Feed Additives: Aromatic Plants and Herbs in Animal Nutrition and Health 0128147016, 9780128147016

Table of contents :
Content: 1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives 2. Innovative uses of aromatic plants as natural supplements in nutrition 3. Herbs and aromatic plants as feed additives: aspects of composition, safety, and registration rules 4. Sustainable use of mediterranean medicinal-aromatic plants 5. Aromatic plants and their extracts pharmacokinetics and in vitro/in vivo mechanisms of action 6. Distribution of aromatic plants in the world and their properties 7. Herbal extracts as antiviral agents 8. Functional ingredients derived from aromatic plants 9. Toxic or harmful components of aromatic plants in animal nutrition 10. Application of aromatic plants and their extracts in diets of broiler chickens 11. Application of aromatic plants and their extracts in the diets of laying hens 12. Application of aromatic plants and their extracts in diets of turkeys 13. Application of plant essential oils in pig diets 14. Application of aromatic plants and their extracts in aquaculture 15. Application of aromatic plants and their extracts in dairy animals 16. The effects of aromatic plants and their extracts in food products 17. The effects of plant extracts on the immune system of livestock: the isoquinoline alkaloids model 18. Effects of phytobiotics in healthy or disease challenged animal 19. Resistance of bacteria, fungi, and parasites to antibiotics or natural substances of botanical origin

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FEED ADDITIVES

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FEED ADDITIVES AROMATIC PLANTS AND HERBS IN ANIMAL NUTRITION AND HEALTH

Edited by

PANAGIOTA FLOROU-PANERI EFTERPI CHRISTAKI ILIAS GIANNENAS

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 © 2020 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: 978-0-12-814700-9 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisition Editor: Patricia Osborn Editorial Project Manager: Susan Ikeda Production Project Manager: Prem Kumar Kaliamoorthi Cover Designer: Christian Bilbow Typeset by TNQ Technologies

Contents Legal status of feed additives Conclusion 51 References 52 Further reading 56

Contributors ix Preface xiii 1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives

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4. Sustainable use of mediterranean medicinal-aromatic plants

Ilias Giannenas, E. Sidiropoulou, Eleftherios Bonos, E. Christaki, and P. Florou-Paneri

Katerina Grigoriadou, Nikos Krigas, Diamanto Lazari, and Eleni Maloupa

Introduction 1 Worldwide use of aromatic plants throughout history 3 Current situation on the use of aromatic plants and herbs in human and veterinary medicine, plant sustainability, and safety issues 9 Future perspectives 13 References 15

Valuable properties of medicinal and aromatic plants (MAPs) and herbal medicinal products 57 European market of medicinal and aromatic plants: trends, challenges, and the value chain 59 Mediterranean medicinal and aromatic plants: wealth, uniqueness, and risks 64 From wild to cultivation: conservation and sustainable exploitation of phytogenetic resources of maps 66 Research on propagation of native maps: a key for sustainable exploitation 69 Pilot fields: a step closer to new crops 70 References 71

2. Innovative uses of aromatic plants as natural supplements in nutrition E. Christaki, Ilias Giannenas, Eleftherios Bonos, and P. Florou-Paneri

Introduction 19 Bioactive compounds of aromatic plants 20 Biological properties of aromatic plants (functional foods) nutrigenomics 21 Aromatic plants as dietary supplements 22 Conclusions 30 References 31

5. Aromatic plants and their extracts pharmacokinetics and in vitro/in vivo mechanisms of action  Ivana Cabarkapa, Nikola Puvaca, Sanja Popovic,  Dusica Colovi c, Ljiljana Kostadinovic, Eleanor Karp Tatham, and Jovanka Levic

3. Herbs and aromatic plants as feed additives: aspects of composition, safety, and registration rules

Introduction 75 Antimicrobial effects of aromatic plants and their EOs 77 Antioxidant effects of aromatic plants 80 Effects on performance, digestibility, and intestinal functions in animals 81 Acknowledgments 85 References 85

Ch M. Franz, K.H.C. Baser, and I. Hahn-Ramssl

Introduction 36 Plants and herbal products used as feed additives 37 Chemistry and activity 45

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CONTENTS

6. Distribution of aromatic plants in the world and their properties Amit Kumar Pandey, Prafulla Kumar, M.J. Saxena, and Prabhakar Maurya

Introduction 89 Historic preview 90 Definition of medicinal and aromatic plants 94 Classification 96 Distribution pattern in the world market 99 Uses of aromatic plants 106 Present status and conservation initiatives 110 Conclusion and way forward 111 References 113

7. Herbal extracts as antiviral agents A.R. Yasmin, S.L. Chia, Q.H. Looi, A.R. Omar, M.M. Noordin, and A. Ideris

Introduction 116 Poultry 116 Swine 121 Ruminants 126 Conclusion 128 References 128

8. Functional ingredients derived from aromatic plants Sonia A. Socaci, Anca C. Farca¸s, and Maria Tofana

Introduction 133 Essential oils 134 Uses and applications 140 Conclusions 143 Acknowledgments 143 References 143 Further reading 146

9. Toxic or harmful components of aromatic plants in animal nutrition Maria Grazia Cappai, and Sabine Aboling

Aromatic plants: toxicological properties in view of their ecological function 147 Sensationdselection behaviordco-existence as essentials of the planteanimal interaction 150

Potential adverse principles/traits of aromatic plants in animal nutrition 152 References 156 Further reading 158

10. Application of aromatic plants and their extracts in diets of broiler chickens Li-Zhi Jin, Yueming Dersjant-Li, and Ilias Giannenas

Introduction 159 Mode of actions of aromatic medicinal plants, spices, or herbs, their extracts or essential oils in poultry 161 Future implementations and conclusions 176 References 179

11. Application of aromatic plants and their extracts in the diets of laying hens David Harrington, Heidi Hall, David Wilde, and Wendy Wakeman

Introduction 187 Aromatic plants in layer well-being Performance 194 Egg characteristics 195 Conclusion 198 References 199

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12. Application of aromatic plants and their extracts in diets of turkeys Mehmet Bozkurt, and Ahmet Engin Tüzün

Introduction 205 Antimicrobial action 207 Plant-derived chemicals contribute to food microbial safety 208 Opportunities to naturally improve antioxidant capacity 210 Initial attempts to enhance immunity 213 Specific effect on gut morphology and function 213 Phytogenic compounds offer novel strategies to control Histomonas meleagridis 214 Effects on growth performance 215 Conclusions and areas for future research 219 References 220

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CONTENTS

13. Application of plant essential oils in pig diets

16. The effects of aromatic plants and their extracts in food products

Hong-Kui Wei, Jun Wang, Chuanshang Cheng, Li-Zhi Jin, and Jian Peng

Bojana Filipcev

Introduction 227 Application of EOs in weaning piglets 228 Application of EOs in sows 233 Application of EOs in boars 233 Effect of oregano oil against transportation stress 234 Conclusions 234 References 235 Further reading 237

14. Application of aromatic plants and their extracts in aquaculture Ángel Hernández-Contreras, and María Dolores Hernández

Introduction 239 Applications of aromatic plants and their extracts in aquaculture 241 Latest properties discovered and possible uses 252 Current regulatory status and future perspectives 254 References 255

15. Application of aromatic plants and their extracts in dairy animals Mariangela Caroprese, Maria Giovanna Ciliberti, and Marzia Albenzio

Introduction 261 Extraction methods of essential oils and use of abioc in animal nutrition 262 Aromatic plants and their extracts as modifiers of rumen fermentation 264 Aromatics plant antimicrobial activities: effect on ruminant immune system 266 Aromatics plant antioxidant activities in dairy animals 268 Aromatic plants and their extracts as enhancer udder health 269 Conclusions and Future Direction 272 References 272

Introduction 279 Application of aromatic herbs in food 280 Conclusion 290 References 291

17. The effects of plant extracts on the immune system of livestock: the isoquinoline alkaloids model Valeria Artuso-Ponte, Anja Pastor, and Manfred Andratsch

AGP removal and gut health 296 The use of antimicrobials and the risk of antimicrobial resistance 296 The public health concern 297 The role of the mucosal immune system 297 The role of NF-kB 298 Causes and consequences of intestinal inflammation 299 Plant metabolites with antiinflammatory properties 300 Nonnitrogen secondary metabolites 300 Nitrogen secondary metabolites 302 Isoquinoline alkaloids 303 Mode of action of isoquinoline alkaloids: inhibition of NF-kB activation 304 Consequences of reducing inflammation with isoquinoline alkaloids 304 Isoquinoline alkaloids, and gut health and stress 306 Regulating intestinal inflammation and the effects on animal performance 307 References 308

18. Effects of phytobiotics in healthy or disease challenged animals Ioannis Skoufos, Eleftherios Bonos, Ioannis Anastasiou, Anastasios Tsinas, and Athina Tzora

Introduction 311 Aromatic and medicinal plants, herbs, their extracts and essential oils as feed additives 313

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CONTENTS

In vitro and in vivo antimicrobial and antiparasitic activities of aromatic and medicinal plants, herbs, their extracts and essential oils in poultry 315 In vitro and in vivo antimicrobial activities of aromatic and medicinal plants, herbs, their extracts and essential oils in pigs 322 In vitro and in vivo antistress activities of aromatic and medicinal plants, herbs, their extracts and essential oils in ruminants 325 Effects of dietary phytobiotics in rabbits 327 Conclusions 328 References 328

19. Resistance of bacteria, fungi, and parasites to antibiotics or natural substances of botanical origin Christos Papaneophytou, Ilias Giannenas, and Catalin Dragomir

Introduction 339 Antibiotics target specific cellular processes 341 Mechanisms of antimicrobial resistance 342 Antimicrobial of botanical origin 344 Plant-derived antiquorum sensing compounds 346 Conclusions 349 References 349 Index 355

Contributors E. Christaki Laboratory of Nutrition, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece

Sabine Aboling Institute of Animal Nutrition, University of Veterinary Medicine, Hannover, Foundation, Hanover, Germany Marzia Albenzio University of Foggia, Department of the Sciences of Agriculture, Food, and Environment, Foggia, Italy Ioannis Anastasiou Cavan, Ireland

Trinity

Nutrition

Maria Giovanna Ciliberti University of Foggia, Department of the Sciences of Agriculture, Food, and Environment, Foggia, Italy  Dusica Colovi c University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia

Ltd.,

Manfred Andratsch Phytobiotics Futterzusatzstoffe GmbH Eltville, Germany Valeria Artuso-Ponte Phytobiotics satzstoffe GmbH Eltville, Germany

Yueming Dersjant-Li Consultant in Animal Nutrition, Nijkerk, The Netherlands

Futterzu-

Catalin Dragomir National Research Development Institute for Animal Biology and Nutrition (INCDBNA), Balotesti, Romania

K.H.C. Baser Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Nicosia, North Cyprus

Anca C. F arca¸s Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania

Eleftherios Bonos Department of Agriculture, School of Agriculture, University of Ioannina, Arta, Greece

Bojana Filipcev University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia

Mehmet Bozkurt Department of Animal Science, Faculty of Agriculture, Adnan Menderes University, Kocarlı, Aydın, Turkey  Ivana Cabarkapa University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia

P.

Maria Grazia Cappai Department of Veterinary Medicine, University of Sassari, Sassari, Italy

Florou-Paneri Laboratory of Nutrition, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece

Ch M. Franz WG Functional Plant Compounds, University of Veterinary Medicine, Vienna, Austria

Mariangela Caroprese University of Foggia, Department of the Sciences of Agriculture, Food, and Environment, Foggia, Italy

Ilias Giannenas Laboratory of Nutrition, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece

Chuanshang Cheng Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, P. R. China; The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, P. R. China

Katerina Grigoriadou Laboratory of Conservation and Evaluation of Native and Floricultural Species-Balkan Botanic Garden of Kroussia, Hellenic Agricultural Organization e DEMETER, Thessaloniki, Greece

S.L. Chia Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

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x

CONTRIBUTORS

I. Hahn-Ramssl WG Functional Plant Compounds, University of Veterinary Medicine, Vienna, Austria

M.M. Noordin Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Heidi Hall Anpario plc, Nottinghamshire, United Kingdom

A.R. Omar Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

David Harrington Anpario plc, Nottinghamshire, United Kingdom Ángel Hernández-Contreras IMIDA - Aquaculture, San Pedro del Pinatar, Region of Murcia, Spain María Dolores Hernández IMIDA - Aquaculture, San Pedro del Pinatar, Region of Murcia, Spain A. Ideris Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Li-Zhi Jin Meritech and Huazhong Agricultural University Cooperative Innovation Center, Wuhan, P. R. China; Guangzhou Meritech Bioengineering Co., Ltd., Guangzhou, P. R. China Ljiljana Kostadinovic Planet Niksic, Montenegro

Fresh

d.o.o.,

Nikos Krigas Laboratory of Conservation and Evaluation of Native and Floricultural SpeciesBalkan Botanic Garden of Kroussia, Hellenic Agricultural Organization e DEMETER, Thessaloniki, Greece Prafulla Kumar Ayurvet Limited Office, Delhi, India Diamanto Lazari Laboratory of Pharmacognosy, School of Pharmacy, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece Jovanka Levic University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia Q.H. Looi Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Eleni Maloupa Laboratory of Conservation and Evaluation of Native and Floricultural SpeciesBalkan Botanic Garden of Kroussia, Hellenic Agricultural Organization e DEMETER, Thessaloniki, Greece Prabhakar Maurya CEHTRA Chemical Consultants Pvt Ltd, Delhi, India

Amit Kumar Pandey Ayurvet Limited Office, Delhi, India Christos Papaneophytou Department of Life and Health Sciences, School of Sciences and Engineering, University of Nicosia, Nicosia, Cyprus Anja Pastor Phytobiotics GmbH Eltville, Germany

Futterzusatzstoffe

Jian Peng Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, P. R. China; The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, P. R. China Sanja Popovic University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia Nikola Puvaca University Business Academy, Faculty of Economics and Engineering Management, Department of Engineering Management in Biotechnology, Novi Sad, Serbia M.J. Saxena Ayurvet Limited Office, Delhi, India E. Sidiropoulou Laboratory of Nutrition, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece Ioannis Skoufos Department of Agriculture, School of Agriculture, University of Ioannina, Arta, Greece Sonia A. Socaci Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania; Institute of Life Sciences, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania Eleanor Karp Tatham University of London, Royal Veterinary College, London, United Kingdom

CONTRIBUTORS

Maria Tofana Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania Anastasios Tsinas Department of Agriculture, School of Agriculture, University of Ioannina, Arta, Greece Ahmet Engin Tüzün Adnan Menderes University, Kocarlı Vocational Scholl, Kocarlı, Aydın, Turkey Athina Tzora Department of Agriculture, School of Agriculture, University of Ioannina, Arta, Greece Wendy Wakeman Anpario plc, Nottinghamshire, United Kingdom Jun Wang Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, P. R. China; Meritech and Huazhong Agricultural University Cooperative Innovation Center, Wuhan, P. R. China

xi

Hong-Kui Wei Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, P. R. China; Meritech and Huazhong Agricultural University Cooperative Innovation Center, Wuhan, P. R. China David Wilde Anpario plc, Nottinghamshire, United Kingdom A.R. Yasmin Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

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Preface Nature has been the source of aromatic plants and herbs for thousands of years, and their use as medicines, enhancers of food aroma, preservatives, or cosmetics is well known from antiquity. Today those aromatic and medicinal plants have gained more recognition as dietary supplements because they are characterized as natural, safe, ecofriendly, and they possess a plethora of health-promoting properties, making their ongoing usage part of an emerging field at the cutting edge of science. It must be emphasized that the relationship between diet and health has been reported 2500 years ago, as initially proposed by Hippocrates, who is quoted as saying “Let food be thy medicine and medicine be thy food.” These benefits depend greatly on the diversity and the number of their bioactive compounds such as phenolics and terpenes. Consumers are becoming increasingly aware of the correlation between diet, health, and disease prevention. A new vista has been opened in the use of aromatic plants in global health care delivery to replace synthetic substances. Therefore, aromatic plants and their derivatives have the potential to become a new research area for human or animal nutrition and health. The compilation of the 19 chapters included in this book describe the use of aromatic plants and their extracts as potential vehicles for natural feed additives, e.g., growth promoters as alternatives to antibiotics, antioxidants, antimicrobials, antivirals, immunostimulators, flavorings, pigments on poultry, pigs, dairy animals, and

aquaculture. Attention was also paid to the historic use of aromatic plants, their production and sustainable use of the self-grown or cultivated plants and their global distribution. Moreover, pharmacokinetics and mechanisms of action of their bioactive compounds are discussed, as well as their applications in food packaging from niche markets to largescale. Additionally, toxic effects, harmful ingredients, risks in use and regulatory rules of aromatic plants are displayed. Finally, resistance of bacteria, fungi, and parasites to antibiotics or natural substances of aromatic plants and their interactions are presented. Recently, there has been considerable interest in the field of nutrigenomics that is helping to understand better the use of aromatic plants as functional ingredients in nutrition, pushing further the boundaries of their potential applications in human and veterinary medicine, pharmaceuticals, as well as feed and food industries. We are very grateful to all the authors around the world who are internationally recognized for their expert contribution, and who have provided their outstanding scientific research conducted in the field of aromatic plants. We sincerely hope that this book can contribute and give impetus to the exploration and utilization of the natural treasure of aromatic plants for the good of both humans and livestock.

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The Editors Professor Panagiota Florou-Paneri Professor Efterpi Christaki Assistant Professor Ilias Giannenas

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C H A P T E R

1 The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives Ilias Giannenas1, E. Sidiropoulou1, Eleftherios Bonos2, E. Christaki1, P. Florou-Paneri1 1

Laboratory of Nutrition, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece; 2Department of Agriculture, School of Agriculture, University of Ioannina, Arta, Greece O U T L I N E

Introduction

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Worldwide use of aromatic plants throughout history

veterinary medicine, plant sustainability, and safety issues

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Current situation on the use of aromatic plants and herbs in human and

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Future perspectives

13

References

15

Introduction Aromatic plants and herbs have been widely used for medical purposes not only for humans but for animals as well. Medicinal plants are mainly considered those used in official and traditional medicine, whereas aromatic plants are those used for their aroma and flavor.

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00001-7

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Copyright © 2020 Elsevier Inc. All rights reserved.

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1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives

The World Health Organization (WHO) defines as “herbal medicines” plant-derived materials or products with therapeutic or other human benefits, which contain either raw or processed ingredients from one or more plants (WHO, 2001). Other specific terms used for medicinal and aromatic plants are (Inoue and Craker, 2014; American Botanical Council, 2019): a) “herb or culinary herb” refers to any aromatic plant material from temperate regions, used in minor quantities to flavor foods and beverages, but has little or no known nutritional value, b) “spice” implies to an aromatic plant material from tropical regions used in minor quantities to flavor foods and beverages, but has little or no known nutritional value, c) “medicinal plant” describes various plants used for treatment of disease or other body afflictions, d) “essential oil” indicates that a volatile oil can be extracted from plants by distillation, solvents or expression, e) “poisonous plant” indicates to plants containing alkaloids or other substances that may produce toxic effects when introduced into the body. The positive effects of aromatic plants, herbs, and their essential oils in various diseases have been evidenced throughout history (Zollman and Vickers, 1999; Giacometti et al., 2018; Oliveira et al., 2018). Aromatic plants and herbs are the first pharmacological compounds that have been used in ancient times to treat diseases or other abnormal conditions and even now are used in folk or as an ethno-type of medicine (Wesley Schultz, 2001; Giannenas, 2008; Christaki et al., 2012; Giannenas et al., 2013, 2018). Almost all ancient civilizations have demonstrated some evidence of awareness of plants’ medicinal use. In ancient civilizations, indigenous people used aromatic plants and herbs to cure not only physical but also mental disorders. At that time, people believed that illness had a supernatural cause or emerged from evil. Therefore, healers were highly respected and played an important role in their communities (Voliotis, 1998; Kankara et al., 2015). Currently, in societies living in isolated, rural and mountainous areas with limited access to official health facilities, such as in developing countries, many physical and spiritual therapies are still relied upon and value the use of aromatic plants (Sen and Chakraborty, 2016; Solomou et al., 2016). Herbal medicine constitutes the main type of traditional medicine, commonly practiced by traditional healers. The Chinese, Native American, Tibetan, and Indian Ayurvedic practitioners are valued not only in countries of origin but also in developed ones, such as the United States or Germany. Often, they use mixtures of unpurified plant extracts, claiming that these may work synergistically so that the effect of the whole herb is greater than the sum total of the effects of individual components. Even more, it is claimed that toxicity is reduced when whole herbs are used instead of isolated active ingredients (Zollman and Vickers, 1999). Similarly, the use of plant-based remedies in vet medicine has been related to traditional knowledge, depending on practical experience and observation being passed from generation to generation, both in verbal and writing form (WHO, 2001). Since the 1990s, it was well understood that the use of drugs, especially antibiotics, as additives in animal feed, was gradually leading to antibiotic resistance of microbe pathogens. This phenomenon, along with concerns that the toxicity of many of these compounds were potentially entering the food chain, raised serious concerns regarding human health and

Worldwide use of aromatic plants throughout history

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safety (Dhama et al., 2015). According to WHO, this has been a major issue as the intensive use of certain antibiotics has been leading to infectious diseases, such as pneumonia or salmonellosis, being difficult to treat due to resistance of microbes (WHO, 2018). The European Union gradually started to phase out the use of antibiotics as growth promoters since 1998 and completely banned their use in 2006. In the United States, antibiotic growth promoter use is still allowed but major restrictions have also been applied following the demands of global market (Gaucher et al., 2015). Similarly, restrictions on the use of antibiotics have also been implemented in Asian countries such as Korea, Vietnam, and China, as well as in Australia, and even in Latin American countries (Hart et al., 2004; Suresh et al., 2018). Currently, due to current restrictions on the use of antibiotics, especially as feed additives, there is a considerable rise on the use of aromatic plants, herbs and essential oils as alternative feed additives in animal nutrition (Franz et al., 2010; Stevanovic et al., 2018). Feed additives are considered any compounds or mixtures, added in animal feed to improve their health status, growth rate, productivity, and performance. Their characteristics may include specific positive effects such as enhancing digestibility, maintaining and stabilizing beneficial microflora in the gut, improving quality of products of animal origin and influencing positively the environment (Huyghebaert et al., 2011). Thus, a huge effort has been made to replace antimicrobial feed additives in animal nutrition with natural plant products that will have the same effect on their performance and health condition. The aims of the current chapter are to present a review on the use of aromatic plants and herbs throughout antiquity, as well as current aspects and future perspectives on their traditional or modern application both in human and animal health.

Worldwide use of aromatic plants throughout history It is rather difficult to claim when and where plants and herbs were used as medicines for first time, especially in prehistoric times (Fig. 1.1). It appears that the very first evidence of humans using plants occurred in a Neanderthal flower burial site in Northern Iraq, approximately 60,000 years ago, as the remains of a body were found surrounded by at least seven medicinal plants, including Ephedra (Solecki and Shanidar, 1975; Jamshidi-Kia et al., 2018). Based on historical reviews, other old references were evidenced by the writings of Zarathustra (1000e500 BCE), during the Aryan civilization of Iran, back in 6500 BCE. A good knowledge of various plants’ medicinal properties was exhibited in Avesta, the holy book of Zoroastrianism (Jamshidi-Kia et al., 2018). The Sumerian civilization followed, presenting written formulas on clay stones, discovered around the Nagpur area, 5000 years ago (Kelly, 2009; Guidi and Landi, 2016). These formulas involved 12 plant preparations, based on more than 200 herbs, including common alkaloids such as poppy, henbane, licorice, and mandrake (Duke, 2002; Kelly, 2009). It is well understood that traditional beliefs and practices were mixed and adapted by ancient civilizations such as the Assyrians, Babylonians, and other inhabitants of Mesopotamia (Elgood, 2010). This is supported by the findings of Cuneiform inscription, a type of writing characters used in Mesopotamia c.2500 BCE, on clay tablets, referring to 1000 plants used to treat various disorders (Hassan, 2015). Cinnamon oil, myrtle, and incense plants were employed in holy ointments, used in rituals, as documented in the Exodus 32:22e26 in the Bible and the holy Jewish book “Talmud” (Smith et al., 2005;

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1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives

FIGURE 1.1

The use of aromatic plants in BCE (Before Christ Era) and CE (Common Era) periods worldwide.

Solomou et al., 2016). A plethora of other aromatic plants mentioned in the Bible include anise, dill, fenugreek, marjoram, mint, frankincense, garlic, juniper, mustard, myrrh, saffron, and poppy while it is mentioned that “wise men offered frankincense and myrrh to newborn child Jesus Christ” (Tucker, 1986; Duke, 2002). In ancient Persia, many plants and herbs were employed as drugs, disinfectants and culinary agents (Jamshidi-Kia et al., 2018). During the 9th to the 13th centuries, Dioscorides and other Greek physicians’ manuscripts were translated into the Arabic language and further enhanced their knowledge and remedies (Duke, 2002; Castleman, 2017). Although Sumerians and ancient Greeks had being using poppy extracts as medicine for long time, the Arabs first noticed opium’s addictive properties c. CE 40e90. In the 8th and 9th century, Iranian medicine was further developed by the physicians Avicenna and Razi, evidenced by their medical books, Canon of Medicine and Al-Hawi, respectively (Jamshidi-Kia et al., 2018). Avicenna, probably the most famous Persian pharmacist, poet, and philosopher, managed to classify the Greek-Roman writings and further advanced medicine in his book Canon of Medicine (Jamshidi-Kia et al., 2018). Thus, it is not of a surprise that the first private drug stores were founded by Arabs during the 8th century (Cragg and Newman, 2013; Jamshidi-Kia et al., 2018). Another important herbalist was Ibn al-Baitar (1197e1248) who extensively characterized the properties of 1400 plants, including tamarind and nux vomica, in his book Corpus of Simples during the 13th century (Gurib-Fakim, 2006; Jamshidi-Kia et al., 2018). In India, there has been a strong culture of using plants and spices as medicine since the ancient times, as they appear in the manuscripts of the holy books of Atharva and Rig Veda, and Sashruta Samhita. Sanskrit writings, among the oldest of Indian culture, were dated approximately in 1500 BCE (Sumner, 2000). Among the most popular plants was snakeroot (Rauvolfia serpentina), which has been used to sedate and treat snakebites or mental diseases for centuries. However, one of its major active compounds, reserpine, was only discovered by

Worldwide use of aromatic plants throughout history

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the Western pharmacy in the last century. Dated from the second year BCE, one of the most ancient Indian therapy systems is Ayurveda, which means “the science of life” (Jaiswal and Williams, 2016). This holistic therapy was based on the writings of Rig Veda and is still practiced nowadays, not only in Hindu communities of India but also in the West. The aromatic plant basil (Ocinum spp), known as Tulsi in India and Nepal, has been commonly employed in Ayurvedic therapy to treat various respiratory, gastrointestinal, kidney, blood, skin, and other ailments (Singletary, 2018). Another important herb, turmeric, which contains the polyphenolic compound curcumin, is still used in preparations of various drug therapies worldwide (Henrotin et al., 2013). Unani, another medical and therapeutic system using aromatic plants in a holistic approach, was flourished in the Persian-Arabic world and based on information obtained from Avicenna’s book (Rahman, 1994). However, as the name reveals since “Unani” means “Greek”, this knowledge was originally derived from the ancient Greek medicine and transferred to Persia by Aristotle, one of the most influential world philosophers, during the Great Alexander campaign. In the 13th century, Unani was introduced as therapeutic medicine in India and the Asia-Islamic world and further developed by the Indian medical treatments of Sushruta and Charaka (Rahman, 1994; Cragg and Newman, 2013). Aromatic plants such as clove (Syzygium aromaticum) and curcuma longa (Haldi), have long been used to treat oral diseases (Hongal et al., 2014). Today, Unani medicine is still practiced in South Asian countries of India, Pakistan, and Bangladesh as well as in Western countries of the United Kingdom, Canada, Germany, and the United States (Hongal et al., 2014). Chinese medicine, a well-known supporter of natural healing, presents its first written scripts from the days of Emperor Shen Nong Ben Jing, dating c.2700 BCE, while the Chinese Materia Medica (1100) listed over 300 plants (Zhu, 1998; Wiart, 2006). In the book “Pen T’ Sao” about roots and plants, 365 dried medicinal plants are mentioned with some of them still in use including Rhei rhisoma, camphor, Theae folium, Podophyllum, the great yellow gentian, ginseng, jimson weed, cinnamon bark, and ephedra (Wiart, 2006). Traditional Chinese medicine generally asserted that synergy was vitally important for the effectiveness of herbal therapy. In China, herbal medicine has existed for more than 5000 years. Nowadays, there are more than 3000 kinds of medicinal herbs that, although each herb has individual indications, in classical Chinese herbal medicinal practice diseases are commonly treated by combining herbs into formulas. In the early Ming dynasty (CE 1368e1644), more than 60,000 herbal prescriptions/formulas were recorded. Li Shizhen (1518e1593), an important Chinese herbalist, wrote the pharmacopoeia, “Pen t’sao kang mu”, which means The Great Herbal. He summarized what was known about herbal medicine, up to the late 16th century and describes more than 1800 plants, along with their medicinal properties and applications. Among them are 365 dried medicinal plants used in ancient times, including Rhei rhisoma, camphor, Theae folium, Podophyllum, the great yellow gentian, ginseng, jimson weed, cinnamon bark, and ephedra (Zhu, 1998; Wiart, 2006). Traditional Chinese medicine generally assessed that synergy was vitally important for the effectiveness of herbal therapy. Nowadays, many traditional herbal formulas are commonly used. For example, one formula named ‘Shi Quan Da Bu Tang’, containing 11 herbs, is used for the treatment of arthritic joint pain; another formula named as ‘Huo Luo Xiao Ling Dan’ that consists of 10 herbs, including Cinnamomum cassia bark and Panax ginseng root, is often used for fatigue and energy, particularly in the elderly. Today, such formulae are being investigated using modern scientific methodology (Yu et al., 2010).

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1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives

Furthermore, the Egyptian civilization was among the first to use formulae of aromatic plants, showing considerable medical awareness of their usefulness c.2900 BCE. In their “Ebers Papyrus” manuscripts, back in around 1800 BCE, a good knowledge of plant species as medicines had been exhibited (Jones, 1996). Specifically, there was a collection of approximately 800 prescriptions, quoting of 700 plants, including garlic, onion, aloe, pomegranate, coriander, and juniper (Glesinger, 1954). Various formulae, such as pills, infusions, ointments, and gargles, were prepared in honey, milk, wine, or beer (Duke, 2002; Cragg and Newman, 2013; Jamshidi-Kia et al., 2018). Known for their preservative properties, cinnamon and its oil were commonly employed in the process of embalming by Egyptians (Smith et al., 2005; Saad and Said, 2011). In Europe, first evidence of a man using plants as medicine was displayed in the findings of Ötzi the Iceman, a frozen body discovered in the Ötztal Alps, dated more than 5000 years ago. It is believed that he consumed mushrooms to heal a whipworm infection (Capasso, 1998). During the ancient Hellenic period (800 BCE), referrals to many medicinal plants and their therapeutic effects were recorded in Homer’s epics, The Iliad and The Odyssey, and retrieved from the Minoan and Assyrian civilizations. Some of them, such as artemisinin (genus Artemisia), were named after the Greek word artemis, which means healthy and was the name of the goddess of forests and hunting, whereas Elecampane (Inula Helenium) was given in honor of Eleni of Trojan war. Other intellectual Greeks, such as Herodotus had noted the castor oil plant, a potent toxicant, Orpheus the fragrant hellebore, and garlic, whereas Pythagoras mentioned cabbage, mustard and sea onion. Other spices of interest in the Mediterranean basin included saffron (Crocus sativus L.) which have been used to treat cardiovascular ailments, and as colorant and flavoring since ancient times (Khorasanchi et al., 2018). It is noteworthy that recent publications provide evidence regarding the effectiveness of hellebore and garlic both used since antiquity, as were found to possess potent anticancer properties (Blowman et al., 2018; Tsiftsoglou et al., 2018). Greek physician Hippocrates (460e377 BCE), who is considered the father of medicine, early acknowledged that disease may occur by natural and not phenomenal causes, such as magic (Jones, 1996). He believed that disease exploits the imbalance of the four humors of the body (Sumner, 2000). His studies involved the understanding of disease development and treatment, through the usage of over 400 plant species, primarily a variety of aromatic medicinal plants, initiating the establishment of Western medicine (Jones, 1996; Solomou et al., 2016). Many of them were emetics and purgatives aiming to reject anything unhealthy out of the body. He early acknowledged the importance of herbs in the food claiming “make food your medicine and medicine your food” (Solomou et al., 2016). It is worth mentioning that Nikander was one of the first to write about poisons and their herbal antidotes, during the second year BCE, followed by King Eupator of Pontus who strongly contributed to that knowledge (Jones, 1996). Galen (CE 130e200), another important Greek philosopher and physician, further introduced the “Galenicals,” plant extract preparations with opium while working in Rome (Rocca, 2003). Accumulating knowledge of aromatic plant medicines from Greeks, Egyptians, Romans, and other Mediterranean civilizations was reflected in the foundation of Alexandrian School of Medicine in the 3rd century BCE, which further influenced the establishment of Western medicine (Solomou et al., 2016). Among the experts of old times, Theophrastus (371e287 BCE) is regarded the father of botany science as he had written the De Causis Plantarium and De Historia Plantarium, which

Worldwide use of aromatic plants throughout history

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stand for Plant Etiology and Plant History respectively. He managed to classify more than 500 plants of that period including cinnamon, mint, pomegranate, fragrant hellebore, black pepper and many others. Furthermore, he noted that an increase of the intake dose of a plant may cause addiction (Hall, 2011). On the other hand, Pedanious Dioscorides, is considered the first authentic medicine botanist (Jones, 1996), as he wrote an encyclopedia, De Materia Medica, referring to the therapeutic properties of over 600 medicinal plants (Jamshidi-Kia et al., 2018). This book contains not only plant descriptions and illustrations of medicinal plants but also references to their preparation, use and possible adverse or toxic effects (Sumner, 2000). He acknowledged cinnamon as breath freshener, digestion enhancer, and noted its antiinflammatory properties in the intestine and kidneys and its diuretic and antipoison properties, back in CE 50 (Sumner, 2000; Smith et al., 2005). Furthermore, parsley was employed as diuretic, fennel to promote milk flow and white horehound with honey for expectorants (Ody, 2017). Thyme, another aromatic plant known from ancient times, was used for embalming (Halmai, 1972) and bathing by Romans soldiers in belief that would become more courageous (Basch et al., 2004). The Greek word for thyme means “to fumigate,” probably because it was used during ritual events in temples of ancient Greece (Singletary, 2016). Alchemists in the Medieval and “Dark” Ages used to refer to De Materia Medica for centuries. At that time, people were aware of the benefits of aromatic plants and herbs but there was no significant progress in that knowledge, lacking any further substantial literature or other information (Guidi and Landi, 2016). However, the renowned medical school in Salerno of Italy presented the Antidotarium Nikolai, a drug textbook written by intellectuals. One of its scholars, Michael Scott (1175e1230) discovered an inhaled anesthetic drug, using a mixture of mandrake, opium and henbane (Sumner, 2000). At that time, in Asia, the Chinese were enhancing Shen Nung’s herbal book and the Arabs were translating Greek and Roman textbooks (Castleman, 2017). During the Middle Ages, physicians followed the philosophy of Doctrine Signatures, according to which, plant’s “shape, color and texture” would imply the suitable body organ to cure. Unfortunately, by the end of the 4th century, the famous library of Alexandria was destroyed and most of the books were burned, losing much of the knowledge of that period. It was not until the Middle Ages (5th to 12th centuries), that monks, alchemists, and wizards began to play an important role in preserving and further advancing botany literature and scientific information of the known world, mainly in England, Ireland, France, and Germany (Guidi and Landi, 2016). However, it was the Arabs who managed to preserve the majority of Greek and Roman herb texts from the Western world, further enhancing it with their experiences and combining it with knowledge gathered from the East, mainly the Chinese and Indian cultures (Cragg and Newman, 2013). New plant species and spices were introduced into the Western world by Marco Polo’s (CE 1254e1324) and Vasco De Gama’s journeys in Asia (Persia, China, India) and America (CE 1492). In the book De Historia Stirpium, physician Leonard Fuchs (1501e1566) described and illustrated over 500 plant species, including imports from the New World, such as pumpkin and corn (Sumner, 2000). Other important writings of this time include, The Herbal or General History of Plants (1597) by English herbalist John Gerard and the English Physician Enlarged by Nicholas Culpeper (Singer, 1923).

