peterson, 2010). Hydroxybenzoic and hydroxycinnamic acids both have been identified for the astringent taste in wine although flavors and taste in wine are a synergistic effect of many components like alcohol, catechins, proteins, etc. (Ferrer-Gallego et al., 2014). Bread made of whole grain and refined wheat flour differs in crumb properties in terms of taste and flavor. This has happened because of the difference in free and bound phenolic acids in whole grain and refined grain. The interaction between Maillard reaction and phenolic acids may alter sensory properties (Jiang & Peterson, 2010). Another hypothesis is, in the oral phase bound phenolic acid may be converted to free phenolic acid by salivary enzymes and taste receptors can interact (Challacombe et al., 2012).

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17.9 Conclusion The research of phenolic acid has broadly focused from two different perspectives. One group of scientists concentrate on their role in plant metabolism while another group investigates their contribution toward food and health. The role in photosynthesis, protein synthesis, nutrient uptake, etc., has already been revealed. Many mechanisms are yet to be discovered and scientists around the world are working restlessly to find the in-depth functionality of plant phenolics and apply them in the human food system. Currently, it’s basically confined to the application as an antioxidant. Recent works indicate their potential application in the food system; phenolic acids are being widely investigated as a functional compound and have already been incorporated in many food systems. Research on their stability in emulsion, hydrogel, colloidal system, bioactive compound, biodegradable films, and interaction with food components like carbohydrate, fat, protein are some of the examples. The interaction of phenolic acid in oral and digestive phases is under scientific scrutiny. How the salivary enzymes react with different phenolic acids and how do they contribute to taste are the new focus for phenolic acids. Future processed foods trend toward a system where food will not be only limited to sensory or satiety value rather it can also serve as a very potential carrier for functional or nutraceutical ingredients that can take part in defense and building up mechanism. Phenolic acids are one of those few components that scientist is aspiring after.

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Chapter 18

Anthocyanins Amir Gull1, Mohd Aaqib Sheikh2, Jasmeet Kour2, Beenish Zehra3, Imtiyaz Ahmad Zargar4, Altaf Ahmad Wani5, Surekha Bhatia6 and Mushtaq Ahmad Lone7 1

Department of Food Science and Technology, University of Kashmir, Srinagar, Jammu and Kashmir, India; 2Department of Food Engineering and

Technology, Sant Longowal Institute of Engineering and Technology, Sangrur, Punjab, India; 3Department of Nutrition and Dietetics, Sharda University, Greater Noida, Uttar Pradesh, India; 4Division of Food Science and Technology, Sher-e- Kashmir University of Agriculture Sciences and Technology, Shalimar, Srinagar, India; 5FAO, Wadura, Sher-e- Kashmir University of Agriculture Sciences and Technology, Shalimar, Jammu and Kashmir, India; 6Department of Processing and Food Engineering, Punjab Agricultural University, Ludhiana, Punjab, India; 7Directorate of Planning and Monitoring, Sher-e- Kashmir University of Agriculture Sciences and Technology, Shalimar, Srinagar, India

18.1 Sources/derivatives/chemistry Anthocyanins are one of the most widely distributed plant pigments belonging to subgroup flavonoids. Being important group of water-soluble plant pigments these are responsible for a wide range of colors. Anthocyanins appear as red pigments in acidic condition while exist as blue pigment in alkaline conditions. Recognized anthocyanin pigments in recent years had also expanded and the molecular genetic control of biosynthesis of anthocyanin is nowadays one of the best understood secondary metabolic pathways (Xie, Zheng, Zheng, & Deng, 2014). Earlier anthocyanins were considered as a useless entity and as of now they are regarded as highly useful plant pigments. These compounds are having a wide range of usage as the colors extracted from these pigments are used as dye and food colorant. The food industry has also been benefited from latest research in anthocyanins as the colors extracted from anthocyanins have opened a new way for their use as natural food colorants. These are considered as potent antioxidants. Recent research focused on the possible benefits of ingestion of anthocyanin-rich foods such as blackcurrants, berries, and other red- or blue-colored fruits to human health (Konczak & Zhang, 2004; Ghosh & Konishi, 2007). Anthocyanin is having molecular heaviness 207.24724 g/mol and empirical formula C15H11Oþ. The aglycone of an anthocyanin is known as anthocyanidin. These are grouped into 3hydroxyanthocyanidins, 3-deoxyanthocyanidins, and O-methylated anthocyanidins. Cyanidin, delphinidin, pelargonidin, peonidin, malvidin, and petunidin are most common anthocyanidins distributed in the plants (Kong et al., 2003). Cyanidin is a major reddish-purple (magenta) pigment in berries, red sweet potato, and purple corn. While as in the plant, blue hue of flowers is due to the delphinidin pigment. However, in fruits and berries pelargonidin imparts red colored pigment. Peonidin is a methylated anthocyanidin colored pigment abundantly found in berries, grapes, and red wines. Malvidin having purple visible color is plentifully found in blue-colored flowers. Petunidin is a methylated anthocyanidin and appears as dark red or purple pigment found in blackcurrants and purple petals of flower (Lo Piero et al., 2005; Zhang et al., 2012). Factors such as pH, temperature, concentration, light, oxygen, solvents, and the presence of enzymes, flavonoids, proteins, and metallic ions affect the stability of anthocyanins. Besides this other factors such as its structure and hydroxyl or methoxyl groups also influence their stability (Grotewold, 2006). Because of its ionic nature, anthocyanin color is pH dependent and is more stable at a low pH.

18.2 Extraction and characterization techniques of anthocyanins Anthocyanins are the phenolic compounds, which are secondary metabolites produced from different pathways such as shikimic acid pathway and melonic pathway in plant tissues. These are present in plant tissues imparting color in the form of red and dark colors. Anthocyanins are the natural source of colors which could be incorporated in the food products. Apart from color, it has medicinal values too as it acts as an antioxidant which helps in reducing free radical stress in the body. Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00018-1 Copyright © 2022 Elsevier Inc. All rights reserved.

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18.2.1 Preextraction treatments Anthocyanins are usually located in the intracellular tissues of the plant matrix which makes them unavailable for direct extraction and isolation. For efficient extraction, rupturing of tissues is required which aids the solubilization of these compounds into the solvent. Extraction of compounds from the plant matrix depends on the ability and the polarity of the solvent which should be able to diffuse into the intracellular tissues for the extraction of compounds and must be able to solubilize the compounds. For increasing the efficiency of the extraction process, the sample is pretreated for efficient extraction by size reduction, freeze-drying, grinding, milling, etc. Size reduction is one of the basic pretreatments which is done to increase the surface area, for exposure of compounds to the solvents. Conversion of the sample into a fine powder is the most common activity for increasing its storage ability by removing moisture of the sample (Silva et al., 2017). Anthocyanins are unstable compounds as they are easily oxidized by the oxygen present in air and conversion of plant material into fine powder makes it more prone to oxidization. In such cases, the plant matrix is then directly homogenized or pretreated with solvent for further extraction. Freeze-drying of the fruit has also been done as pretreatment before its conversion to a pulp (Antolovich et al., 2000).