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1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives

In the 18th century, Carl Linnaeus contacted a systematic botanology and taxonomy classification. In his textbook Species Plantarum, he classified and named in Latin many identifiable plants, including medicinal herbs known at that time. This book is still used as a reference for many plants. Previously, many of the medicinal uses of herbal plants were reported in Materia Medica (1749), a book useful for physicians and doctors (Sumner, 2000). Native Americans used to believe and follow the remedies of a healer or “shaman” who used the smoke of various plants such as tobacco or peyote, and other rituals to enable them to reach and heal the soul of a sick person (Ody, 2017). Even now, in some parts of South America, healers use plant vines, called yage in Colombia and ayahuasca in Peru and Ecuador, to treat their patients. Native American Indians of the New World also knew the benefits of twinflower, a woodland wildflower that was named after Linnaeus (Linnaea Borealis). They used it during pregnancy and or to treat feverish conditions in children. This renowned plant was traced around the area of North Pole, including the countries of United States, Greenland, Labrador, Alaska, Newfoundland, and Nova Scotia (Sumner, 2000). In the 19th century, botanist Asa Gray wrote a significant book about the flora of Northeast America and the followers of a religious community, named Shakers. These people used to grow and gather medicinal plants in a systematic way, one at a time, to avoid confusion, prepared their products with accuracy and sold them only to pharmacists and doctors. They succeeded in writing catalogs with over 200 plant species, including the discovery of Oswego tea or American bee balm with beneficial effects in digestive problems and antiseptic with mild sedative ability, due to its thymol extract. It is notable that they also cultivated poisonous plants, such as black henbane (Hyoscyamus niger) and thorn apple (Datura stramonium), which were used only after prescription by physicians (Sumner, 2000; Priyanka et al., 2012). Further, early in the nineties, Samuel Thomson founded a botanical system of remedies, based on traditional and herbal medicine, presented in his book, Thomson’s Improved System of Botanic Practice of Medicine, with many followers at that time (Ody, 2017). In Russia, it was until the 10th century that an exchange of medicinal herbal information was evidenced. Literature on medicinal plants mainly was introduced by visiting monks from Greek monasteries (Shikov et al., 2014). It appears that herbal medicine was well established, and the first pharmacy opened in a Kiev hospital by a monk from Athos in CE 1010. Moreover, Russian herbalists claim that they were aware of the properties and used mold to cure infected wounds long before the discovery of penicillin in England (Solovieva, 2005). Later, during the 15the16th centuries, when the Russian Empire flourished, additional knowledge was introduced from Asian, Arabic and Western European cultures. In the 19th century, while European physicians abandoned herbal medicine and turned mainly to synthetic drugs and Chinese healers mainly practiced their herbal traditions, Russian physicians combined both, traditional and Western medicine (Shikov et al., 2014). Nowadays, botanical therapy still plays an important role in their medicine, as herbal preparations are considered official drugs. In African countries, such as Nigeria, where many herbal medicaments have been used for long time, information has been passed from generation to generation by the word of mouth yet not documented (Kankara et al., 2015). Even today, in several parts of Africa, a major percentage of the rural population depends on medicinal plants as their primary healthcare option (Kankara et al., 2015).

Current situation on the use of aromatic plants and herbs in human and veterinary medicine, plant sustainability, and safety issues

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In Europe, during the 18th century, the therapeutic effect of willow bark (Cortix salignus) in disorders was documented by Rev. Edward Stone, in the Philosophical Transactions of the Royal Society of London. He noticed that this herbal drug had similar bitter taste with the Peruvian bark, showing positive effects on a disease, now known as malaria. Furthermore, foxglove therapeutic effects of an herbal tea had been noticed by William Withering back in 1775. Thymol and carvacrol, derivatives of thyme, have been used in the therapy of oral abscesses and other conditions during the 19th century and even now are added in mouthwashes, cosmetics, pesticides, and commonly used as culinary and feed additives (Meeker and Linke, 1988). Based on the discovery of plant-derived natural remedies, in CE 1897, Victorian chemists Eichengrun and Hoffmann developed salicylic acid, with acetyl acid, as a less gastric irritating medicine. It is one of the worldwide known synthetic medicines manufactured by the German company Bayer AG, aspirin (Schmidt et al., 2008). This was the beginning of a new era in the pharmaceutical industry, focusing in creating new synthetic drug medicines. Furthermore, the ability of microorganisms to produce drugs such as penicillin was discovered by Alexander Fleming in 1928, showing the perspectives of other sources of medicines (Schmidt et al., 2008). Another astonishing discovery was the use of the alkaloid compound hyoscine (scopolamine), found in the mandrake of potatoes, and applied as a relaxant before operations. Despite its analgesic properties, its use was restricted in the past because of its toxicity, as an overdose could be fatal for the patient (Jones, 1996). Thus, in the 19th century, the discovery and isolation of compounds retrieved from medicinal plants resulted in the production of drugs, such as opium, cocaine, codeine, digitoxin, quinine, and morphine (Balunas and Kinghorn, 2005). For example, the herb foxglove (Digitalis purpurea) is currently employed to obtain its active ingredients, the cardiac glycosides digitoxin and digoxin, to treat congestive heart failure. Most important, the discovery of artemisinin, a natural product derived from plants that significantly reduces the mortality rate of patients suffering from malaria, shows the importance of herbal plants in medicine (Tu, 2011). However, in the late 20th century, the industrial revolution, and chemical and mechanical engineering, led to the production of mainly synthetic chemicals.

Current situation on the use of aromatic plants and herbs in human and veterinary medicine, plant sustainability, and safety issues The use of plants for healing purposes predates history and forms the origins of modern medicine. It has been well documented that plants have been employed as medicines and still used as model-compounds in the pharmaceutical industry from historical textbooks worldwide and other written evidence throughout the centuries (Schmidt et al., 2008). Many conventional drugs originate from plant sources and a century ago, most of the few effective drugs were plant-based. Examples include aspirin (from willow bark), digoxin (from foxglove), quinine (from cinchona bark), and morphine (from the opium poppy) (Vickers and Zollman, 2001). However, today it is necessary that the discovery of new plant origin drugs and plant extract feed additives will combine the knowledge of herbal medicine and recent technology (Shen, 2015).

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1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives

Based on inherited knowledge and long-term usage for the treatment of various ailments over the centuries, aromatic plants and herbs are considered natural and therefore safer than conventional synthetic pharmaceuticals. On the other hand, plant and herbal-based medicine may present a greater risk of adverse effects and interactions than any other complementary therapy. There are several limitations around the scientific evidence supporting the belief that plant extracts can be regarded as absolutely safe (Raskin et al., 2002). There are indications that toxicity of various herbs was known from ancient times. Serious adverse effects after administration of herbal products have been reported and recent scientific evidence has revealed that many plants considered to be medicinal are potentially toxic, mutagenic, and carcinogenic (Fennel et al., 2004). Poisoning by medicinal plants may be attributed to misidentifications, incorrect preparation, or inappropriate administration and dosage. Information from health centers and emergency rooms has reported many dangerous and lethal effects from the use of plant and herbal products, especially in developing countries (Rodriguez-Fragoso et al., 2008). Plants may be regarded as toxic whether they produce compounds that interfere directly or indirectly with the metabolism of living organisms and exert toxic actions. There are more than 750 naturally occurring poisonous substances in more than 1000 plant species (Frohne and Pfander, 2005). However, only a small group of plants may cause severe poisoning after ingestion of a limited amount (Frohne and Pfander, 2005). Usually, most of the toxic plants may cause poisoning only under certain circumstances (Kankara et al., 2015). Thus, researchers have suggested that food and drug interactions should be checked through alterations of pharmacokinetics, including absorption, distribution, metabolism and excretion of a drug, or a compromise in nutritional effects of dietary components (Genser, 2008; Ribeiro dos Santos et al., 2017). In veterinary medicine, aromatic plant and herbal extracts may become easily popular, because they are not synthetic products. However, they must be extensively investigated in terms of their mechanisms of action, efficacious level of administration and clinical effects. Halofuginone, for example, is derived from an extract of the Dichroa febrifuga. The original extract was known for its antimalarial and coccidiostatic activity but was not marketed for long, because of a very narrow safety margin at the dose of 3 ppm (Youn and Noh, 2001). Issues of safety, toxicity, and side effects for medicinal plants should be standardized and their extraction should also be properly controlled and manufactured before wide use in animal diets (Chang, 2000; Giannenas et al., 2003). Animals do not consume voluntarily poisonous plants as they usually have bitter taste (Sumner, 2000; Vickers and Zollman, 2001). However, reported intoxications of animals occur mainly by accidental ingestion of drugs, metals, or other toxic substances. In the last decades, there is a considerable rise on the use of plant extracts as feed additives in animal nutrition. In Europe, the ban on the use of antibiotics routinely added in feeds and their potential prohibition worldwide, led to an increased interest in finding alternatives to antibiotics for farm animals, especially broiler chickens and pigs. In a pioneer study, about 40 years ago, herbs and spices were used in chicken nutrition to support growth performance (Vogt et al., 1989). The hypothesis of rearing chickens without coccidiostat compounds was introduced 30 years ago (Ekstrand et al., 1994) and appeared to be promising, but further investigation is needed (Giannenas et al., 2003, 2018; Gaucher et al., 2015). Aromatic medicinal plants have shown the ability not only to treat diseases but also to support growth performance in farm animals (Windisch et al., 2008; Franz et al., 2010). Currently, research is

Current situation on the use of aromatic plants and herbs in human and veterinary medicine, plant sustainability, and safety issues

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mainly focused on the usage of aromatic plants as feed additives that may be used to alleviate the problems associated with the withdrawal of antibacterial growth promoters and anticoccidials (Florou-Paneri et al., 2004; Christaki et al., 2012; Giannenas et al., 2013). Therefore, additional studies are required to investigate the potential properties of various aromatic plants and their extracts, the dose levels needed in feed and their efficacy against parasitic and bacterial species (Christaki et al., 2012; Giannenas et al., 2018; Oliveira et al., 2018). Moreover, research should be focused on examining the composition of the mixture of herbal extracts, identifying the active components and elucidating their mechanisms of action. Otherwise, plant and herbal feed additives may be used as food materials to enhance appetite. Furthermore, an effective concomitant use of aromatic plants or their extracts as alternative mean of controlling diseases will be attained by extensive multidisciplinary prospective research. In the United States, Health organizations, under the Congress of the Human Dietary Supplement Health and Education Act, determined that botanical substances would be classified as food supplements and their use would not require prescription since 1994. However, in Europe, companies and manufacturers of dietary feed additives should apply for an authorization by the European Commission and the European Food Safety Authority (EFSA). Although, requirements concerning quality control of these products are appointed by the manufacturer, medical claims on labels are largely restricted (Stevanovic et al., 2018). Medicinal plants and herbs are traditionally obtained from the wild, where they grow naturally. However, due to many harmful human activities and environmental factors, such as overharvesting, deforestation, desertification, and global warming, medicinal plants have been facing the serious problem of extinction, especially in the last decade. It has been reported that approximately 15,000 medicinal plant species are at risk of becoming extinct due to habitat destruction, overharvesting and big business throughout the world (Naguib, 2011). This is further compounded by the fact that many medicinal plants are also useful as raw material for other industries, such as culinary, cosmetics, textiles, and biomass; thus, the pressure on diversity of medicinal plants is extremely high. Other issues regarding the use of herbal medicinal plants and their products may include contamination, adulteration, or misidentification (Posadzki et al., 2013). Adverse effects seem more common with herbs imported from countries other than those of Europe and North America. The increased use and demand of wild aromatic plant species led the pharmaceutical and market industry to put pressure on their systematic cultivation. Among the advantages of cultivating wild plant species are the reliable botanical identification, regularity in quantity supplies, control of preferable genotypes and control of quality and safety issues that may occur from wild plants (Posadzki et al., 2013). On the other hand, cultivation of wild plant species may lead to a disruption of environmental equilibrium or alterations on genetic dynamics of plants. Another aspect of this issue is that cultivation and plant production could have major consequences not only on conservation of species but to local people’s income and life as well. On the other hand, when pharmaceutical companies stop acquiring their products from local communities, certain plant species will be abandoned by local harvesters with the imminence danger of extinction. Thus, it is very important for wild plant species to be preserved by sustainable harvest from wild populations (Schippmann et al., 2006). Due to overexploitation of many wild aromatic plant species used as medicines, several regulatory agencies and scientific organizations have recommended that wild species should be cultivated. The term “endangered plant” refers to plant species of which few remain, are in

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1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives

danger of becoming extinct and are generally protected by law and trade restrictions. In addition, a part of botanists and economists argue that sustainable harvest should be the most important conservation strategy for wild-harvested species, contributing to local economies and offering greater value to harvesters over the long term. Besides poverty and the breakdown of traditional controls, the major challenges for sustainable wild collection include lack of knowledge about sustainable harvest rates and practices, undefined land-use rights and lack of legislative and policy guidance. Identifying the conservation benefits and costs of the different production systems for medicinal aromatic plants and herbs should help guide policies as to whether species conservation should take place in nature, nursery, or both. Collection and trading of high-value products such as medicinal aromatic plants even in developed countries is based on both cultural and economic reasons (Lambert et al., 1997; BAH, 2002; Jones et al., 2002). Sustainability should be viewed from an ecological perspective in terms of plant populations. Comparison of cultivation and wild collection of medicinal and aromatic plants under sustainability aspects (Schippmann et al., 2006). Aromatic plants and herbs like tea, oregano, turmeric, garlic, ginger, and ginseng are the most commercially precious ones. This is because they are claimed to possess some excellent properties, although not fully justified by relative publications. In most cases, information obtained from extrapolated data regarding preliminary reports or from in vitro results on cellular lines and not by in vivo clinical trials. Among those of the most common plants used as medicinal plants are: a) garlic, which contains vital nutrients, including flavonoids, oligosaccharides, selenium and a high level of sulfur, is used against diabetes and inflammation, or to regulate blood pressure and boost the immune system (Bongiorno et al., 2008), b) ginger, which is the most widely used dietary compliment, contains gingerols and acts as a highly potent antioxidant and antiinflammatory agent (Grzanna et al., 2005), c) turmeric, which contains curcumin, has a long history dating back nearly 4000 years, has antiinflammatory, antioxidant, antimutagenic, antimicrobial, and anticancer properties (Garg, 2018), d) ginseng, which is one of the most popular herbs in Asia and North America, is used to treat diabetes, stress, and lung dysfunction, boosts the immune system and reduces inflammation (Shergis et al., 2013), e) ginkgo biloba has been used in traditional Chinese medicine for thousands of years to heal various ailments, including neurological impairment, anxiety, and depression (Mahadevan and Park, 2008), f) Aloe Vera, in the Western world is used in cosmetic, pharmaceutical and/or food industry while in Indian medicine to treat skin diseases and in Chinese medicine fungal diseases (Ahlawat and Khatkar, 2011), g) saffron, which contains potent antioxidants such as safranal, promotes mental health and good digestion, helps preventing degeneration, supports the skin, respiratory and heart health (Melnyk et al., 2010), k) oregano, which contains thymol and carvacrol, is used worldwide as potent antibacterial, antioxidant, antiinflammatory and antifungal agent (Giannenas et al., 2018; Mathesius, 2018), l) tea, which is mostly used as an infusion, may promote digestion (Chandrasekara and Shahidi, 2018).

Future perspectives

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Future perspectives The historic review of aromatic plants and herbs as medicines showed their importance in diversity and wide use as healing agents in both human and veterinary medicine. It has been reported that more than 50% of available medicine drugs originate from plants (Jamshidi-Kia et al., 2018). Despite the fact that pharmaceutical industry focused on the discovery of new technology synthetic drugs, naturally based products remain a source for novel compounds (Schmidt et al., 2008). Medicinal plants are essential natural resources, which constitute one of the potential sources of new products and bioactive compounds for drug development (Cragg and Newman, 2013). Traditional medicinal uses contribute significantly to such drug development. Among these uses, medicinal plants play a central role, as not only traditional medicines used in many cultures, but also as trade commodities that meet the demand of often-distant markets. WHO has reported that 80% of human population uses traditional drugs, mainly plants to cure various ailments, and it is estimated that about 60% of the world population and 80% of the population of developing countries rely on traditional medicine for their primary health care needs (WHO, 2018). Many aromatic and herb plants and/or their essential oils have been used throughout the centuries in animal health management, especially in ethnoveterinary practice (Franz et al., 2010). The last 2 decades, the popularity of natural alternatives versus synthetic feed additives has been fast growing as the future of animal industry and mostly orientated by consumer demands. Antibiotic-free animal products seem to be the next big target in animal industry. The question of how to succeed with antibiotic-free production is raised worldwide, and its application requires a new approach on animal management and breeding, mainly focused on the stressful periods of rearing. It is well known that normal beneficial microflora may support high rates of growth in animals. Therefore, effort should be focus on the establishment and maintenance of the animal intestinal equilibrium gut microflora, which is also affected by their diet composition. Further, the discovery of new natural plant products with potent antimicrobial and antioxidant effects should be expected, as they are environmentally friendly and more desirable for consumers as well. In the recent years, there is an enormous additional use of plant extracts as feed additives (Windisch et al., 2008; Christaki et al., 2012). Research on these subjects had intensified, especially during the last decade (Fig. 1.2). It has been observed that plants, herbs and their extracts are increasingly being used and informally marketed not only as sensory additives, but also for other purposes not covered by the legislation, notably better growth or feed conversion, improved meat quality and prophylactic purposes. There are strong indications that such use has been further increased in the last decade, as replacement additives for the antibiotic growth promoters (Pena et al., 2005). A recent survey on the use of phytogenic feed additives (PFA) on livestock showed that over half of the respondents from 80 countries use them as part of their feeding program. Most of them also indicated that they plan to increase the use of PFA’s over the next rearing periods (BIOMIN, 2018). The prediction of high consumption of plant-derived products is based not only on future population increase but also on the growing popularity of natural-based and environmentally friendly products. One of the major issues regarding the use of medicinal aromatic plants and herbs is to preserve them and keep a sustainable agriculture in the European Union. Therefore, there is a

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1. The history of herbs, medicinal and aromatic plants, and their extracts: past, current situation and future perspectives

FIGURE 1.2 Published documents in Scopus abstract and citation database (www.scopus.com) on the subjects of medicinal/aromatic plants and their substances (keywords searched in Titles, Abstracts, and Keywords lists).

need for the state to develop a conservation strategy to sustain those natural reserves (Haslett et al., 2010). The majority of medicinal aromatic plants and herbs are harvested from wild. Thus, an inventory of the most popular aromatic plant species should be composed, in order to enhance our knowledge regarding their genetic identity and abundance. This would contribute to their classification and the data collection would address conservation matters especially of any endanger species (Haslett et al., 2010). Another important issue regarding the use of aromatic medicinal plants and herbs is the future development of traditional medicine under the perspectives of safety, efficacy and quality that would help not only to preserve the traditional heritage but also to rationalize the use of herbal medicine in human and veterinary healthcare (Mukherjeee et al., 2018). Safe, efficient, practical, and preferably inexpensive ways for supporting growth performance of animals are mostly required. In conclusion, applications of aromatic plants and herbs in various forms (whole, ground, extracts or essential oils), will be continued either as therapeutic compounds in pharmaceutical industry or as flavoring, antimicrobial and antioxidant agents in food industry, and cosmetics. In addition, their use will be further expanded by the demand of new products and applications in the feed industry in future years. Scientific information gathered though peerreviewed literature will be the basal approach for the administration of aromatic plants and their extracts in farm animals as a fundamental alternative to antibiotics. However, extraction and compounding of aromatic plant products should be properly produced under controlled and regulated methods to obtain accurate and reproducible data. Further, in vitro and in vivo experimental and pharmacological studies should be focused on transforming aromatic

References

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plants and their extracts into alternative medicinal products. The application of ethnoveterinary knowledge may contribute to modern animal production systems. Finally, a better understanding of the mechanisms of action and assessment of possible interactions with food, feed and other drugs is necessary for their thorough exploitation. It appears that medicinal botany remains a huge field to discover.

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C H A P T E R

2 Innovative uses of aromatic plants as natural supplements in nutrition E. Christaki1, Ilias Giannenas1, Eleftherios Bonos2, P. Florou-Paneri1 1

Laboratory of Nutrition, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece; 2Department of Agriculture, School of Agriculture, University of Ioannina, Arta, Greece O U T L I N E

Introduction

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Bioactive compounds of aromatic plants

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Biological properties of aromatic plants (functional foods) nutrigenomics Modes of action

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Aromatic plants as dietary supplements Aromatic plants as growth promoters Aromatic plants as antimicrobials

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Aromatic Aromatic Aromatic Aromatic Aromatic

plants plants plants plants plants

as immunostimulators as antioxidants as flavourings as pigments of preservatives

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Conclusions

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Introduction Consumer demand for safe and natural animal foods with increased nutritional value is a challenge for animal nutrition. There is a trend toward using feed additives of natural origin versus potentially harmful synthetics. For this reason, a great deal of interest has been expressed for the aromatic plants and herbs, since they are considered as an untapped reservoir of valuable substances and their research is an ongoing discipline.

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00002-9

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Copyright © 2020 Elsevier Inc. All rights reserved.

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Herbal plants and their essential oils, also referred as phytobiotics, phytogenics, phytochemicals, spices, or botanicals, represent a wide range of biologically active compounds which may have positive effects on animal growth and health, although they are not established as essential ingredients (Hashemi and Davoodi, 2011; Lavecchia et al., 2013; Zeng et al., 2015; Gadde et al., 2017). Usually, they are defined by their low abundance, less than 1%e5% of dry weight (Hashemi and Davoodi, 2011). It is reported that some environmental factors such as geographical origin, harvest season, nutrient supply, temperature changes, light density, and atmospheric carbon dioxide concentrations can influence their quantity (JimenezGarcia et al., 2013; Hunter, 2014; Diaz-Sanchez et al., 2015). Many aromatic plants and herbs exist worldwide, particularly originating from the Mediterranean area, either self-grown or cultivated, such as oregano, rosemary, sage, thymus, peppermint, anise, and garlic. They can be used in dried and ground powder form or as extracts (Hunter, 2014; Martinez-Gracia et al., 2015; Gadde et al., 2017). Besides, from a biotechnological point of view, the aromatic plants can be distilled to extract essential oils (Ghanmi et al., 2014), which are odorous, volatile, hydrophobic, and high concentrated substances, also called volatile oils. These substances, responsible for the flavor and fragrance can be obtained from several parts of the plants including flowers, buds, seeds, leaves, twigs, bark, fruits, and roots. In the plants they are stored in secretary cells, cavities, canals, epidermic cells, or glandular trichomes (Bakkali et al., 2008; Brenes and Roura, 2010; Christaki et al., 2012; Rehman et al., 2016). Aromatic plants that produce essential oils usually belong to various families such as Alliaceae, Asteraceae, Apiaceae, Lamiaceae (or Labiaceae), Myrtaceae, Poaceae, and Rutaceae (Raut and Karuppayil, 2014). From antiquity, aromatic plants and herbs have played a crucial role in primary healthcare of humans as therapeutic agents for treatment of many illnesses. Therefore, some of them can also be medicinal plants, although they have much broader applications (Efferth and Greten, 2012). Currently, their use continues undiminished for medical purposes, especially in developing countries, where up to 80% of the people are dependent on herbal drugs, as well as over 25% of medicines in developed countries have plant origin (Chen et al., 2016). Additionally, the aromatic plants and their essential oils can be applied in animal health management or to improve their productivity (Franz et al., 2010; Yang et al., 2015; Prakash and Kiran, 2016). An important consideration is that the animals’ good health can be translated to better animal product quality, which is also the consumers’ demand. Therefore, the aromatic plants and herbs besides being a promising approach as drug candidates in modern medicine, they can also be used in the cosmetic industry, e.g., aromatotherapy, hair, and skin products, and as dietary supplements both in the food industry, e.g., soft drinks, food confectionary, and the feed industry, e.g., growth promoters, antioxidants (Christaki et al., 2012; Ismail and Imam, 2014; Antolak and Kregiel, 2017).

Bioactive compounds of aromatic plants The aromatic plants and their essential oils usually belong to diverse classes of organic molecules, according to their biosynthetic pathways. They are not single compounds, but rather mixtures of which the main are phenolic compounds e.g., carvacrol and thymol, as well as terpenoids, e.g., linalool and menthol. The term “phenolic” or “polyphenolic” is

Biological properties of aromatic plants (functional foods) nutrigenomics

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referred chemically as a component comprised of an aromatic ring bearing one (phenol) or more (polyphenol) hydroxyl substituents esters, methylesters, etc. Moreover, phenolics are divided into two groups: nonsoluble compounds, e.g., condensed tannins and lignins, and soluble compounds, e.g., phenolic acid (gallic acid, rosmarinic acids, etc.), flavonoids (catechin, quercetin, etc.), quinones, phenolic diterpenes (carnasol and carnosic acid) (Jimenez-Garcia et al., 2013; Puvaca et al., 2015; Jiang and Xiong, 2016; Rahman et al., 2017). Furthermore, some other oxygenated aromatic compounds prevalent in plants and their essential oils can be found, such as alcohols (e.g., borneal), aldehydes (e.g., cinnamaldehyde), and ketones (e.g., carvone) (Sharopov et al., 2015; Pandini et al., 2018). Terpenes and terpenoids are another groups of bioactive compounds, being the basic constituents of many aromatic plants essential oils, e.g., linalool and menthol (Bakkali et al., 2008; Raut and Karuppayil, 2014). They are lipid-soluble comprising hydrocarbons and their structure has one or more 5-carbon isoprene units, which are synthesized possibly through two pathways: mevalonate and deoxy-D-xylulose. These compounds are divided according to the isoprene units they contain, as hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, sesterpenes, triterpenes, and tetraterpenes (Jimenez-Garcia et al., 2013; Raut and Karuppayil, 2014). The bioactive compounds usually present in low quantities in the aromatic plants and their essential oils are influenced by the part of the plant used and the method applied to extract them effectively (Jimenez-Garcia et al., 2013; Hunter, 2014; Diaz-Sanchez et al., 2015). The diversity and the plethora of these compounds makes their analysis a challenging task. The techniques used to release the compounds from the matrix are conventional such as steam distillation or hydrodistillation, and advanced such as pressurized liquid extraction, subcritical and supercritical extraction, microwave, ultrasound, and enzyme-assisted extractions (Gil-Chavez et al., 2013; Ribeiro dos Santos et al., 2017). Therefore, novel approaches, such as the “omics” are required for the separation of the bioactive substances. The most common technique for their analysis is gas-chromatography (GC), coupled with mass spectrometry (MS). Moreover, this separation can be achieved by some other biomic technologies such as ultra-high-performance liquid chromatography (UHPLC), capillary zone electrophoresis (CZE), capillary electrochromatography (CEC), DNA barcoding technologies, as well as nuclear magnetic resonance (NMR) spectroscopy (Efferth and Greten, 2012; Ghanmi et al., 2014; Ribeiro dos Santos et al., 2017; Sanchez-Vidana et al., 2017). It is remarkable that next generation sequencing (NGS) strategies reveal links between nutrition and genes, while the newer RNA sequencing analysis has the ability to investigate both known and unknown transcriptional units, so they create new possibilities for understanding animal growth, health, and performance (Sabino et al., 2018).

Biological properties of aromatic plants (functional foods) nutrigenomics The addition of aromatic plants and their derivatives to livestock nutrition is an interesting tool for providing supplements with biologically active compounds. These reveal considerable properties such as antimicrobial, antiviral, antifungus, antioxidant, antiinflammatory, and immunostimulatory (Diaz-Sanchez et al., 2015; Adaszynska-Skwirzynska and Szczerbinska, 2017; Ribeiro dos Santos et al., 2017). Subsequently, the use of aromatic plants

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2. Innovative uses of aromatic plants as natural supplements in nutrition

due to their valuable compounds is fundamental for successful development of novel, healthy foods, the functional foods. These foods beyond their nutritional effects have demonstrated benefits to the human organism by improving the state of health or well-being. They may reduce the risk of chronic diseases such as cardiovascular, neurodegenerative, bone metabolism, cancer and may find application for the treatment of respiratory and inflammatory disorders, allergies and diabetes (Prescott and Saffery, 2011; Ismail and Imam, 2014). Accordingly, the interest of the dietary use of the compounds of aromatic plants as functional ingredients or nutraceuticals has been enhanced by the recent advances in genetics. In relevant studies, an interaction between dietary components and the genome has been highlighted, which is mandatory to affect metabolic pathways and homeostasis in the human body. Hence, a new concept the “nutrigenomics” has been revealed. The nutrigenomic actions exerted by the aromatic plants could be a preventive approach for optimizing health, delaying chronic disorders or minimizing their intensity or severity, since many diseases have a genetic predisposition (Simopoulos, 2010; Ismail and Imam, 2014; Carrasco Lopez, 2015; Pavlidis et al., 2015; Elsamanoudy et al., 2016).

Modes of action Although the precise modes of action of the phytogenics are not elucidated yet, studies have shown their beneficial effects on productive animals, concerning growth performance, carcass characteristics, and meat quality. Generally, the benefits of aromatic plants and their essential oils depend greatly on the diversity and number of aromatic compounds responsible for their biological activities, their synergistic effect, the origin of the plants, the inclusion level in the diet and their pharmacokinetics (Franz et al., 2010; Diaz-Sanchez et al., 2015; Gadde et al., 2017; Sanchez-Vidana et al., 2017).

Aromatic plants as dietary supplements The recent applications of aromatic plants as source of growth promoters, antimicrobials, immunostimulators, antioxidants, flavorings, pigments, and preservatives, in animal nutrition, especially those that can satisfy the increasing consumers’ demands for natural products and functional foods in relation with human health (Fig. 2.1).

Aromatic plants as growth promoters In-feed antibiotic use has become a common practice in the animal production since the antibiotics at subtherapeutic doses play a crucial role to the improvement of growth and feed conversion efficiency by reducing the activities of the microbial in the gastrointestinal tract. However, the continuous use of antibiotics as growth promoters was linked to the development, transmission and proliferation of resistant microbes via the food chain. Accordingly, consumers’ awareness of the potential health negative effects and environmental problems has increased. Consequently, after the ban in 2006 of the use of antibiotics as feed additives in the European Union countries and the overall shift toward less antibiotic use worldwide, novel methods for promoting growth in productive animals have appeared.

23

Aromatic plants as dietary supplements

As growth promoters

As preservatives

As antimicrobials

Innovative uses of aromatic plants

As pigments

As flavourings

As immunostimulators

As antioxidants

FIGURE 2.1 Innovative uses of aromatic plants as natural supplements in nutrition.

Among them, aromatic plantebased feed additives are a promising solution for both conventional or antibiotic-free livestock production systems (Diaz-Sanchez et al., 2015; Steiner and Syed, 2015; Gadde et al., 2017; Valenzuela-Grijalva et al., 2017). The dietary use of aromatic plants is expressed as significant improvement in body weight gain, feed intake and feed conversion ratio, as well as reduction of morbidity and mortality. It is also reported that essential oils improve flavor and palatability of the feed so they increase feed intake and performance. Indeed, a range of studies have shown the growth of promoting effects of herbal products when were supplemented in a single phase (e.g., essential oils) or as isolated compounds (e.g., quercetin) on poultry and swine performance by ensuring optimum gastrointestinal functionality. In other words, to achieve the desired ratio between the good bacteria and pathogens (Franz et al., 2010; Christaki et al., 2012; AdaszynskaSkwirzynska and Szczerbinska, 2017; Gadde et al., 2017; Giannenas et al., 2018). Nevertheless, there are some studies which have not reported any significant effects of dietary phytochemicals on growth performance of the animals. This could be due to the efficiency of the phytochemicals which is dose-dependent or the environmental and dietary conditions (Botsoglou et al., 2002; Khattak et al., 2014; Diaz-Sanchez et al., 2015; Valenzuela-Grijalva et al., 2017). Aromatic plants (Fig. 2.2), may act as growth enhancers of the animals, mainly by increasing digestive secretions (endogenous digestive enzymes, saliva, bile and mucus), decreasing the bacterial populations in the gastrointestinal tract or improving gut morphology due to antioxidant and antiinflammatory activities (Hashemi and Davoodi, 2011; Criste et al., 2017; Gadde et al., 2017; Gheisar and Kim, 2017; Valenzuela-Grijalva et al., 2017).

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2. Innovative uses of aromatic plants as natural supplements in nutrition

Aromatic plants and their derivatives

Improve gut microbiota

Contribute to digestibility

Increase digestive secretions

Higher nutrient digestibility Less competition for nutrients Decrease of potential pathogens

Enhance performance of animals

FIGURE 2.2

Possible modes of action of aromatic plants as growth promoters.

Evidence for aromatic plants as growth enhancers in ruminants has not been extensively studied; there is limited knowledge about the effect of aromatic plants on feed intake and palatability of ruminants (Franz et al., 2010). Later reports have described a relation between selection of some roughages and the well-being of ruminants (Faehnrich et al., 2015). According to Kolling et al. (2016), essential oils may affect the neuronal activity through modifying of neurotransmitters, which could influence animal feeding behavior and feed intake, and therefore their performance. Nevertheless, other researchers did not find any positive effect of dietary oregano in the performance parameters of growing lamps (Bampidis et al., 2005) or in bovines (Valenzuela-Grijalva et al., 2017). In addition, it is reported that high levels of dietary herbal additives in ruminant diets can inhibit the growth of various microorganisms and the process of ruminant fermentation which are necessary activities in order to improve feed efficiency and animal productivity (Christaki et al., 2012; Valenzuela-Grijalva et al., 2017). The variability of the published data may be mainly due to the complex gastrointestinal tract of the ruminants, as well as the bioactivity of the various phytochemicals (Kolling et al., 2016). Moreover, according to some animal studies concerning carcass quality, the dietary use of aromatic plants resulted in improved carcass weight, breast weight, tenderness and juiciness of the meat, reduction of lipid oxidation (poultry and pigs), as well as, a decrease of abdominal fat (mainly pigs and cattle) that satisfy consumer preferences for lean meat. Nevertheless, the impact of aromatic plants and their derivatives on meat and carcass quality is still a topic of discussion (Steiner and Syed, 2015; Valenzuela-Grijalva et al., 2017).

Aromatic plants as antimicrobials The antimicrobial activity of aromatic plants and their derivatives whether bacteriostatic or bactericidal against food-borne pathogenic and spoilage microorganisms has been

Aromatic plants as dietary supplements

25

documented by several researchers (Franz et al., 2010; Giannenas et al., 2013; Gheisar and Kim, 2017). Mainly, the essential oils have been shown to exhibit broad spectrum inhibitory activities against Gram negative (Escherichia coli, Campylobacter jejuni, etc.) and Gram positive bacterial pathogens (Bacillus subtilis, Clostridium coliforms, etc.). Generally, Gram negative bacteria are less sensitive to the antimicrobials, due to their lipopolysaccharide outer membrane which restricts diffusion of phenolics. But Gram positive bacteria are usually more susceptible to the phytobiotics because of the direct interaction of their cell membrane with these lipophilic components (Martinez-Gracia et al., 2015). As it is already reported, the mechanism of action of essential oils is not full elucidated due to the complexity of their composition. It has been noticed that the antimicrobial activity of the essential oils is not the results of one specific mode of action, but it is possibly the synergistic effect of the different components on various targets in the different organelles of the microbial cell (Burt, 2004; Giannenas et al., 2018). According to the literature, several in vivo and in vitro studies have reported that due to the lipophilic nature of the phenolics present in the essential oils, these can accumulate in the lipid bilayer of the bacterial cell membrane and mitochondria, rendering them more permeable and disrupting their normal function. Furthermore, some essential oils can disrupt the cell homeostasis and cause loss of ions and cell content, finally leading to cell death. The hydroxyl (eOH) groups in phenolic compounds seem to be responsible for the inhibitory action against microorganisms. In addition, denaturation of cytoplasmic proteins and enzymes lead to bacterial cell death (Raut and Karuppayil, 2014; Yang et al., 2015; Gheisar and Kim, 2017). Moreover, it has been found that the decrease of undesirable bacteria, including coliforms, clostridia, staphylococci, etc., in the gut of host animals can help the growth of beneficial bacteria including lactobacilli, which may exclude pathogens from colonization in the gut (Steiner and Syed, 2015; Gheisar and Kim, 2017; Valenzuela-Grijalva et al., 2017). In other words, aromatic plants may have a prebiotic effect. In vivo studies in broilers demonstrated that dietary essential oils, alone or as a blend of herbals, can act against intestinal colonization of E. coli and Clostridium perfringens due to the antimicrobial action of their phenolic compounds (e.g., carvacrol and thymol) (Christaki et al., 2012; Steiner and Syed, 2015). Although, antimicrobial activity has been shown by a variety of non-phenolic compounds, e.g., limonene, cinnamaldehyde, etc. (Diaz-Sanchez et al., 2015; Gheisar and Kim, 2017). Moreover, phytogenics can prevent the negative impacts of Eimeria experimental infection (E. acervulina, E. maxima, and E. negatrix) in broiler chickens (Giannenas et al., 2003, 2018; Christaki et al., 2004; Bozkurt et al., 2013; AdaszynskaSkwirzynska and Szczerbinska, 2017). In addition, it has been reported that essential oils and mainly oregano oil is a new potent bioactive substances against gut parasites, especially Cryptosporidium spp. (Gaur et al., 2018). Also, dandelion herb has been found to have effective antibacterial activity against Staphylococcus aureus and E. coli in poultry (Qureshi et al., 2017). Nowadays, in order to retain longer the antimicrobial activity of the essential oils, microencapsulation has been investigated as a tool to delay the absorption of the phytobiotics, to protect them from the environmental agents and to minimize any negative palatability or strong odor (Diaz-Sanchez et al., 2015).

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Aromatic plants as immunostimulators Several aromatic plants and their essential oils have been found that can promote multiple immunomodulatory actions, when animals are in immune-suppressed conditions. They can improve phagocytosis, modulate immunoglobin and cytocine secretion, enhance lymphocyte expression and boost the release of interferone-g. Such activities may promote duodenal function and increase the availability of absorbed nutrients, which result in an amelioration of the health status of the animal and finally support the genetic potential for better growth (Adaszynska-Skwirzynska and Szczerbinska, 2017; Sheoran et al., 2017; ValenzuelaGrijalva et al., 2017). According to some researchers, the dietary use of dandelion, a medicinal plant used in herbal remedy or curcumin (carotenoid derived from turmeric) caused inhibition of the proliferation of tumor cells by suppressing the necrosis factor (TNF) (Kim et al., 2012; Rajput et al., 2013; Qureshi et al., 2018).

Aromatic plants as antioxidants The aromatic plants can express their antioxidant capacity mainly due to their phenolic compounds, which can act as scavengers of free radicals and reactive oxygen species (ROS) such as single oxygen and superoxide or hydroxyl radicals, hydrogen donators, metal-chelators, and inhibitors of the enzymatic systems, which are responsible for initiating oxidation reactions (Babbar et al., 2011; Mimica-Dukic et al., 2016; Rahman et al., 2017). Since oxidation is linked with loss of nutritionally valuable ingredients and the formation of potential toxic compounds, it is obvious that the food matrix must be protected against oxidative damage. Nowadays, there is much interest into the replacement of traditionally used synthetic antioxidants with natural, since the use of the synthetics is associated with human and animal health risks (Prakash et al., 2015; Jiang and Xiong, 2016). Inclusion of aromatic plants such as oregano, rosemary, thyme, saffron, and sage in animal nutrition has been reported as a simple and conventional method to incorporate natural antioxidants in the animal food products in order to inhibit or delay the lipid oxidation (oxidative rancidity) by terminating the start or propagation of oxidizing chain reactions (Christaki et al., 2012; Loizzo et al., 2016; Wilson et al., 2017). The dietary phytochemicals may be able of reverse the endogenous a-tochopherol in the phospholipid bilayer of the membrane back to its active antioxidant form (Rice-Evans et al., 1996; Botsoglou et al., 2009). The supplementation of aromatic plants or essential oils in animal nutrition could improve the oxidative stability or egg yolk or raw and precooked chicken, turkey, pork, and rabbit meat, as well as meat products during refrigerated or long-term frozen storage (Botsoglou et al., 2003; Franz et al., 2010; Jiang and Xiong, 2016; Adaszynska-Skwirzynska and Szczerbinska, 2017; Alarcon-Rojo et al., 2017). Additionally, the dietary use of aromatic plants in sheep and goats presented higher oxidative stability of the meat, and increased the content of polyphenolic antioxidants in both meat or milk and cheese (Franz et al., 2010). The ability of the aromatic plants to neutralize free radicals, as it has been already mentioned is linked mainly to the phenolics which are known as powerful antioxidants that contribute alone or synergistically to the prevention of some oxidative-stress related chronic animal conditions, such as cancer, diabetes, cardiovascular, neurodegenerative (e.g., dementias) and bone metabolism disorders (Fig. 2.3). These health-promoting

27

Aromatic plants as dietary supplements

Low levels

Reactive Oxygen Species (ROS)

High levels

Oxidative stress

Prevent or inhibit

Cell damage Apoptosis Promote tissue injuring

Neutralize Cell growth Immunity Stress adaptation

Aromatic plants as antioxidants

Neurodegenerative diseases Cardiovascular diseases Liver diseases Cancer Aging

Diagram presenting the effects of aromatic plants as antioxidants in modulating the damaging effects of reactive oxygen species (ROS).

FIGURE 2.3

properties of phytobiotics and their functional molecules are now supported by a first set of data coming from nutrigenomic investigations, which aim to clarify the interaction between genes and bioactive compounds from food sources (Brewer, 2011; Ismail and Imam, 2014; Piroddi et al., 2016; Gheisar and Kim, 2017). Furthermore, the phytobiotics may affect lipid metabolism in living organisms’ tissues, in order to protect them by showing beneficial effects on the activity of antioxidative enzymes, such as superoxide-dismutase (SOD), glutalthione peroxidase (GSH-PX), and catalase (CAT) (Christaki et al., 2012; Gheisar and Kim, 2017; Qureshi et al., 2017). However, there are few in vivo studies in animal nutrition concerning the health status after dietary intake of antioxidants from herbs and spices e.g., in poultry the oxidative stress is limited due to their short life span, nevertheless it is regarded as one of the causes leading to biological damage and affects poultry growth (Florou-Paneri et al., 2006). In dairy cows, phytobiotics may inhibit metabolic disorders such as fatty liver disease and ketosis or improve their health status by alleviating the consequence of mastitis (Faehnrich et al., 2015). However, it is noticed that in the ruminants the antioxidant activity of the phytobiotics depends on the ruminal pH, which must be low in order to exert it (ValenzuelaGrijalva et al., 2017). Besides secondary metabolites, the aromatic plants and their essential oils include other compounds, such as vitamins (vitamin E or a-tocopherol and vitamin C or ascorbic acid), reducing sugars (glucose and fructose), and polyunsaturated fatty acids (linoleic acid and a-linolenic acid) which can act as natural antioxidants, alone or in combination with the phytochemicals against various oxidative-stress related diseases (Guimaraes et al., 2010; Jiang and Xiong, 2016; Alarcon-Rojo et al., 2017; Mohiseni, 2017). Recently, some researches have shown that antioxidants due to their flavonoid content can act as prooxidants under some conditions, especially when they possess multiple hydroxyl groups (Miguel, 2010; Eghbaliferiz and Iranshahi, 2016). When they enter the inner cells membrane, flavonoids can be oxidized by ROS, to prooxidants forms that can oxidize lipids

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2. Innovative uses of aromatic plants as natural supplements in nutrition

and proteins, as well as can induce nuclear DNA damage. This mechanism may be related to a late apoptosis of the affected cells, which can play a “protective” role by killing the potential mutants (Bakkali et al., 2008; Christaki et al., 2012). Furthermore, vitamin C has both antioxidant and prooxidant properties, depending on the dose taken. Relatively low concentrations my confer benefits, while very high concentrations can exert prooxidant activity due to involvement in the initiation of reactions resulting in harmful long-term effects (Finley et al., 2011; Christaki et al., 2012).

Aromatic plants as flavourings The organoleptic characteristics of the food are very important attributes influencing the consumers preference. Herbs, spices, and essential oils have been playing a key role since antiquity in animal nutrition to enhance feed flavor and thus improve the palatability (DiazSanchez et al., 2015; Steiner and Syed, 2015; Valenzuela-Grijalva et al., 2017). According to a recent study (Upadhaya and Kim, 2017), the dietary supplementation of a blend of essential oils (basil, carawasy, laurel, lemon, oregano, sage, tea, and thyme) resulted in improvement of weight gain of monogastric animals, because of the improvement of the flavor and the palatability of the feed. However, there are differences between the various species of animals, poultry being less sensitive to taste compared to pigs or cattle (Valenzuela-Grijalva et al., 2017). Likewise, the properties of ameliorated flavors and palatability are variable, depending on the type as well as the dose of the essential oils used (Upadhaya and Kim, 2017). In addition, the use of spices as natural flavors of foods is very common all over the world. Different spices as cinnamon, vanilla, oregano, pepper, rosemary, thyme, etc. use as flavorings, is well known for culinary purposes. Natural flavoring provide the consumers the sensory experience, consistency of flavor and safety (Burt, 2004; De la Torre Torres et al., 2017). The sensory properties of aromatic plants and their derivatives are mainly due to their phenolic compounds (Burt, 2004). The stability of dietary phytobiotics must be taken under consideration due to processing of the feed, such as pelleting, which may lead to reduction of their activity (Upadhaya and Kim, 2017). Concerning the dietary use of flavorings originated from aromatic plants and their derivatives, the European Commission (EC) and the Food and Drug Administration (FDA) have approved them as generally recognized as safe (GRAS) (Ribeiro dos Santos et al., 2017). Synergism or antagonism between the compounds of the essential oils and food constituents, as well as higher concentrations of essential oils as flavors need further study.

Aromatic plants as pigments Color may be considered as one of the most impressive and enjoyable qualities of foods which can be more attractive for the consumers. During the past decades, the food industry used mainly synthetic pigments, while currently some of them are suspicious for their possible toxicological effect in humans. Therefore, there is a growing interest in using natural pigments of plant origin as new food additives, which can grand their functional properties to food products. They are demanded by the market as renewable natural color enhancers for

Aromatic plants as dietary supplements

29

foods and feeds. Consequently, there is an economic potential to promote the addition of herbal pigments, since some of them act as natural antioxidants to ameliorate the quality of animal products, while at the same time provide certain health benefits (Faehnrich et al., 2015). Some of the plant-derived pigments are flavonoids, such as anthocyanins, the most widely studied natural pigments. Their color may vary from red to purple and blue, depending on pH, light, oxygen, presence of enzymes, metal ions and temperature during storage (Martins et al., 2016; Rodriguez-Amaya, 2016; Cortez et al., 2017). Other compounds with coloring properties are carotenoids such as crocin derived from saffrondone of the richest spices in carotenoids, with renowned demand, mainly because of their strong antioxidant activity (Botsoglou et al., 2005; Martins et al., 2016). There are many herbs and spices which can be used as pigments in human and animal nutrition, such as the parsley with the yellow colored apigenin, and the turmeric with the bright yellow colored curcumin as principle active substances, with many other activities in mammals, mainly including antiinflammatory, antioxidant, and anticancer (Kim et al., 2012; Faehnrich et al., 2015; Vikram et al., 2015). The pigments derived from aromatic plants (Fig. 2.4), when consumed by the animal with the feed, are absorbed through the digestive gut, metabolized and then deposited in animal tissues or excreted via urine or feces (Faehnrich et al., 2015). Nevertheless, the in vivo Pigments originating from aromatic plants

Intake with feed

Degradation in the gut

Uptake

Metabolism in the cells

Deposition in animal products

Excretion via urine and faeces into the environment

FIGURE 2.4 Metabolism of pigments originating from aromatic plants in animals.

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2. Innovative uses of aromatic plants as natural supplements in nutrition

experiments using phytogenic pigments in animal nutrition are scarce. Various studies presented that the utilization of herbal pigments in nutrition has been limited because of their low stability during feed processing e.g., pelleting, and interaction with other components in the food matrix (Lubbe and Verpoorte, 2011; Zeng et al., 2015; Rodriguez-Amaya, 2016; Cortez et al., 2017). There are some strategies to overcome the problem of stabilization with the addition of metallic ions (e.g., iron) or with microencapsulation and nanoencapsulation techniques. Another disadvantage of natural colorants seems to be their cost, which is two to 10 times higher than that of the synthetics. Nevertheless, these bottlenecks need to be solved before pigments of aromatic plant origin can be moved from the niche market to large-scale use.

Aromatic plants of preservatives Fresh foods such as seafood, meat, and horticultural products can be protected through the use of preservatives from a variety of pathogenic microorganisms (causing food-borne diseases) or spoilage (causing significant economic loses for the food industry, because of the undesirable effects on the food properties). The current trends focus on the use of natural compounds in order to sufficiently extend the shelf-life of the food products, which can be considered as safe alternatives to synthetic preservatives and satisfy the consumers’ preferences for more “green” foods. The plant-derived essential oils, beyond their antimicrobial and antioxidant properties, can play a promising role for food preservation by retarding microbial growth and oxidative deterioration (Martinez-Gracia et al., 2015; Pandey et al., 2016; Prakash and Kiran, 2016). Nevertheless, until today the use of essential oils as preservatives is limited, due to the high concentrations needed to reach sufficient antimicrobial activity, which are not always organoleptically acceptable by the consumers. Consequently, the strong aroma of the plant-derived food preservatives can be reduced by applying them in combination with other substances with antimicrobial properties to act in a synergistic way. Other options for the future use of essential oils in food preservation systems may be better highlighted through the use of modern encapsulation techniques (Burt, 2004; Hyldgaard et al., 2012; MartinezGracia et al., 2015; Prakash et al., 2015).

Conclusions Aromatic plants and herbs could be a leading natural resource for potential innovative supplements in nutrition. Nowadays, the popularity of feed additives of herbal origin such as growth promoters, antimicrobials, immunostimulators, antioxidants, flavorings, pigments, and preservatives is increasing compared to synthesized substances. These herbal feed additives provide new perspectives and can be used in the diets of humans and animals, because they have a particular role for the health status, and they can satisfy the increasing consumers’ demand for natural substances with functional properties. The study of aromatic plants is still an evolving field of study that has yet to realize its full potential.

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Rice-Evans, C., Miller, N.J., Paganga, G., 1996. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biol. Med. 20, 933e956. Rodriguez-Amaya, D.B., 2016. Natural food pigments and colorants. Curr. Opin. Food Sci. 7, 20e26. Sabino, M., Capomaccio, S., Cappelli, K., Verini-Supplizi, A., Bomba, L., Ajmone-Marsan, P., Cobellis, G., Olivieri, O., Pieramati, C., Trabalza-Marinucci, M., 2018. Oregano dietary supplementation modifies the liver transcriptome profile in broilers: RNAeq analysis. Res. Vet. Sci. 117, 85e91. Sanchez-Vidana, D.I., Rajwani, R., Wong, M.S., 2017. The use of omic technologies applied to traditional Chinese medicine research. Evid. Based Complement. Alt. Med. 2017, 6359730. Sharopov, F., Braun, M.S., Gulmurodov, I., Khalifaev, D., Isupov, S., Wink, M., 2015. Antimicrobial, antioxidant, and anti-inflammatory activities of essential oils of selected aromatic plants from Tajikistan. Foods 4, 645e653. Sheoran, N., Kumar, R., Kumar, A.A., Batra, K., Sihag, S., Maan, S., Maan, N.S., 2017. Nutrigenomic evaluation of garlic (Allium sativum) and holy basil (Ocimum sanctum) leaf powder supplementation on growth performance and immune characteristics in broilers. Vet. World 10, 121e129. Simopoulos, A.P., 2010. Nutrigenetics/nutrigenomics. Annu. Rev. Public Health 31, 53e68. Steiner, P., Syed, B., 2015. Phytogenic feed additives in animal nutrition. In: Mathe, A. (Ed.), Medicinal and Aromatic Plants of the World. Springer, London, UK. Upadhaya, S.D., Kim, I.H., 2017. Efficacy of phytogenic feed additive on performance, production and health status of monogastric animals - a review. Ann. Anim. Sci. 17, 929e948. Valenzuela-Grijalva, N.V., Pinelli-Saavedra, A., Muhlia-Almazan, A., Dominguez-Diaz, D., Gonzalez-Rios, H., 2017. Dietary inclusion effects of phytochemicals as growth promoters in animal production. J. Anim. Sci. Technol. 59, 8. Vikram, N., Kewat, R.N., Singh, R.P., Ramesh, P.S., Singh, P., 2015. Natural edible colour and flavours used as human health. Int. J. Pharm. Sci. Res. 6, 4622e4628. Wilson, D.W., Nash, P., Buttar, H.S., Griffiths, K., Singh, R., De Meester, F., Horiuchi, R., Takahashi, T., 2017. The role of food antioxidants, benefits of functional foods, and influence of feeding habits on the health of the order person: an overview. Antioxidants 6, 81. Yang, C., Chowdhury, K., Hou, Y., Gong, J., 2015. Phytogenic compounds as alternatives to in-feed antibiotics: potentials and challenges in application. Pathogens 4, 137e156. Zeng, Z., Zhang, S., Wang, H., Piao, X., 2015. Essential oil and aromatic plants as feed additives in non-ruminant nutrition: a review. J. Anim. Sci. Biotechnol. 6, 1e10.

C H A P T E R

3 Herbs and aromatic plants as feed additives: aspects of composition, safety, and registration rules Ch M. Franz1, K.H.C. Baser2, I. Hahn-Ramssl1 1

WG Functional Plant Compounds, University of Veterinary Medicine, Vienna, Austria; Department of Pharmacognosy, Faculty of Pharmacy, Near East University, Nicosia, North Cyprus

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O U T L I N E Introduction

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Plants and herbal products used as feed additives 37 Plant species 37 Herbal preparations 38 Identification of the starting material 40 Natural variation of the plant material 41 Agricultural practices and harvesting 42 Contaminations 42 Identification of the “active” substances 42 Product consistency and stability 43 Hazard identification and characterization 44 Risk characterization 44 Chemistry and activity Carvacrol and thymol

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00003-0

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Camphor and 1,8-cineole Cinnamaldehyde Eugenol Essential oils or aromachemical blends Organic acids Fatty acids Inulin Rosemary Capsaicin Flavonoids Betaine Polysaccharides Resveratrol Artemisinin Tocopherol and turmeric Saffron Sage Mulberry Leaf, Japanese Honeysuckle, Goldthread

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Copyright © 2020 Elsevier Inc. All rights reserved.

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Legal status of feed additives

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Conclusion

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Introduction Humankind has used herbs and herbal products in animal health, welfare, and nutrition, presumably since early domestication of farm as well as pet animals. Ancient reports exist especially on the phytotherapy of horses (Dum-Tragut, 2005), as also the first veterinary schools have been established for military, transport, and riding horses (Lorenz et al., 1997). Ethnoveterinary-Botany refers in addition to the widespread use of herbal preparations in traditional veterinary medicine (Pieroni, 1999). Similar to human nutrition, animal husbandry and nutrition has been focused within the last more than 50 years primarily on the supply with macro- and micronutrients (protein, carbohydrates, fatty acids, trace elements, and vitamins) followed by crude fiber. Performance, especially weight gain and productivity of farm animals should be improved, and an increasing amount of in-feed antibiotics has been applied as so-called growth enhancers or growth promoters. Due to increasing technicality and exact calculation of the feed (diet) composition in order to optimize feed intake and productivity of farm animals on one side, and of convenience food for pets on the other side, the value of herbs and aromatic plants had been more and more neglected. The last two decades have brought, however, a change in paradigm: secondary plant products in the diet are no longer seen as antinutritive only (as e.g., high concentrations of tannins, saponins or toxic alkaloids in food or feed) but they have been rediscovered acting as nonnutritive substances with differentiated added value. This starts from flavoring and appetizing, improving the digestion and performance, stress modifying, animal welfare enhancers including stable odor and climate improvement up to antimicrobial activity of, e.g., essential oils able to substitute in-feed antibiotics (Franz et al., 2010). Due to the fact that antibiotics are principally very valuable and almost indispensable drugs in human and veterinary medicine, the excessive and partly inappropriate use in animal nutrition as growth promoters and generally in veterinary medicine has been observed very critically over the last two decades (WHO, 2015). Especially the noticed increasing antimicrobial resistance (AMR) of pathogenic microorganisms had led already to first restrictions in all EU Member States as, e.g., the ban of in-feed antibiotics in animal nutrition by January 1, 2006 following Reg. (EC) No. 1831/2003. This resulted on one side in search of alternatives to antibiotics and on the other side, however, in partly increasing use of antimicrobials as veterinary drugs. One point should not be neglected in this context: animals in large-scale livestock farming are quite often stress exposed which influences the immune status and thereby the gut microbiome. Stressors may induce discomfort and affect the welfare and disease resilience of animals, and the most commondbut questionabledsolution had been treating the animals with antibiotics and other veterinary drugs (Wegener, 2012).

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The increased concerns over the spread of antibiotic resistance has in the meantime raised the attention in functional plant products in animal health and veterinary phytotherapy due to the fact that many plant metabolites are well known natural (mild) biocontrols of microorganisms. The reason to use herbs and spices and their extracts in animal nutrition is, however, not reduced to their possibly antimicrobial activity to substitute in-feed antibiotics, which is mainly the domaine of essential oil-bearing plants (e.g., oregano, thyme, rosemary, tea tree, cloves). Plant extracts and the phytochemicals therein are multi-talented improving e.g., the nutrient uptake and digestion and are therefore growth and productivity enhancers per se (e.g., hops, gentian, nettle, licorice). They may have antispasmodic, antiinflammatory, immunomodulatory or hepatoprotective effects (e.g., fennel, caraway, camomile, milk thistle), could inhibit the biofilm formation, are acting antiadhesive influencing the gut microbiome (e.g., fenugreek, pomegranate, ginseng, hot pepper), and in general they are often able to reduce free radical oxidation stress. Learning from human medicine, human nutrition and the increasing significance of plant food supplements (Restani et al., 2017), a high number of herbs and herbal products is therefore actually applied in animal nutrition all over the world. However, to the best of our knowledge, no general compilations or studies concerning the use and prescription of herbal (medicinal) products for food-producing and/or companion animals by veterinarians are available, even in Europe. Despite the fact that there are several products and published studies (Christaki et al., 2012). One review of the European Ethnoveterinary Research reported that almost 600 plant species, deriving from more than 100 different plant families, are investigated for respective use in animal treatments in the EU, but only very few are registered for application in livestock (Mayer et al., 2014). Thus, the use of herbal medicinal products in farm practice is limited. Even in organic agriculture, where phytotherapeutic treatments are preferably requested by the EU Regulation EC No. 889/2008, herbal remedies are administered to only a negligible extent in comparison to antibiotics or antiparasitics, mainly due to economic reasons and the cascade regulation concerning the use of veterinary medicinal products being in favor of allopathic synthetic drugs. The only legal possibilities to apply herbal products in animal health have therefore be seen in phytogenics as feed additives according to Reg. 1831/2003, in feed/dietary supplement for particular nutritional purpose (¼ “PARNUTS“) according to Reg. 94/39 (EC) or finally in plant derived medical devices (Reg. (EU) 2017/745) as far as applicable also to animal health care products.

Plants and herbal products used as feed additives Plant species A very large number and diversity of herbs and spices as well as extracts, tinctures, and essential oils as herbal products has been notified in the European Union since establishing the Community Register of Feed Additives following Reg. (EC) 1831/2003. At the beginning the register counted more than 500 entries notified under “natural productsdbotanically defined,” from Abelmoschus to Zizyphus japonicus. In the actual version 7/2018 only 156 entriesdAbies alba to Zingiber officinaledcan be found, all others have been withdrawn due to economic, functional, safety, or EU regulatory reasons. Similar Registers or Compendia

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could not be identified neither in the United States nor India nor China. But at least as many herbs and herbal products are used as feed additives in the respective countries according to the widespread use of traditional medicine (TCM, Ayurveda) and corresponding food supplements. In addition to the Register of Feed Additives, there exists also an official EU Catalog of Feed Materials (Reg. (EU) 2017/1017) and a Feed Materials Register of the EU organizations representing the feed business in Europe (www.feedmaterialsregister.eu). A further number of herbal materials used as single substances can be found there, increasing the total number of herbs and herbal products in animal nutrition. Taking these documents in mind, the relevant publications and some market observations into consideration, the most fequently used and important species belong to the plant families Lamiaceae (Thymus sp., Origanum sp., Rosmarinus officinalis., Mentha sp., Salvia sp. and others), Apiaceae (Carum carvi, Foeniculum vulgare, Levisticum officinale, Pimpinella sp. and others), Asteraceae (Artemisia sp., Cynara sp., Echinacea sp., Inula helenium, Silybum marianum, Stevia rebaudiana, Tagetes sp., Taraxacum officinale, etc.) and Rosaceae (Crataegus sp., Filipendula ulmaria, Malus sylvestris, Prunus sp., Rosa sp., Rubus fruticosus and many others). From many other plant families only a few species are used as feed additives, as e.g., Alliaceae (Allium sp.), Brassicaceae (Armoracia sp., Brassica sp.), Cannabaceae (Cannabis sp., Humulus lupulus), Fabaceae (Glycyrrhiza glabra, Trigonella sp. etc.), Gentianaceae (Gentiana sp.), Lauraceae (Cinnamomum sp., Laurus nobilis), Myrtaceae (Melaleuca alternifolia, Pimenta sp., Syzygium sp.), Papaveraceae (Eschscholtzia californica, Macleaya cordata, Papaver sp.), Rutaceae (Citrus sp.), Solanaceae (Capsicum sp. et al.) or Zingiberaceae (Curcuma sp., Elettaria cardamomum, Zingiber officinale). Finally, some single scattered species might also be found among the herbal feed additives, as e.g., Equisetum arvense, Ginkgo biloba, Olea europaea, Punica granatum, Schisandra chinensis or Uncaria tomentosa. It is interesting to learn that some of the herbs have a long tradition as feed additives, as e.g., chamomile (Matricaria recutita), linseed (Linum usitatissimum) or nettle (Urtica dioica), some are new or coming from exotic ethnobotanical knowledge (e.g., Echinacea sp., Schisandra chinensis or Uncaria tomentosa) and some are re-discovered‘, especially several essential oils as antimicrobials and antioxidants (e.g., oregano-, thyme-, cinnamon- or cloveoil). The diversity of all the phytochemicals in feed additives is correspondingly high, especially if taking also the inter- and infraspecific variation into consideration.

Herbal preparations Herbs are used: - in solid, dry, comminuted or ground form (herbal materials), - as extracts (crude, concentrated or dry). Further, they can be classified into: - essential oils (volatile lipophilic substances obtained by physical means only: pressing or steam distillation), - oleoresins (extracts derived by polar or apolar solvents or supercritical CO2), - extracts/tinctures (aqueous or lipophilic, obtained usually by maceration, percolation or supercritical CO2 extraction).

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Another very specific group of useful but often neglected herbal materials are the residues, byproducts, and waste materials from herbal or other plant material processing. Especially extraction and distillation residues should be taken into consideration, which have shown very interesting results as herbal feed additives. Stockhammer et al. (2009) investigated the antioxidant activity of a number of residues and byproducts, e.g., echinacea, carrot pulp, blueberry peel, grape seed; Tedesco et al. (2015) investigated the effect of larch sawdust on dairy cows; Monino et al. (2008) investigated the effect of rosemary distillation waste on lamb meat quality and shelf-life, and phytogenous industrial waste, finally, has been studied to keep the health status of piglets in the critical postweaning phase (Hagmueller et al., 2011), to mention some examples. Herbal ingredients, extracts and botanical preparations used as plant feed additives (PFA, phytogenics) have to comply with all appropriate requirements of national (in Europe also EU) food and feed legislation with regard to composition and safety. The quality of the extract and the consistency of production is particularly relevant where a quantitative claim is made for a botanicaldor specific constituents of it. Identity and quality control are therefore of high relevance. The observed substantial increase in the use of herbs/botanicals and their products, especially extracts for PFA in the last two decades were not effectively accompanied by quality and safety control. There exist a number of standard and reference methods for the analysis of phytochemicals on one side and contaminations as well as undesired substances in plants. The most common ones are ISO Standards, Codex Alimentarius, the current European Pharmacopoea methods (2016), and the WHO Quality Control Methods (2011). Considering the multi-component nature and variable bioactivity of PFA, a proper and meaningful quality control must also include the analysis of a broad spectrum of potential physiologically active phytochemicals and biomarkers as well, including possibly hazardous substances as shown for plant food supplements by Restani et al. (2017). More recently, EFSA (the European Food Safety Authority) published a guidance on the identity, characterization and conditions of use of feed additives (2017). There one can read under Identity, characterization and conditions of use of the additive: For additives of plant origin, the characterisation should include the scientific name of the plant of origin and its botanical classification (family, genus, species, if appropriate subspecies). The parts of the plant used to obtain the active substance(s) (e.g. leaves, flowers, seeds, fruits, tubers, roots) should be indicated. The identification criteria and other relevant aspects of the plants should be indicated. For complex mixtures of many compounds obtained by an extraction process, it is recommended to follow the relevant terminology such as essential oil, absolute, tincture, extract and related terms widely used for botanically defined flavouring products to describe the extraction process. Reasonable efforts should be made to identify and quantify all components of the mixture. One or more marker compounds should be selected, which will allow the additive to be identified in the different studies. Information on the variability in composition of comparable products should be provided. This could be done by reference to published literature. EFSA FEEDAP Panel (2017)

Taking into account the large number of aromatic plants, herbs and their products (botanicals or phytogenics) widely available in animal nutrition through several distribution channels

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3. Herbs and aromatic plants as feed additives: aspects of composition, safety, and registration rules

and marketed actually for a variety of uses, several issues have arose mainly relating to the quality assurance and safety: 1. 2. 3. 4. 5. 6. 7. 8.

identification of the starting material natural variation of the plant material agricultural and harvesting practices contaminations or adulterations identification of the “active substance(s)” product consistency and stability hazard identification and characterization risk characterization

Identification of the starting material PFAs are characterized by the enormous diversity of plant species used in a diversity of different product preparations. Many of the species used originate from taxonomically delicate plant species and are therefore often subject to misidentification. One major issue when dealing with plants and herbal products is the correct identification of the species and chemotype. Quite often closely related species are used with different chemical outfit, as e.g., thyme (Thymus sp.) consisting of several small and/or subspecies of different ploidy level, with distinct chemical composition. In other cases, various chemotypes occur within a species, of which only one is appropriately effective, as known for chamomile (Matricaria recutita) or hemp (Cannabis sativa). This may be challenging, especially if the chemical variation results in adverse effects. For example, bitter substances may differ considerably within the Lamiaceae family and their acceptance by animals (espec. chicken) may cause only minor uptake and losses (Crawford and Rudgers, 2012). Exotic herbs are often known under different common/vernacular names and the correct botanical source is sometimes unclear, as shown e.g., by Lawrence and Reynolds (1984) for species used commercially as oregano. Consequently, also mixtures with poisonous plants might and do occur (e.g., chamomile with allergenic Anthemis sp.). Sometimes raw materials are deliberately substituted by similar, cheaper materials. In that respect, this specific problem is related to wild collected herbs (e.g., Gentiana vs. Veratrum roots). To avoid confusion, a clear description to ensure the authenticity is therefore an indispensable prerequisite (Orhan et al., 2016). PFAs are frequently confronted with the prejudice to have low quality, to contain admixtures, adulterations or illicit additives, or even to be “fake products” as known from plant food supplements (O’Connor, 2013). DNA based identification (DNA barcoding) is since few years the “gold standard” for authentication of biological material to avoid misidentification, admixtures and adulterations (Hebert et al., 2003). Although this is very important in herbal materials, for many plant species appropriate methods do not exist yet. Starting (herbal) materials for PFA have to be controlled in the form of crude drug - or extracts, which latter need specific preparation. DNA barcoding of plant extracts was introduced by Novak et al. (2007). With chamomile, a comparison of multiplex Polymerase Chain Reaction (PCR) and High Resolution Melting Curve Analysis (HRM) offered optimal methods to differentiate between closely related species, while Passiflora incarnata can clearly be separated from other Passiflora sp. by use of a Neighbor Joining Tree (NJT) of Internal

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Transcribed Spacer (ITS) sequences, to mention only some examples (Novak et al., 2018; Schmiderer and Novak, 2014). The acceptance of molecular methods depends, however, on the repeatability of the results which is quite often in question if the number of individuals and populations investigated is rather small. And it is also necessary to identify specific primers first from a standardized conservative region of every genome of plants. The heavy discussion on a paper of Newmaster et al. (2013) made the sensibility of this topic obvious (Gafner et al., 2013). Due to these reasons, methods for a clear identification are necessary. DNA based identification of plants used as food or feed is becoming increasingly popular because of its ability to identify the plant species in a state difficult to identify with morphological or chemical methods, e.g., identification of root drugs or of processed materials (Novak et al., 2007). These methods have therefore the potential to complement existing methods for the identification of PFA. An important task is also to ensure that the plant material is free of any toxic admixtures. The American Botanical Council started therefore its Botanical Adulterants Prevention Program end of 2011 (Foster, 2011). As regards phytochemistry, by testing different batches of the same species it becomes obvious that in general a large infraspecific variation exists influencing the quality and efficacy of PFAs considerably. This is extremely important if the species in question might contain substances of health concern as, e.g., alkenylbenzenes or pyrrolizidine alkaloids (the latter ones detected in several herbal materials contaminated with PA-containing weeds as e.g., Senecio sp.; see also The EFSA Compendium of Botanicals, Version, 2016). As a consequence, not the species only, but the chemotype, plant part and development stage is decisive for the quality of the herbal product, and a batch-to-batch variation might occur and has to be taken into consideration respectively. Also a thermoluminescence method exists for the detection if the plant starting material was irradiated to reduce microbial load of herbal materials not proper produced and stored according to GAP (Good Agricultural Practice) for medicinal and aromatic plants (Restani et al., 2017). Finally, the “hot topic” quality of herbal materials and extracts for PFA discussed in several meetings of the European Feed Additives Associations, at ISEO and HerbAn Conferences resulted in the demand for a respective Guideline which should be developed “bottom up,” i.e., under participation of the parties concerned. Natural variation of the plant material It is well known that the chemical composition of plants varies from year to year. Therefore, prerequisite for a batch-to-batch consistency is to manage obtaining similar plant constituents. In particular, attention should be given to the precise (chemo-) variety, since many plants, especially essential oil-bearing species, show not only a high interspecific phytochemical diversity but also an intraspecific chemical variation. Moreover, the respective phytochemicals are not equally distributed in the plant but may accumulate in specific plant parts, organs and/or tissues. Therefore, a clear description and specification of the plant part used is necessary. For example, milk thistle (Silybum marianum) contains the effective flavonolignans in the fruit husk, while thistle oil and thistle herb are totally different products. In cinnamon (Cinnamomum zeylanicum), the root-, bark-, and leaf-oil composition differs significantly (Wijesekera, 1974). The chemical composition of herbal materials depends also from growth phase. Secondary plant products are not at all quantitatively or qualitatively present uniformly over the life

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cycle of the plant, but depending on the development stage, which is influenced by the environment, especially day length, temperature, water and nutrition. Thus, oregano oils harvested from plants in early spring or late autumn contain much more p-cymene instead of carvacrol (Kokkini et al., 1997). As a result, the quality of the product differs on the harvest date and the ecological impacts at production site (Franz and Novak, 2016), which should be documented by the producer, taken into consideration by the customer and finally the applicant. Agricultural practices and harvesting Since the above problematic of batch-to-batch conformity is well recognized for medicinal and aromatic plants, the European Herb Producers Association (EUROPAM/EHGA) elaborated already in the 1990s “Guidelines for Good Agricultural Practice” and “Guidelines for Good Wild Collection Practice of Medicinal and Aromatic Plants” (GAP/GCP-Guidelines 8.0, EUROPAM, 2018), adopted also by EMEA and WHO. The respective guidelines refer to the following items (not dealt with in detail in the present context): - seeds and propagation material - cultivation, including soil and fertilization, irrigation, crop maintenance, and plant protection (products) - wild collection - harvest - primary processing and packaging - storage and distribution - quality assurance - personnel and education - building and facilities - equipment - documentation Therefore, the principles stated on these guidelines should also apply at plants/herbs used as additives in animal nutrition. Contaminations Herbal feed additives (phytogenics) have to consider all national regulations regarding residues of possible contaminants in food and feed. This includes heavy metals, plant protection products, microbiological and botanical contamination, mycotoxins, polycyclic aromatic hydrocarbons (PAH), dioxins and dioxin-like polychlorinated biphenyls (PCBs), to mention the most important items. Limits for nicotine as well as for pyrrolizidine alkaloids especially from contamination of toxic weeds (Crotalaria-, Echium-, Heliotropium-, Myosotis-, Senecio sp., and others) are in discussion. Identification of the “active” substances Phytogenics used as sensory feed additives or zootechnicals (including products enhancing animal welfare) are in general characterized with regard to their major compounds. Sometimes, however, the “active” principle is not definitely identified yet, as in

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the case e.g., of valerian, or other compounds acting as precursor (for example salicin from willow bark being metabolized first to salicylic acid derivatives). Quite frequently also marker substances, without direct relation to the activity but characteristic for the species, are used for product identification. In plant preparations, usually, more than a single compound is functional and the interaction of several substances is evident, as additive or adverse effects may occur, complicating the situation even more. Therefore, standardized extracts based on a level of several active constituents are considered as the “active principle.” Especially ingredients with known adverse effects should be assessed under standardization criteria. In cases where neither appropriate marker nor active compounds are known, a chemical fingerprint should be required with restrictions on its variability (Wallace et al., 2010). Quality assessment is normally relied on methods such as chromatographic techniques to adequately characterize the material. In a first approach, thin layer chromatography (TLC) may be the appropriate analytical method to validate authenticity and to detect any adulterations. But for more detailed information, including determination of residues, a battery of analytical techniques may be useful (IR, UV, GLC, HPLC, and coupled with MS). To identify herbal compounds in feed mixtures, new approaches are based on molecular biological methods such as PCR (Heubl, 2010). Product consistency and stability Product stability and consistent quality of herbal products may only be assured if the initial materials are adequately defined. In particular, the specific botanical and phytochemical identification of the plant material used should be precise. Essential elements should be reproducible in various batches, with special care when originate from different sources (environments, varieties, etc.), as they may show inconsistency, if different plant parts are used in their production (herbs consist of leaves/stems, flowers with/without vegetation). Stability is generally measured by the analytical follow-up of the active substance(s) (e.g. mg/kg) or agent(s) (e.g. CFU/kg) or its activity (e.g. units of catalytic activity/kg) or effects (e.g. pellet durability) during time. When the additive contains more than one active substance/agent, stability should be assessed for each of the active substance(s)/agent(s). If specific effects are claimed for a particular form of the additive (e.g. chelation), the stability of that specific form of the additive should be followed. For some chemical mixtures/ extracts, stability may be assessed by monitoring the concentration of one or more appropriate marker substances. Data should include at least one observation at the beginning and one at the end of the storage period. Where appropriate, potential degradation or decomposition products should be characterized. EFSA (2017)

Among the most important factors that may influence the stability of aromatic plant constituents are the degree of comminution (cutting, crushing, and milling), the storage conditions and the type of active substances. Thus, herbal products should be stored up to room temperature and humidity of the products should not exceed 14% moisture to avoid microbial growth. Another factor to consider especially for herbs and flowers is that their essential oils, as volatile compounds, will be reduced during storage. This is not so relevant for fruits/ seeds and roots. The more the product is processed/comminuted and the smaller the particle size is, the higher are the losses of volatiles. Another way of processing animal feed additives is by microencapsulation. Otherwise, a short-term use (consumption) should be advised to ensure the same level in constituents. Although oxidation processes may affect the quality of nonvolatile compounds, such as flavonoids, alkaloids, and glycosides, they commonly exhibit better stability (Thakur et al., 2011).