18.2.2 Soluteesolvent interaction Basic extraction process comprises interactive outcomes of solute and solvent. In such an interactive case, the plant matrix is kept in contact with the solvent for the solubilization of the bioactive compounds present in that plant matrix. The interaction of the compounds present in the form of solute depends on the polarity of the solution. The solubilization of the compounds can also be due to the formation of different types of bonds between the functional group of bioactive compounds and the solvent. The solvent used must be able to solubilize the required compounds with help of external aid in terms of temperature or pressure. To speed up the solubilization process, many other external factors are taken into consideration. Different techniques are used by using different types of process parameters for the extraction of bioactive compounds from the plant matrix. These extraction processes can be classified into conventional, which include soxhlet extraction, hot water extraction, extraction using magnetic stirring, etc., and nonconventional techniques, which are ultrasound-assisted extraction, microwave-assisted extraction, supercritical fluid extraction (SFE), high pulse electric field extraction, etc.

18.2.3 Solvent extraction Simplest and conventional technique used for extraction of bioactive compounds is the solvent extraction which particularly depends on the type and ability of the solvent for the extraction. Polarity of solvent is considered for the solubilization of the compound, apart from polarity, acidified solvents are made with the help of addition of acids such as HCl in ethanol, methanol, water, etc. (Ali et al., 2016, pp. 1e24). Addition of acids helps in diffusion of the compounds in the solvent. With the decrease in the pH value anthocyanins were found to be more stable as compared to neutral and alkaline pH. With increase in temperature, chances of decrease in the anthocyanin content in extract is there as thermal degradation can cause loss of the extracted compound. Repetition of the process is done for complete extraction of anthocyanin from the sample matrix, but such repetition extends the length of the whole extraction process and complete removal of solvent from the extract is difficult. Due to such reasons, conventional extraction process is now in process of replacement by green and novel extraction techniques.

18.2.4 Ultrasound-assisted extraction Ultrasound-assisted extraction is the novel technique of extraction of bioactive compounds from the plant tissues which enhances the extraction by using ultrasound waves for the rupturing of the sample matrix. From microscopic evaluation rupturing of the surface of the matrix has been justified (Vinatoru, 2001). Ultrasound waves frequency is mostly used in ultrasound power region of 20e25 kHz, which produces mechanical, chemical, and biochemical action by continuous production and rupturing of cavitation bubbles which increases their sizes up to a range of diameter and then burst out when internal pressure increased to maximum limit (Paniwnyk, 2017). The increase in the size of the cavity bubble is due to rectified diffusion of the gases present in the liquid medium which in turn makes it unstable and the bubbles collapse with high temperature and pressure releasing high shear forces on the surface of the solid matrix causing surface rupturing that aids the solubilization of the compounds in the solution medium. The sound waves pass through liquid with series of subsequent compression and rarefaction in the medium causing rise in temperature hotpots in the liquid which helps in

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mass transfer, homogenization, and efficient mixing of compounds (Paniwnyk, 2017). Increases in temperature increase the diffusion efficiency of anthocyanins, which might be due to the rise in internal energy and their mobility which in turn reduces the viscosity coefficient. In ultrasound-assisted extraction maximum anthocyanin extracted in fast diffusion stage of extraction and the rest was extracted in slow transfer rate (Silva et al., 2017).

18.2.5 Microwave-assisted extraction Microwave extraction is also one of the novel extraction techniques. The extraction of bioactive compounds from sample matrix is placed into the cavity of the system where it is exposed to the microwaves for the selected time period and at specified temperature according to the sensitivity of the bioactive compound to be extracted. The extraction process works on the principle of conduction of microwaves to raise the temperature of the solid matrix due to ionic conduction and dipole rotation. During the ionic conduction, the ions tend to move under the influence of electromagnetic fields, which could be resisted in a solid matrix and thus creating friction and dissipating heat. Dipole rotation accelerates the bending of the molecules in the applied field; with such frequent alignment and molecular movement, heat is generated in the matrix. The sample matrix is placed in the flask with solvent and the loaded flask is then placed in the microwave system cavity and the prerequisite heating is done which almost took 2 min and sample exposure to microwave for the required time and temperature at particular microwave frequency. The requirement of process parameters depends on the sensitivity of the sample and the solvent added. The extract obtained then kept to cool down to temperature to handle. The extraction process is affected by the solvent, temperature, matrix characteristics, extraction time, etc. Solvents such as ethanol, methanol, water, acetone, etc., are used as these are polar in nature. Polarity of solvent affects the absorption of microwaves as polar solvents have permanent dipole moment which makes them favorable for the extraction process whereas nonpolar solvent doesn’t heat up when exposed to microwaves. The dissolution of the compound from the matrix also depends on the polarity of the solvent. Temperature of the process is the most considerable parameter for effective yield of extract. Pretreatments are required in case of coarse material such as grinding, milling, etc., to increase the surface area of the sample matrix, and drying methods such as oven-drying, freeze-drying, etc., are also used to remove moisture to prevent the degradation of the compounds due to the high water activity. As in the terms of extraction water can improve extraction of bioactive compounds as it can increase the polarity of the solvent for the solubilization of the compounds.

18.2.6 High-pressure processing High-pressure processing (HPP) used high pressure as the basic principle of action on the extraction of compounds from the sample. Any process such as change in phase, chemical reaction, or molecular configuration change which is dependent on volume change can be enhanced by increase in pressure as it causes decrease in volume of the medium. Increase in pressure breaks covalent bonds in high-molecular-weight components which are more affected by high pressure in comparison to low-molecular-weight components which are less sensitive to high pressure. High pressure is mostly used for the preservation and microbial inactivation in the food industry. Application of HPP is not as much worked out in the extraction.

18.2.7 Enzymatic-assisted extraction Enzymatic extraction is a greener technology for the extraction as enzymes are the main ingredient for the solubilization and extraction of bioactive compounds from sample matrix. Enzymes are used for the disruption of the cell wall of the tissues or cells where anthocyanins biomolecules are trapped. Mixtures of different enzymes are used for the purpose; thick cell walls might be due to the pectin or other wall material (Silva et al., 2017). Many enzyme mixtures such as pectinase, cellulases, etc., are commercially available for the extraction of anthocyanin in juice (Buchert et al., 2005). All the blends of enzymes might not be able to give the similar results as increase in the anthocyanin content. In some cases, there might be the reduction in its content as enzymes may hydrolyze the anthocyanin forming the simpler product after hydrolysis (Buchert et al., 2005). This extraction method can be applied in two ways such as enzyme-assisted extraction and enzymeassisted cold pressing. However, enzyme-assisted extraction method is mostly applicable for the extraction of oil from the various seeds. While enzyme-assisted cold pressing is a good replacement technique for the extraction of bioactive compounds from the oil seeds as being nontoxic and nonflammable. Also this technology uses water as the main solvent instead of chemicals which makes it eco-friendly (Swami et al., 2019).

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18.2.8 Supercritical fluid extraction SFE uses supercritical fluids at their critical temperature and pressure, such as carbon dioxide for the extraction process (Silva et al., 2017). SFE technique is also an alternative technique for conventional solvent extraction technique. SFE is preferable for heat-labile plant compounds which could be lost due to thermal degradation. Carbon dioxide is most commonly used as the extraction fluid for the extraction of heat-labile biomolecules which after extraction, further separated from carbon dioxide (Roh et al., 2008). SFE removes nonpolar substances which could create hindrance for the extraction of polar bioactive compounds. Solvent plays a major role in the extraction process as its selectivity depends on the polarity of the bioactive compound which to be extracted. In some cases, along with CO2, polar solvents such as ethanol, methanol, acetone, etc., are used for extraction of polar compounds such as anthocyanin polyphenols (Silva et al., 2017). Extract obtained from this extraction technique contains solvent residues which could be separated later using purification method. SFE unit consists of an extraction cell which could resist high pressure, heating system to control extractor temperature, temperature measuring devices such as thermocouple or thermistors, pressure gauges, cooling system to maintain the temperature of CO2 at inlet of pump, heating bath, etc. Heating exchange coils in heating baths are meant for maintaining the supercritical stage of CO2. Micrometer valve maintains the flow of CO2, where it is depressurized and extract is separated in a separating vessel (Roh et al., 2008). The pressurized CO2 is transferred to the extraction vessel where the extraction of desired bioactive compound from sample matrix takes place. The mixture of extract and the CO2 is then passed to the separator where the CO2 is depressurized and the extract is separated in a separating vessel and CO2 is then recycled for the next run. SFE has been used for the extraction of polyphenols, essential oils, caffeine, etc. For the optimized extraction or maximized extraction of anthocyanins, many studies have been conducted using different cosolvents, temperature, and pressure combinations. Even though higher temperature could be used for the extraction of polyphenols but temperature for the extraction of anthocyanin was set up to or below 40 C, high temperature may degrade its content in the extract. Anthocyanins are heat sensitive in nature.