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3. Herbs and aromatic plants as feed additives: aspects of composition, safety, and registration rules

Hazard identification and characterization Identification of the inherent biological activity of the material and assessment of relevance to animals and humans are necessary before the exposure depending risk can be characterized. Of most of the herbs in question, human safety data are available. However, little information is available on their activity and metabolism in target animals and especially on the carry-over of single compounds into the food chain. Even though absorption, distribution, metabolism, and excretion (ADME) of herbal products in food producing animals is scarce, that would be of significance only if possibly hazardous substances are contained in the starting material. Especially, matrix effects would play an important role in the safety assessment of aromatic plants and herbal preparations used in animal nutrition. The herbs themselves, as already mentioned, consist of a complex mixture of macro- and micro-molecules of various nutritive values, flavor compounds, and other secondary metabolites responsible for either beneficial and/or adverse effects. Interactions due to the complex matrix will result in only partly comparable data to results from ADME studies performed on isolated individual substances. Therefore, the extent of the necessary experimental investigations depends on the adequacy of the history of use and on the product comparability to traditional food/feed ingredients. Regarding extracts and essential oils, it must be safeguarded that substances of possible concern are not enriched in the product by the type of extraction (Schilter et al., 2003). Another issue to consider may be the safety in handling plants and herbal products, due to their allergenic capacity. Since this is well documented for herbs used by humans, precautions should also be taken in their application as herbal feed additives (Schilter et al., 2003). Risk characterization Any risk characterization has to be considered predominantly from the point of view of safety for the target animal and the consumer of food of animal origin as well. Although data are available on the pharmacological effects of herbal products and secondary plant substances in laboratory animals, toxicology on target animals is randomly examined. There is obviously a wide range of knowledge on the activity and safety of herbs and aromatic plant products to the target animals from “traditional use” (from field experience to experimental data, up to well-documented scientific results). However, most of published data are probably limited to zootechical parameters (i.e., weight gain and feed conversion) not to endanger further use or future authorization by demonstrating metabolic, microbial, or even pharmacological effects. Along with the history of use, different levels of evidence may be requested to decide if an herbal product may pose any risks or be beneficial for animals and humans (Franz et al., 2006; Restani et al., 2017). Any outcomes and information on aromatic plants and herbal products for human used either as food, food additive, dietary product, or medical usedmay also be valuable for decision makings and regulations on their application in animal nutrition. Therefore, in addition to the existing guidelines, EFSA has issued a new guidance for the assessment and/or application of herbs and plant products used as feed additives in animal nutrition (EFSA, 2017).

Chemistry and activity

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Chemistry and activity In the following part, essential oils and other phytogenics shall be discussed separately for their effects on animals.

Carvacrol and thymol Carvacrol reduced feed intake, weight gain and feed conversion rate and thymol showed no effect, when broilers were fed with 200 mg/kg carvacrol and thymol (Lee et al., 2003). Thymol or thyme oil showed beneficial effects on the antioxidative enzymes superoxide dismutase and glutathione peroxidase as well as on polyunsaturated fatty acid composition in various tissues of aging rats (Youdin and Deans, 1999). Carvacrol (200 mg/kg) caused the reduction of ruminal pH and increase of volatile fatty acids concentration on lambs. No effect was observed on other rumen parameters (A:P ratio, NH3), dry matter intake, average daily gain, carcass characteristics, and meat characteristics (Chaves et al., 2008a). Oxidative stability of eggs was improved by thymol (Botsoglou et al., 1997; Liu et al., 2009). The same effect was also observed with oregano (Radwan et al., 2009), sage (LopezBote et al., 1998), turmeric (Curcuma longa; Radwan et al., 2009), rosemary (Lopez-Bote et al., 1998; Florou-Paneri et al., 2005; Radwan et al., 2009), tea catechins (Yilmaz, 2006), mulberry leaf, Japanese honeysuckle, and goldthread (Liu et al., 2009). In a similar study, it was found that saffron constituents delayed lipid oxidation in shell eggs, as this was indicated by the formation of malondialdehyde, despite its steadiness with duration of storage. In stored shell eggs, pH and iron influenced lipid oxidation but saffron constituents showed a dosedependent antioxidative capacity (Botsoglou et al., 2005). Both carvacrol and eugenol showed potent antimicrobial activity against several microbes such as Staphylococcus aureus, Clostridia spp., E. coli, and Salmonella pullorum (Dahiya et al., 2006). Carvacrol and thymol-rich essential oil showed higher inhibitory activity compared to linalool against avian E. coli (Penalver et al., 2005a). Thymol, carvacrol, and eugenol mixtures, astaxanthin isolated from microalgae Haematococcus pluvaris and lupulone were effective in controlling Clostridium perfringens colonization and proliferation in the guts of broilers (Mitsch et al., 2004; Siragusa et al., 2008). Carvacrol also acts as antibacterial by preventing the synthesis of flagellin, causing bacteria to be nonmotile (Burt et al., 2007). Antiflagellate activity on Tetratrichomonas gallinarum and Histomonas meleagridis by essential oils obtained from Cinnamomum aromaticum leaves, Citrus limon pericarps and Allium sativum bulbs was also reported (Burt et al., 2007). Synergism between carvacrol and its precursor p-cymene and between cinnamaldehyde and eugenol has been reported. Due to hydrophobicity of essential oils, they are partitioned between the lipids of the cell membrane and mitochondria rendering them permeable and causing the leakage of cell contents, leading to cell death (Burt, 2004). Carvacrol and its isomer thymol exert an hydrophobic character at a different level. Invasion of MadineDarby bovine kidney (MDBK) epithelial cells by Eimeria tenella sporozoites is inhibited in the presence of carvacrol, curcumin, or Echinacea purpurea extract (EP) and enhanced by betaine. Combining carvacrol with EP inhibited E. tenella invasion more

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3. Herbs and aromatic plants as feed additives: aspects of composition, safety, and registration rules

effectively than applying the compounds individually, but the further addition of curcumin did not reduce invasion further (Burt et al., 2012). This in vitro assay has been proved to be a fast and inexpensive tool to screen natural substances against coccidian oocysts in order to discover effective anticoccidials and confirm their inhibiting action (Hessenberger et al., 2016). Combination of oregano essential oil with antibiotics to treat Escherichia coli bacteria characterized as extended-spectrum beta-lactamase (ESBL)eproducing bacteria. Oregano in combination with antibiotics lowered the effective dose of antibiotics, thus minimized their adverse effects on ESBL-producing E. coli (Si et al., 2006). Carvacrol, cinnamaldehyde, oregano oil, and thymol were also found to inhibit C. perfringens spore germination and growth in ground turkey during chilling (Juneja and Friedman, 2007). Carvacrol, thymol, transcinnamaldehyde, and tetrasodium pyrophosphate on the radio sensitization of E. coli and Salmonella typhi in chicken breast significantly reduced the numbers of these pathogens (Lacroix et al., 2004). The effect of oregano (Origanum onites) essential oil rich in carvacrol on the extension of shelf life of overwrap packed fresh chicken drumsticks was investigated. Oregano essential oil extended product shelf life by approximately two days (Oral et al., 2009). This has been also confirmed in studies with Greek oregano oil (Giannenas et al., 2005, 2016). Similarly, dietary Sideritis scardica known as Greek Mountain Olympus Tea gave positive effects on chickens challenged with the pathogen Eimeria tenella (Florou-Paneri., 2005).

Camphor and 1,8-cineole The use of camphor and 1,8-cineole sustained weight gains and reduced E. tenella lesion totals. Furthermore, camphor was also found to decrease E. acervulina lesions (Allen et al., 1997, 1998).

Cinnamaldehyde Cinnamaldehyde was significantly more effective against Cl. perfingens compared to other plant derivatives at a lower concentration (0.5%) and at the most abusive chilling rate in ground turkey meat (Juneja and Friedman, 2007). Cinnamaldehyde (200 mg/kg) when fed to lambs reduced ruminal pH, increased volatile fatty acids concentration and no effect was obseved on other ruminal parameters (A:P ratio, ammonia), dry matter intake, average daily gain, carcass characteristics and meat characteristics (Chaves et al., 2008a). In another study, cinnamaldehyde supplementation had a positive effect on average daily gain (Chaves et al., 2008b). Cinnamaldehyde and eugenol when fed to dairy cows had no effect on dry matter intake, feed efficiency, volatile fatty acids concentration, ammonia, A:P ratio, ruminal pH, nutrients digestibility and milk yield (Tager and Krause, 2011).

Eugenol Eugenol (50 mg/kg) when fed to dairy cows showed no effect on dry matter intake, nutrients digestibility, rumen parameters and milk yield (Benchaar et al., 2012).

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Essential oils or aromachemical blends Essential oil blend (24 mg/kg complete feed), consisting of oregano, laurel leaf oil, sage leaf oil, myrtle leaf oil, fennel seed oil and citrus peel oil, improved egg production and reduced the incidence of broken-cracked eggs when supplied in laying hens (Cabuk et al., 2006). However, two other studies showed that the same mixtures (24 mg/kg and 36 mg/ kg) didn’t have any impact on liveweight gain (LWG), livability, hen-day egg production, egg weight, egg mass, feed intake, feed conversion ratio and cracked-broken egg ratio but only a tendency to increase egg weight were observed. Increased albumen height and Haugh unit (with 36 mg/kg) and increased eggshell weight (24 mg/kg) were reported (Özek et al., 2011; Bozkurt et al., 2012). Chickens supplemented with an essential oil blend of star anise, rosemary, thyme, and oregano, and a Quillaja saponin blend, or a combination of both phytogenic preparations showed a comprehensively and significantly improved apparent ileal digestibility of crude protein and amino acids compared to control birds (Zentek et al., 2017). The individual and combined effects of rosemary, fennel and oregano essential oil supplementation on the performance and ilio-caecal bacteriological flora of broiler chickens were investigated on a total of 800 male Ross-308 broiler chickens. The blend of oregano, rosemary and fennel essential oils at higher concentrations (400 mg/kg concentration) in diets stimulated the growth and improved the intestinal microbial balance, including a reduction of coliform bacteria and an increase in Lactobacillus spp. counts of broiler chickens (Cetin et al., 2016). Mixture of linalool, alpha-pinene, beta-pinene and p-cymene (0.043 or 0.43 g/kg) supplementation to dairy goats had no effect on dry matter intake, nutrients digestibility, volatile fatty acids concentration, A:P ratio and milk yield (Malecky et al., 2009). Organic acids Benzoic acid

Benzoic acid (0.1%e0.2%) supplementation to broilers suppressed the growth in broiler chickens, the dry matter of the digesta increased in the crop and caeca, and decreased lactic acid bacteria and coliform bacteria (Jozefiak et al., 2010). However, benzoic acid in combination with carvacrol and thymol improved growth performance of broilers and turkeys, shifted the balance of intestinal microbiota by increasing the number of benfecicial lactobacilli and enhanced functionality of intestinal cells (Giannenas et al., 2014a,b). Rosmarinic acid

Rosmarinic acid-rich extracts of sage, thyme, and rosemary had no effect on feed conversion and feed intake but 14e21 day broilers grew faster (Hernandez et al., 2004). Fatty acids Lauric acid

Lauric acid as a feed additive has been shown to improve food safety by reducing the numbers of Campylobacter coli in broiler meat (Zeiger et al., 2017).

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3. Herbs and aromatic plants as feed additives: aspects of composition, safety, and registration rules

Inulin Bifidobacterium and Lactobacillus strains were rapidly proliferated when inulin was added into poultry diets, contributing to a positive modulation of intestinal microbiota through the promotion of beneficial microbes and deterring the growth of pathogenic microbes. However, research reports on inulin effects on the body and performance of poultry are frequently inconsistent, as the efficiency of this prebiotic is strongly depended on the type, dose and duration of its administration (Buclaw, 2016). Rosemary Oxidative stability of eggs were improved by rosemary (Lopez-Bote et al., 1998; FlorouPaneri et al., 2005; Radwan et al., 2009). Capsaicin Capsaicin (5e20 mg/kg) supplementation in broilers caused the reduction of Salmonella typhimurium counts (Orndorff et al., 2005). Flavonoids Extracts containing isoflavones significantly risen serum testosterone levels in male chickens and decreased serum uric acids and abdominal fat (Zhengkang et al., 2006). Supplementation with daidzein (3 mg/kg/day) considerably increased laying rate, average egg weight, and egg cholesterol level in laying hens and ducks (Wang et al., 1994). Catechins and their complexes, proanthocyanidins, flavolignans, tannins, and phenolic acids. Green tea (Camellia sinensis) extract containing catechins and their gallates minimized hyperlipidemia and oxidative stress induced by corticosterone treatment in broiler chickens (Eid et al., 2003). The addition of 300 mg/kg of flavonoids (rutin, hesperidine, quercetin, and naringenin) together with mannanoligosaccharides (MOS) in feed had a significant stimulatory effect on feed conversion ratio (Batista et al., 2007; Burda and Oleszek, 2001). Hesperidin and naringin, positively affected meat antioxidative properties without negative implications on growth performance and meat quality characteristics in poultry (Goliomytis et al., 2015). Genistein and hesperidin supplementation to broilers improved meat quality in a dosedependent mode, with pronounced effects of combined treatment (Kamboh and Zhu, 2013a,b). Silymarin supplementation (40 and 80 mg/kg) in broiler chickens had no effect on growth performance but decreased slaughter yields, lipid content of breast and thigh (Schiavone et al., 2007). Silymarin phytosome provided protection against the bad effects of aflatoxin B1 in broiler chickens (Tedesco et al., 2004). Grape seed proanthocyanidins extract (12 mg/kg) supplementation to broilers increased body weight gain, reduced lesion scores and restored antioxidant/oxidant system balance after the parasite infection (Wang et al., 2008). Tannins

Tannins are plant derived compounds that are being successfully used as additives in poultry feed to control diseases and to improve animal performance. Chestnut (hydrolizable)

Chemistry and activity

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and Quebracho (condensed) tannins are probably the most readily available commercial products that are being used and cover those needs as well as there is an important number of data supporting their usage (Redondo et al., 2014). Oxidative stability of eggs were improved with tea catechins (Yilmaz, 2006). Betaine Betaine prevents heat stress, inhibits Eimeria infections and leads to improved nutrient digestibility and growth performance when used in feeds (Metzler- Zebeli et al., 2009). Polysaccharides Cellular and humoral immune responses of E. tenella-infected chickens were improved by the supplementation of an Astragalus membranaceus polysaccharide extract, especially when used in conjunction with vaccination (Guo et al., 2004a,b). Galactoglucomannan oligosaccharides (GGMs) extracted from red pine wood (Pinus brutia) showed effect on Salmonella typhimurium colonization, growth performance and intestinal morphology in broiler chickens. Birds give GGMs or commercial mannanoligosaccharides (MOS) showed better growth performance, increased villus height and villus surface area and decreased S. typhimurium colonization than the positive control birds (Rajani et al., 2016). Resveratrol Resveratrol had no effect on dry matter intake, ruminal pH, ammonia and volatile fatty acids concentration on sheep. Positive effects were observed on nutrients digestibility, decrease of methane production and A:P (acetate:propionate) ratio (Ma et al., 2015). Artemisinin Artemisinin lowered lesions and reduced oocyte output from Eimeria tenella (Allen and Fetterer, 2002; Arab et al., 2006; Allen et al., 2000). Tocopherol and turmeric g-Tocopherol and turmeric or curcumin supplemented diets reduced small intestinal lesion scores and improved weight gains during Eimeria acervulina and E. maxima infections. 1,8-cineole and camphor also protected weight gain and reduced the lesions caused by E. tenella and E. maxima (Allen and Fetterer, 2002). Oxidative stability of eggs were improved with turmeric (Curcuma longa) (Radwan et al., 2009). Saffron Oxidative stability of eggs were improved with constituents of saffron such as Crocus sativus L (Botsoglou et al., 2005). Yolks from eggs of layer hens fed with saffron constituents presented considerable lower values of malondialdehyde, main indicative compound of lipid oxidation during storage or after propagation of iron-induced lipid oxidation. Sage Oxidative stability of eggs were improved with sage (Lopez-Bote et al., 1998).

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3. Herbs and aromatic plants as feed additives: aspects of composition, safety, and registration rules

Mulberry Leaf, Japanese Honeysuckle, Goldthread Oxidative stability of eggs were improved with Mulberry leaf, Japanese honeysuckle, Goldthread (Liu et al., 2009).

Legal status of feed additives Feed additives are intended to correct nutritional deficiencies, sustain an adequate intake of certain nutrients, or support specific physiological functions. Thus, they are not medicinal products and as such cannot allege to exert any pharmacological, immunological or metabolic action. Therefore, their use should not be aimed to treat or prevent diseases in animals. Although herbs and aromatic plants are used to a large extent as feed additives and occasionally even as feed materials, dietary supplements or other products influencing, e.g., the intestinal microbiome or enhancing/improving animal welfare, the legal status, and registration of the different products is rather unclear. Principally, one applies feed additives at healthy animals to achieve one of the above-mentioned positive effects over longer periods or even throughout the whole life cycle. Applicant is usually the animal keeper or owner. Medicinal productsdalso herbal medicinal productsdon the contrary, are utilized short term to treat sick animals by or under supervision of a veterinarian only. Therefore, pathology related pharmacological health claims are not permitted for phytogenic feed additives. They are, however, authorized for feed/dietary supplements for particular nutritional purpose (“PARNUTS“) according to Reg. 94/39 (EC), but under four obligatory provisions only: (1) nutritional purpose, (2) specific nutrition-physiological disturbances, (3) species, (4) duration of use/application. Actually, the total number of approved claims is 35, of which typical indications are: function of liver, kidney, skin, intestine; nutrition intolerances, metabolism problems, obesity, diarrhea, obstipation; heart insufficiency, renal, and other stones. As regards feed additives in a strict sense, their processing, marketing, and use within the European Union is laid down in Reg. (EC) 1831/2003 followed by detailed rules for the implementation according to Reg. (EC) 429/2008, finally the EFSA Guidance on identity, characterization and conditions of use (2017) and the EFSA Guidance on the assessment of the efficacy of feed additives (2018). Any feed additive needs to be authorized by the European Commission displaying the following presuppositions: having no negative effect on animal or human health or on the environment, misleading or harm the user or finally the consumer. A feed additive shall, however: (1) favorably affect the characteristics of feed (e.g., antioxidants: substances prolonging the storage life of feedingstuffs and feed materials by protecting them against deterioration caused by oxidation), (2) favorably affect the characteristics of animal products (e.g., colorants: substances that add or restore color in feedingstuffs; substances which, when fed to animals, add colors to food of animal origin), (3) favorably affect the color of ornamental fish and birds, (4) favorably affect the environmental consequences of animal production, (5) favorably affect animal production, performance or animal welfare, particularly by affecting the gastrointestinal flora or digestibility of feedingstuffs, or (6) have a coccidiostatic or histomonostatic effect.

Conclusion

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Depending on its functions and properties, the following categories are especially important for herbal feed additives (phytogenics): 1. technological additives: any substance added to feed for a technological purpose; 2. sensory additives: any substance, the addition of which to feed improves or changes the organoleptic properties of the feed, or the visual characteristics of the food derived from animals; 3. zootechnical additives: any additive used to affect favorably the performance of animals in good health or used to affect favorably the environment. According to the regulations, any additive has to be fully identified and characterized. Additives in which not all constituents can be identifieddas especially plant extractsd should be characterized by the secondary plant products contributing to its activity. The activity itself should be shown, in addition, by studies that demonstrate the efficacy for each proposed use and satisfy at least one of the characteristics according to the categories and functional groups of feed additives. There is an ongoing discussion concerning the categories and functional groups due to the fact that Regulation (EC) 1831/2003 is under revision. One hot topic is Animal Welfare, which is defined by conditions that an animal is healthy, comfortable, well nourished, safe, able to express innate behavior, and does not suffer from unpleasant states such as pain, fear, and distress. For additives favorably affecting welfare, the choice of long-term or short-term studies to demonstrate the efficacy will depend on the nature of the substance and their intended purpose (EFSA, 2018). If a new category as e.g., Animal Welfare Products, Animal Welfare Stabilizers, or Animal Welfare Enhancers will be implemented into European regulations, or a functional group Physiological Condition Stabilizers/Enhancers within the category Zootechnical Additives only, remains to be seen in future. It should in any case not hindering but promoting innovations.

Conclusion Herbs, aromatic plants, and herbal products play a prominent role in animal nutrition and have a high impact on animal health, performance, and welfare. A number of regulations exist concerning identity, quality, activity, and efficacy, safety, and finally also marketing of the products, complemented by respective guidance to enable the applicant to meet all actual requirements. Due to a progressive development of analytical methods and highly advanced research one has to keep in mind, however, the critical and by all means essential focal points for herbal feed additives: 1. safety for the animal, the user, and in case of food producing animals the consumer, 2. animal welfare and environmental compatibility. There should remain sufficient space for innovations, especially with respect to further avoid the use of in-feed antibiotics and to develop animal health promoting feed additives also from up-to-now unconventional and “exotic” plants.

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Ma, T., Chen, D.D., Tu, Y., Zhang, N.F., Si, B.W., Deng, K.D., Diao, Q.Y., 2015. Effect of dietary supplementation with resveratrol on nutrient digestibility, methanogenesis and ruminal microbial flora in sheep. J. Anim. Physiol. Anim. Nutr. 99, 676e683. Malecky, M., Broudiscou, L.P., Schmidely, P., 2009. Effects of two levels of monoterpene blend on rumen fermentation, terpene and nutrient flows in the duodenum and milk production in dairy goats. Anim. Feed Sci. Technol. 154, 24e35. Mayer, M., Vogl, C.R., Amorena, M., Hamburger, M., Walkenhorst, M., 2014. Treatment of organic livestock with medicinal plants: a systematic review of European Ethnoveterinary research. Forschende Komplementärmed. 21, 375e386. Metzler-Zebeli, B.U., Eklund, M., Mosenthin, R., 2009. Impact of osmoregulatory and methyl donor functions of betaine on intestinal health and performance in poultry. World’s Poult. Sci. J. 65, 419e441. Mitsch, P., Zitterl-Eglseer, K., Kohler, B., Gabler, C., Losa, R., Zimpernik, I., 2004. The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens. Poultry Sci. 83, 669e675. Monino, I., Martìnez, C., Sotomayor, J.A., Lafuente, A., Jordàn, M.A., 2008. Polyphenolic transmission to Segureño lamb meat from ewes diet supplemented with the distillate from rosemary (Rosmarinus officinalis) leaves. J. Agric. Food Chem. 56, 3363e3367. Newmaster, S.G., Grguric, M., Shanmughanandhan, D., Ramalingam, S., Ragupathy, S., 2013. DNA barcoding detects contamination and substitution in North American herbal products. BMC Med. 11, 222. Novak, J., Ruzicka, J., Schmiderer, C., 2018. How far advanced is the DNA-based identification of the BELFRIT-list? In: Restani, P. (Ed.), Food Supplements Containing Botanicals: Benefits, Side Effects and Regulatory Aspects. Springer, Berlin, pp. 227e301. Novak, J., Grausgruber-Gröger, S., Lukas, B., 2007. DNA-based authentication of plant extracts. Food Res. Int. 40, 388e392. O’Connor, A., November 3, 2013. Herbal Supplements Are Often Not what They Seem. The New York Times. Oral, N., Vatansever, L., Sezer, C., Aydin, B., Guven, A., Gulmez, M., Baser, K.H.C., Kurkcuoglu, M., 2009. Effect of absorbent pads containing oregano essential oil on the shelf life extension of overwrap packed chicken drumsticks stored at four degrees Celsius. Poultry Sci. 88, 1459e1465. Orhan, I.E., Senol, F.S., Skalicka-Wozniak, K., Georgiev, M.I., 2016. Adulterations and safety issues in nutraceuticals and dietary supplements: innocent or risky?. In: Grumezescu, A.M. (Ed.), Nutraceuticals, Nanotechnology in the Agri-Food Industry, vol. 4. Academic Press, Amsterdam, The Netherlands, pp. 153e182. Orndorff, B.W., Novak, C.L., Pierson, F.W., Caldwell, D.J., McElroy, A.P., 2005. Comparison of prophylactic or therapeutic dietary administration of capsaicin for reduction of Salmonella in broiler chickens. Avian Dis. 49, 527e533. Özek, K., Wellmann, K.T., Ertekin, B., Tarım, B., 2011. Effects of dietary herbal essential oil mixture and organic acid preparation on laying traits, gastrointestinal tract characteristics, blood parameters and immune response of laying hens in a hot summer season. J. Anim. Feed Sci. 20, 575e586.

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Penalver, P., Huerta, B., Borge, C., Astorga, R., Romero, R., Perea, A., 2005. Antimicrobial activity of five essential oils against origin strains of the Enterobacteriaceae family. APMIS 113, 1e6. Pieroni, A., 1999. Herbs, Humans and Animals. Experiences Publ, Cologne, 198 pp. Radwan, N.L., Hassan, R.A., Quota, E.M., Fayek, H.M., 2009. Effect of natural antioxidant on oxidative stability of egg and productive and reproductive performance of laying hens. Int. J. Poult. Sci. 7, 134e150. Rajani, J., Dastar, B., Samadi, F., Karimi Torshizi, M.A., Abdulkhani, A., Esfandyarpour, S., 2016. Effect of extracted galactoglucomannan oligosaccharides from pine wood (Pinus brutia) on Salmonella typhimurium colonisation, growth performance and intestinal morphology in broiler chicks. Br. Poult. Sci. 57, 682e692. Redondo, L.M., Chacana, P.A., Dominguez, J.E., Miyakawa, M.E.F., March 27, 2014. Perspectives in the use of tannins as alternative to antimicrobial growth promoter factors in poultry. Front. Microbiol. Accessed date: 16 May 2019 https://doi.org/10.3389/fmicb.2014.00118. Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on Additives for Use in Animal Nutrition. Regulation (EC) No 429/2008 of 25 April 2008 on Detailed Rules for the Implementation of Regulation (EC) No 1831/ 2003 of the European Parliament and of the Council as Regards the Preparation and the Presentation of Applications and the Assessment and the Authorization of Feed Additives. Restani, P., 2017. In: Restani, P. (Ed.), Food Supplements Containing Botanicals: Benefits, Side Effects and Regulatory Aspects. The Scientific Inheritance of the EU Project PlantLIBRA. Springer, Berlin, 467 pp. Schiavone, A., Righi, F., Quarantelli, A., Bruni, R., Serventi, P., Fusari, A., 2007. Use of Silybum marianum fruit extract in broiler chicken nutrition: influence on performance and meat quality. J. Anim. Physiol. Anim. Nutr. 91, 256e262. Schilter, B., Andersson, C., Anton, R., Constable, A., Kleiner, J., O’Brien, J., Renwick, A.G., Korver, O., Smit, F., Walker, R., 2003. Guidance for the safety assessment of botanicals and botanical preparations for use in food and food supplements. Food Chem. Toxicol. 41 (Issue 12), 1625e1649. Schmiderer, C., Novak, J., 2014. PCR-basierende Identitätsprüfung von Kamille (Matricaria recutita L.) und Nachweis von Verunreinigungen mit Anthemis-Arten in Kamillenprodukten, 7. Si, W., Gong, J., Tsao, R., Zhou, T., Yu, H., Poppe, C., Johnson, R., Du, Z., 2006. Antimicrobial activity of essential oils and structurally related synthetic food additives towards selected pathogenic and beneficial gut bacteria. J. Appl. Microbiol. 100, 296e305. Siragusa, G.R., Haas, G.J., Matthews, P.D., Smith, R.J., Buhr, R.J., Dale, N.M., Wise, M.G., 2008. Antimicrobial activity of lupulone against Clostridium perfringens in the chicken intestinal tract jejunum and caecum. J. Antimicrob. Chemother. 61, 853e858. Stockhammer, S., Stolze, K., Rohr-Udilova, N., Chizzola, R., Zitterl, K., Franz, C., 2009. Antioxidant activity of phytogenous industrial waste and derived extracts for the production of feed and food additives. Food Sci. Technol. 44, 703e710. Tager, L.R., Krause, K.M., 2011. Effects of essential oils on rumen fermentation, milk production, and feeding behavior in lactating dairy cows. J. Dairy Sci. 94, 2455e2464. Tedesco, D., Steidler, S., Galletti, S., Tameni, M., Sonzogni, O., Ravarotto, L., 2004. Efficacy of silymarin-phospholipid complex in reducing the toxicity of aflatoxin B-1 in broiler chicks. Poultry Sci. 83, 1839e1843. Tedesco, D., Garavaglia, L., Spagnuolo, M.S., Pferschy-Wenzig, E.M., Bauer, R., Franz, C., 2015. In vivo assessment of an industrial waste product as a feed additive in dairy cows: effects of larch (Larix decidua L.) sawdust on blood parameters and milk composition. Vet. J. 206, 322e326. Thakur, L., Ghodasra, U., Patel, N., Dabhi, M., 2011. Novel approaches for stability improvement in natural medicines. Pharmacogn. Rev. 5 (9), 48e54. Wallace, R.J., Oleszek, W., Franz, C., Hahn, I., Baser, K.H.C., Mathe, A., Teichmann, K., 2010. Dietary plant bioactives for poultry health and productivity. Br. Poult. Sci. 51 (4), 461e487. Wang, G.J., Han, Z.K., Chen, J., Chen, W.H., 1994. Effect of daidzein on muscle growth in broilers and mechanism involved. Sci. Technol. Anim. Husbandry Vet. Med. 19, 4e6. Wang, M.L., Suo, X., Gu, J.H., Zhang, W.W., Fang, Q., Wang, X., 2008. Influence of grape seed proanthocyanidin extract in broiler chickens: effect on chicken coccidiosis and antioxidant status. Poultry Sci. 87, 2273e2280. Wegener, H.C., 2012. Antibiotic resistance-linking human and animal health. In: Improving Food Safety through a One Health Approach: Workshop Summary. National Academies Press. WHO, 2015. Global Action Plan on Antimicrobial Resistance. World Health Organization.

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Wijesekera, R.O.B., Jajewardene, A.I., Rajapakse, I.S., 1974. Composition of the essential oil from leaves, stem bark and root bark of two chemotypes of cinnamon. J. Sci. Food Agric. 25, 1211e1218. Yılmaz, Y., 2006. Novel uses of catechins in foods. Trends Food Sci. Technol. 17, 64e71. Youdim, K.A., Deans, S.G., 1999. Beneficial effects of thyme oil on age related changes in phospholipid C20 and C22 polyunsaturated fatty acid composition of various rat tissues. Biochim. Biophys. Acta 1438, 140e146. Zeiger, K., Popp, J., Becker, A., Hankel, J., Visscher, C., Klein, G., Meemken, D., 2017. Lauric acid as feed additive e an approach to reducing Campylobacter spp. in broiler meat. PLoS One 12 (4), e0175693. https://doi.org/10.1371/ journal.pone.0175693. Zentek, R.H., Maenner, K., Youssef, I.M.I., Aumiller, T., Weghuber, J., Wimmers, K., Mueller, A.S., 2017. Possible molecular mechanisms by which an essential oil blend from star anise, rosemary, thyme and oregano and saponins increase the performance and ileal protein digestibility of growing broilers. J. Agric. Food Chem. 16, 6821e6830. Zhengkang, H., Wang, G., Yao, W., Zhu, W.Y., 2006. Isoflavonic phytoestrogens d new prebiotics for farm animals: a review on research in China. Curr. Issues Intest. Microbiol. 7, 53e60.

Further reading Baser, K.H.C., Franz, C., 2016. Essential oils used in veterinary medicine. In: Handbook of Essential Oils, second ed. CRC Press, pp. 655e668. Bento, M.H.L., Ouwehand, A.C., Tiihonen, K., Lahtinen, S., Nurminen, P., Saarinen, M.T., Schulze, H., Mygind, T., Fischer, J., 2013. Essential oils and their use in animal feeds for monogastric animals e effects on feed quality, gut microbiota, growth performance and food safety: a review. Vet. Med. 58 (9), 449e458. Blanco-Penedo, I., Fernández González, C., Tamminen, L.M., Sundrum, A., Emanuelson, U., 2018. Priorities and future actions for an effective use of phytotherapy in livestockdoutputs from an expert workshop. Front. Vet. Med. 4, 248. Chao, S.C., Young, D.G., Oberg, C.J., 2000. Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses. J. Essent. Oil Res. 12, 639e649. Dhama, K., Latheef, S.K., Mani, S., Samad, H.A., Karthik, K., Tiwari, R., Khan, R.U., Alagawany, M., Farag, M.R., Alam, G.M., Laudadio, V., Tufarelli, V., 2015. Multiple beneficial applications and modes of action of herbs in poultry health and production e a review. Int. J. Pharmacol. 11, 152e176. Diaz Carrasco, J.M., Redondo, L.M., Redondo, E.A., Dominguez, J.E., Chacana, A.P., Fernandez Miyakawa, M.E., 2016. Use of plant extracts as an effective manner to control Clostridium perfringens induced necrotic enteritis in poultry. BioMed Res. Int. 2016, 3278359. https://doi.org/10.1155/2016/3278359. Fink-Gremmels, J., 2017. Recent Challenges in Veterinary Pharmacotherapy e Could Medicinal Plants Be an Option? Plenary Lecture, GA-Congress Sept. 2017 Basel/CH. Florou-Paneri, P., Christaki, E., Giannenas, I.A., Papazahariadou, M., Botsoglou, N.A., Spais, A.B., 2004. Effect of dietary Olympus tea (Sideritis scardica) supplementation on performance of chickens challenged with Eimeria tenella. J. Anim. Feed Sci. 13, 301e311. Franz, C., Hahn, I., 2010. Veterinärmedizin und Tierernährung. In: Handbuch des Arznei- und Gewürzpflanzenbaus, vol. 1. Saluplanta, Bernburg, pp. 438e471. Granados-Chinchilla, F., 2017. A review on phytochemicals (including essential oils and extracts) inclusion in feed and their effects on food producing animals. Dairy Vet. Sci. J. 3 (4), 555e620. Hahn-Ramssl, I., Franz, C., Kranzl, A., 2014. Einsatz von Malventee bzw. NaCl bei Vollhautwunden im klinischexperimentellen Mäuseversuch zum Vergleich der Heilungsdauer. In: Internationale Tagung Phytotherapie 2014, Klinik und Praxis, Winterthur, Switzerland, June 18e21, 2014. Forschende Komplementärmed./Res. Complementary Med., pp. 63e64. Lillehoj, H., Liu, Y., Calsamiglia, S., Fernandez-Miyakawa, M.E., Chi, F., Cravens, R.L., Oh, S., Gay, C.G., 2018. Phytochemicals as antibiotic alternatives to promote growth and enhance host health. Vet. Res. 49 (1), 76. Máthé, Á., 1997. Essential oils as phytogenic feed additives (PFA). In: Franz, C., Máthé, Á., Buchbauer, G. (Eds.), Essential Oils: Basic and Applied Research. Proc. 27th ISEO 1996. Allured Publ. Carol Stream, pp. 315e321. Saranraj, P., Veerajan, D.D., 2017. Essential Oils and its Antibacterial Properties e A Review researchgate.net/publication/319302027. Yang, C., Chowdhury, M.A.K., Hou, Y., Gong, J., 2015. Phytogenic compounds as alternatives to in-feed antibiotics: potentials and challenges in application. Pathogens 4, 137e156.