18.3 Health and pharmacological benefits of anthocyanins Anthocyanins being a combination of two words “Anthos” and “kianos”, the former meaning flower and the latter relating to blue, pertain to the most important class of pigments of vascular plants and water-soluble coloring agents of natural origin which impart red and blue colors to various fruits and vegetables (Shaik et al., 2018). Anthocyanins belong to an important subclass of flavonoids which are under the classification of compounds called as polyphenols and constitute most prevalent water-soluble naturally occurring pigments in plant kingdom (Martín et al., 2017). These pigments exist in diversified colors in plants ranging from blue, red, and purple with major occurrence in flowers, fruits, as well as tubers (Khoo et al., 2017). In nature, anthocyanins occur prominently in the form of heterosides. Anthocyanins derived from red wine exhibit tremendous relative bioavailability along with glycosides derived from peonidin, cyanidin, malvidin, delphinidin, and petunidin having impeccable relative bioavailability (Frank et al., 2003). More than 650 types of anthocyanins including aglycan form of anthocyanins known as anthocyanidins such as cyanidin, petunidin, pelargonidin, malvidin, peonidin, and delphinidin have been identified depending on the position and nature of methoxyl and hydroxyl groups (Khoo et al., 2017). Anthocyanins are credited to harmonize various cognitive and motor functions and to prevent hampering in neural function (Lila, 2004). There are numerous anthocyanins with acyl substituents linked to sugars, pigments possessing aliphatic and aromatic substituents, cinnamic acids like p-coumaric, ferulic, or sinapic acid, and aliphatic acids like malonic, succinic, malic, and acetic acids (He & Giusti, 2010). An additional complexity in their structure results due to the occurrence of manifold acylated sugars and these types have sometimes been known as polyglycosides. The different colors of various fruits and vegetables such as orange, purple, red, and blue are mainly produced by the presence of anthocyanins in them (Horbowicz et al., 2008). Moreover, anthocyanins not only impart color but correspondingly possess potential health benefits (Table 18.1). It has been demonstrated over the years that they possess antioxidative, antiinflammatory, cardioprotective, neuroprotective, chemotherapeutic, and hepatoprotective properties as well as exhibit therapeutic effects to counter various other chronic diseases (Winter et al., 2017). These pigments being less bioavailable absorb less into the blood leading to their high excretion in urine and feces, thereby resulting in reduced free radical scavenging efficacy (Khoo et al., 2017). Various in vitro as well as in vivo and human studies have reported that anthocyanins enhance the antioxidative value apart from improving vision, maintaining dermal health and exhibiting neuroprotective, anticarcinogenic, cardiovascular, and antidiabetic properties (Zafra-Stone et al., 2007). There has been a rising concern with the recognition of anthocyanins as one of promising health benefactors and their incorporation from several sources into eatables such as soft drinks, jams, dairy products, beverages, confectionery, etc., and it has been estimated that 3e215 mg of anthocyanins are consumed daily (Sivamaruthi et al., 2020). Extracts of prominent berries

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TABLE 18.1 Health beneficiary properties of various anthocyanins. Type of anthocyanin

Property

References

Delphinidin and cyanidin

Prevents atherosclerosis by inhibiting vascular endothelial growth factor expression

Oak et al. (2006)

Pelargonidin and cyaniding

Make myocardium less susceptible to ischemia injury

Curtis et al. (2009)

Cyanidin, pelargonidin, delphinidin, petunidin, peonidin, and malvidin

Prevents lipid peroxidation and damage of subcellular organelles

Martin et al. (2017)

Malvidin 3-O-galactoside, petunidin 3-O-galactoside, delphinidin 3-O-galactoside, delphinidin, and cyanidin 3-glucoside

Antiproliferative and anticancer effects on myeloma, breast and colon cancer cells

Lin et al. (2016)

Delphinidin 3-O-glucoside, cyanidin 3-O-glucoside

Antiinflammatory property by inhibiting NF-kB and COX-2 expression

Taverniti et al. (2014)

Cyanidin 3-O-glucoside

Stimulate PKC activation which is the target in prevention and treatment of obesity and diabetes in HepG2 cells by increasing its phosphorylation and membrane translocation from endoplasmic reticulum to outer mitochondrial membrane to phosphorylate the mtF0F1-ATPase b-subunit

Guo et al. (2008)

Cyanidin 3-O-glucoside

Inhibits cell damage, neurological dysfunction, and superoxide production

Kim et al. (2012)

such as blueberry, bilberry, elderberry, cranberry, raspberry, and strawberry have been proven to be potent antioxidants possessing antiangiogenic and antibacterial potential along with proanthocyanidin extract of grape seed exhibiting tremendous antioxidative value (Yasmin et al., 2003). Anthocyanins possess immense functional properties which have the potential to combat various dreadful diseases (Khoo et al., 2017). In this we summarize the utmost current literature concerning the health benefits of anthocyanins.

18.3.1 Antioxidant activity Reactive oxygen species (ROS) are produced naturally by metabolic reactions and act as signaling molecules at low concentration but are toxic at high concentration (Huang et al., 2019). Too much ROS can cause oxidative stress, leading to lipid membranes peroxidation and damage to subcellular organelles. Antioxidants are the compounds which provide defense against harmful effects of ROS by scavenging them. Plant-derived anthocyanins can counteract oxidative stress due to their antioxidant property. It has been reported that some common anthocyanins such as cyanidin, pelargonidin, delphinidin, petunidin, peonidin, and malvidin acted as strong antioxidants in emulsion and low-density lipoprotein and many of them showed activity equivalent to well-known antioxidants alpha-tocopherol, trolox, catechin, quercetin (Tena et al., 2020). Anthocyanins have an antioxidant potential twice that of other known synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) generally used in various food products. Antioxidant activity of various anthocyanins varies with the glycosylation patterns. Anthocyanins have the property very similar to ascorbate like preventing the biomembranes peroxidation by proficiently deceiving peroxyl radicals (Skrovankova et al., 2015). The antioxidant capacity of 14 anthocyanins was studied by oxygen radical absorbance capacity method and it was found to be more than 3.5 times higher than those of trolox (Wang & Goodman, 1999). Anthocyanins derived from berries, blackcurrants, and various fruits with red to blue color have been known to report strong antioxidant potential (Khoo et al., 2017).

18.3.2 Cardiovascular effects Anthocyanins have been reported to possess the defending action against atherosclerosis by inhibiting TNF-a-induced chemokine monocyte chemotactic protein 1 secretion in primary human endothelial cells which is directly involved in atherogenesis by recruiting macrophages at the sites of inflammation (Krga & Milenkovic, 2019). Anthocyanins, specially delphinidin and cyanidin, have been reported to prevent the vascular endothelial growth factor expression which is the

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foremost proangiogenic and proatherosclerotic factor thereby averting c-Jun N-terminal kinase and p38 mitogen-activated protein kinases activation (Oak et al., 2006). Due to high anthocyanin content of black raspberry these have been shown to provide significant antioxidant protection in the gut epithelium of weanling pigs (Wu et al., 2006). Endothelium-dependent relaxation of coronary arteries has been shown by the anthocyanins extracted from elderberry, chokeberry, and bilberry. Necrosis and apoptosis of myocyte cell is caused by ischemia and it has been studied that anthocyanin such as delphinidin inhibits cell death either due to necrosis or apoptosis in cultured cardiomyocytes of rats (de Pascual-Teresa et al., 2010). Moreover, long-term ingestion of anthocyanins such as pelargonidin and cyanidin derived from corn leads to the decreased susceptibility of myocardium to ischemia injury in vivo as well as ex vivo.

18.3.3 Anticancerous activity In a body, mutation causes uncontrolled cell division and growth resulting in formation of tumors or cancer. Cancer cells develop some imperative features to escape immune responses, local incursion of tissue, reserved metastasis, and repel cell death or apoptosis (Gonzalez et al., 2018). Chemotherapy, radiotherapy, and transplantation of stem cells are the leading clinical treatments against cancer, in spite of having countless side effects. By targeting specific checkpoints of cancer signaling pathways, ingestion of plant-derived anthocyanins in diet has been exposed to possess cytotoxic or inhibitory effects in the tumor cells eradication by reducing inflammation, cancer cell proliferation, and inducing apoptosis (Chirumbolo et al., 2018). Afaq et al. (2005) described that animals pretreated with anthocyanin-rich extract from pomegranate bring about noticeable reduction in tumor cells by phosphorylating extracellular signal-regulated kinase (ERK)1/2, p38, and JNK1/2. For instance, anthocyanins extracted from blackberry showed antiproliferative effects on colon, lung, breast cancer and leukemia cells. It has also been demonstrated that anthocyanin-rich extract from Pourouma cecropiifolia considerably decreased the laryngeal cancer, gastric cancer, and breast cancer cells viability (Barrios et al., 2010). Anwar et al. (2016) reported the antiproliferation of Caco-2 cells by standardized berry anthocyanin-rich extract by upregulating the expression of p21Waf/Cif1 and blocking the cell cycle along with bringing about apoptosis. Blueberry-derived anthocyanins pointedly persuaded apoptosis of melanoma and colon cancer cells in mice and humans, respectively (Wang & Goodman, 1999).

18.3.4 Antiinflammatory properties Antiinflammatory responses are a sequence of well-ordered reactions controlled by numerous factors, such as enzymes, vasoactive mediators, cytokines, and lipid mediators (Abdulkhaleq et al., 2018). Cyclooxygenases (COXs) are chief proinflammatory enzymes involved in metabolism of arachidonic acid and lipid mediators such as prostaglandin E2 (Hanna & Hafez, 2018). Cyanidin 3-glucoside extracted from mulberry has been reported as a supplement in diet to downregulate the expression of COX especially COX-2, ultimately inhibiting prostaglandin production. Unnecessary nitric oxide production is also linked to the inflammatory responses and it has been observed that cyanidin 3-O-b-glucoside inhibits the expression of inducible nitric oxide synthase (iNOS) which is responsible for nitric oxide production, interleukins, and tumor necrosis factor in rats (Soufli et al., 2016). Likewise, NF-kB pathway is another metabolic process which plays an imperative part in eliciting and regulating various inflammatory processes such as activating iNOS and COX-2 expression. Blueberry- and bilberry-derived anthocyanins exposed antiinflammatory activity by inhibiting NF-kB pathway in human intestinal cells and mice, respectively, under pro-inflammatory stimulus. Likewise, COX-2 expression was inhibited by delphinidin by pointing NF-kB transcription factors in macrophages. The distribution of cholesterol in lipid rafts is reduced by cyanidin 3O-b-glucoside ultimately inhibiting inflammatory responses. Moreover, continuing ingestion with purified extract of bilberries and blackcurrants derived delphinidin 3-O-glucosides and cyanidin 3-O-glucoside repressed demagogic response in adults suffering from hypercholesterolemia (Desjardins et al., 2012). However, additive effect of black soybean derived anthocyanins and antimicrobial ciprofloxacin has also been shown in treating chronic bacterial prostatitis than alone antimicrobial treatment (Yoon et al., 2013).

18.3.5 Antiobesity and antidiabetic activity Overconsumption and outflow of energy cause disproportionate accumulation of adipose tissue consequently leading to obesity. It affects several metabolic processes and regulatory pathways and makes them more prone to diseases and sometimes its long-lasting effects are very severe (Singla, 2010). It has been reported by various studies in the literature that consumption of anthocyanin as dietary supplement ameliorates the protective responses against the excessive deposition of fatty acids, for example, Kamchatka honeysuckle berry derived anthocyanins have been shown to amend various

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metabolic disturbances associated with carbohydrates and lipid metabolism and ultimately repressed increase in body weight, improved liver functioning, and significantly decreased hepatic and serum lipid profiles in the mouse supplemented with diet enriched in triacylglycerols (Jurgo nski et al., 2013). One of the ways to prevent the obesity is to reduce the intake of glucose by the adipocytes and it has been demonstrated that supplementation of anthocyanin-rich diet has a significant impact on the reduction of glucose uptake by the cells by inhibiting the activity of glucose transporters (Solverson, 2020). Tsuda et al. (2003) treated mice with anthocyanins extracted from purple corn along with a diet rich in lipids which led to an overall decrease in body weight and adipose tissue concluding that anthocyanins can act as a functional food preventing obesity and diabetes eradicating symptoms such as hyperglycemia, hyperinsulinemia, and hyperleptinemia induced by a high-fat diet. Diabetes being a metabolic disorder is associated with the malfunctioning of receptors of insulin or long lasting little production of insulin. Among various reasons for diabetes, the most common are hyperglycemia, intolerance of glucose, and disruption of metabolic processes associated with amino acids, lipids, and carbohydrates. It has been reported that black rice derived anthocyanins in rats supplemented with fructose-rich diet inhibits oxidative stress, avoids insulin resistance ultimately refining the counter of plasma lipids (Guo et al., 2008). Anthocyanins inhibit dipeptidyl peptidase-4 (DPP-IV) activity which inactivates hormones such as glucagon-like peptide-1 (GLP-1) and glucosedependent insulin tropic polypeptide that stimulate insulin secretion in body (Sharma et al., 2019). Additionally, bilberry derived anthocyanins in mice suffering from type II diabetes decreased the level of glucose in blood and enhanced sensitivity of insulin through AMPK activation (Takikawa et al., 2010). Anthocyanin Cy-3-g enhanced AMP-activated protein kinase which helps in inhibiting liver fat downstream target ACC phosphorylation and inactivation stimulating CPT-1 expression and increasing fatty acid oxidation in HepG2 cells.