C H A P T E R

4 Sustainable use of mediterranean medicinal-aromatic plants Katerina Grigoriadou1, Nikos Krigas1, Diamanto Lazari2, Eleni Maloupa1 1

Laboratory of Conservation and Evaluation of Native and Floricultural Species-Balkan Botanic Garden of Kroussia, Hellenic Agricultural Organization e DEMETER, Thessaloniki, Greece; 2 Laboratory of Pharmacognosy, School of Pharmacy, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece O U T L I N E Valuable properties of medicinal and aromatic plants (MAPs) and herbal medicinal products

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European market of medicinal and aromatic plants: trends, challenges, and the value chain

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Mediterranean medicinal and aromatic plants: wealth, uniqueness, and risks

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From wild to cultivation: conservation and sustainable exploitation of phytogenetic resources of maps

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Research on propagation of native maps: a key for sustainable exploitation

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Pilot fields: a step closer to new crops

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References

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Valuable properties of medicinal and aromatic plants (MAPs) and herbal medicinal products From ancient times to date, natural plant products have been used as a source of nutrition and traditional therapy. People have used plants to apply traditional recipes for their

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00004-2

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Copyright © 2020 Elsevier Inc. All rights reserved.

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4. Sustainable use of mediterranean medicinal-aromatic plants

nutrition and for their health. Many modern medicines are based on traditional knowledge reflecting the therapeutic value of plants. The majority of the world’s population (approximately 80%), especially in undeveloped countries, still rely on plant-based traditional medicines as part of their primary health care (WHO, 2008). Moreover, most people in developed countries use plant-based products for their basic health care or as an alternative or complementary medicine (WHO, 2008). Secondary metabolites are the chemical compounds synthesized during plant metabolic procedures not directly involved in the normal growth, development, or reproduction of the organism, stored sometimes in special organs or parts. Plants produce a large number of such organic compounds, estimated over 100,000, but only 10% of them have been isolated. These metabolites belong mainly in three groups: terpenes, phenolics, and alkaloids (nitrogen-containing compounds) with medicinally useful properties. The active compounds that are present in medicinal and aromatic plants (MAPs) originate from plant metabolic pathways, and their chemical composition may vary considerably according to the part of the plant (inflorescence, bracts, leaves, stems), the stage of development, the time of the day or year (seasonal variability), and the environmental conditions (i.e., soil and climate) in which an aromatic-medicinal plant was cultivated or was growing in the wild habitats (Kokkini et al., 1997; Góra et al., 2002). It has been found that a qualitative as well as a quantitative variety of essential oils is produced by several MAPs, and this may vary in populations that belong in the same species and can differ even between individuals of a single population (Kokkini et al., 1991, 1997; Karousou and Kokkini, 1997; Karousou et al., 1998, 2012). In addition, the methods of processing and extraction may also affect the composition and concentration of the obtained extracts (Azmir et al., 2013; Moore et al., 2014; Bruni et al., 2009). Native MAPs have been used in the Mediterranean since antiquity for nutritional purposes and for the treatment of various diseases and disorders in humans and animals. Some MAPs have well-established use and significant health claims associated with sufficient number of studies demonstrating their properties (even if there is no evidence of long-term use). According to the Committee on Herbal Medicinal Products (HMPC), which is the committee of the European Medicines Agency (EMA) (see www.ema.europa.eu) responsible for compiling and assessing scientific data on herbal substances, preparations and combinations, such herbal medicinal products (as well as their preparations) are classified under the label of “Traditional Use” or “Well-established Use” European Union (EU) herbal monographs (formerly known as Community herbal monographs) are species-specific or drug-specific, contain the HMPC’s scientific opinion on safety and efficacy, and critically deliver the available data about specific herbal substances and their preparations intended for medicinal use. The available knowledge and information are evaluated by HMPC, taking into account documented long-standing use and traditional experience in the EU, including also clinical and nonclinical data. Herbal Medicinal Products are defined as “any medicinal product, exclusively containing as active ingredients one or more herbal substances, or one or more herbal preparations, or one or more such herbal substances in combination with one or more such herbal

European market of medicinal and aromatic plants: trends, challenges, and the value chain

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preparations” Traditional Herbal Medicinal Products for human use are those that fulfill the following conditions: - they are made from traditional herbal medicinal products and they will be used without the supervision of a doctor for diagnostic purposes and without prescribing or monitoring of treatment - they are exclusively for administration in accordance with a specified strength and posology; - they are used for oral, external and/or inhalation preparation; - the herbal medicinal product has been used for at least 30 years before the official request of use permission, at least 15 of which in the EU - the information included in the application form sufficiently prove the traditional use of the herbal product as medicine (especially because it cannot cause any kind of damage under certain conditions of use); furthermore, the efficacy of the product is reasonable under the experience over the above-mentioned time period. MAPs, herbal medicinal products and their preparations have been increasingly used to either treat or cure or prevent diseases of humans or animals. The Mediterranean flora and especially the native flora of Greece are quite rich in MAPs, which contain plenty of significant biologically active ingredients (e.g., phenolic derivatives). Many contemporary studies show that these ingredients have potential health benefits as they protect and prevent the onset of serious conditions such as diabetes, cardiovascular diseases, aging, etc (Lazari et al., 2006; Matta et al., 2007; Papaioannou et al., 2007; Kontogiorgis et al., 2016a, 2016b; Vartholomatos et al., 2016). To date, >75 plant species of native Greek flora have a finalized herbal monograph adopted and published by EMA (Table 4.1). These are mainly plants with expectorant properties, as well as softening, mucolytic, and antiinflammatory actions, and are most commonly used to treat various diseases as their benefits are well-established. Some of them are confined only to small geographical regions such as Peloponnese, Greece (Sideritis clandestina subsp. clandestina and subsp. peloponnesiaca; Taygetus Mountain Tea and Kyllini Mountain Tea, respectively) or to the island of Crete (single-island endemics), i.e., Origanum dictamnus (Dittany of Crete) and Sideritis syriaca subsp. syriaca (Malotira, Cretan Mountain Tea).

European market of medicinal and aromatic plants: trends, challenges, and the value chain Consumers worldwide have a strongly increased interest in a healthy lifestyle and, consequently, in the consumption of food or nonfood products with health benefits. The “natural” claim includes ‟no additives/preservatives,” “organic,” and “wholegrain” as well as “totally natural itself” (Barata et al., 2016). MAPs are used for the production of “natural” goods due to their properties such as functional foods, cosmetics and healthcare products, herbal-based medicines and agriculture and veterinary medicine or feeds. They are also used nowadays as natural preservatives for many food products as for example the extended use of oregano and rosemary not only as flavor additives in sausages and other meat products but also for their antioxidant properties (Krishnaiah et al., 2011; Lucera et al., 2012). In the nonfood sector, alternative medicines using MAPs have increased in most EU countries over the last few years (Ekor, 2013).

60 TABLE 4.1

4. Sustainable use of mediterranean medicinal-aromatic plants

Greek native plants (alphabetically) with finalized herbal monograph and approved indications by the European Medicines Agency.

Botanical name of plant

Name of medicinal herbal substance and English common name

Approved indications

Achillea millefolium L.

Millefolii flos (yarrow flower); Millefolii herba (yarrow)

A, E, F, K

Aesculus hippocastanum L.

Hippocastani cortex (Horse-chestnut bark)/Hippocastani semen (Horse-chestnut seed)

F, H/H

Agrimonia eupatoria L.

Agrimoniae herba (Agrimony)

B, E, K

Elytrygia repens (L.) Nevski [¼Agropyron repens (L.) P. Beauv.]

Agropyri repentis rhizoma (Couch grass rhizome)

F

Althaea officinalis L.

Althaeae radix (Marshmallow root)

B, G, K

Arctium lappa L.

Arctii radix (Burdock root)

A, E, F

Arctostaphylos uva-ursi (L.) Spreng.

Uvae-ursi folium (Bearberry Leaf)

F

Artemisia absinthium L.

Absinthii herba (Wormwood)

A, K

Avena sativa L.

Avenae herba (Oat herb)/Avenae fructus (Oat fruit)

C, J/E

Capsella bursa-pastoris (L.) Medikus.

Bursae-pastoris herba (Shepherds Purse)

F

Centaurium erythraea Rafn. s.l.

Centaurii herba (Centaury)

K

Cichorium intybus L.

Cichorii intybi radix (Chicory root)

A, K

Crataegus spp.

Crataegi folium cum flore (Hawthorn Leaf and flower)

H, J

Epilobium angustifolium L. or E. parviflorum Schreb.

Epilobii herba (Willow herb)

F

Equisetum arvense L.

Equiseti herba (Horsetail herb)

F

Filipendula ulmaria (L.) Maxim.

Filipendulae ulmariae flos (Meadowsweet flower); filipendulae ulmariae herba (Meadowsweet)

D, G

Foeniculum vulgare Mill. [var. dulce (Miller) Thellung.]/ [var. vulgare].

Foeniculi dulcis fructus (Sweet fennel) & foeniculi amari fructus (Bitter fennel)/Foeniculi amari fructus aetherolaeum (Bitter fennel fruit oil)

F, G, K/G

Fraxinus excelsior L. or F. angustifolia Vahl

Fraxini folium (Ash Leaf)

D, F

Fumaria officinalis L.

Fumariae herba (fumitory)

K

Gentiana lutea L.

Gentianae radix (Gentian root)

K

Glycyrrhiza glabra L.

Liquiritiae radix (Liquorice root)

K

Hedera helix L.

Hederae helicis folium (Ivy leaf)

G

Humulus lupulus L.

Lupuli flos (Hop strobile)

A, C, J

Hypericum perforatum L.

Hyperici herba (St. John’s Wort)

E, J, K

Juglans regia L.

Juglandis folium (Walnut leaf)

E

Juniperus communis L.

Juniperi aetherolaeum (Juniper oil)

D, F, K

61

European market of medicinal and aromatic plants: trends, challenges, and the value chain

TABLE 4.1

Greek native plants (alphabetically) with finalized herbal monograph and approved indications by the European Medicines Agency.dcont'd

Botanical name of plant

Name of medicinal herbal substance and English common name

Approved indications

Juniperus communis L.

Juniperi pseudo-fructus (Juniper berry)

F, K

Leonurus cardiaca L.

Leonuri cardiacae herba (Motherwort)

H, J

Linum usitatissimum L.

Lini semen (Linseed)

I, K

Marrubium vulgare L.

Marrubii herba (White horehound)

A, G, K

Matricaria recutita L.

Matricariae flos (Matricaria flower)/Matricariae aetherolaeum (Matricaria oil)

B, E, G, K/E

Melilotus officinalis (L.) Lam.

Meliloti heraba (Melilot)

E, H

Melissa officinalis L.

Melissae folium (Melissa leaf)

C, J, K

Olea europaea L.

Oleae folium (Olive leaf)

F

Origanum dictamnus L.

Origani dictamni herba (Dittany of Crete herb)

E, G, K

Origanum majorana L.

Origani majoranae herba (Majoram)

E, K

Plantago afra L. or P. indica L.

Psyllii semen (Psyllium seed)

I

Plantago lanceolata L.

Plantaginis lanceolatae folium (Ribwort Plantain)

G

Polygonum aviculare L.

Polygoni avicularis herba (Knotgrass herb)

B, F, G

Potentilla erecta (L.) Raeusch.

Tormentillae rhizoma (Tormentil)

K

Primula veris L. or P. elatior (L.) Hill.

Primulae radix (Primula root); Primulae flos (Primula flower)

G

Quercus robur L., Q. petraea (Matt.) Liebl., Q. pubescens Willd.

Quercus cortex (Oak Bark)

E, H, K

Frangula alnus Mill. (¼Rhamnus frangula L.)

Frangulae cortex (frangula bark)

I

Rosmarinus officinalis L.

Rosmarini folium (rosemary leaf); Rosmarini aetherolaeum (rosemary oil)

D, H, K

Rubus idaeus L.

Rubi idaei folium (Raspberry leaf)

B, D, K

Ruscus aculeatus L.

Rusci i rhizoma (Butcher’s Broom)

H

Salvia officinalis L.

Salviae officinalis folium (Sage Leaf)

B, E, K

Salix purpurea L. or S. fragilis L.

Salicis cortex (Willow Bark)

D, G

Sambucus nigra L.

Sambuci flos (Elder flower)

G

Sideritis scardica Griseb., S. clandestina (Bory & Chaub.) Hayek, S. raeseri Boiss. & Heldr., S. syriaca L.

Sideritis herba (Ironwort)

G, K

Sisymbrium officinale (L.) Scop.

Sisymbrii officinalis herba (Hedge mustard)

G (Continued)

62 TABLE 4.1

4. Sustainable use of mediterranean medicinal-aromatic plants

Greek native plants (alphabetically) with finalized herbal monograph and approved indications by the European Medicines Agency.dcont'd

Botanical name of plant

Name of medicinal herbal substance and English common name

Approved indications

Solanum dulcamara L.

Solani dulcamarae stipites (Woody nightshade stem)

E

Solidago virgaurea L.

Solidaginis virgaureae herba (European Goldenrod)

F

Tanacetum parthenium (L.) Schultz Bip.

Tanaceti parthenii herba (feverfew)

D

Tilia cordata Mill. or T. platyphyllos Scop.

Tiliae flos (Lime flower)

G, J

Trigonella foenum-graecum L.

Trigonellae foenugraeci semen (fenugreek)

A, E

Urtica dioica L. or U. urens L.

Urticae folium (Nettle Leaf), Urticae herba (Nettle herb)/Urticae radix (Nettle root), Urticae folium (Nettle Leaf), Urticae herba (Nettle herb)

D, E/F

Vaccinium myrtillus L.

Myrtilli fructus siccus (dried Bilberry fruit)/Myrtilli fructus recens (fresh Bilberry fruit)

B, K/H

Valeriana officinalis L.

Valerianae radix (Valerian root); Valerianae aetherolaeum (Valerian essential oil)

C, J

Verbascum densiflorum Bertol. or V. phlomoides L.

Verbasci flos (Mullein flower)

B, G

Viola macedonica Boiss. & Heldr. in Boiss. (¼V. tricolor L.) or V. arvensis Murray.

Violae tricoloris herba cum flore (wild Pansy)

E

Vitex agnus-castus L.

Agni Casti fructus (Agnus Castus fruit)

F

Vitis vinifera L.

Vitis viniferae folium (Grapevine Leaf)

H

Combination: Valeriana officinalis L. and Humulus lupulus L.

Valerianae radix and Lupuli flos (Valerian root and Hop Strobile)

C, J

A, loss of appetite; B, mouth and throat disorders; C, sleep disorders and temporary insomnia; D, pain and inflammation; E, skin disorders and minor wounds; F, urinary tract and genital disorders; G, cough and cold; H, Circulary disorders; I, constipation; J, mental stress and mood disorders; K, gastrointestinal disorders.

Today’s consumers are value-conscious, interactive, multicultural, health-driven, socially responsible, and always interconnected. In developed countries quality of life, personal appearance, healthy lifestyle proceeds to acquisition of goods. MAP-based products are associated with an increasing demand because of a growing awareness about their benefits and efficacy, which in turn leads to a growing acceptability by consumers all over the world. Furthermore, there is an increasing awareness from the consumers’ side related to biodiversity issues, conservation and sustainable use of plant resources. A new group of consumers is rapidly emerging which is strongly committed to healthy and sustainable lifestyle. Increasingly, these are referred to as Lifestyle of Health and Sustainability (LOHAS) consumers (CBI Market Survey, 2009). They are focused on health and fitness, personal development, sustainable living, and they care about environmental issues and social justice. The

European market of medicinal and aromatic plants: trends, challenges, and the value chain

63

emergence of this new group of consumers is one of the main underlying forces for organic as well as fair trade market growth. “Fair Trade” is an approach to business and to development based on dialogue, transparency, and respect that seeks to create greater equity in the international trading system, the overall market for products that are ethically sourced (Witkowski, 2014; Dragusanu et al., 2014). A “Value Chain” can be defined as a strategic partnership among interdependent businesses that collaborate to progressively create value for the final consumer resulting in a collective competitive advantage (Poter and Kramer, 2011). The value chain for MAPs is complex as it is constituted by several intermediaries. Additionally, many different industries are involved which accept primary and secondary processed products as well as final, more sophisticated and innovative products. Usually value chains are presented in a linear form; however, MAPs value chain links refer to different industries producing with different products considered as first material for further processing (raw materials, natural ingredients, essential oils, etc.), secondary processed products (cosmetics, food and beverage, animal feed, medicines, agrochemicals and veterinary medicines etc.), trade patterns and services, all of which are connected in a complex and complementary way (Fig. 4.1). In the framework of international initiatives such as Convention on Biological Diversity, the Global Strategy for Plant Conservation, the Nagoya Protocol and the European Regulation 511/2014, the European Directive 92/43/EEC, the following main goals were established: the conservation of biological diversity, the sustainable use of its components, and the fair and equitable sharing of the benefits from the use of genetic resources. Priority was given both to in situ protection of wild populations of rare and threatened species in protected areas and to their ex situ (out-of-site) conservation in seed banks and botanical gardens (https://www.cbd.int/convention/articles/default.shtml?a¼cbd-09).

FIGURE 4.1 MAPs’ value chain is presented in circular form. Inputs, primary and secondary production, and process (raw materials, natural ingredients, essential oils, cosmetics, food and beverage, animal feed, medicines, agrochemicals, human and veterinary medicines, etc.) are connected in a complex, complementary, and global way with ethics, environmental issues, trade, and services.

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4. Sustainable use of mediterranean medicinal-aromatic plants

MAPs are threatened worldwide by overexploitation, destructive harvesting techniques, habitat loss, and alteration, largely as a result of increased urbanization and changing agricultural practices during the past 100e200 years (Misra, 2009). Their trade is difficult to accurately estimate and regulate. In Europe, many countries regulate or prohibit trade of species whose wild populations have evidenced decline in recent years; however, in many countries where threatened MAPs are found, there is insufficient protective legislation and the existing laws are often inadequate enforced (Marshal, 2012). Sustainable exploitation from the wild is difficult to achieved and certified. There are several certification systems to assure food quality, environmental protection, occupation safety and stuff care. These systems focus on reducing risks for food safety in the primary production, minimizing adverse impact on the environment and improving effectiveness of activities of the agricultural primary production along the value chain of MAPs. Four of them are the most relevant for MAPs products and they are used as a competitive advantage between supplierecustomer agreements: the forest management certification (e.g., Forest Stewardship Council [FSC]), the social certification (e.g., Fair Trade Federation [FTF]), the organic certification (e.g., International Federation of Organic Agriculture [IFOAM]), and the product quality certification (Fig. 4.1). A special certification on Good Harvesting Practices (GHP) for plant material collected from the wild aims for environmental protection and minimized damage on biodiversity; however, greater consideration must be given in these aspects to ensure sustainability (Walter, 2002). Protection of ecosystems as well as the use of renewable energy sources lead farmers to certify not only their products but the whole production procedures (Schippmann et al., 2006; Lubbe and Verpoorte, 2011). Domestication/introduction into cultivation of MAPs is considered to be one of the most significant methods that could secure the reliable raw material supplies. Furthermore, this is one of the most important conservation options, since constant drain of material from their wild populations is much higher than the annual yield produced (Maloupa et al., 2008).

Mediterranean medicinal and aromatic plants: wealth, uniqueness, and risks The Mediterranean region is currently considered as a conservation sanctuary and a global biodiversity hot-spot (Myers et al., 2000). About 10% of the world’s vascular plants can be found in this area, which represents only 1.6% of the Earth’s surface (Medail and Quézel, 1999); this includes around 25,000 species naturally found in the Mediterranean countries, at least half of which are endemic to this region (Heywood, 1999; Greuter, 1991). Within the Mediterranean context, Greece alone hosts at least 6620 taxa (species and subspecies), among which 1459 single-country endemics not to be found elsewhere (Dimopoulos et al., 2013, 2016); this is the highest endemism rate per unit area in Europe and the Mediterranean Basin (Georghiou and Delipetrou, 2010). This outstanding center of biodiversity is also considered to be the most threatened one, mainly due to human activities. The Mediterranean MAPs constitute a unique, highly diverse and valuable phytogenetic resource which is largely exploited since ancient times. Overexploitation of natural resources, often accompanied by illegal activities, has been identified as one of the principal causes of biodiversity loss (Bradshaw et al., 2009; St. John et al., 2012). Currently, over 400,000 tons of MAPs are traded every year and rapidly growing

Mediterranean medicinal and aromatic plants: wealth, uniqueness, and risks

65

demand is putting severe pressure on natural resources. In Europe, 90% of 1200e1300 different MAPs are still collected from the wild; this situation, coupled with land conversion or habitat loss, is currently threatening 25% of MAPs species with extinction (Schippmann et al., 2006). Increasing demand has also led to inconsistency and deterioration of the quality of the material harvested from the wild. To date, about 150 medicinal plant species have been reported to be threatened in at least one European country, as a result of over collection from the wild. The uncontrolled harvesting in the Mediterranean area has been identified as a major threat for the populations of the MAPs and it has laid several species in the verge of extinction, such as the rosemary in Sardinia (Mulas and Mulas, 2005), Gentiana acaulis and Arnica
montana in Croatia (Satovi c, 2004). In the latter country, at least 17 MAPs are already threat
ened by overharvesting from the wild (Satovi c, 2004), while other studies reveal that at least 30% of the rare and threatened plants of Greece actually suffer by uncontrolled collections (Krigas et al., 2014a). In fact, overcollection from the wild both in Greece and Cyprus is currently included among the major exogenous threats affecting the survival of the wild populations of these plants, together with grazing, land use changes and urban development (Krigas et al., 2014a; Tsintides et al., 2007). In Greece, as well as in other Balkan countries, Primula spp., Gentiana spp. and Sideritis spp. (10 perennial taxa in Greece) are often overharvested illegally from the wild (Krigas et al., 2014b). For example, the wild populations of the local Balkan endemic Sideritis scardica (Greek mountain tea, “Tsai Olympou”; also cultivated in Greece and Bulgaria) is already assessed as Near Threatened by the IUCN Global Red List and the populations of the Cretan endemic Sideritis syriaca subsp. syriaca (Cretan Mountain tea or Malotira) have severely declined which has been launched recently in Crete (since 2017), leading to a population monitoring program which has been launched recently in Crete. Some wild orchids like Orchis spp., Dactylorrhiza spp. and Anacamptis spp. are also overharvested from the wild in the Balkans and Turkey (Kreziou et al., 2015) and are illegally traded at local or regional scale (their dried tubers are consumed widely as a hot traditional beverage called “salepi,” also known as “salep” in Turkey). Furthermore, there has been an increase in the electronic trade of single-country endemic plants, among which the majority are MAPs and many of them are threatened with extinction e.g., the Greek endemics Origanum dictamnus and O. calcaratum (Krigas et al., 2014b), as well as the Cypriot endemics Origanum cordifolium, Scutellaria cypria subsp. cypria and Sideritis cypria (Krigas et al., 2017). The great majority of MAP species in trade are still wild-collected (Lange and Schippmann, 1997; Srivastava et al., 1996; Xiao, 1991). In the International Standard for Sustainable Wild Collection of Medicinal and Aromatic Plants, it is estimated that this trend is likely to continue over the long term due to numerous factors (Medicinal Plant Specialist Group, 2007), including: • there is not enough experience on cultivation of MAP species and knowledge is limited on their special requirements for development and reproduction as the most of them belong to taxonomy groups different of the major crops, • limited MAP species have the market impact to support high costs of research and experience from collection of the wild, domestication and developing an effective cultivation protocol, • there are geographic areas, where collection of plant material from the wild is traditional and plays important role as a source of income to the local people, in cases there are not enough arable land.

66

4. Sustainable use of mediterranean medicinal-aromatic plants

In comparison with the MAPs traded internationally the cultivated ones are very limited. Over 3000 species can be found in the world market, but greater numbers are sold in local/ regional areas (Lange and Schippmann, 1997). There is a contradiction that prohibition of wild collection could remove a valuable income from sensitive social groups like older people or housewives, especially in developing countries. Replacing wild collection with cultivation may not always be possible and may not have the same financial results (Schippmann et al., 2006). Cultivation of certain MAPs has certainly provided an alternative to wild harvesting, but this has also been severely compromised by inconsistency in the organoleptic properties of the final products, resulting in consumer complaints for loss of typical flavor and aroma of the cultivated material. This is presumably due to the arbitrary selection of clones for cultivation, and the nonstandardized variations in cultivation techniques, as well as the harvesting and postharvesting methods followed by farmers, retailers, and traders. Although there is significant genetic and phenotypic variability present in in situ (wild habitats and protected areas) and in ex situ genetic repositories (botanic gardens, gene banks, seed banks) that could be used as a source of donor material for cultivation, selection, and appropriate breeding (cf. Krigas et al., 2016), this is only partly characterized, while documentation among the different collections lacks uniformity and standardization (Schippmann et al., 2006). Nevertheless, cultivation of MAPs seems to be the only plausible way for sustainable use of phytogenetic resources in the long term, especially regarding the big quantities of MAPs needed to be used as animal feed additives. To date there is a need for checklists of MAPs distributed in different countries with annotation regarding the status of their local wild populations. Such conservation needs have not yet been determined and they are not included in the conservation agendas of the international organizations, public authorities, research foundations and NGOs. Approaches to wild collection of MAPs that engage local, regional, and international collection enterprises and markets along with governments and healthcare providers are urgently needed in the work of conservation and sustainable use of MAP resources. Sustainable conservation schemes bringing new MAPs into fast domestication and cultivation with low cost and high added value are also needed.

From wild to cultivation: conservation and sustainable exploitation of phytogenetic resources of maps The major challenges for sustainable wild collection include: lack of knowledge about sustainable harvest rates and practices, undefined land use rights, and lack of legislative and policy guidance (Blackmore and Rae, 2011). Identifying the conservation benefits and costs of the different production systems for MAPs, guide policies would be needed as to whether ex situ conservation of species should take place (Schippmann et al., 2006). The most suitable way to channel the MAPs raw material to industry is to cultivate the plants. Cultivation also has quality advantages over wild-collecting. Wild-collected plants normally vary in quality and composition due to environmental and genetic differences. In cultivation, such variation is much reduced and managed. The plants can be grown in areas of similar climate and soil, they can be irrigated to increase yields and they can be harvested at the right time. Cultivation also greatly reduces the possibility of misidentification and adulteration (Marshal, 2012).

From wild to cultivation: conservation and sustainable exploitation of phytogenetic resources of maps

67

The Laboratory for the Protection and Evaluation of Native and Floricultural Species (LPENFS) and the Balkan Botanical Garden of Kroussia (BBGK), which belongs to the Institute of Plant Breeding and Genetic Recourses of Hellenic Agricultural Organizatione DEMETER, specializes in ex situ conservation and protection of native plants of Greece. Its approach is innovative at national and international level (Maloupa et al., 2008) and has been developed around the “documentation-conservation-evaluation-sustainable exploitation of phytogenetic resources” axis (Fig. 4.2). Priority in LPENFS-BBGK has been given to the important plant species, i.e., endemic, threatened (critically endangered, endangered and vulnerable), and/or protected plants by national legislation or international conventions, as well as plants with aromatic-pharmaceutical properties, edible parts, beekeeping uses, and/or potential ornamental-horticultural value (Maloupa et al., 2008).

FIGURE 4.2 Concept map of the integrated step-by-step approach for the sustainable exploitation of Medicinal

and Aromatic Plants (MAPs), illustrating the parallel advance of interconnected conservation actions (in situ, ex situ) and sustainable management activities related to MAPs. Prerequisite steps 1 to 3 can lead either to: (A) conservation translocations (neopopulations) of threatened taxa established at protected areas allowing future reintroduction of plant material to wild habitats, or (B) conservation in ex situ facilities, experimental cultivation, development of domestication process, documentation and selection of clones, contract farming and fair trade and development of new products, after phytochemical analyses and comparative evaluation of wild populations and cultivated material.

68

4. Sustainable use of mediterranean medicinal-aromatic plants

Target plants are collected from the natural environment using special collection permit (Fig. 4.2), which is renewed annually by the Greek Ministry of the Environment and Energy (Directorate of Forest Management and Forest Environment). To this end, several specialized botanical expeditions are organized every year in various regions of the Greek territory, to explore the natural populations of specific plant species, during which appropriate propagating material is collected (seeds, cuttings, rhizomes, tubers, live plants). Plant material is transferred to LPENFS where samples are sorted and each one is assigned with a unique, special “accession code”, which accompanies the plant material in all future steps i.e., experiments, maintenance, conservation, reproduction, cultivation, evaluation, and sales. To allow ex situ cultivation of previously unknown MAPs, GIS ecological profiling is suggested (Krigas et al., 2010, 2012). Georeferenced populations of specific MAPs can be linked accordingly with thematic maps in a GIS environment. The thematic maps are produced using open access geodatabases (e.g., Worldclim geodatabase, European Soil geodatabase). The generated linkage for the plant distribution localities across the thematic maps allows the data-mining of species-specific ecological information with point sampling techniques. The ecological information extracted can illustrate quantitatively and qualitatively the soil and climate profiles (temperature and precipitation attributes) prevailing at the localities where the prioritised plants occur in the wild and can be used to outline their species-specific in situ requirements (Krigas et al., 2010, 2012). This information, if properly exploited, can be very useful for the ex situ conservation of prioritized MAPs in man-made habitats such as botanic gardens and field cultivations. Native species collected from the wild are then maintained in one or more of the following: (1) Conservation collection: A living plant collection aiming to conserve populations of rare and threatened species. These collections contribute to the implementation of Article 9 of the Convention on Biological Diversity and Goal 8 of the Global Plant Conservation Strategy and they may potentially be used for pilot reintroduction activities in the natural environment. Currently, this conservation collection of LPENFS-BBGK contains some thousands of plant individuals (more than 540 accession codes) which correspond to more than 300 Greek species and subspecies, thus presenting a unique conservation collection in Greece. (2) Seed Bank: Seeds are gathered annually either from the conservation collection or from wild populations accessed during the organized botanical expeditions and they are kept in the seed bank, at 4 C, after cleaning and dehumidification. Seed bank serves long-term conservation storage, sexual reproduction’s experimentation needs, including breeding procedures, but also seed exchange with other research institutes or botanic gardens working on conservation of plant genetic resources. Currently, more than 1500 accession codes are maintained there, corresponding to more than 800 native Greek species and subspecies. (3) Tissue culture: Some important species (rare, endemic, threatened etc.) and species of research interest are maintained in vitro. In total, 25 accession codes are established in vitro corresponding to 23 Greek taxa. Tissue culture methods are used for rapid propagation of healthy plant material and experimentation under stress conditions (salt tolerance, extreme temperatures etc.).

Research on propagation of native maps: a key for sustainable exploitation

69

(4) Botanic Gardens: In the two botanic gardens which belong to the LPENFS i.e., the Garden of Environmental Awareness (0.8 ha, situated at sea level in the surround area of LPENFS and/or in the BBGK (30 ha, Kilkis perfecture, near the boarders with FUROM, 600 m altitude) more than 760 Greek native taxa are ex situ conserved, thus representing the richest collection in Greek native perennial plants in the country.

Research on propagation of native maps: a key for sustainable exploitation There are usually large gaps in knowledge on cultivation of native MAPs and the treatment of the plant material collected from the wild, although many species have special properties and they are candidate to commercial cultivation for different uses. To understand and simulate the soil/climate requirements of the collected plants (as far as possible) in the anthropogenic environment, the GIS application previously mentioned has proved to be fast enough, leading from experimentation with selected MAPs originally collected from the wild to effective propagation and pilot cultivations in less than 2 years (Krigas et al., 2010; Grigoriadou et al., 2011, 2014; Gkika et al., 2013). These data are also used in the study of the biological cycle of the native plants and the propagation/breeding process. The plant material is used for the development of special species-specific propagation protocols (Grigoriadou et al., 2011, 2014; Tsoktouridis et al., 2013; Sarropoulou and Maloupa, 2015). Mother plants of the collected species are kept in different areas of the nursery in pots or in the field according to their special needs (irrigation, soil, light density, temperature). The methodological approach for the development of their propagation protocols depends on the nature of the species and it is realized in the following ways (Step 2, Fig. 4.2). (1) Sexual reproduction (seeds): Used mainly in annual species, to raise mother plants for conservation purposes and in cases of plants which are difficult or impossible to be reproduced in other ways. Temperature, humidity and light conditions which affect seed germination are studied. Data derived from the GIS application are exploited to modify and improve propagation protocols (Grigoriadou et al., 2014). (2) Asexual reproduction (cuttings, layering, rhizomes, tubers): Applied widely to maintain the genotypes of the selected species. Identical clones of selected species are necessary when it comes to cultivation and production of first material of MAPs. Analytical studies are conducted focusing on factors affecting the physiology of root and shoot formation during propagation procedures and every species is a subject of different research study (Fig. 4.3). (3) Tissue culture: Used for plants species they do not root easily. Propagation is carried out under aseptic conditions, free from pathogens, resulting in the production of large amount of plants in a limited time period, regardless the environmental conditions. Priority is given to specific categories of plants (e.g., threatened species or those with reproduction problems) (Fig. 4.3). This biotechnological approach of propagation provides solution in cases when it is difficult to imitate species-specific environmental conditions such as subalpine species of high altitude habitats (Grigoriadou et al., 2014).

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4. Sustainable use of mediterranean medicinal-aromatic plants

FIGURE 4.3 Successful domestication procedure followed for sustainable exploitation of selected medicinalaromatic plants of Greece. Above: Crithmum maritimum (rock samphire, Apiaceae family) domesticated from wild habitats (left) through propagation with cuttings and acclimatization-experimental cultivation (middle) to large-scale field cultivation (Chalkidiki region, Northern Greece). Below: Thymus sibthorpii (Sibthorp’s wild thyme, Lamiaceae family) domesticated from native wild populations (left) through in vitro propagation and acclimatization (middle) to small-scale field cultivation (Serres region, Northern Greece).

To date, with the aim to exploit the potential of the Greek biodiversity of MAPs, several valuable MAPs which are native to Greece are under investigation or pilot cultivations in order to be sustainably exploited as alternative crops (Table 4.2).

Pilot fields: a step closer to new crops Pilot cultivations of selected MAPs and genotypes are established and special cultivation practices are followed to develop specific cultivation protocols (Fig. 4.2, Step 3, Fig. 4.3). Phenotypic data are recorded together with plant responses to abiotic and biotic factors. Plant material is collected for phytochemical screening and analysis. Qualitative and quantitative assessment of the produced secondary metabolites is conducted. Essential oils, extracts and other products are evaluated to produce other products. Often residues of the MAPs are used as feed additives in collaboration with feedstuff industries. These steps are considered necessary for the sustainable exploitation of MAPs phytogenetic resources in order to create new products and alternative crops (Figs. 4.2 and 4.3). Since 2015, more than 80 speciesesubspecies of the Greek flora with aromatic, medicinal, and/or beekeeping use were selected and they are available for sale to commercial nurseries for further propagation and use from citizens and farmers. All of them are accompanied with their accession code, the scientific name, the origin of the species (collection information), and the special propagation and cultivation protocol developed in LPENFS-BBGK.

References

TABLE 4.2

71

Commercial Lamiaceae medicinal-aromatic plants and examples of other closely related Greek native taxa under investigation or in pilot cultivations in Greece (in bold letters) with restricted natural distribution range. Commercially valuable taxon

Other related Greek native taxa with restricted distribution range

Greek oregano

Origanum vulgare subsp. hirtum

O. onites

Cretan dittany

Origanum dictamnus

O. calcaratum*, O. lirium*, O. scabrum*, O. sipyleum^, O. symes*, O. vetteri*

Spanish oregano

Thymbra capitata

Thymbra calostachya*

Majoram

Origanum majorana

O. microphyllum*

Lemon balm

Melissa officinalis subsp. ofiicinalis

Melissa officinalis subsp. altissima

Greek mountain tea

Sideritis scardica/S. raeseri subsp. raeseri

Sideritis euboea*, S. clandestina subsp. clandestina*, S. clandestina subsp. peloponnesiaca*, S. perfoliata subsp. perfoliata^, S. perfoliata subsp. athoa^, S. raeseri susbp. attica*, S. sipylea^, S. syriaca subsp. syriaca*

Thyme

Thymus vulgaris

Thymus degenii˅˅, T. holosericeous*, T. longicaulus subsp. chaubardii˅˅, T. plasonii*, T. sibthorpii˅˅, T. sipyleus^, T. striatus˅˅, T. thracicus

Winter savory

Satureja montana subsp. montana

Satoureja athoa^, S. cuneifolia˅˅, S. hellenica*, S. horvatii subsp. macrophylla*, S. icarica*, S. montana subsp. macedonica*, S. paranassica subsp. parnassica*, S. pilosa˅, S. spinosa˅

Commercial name

*, Greek endemic;^, East Mediterranean endemic; ˅, Local Balkan endemic or Balkan endemic extending to Anatolia in Turkey or to Italy; no indication, endemic to the Mediterranean Region.

References Azmir, J., Zaidul, I.S.M., Rahman, M.M., Sharif, K.M., Mohamed, A., Sahena, F., 2013. Techniques for extraction of bioactive compounds from plant materials: a review. J. Food Eng. 117, 426e436. Barata, A.M., Rocha, F., Lopes, V., Carvalho, A.M., 2016. Conservation and sustainable uses of medicinal and aromatic plants genetic resources on the worldwide for human welfare. Ind. Crops Prod. 88, 8e11. Blackmore, S., Rae, M.G.D., 2011. Strengthening the scientific contribution of botanic gardens to the second phase of the Global Strategy for Plant Conservation. Bot. J. Linn. Soc. 166 (3), 267e281. Bradshaw, C.J.A., Sodhi, N.S., Brook, B.W., 2009. Tropical turmoil: a biodiversity tragedy in progress. Front. Ecol. Environ. 7, 79e87. Bruni, R., Sacchetti, G., 2009. Factors affecting polyphenol biosynthesis in wild and field grown St. John’s Wort (Hypericum perforatum L. Hypericaceae/Guttiferae). Molecules 14, 682e725. CBI Market Survey, 2009. The Spices and Herbs Market in EU 2009. https://www.scribd.com/document/75195767/ The-Spices-and-Herbs-Market-in-the-Eu-2009. Dimopoulos, P., Raus, T., Bergmeier, E., Constantinidis, T., Iatrou, G., Kokkini, S., Strid, A., Tzanoudakis, D., 2013. Vascular Plants of Greece: An Annotated Checklist. Berlin: Botanic Garden and Botanical Museum Berlin-Dahlem, Athens: Hellenic Botanical Society. [Englera 31]. Dimopoulos, P., Raus, T., Bergmeie, r E., Constantinidis, T., Iatrou, G., Kokkini, S., Strid, A., Tzanoudakis, D., 2016. Vascular plants of Greece: an annotated checklist. Supplement. Willdenowia 46, 301e347.

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Dragusanu, R., Giovannucci, D., Nunn, N., 2014. The economics of fair trade. J. Econ. Perspect. 28, 217e236. Ekor, M., 2013. The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front. Pharmacol. 4, 177. Georghiou, K., Delipetrou, P., 2010. Patterns and traits of the endemic plants of Greece. Botanical Journal of the Linnaean Society 162 (2), 130e422. Gkika, P.I., Krigas, N., Menexes, G., Eleftherohorinos, I.G., Maloupa, E., 2013. Conservation of the rare Erysimum naxense Snogerup and the threatened Erysimum krendlii Polatschek: effect of temperature and light on seed germination. Open Life Sciences (Central European Journal of Biology) 8 (12), 1194e1203. Góra, J., Lis, A., Kula, J., Staniszewska, M., Wołoszyn, A., 2002. Chemical composition variability of essential oils in the ontogenesis of some plants. Flavour Fragrance J. 17, 445e451. Greuter, W., 1991. Botanical diversity, endemism, rarity, and extinction in the Mediterranean area: an analysis based on the published volumes of Med-Checklist. Botanika Chronika 10, 63e79. Grigoriadou, K., Krigas, N., Maloupa, E., 2011. GIS-facilitated in vitro propagation and ex situ conservation of Achillea occulta. Plant Cell Tissue Organ Cult. 107, 531e540. Grigoriadou, K., Krigas, N., Maloupa, E., 2014. GIS-facilitated ex situ conservation of the rare Greek endemic Campanula incurva Aucher: seed germination requirements and effect of growth regulators on in vitro proliferation and rooting. Plant Biosyst. 14 (6), 1169e1178. Heywood, V.H., 1999. The Mediterranean region a major centre of plant diversity. In: Heywood, V.H., Skoula, M. (Eds.), Wild Food and Non-food Plants: Information Networking. CIHEAM-IAMC, Chania, pp. 5e13. Karousou, R., Kokkini, S., 1997. Distribution and clinal variation of Salvia fruticosa Mill. (Labiatae) on the island of Crete (Greece). Willdenowia 27, 113e120. Karousou, R., Vokou, D., Kokkini, S., 1998. Variation of Salvia fruticosa essential oils on the island of Crete (Greece). Bot. Acta 111, 250e254. Karousou, R., Efstathiou, C., Lazari, D., 2012. Chemical diversity of wild growing Origanum majorana in Cyprus. Chem. Biodivers. 9 (10), 2210e2217. Kokkini, S., Karousou, R., Dardioti, A., Krigas, N., Lanaras, T., 1997. Autumn essential oils of Greek oregano. Phytochemistry 44, 883e886. Kokkini, S., Vokou, D., Karousou, R., 1991. Morphological and chemical variation of Origanum vulgare L. in Greece. Botanica Chronika 10, 337e346. Kontogiorgis, C., Ntella, M., Mpompou, L., Karalaaki, F., Papadopoulos, A., Hadjipavlou-Litina, D., Lazari, D., 2016a. Study of the antioxidant activity of Thymus sibthorpii Bentham (Lamiaceae). Journal of Enzyme Inhibition and Medicinal Chemistry 31 (S4), 154e159. Kontogiorgis, C., Mpompou, E.M., Papajani-Toska, V., Hadjipavlou-Litina, D., Lazari, D., 2016b. Chemical composition and antioxidant activity of the essential oils isolated from Greek and Albanian Thymus species. J. Chem. Pharm. Res. 8 (9), 180e184. Kreziou, A., de Boer, H., Gravendee, B., 2016. Harvesting of salep orchids in north-western Greece continues to threaten natural populations. Oryx 50 (3), 393e396. Krigas, N., Mouflis, G., Grigoriadou, K., Maloupa, E., 2010. Conservation of important plants from the Ionian islands at the Balkan Botanic Garden of Kroussia, N Greece: linking with GIS the in situ collection data with plant propagation and the ex situ cultivation. Biodivers. Conserv. 19, 3583e3603. Krigas, N., Papadimitriou, K., Mazaris, A.D., 2012. GIS and ex situ plant conservation. In: Alam, B.M. (Ed.), Application of Geographic Information Systems. InTechopen.com, Rijeka, Croatia, pp. 153e174. Krigas, N., Bandi, A., Vokou, D., 2014a. Identified threats and proposed protection measures for the Rare and Threatened Plants of Greece: classification, ranking and mismatches. In: Krigas, N., Tsoktouridis, G., Cook, C.M., Mylona, P., Maloupa, E. (Eds.), Botanic Gardens in a Changing World: Insights into Eurogard VI. Botanic Gardens Conservation International, pp. 43e52. http://www.botanicgardens.eu/eurogard/eurogard6/krigas_ threatened.pdf. Krigas, N., Mendeli, V., Vokou, D., 2014b. The electronic trade in Greek endemic plants: biodiversity, commercial and legal aspects. Econ. Bot. 68 (1), 85e95. Krigas, N., Mendeli, V., Vokou, D., 2016. Analysis of the ex situ conservation of the Greek endemic flora at national European and global scales and of its effectiveness in meeting GSPC Target 8. Plant Biosyst. 150 (3), 573e582. Krigas, N., Mendeli, V., Chrysanthou, P., Vokou, D., 2017. The electronic trade in endemic plants of Cyprus through the Internet. Plant Biosyst. 151 (3), 383e393.

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Krishnaiah, D., Sarbatly, R., Nithyanandam, R., 2011. A review of the antioxidant potential of medicinal plant species. Food Bioprod. Process. 89 (3), 217e233. Lange, D., Schippmann, U., 1997. Trade Survey of Medicinal Plants in Germany: A Contribution to International Plant Species Conservation. BundesamtfürNaturschutz, Bonn. Lazari, D.M., Matta, M.K., Sylignaki, G.I., Panagiotidis, C.A., 2006. Evaluation of the antiherpetical activities of Sideritis perfoliata subsp. perfoliata (Lamiaceae). Planta Med. 72 (11), 90. Lubbe, A., Verpoorte, R., 2011. Cultivation of medicinal and aromatic plants for specialty industrial materials. Ind. Crops Prod. 34, 785e801. Lucera, A., Costa, C., Conte, A., Del Nobile, M.A., 2012. Food applications of natural antimicrobial compounds. Front. Microbiol. 3, 287. Maloupa, E., Krigas, N., Grigoriadou, K., Lazari, D., Tsoktouridis, G., 2008. Conservation strategies for native plant species and their sustainable exploitation: case of the Balkan Botanic Garden of Kroussia, N. Greece. In: Teixeira da Silva, J.A. (Ed.), Floriculture Ornamental Plant Biotechnology: Advances and Topical Issues, vol. 4. Global Science Books, Isleworth, UK, pp. 37e56. Marshall, E., 2012. Health and Wealth from Medicinal Aromatic Plants. FAO. http://www.fao.org/3/a-i2473e.pdf. Matta, M.K., Paltatzidou, K., Triantafyllidou, H., Lazari, D.M., Karioti, A., Skaltsa, H., Panagiotidis, C.A., 2007. Evaluation of the anti-herpes simplex virus activity of Thymus longicaulis L. (Lamiaceae). Planta Med. 73 (9), 988e998. Medail, F., Quézel, P., 1999. Biodiversity hotspots in the Mediterranean Basin: setting global conservation priorities. Conserv. Biol. 13 (6), 1510e1513. Medicinal Plant Specialist Group, 2007. International Standard for Sustainable Wild Collection of Medicinal and Aromatic Plants (ISSC-MAP). Version 1.0. Bundesamtfür Naturschutz (BfN). MPSG/SSC/IUCN, WWF Germany, and TRAFFIC, Bonn, Gland, Frankfurt, and Cambridge (BfN-Skripten 195). Misra, A., 2009. Studies on biochemical and physiological aspects in relation to phyto-medicinal qualities and efficacy of the active ingredients during the handling, cultivation and harvesting of the medicinal plants. J. Med. Plants Res. 3 (13), 1140e1146. Moore, B.D., Andrew, R.L., Kulheim, C., Foley, W.J., 2014. Explaining intraspecific diversity in plant secondary metabolites in an ecological context. New Phytol. 201, 733e750. Mulas, M., Mulas, G., 2005. Cultivar selection from rosemary (Rosmarinus officinalis L.) spontaneous populations in the Mediterranean area. Acta Horticulturae 676, 127e133. Myers, N., Mittermeier, R.A., Mittermeier, C.G., da Fonseca, G.A.B., Kent, J., 2000. Biodiversity hotspots for conservation priorities. Nature 403, 853e858. Papaioannou, P., Lazari, D., Karioti, A., Souleles, C., Heilmann, J., Hadjipavlou-Litina, D., Skaltsa, H., 2007. Phenolic compounds with antioxidant activity from Anthemis tinctoria L. subsp. tinctoria var. pallida DC. (Asteraceae). Z. Naturforsch. C Biosci. 62c, 326e330. Porter, E., Kramer, M., 2011. The big idea: creating shared value. Harv. Bus. Rev. 1, 2e17. Sarropoulou, V., Maloupa, E., 2015. Effect of exogenous dikegulac on in vitro shoot proliferation of Sideritis raeseri L. e Greek mountain tea species. Agric. For. 61 (4), 153e159.
Satovi c, Z., 2004. Legal protection, conservation and cultivation of medicinal and aromatic plants in Croatia. In: International Plant Genetic Resources Institute (Ed.), Report of a Working Group on Medicinal and Aromatic Plants, pp. 34e38. Rome, Italy. Schippmann, U.D., Leaman, B., Cunnigham, D., 2006. Cultivation and wild collection of medicinal and aromatic plants under sustainability aspects. In: Bogers, R.J., Craker, L.E., Lange, D. (Eds.), Medicinal and Aromatic Plants. Springer, Dordrecht. http://library.wur.nl/frontis/medicinal_aromatic_plants/05_schipmann.pdf. Srivastava, J., Lambert, J., Vietmeyer, N., 1996. Medicinal Plants: An Expanding Role in Development. World Bank, Washington, DC. World Bank Technical Paper 320. St John, F.A.V., Edwards-Jones, G., Jones, J.P.G., 2012. Opinions of the public, conservationists and magistrates on sentencing wildlife trade crimes in the UK. Environ. Conserv. 39, 154e161. Tsintides, T., Christodoulou, C.S., Delipetrou, P., Georghiou, K. (Eds.), 2007. The Red Data Book of the Flora of Cyprus. Cyprus Forest Association, Lefkosia, Cyprus. Tsoktouridis, G., Grigoriadou, K., Doua, E., Nikolaidou, A., Menexes, G., Maloupa, E., 2013. In vitro shoot proliferation, rooting, and acclimatization of four diverse Dianthus petraeus W. ET K. genotypes using TDZ, NAA, and IBA. Propagation of Ornamental Plants 13 (4), 181e188.

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Vartholomatos, E., Lazari, D., Alexiou, G., Tsiftsoglou, O., Galani, V., Markopoulos, G., Kyritsis, A., 2016. Study on the effect of Polypodin-B in cell lines of glioblastoma. In: 14th Panhellenic Congress of Clinical Chemistry and Clinical Biochemistry Society, 29/9-1/10/2016, Ioannina, Greece. Walter, S., 2002. Certification and benefit-sharing mechanisms in the field of non-wood forest products: an overview. Medicinal Plant Conservation 8, 3e9. Witkowski, T.H., 2014. Fair Trade Marketing: an alternative system for globalization and development. J. Mark. Theory Pract. 13, 22e33. World Health Organization (WHO), 2008. Traditional Medicine. Fact Sheet No. 134. Xiao, P.G., 1991. The Chinese approach to medicinal plants e their utilization and conservation. In: Akerle, O., Heywood, V., Synge, H. (Eds.), Conservation of Medicinal Plants. Cambridge University Press, Cambridge, UK.

C H A P T E R

5 Aromatic plants and their extracts pharmacokinetics and in vitro/in vivo mechanisms of action 1  Ivana Cabarkapa , Nikola Puvaca2, Sanja Popovic1,  Dusica Colovi c1, Ljiljana Kostadinovic3, Eleanor Karp Tatham4, Jovanka Levic1 1

University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia; 2University Business Academy, Faculty of Economics and Engineering Management, Department of Engineering Management in Biotechnology, Novi Sad, Serbia; 3Planet Fresh d.o.o., Niksic, Montenegro; 4 University of London, Royal Veterinary College, London, United Kingdom O U T L I N E Introduction

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Antimicrobial effects of aromatic plants and their EOs

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Acknowledgments

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Antioxidant effects of aromatic plants

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References

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Introduction The last sixty years usage of antibiotics in food animal nutrition as an antimicrobial growth enhancer were justified for the improvement of an animal’s productive performance, prevention, and occurrence of possible diseases. High usage of antibiotics as growth promoters in

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00005-4

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Copyright © 2020 Elsevier Inc. All rights reserved.

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5. Aromatic plants and their extracts pharmacokinetics and in vitro/in vivo mechanisms of action

animal nutrition lead to bacterial resistance to antibiotics. In addition, due to the presence of residues of antibiotic drugs in food originating from animals (Bampidis et al., 2005; Popovic et al., 2016; Puvaca et al., 2013; Stanacev et al., 2012), the goal is set to remove antibiotics as a growth enhancer from food-animal diets. In 2006, the EU, banned use of antibiotics as a growth promoters in accordance to regulation EC 1831 created in 2003. As a result, the demand for alternative natural products or replacement additives to antibiotics that can be used in prophylactic purposes and as a growth promoting agents has become of great importance. Animal production industries tending to improve animal production performances and minimize economic losses as well as ensuring the safety of products intended for human consumption, such as meat, table eggs, and milk, through the control of food-borne pathogens (Kostadinovic and Levic, 2018; Levic et al., 2011). The beneficial potential of various microbes and bioactive compounds has been highlighted in enhancing animal performance and health. Examples include probiotics, prebiotics, enzymes, organic acids and phytogenic compounds (Levic et al., 2011; Puvaca et al., 2015; Puvaca, 2018). Parts of medicinal, aromatic, and spice plants (garlic, oregano, thyme, rosemary, coriander, and cinnamon) such as seeds, roots, and leaves, or their respective extracts in the form of essential oils (EOs) are commonly named as phytogenic additives or phytobiotics (Windisch et al., 2008). Bioactive compounds such as carvacrol, thymol, cineole, linalool, anethole, allicin, capsaicin, allyl isothiocyanate, and piperine represents the main active ingredients which act beneficially and makes this plants as a very important and valuable asset of the nature itself. During the last three decades different test and experiments in vitro and in vivo was conducted with aim to investigate antibacterial and antioxidant properties which today has been proved, but the lack of its mode of action is not jet clearly defined (Burt, 2004; Puvaca et al., 2018; Windisch et al., 2008). In addition to all of these properties, an investigation conducted by Burt (2004) confirmed also their antitoxigenic, antiviral, but also an antiparasitic, antiacaricidal, and insecticidal properties. Currently, there is a rising interest in EOs for food animal nutrition, as they have a much higher biological activity compared to the raw material they were extracted from. An issue with EOs is their complexity of mixtures of plant bioactive compounds along with variable chemical composition and concentrations (Puvaca, 2018). EOs consist basically of two classes of compounds, the terpenes and phenylpropenes (Lee et al., 2004). There is a dependency regarding the content and composition of EOs on the interaction of many factors in the plant raw material and the EO production process. For example, plant species and growth stage, the environment, agricultural practices, and growing region will all affect EOs content and composition of the raw extract (Daferera et al., 2003; Mert, 2002; Popovic et al., 2018). As a result, the raw materials for EOs production contain considerable variation, and the same applies to the resulting EO products (Levic et al., 2011). The processing methods applied in EOs production, such as hydrodistillation or solvent extraction, can have a significant effect on the amount and composition of the extracted oil (Russo et al., 1998). During the past two decades, phytobiotics have been in focus of scientific investigation regarding their potential role as natural alternatives to antibiotic growth enhancer in animal food nutrition. During that period, phytobiotics have become popular as natural feed additives in poultry, pig, and cow diets, and in aquaculture production as well.

Antimicrobial effects of aromatic plants and their EOs

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Antimicrobial effects of aromatic plants and their EOs Essential oils are aromatic and volatile liquids derived from different part of plant material, such as flowers, roots, bark, leaves, seeds, peel, fruits, wood, and whole plants, mainly by steam distillation (Roldán et al., 2010). EOs represents a complex mixture of low molecular weight organic compounds with large differences in antimicrobial activity. In correspondence to their chemical structure, the active compounds can be divided into diverse groups: alcohols, phenols, aldehydes, ketones, hydrocarbons, and ethers. The concentration of components in EOs is quite varied and major components may represent up to 85% of the EOs,  while the remaining components can be found in traces (Cabarkapa et al., 2011). The major components of EOs are usually responsible for their biological properties. The chemical compositions of EO in aromatic plants are subjected to seasonal variations and depend on phenophase of the plant. In addition to phenophase, chemical composition, and content of EO could be under the influence of environmental factors (climate and habitat conditions), postharvest techniques (drying methods, the technique of extraction, distillation time), and quan tification methods (Bakkali et al., 2008; Cabarkapa et al., 2016). The largest antimicrobial effect exhibit phenols which have been mainly present in the highest percentage in the EOs, followed by alcohols, aldehydes, ketones, and ethers, while the antibacterial effect of hydrocarbons is low (Dorman and Deans, 2000). Despite the fact that antimicrobial effect of EOs is mainly attributed to phenols, the influence of components present in traces should not be disregarded due to their potential interactions which may have an influence on antimicrobial activity. A number of studies have been shown that EO or a mixture of EO components may have a greater antimicrobial effect in comparison  to the individual components of EOs (Bassolé et al., 2010; Cabarkapa et al., 2016; Pei et al., 2009; Zhou et al., 2007). These studies suggest that the antimicrobial activity of the EOs is a result of interactions between different classes of compounds present in the EO, although, in some research, the activity of the EO is closely associated with the activity of the main  components of EO (Burt, 2004; Cabarkapa et al., 2016). Interactions between different classes of compounds present in the EO can be described as an additive, antagonistic, and synergistic. According to Bassolé and Juliani (2012), the additive effect usually occurs in case of the combined effect of the components is alike or equal to the sum of the effects each of the single compound. Synergism is registered when the activity of the combined substances are more efficient than the sum of the individual activities. The antagonistic effect can be recorded when the activity of components in combination are lower in comparison with their individual activity. Data of antimicrobial activity of EOs and their active components  tested in vitro summarized by Cabarkapa et al. (2016) are presented in Tables 5.1 and 5.2. Burt (2004) assumed that one of the principles of antimicrobial effect of EOs is based on their hydrophobic feature, through to easier incorporation within the lipid bilayer cellular membranes of bacteria causing disturbances in its structure, permeability, and flow of protons with a decline in membrane potential, intracellular pH, and synthesis of ATP. Sikkema et al. (1995) suggested that when osmotic cell equilibrium is disturbed in this manner, the secondary effect is cell death. This mode of action of EOs has been confirmed by electron microscopy in Escherichia coli (Burt and Reinders, 2003).

78 TABLE 5.1

5. Aromatic plants and their extracts pharmacokinetics and in vitro/in vivo mechanisms of action

Selected MICs of essential oils tested in vitro against different bacterial pathogens.

Plant from which EO is derived

Species of bacteria

MIC, approximate range (mL/mLL1)a

Origanum heracleoticum

Salmonella enteritidis

0.025e0.625

Roldán et al., 2010; Stankovic et al., 2011

Origanum vulgare

Salmonella typhimurium

1.2e0.312

Hammer et al., 1999; Derwich et al., 2010; Roldán et al., 2010

Ocimum basilicum

Escherichia coli Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa

1.6e2.6 0.9e1.5 0.8e1.4 1.7e2.3

Hussain et al. (2008)

Cinnamomum spp.

Escherichia coli Staphylococcus aureus

1.0 1.0

Zhang et al., 2016

Rosmarinus officinalis

Escherichia coli Salmonella enteritidis Salmonella typhimurium Salmonella choleraesuis Staphylococcus aureus

1.25 0.63 0.63 0.63 0.63

Saric et al., 2014

Syzygium aromaticum

Staphylococcus aureus

0.39

Budri et al. (2016)

Reference

a In the references MICs have been reported in the units mg/mL 1 and mg/mL 1. For ease of comparison these have been converted to mL/ mL 1, whereby it was assumed that EOs have the same density as water.

TABLE 5.2

Selected MICs of essential oil components tested in vitro against food borne pathogens.

Essential oil component

Species of bacteria

MIC, approximate range (mL/mLL1)a

Thymol

Salmonella enteritidis Staphylococcus aureus Escherichia coli

0.2 0.31 0.4e5.0

Trombetta et al., 2005; Lu and Wu, 2010

Carvacrol

Salmonella typhimurium Escherichia coli

0.2 0.4

Pei et al., 2009; Lu and Wu, 2010

Eugenol

Escherichia coli Staphylococcus aureus

1.6 0.2

Pei et al., 2009; Budri et al., 2016

Cinnamaldehyde

Escherichia coli Staphylococcus aureus

0.4 0.19

Pei et al., 2009; Budri et al., 2016

Linalool

Escherichia coli Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa Salmonella choleraesuis

0.4 0.9 0.3 0.9 0.4

Kubo et al., 2004; Hussain et al., 2008

References

a In the references MICs have been reported in the units mg/mL 1 and mg/mL 1. For ease of comparison these have been converted to mL/mL 1, whereby it was assumed that essential oil components have the same density as water.

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The mechanism of antimicrobial effect of EOs was the subject of numerous studies during the decades; however, the connection between antimicrobial activity of EOs and their chemical structure is still not completely clarified. Bearing in mind that constituent of EOs represents a large number of different chemical compounds; the assumption is that the antimicrobial activity of EOs is not based on just one specific mechanism. Previous studies have suggested and described several target positions and their mechanisms of antimicrobial action, such as degradation of the cell wall, damage to membrane proteins, damage to cytoplasmic membrane, leakage of cell contents, coagulation of cytoplasm, and depletion of the  proton motive force (Burt, 2004; Cabarkapa et al., 2012). Taking into consideration that most of antimicrobial effect is an exhibit by phenols, considerable research is directed toward testing their antimicrobial activity and mechanisms of action. Furthermore, it has been supposed that most of antimicrobial components of the EOs represent phenolic compounds, and their mechanism of action is similar to phenols. It is known that phenolic compounds show antimicrobial activity in a concentration-dependent manner; at lower concentrations, they may inhibit enzyme activity while at high concentration they usually cause protein denaturation. Phenols also can cause alteration of bacterial cell membrane permeability leading to the loss of macromolecules. Reduction of macromolecules negatively effects the microbial growth and energy production, provoking cell death (Carson et al., 2002). Monoterpenoid phenol, carvacrol has a hydroxyl group (eOH) and delocalized electron system that contributes to its antimicrobial effect. The functional principle of carvacrol, one of the major components of oregano and thyme oils, appears to have received the most attention from researchers. Thymol, similarly to carvacrol, has eOH at a different location on the phenolic ring. Both compounds have an influence on the permeability of cell membrane (Lambert et al., 2001). In comparison antimicrobial effect of carvacrol with his isomer thymol, which also possesses eOH group, with the system of the delocalized electrons (the double bond) but in meta position, there are no established differences in their antimicrobial activity. In the case of the methyl ester of carvacrol comprising the ethyl ester instead of the eOH, and pcymene which lacks eOH, antimicrobial effect was not noticed. Importance of the eOHs and system of delocalized electrons can be viewed at a lower activity of menthol with respect to carvacrol. Menthol possesses eOH in its ring, but its antimicrobial activity was not expressed. It is postulated that the absence of antimicrobial activity arises from the lack of a system of delocalized electrons (double bonds), due to its eOH that is inability to dismiss protons (Ultee et al., 2002). These support the findings from Veldhuizen et al. (2006), who were comparing the activity of carvacrol and 2-amino-p-cymene, an analog of carvacrol, to confirm the significance of the eOHs on the activity of carvacrol. This study discovered threefold higher activity of carvacrol compared to 2-amino-p-cymene and showed that the -OH group influenced on the antimicrobial activity of carvacrol. A number of previous studies have shown that phytophenols have an effect on proteins of the cytoplasmic membrane and transport of proteins through the channels. The interaction between these compounds and membrane proteins leads to reduction and inhibition of their activity. Therefore, the next possible mechanisms have been suggested. The first mechanism occurs as a result of distortion of lipid bilayer due to the accumulation of lipophilic molecules thus disrupting the lipid-protein interactions.

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A second mechanism occurs as a result of the direct interaction of a lipophilic component with a hydrophobic part of the protein (Burt, 2004). Hydrocarbon monoterpenes p-cymene and g-terpinene are biochemical precursors of carvacrol and thymol that do not exhibit antimicrobial activity, but it has been noticed that their presence enhances the antimicrobial effect of carvacrol and thymol (Burt et al., 2005). Comparing to the treatment of bacteria with carvacrol, p-cymene leads to a stronger swelling of the cytoplasmic membrane. As the antimicrobial activity of the mixture is greater than the antimicrobial effect of the individual components, it is clear that between these two compounds exists synergism. However, it should also be noted that p-cymene appears to incorporate into cytoplasmic membrane causing its swelling and facilitates transport of carvacrol across cytoplasmic membrane. The result of the joint action of two compounds leads to destabilization of cytoplasmic membrane, fall of membrane potential, reduction of intracellular pH and disruption of ATP synthesis, which together contribute cell death. Ultee et al. (2000) showed that p-cymene in the absence of carvacrol, except swelling of the cytoplasmic membrane, causes only a slight decrease of membrane potential. Phenylpropenes, eugenol, and cinnamaldehyde are synthesized from their precursor phenylalanine. Eugenol showed antibacterial activity, similarly to thymol and carvacrol. After the exposition of bacterial cells to eugenol, they have incorporated into the cellular membrane and alter the surface and structural proteins (Budri et al., 2016). It’s believed that both compounds inhibit cellular metabolism and potentially serve as ATPase inhibitors. Cinnamaldehyde may also act through membrane disruption (Gill and Holley, 2006; Hyldgaard et al., 2012). According to another theory deemed that antibacterial potential of EOs includes the inhibition of enzymatic synthesis or activity required for energy generation, disruption in the generation process of ATP, or depletion of ATP already present within the cell (Helander et al., 1998). From a food safety point of view, it is important to note that the majority of EOs is classified as Generally Recognized As Safe (GRAS).

Antioxidant effects of aromatic plants Many aromatic plants and their EOs are known for their antioxidative properties, based mainly on phenolic compounds in the oil (Kostadinovic, 2013) or in other phytochemical fractions. Some nonphenolic substances may also show considerable antioxidative potential (Puvaca, 2016; Puvaca et al., 2018). Mostly phenolic compounds are the main contributors in protection of feed peroxidation process, acting in a way of partial substitute and use of vitamin E acetate (C31H52O3) with related compounds as feed additives or preservatives. EOs may also affect lipid metabolism in the animal body. For example, a dietary supplement of thyme essential oil or thymol to laboratory animals showed beneficial effects on the antioxidative enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as on polyunsaturated fatty acid (PUFA) composition in various tissues. Experimental laboratory animals receiving these supplements had higher enzyme levels and higher concentrations of PUFAs in phospholipids of the brain compared to untreated controls treatments (Youdim and Deans, 2000). Oregano EO added in doses of 0.005%e0.01% to the diet of chickens exerted an antioxidant effect in the white and red meat (Popovic et al., 2016), while the pattern of fatty acids (FA) of the abdominal fat of chicken was also altered by oregano EO

Effects on performance, digestibility, and intestinal functions in animals

81

dietary addition (Lee et al., 2003). Also, the same research has shown a positive influence from dietary carvacrol additions to chicken nutrition, which have led to lower concentration of total triglycerides in blood plasma. In food-producing animals, such effects are important for product quality: they can improve the dietary value and lead to better oxidative stability and longer shelf life of fat which brings longer shelf life to meat and table eggs (Nikolova and Kocevski, 2018; Popovic et al., 2018). Oxidation of meat and membrane phospholipids from chickens fed with 0.05% diet Rosmarinus officinalis and Salvia officinalis extracts was significantly lower after nine days refrigerated storage compared to 0.02% vitamin E and control treatments, respectively (Botsoglou et al., 1997; Govaris et al., 2005). The concentration of total cholesterol oxidation products was also reduced, and a similar trend was observed in microsomal fraction isolates, in which the rate of metmyoglobin/hydrogen peroxideecatalyzed lipid peroxidation was lower in birds receiving aromatic plants than in controls fed on basal diet according the research of Lopez-Bote et al. (1998). A diet containing 1% Salvia officinalis or Origanum vulgare crude herbal drug, either alone or in a 1:1 mixture, was tested with pigs. Raw belly bacon produced from animals fed with only oregano as the additive showed significantly improved stability and lower cholesterol oxide content compared to controls after 34 weeks of storage (Bauer, 2001). Sage as the additive, in contrast, had a much lower impact. The effect of dietary thyme in the concentration of 3% in form of ground herb of Thymus vulgaris as a feed additive for laying hens on the oxidative stability of eggs over 60 days storage in the refrigerator was evaluated by Botsoglou et al. (1997). Dietary addition of thyme to laying hens led to significant a reduction of lipid yolk oxidation, while some comparative investigation of different antioxidants activity, added to yolk suggested that thymol alone could not be responsible for the oxidative resistance of table eggs from thyme added to laying hen’s daily diet. The fact that substances other than the EO components such as rosmarinic acid, carnosol, and carnosic acid, are at least as important as antioxidants were clearly demonstrated by feeding experiments with distillation residues on small ruminants (Jordán et al., 2007). High antioxidant stability of the meat obtained from goats and sheep fed with dietary rosemary or thyme for several months showed high concentration of polyphenolic antioxidants in the meats as well as high concentration of polyphenolic antioxidants in the milk. During the experiment lower susceptibility of oxidative stress in suckling goat kids and increased concentration of polyphenols with antioxidant capacity in the cheese was also recorded by Martinez et al. (2007) and Monino et al. (2007). Results obtained by the EU funded research and development project (Franz et al., 2008) demonstrated that residues of several aromatic plants, after distillation, could be useful in animal feeding as nutritional additives. Traditionally known in this respect is the use of dried parsley leaves in dairy cows to enhance milk production. However, appropriate caution is critical, since EOs and other compounds, such as phenolic substances, could influence the flavor and taste of the products or even cause an “off flavor.” The taste and flavor of the animal product can also be positively affected by aromatic plants and EOs (Puvaca et al., 2016).

Effects on performance, digestibility, and intestinal functions in animals Many aromatic plants and EOs are used for improving the flavor and palatability of feed or to affect other parameters in animal production (Puvaca, 2016; Puvaca et al., 2013). Large

82 TABLE 5.3

5. Aromatic plants and their extracts pharmacokinetics and in vitro/in vivo mechanisms of action

Effect of selected phytogenic additives on the broiler chickens performances (Puvaca et al., 2015, Popovic et al., 2016).

Feed additive

Dietary dose, %

Body weight, g

Feed conversion ratio, kg/kg

European broiler index

Herbs and spices in powder form Garlic

0.5

2371.0

1.8

295

Black pepper

0.5

2077.0

1.9

244

Red hot pepper

0.5

2461.0

1.9

299

Mix of oregano, thyme and rosemary

0.05

2087.8

1.55

298.2

Mix of oregano, thyme and rosemary

0.1

2096.0

1.56

289.2

Essential oils

numbers of feeding trials have been performed with such additives, but most of the results are reduced to the growth-promoting parameters such as feed intake, weight gain, and feed conversion ratio (Table 5.3). In poultry, most studies have shown no significant change in feed intake caused by aromatic plants or EO additives, although growth was often enhanced and the feed conversion rate improved (Table 5.3). Since poultry are known to adjust feed intake according to energy demand, the feed conversion rate is, therefore, a better parameter of the effects of growth promoters. Published results are, however, contradictory. Addition of carvacrol or thymol, carvacrol mixture in concentration of 0.02% to broiler chicken nutrition led to a decreased feed intake, lower weight gain, and increased feed conversion rate (Halle et al., 2001). Addition of oregano herb in quantities of 2e20 g/kg feed or of oregano oil (100e1000 mg/kg feed) resulted, in contrast, in all cases in better performance of broiler chicks (Halle, 2004). Another trials conducted by Westendarp et al. (2006) with pure active substances derived from oregano EO and thyme EO in approximately 50 (carvacrol) or 25 (terpinene, p-cymene) mg/kg did not show any significant effect (Haselmeyer et al., 2015). studied the effect of thymol in four concentrations from 0.1% to 1.0% as a feed additive in broilers. No significant difference in performance was observed over the experimental period of 35 days. Turkeys fed with 1.25e3.75 g/kg dried oregano leaves showed, in contrast, a clearly improved feed conversion rate according to the investigation of Bampidis et al. (2005). An investigation by Denli (2004) showed that the addition of 60 ppm thyme EO to the diet of quails resulted in significantly higher body weight gain and better feed efficiency as well as decreased abdominal fat weight. Similarly, the addition of 250 mg/kg hops to the diet of broilers resulted in significant improvements in feed conversion ratio and feed efficiency at all ages (Cornelison et al., 2006). The different results are due to the type and origin of the EO or aromatic plants, the quantity added to the feed, and the environmental conditions of the trial. Investigations under practical conditions of large scale animal production have shown better responses to the

Effects on performance, digestibility, and intestinal functions in animals

FIGURE 5.1

83

Effect of EO on the average daily gain and feed intake of rearing piglet’s diet (Puvaca, 2016).

treatment (Kyriakis et al., 1998; Tsinas et al., 1998) compared to studies completed under controlled experimental conditions with a higher level of hygiene (Gollnisch et al., 2001) as shown in Fig. 5.1. Furthermore, in large-scale pigs fattening facilities, supplementation with 250e500 mg oregano oil/kg of complete feed showed improvements on animal production parameters of up to 20% and a heavy decrease in mortality of weaning piglets (Kyriakis et al., 1998). On the contrary, no significant differences were observed in an experimental station between control and the addition of either antibiotics or several EOs (Gollnisch et al., 2001). Medicinal and aromatic plants, spices, and herbs and their EOs are often claimed to improve the flavor and palatability of feed, thus enhancing animal performance. Indeed, there are reports of higher feed intake of piglets through flavoring additives, with evident decrease in feed conversion ratio and improved feed digestibility. However, an increase in feed intake is commonly observed with growth promoting feed additives and primarily reflects the higher consumption capacity of larger grown animals compared to untreated individuals, but not necessarily a specific enhancement of voluntary feed consumption due to improved palatability. A small number of experimental assessments of feed acceptance, preference and palatability affected by flavoring additives have been reported so far, indicating reduction of voluntary feed intake in piglets through increasing amounts of fennel and caraway essential oils (Schöne et al., 2006) and thyme and oregano plants (Jugl-Chizzola et al., 2006), respectively. Many more positive results on animal performance are reported with aromatic plants or EOs mixtures. From a scientific point of view many of these reports are difficult to assess since detailed information on the formulation used and on the phytochemical and sensorial quality is missing. However, even if there are some uncertainties associated with the reporting of studies, there is sufficient evidence that aromatic plants and EOs are able to improve animal performance in piglets and poultry.