18.3.6 Neuroprotective properties It has been evident from the literature that anthocyanins extracted from various fruits and vegetables showed neuroprotective properties signifying their prospective use for the cure of Parkinson’s and Alzheimer’s disease (Magendira Mani et al., 2014). Being a neurodegenerative syndrome, Parkinson’s disease results in the damage of midbrain neurons leading to dopaminergic cell death. It has been reported that anthocyanins extracted from various fruits and vegetables, such as blackcurrant, grape seed, Chinese mulberry, and blueberries, considerably repressed dopaminergic cell death through improvement of dysfunction of mitochondria which is caused by oxidative stress (de Andrade Teles et al., 2018). Likewise, cyanidin 3-O-glucoside showed the neuroprotective properties in mice by preventing mitochondrial dysfunction during stroke or oxidative stress (Min et al., 2011). Di Giacomo et al. (2012) investigated the effect of cyanidin 3-O-b-glucoside on cerebral ischemia in rats. Cyanidin 3-O-b-glucoside-pretreated rats were injected with 10 mg/kg in posttreated rats suffering from cerebral ischemia. This study indicated that cyanidin enhanced the nonprotein thiol groups apart from significantly reducing the lipid hydroperoxides which highlighted the pivotal role of anthocyanins in both prevention and treatment of postischemic reperfusion brain damage. Anthocyanins extracts from black soybeans exhibited a protective effect on U87 cells which is associated with an increase in autophagy induction under conditions of hypoxic stress. Health properties of various anthocyanins are also listed in Table 18.1.

18.4 Bioavailability Anthocyanins are the bioactive compounds occurring naturally and are substantially dispersed in food items derived from plants and consumed by human beings on a large scale. The intake of anthocyanin-rich food items is associated in decreasing the risk of progression of heart-related diseases and cancers. In spite of the advantageous properties of anthocyanins, their efficacy at stopping or treating an extent of diseases depending on their bioavailability. Scientific growth has brought about some methods that mimic the digestion process, facilitating a development in the knowledge regarding bioavailability of anthocyanins. Lately, in vivo and in vitro models imitating digestion have been reported, which have come up with a greater level of comprehension of anthocyanin bioavailability, but every experiment has essential benefits and limitations. These subdivisions of flavonoids are accountable for the color blue, purple, and red of numerous leaves, fruits, and flowers. Eating food items that are abundant in anthocyanins are linked to decreased risk of cardiovascular disease and various types of cancers. Postoral administration, the path of anthocyanins follows a unique pattern which is quite different from those of other flavonoids. Besides the stomach, intestines too can absorb anthocyanins. In absorption process of anthocyanins active transporters play a major role. The gastrointestinal wall can contain the cyanidin 3glycoside and the pelargonidin-3-glucoside (anthocyanins), as rigorous as the metabolism of the first pass, and the systems as metabolites can also be absorbed in its entirety. These phenolic acid metabolites are as high in the blood stream as their parent compounds. Because of the health benefits provided by anthocyanins, the current metabolites may be the

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cause. Many anthocyanins can pass in large quantities into the large intestine and undergo decomposition catalyzed by microbiota. As a result, these decomposition products may be to blame for the anthocyanin-related health benefits in the large intestine. Bioavailability is defined as the rate and extent to which the active ingredient or moiety is absorbed from a drug product and becomes available at the site of action, according to the Food and Drug Administration. This definition is provided in the context of bioequivalence criteria, which are available for the acceptance of new generic items and are taken into account not only in terms of extent but also in terms of absorption rate. Bioavailability refers to the extent to which a xenobiotic can be used by the body. Systemic availability refers to the amount of a xenobiotic that enters the systemic circulation without being broken after ingestion. Apparent bioavailability in the body (apparent bioavailability): When it comes to xenobiotics that undergo extensive first-pass metabolism, apparent bioavailability refers to the amount of the dose that remains intact in the systemic circulation after oral administration. Total systemic bioavailability (total bioavailability): the proportion of a xenobiotics intake that is absorbed through the gastrointestinal wall and circulated in its native form, as well as its metabolite(s) generated by first-pass metabolism. Disposition: The process of getting a xenobiotic or its active metabolite(s) to their specific site(s) of action(s) in the body in the right concentration. All of these terminology as well as bioavailability and disposition are appropriate terms to use to describe the complex processes that play a role in anthocyanin fate in the gastrointestinal tract and throughout the body. First-pass effect and bioavailability: When only intravenous administrations were used for referencing and comparisons, the systemic bioavailability of anthocyanins was only 0.26%e1.8% in animal studies (Borges et al., 2013; Ichiyanagi et al., 2006; Marczylo et al., 2009; Matsumoto et al., 2006).

18.5 Stability Anthocyanins are exceedingly unstable and highly receptive to degradation. Color stability of anthocyanins is afflicted by various elements such as pH, chemical structure, moisture content, concentration, temperature, light, and oxygen (Hellström et al., 2013). Furthermore, the technologies used such as ultrasound, microwave, solvent extraction, and micellar encapsulation have a significant impact on the anthocyanin stability. In the course decades, anthocyanins have found to be increased a great deal not only established on knowledge about the pigments being used as feasible options for artificial food colorants and bioactive properties, but relating to their purification, stability, and extraction. A fundamental anthocyanidin form can be altered by adding wide variation of chemical groups, specifically through the several processes such as hydroxylation, acylation, and methylation. Consequently, there are a small number of types of anthocyanidins which are pointed out as basis of the successive lots of widely known anthocyanins with varying glycosylation and acylation forms, comprising anthocyanidins such as the proanthocyanidins and riccionidin A with supplementary additional incorporated rings (van Duynhoven et al., 2012). Currently, it is a widely known fact that some positions in anthocyanins are sensitive in reacting with numerous nucleophilic and electrophilic compounds. Also through nucleophilic attack in their positively charged carbons 2 and 4 of their pyranic ring anthocyanins hydration in carbon 2 generates the colorless hemiacetal form. Because of its positive charge anthocyanins appeared can also react with electrophilic compounds by its hydroxyl groups and will likely have an involvement of the uncharged hemiacetal form. The presence of the 5-OH group is necessary for the reactivity of these pigments alongside more compounds which occur during anthocyanin-rich food processing and aging. Normally di-, tri-, or polyacylated anthocyanins are better in stability in neutral and slightly acidic environments compared to monoacylated anthocyanins (Skrede & Wrolstad, 2002).

18.5.1 Factors affecting anthocyanins stability Research studies on major physical and chemical factors which play role in the anthocyanin degradation in the model structure and food extracts have been conducted. Anthocyanin color stability also depends on other factors such as nature and number of sugars bonded to the flavylium ion and acids linked to the glycosylic moiety. Food implementation of anthocyanins is constricted because of their ability to take part in reactions arising in its decolorization (Lee & Khng, 2002). Anthocyanins can be the perfect replacements for the synthetic red colorants. Although their utilization in food items has been hindered due to poor stability (Troise & Fogliano, 2013). Hence studies have been carried out on strong anthocyanin stabilization (Troise & Fogliano, 2013).

18.5.1.1 pH Anthocyanins are very responsive to pH modifications. When a particular anthocyanin is made to dissolve in water the secondary structure sequences are established from the flavylium cation with different acidebase, hydration, and

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tautomeric reactions (Chandrapala et al., 2012). The flavylium cation has been the principal balance form in strongly acidic solutions. Furthermore, the presence of tautomeric quinonoidal bases obtained from the flavylium cation by deprotonation and hemiacetal and chalcone configurations associated to the flavylium form by nucleophilic reaction with water under several pH environments is narrated (Andersen, 2008). Furthermore, anthocyanin color also differs with change in the pH, as it gives an intense red color at pH 1 or below but turns out to be colorless or purple with increase in the pH. Henceforth, its color turns deep blue between pH 7 and 8. Besides this increasing the pH turns the anthocyanin pigment from blue to green and further to yellow. The difference in the color is linked with the structure transformation with regard to alterations in the pH value (Lee & Khng, 2002). Also anthocyanin configuration is highly dependent on the pH of the solution and as a result its color stability which is strongly related to the deprotonation and hydration equilibrium reaction constant values (Ka and Kh) (Troise & Fogliano, 2013).