84

5. Aromatic plants and their extracts pharmacokinetics and in vitro/in vivo mechanisms of action

In view of focusing on ruminants, a small number of studies were conducted about the effect on feed intake and palatability of aromatic plants or volatile compounds (Greathead, 2007; Rochfort et al., 2008). Some reports have shown that terpene volatiles could affect feed intake in sheep, which is important when grazing in Mediterranean pastures (Estell et al., 1998). Better knowledge of specific chemical interactions with feed intake would be useful for altering feeding management. In addition, the potential of EOs as manipulators of rumen metabolism is of high significance. The primary mode of action of growthpromoting feed additives appears to arise from stabilizing feed hygiene and from beneficially affecting the ecosystem of gastrointestinal microflora by controlling potential pathogens. This applies especially to those critical phases of the animals’ development where a higher susceptibility to digestive disorders may be present in the weaning phase of piglets, the early life span of poultry, or restocking at young bull fattening. Improved intestinal health flavored by feed additives may mean that animals are less exposed to microbial toxins or other undesired metabolites such as ammonia and biogenic amines. Natural feed additives such as aromatic plants, medicinal plants, herbs and spices, or volatile oils may relieve the animals from having to mount an enhanced immune defense during critical periods, increasing the intestinal availability for nutrient absorption with an increased surface of intestinal villi trough improvement of animal’s productive performance and showing their maximal genetic potential. A large number of aromatic plants, spices, and EOs are known for digestive or carminative activity. This is due to the stimulation of digestive secretions, saliva, bile, mucus, as well as enhanced enzyme activity being a core mode of beneficial nutritional action (Puvaca et al., 2013). An investigation of Manzanilla et al. (2004) based on the combination of EOs with carvacrol and cinnamaldehyde as the main compounds together with capsaicin used in piglets diet, reported an increased gastric retention time of ingested feed, resulting in better nutrient absorption and favoring intestinal stability against digestive disorders. Thus, there is evidence that EOs and aromatic compounds may favorably affect gut functions. EOs used as feed additives for broilers were shown to enhance the activities of trypsin and amylase in tissue homogenates of the pancreas, as well as the jejunal chyme content (Lee et al., 2003). Intestinal mucus secretion could also be stimulated by dietary addition of bioactive compounds such as carvacrol, cinnamaldehyde, and capsaicin. Jamroz et al. (2006) showed an increased release of large amounts of mucus and the creation of a thick layer of mucus on the glandular stomach and jejunum wall in chicks fed with addition of carvacrol, cinnamaldehyde, and capsaicin, which could be explained by the reduced adherence of pathogens such as E. coli and Clostridium perfringens to the intestinal epithelium. There has been a little investigation and published papers on the effects of EOs and aromatic plants on rumen metabolism. Broudiscou et al. (2000) observed in vitro that Lavandula officinalis promoted the extent of rumen fermentation and that Salvia officinalis had a possible inhibitory effect on methane production. Some EOs have been reported to inhibit enzyme activity a well (Perry et al., 2000), such as thymol which is a strong deaminase inhibitor. Cardozo et al. (2006) found, furthermore, that higher doses of cinnamaldehyde decreased ruminal L-lactate concentration.

References

85

Acknowledgments This paper is part of the project III 46012 which is financed by the Ministry of Education, Science, and Technological Development of the Republic of Serbia.

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Russo, M., Galletti, G.C., Bocchini, P., Carnacini, A., 1998. Essential oil chemical composition of wild populations of Italian oregano spice (Origanum vulgare ssp. hirtum (link) Ietswaart): a preliminary evaluation of their use in chemotaxonomy by cluster Analysis. 1. Inflorescences. J. Agric. Food Chem. 46, 3741e3746.    c, B., Plavsic, D., Levic, J., Pavkov, S., Kokic, B., 2014. Composition and antimicrobial Saric, Lj., Cabarkapa, I., Sari activity of some essential oils from Serbia. Agro Food Ind. Hi-Tech 25 (1), 40e43. Schöne, F., Vetter, A., Hartung, H., Bergmann, H., Biertüpfel, A., Richter, G., Müller, S., Breitschuh, G., 2006. Effects of essential oils from fennel (Foeniculi aetheroleum) and caraway (Carvi aetheroleum) in pigs. J. Anim. Physiol. Anim. Nutr. 90, 500e510. Sikkema, J., De Bont, J.A.M., Poolman, B., 1995. Mechanisms of membrane toxicity of hydrocarbons. Microbiol. Rev. 59, 201e222. Stanacev, V., 2012. Influence of garlic (Allium sativum L.) and copper as phytoadditives in the feed on the content of cholesterol in the tissues of the chickens. J. Med. Plants Res. 6, 2816e2819.  Stankovic, N., Comi c, L., Kocic, B., Nikolic, D., Mihajilov-Krstev, T., Ilic, B., Miladinovic, D., 2011. Antibacterial activity chemical composition relationship of the essential oils from cultivated plants from Serbia. Chem. Ind. 65 (5), 583e589. Trombetta, D., Castelli, F., Sarpietro, M.G., Venuti, V., Cristani, M., Daniele, C., Saija, A., Mazzanti, G., Bisignano, G., 2005. Mechanisms of antibacterial action of three monoterpenes. Antimicrob. Agents Chemother. 49, 2474e2478. Tsinas, A.C., Giannakopoulos, C.G., Papasteriades, A., Alexopoulos, C., Mavromatis, J., Kyriakis, S.C., 1998. Use of Origanum essential oil as growth promoter in pigs. In: Proc. 15th IPVS Congr, Birmingham, UK, p. 221. Ultee, A., Bennik, M.H., Moezelaar, R., 2002. The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl. Environ. Microbiol. 68, 1561e1568. Ultee, A., Slump, R.A., Steging, G., Smid, E.J., 2000. Antimicrobial activity of carvacrol toward Bacillus cereus on rice. J. Food Prot. 63, 620e624. Veldhuizen, E.J., Tjeerdsma-van Bokhoven, J.L., Zweijtzer, C., Burt, S.A., Haagsman, H.P., 2006. Structural requirements for the antimicrobial activity of carvacrol. J. Agric. Food Chem. 54, 1874e1879. Westendarp, H., Klaus, P., Halle, I., Köhler, P., 2006. Effect of carvacrol, g-terpinene and p-cymene-7-ol in broiler feed on growth traits and N-metabolism. Landbauforschung Volkenrode 56, 149e157. Windisch, W., Schedle, K., Plitzner, C., Kroismayr, A., 2008. Use of phytogenic products as feed additives for swine and poultry. J. Anim. Sci. 86, E140eE148. Youdim, K.A., Deans, S.G., 2000. Effect of thyme oil and thymol dietary supplementation on the antioxidant status and fatty acid composition of the ageing rat brain. Br. J. Nutr. 83, 87e93. Zhang, Y., Liu, X., Wang, Y., Jiang, P., Quek, S.-Y., 2016. Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Food Cont. 59, 282e289. Zhou, F., Ji, B., Zhang, H., Jiang, H.U.I., Yang, Z., Li, J., Li, J., Yan, W., 2007. The antibacterial effect of cinnamaldehyde, thymol, carvacrol and their combinations against the foodborne pathogen Salmonella typhimurium. J. Food Saf. 27, 124e133.

C H A P T E R

6 Distribution of aromatic plants in the world and their properties Amit Kumar Pandey1, Prafulla Kumar1, M.J. Saxena1, Prabhakar Maurya2 1

Ayurvet Limited Office, Delhi, India; 2CEHTRA Chemical Consultants Pvt Ltd, Delhi, India O U T L I N E

Introduction

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Uses of aromatic plants

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Historic preview

90

Present status and conservation initiatives

110

Definition of medicinal and aromatic plants

94

Conclusion and way forward

111

Classification Classification of aromatic plants

96 97

References

113

Distribution pattern in the world market

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Introduction Aromatic plants come in Medicinal plants category and collectively they are known as Medicinal and Aromatic Plants (MAP). These are plants, which provide medicines or help in preservation of health. With the development of science, there is a tremendous increase in scientific knowledge of these plant species that open new paths for their use in many facets of our life (e.g., cosmetics, medicinal products, food and feed additives). According to the World Health Organization (WHO) 30% of the drugs sold worldwide contain compounds

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00006-6

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Copyright © 2020 Elsevier Inc. All rights reserved.

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6. Distribution of aromatic plants in the world and their properties

derived from plant materials, which include more than 21,000 taxa (Orzechowski et al., 2002; Giannenas, 2008). Aromatic and Medicinal plants are widely distributed globally including south and southeast Asia, as well as America, Europe, Africa and Australia. In India, more than 7500 species are used in ethnomedicines (Shankar and Majumdar, 1997) which is half of country’s Indian native plant species. China has around 6000 species in use that have medicinal properties (Xiao, 1991). In Africa, over 5000 plant species are used for medicinal purpose (Iwu, 1993). In Europe at least 2000 MAPs are being used and two-thirds of those (1200e1300) are native in the European continent. Most of these plants are still collected in the wild. The pharmacological activity of medicinal and aromatic plants (MAPs) is due to their biologically active ingredients classified into four groups: alkaloids, glycosides, essential oils and other miscellaneous active substances. Secondary metabolites, being end products of metabolic processes of the plants, can be observed through color, taste, or odor, and be determined by chemical analysis. Growth and development of MAPs and their metabolites are influenced by the physical environment, including light, temperature, rainfall, and soil properties. Changes can occur in the course of the plant cycle and even during one single day known as diurnal variations. These aromatic plants are distributed in the whole word and they can be classified according to their cultivation into different climatic zones of the world like tropical, equatorial, temperate etc. The chemical properties/active ingredients of the same species of a plant differs in different climatic zones due to the change in the micro and macro environment of the plant.

Historic preview Plants are the basic lifeline for survival of human life on earth throughout centuries. Plants have been studied by humans from ancient times and are an integral part of the ecosystem for continuance of life cycle running on the earth are the basic producers in the food chain. The knowledge of scientific properties of plants is fundamental to human life on earth. Plants are used in all aspects of our everyday life and humans are dependent on plants for almost every aspect of lifedfrom the breathing air, the food that is consumed, the clothes that are worn, the substances that are being employed in medicine. These essential services provided by plants are far too often taken for granted (World’s Plant Report, 2017). The irony is that for the advancement of human race, the very same plants are used indiscriminately. However, it is undoubtable that whether species of plant Kingdom getting extinct or lost, humanity and the world lose a great deal of scientific knowledge and natural resource. Plants are known to hold keys to some of the most valuable medicinal compounds and it is essential to realize that conservation and propagation of not only domestic but even wild varieties of plants is essential for the continuance and survival of human race (Table 6.1). The agriculturists, pastoralists, famers, animal herders, herbalists of ancient times gathered some knowledge about plants which was passed on from generation to generation and botany as a scientific discipline only emerged much later. There is evidence of knowledge of plants and their properties in ancient Mesopotamian (Milton-Edwards, 2003) and Harappa civilizations (Weber, 1991). Plants have been classified from ancient times into various categories based on their appearance, usages, properties, geographical location and any such

TABLE 6.1 Time-line in history of medicinal and aromatic plants. Book/Monograph/ Treatise/Document

Author(s)

Importance

Geography

800e1000 BC

Sushrut Sutra

Sushruta

Classified plants in 4 categories on basis of flowering pattern structure and life span viz. Vanspataya, Vruksha, Virudh, Aushodh

India

500e300 BC

Hippocratic corpus

Hippocrates

Formalized medicine practices in diagnosis and treatments; list of medicinal plants and application

Greek

372e287 BC

De Causis Plantarum and Historia Plantarum

Theophrastus

Formalized medicine practices in diagnosis and treatments; list of medicinal plants and application

Greek

w150e100 BC

Huangdi Neijing

Author unknown

Theoretical foundation of Chinese medicine, diagnostic methods, and acupuncture

China

w100 BCe100 AC

De Medicina

Celsus

Alexandrian medicine; pharmacopoeia of herbs and the medicines

Greek

w40e90 AD

De Materia Medica

Dioscorides

w23e79 AD

Naturalis Historia

Plinius

A work of 160 volumes, in which he described several plants and gave them Latin names. Many of these names we still recognize, like Populus alba and Populus nigra, and since Latin was later kept for botanical science, we may call him the father of Botanical Latin.

Roman

w200e300 AD

Shennong Bencaojing

Author unknown

Agriculture and medicinal plants; 113 herbal prescriptions and six stages of disease

China

Shanghan Lun

Shang Zhongjing

w500 AD

Treatise on agriculture

Parasara

Classified the plants into many “ganas” or families giving clear picture of the morphology of flowers and fruits.

India

w750e800 AD

The Classic of Tea

Lu Yu wrote

Tea tree, making tea, and tea ceremony

China

91

(Continued)

Historic preview

Time period

92

TABLE 6.1 Time-line in history of medicinal and aromatic plants.dcont'd Time period

Book/Monograph/ Treatise/Document

Importance

Geography

Book of Healing and Canon of Medicine

Avicenna

Clinical trials on medicines; Encyclopedia of medical practices; Scientific and medicinal properties of various plants; Medical encyclopedia

Arabic countries and Germany

Kitab al-Tasrif

Albucasis

Ohysica

Hildegard of Bingen

Kulliyat

Averroes

w1000e1500 AD

Compendium on Simple Medicaments and Foods

Ibn al-Baitar

Pharmacopoeia listing 1400 plants

Arabic countries

w1100 AD

Upaban Vinoda

Sarangadhara

Dealt with different aspects of plant life and classification of plants.

India

w1200e1300 AD

De Vegetabilis

Albert Magnus

Difference in the stem structure of di-cotyledons and Monocotyledons was shown and the two groups were given the terms Tunicate and Corticate.

German

w1530e36 AD

Herbarium vivae Eiconis (3 volumes)

Otto Brunfels

Profusely illustrated with good figures

German

w1498e1554 AD

Nue Kreuterbuch

Jerome Bock

Accurate descriptions of about 600 species of flowering plants.tried to trace the natural relationship of plants while classifying them into 3 major groups, viz., herbs, shrubs, and trees and also noted the original distribution of each species.

German

w1519e1603 AD

De plants in 16 volumes

Andrea Caesalpino Classified the plants on the character of their habit, viz., trees, shrubs, and herbs but also took into account the characters of ovary, fruit, and seed.

Italy

w1545e1612 AD

Herball, or Generall Historie of Plantes

John Gerard

England

w800e1000 AD

Heavily illustrated of 1000 plants

6. Distribution of aromatic plants in the world and their properties

Author(s)

Historia plantarum universalis

Bauhin brothers Jean (Johna) Bauhin

Made use of the habit-character of plants in classifying them.

France eSwitzerland

w1560e1624 AD

3 botanical treatises the third one of which, viz., Pinax theatri Botanic

Gaspard (Casper) Bauhin

Formulated the idea of a genus and in many cases gave binary nomenclature to his plants. He also collected all names of plants published in different botanical works till his time and referred them as synonyms along with names he used as correct ones.

w1628e1705 AD

Historia plantarum

John Ray

First to recognise 2 major taxa of flowering plants, viz., Dicotyledons and Monocotyledons. He also tried to group the plants into several families which he called “classes”.

England

w1707e1778 AD

Systema Naturae, Species plantarum, Philosophia Botanica

Carolus Linnaeus

He used the character of stamens, i.e., the number and nature of stamens, to distinguish the 20 classes in which he divided the plant kingdom. He also used the number and nature of carpels to distinguish the orders, i.e., subdivisions of his classes.

Sweden

w1862e83 AD

Genera Plantarum

George Bentham and Joseph Dalton Hooker

Elaborate descriptions of each and every genera and of the natural orders were given together with names of all species under each genus, the synonyms, localities and reference to literature.

British

1887e99 AD

Die naturlichen Pflanzenfamilien

Adolf Engler and Karl Prantl

All the genera of plants were arranged and described systematically. In doing so they proposed a new system of classification for the whole plant kingdom-true phylogenetic system.

Germany

w1900e2000 AD

Aromathérapie

René-Maurice Gattefossé

Aroma and essential oil for medicine

France

w1900e2000 AD

Aromathérapie

René-Maurice Gattefossé

Aroma and essential oil for medicine

France

Historic preview

w1541e1631 AD

BC, before Christ; AD, after Christ.

93

94

6. Distribution of aromatic plants in the world and their properties

parameter which was simple to identify useful and edible plants and avoid poisonous ones. It was by Theophrastus (372e287 BC), the Greek philosopher-scientist who placed the knowledge of plants on a scientific footing. In “De Causis Plantarum” and “Historia Plantarum” he dealt with the plants at large and attempted to arrange the plants in several groups. For his contributions, he is rightly called the “Father of Botany”. Hippocrates (500e300 BC) in his treatise “Hippocratic corpus” formalized medicine practices in diagnosis and treatments and gave a list of medicinal plants and their application. After him, many philosophers, academics and alike have attempted to understand and classify plants into a reasonable taxonomical classification. Pliny who wrote “Historia Naturalis”, Dioscorides who wrote “Materia Medica” where he described about 600 species of plants mentioning their local name and giving their medicinal properties along with their sketches making the identification much easier, has made the major contributions. In the 13th century, Albert Magnus wrote “De Vegetabilis” where the difference in the stem structure of Dicotyledons and Monocotyledons was mentioned. Printed books on plants were available toward the close of the 15th century, which made the study of Botany quite popular. The attempts by Charles Darwin through his book “Organic Evolution” and classification systems by Carolus Linnaeus (1707e78), a Swedish naturalist and Whittaker were the most significant landmarks in the history of understanding of the science of plants. Healing with medicinal plants is as old as mankind itself (Petrovska, 2012). Ancient people became aware of the worth and charm of aromatic and medicinal plants. One guidance for the efficient use of these plant materials is the knowledge retrieved from historical books. Initially, books on use on medicinal and aromatic plants were sourced in various parts of the world, such as the Middle East, Greece, China, and India, indicating that these ancient civilizations used indigenous aromatic and medicinal plants to improve lives in their own separate ways before ideas were shared (Inoue et al., 2017). There is mention in Bible and the holy Jewish book the Talmud, during various rituals accompanying a treatment, aromatic plants were utilized such as myrtle and incense (Dimitrova, 1999). In ancient Persia, plants were commonly used as a drug and disinfectant and aromatic agent (Hamilton, 2004). As per an estimate, 70,000 plant species were used in traditional medicine in 1990s (Farnsworth and Soejarto, 1991). Nowadays, this number has considerably increased.

Definition of medicinal and aromatic plants Aromatic plants as the name suggests are the plants that exude aroma. Aromatic plants are mostly described in association with medicinal plants as together they form a special category described by ethnobotanists as Medicinal and Aromatic Plants abbreviated as MAP. Different authors have tried to describe aromatic plants on different basis. The standard definitions are described in this review. Aromatic plants are a special class of plants mainly used for their aroma and flavor. Many of them are exclusively used also for medicinal purposes in aromatherapy as well as in various systems of medicine (Maiti et al., 2007). Plants that produce and exude aromatic substances (largely ether oils), which are used in making perfumes, in cooking, and in the food, pharmaceutical, and liquor industries. Many aromatic plants are species of the Lauraceae, Umbelliferae, Myrtaceae, and Labiatae families. In the USSR roses,

Definition of medicinal and aromatic plants

95

geraniums, laurel, lavender, and rosemary are among the plants used in industry (The Great Soviet Encyclopedia, 2010). Some plants are endowed with specific aroma characteristics. Such particularities are due to the presence of volatile compounds known as essential oils. Consequently, aromatic plants, herbs and herbal extracts have always constituted the most characteristic elements of the Mediterranean Cuisine and in this area, those mainly used in the local gastronomical traditions are Ocimum basilicum (basil), Rosmarinus officinalis (rosemary), Salvia officinalis (sage), Allium schoenoprasum (chives), Origanum majorana (oregano), Allium sativum (aglio) (Orto Botanico di Napoli, 2018). Aromatic plants are a section of plants mainly used for their aroma and flavor. Many of them are also have medicinal use. They represent a large group of economically important plants. There are growing needs for plant compounds, essential oils, aromatic chemicals and pharmaceuticals in the world market for the last 2 decades. These needs will cover the demand to replace drugs with nature identical compounds. Aromatic compounds are present in plants i.e., in root, wood, bark, foliage, flower, fruit, seed etc (Medicinal and Aromatic Plants, 2018). An aromatic plant or food has a strong, pleasant smell of herbs or spices (Collins English Dictionary, 2018). Aromatic plants possess odorous volatile substances which occur as essential oil, gum exudate, balsam and oleoresin in one or more parts, namely, root, wood, bark, stem, foliage, flower and fruit (Joy et al., 2001a,b, 2014). Aromatic plant - a plant with an aroma, something we can smell, has aromatic qualities, not all aromatic plants are essential oil crops (Jeljazkov and Cantrell, 2016). In 1997, the International Standards Organization (ISO) defined an essential oil as a “product obtained from vegetable raw material, either by distillation with water or steam, or from the epicarp of citrus fruits by a mechanical process, or by dry distillation.” A range of essential oils have been found to have various degrees of antimicrobial activity and are believed to have antiviral, nematocidal, antifungal, insecticidal, and antioxidant properties. Medicinal and aromatic plants is a category assigned to plants, which are being traditionally used since ancient times for medicinal usage and are having some sort of aroma mostly a pleasing one, which is utilized in essential oils and toiletries. The nomenclature “Medicinal and aromatic plants” is based on traditional and conventional usage. It is interesting to be noted that there is no solid scientific base as prerequisite as some nonaromatic plants can also be classified as of medicinal usage and some aromatic plants may not have any medicinal usage. This group of plants plays an important role in the life of people and their usage dates back to ancient times for which a time-line is not been recorded, hence difficult to establish one. As per second annual State of the World’s Plants Report-2017; 3,900,900 plant species are known to science and of those approximately 3,690,400 (94%) are flowering and at least 28,187 (7.21%) plant species are currently recorded as being of medicinal use which seems a very conservative figure indeed. Based on the best available estimate, scientists say that 21% of all plant speciesdor one in every five-plant speciesdis likely threatened with extinction. The report highlights that fewer than 16% (4478) of the species used in plant-based medicines are cited in medicinal regulatory publications. Interestingly, there are currently 15 alternative names for each medicinal species, causing confusion and risk in the sector. The report suggests how this can be streamlined and improved in databases like Kew’s Medicinal Plant Names Service (MNPS) (World’s Plants Report, 2017).

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However, there is no clear solution on the elusive problem of the definition of “Medicinal and Aromatic Plants”. As per one definition, MAPs are botanical raw materials, also known as herbal drugs that are mostly used for aromatic, therapeutic and/or culinary purposes as components of cosmetics, medicinal products, health foods and other natural health products. Also, they can be regarded as the basic materials for value-added processed natural ingredients such as essential oils, dry and liquid extracts and oleoresins (International Trade Center, 2018). An older definition of Medicinal Plants is a large group of plants used in medicine or veterinary practice for therapeutic or prophylactic purposes (The Great Soviet Encyclopedia, 2010). As per modernized definitions, Medicinal Plants can be regarded as: “Medicinal plants are those plants which are used in official and various traditional systems of medicines throughout the world”. In analogous way, other definition could be “Medicinal plants are plants that provide people with medicines - to prevent disease, maintain health or cure ailments” (Maiti and Geetha, 2007). Medicinal plants are plants used as natural medicines. Many modern medicines had their origin in medicinal plants. Examples include aspirin from willow bark (Salix spp.), digitalis from foxglove (Digitalis purpurea), and vinblastine from Madagascar periwinkle (Vinca rosea) for the treatment of childhood leukemia (The Columbia Electronic Encyclopedia, 2013).

Classification Aromatic plant essences offer an infinite variety of possibilities for regeneration, revitalizing and healing Rodolphe Balz

Conventionally, aromatic plants have been categorized along with medicinal plants since most of the aromatic plants have found their usage in medicines in addition to other uses. Aromatic plants have been used traditionally in perfumery and cosmetics because of the rich aroma that they produce which is due to essential oils as a phytoconstituent of these plants. Aromatic plants have a great importance in commerce and trade because of such properties and the continuous scientific investigations have opened up new avenues in the aromatic plant research. The most important usage of aromatic plants is still in the field of medicines and pharmaceutical industries continuously search for new effective drug molecules at a time when antibiotic resistance is taking new leaps. Aromatic plants have rich usage in traditional systems of medicine such as Ayurveda (India), an ancient Chinese system of medicine and many other traditional systems of medicine. Aromatherapy is one of the alternative therapy systems in natural medicine, which fully relies on aromatic plants and uses essential oils for therapy. Essential oils have been used since ancient period, with the purpose of improvising a person’s health or mood. The National Association for Holistic Aromatherapy (NAHA) defines aromatherapy as “the therapeutic application or the medicinal use of aromatic substances (essential oils) for holistic healing.” Aromatherapy applications include massage, topical applications, and inhalation. However, we should not forget that natural products are also chemicals and they can be hazardous if used wrongly.

Classification

97

As aromatic plants have been grouped with medicinal plants for identification and taxonomical purposes, the general classification is also more feasible under the category of Medicinal and Aromatic Plants. For the purpose of study and utilization of MAPs, the primary step is the identification of the plants and then their taxonomical classification. Earlier the identification of plants was solely based on senses of vision, olfaction and touch in absence of any scientific precedent hence the main basis for classification of aromatic plants was their external morphology. With the advent of science and the identification of large number of plants with similar morphologies it not only became difficult to classify them morphologically but it also pushed for the need of a more standard classification system where new species identified in future could be accommodated and hence classification system used other characters such as chemical traits such as phytochemical profiling, DNA markers, biosynthetic causes of chemo-differentiation, and now even the plant metabolome is used as a tool for identification and classification. Thus, botany assisted by other scientific achievements seems to open up promising perspectives for the breeding of new, highly powerful chemo-cultivars of medicinal and aromatic taxa (Shukla et al., 2009; Mathe, 2015). Out of a total of about 1500 species of aromatic plants known, only a little over 500 species have been studied in some detail. Of the 50 species which find use as a commercial source of essential oils and aroma-chemicals, the total number of those having regular and large-scale utilization hardly exceeds two dozen (Joy et al., 2014; Inoue et al., 2017). It is not possible to ascertain the exact number of species of aromatic plants or MAP material in use in the world. This is because firstly, some aromatic and medicinal plant material is used in minute amounts and will therefore not be listed in trader’s catalog. Secondly, an aromatic or MAP commodity may come from several species yet be traded under a trade name which obscures the various specific origins. Lastly, many species are only used at local the level and their use is not comprehensively documented (Lange, 1998). According to a very recent review, a comprehensive classification has been suggested for medicinal including aromatic herbs or plants (Alamgir, 2017). They may be classified in various ways according to Fig. 6.1. According to another system of classification (Fig. 6.2) for aromatic plants exclusively, the following system has been proposed (Joy et al., 2014) The main points of MAPs classification are:

Classification of aromatic plants 1. Based on importance Plants grown exclusively for extraction of aromatic principles for use in perfumery cosmetics are classified as major whereas those in which volatile oil and aroma principles are by-products or secondary products, are classified as minor aromatic crops. (i) Major aromatic crops: e.g., Chamomile, Vetiver, Lemongrass, Patchouli, Tea tree, Eucalyptus (ii) Minor aromatic crops: e.g., Cinnamon, Marigold, Dill, Ambrette, Celery 2. Based on the part used (i) Herbage: e.g., Patchouli, Citronella, Sweet basil, Geranium, Rosemary (ii) Root: e.g., Vetiver, Sassafras albidum, Sandalwood, Camphor

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FIGURE 6.1 Classification system of aromatic and medicinal plants (Alamgir, 2017).

FIGURE 6.2

Classification system of aromatic plants (Joy et al., 2014).

Distribution pattern in the world market

3.

4.

5.

6.

7.

99

(iii) Wood: e.g., Ocotea cymbarum, Ocotea pretiosa, Sandalwood, camphor, Linaloe. (iv) Bark: e.g., Cinnamomum verum, C. burmannii (v) Leaf: e.g., Eucalyptus, Tea tree, Skimmia laureola, Doryphora sassafras, Laurus nobilis, Lemon grass, Mint, Camphor (vi) Flower: e.g., Jasmine, Rose, Marigold, Chamomile, Champak, Tuberose, Ylang ylang (vii) Flowering tops: e.g., Davana, Palmarosa, Thyme (viii) Fruit: e.g., Dill, Litsea cubeba, Linaloe (ix) Seed: Ambrette, Ajowan, Celery, Clarysage Based on growth habitat (i) Grasses: Palmarosa, Rosha grass, Lemongrass, Vetiver, Citronella (ii) Herbs: Sweet basil, Tuberose, Thyme, Rosemary, Chamomile, Ajowan, Davana, Marigold, Mint (iii) Shrubs: Skimmia laureola, Patchouli, Rose, Geranium, Jasmine (iv) Trees: Eucalyptus, Tea tree, Camphor, Champak, Cinnamon, Linaloe, Ylang ylang Based on habitat (i) Tropical: Lemon grass, Ocimum, Cinnamon, Linaloe, Sandalwood, Eucalyptus, Citronella, Palmarosa, Patchouli, Vetiver, Ylang ylang (ii) Sub-tropical: Vetiver, Mint, Eucalyptus, Ajowan, Thyme, Rosemary, Citronella, Davana, Fennel, Japanese mint (iii) Temperate: Chamomile, Ajowan, Fennel, Pepper mint, Spear mint, Bergamot mint Based on crop duration (i) Annuals: Chamomile, Ocimum basilicum, Ajowan, Davana (ii) Biennials: Vetiver, Celery (iii) Perennials: Lemon grass, Geranium, Lemon grass, Mint, Palmarosa, Rose, Cinnamon, Ylang ylang, Tea tree Based on method of propagation (i) Vegetatively propagated: Citronella, Geranium, Jasmine, Patchouli, Rose, Tuberose (ii) Sexually (seed) propagated: Clarysage, Cumin, Davana, Camphor, Eucalyptus, Sandalwood, Palmarosa, Ylang Ylang (iii) Both vegetatively and sexually propagated: Lemon Grass, Linaloe, Marigold, Palmarosa, Rosemary, Thyme, Vetiver Based on Botanical classification Following Fig. 6.3 explains in brief, the botanical system of classification. The detailed botanical classification has been explained in Table 6.2.

Distribution pattern in the world market The knowledge of important herbs, their usage in common household and their importance in dealing with common diseases are passed from generation to generation either in the form of written documents or in the form of practice. Earlier society was totally depending on the wild harvest for their need of medicinal and aromatic plants. When we are discussing about aromatic plants or Medicinal and Aromatic Plants (MAP), the role of wild harvest should not be neglected. Even today also wild harvest is contributing

100

6. Distribution of aromatic plants in the world and their properties

FIGURE 6.3 Botanical classification of aromatic plants.

a great portion in the availability of medicinal and aromatic plant. In developing countries, wild crafting is of two types based on the uses. 1. Small-scale wild plant harvesting for the purpose of local use (health practitioner collect plant for their own use or depend on collectors for the plant for improving the livelihood) 2. Large-scale wild plant harvesting to comply the need of global market on a commercial basis. In the Indian context the record of MAPs based on old literature 7500 to 8000 spices are in use (Sati, 2013). For fulfilling most of the demands of aromatic plants, suppliers are depended mostly on wild plants of highland region. Although the domestic cultivation of aromatic plants increases still the proportion of cultivated in comparison with the wild plants remain low. In Pithoragarh alone (Himalayan region), more than 1300 tons are collected annually. Over half a million tons of dry raw material is collected from the wild every year (Tandon, 2006). The below average economic condition of the people stimulates wild harvesting which provides income. Still the gap between the demand and supply is very huge which is estimated to be about (about 162 species),200,000e400,000 tons (Sati, 2013). In Yunnan Province in southwestern China, well known for its richness and diversity of medicinal plants, 216 medicinal species belonging to 194 genera in 98 families were recorded in the local markets (Lee et al., 2008). 173 species (80.1%) are wild and 43 (19.9%) are cultivated in gardens or semicultivated in wild habitats. The wild plant’s species, which are included in the China Red list of endangered species.

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Distribution pattern in the world market

TABLE 6.2

Botanical classification of aromatic plants. DIVISION: EMBRYOPHYTA

Family

Genus (species names given in parentheses)

SUBDIVISION: I. GYMNOSPERMAE Class: Coniferae Podocarpaceae

Dacrydium (franklini)

Pinaceae

Picea (abies, alba, canadensis, excelsa, glauca, jezoensis, mariana, nigra, obovata, vulgaris) Tsuga (canadensis, douglasii, heterophylla) Pseudotsuga (douglasii, glauca, mucronata, taxifolia) Abies (alba, balsamea, balsamifera, douglasii, excelsa, mayriana, mucronata pectinata, picea, sachalinensis, sibirica) Cedrus (atlantica, deodara, libani, libanotica) Pinus (albicaulis, aristata, attenuata, ayacahuite, balfouriana, balsamea, banksiana, caribaea, cembra, clausa, contorta, coulteri, echinata, edulis, flexilis, glabra, jeffreyi, lambertiana, longifolia, monophylla, montana, monticola, mugo.)