18.5.1.2 Temperature In the extracts as well as in the model systems anthocyanins thermal breakdown follows first-order reaction kinetics. As reported temperature decreases the anthocyanin stability (Troise & Fogliano, 2013). Studies showed that the anthocyanin stability depends on its configuration with the sugar moiety (Lee & Khng, 2002). However, the polymer fraction (brown pigments) exhibited the reverse. Troise and Fogliano (2013) reported with increase in temperature the half-life of juices and concentrates decreased. For maximum anthocyanin retention of foods carrying anthocyanins high-temperature short-time processing is preferred (Skrede & Wrolstad, 2002).

18.5.1.3 Oxygen Oxygen showed great impact on anthocyanins stability as vacuum, nitrogen, or argon atmosphere stored anthocyanins were stable than exposed to molecular oxygen (Skrede & Wrolstad, 2002). This could be ascribed due to the reaction of anthocyanins to hinder the radical activity at high concentration of oxygen resulting in the depletion of the antioxidant pigments (Gonzalez et al., 2018).

18.5.1.4 Light Anthocyanins being excellent visible light absorbers appear as colored substances. The pigment is based on the substitution pattern of the B-ring of the aglycon compared with the form of glycosylation of the flavan structure, which influences color formation to a smaller extent (Skrede & Wrolstad, 2002). Light-induced degradation depends upon the concentration of presence of the molecular oxygen. The strongest anthocyanin loss occurs when the pigments get exposed to fluorescent light (Giusti & Wallace, 2009). By selecting a proper packaging material may help to protect toward light and mainly in the spectrum of ultraviolet range. Giusti & Wallace (2009) reported improved light stability of anthocyanins by processes like glycosylation, acylation, and copigmentation.

18.5.1.5 Enzyme Ducamp-Collin et al. (2007) reported anthocyanins degraded by action of polyphenol oxidase (PPO) and peroxidase (POD) are essentially membrane bound. However, degradation of cyanidin 3-glucoside occurs in the absence of phenols found in the skin of sweet cherries; on the contrary only in presence of phenols the pulp homogenate degrades anthocyanin. Anthocyanin decoloration was affected by the pH and nature of the quinone acquired by enzymatic oxidation. Anhydrobase appears to be the most vulnerable anthocyanin form to oxidation. Anthocyanin degradation occurs due to the oxidation of the phenol substrate and is inhibited by ascorbic acid. Degradation of anthocyanin pigments found in fruit under specific enzyme preparations may be caused by hydrolyzing glycoside substituents. As these anthocyanins in their pure aglycon form are unstable and get degraded quickly (Giusti & Wallace, 2009).

18.5.1.6 Effect of concentration Anthocyanins are generally highly stable at higher concentrations. Skrede and Wrolstad (2002) reported concentration to be more important than the variation in stability caused by differences in anthocyanin structure.

18.5.1.7 Impact of water activity (aw) Studies show that anthocyanin stability increases with decrease in water content. As dry powdered anthocyanins (aw  0.3) can remain stable for many years when kept in the hermetically secured containers.

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18.6 Safety and toxicology Anthocyanins alter the appearance and actions of cellular and tissue targets influencing numerous systems linked with benefits to health, which includes anticancer, vascular, metabolic, neuronal health, etc. Evaluation of anthocyanin safety and toxicology suggests that acute toxicity is found to be very low in animals and no reports indicate any adverse health effects in humans with intake of anthocyanins usual dietary consumption amount. At present, there has been no recommended consumption amount of anthocyanins for ideal health or to abstain from negative effects; although, the potential research and good consumer demand will surely show the chances for following the dietary guidelines recommendations. The anthocyanins are widely used as food additives in production of colored jam, confectionaries, and drinks. Lately, synthetic food dyes have attracted public attention with regard to safety and their adverse effect on human health, especially with regard to neurological functions. The utilization of anthocyanin-based colorants in yogurt or yoghurt containing drinks and some mixed fruit juices is becoming more and more trending. Of lately acylated anthocyanins food colorants are being used in the food industry because of their high stability over nonacylated anthocyanins. Anthocyanins pigments extracted from the plants have an attractive hue with no toxicity even consumed at high doses compared to the synthetic colorants. These are also having value-added properties like being an antioxidant, nutraceutical and are having antimicrobial effect. However, the usage of organic solvents to draw out the anthocyanin pigment causes a toxicity issue. Despite the fact ethanol is generally considered to be a secure medium of extraction. One more extraction method known as subcritical water-based extraction has been tested for anthocyanin removal from berries and is regarded as efficient anthocyanin extraction method. Yet anthocyanin toxicity isn’t shown in published findings. The acceptable daily intake of 2.5 mg/kg/day for anthocyanins through grape skin extracts as confirmed by Joint FAO/WHO Expert Committee on Food Additives. The first country who mentioned recommended intake for anthocyanins was China, but they did not mention its tolerable upper intake level. Till date no toxicity effect of this pigment has been identified from animal studies. The aglycones proanthocyanidins of methyl pyran anthocyanins were considered to be safe and healthy functional ingredients incorporated as food additives. Although, the toxicological assessment of proanthocyanidins remains to be investigated in the near future.

18.7 Conclusion Anthocyanins are the phenolic compounds which could be incorporated in the food such as soft drinks, jams, dairy products, beverages, and confectionery as food additive, as anthocyanin-rich food decreases the risk of progression of several diseases. However, anthocyanin is exceedingly unstable and highly receptive to degradation. Several novel techniques could be employed for extraction of bioactive compounds especially anthocyanin. Each technique has its merits and limitations. Anthocyanins pigments have an attractive hue with no toxicity even consumed at high doses.

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Chapter 19

Genistein and daidzein Loveleen Sarao1, Sandeep Kaur2, Tanu Malik3 and Ajay Singh4 1

Department of Microbiology, Punjab Agricultural University, Ludhiana, Punjab, India; 2Department of Agriculture, MM University, Ambala,

Haryana, India; 3Centre of Food Science and Technology, CCSHAU, Hisar, Haryana, India; 4Department of Food Technology, Mata Gujri College, Fatehgarh Sahib, Punjab, India

19.1 Introduction Genistein (4,5,7-trihydroxyisoflavone) (Fig. 19.1) and daidzein (7-hydroxy-3-(-4-hydroxyphenyl)-4H-chromen-4-one) (Fig. 19.2) are isoflavones commonly found in soybeans and other legumes. Genistein got its name from the dyer’s broom, Genista tinctoria in 1899. Soybean has been reported to contain the majority of genistein which is the highest protein containing vegetables. Garbanzo beans (chickpeas) contain pint-sized amount of genistein. Soy milk, soy flour, soy protein isolates, textured soy protein, tofu, child formula, tempeh, and miso are the soy-based edibles containing genistein in variable quantity. Genistein is also found in Alfalfa and clover sprouts, barley meal, broccoli, cauliflower and sunflower, caraway, and clover seeds. Generally 0.2e1 mg/g in various forms of glycosidic conjugates is the concentration of genistein in the bulk part of soy nourishment materials. Genistein have also been concluded to found in the trifolium species. Various kinds of microorganisms such as Streptomyces sp. and Pseudomonas sp. have been reported to provide genistein from the fermentation broth. Daidzein is also called an inactive analog of genistein. It is a secondary metabolite formed with the help of phenylpropanoid pathway in plants and is used in defense response against pathogens. In humans, it is used as phytoestrogen for menopausal relief and also used in medicines for osteoporosis, blood cholesterol, and lowering the risk of some hormonerelated cancers and heart disease.

19.2 Sources 19.2.1 Genistein sources Soy-based foods are the best-known sources of genistein. These include soy cheese or soy drinks such as soy milk and soy-based beverages. Bhagwat et al. (2008) reported that the mature soybeans constituted 5.6e276 mg/100 g of genistein with an average content of 81 mg/100 g often described for comparative purposes. Besides containing genistein, soy foods also contain daidzein, which is another major isoflavone, differing from genistein by the lack of the hydroxyl group at position 5 (Fig. 19.1). These isoflavones may either exist in their aglycone or glycoside forms. The glycoside forms most commonly found of genistein and daidzein are those of O-b-D-glucoside derivatives at position 7 in both compounds. Soybean is used in the preparation of several traditional Asian foods, resulting in the average dietary isoflavone intake of 25e50 mg/day in Asian countries (Messina et al., 2006; Van Erp-Baart et al., 2003). This intake is as low as 2 mg/day on an estimate in Western countries. Second major source of genistein having 0.2e0.6 mg/100 g together with the other related isoflavone, daidzein is legumes (Liggins et al., 2000a). A typical example of legume is the genus Lupinus (commonly known as lupin) possessing nutritional value similar to soybean and is now being widely cultivated for its seeds. A considerable amount of genistein is found in fruit, nuts, and vegetables. Liggins et al. (2000c) reported the estimated range from 0.03 to 0.2 mg/100 g. Genistein concentrations up to 4.4 mg/100 g have been recorded in some of the native cherry cultivars of Hungarian origin. Various trends are adopted to enhance the nutritional and medicinal values of certain foods; the biotechnological approach used to maximize the isoflavonoid yield by sprouting seeds is the commonest method. The nutritional value is Nutraceuticals and Health Care. https://doi.org/10.1016/B978-0-323-89779-2.00016-8 Copyright © 2022 Elsevier Inc. All rights reserved.

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FIGURE 19.1 Chemical structure of genistein.

FIGURE 19.2 Chemical structure of daidzein.

also improved by the metabolic processes of seed germination, characterized by the degradation of food reserves and anabolic processes devoted to the developing embryo. Ahmad and Pathak (2000) reported that this leads to an increase in the content of vitamins and plant secondary metabolites, such as isoflavonoids. In the germinated soybean seeds and related products, the increased content of genistein and other isoflavonoid aglycones has been well reported by Quinhone and Ida (2015).

19.2.2 Daidzein sources Soybeans are a rich source of genistein and daidzein containing approximately 670 and 540 mg/kg, respectively, of both compounds. Daidzein is found in measurable quantities in oatmeal, wheat bran, corn flour, fruits like mango, melon, peaches, plums, prunes, strawberry, coconut, etc. (Liggins et al., 2000b). Fermented food products from soy are also a rich source of these isoflavones, e.g., Japanese Miso, doenjang (Korea), douchi (China), tempeh (Indonesia). Plants of the family Fabaceae are also rich in isoflavones, e.g., red clover and hop clover, which is an important part of animal feed (Daniel & Marius, 2017). Daidzein is considered weak phytoestrogen because of its 1000 times lower affinity for the estrogen receptor (Shutt & Cox, 1972). Lee et al. (2014) documented that the content of genistein and related aglycones increases during the process of fermentation of soybean products; it is also possible to obtain genistein from nonlegume plants, such as rice, through genetic manipulation. Transgenic rice lines with 30-fold more genistein content were reported by Sohn et al. (2014) by cloning the enzyme IFS from a genistein-rich soybean cultivar. In Europe and the United States, soy-based meat substitutes, soy milk, soy cheese, and soy yogurt have recently gained popularity owing to the recognition of the medicinal value of genistein and related isoflavonoids.

19.3 Extraction and characterization techniques The aglycone derivatives can be extracted from their sources through various means such as treatment with enzyme b-glucosidase; acid treatment of soybeans, followed by solvent extraction; or the compound can be chemically synthesized (Prakash & Tanwar, 1995). Acid treatment is a very harsh method as concentrated inorganic acids are used. Both enzyme treatment and chemical synthesis methods are costly. Due to this, more economical processes are sought. One such approach consists of using fermentation to isolate genistein. As genistein is present mostly in its glycoside forms, the enzyme b-glycosidase was used to convert genistein glycosides to aglycone genistein. b-Glucosidase is an expensive enzyme, hence microorganisms, which can use soy as a substrate to grow and secrete b-glucosidase, were used. Thus biotransformation of genistein glycosides to genistein aglycone using b-glucosidase secreting microorganisms was thought to be an economically feasible process, which could be adapted to large-scale production of genistein.

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19.3.1 Analytical methods for determination and characterization of genistein and daidzein There are several analytical methods developed for the identification and quantification of genistein and daidzein. Highperformance liquid chromatography (HPLC), Raman spectroscopy, FTIR spectroscopy, and polarimetry methods have been used by different scientists for detection and analysis (Sulistyowati et al., 2019). Thin-layer chromatography and polyamide chromatography have been popular techniques in past for isoflavone separation. During current times reversephase separation is the most preferred one. Few techniques have been discussed below.

19.3.1.1 UVevisible spectroscopy A simple and rapid UVevisible method was developed and validated by da Costa César et al. (2008) for the quantification of genistein and its glycoside genistein in soy dry extracts, on reaction with AlCl3. An intense absorption band with a maximum wavelength at 382 nm was observed in the UVevisible spectrum for a solution of genistein after reaction with AlCl3.

19.3.1.2 Mass spectrometry Chang et al. (2000) developed and validated an analytical methodology that was used to determine the pharmacokinetics in blood and distribution of genistein in tissues from rats exposed through the continuous dietary intake in a multigeneration test.

19.3.1.3 Liquid chromatography/tandem mass spectrometry To quantify genistein in dog plasma, an accurate and sensitive analytical method has been developed by Feng et al. (2013) using HPLC/tandem mass spectrometry. For a pharmacokinetic comparison of immediate and extended-release tablets in beagle dogs after oral administration, this method was successfully applied. Rapid genistein absorption was shown by immediate-release tablets. Feng et al. (2013) reported considerably slower absorption of genistein and more sustainability for extended-release tablets. Holder et al. (1999) developed and validated a simple and sensitive analytical method based on LC/ES-MS for the determination of genistein in the blood of rats receiving dietary genistein. The method uses serum/plasma deproteination, liquideliquid extraction, deuterated genistein and daidzein internal standards, isocratic LC separation, and electrospray mass spectrometric quantification using selected ion monitoring. As compared to the previous LC/MS methods, the sensitivity of LC/ES-MS detection in combination with isotopic labeled internal standards adds additional accuracy and precision of determinations as it directly provides quality control and assurance information in every sample throughout large sample sets.

19.3.1.4 Ultra performance liquid chromatography/mass spectroscopy This is a highly sensitive, accurate, and robust method used to determine genistein and its four metabolites’ concentrations in the blood of FVB mice. The method was successfully applied for mouse bioavailability study by using only 20 mL of a mouse blood sample, hereby, giving a complete pharmacokinetic profile (Yang et al., 2010). HPLC a wide variety of analytical techniques has been applied to the quantitation of soy isoflavones in foods and biological fluids. However, methodological improvements for the quantitative analysis of the free plus conjugated forms of isoflavones in human urine and plasma continue to be needed for clinical trials aimed at defining single- and multiple-dose safety, pharmacokinetics, and efficacy profiles. Validated analytical methods were developed using HPLC with UV detection which enabled the measurement of (1) the free, nonconjugated molecules, (2) the combined free plus the sulfate-conjugated molecules, or free plus sulfate fraction, and (3) the total conjugated and free molecules of genistein, daidzein, and glycitein in human plasma and urine. The development of these validated HPLCeUV assay provided novel analytical methods with which the pharmacokinetics and pharmacodynamics of the principle active forms of soy isoflavones can be studied.

19.3.1.5 Reverse-phase high-performance liquid chromatography Reverse-phase separation is based on hydrophobic interactions of isoflavones with the stationary phase (Vacek et al., 2008). The retention time of separated compounds is affected by their solubility in water. Free glycone form of daidzein and genistein was determined by Sulistyowati et al. (2019) by using reverse-phase high-performance liquid

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chromatography (RP-HPLC). They used a binary mobile phase consisting of methanol and 0.1% acetic acid (53:47). The recovery rate for daidzein was 83.0%e100.95% and genistein was 80.07%e108.79%. Khoo et al. (2008) reported reversephase column was best to determine isoflavone contents, out of various HPLC techniques. They also reported that 90 min extraction time increased isoflavone aglycone contents.

19.3.1.6 Capillary zone electrophoresis Luan et al. (2017) used capillary zone electrophoresis to determine genistein, daidzein, and formononetin content in coffee. The samples were extracted with acid hydrolysis and purified with ether and butylated hydroxytoluene. The average recovery of compounds was 99.39%. Electromigration techniques like capillary electrochromatography and micellar electrokinetic capillary chromatography have also been used for isoflavonoid detection and analysis (Vacek et al., 2008). In the capillary electrochromatography technique separation of analytes occurs in a capillary filled with sorbent and separation is done by an electric field.

19.4 Chemistry of genistein 19.4.1 Chemical nature The biologically active glucoside genistein is the prime dietary source of genistein. When soybeans or soy products are fermented/digested, it results in the release of the sugar molecule from the isoflavone glycoside, genistein leaving the isoflavone aglycone, genistein (Table 19.1) (Markovits et al., 1989).

19.4.2 Chemical structure Genistein has a diphenol structure as reported by Cassidy (1999). It resembles human endogenous estrogen (E2) stereochemically. The similar distance between the OH groups on the opposite sides of genistein and E2 molecules makes also genistein capable of binding to ER subtypes a and b. The structure of daidzein resembles mammalian estrogens and it has a dual-directional function as it replaces/interferes with estrogen and estrogen-receptor complex. It has a C6eC3eC6 structure (phytochemicals) and is an aglycone (molecule without sugar) of glycoside daidzein. It has poor water and lipid solubility because of its chemical structure (Wang et al., 2019). It contains 31 bonds in total out of which 12 are aromatic bonds. It contains three 6-membered rings, one 10-membered ring, one aromatic ketone, two aromatic hydroxyls, and one aromatic ether. Daidzein contains a 2-isoflavene skeleton bearing a ketone group at C4 carbon atom, hence considered a flavonoid lipid molecule.

19.4.3 Active principles 19.4.3.1 Phytoestrogens and mammalian estrogens The major source of plant-derived phytoestrogen compounds is soy (genistein). It has been used as a traditional food for a long (Farina et al., 2006). Phytoestrogen is plant-derived secondary metabolites that mimic mammalian estrogen

TABLE 19.1 Chemical nature of genistein and daidzein. R1

R2

R3

Glucoside

H

H

H

Daidzein

OH

H

H

H

H

COCH3

Acetyldaidzin

OH

H

COCH3

Acetylgenistin

H

H

COCH2COOH

Malonyldaidzin

OH

H

COCH2COOH

Malonylgenistin

Acetylglucoside

Malonylglucoside

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17b-estradiol structurally and functionally (Barnes et al., 2000; Dixon & Ferreira, 2002; Magee & Rowland, 2004). Hence, for overcoming breast cancer, genistein may be used. Several subtypes of phytoestrogens, including isoflavones, coumestans, lignans, chalcones, flavones, and prenylflavonoids, are known. The most common form of phytoestrogens is isoflavones (Matsuda et al., 2001; Milligan et al., 1999; Ososki & Kennelly, 2003; Rafi et al., 2000). A common diphenolic structure resembling the structure of the potent synthetic estrogens diethylstilbestrol and hexestrol is possessed by them. Aglycones and glycosides are the two basic subgroups of isoflavones. Genistein belongs to the aglycone subgroup of isoflavones.

19.4.3.2 Anticancer effects Peterson and Barnes (1991) and Zhou et al. (1999) reported that genistein and daidzein inhibit the proliferation of different types of cancer cells. Although the mechanism of action is unknown, according to a hypothesis soy isoflavones were shown to activate estrogen receptors (Messina, 1999) and inhibit enzymes progesterone 5a-reductase and 17b-hydroxysteroid dehydrogenase hence inhibiting the activity of growth-promoting steroid hormones (Pereira et al., 1994). Besides this antioxidant activity (Wei et al., 1996), inhibition of protein-tyrosine kinaseemediated signal transduction (Akiyama et al., 1987), DNA topoisomerase (Constantinou et al., 1995), transforming growth factor 1emediated signal transduction (Nilsson et al., 2011), and angiogenesis inhibition have been reported by many scientists (Zhou et al., 1999).

19.4.3.3 Stability The stability of compounds is affected by several physical and chemical factors like light, temperature, pH, solvent, etc. Hence processing and storage conditions affect the form and availability of isoflavones in meals and pharmaceutical preparations. Isoflavones have been reported to be sensitive to irradiation (UV photodegradation) (Vacek et al., 2008). Dunford et al. (2003) reported that a solution of daidzein in organic solvents (acetonitrile, methanol, hexanol, ethanol, etc.) undergoes photodegradation on exposure to solar-type radiation. The photostability increases with an increase in solvent polarity and daidzein has relatively higher photostability because of its antioxidant nature. The degradation can be detected by increasing absorption maximum and changes in fluorescence emissions (Vacek et al., 2008). In their study on the stability of malonyl and acetyl conjugates of daidzein and genistein (Table 19.2), Mathias et al. (2006) checked the stability of compounds at 25, 80, and 100
C and different pH values. They reported that b-glucosides and their derivatives undergo degradation at higher pH (þ10) and negligible degradation occurs at room temperature. Delmonte et al. (2006) observed genistein degradation and a slow decrease in its concentration with the heating of soy milk (Table 19.2).

TABLE 19.2 Acetyl and malonyl derivatives of genistein and daidzein. Common name

Genistein

Daidzein

IUPAC name

5,7-Dihydroxy-3-(4-hydroxyphenyl) chromen-4-one

7-Hydroxy-3-(-4-hydroxyphenyl)-4H-chromen-4-one

Other names

4,5,7-Trihydroxyisoflavone

40 ,7-Dihydroxyisoflavone, Daidzeol, Isoaurostatin

Molecular formula

C15H10O5

C15H10O4

Formula weight

270.24

254.24

Physical state

Solid

Solid

Melting point




330 C

297e298
C

Water solubility

Insoluble

Insoluble

Solubility

DMSO

DMSO, ethanol, dimethyl formamide

Color

Off-white

Pale yellow




Storage temperature

20 C