Taxodiaceae

Sciadopitys (verticillata) Cryptomeria (japonica)

Cupressaceae

Callitropsis (araucarioides) Thujopsis (dolabrata) Thuja (plicata) Cupressus (fastigiata, glauca, Japonica, lambertiana, lawsoniana lusitanica, macrocarpa, pendula, sempervirens, sinensis, torulosa) Chamaecyparis (lawsoniana, obtusa, taiwanensis, thyoides) Juniperus (communis, mexicana, oxycedrus, phoenicea, procera, sabina, thurifera, virginiana)

SUBDIVISION: II. ANGIOSPERMAE Class: 1. Monocotyledonae Graminae (Poaceae)

Elyonurus (latiflorus, tripsacoides) Vetiveria (zizanioides) Cymbopogon (afronardus, caesius, citratus, clandestinus, coloratus, confertiflorus, densiflorus, exaltatus, flexuosus, georingii, giganteus, jwarancusa, rectus, martinii, nardus, nervatus, polyneuros, procerus, proximus, schoenanthus, senaarensis, stipulatus,virgatus, winterianus) Andropogon (aciculatus, connatus, fragrans, intermedius, kuntzeanus, muricatus, nardoides, odoratus, versicolor)

Cyperaceae

Cyperus (rotundus)

Palmae (Palmaceae)

Cocos (nucifera)

Araceae

Acorus (calamus) Liliaceae Allium (cepa, sativum) Lilium (candidum) Hyacinthus (non-scriptus, orientalis) Convallaria (majalis)

Amaryllidaceae

Narcissus (jonquilla, poeticus, tagetta) Polyanthes (tuberosa)

Irridaceae

Crocus (sativus) Iris (florentina, germanica, pallida) (Continued)

102 TABLE 6.2

6. Distribution of aromatic plants in the world and their properties

Botanical classification of aromatic plants.dcont'd DIVISION: EMBRYOPHYTA

Family

Genus (species names given in parentheses)

Zingiberaceae

Hedychium (flavum) Kaempferia (galanga, rotunda) Curcuma (amada, aromatica, caesia, domestica, longa, xanthorrhiza, zedoaria, zerumbet) Alpinia (alleghas, galanga, officinarum, calcarata, khulanjan, malaccensis, nutans) Zingiber (mioga, nigrum, officinale) Amomum (angustifolium, aromaticum, cardamom, globosum, hirsutum, korarima, melegueta) Elettaria (cardamomum)

Class: 2. Dicotyledonae Piperaceae

Piper (acutifolium, angustifolium, asperifolium, camphoriferum, clusii, crassipes, cubeba, guineense, lineatum, longum, lowong, mollicomum, molissimum, nigrum, officinarum, ribesioides)

Betulaceae

Betula (alba, brea, dulce, lenta, papyrifera, pendula, pubescens)

Moraceae

Humulus (americanus, lupulus)

Santalaceae

Osyris (tenuifolia) Santalum (album, lanceolatum, preissianum, spicatum, zygnorum) Fusanus (spicatus)

Aristolochiaceae

Asarum (canadense, europaeum)

Chenopodiaceae

Chenopodium (ambrosioides)

Caryophyllaceae

Dianthus (caryophyllus)

Ranunculaceae

Nigella (damascena)

Magnoliaceae

Magnolia (grandiflora) Michelia (champaca, longifolia, excelsa, figo, kisopa, nilagirica, rheedi) Illicium (anisatum, japonicum, religiosum, verum)

Anonaceae

Cananga (odorata)

Myristicaceae

Myristica (argentea, fragrans, malabarica, succedanea)

Lauraceae

Cinnamomum (aromaticum, infers, glanduliferum, camphora, cassia, culilawan, kanahirai, loureirii, micranthum, obtusifolium, xanthoneuron, zeylanicum, tamala) Ocotea (caudata, cymbarum, parviflora, pretiosa, sassafras) Sassafras (albidum) Cryptocaria (massoia) Laurus (nobilis) Umbellularia (californica) Aniba (parviflora, rosaeodora)

Cruciferae

Cochlearia (armoracia) Brassica (alba, juncea, napus, nigra) Raphanus (sativus)

Resedaceae

Reseda (odorata)

Saxifragaceae

Philadelphus (coronarius)

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Distribution pattern in the world market

TABLE 6.2

Botanical classification of aromatic plants.dcont'd DIVISION: EMBRYOPHYTA

Family

Genus (species names given in parentheses)

Hamamelidaceae

Hamamelis (virginiana) Liquidambar (orientalis, styraciflua)

Rosaceae

Spiraea (ulmaria) Rosa (alba, canina, centiflolia, damascena, gallica, indica, glandulifera, moschata, pubescens) Prunus (amygdalus, laurocerasus)

Leguminosae

Acacia (cavenia, dealbata, decurrens, farnesiana, floribunda) Copaifera (coriacea, glycycarpa, guianensis, martii, multijuga, officinalis, reticulata) Myroxylon (balsamum, pereirae) Lupinus (luteus) Genista (sibirica, tinctoria) Spartium (junceum) Wistaria (sinensis) Hardwickia (mannii) Myrocarpus (fastigiatus, frondosus,)

Geraniaceae

Geranium (lugubre, macrorrhizum) Pelargonium (capitatum, fragrans, graveolens, odoratissimum, radula, roseum, terebinthinaceum)

Zygophyllaceae

Bulnesia (sarmienti)

Rutaceae

Xanthoxylum (piperitum) Ruta (angustifolia, bracteosa, graveolens, montana) Pilocarpus (jaborandi, microphyllus, racemosus, spicatus) Cusparia (trifoliata) Boronia (megastigma) Barosma (betulina, crenulata, serratifolia) Amyris (balsamifera) Clausena (anisata, anisum-olens, excavata) Citrus (acida, aurantifolia, decumana, aurantium, deliciosa, limetta, limon, medica, nobilis, paradisi, reticulata, sinesis, unshiu)

Burseraceae

Boswellia (carterii) Bursera (aloexylon, delpechiana, fragroides, glabrifolia) Commiphora (abyssinica, erythraea, myrrha, schimperi) Canarium (luzonicum)

Euphorbiaceae

Croton (eluteria)

Anacardiaceae

Pistacia (lentiscus) Schinus (molle)

Tiliaceae

Tilea (cordata, platyphyllos, tomentosa,)

Malvaceae

Hibiscus (abelmoschus)

Dipterocarpaceae

Dryobalanops (aromatica, camphora) Dipterocarpus (tuberculatus, turbinatus)

Cistaceae

Cistus (ladaniferus)

Violaceae

Viola (odorata) (Continued)

104 TABLE 6.2

6. Distribution of aromatic plants in the world and their properties

Botanical classification of aromatic plants.dcont'd DIVISION: EMBRYOPHYTA

Family

Genus (species names given in parentheses)

Myrtaceae

Myrtus (aeris, caryophyllata, communis, pimenta) Pimenta (acris, citrifolia, officinalis, racemosa) Eugenia (acris, caryophyllata, pimenta,) Leptospermum (citratum, flavescens) Melaleuca (alternifolia, bracteata, cajeputi, leucodendron, linariifolia, maideni minor, smithii, trichyostachya, viridiflora) Eucalyptus (amygdalina, australiana, bicostata, citriodora, cneorifolia, dives, dumosa, elaeophora, fruticetorum, globulus, leucoxylon, lindleyana, macarthuri, maculosa, numerosa, phellandra, polybractea, radiata, sideroxylon, smithii, viridis)

Umbelliferae (Apiaceae)

Coriandrum (sativum) (Apiaceae) Cuminum (cyminum) Apium (graveolens, petroselinum) Petroselinum (hortense, sativum) Carum (ajowan, bulbocastanum, carvi, copticum, petroselinum, verticillatum) Pimpinella (anisum, diversifolia, saxifragra) Foeniculum (vulgare,) Anethum (graveolens, sowa) Oenanthe (phellandrium) Levisticum (officinale) Angelica (archangelica, atropurpurea, glabra, levisticum Ferula (alliacea, asafoetida, badra-kema, ceratophylla, foetida, galbaniflua, rubricaulis, suaveolens, sumbul) Peucedanum (ostruthium) Daucus (carota) Crithmum (maritimum)

Ericaceae

Gaultheria (procumbens)

Primulaceae

Cyclamen (europaeum)

Oleaceae

Syringa (vulgaris) Jasminum (officinale, grandiflorum, auriculatum, sambac, undulatum)

Verbenaceae

Lippia/Aloysia (citriodora)

Labiatae (Lamiaceae) Rosmarinus (flexuosus, lavandulaceus, laxiflorus, officinalis, tournefortii) Lavandula (barmanni, dentata, hybrida, intermedia, latifolia, officinalis, pedunculata, spica, stoechas, vera, viridis) Nepeta (cataria, liniaris, spicata) Salvia (carnosa, espanola, hiemalis, hispanorum, lavandulaefolia, leucophylla, moscatel, officinalis, sclarea, triloba, verbenaea) Monarda (citriodora, fistulosa, menthaefolia, pectinata, punctata) Melissa (officinalis) Hedeoma (pulegioides) Satureia (hortensis, montana) Hyssopus (officinalis) Origanum (compactum, elongatum, fort-queri, grossi, majorana, virens, vulgare) Marjorana (silvestre, hortensis) Thymus (capitatus, cephalotus, hiemalis, hirtus, loscossi, mastichina, serpyllum, virginicus, vulgaris, zygis)

Distribution pattern in the world market

TABLE 6.2

105

Botanical classification of aromatic plants.dcont'd DIVISION: EMBRYOPHYTA

Family

Genus (species names given in parentheses)

Myoporaceae

Mentha (aquatica, arvensis, cablin, canadensis, citrata, japonica, longifolia, piperita, pulegium, rotundifolia, spicata, sylvestris, verticillata, viridis) Perilla (citriodora, frutescens, nankinensis, ocymoides) Pogostemon (cablin, heyneanus, hortensis, patchouli) Ocimum (americanum, basilicum, canum, carnosum, gratissimum, kilimandscharicum, album, anisatum, menthaefolium, micranthum, minimum, nakurense, pilosum, sanctum, suave, viride.) Mosla/Orthodon (angustifolia, chinesis, formosana, hadai, japonica, lanceolata, lysimachiiflora, punctata, thymolifera) Pycnanthemum (incanum, lanceolatum, muticum, pilosum) Coridothymus (capitatus) Eremophila (mitchelli)

Rubiaceae

Gardenia (citriodora, florida, grandiflora, longistyla, resinifera, floribunda, latifolia) Leptactina (senegambica)

Caprifoliaceae

Lonicera (caprifolium, gigantea, japonica)

Valerianaceae

Valeriana (celfica, officinalis, wallichii, brunoniana, hardwickii)

Compositae

Solidago (odora)

Asteraceae

Erigeron (canadensis) Blumea (balsamifera, lacera, ampletectens, densiflora, aurita, glabra) Helichrysum (angustifolium, arenarium, italicum, stoechas) Inula (helenium) Tagetes (glandulifera, minuta, erecta, patula) Santolina (chamaecyparisus) Anthemis (nobilis) Achillea (millefolium, moschata) Matricaria (chamomilla, inodora) Artemisia (absinthium, cina, dracuculus, maritima, pallens, pontica, tridentata, vulgaris, vestita, scoparia, parviflora) Arnica (montana) Saussurea (lappa) Tanacetum (vulgare)

Source: Guenther, E., 1952. The Essential Oils. vol. 5. Van Nostrand Co. Inc., New York, Modified; Joy, P.P., Thomas J., Mathew S., Jose, G., Johnson J., 2014. Aromatic and Medicinal Plants, Research Station, Odakkali Asamannoor e 683 549, Kerala, India.

Africa is also very rich in terms of diversity of the aromatic plants. It is also exporter of many wild species of plants e.g., Aloe faro, Aloe sinkatana, Aloe scabrifolia, Prunus africana, Garcinia afzelii, Randia acuminate Warburgia salutaris, Warburgia ugandensi. Some species have now become subject to international trade controls under CITES (Maundu et al., 2006). In South Africa, nearly 4000 species are used. About 700 indigenous species are traded locally. 20,000 tons of plant material is marketed yearly (McGaw et al., 2005). Ghana alone sold 133,951 tons of medicinal and aromatic plants annually of about 7.8 million dollars in addition to that 6 tons used locally (Van Andel et al., 2012). The list also includes some rainforest species and species harvested in officially protected areas, which are also enlisted in the International Union for Conservation of Nature (IUCN) red list of Ghana.

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6. Distribution of aromatic plants in the world and their properties

Moreover, in Latin America and especially in northern Peru, the maximum quantity of plant materials marketed is mostly harvested from wild where cultivation is negligible (Bussmann and Sharon, 2009). Here more than two-third of the species are originated from the highlands and more than 40% of the dales is represented by seven native species (Croton lechleri, Uncaria tomentosa, Equisetum giganteum, Peumus boldus, Erythrina spp., Buddleja utilis and Piper aduncum) and three exotic species (Chamomilla recutita, Ruta graveolens, Eucalyptus globulus). On the other hand, in Ukraine, the trade of medicinal and aromatic plants declines, partly due to exhausted natural stocks because of excessive past exploitation. The change in ecosystem also contributes in the decline of MAP trade. According to one study, about 600 tons of wild materials and 400 tons of cultivated materials are used in Ukraine (Minarchenko, 2011). In Turkey, collection in protected areas (e.g., national parks), is still legal (Baser and Franz, 2009). Collectors pay a fee to General Directorate of Forestry which issues licenses (Cetinkaya, 2010). Approximately some 472 tons of aromatic and medicinal plants were collected from wild (reported to be sustainable) in Kanyon National Park in 2005, in Turkey, alone (Baricevic et al., 2015).

Uses of aromatic plants The aromatic plants are mainly used for the purpose of extraction of essential oils in the world. Essential oils are also termed as volatile oils, as name also implies, they are volatile in steam. These essential/volatile oils are accumulated in oil cells, in secretion ducts or cavities or in glandular hair of plants (Mathe, 2015). The production and consumption of essential oils is increasing rapidly because of their multipurpose uses. Essential oils are used in perfumery, the food industry, household industry, condiment industry, in making sweets and beverages as well as pharmaceutical and aroma therapeutic products of plants origin (Bernath, 2009). MAPs exhibit many bioactive compounds who are responsible of their biological properties, such as antimicrobial, antioxidant, antiparasitic and other health promoting properties (Christaki et al., 2012; Giannenas et al., 2013). The beneficial use of aromatic and medicinal plants, their extracts, essential oils or herbal constituents are clearly presented in Fig. 6.4. In Table 6.3, some MAPs and their main compounds are listed. Various species of plants are used to produce essential oils, which can be categorized based on the regions they belong. The different climatic and ecological conditions, in which these plants grow, are the base of their classification. The three climatic zones, which is considered for this classification is tropical temperate and Mediterranean. Based on these climatic and ecological conditions some of the important species of plants and their important phytochemical constituents are classified in Table 6.3. The fragrance of an aromatic plant is due to the presence of traces of essential oils in different parts. Numerous fragrant materials are present in roots, stems, barks, leaves, flowers, fruits and heartwoods. Gums, balsams and oleoresins are also valuable raw materials for perfumes by virtue of their tenacious but soft odor. Several processes like hydro

Uses of aromatic plants

107

FIGURE 6.4 Effect of herbal feed additives to support health and performance of animals as indicated by the philosophy of Ayurvet limited.

distillation, steam distillation, hydro diffusion, enfleurage, maceration, expression and solvent extraction are available for the extraction of aroma principles. Application of these processes, either singly or in combination, depends upon the nature of the material and of the essential oil or absolute intended to be recovered. 1. Distillation: The majority of essential oils are produced by distillation. There are three types of distillation process-hydro, hydro-steam and steam distillation. 2. Unconditional flower oils: Flower oils are obtained by maceration, expression, enfleurage and extraction with volatile solvents. 3. Supercritical Fluid Extraction (SCFE): This is flexible and important tool to separate components that are susceptible to thermal degradation. It is engaged for the extraction of perfumes, flavors and fragrances and from a wide variety of natural products. This method of extraction is better and take less time than distillation. 4. Natural Aroma Products: Essential oils production helps in developing agroindustry into an exceptionally cost-effective business with high profitability. This trend started only very recently. It has provided plethora of diversification to the agricultural sector and is become very popular on the rural front. This is because the demand of natural products has been increased in developed countries, along with its constant consumption in developing countries. Due to the availability of essential oils more freely in the market, not only their direct uses as attars, floral and aromatic waters, perfumery grade alcohol and in flavor encapsulation, but also the end uses have been widening. As it has been already described, the essential oils are today used in soaps, perfumery, cosmetics, incense sticks, disinfectants, deodorants, mosquito repellents, flavoring of foods and pharmaceuticals and a range of allied products.

TABLE 6.3 Essential oils of aromatic and medicinal plants and their phytochemical bioactive components (Bernath, 2009). Scientific name

Main components of essential oil

Essential oil producing species of tropical origin Cananga (ylang-ylang)

Cananga odorata

Linalool, beta-caryophyllene, farnesene

Pelargonium (geranium)

Pelargonium spp.

Linalool, isomenthone, citronellol, geraniol, citronellyl formate

Citronella

Cymbopogon spp.

Citronellal, citronellol, geraniol

Cymbopogon citratus Cymbopogon flexuosus

Neral, geranial

Palmarosa (gingergrass)

Cymbopogon martinii

Linalool, geranyl acetate, geraniol

Vetiver

Vetiveria zizamoides

Vetiverol

Patchouli

Pogostemon cabin

Camphor

Cinnamomum camphora

Patchoulol, norpatchoulenol, azulene Camphor, safrole, cineol

Cassia

Cinnamomum cassia

Cinnamaldehyde

Cinnamon

Cinnamomum verum

Eugenol, beta-caryophyllene,

Sassafras albidum

Safrole, phellandrene, pinene

Sassafras Cubeba

Litsea cubeba

Nutmeg

Myristica fragrans

Clove

Syzygium aromaticum

Citral Alpha-pinene, beta-pinene, sabinene Eugenol, beta-caryophyllene

Eucalyptus

Eucalyptus spp.

Cineole, phellandrene, piperitone

Melaleuca

Melaleuca alternifolia

Terpinen-4-ol, gamma-terpinene

Cajuput

Melaleuca cajuputi

Jasmine

Jasminum spp.

Pepper

Piper nigrum

Lime

Citrus aurantifolia

Sweet orange

Citrus sinensis

Sandalwood

Santalum album

Ginger

Zingiber officinale

Cineole benzyl acetate, phytol, isophytol Limonene, sabinene, pinene, caryophyllene Limonene, gamma-terpinene, alpha-terpinrol Limonene, myrcene Santalol, santalyl acetate, santalene Monoterpenoid and sesquiterpenoid hydrocarbons, zingiberene, geraniol, nerol

6. Distribution of aromatic plants in the world and their properties

Lemongrass

108

Common names

Essential oil producing species of Mediterranean origin Myrte

Myrtus communis

Rose

Rosa damascena

Bitter orange Bergamot orange Lemon Fennel

Lavender Sage

Linalool, linalyl acetate, geraniol, limonene

C. aurantium subsp. bergamia

Linalyl acetate, linalool, D-limonene, pinene

Foeniculum vulgare a

a a

Garden thyme

Stearoptene, nerol, geraniol, citronellol

Citrus aurantium

Citrus limon

a

Alpha-pinene, limonene, linalool

Limonene, beta-pinene, gamma-terpinene Anethole, fenchon, methyl chavicol

Lavandula spp.

Linalyl acetate, borneol, camphor, geraniol

Salvia officinalis

Thujone, cineol, camphor, borneol

Thymus vulgaris

thymol, carvacrol, borneol, cymen

Salvia sclarea

Linalyl acetate, sclareol, linalool, nerol, pinene, thujone, borneol

Corianderb

Coriandrum sativum

Alpha-linalool, geraniol, geranyl-acetate

Anethum graveolens

D-carvone, D-limonene,

Dill

b

Anise

Pimpinella anisum

phellandrene

Methylcavicol, anisaldehyde, aniscetone

Angelica

Angelica archangelica

Alpha-pinene, alpha-phellandrene, camphene

Lovage

Levisticum officinale

n-butylidene phtalide, alpha-terpineol, carvacrol, eugenol

Carawayb

Carum carvy

Pepermint

Mentha piperita

Menthol, menthone, pinene, menthyl acetate

Spearmint

Mentha spicata

L-carvone

Camomile

b

Tarragon a

Matricaria recutita Artemisia dracunculus

D-carvone,

Uses of aromatic plants

Clary sagea

limonene, dihydrocarvone

Chamazulene, alpha-bisabolol, pharnesene, bisabolol oxide Methyl chavicol, anethole, camphene, ocimene, sabinene, anisol

Cultivated in the temperate regions, too. Cultivated in the Mediterranean, too.

b

109

110

6. Distribution of aromatic plants in the world and their properties

Present status and conservation initiatives Many initiatives have been taken from researchers, scholars and scientists all over the world for the conservation efforts of plants, and a decent number of conferences have been organized for the discussion and deliberation on the same. Research institutes dedicated to aromatic and medicinal plants have been doing breakthrough research to unravel the active ingredients present in plants. Every year new species of plants are being discovered but at the same times, many plant species were lost due to extinction. Rapid urbanization, industrialization and the growing pollution are contributing to deforestation and habitat loss along with making the natural habitats of the plants no more sustainable by causing change in the chemical profile of the soil and water. The Global Strategy for Plant Conservation (GSPC), with its 16 plant conservation targets which was originally adopted by the Parties to the Convention on Biological Diversity (CBD) in 2002 and has targets set to be achieved by 2020. With the GSPC reaching the end of its second phase in 2020, it is important to consider how plant conservation can enhance its visibility and generate support in the future. The 2030 Sustainable Development Agenda and associated Sustainable Development Goals (SDGs) were developed to succeed the Millennium Development Goals and were adopted in 2015 by the international community through the United Nations. It is projected that the SDGs will provide concrete shape to the actions taken by the governments in the future. The SDG framework delivers a supportive point of reference to show the fundamental importance of plants for the planet, and the achievement of the GSPC targets up to and beyond 2020 and can play a valuable and sometimes a central role in helping the achievement of several SDGs. In an up to date review about Botanic Gardens Conservation International (BGCI, 2019) it has been reported how and where plant conservation actions contribute to the achievement of the SDGs. While the closest linkages are with SDG15 e Life on Land e there are also clear linkages with goals to end poverty and hunger and to ensure good health (SDGs 1, 2 and 3), as well as those focusing on clean water, renewable energy, sustainable cities, responsible consumption and climate action (SDGs 6, 7, 11, 12 and 13). The above data are shown in Fig. 6.5.

FIGURE 6.5

Number of CITES listed species of plants in each country (World’s Plants Report, 2017).

Conclusion and way forward

111

Trade in rare species of plants shows no signs of abating and cites plant trade into the EU in 2014 was worth US$ 286 million, with 2320 plant taxa imported. The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) is known as the Washington Convention due to an international agreement between governments. Its aim is to ensure that international trade does not threaten the survival of plants and animals that are traded. Every 3 years, there is a meeting conducted between 183 countries (or Parties to the Convention) to add, delete or amend species listings on the CITES Appendices. CITES regulates the trade in endangered plant species under three Appendices, and species must meet certain biological and trade criteria in order to be listed. As a result of CoP17, an additional 304 species have been added to the over 31,517 plant species currently listed on the CITES Appendices. In EU member states, these new CITES listings are legally enshrined by a new EU regulation, and there are similar laws in other countries (World’s Plants Report, 2017). However, it must be noticed that there is a need, along with international efforts, the local governments to take further concerted efforts for protection and conservation of aromatic plants. It should be also founded a digital repository of all such information which can be accessed by researchers all over the world in order collaborative research projects could be developed to advance research on the topic. Aboriginal and tribal population in many parts of world have better access to information about locally and wildly available aromatic and medicinal plants and they tapping their traditional knowledge can be of great help in documenting these plants as well. However, they are in need to be assisted to develop such foundations or projects to carry of efforts of both preservation and development of the sector of aromatic and medicinal plants. Moreover, the intellectual property rights benefit can be helpful for these communities to economically uplift them.

Conclusion and way forward The development of tools to understand nature in a better way provided by scientists has given human race an opportunity to understand the hidden treasure in the natural resources. The ancient knowledge of Ayurveda with modern research has provided the right tool to enlighten the beneficial properties of the phytoconstituents to this world by scientific processing and efficacy evaluation of different herbs. The aggressive and extensive trade of aromatic plants in the world has become a prime threat to the survival of aromatic plants. The excessive dependence on wild harvest and very less organized cultivation of aromatic plants have become a prime threat to the survival of aromatic herbs. For effective conservation and management of aromatic plants, there is a need for proper identification and exploring biogeographic distribution. We can also use the scientific tools like global positioning system (GPS) mapping in order to accurately study the loss of endangered species and incidences of plant invasion. This investigation will provide a plan that is necessary for creation of successful strategies for the protection of important aromatic plants, which are on risk of extinction. Active contribution of all the stake holders and use of modern technological intervention can effectively manage the aromatic plant resources in the world. Aromatic plants have

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been identified since ancient time and have been in use for the benefits of humankind. Since last 500 years taxonomists, botanists, and various scientists have attempted to arrive at standard classification for aromatic plants. Aromatic plants have been classified along with medicinal plants due to their similar nature, property and usage. However, due to lack of coordination and international initiatives for a common/standard classification of aromatic plants as well as due to the vast geographical diversity of the natural habitats of aromatic plants, it has not been possible to arrive at up to date distribution pattern of aromatic plants. It is necessary that information related to aromatic plants, represented by many different concepts that is scattered among many information resources presenting end users with an indomitable task of obtaining comprehensive information to be aggregated in a new global base. A holistic scientific approach is required to taxonomically document the aromatic plants from all over the world growing domestically as well as in the wild and make a repository of active ingredients with detailed phytochemical and genomic analysis. Aromatic plants may have some unexplored medicinal properties which may be of great use to the humankind in near future and to tap the potential of aromatic plants we need a coordinated approach and interdisciplinary research initiatives. In conclusion. aromatic plants are known to possess distinctive and mostly pleasant aroma and have been used since ancient times for the variety of purposes such as in medicines and perfumery and used as decoctions, powder and other forms. Aromatic plants possess odorous volatile substances, which occur as an essential oil, gum exudate, balsam and oleoresin in one or more parts of the plant, namely in root, wood, bark, stem, foliage, flower or fruit. Many of them are exclusively used for medicinal purposes in aromatherapy as well as in various systems of medicine. They have been even mentioned in ancient texts. The term essential oil is concomitant to fragrance or perfumes because these fragrances are only oily in nature and they represent the essence of the active constituents of the plants. They are called volatile or ethereal oils as they evaporated when exposed to air at ordinary temperature. Essential oils are highly concentrated, low volume, high-value products. As such, they have high trade value that has been tremendously increased in the current century. Aromatic plants have been classified along with medicinal plants as MAP. Most of the aromatic plants have been growing in wild and only a limited knowledge of the aromatic plants which have been domesticated is available till now. The taxonomic and geographical classification of aromatic plants needs more impetus. Strategic policy interventions are required along with the scientific coordination of researchers across the world for the conservation efforts of aromatic plants. The last two decades have seen a substantial increase in the use of aromatic herbs and essential oils and aromatherapy has been promoted as a natural therapy system. The antimicrobial effects of several essential oils and oil compounds against enteropathogenic organisms were proven in recent investigations. In addition, the antioxidative activity of aromatic plants and essential oil compounds contributes to the stability and cosmetics and has resulted in improved shelf life and quality of products, due to reduced oxidation. Nonetheless, the overall efficacy of essential oils and aromatic herbs, especially their nonnutritive value with impact on the health status and benefit of animals and humans, is encouraging further research and development in this field.

References

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References Alamgir, A.N.M., 2017. Therapeutic use of medicinal plants and their extracts. Prog. Drug Res. 1, 73. Baricevic, D., Mathe, A., Bartol, T., 2015. Conservation of wild crafted medicinal and aromatic plants and their habitats. In: Máthé, Á. (Ed.), Medicinal and Aromatic Plants of the World, vol. 1. Springer, Amsterdam, The Netherlands, pp. 131e144, 7. Baser, K.H.C., Franz, C., 2009. Essential oils used in veterinary medicine. In: Hüsnü, K.C.B., Buchbauer, G. (Eds.), Handbook of Essential Oils, second ed. CRC Press, pp. 655e668. Bernath, J., 2009. Cultivated plants, primarily as food sources. In: Fuleky, G. (Ed.), Aromatic Plants, 329, vol. 2. UNESCO-EOLSS Publishers Co Ltd., Oxford, UK. Botanic Gardens Conservation International, 2019. Accessible at: https://www.bgci.org/poli-cy/plantconservationsdgs/. Bussmann, R.W., Markets, D.S., 2009. Healers, vendors, collectors: the sustainability of medicinal plant use in Northern Peru. Mt. Res. Dev. 29 (2), 128e134. Cetinkaya, G., 2010. Conservation and sustainable wild-collection of medicinal and aromatic plants in Keoprülü Kanyon National Park, Turkey. J. Med. Plants Res. 4 (12), 1108e1114. Collins English Dictionary, 2018. Accessible at: https://www.collinsdictionary.com/dictiona-ry/english/aromatic. Christaki, E., Bonos, E., Giannenas, I., Florou-Paneri, P., 2012. Aromatic plants as a source of bioactive compounds. Agriculture 2, 228e243. https://doi.org/10.3390/agriculture2030228. Dimitrova, Z., 1999. The History of Pharmacy. St Clement of Ohrid, Sofija, pp. 13e26. Farnsworth, N.R., Soejarto, D.D., 1991. Global importance of medicinal plants. In: Akereb, O., Heywood, V., Synge, H. (Eds.), Conservation of Medicinal Plants, 362. Cambridge University Press, Cambridge. Giannenas, I., 2008. How to use plant extracts and phytogenics in animal diets. In: Binder, E.M., Schatzmayr, G. (Eds.), World Nutrition Forum, the Future of Animal Nutrition. Nottingham University Press, Nottingham. Guenther, E., 1952. The Essential Oils, vol. 5. Van Nostrand Co. Inc., New York. Giannenas, I., Bonos, E., Christaki, E., Florou-Paneri, P., 2013. Essential oils and their applications in animal nutrition. Med. Aromat. Plants 2, 1e12. Hamilton, A.C., 2004. Medicinal plants, conservation and livelihoods. Biodivers. Conserv. 13 (8), 1477e1517. Inoue, M., Hayashi, S., Craker, L.E., 2017. Culture, History and Applications of Medicinal and Aromatic Plants in Japan, vol. 5, pp. 95e110. International Trade Centre, 2018 accessible at: http://www.intracen.org/itc/sectors/medicinal-plants/. Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press, Boca Raton. Jeliazkov, V.D., Cantrell, C.L., 2016. Overview of medicinal and aromatic crops, medicinal and aromatic crops: production, phytochemistry, and utilization. In: ACS Symposium Series. American Chemical Society, Washington, DC. Joy, P.P., Thomas, J., Mathew, S., Jose, G., Joseph, J., 2001a. Aromatic plants. In: Bose, T.K., Kabir, J., Das, P., Joy, P.P. (Eds.), Tropical Horticulture, vol. 2. Naya Prokash, Calcutta, India, pp. 633e733. Joy, P.P., Thomas, J., Mathew, S., Skaria, B.P., 2001b. Medicinal plants. In: Bose, T.K., Kabir, J., Das, P., Joy, P.P. (Eds.), Tropical Horticulture. Naya Prokash, Calcutta, pp. 449e632. Joy, P.P., Thomas, J., Mathew, S., Jose, G., Johnson, J., 2014. Aromatic and Medicinal Plants, Research Station. Odakkali Asamannoor e 683 549, Kerala, India. Lange, D., 1998. Europe’s Medicinal and Aromatic Plants: Their Use, Trade and Conservation. TRAFFIC, Europe. Lee, S., Xiao, C., Pei, S., 2008. Ethnobotanical survey of medicinal plants at periodic markets of Honghe Prefecture in Yunnan Province, SW China. J. Ethnopharmacol. 117, 362e377. Maiti, S., Geetha, K.A., 2007. FLORICULTURE (Ornamental, Medicinal & Aromatic Crops)-Medicinal and Aromatic Plants in India. Mathe, A., 2015. Botanical Aspects of Medicinal and Aromatic Plants, Medicinal and Aromatic Plants of the World, vol. 1, pp. 13e33. Ch: 2. Maundu, P., Kariuki, P., Eyog-Matig, O., 2006. Threats to medicinal plant speciesdan African perspective. In: Miththapala, S. (Ed.), Conserving Medicinal Species: Securing a Healthy Future, 184. IUCN: Ecosystems and Livelihoods Group, Asia, pp. 47e63. McGaw, L., Jäger, A., Grace, O., Fennel, C., van Staden, J., 2005. Medicinal plants. In: van Niekerk, A. (Ed.), Ethics in AgricultureeAn African Perspective. Springer, Nether-lands, pp. 67e83.

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Medicinal and Aromatic Plants, 2018. Accessible at: https://www.omicsonline.org/scholarly/-aromatic-cropsjournals-articles-ppts-list.php. Milton-Edwards, B., 2003. Iraq, Past, Present and Future: A Thoroughly-Modern Mandate? History & Policy, United Kingdom. History & Policy. Minarchenko, V., 2011. Medicinal plants of Ukraine: diversity, resources, legislation. In: Medicinal Plant Conservation, Medicinal Plant Specialist Group of the IUCN Species Survival Commission, pp. 7e13. Ottawa, Canada. Orto Botanico di Napoli, 2018. Università degli Studi di Napoli Federico II. Accessible at: http://www.ortobotanico. unina.it/ApprofondimentiE/PianteOfficinali_aromaticheE.htm. Orzechowski, A., Ostaseweski, P., Jank, M., Berwid, S.J., 2002. Bioactive substances of plant origin in food-impact on genomics. Reprod. Nutr. Dev. 42, 461e477. Petrovska, B.B., 2012. Historical review of medicinal plants’usage. Pharmacogn. Rev. 6 (11), 1e5. Sati, V.P., 2013. Cultivation of medicinal plants and its contribution to livelihood enhancement in the Indian Central Himalayan Region. Adv. Med. Plant. Res. 1 (1), 17e23. Shankar, D., Majumdar, B., 1997. Beyond the biodiversity convention: the challenge facing the biocultural heritage of India’s medicinal plants. In: Bodeker, G., Bhat, K.K.S., Burley, J., Vantomme, P. (Eds.), Medicinal Plants for Forest Conservation and Health Care. FAO, Rome, pp. 87e99. Non-wood forest products 11. Shukla, Y.M., Dhruve, J.J., Patel, N.J., Bhatnagar, R., Talati, J.G., Kathiria, K.B., 2009. Plant Secondary Metabolites. New India Publishing, pp. 2009e2306. Tandon, V., 2006. The risks of the loss of medicinal plants for livelihood and health security in South Asia. In: Miththapala, S. (Ed.), Conserving Medicinal Species: Securing a Healthy Future, 184. IUCN: Ecosystems and Livelihoods Group, Asia, pp. 32e39. The Columbia Electronic Encyclopedia, 2013. Columbia University Press, Licensed from Columbia University Press. Accessible at: www.cc.columbia.edu/cu/cup/. The Great Soviet Encyclopedia, 1970e1979, 2010. The Gale Group. Accessible at: https://-encyclopedia2. thefreedictionary.com/AromaticþPlants. Van Andel, T., Myren, B., Van Onselen, S., 2012. Ghana’s herbal market. J. Ethnopharmacol. 140, 368e378. Weber, S.A., 1991. Plants and Harappan Subsistence. Westview, Washington State University. World’s Plants Report, 2017. Royal Botanic Gardens, Kew accessible at: https://stateofthe-worldsplants.org/2017/ report/SOTWP_2017.pdf. Xiao, P., 1991. The Chinese approach to medicinal plantsdtheir utilization and conservation. In: Akerele, O., Heywood, V., Synge, H. (Eds.), The Conservation of Medicinal Plants. Cambridge University Press, Cambridge, pp. 305e313.

C H A P T E R

7 Herbal extracts as antiviral agents A.R. Yasmin1, S.L. Chia2, Q.H. Looi3, A.R. Omar1, 3, M.M. Noordin1, A. Ideris1 1

Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; 3Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia 2

O U T L I N E Introduction

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Poultry Newcastle disease virus Aloe species Azadirachta indica (neem) Commiphora swynnertonii (Burtt) Avian influenza virus Camellia sinensis (green tea) Eugenia jambolana Lam NAS preparation Echinacea purpurea (purple coneflower) Sambucus nigra L. (elderberries) Infectious bursal disease virus (IBDV) Ocimum sanctum and Argemone mexicana

116 116 118 118 118 119 119 119 120

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00007-8

120 120

120 121

115

Combined extracts of rhizoma Dryopteridis crassirhizomatis and Fructus mume (RDCFM) Other medicinal plants

121 121

Swine 121 Swine flu 122 Liquorice 122 Giloy (Guduchi) 122 Neem 123 Ginger 123 Garlic 124 Porcine circovirus (PCV) 125 Sickle-leaved hare’s ear 125 Porcine epidemic diarrhea virus (PEDV) 125 Horny goat weed 125 Porcine reproductive and respiratory syndrome virus (PRRSV) 126

Copyright © 2020 Elsevier Inc. All rights reserved.

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Tea seed Ruminants Foot and mouth disease (FMD) Kombucha Honey Bovine viral diarrhea

Basil Peste des petits ruminants Goat weed

126 126 127 127 127 127

128 128 128

Conclusion

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Introduction The use of plants as traditional medicine against viral diseases in the production of animals have been described and practiced worldwide. The use of herbs and their extracts as antiviral agents began following World War II in Europe, and the research was later developed worldwide. Briefly in this chapter, we will discuss common herbal extracts used as antiviral agents in treating or preventing virus diseases of farm animals such as poultry, swine, and ruminants (Table 7.1).

Poultry The poultry industry is one of the most important agricultural industries, providing food to almost 7 billion people worldwide. The demand for chicken meat has been steadily increasing and is expected to reach 131,607.3 thousand tonnes in the year 2026 (data obtained from https://data.oecd.org/agroutput/meat-consumption.htm). Disease causing microorganisms in the poultry industry includes various virus, bacteria and protozoa. The most challenging pathogens among these is the virus pathogen which continue to emerge through various genetic modification such as mutations, recombinations or co-evolution with vaccines (Bagust, 2008). The most destructive avian viral diseases are Newcastle disease virus (NDV), avian influenza virus (AIV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), egg drop syndrome avian adenovirus, and fowl pox virus. Vaccination programmes against these viruses has been applied in many countries worldwide (Marangon and Busani, 2006). However, the problems arise from backyard-reared chicken infections, which are normally not vaccinated, but still prevalent, leading to the spread of the virus that eventually causes outbreak in the community (Bagust, 2008). Modern treatments of the infected avian species are laborious and expensive. Treatments with medicinal plants have been practiced traditionally to overcome the virus infection. In this section, we will briefly discuss three major avian viruses, i.e., NDV, AIV, and IBDV, in terms of disease symptoms, some medicinal plants used for the treatment, and their mechanism of action where available.

Newcastle disease virus Newcastle disease virus (NDV) is an avian paramyxovirus type-I (APMV-1) under the Genus Avulavirus, Subfamily Paramyxovirinae, Family Paramyxoviridae, and Order

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TABLE 7.1

List of herbal extracts acting as antiviral agents against viral diseases of livestock.

No.

Species

Common diseases

Examples of herbs

1

Poultry

Newcastle disease

• • • •

aloe species Azadirachta indica (neem) Commiphora swynnertonii (Burtt) others

Avian influenza

• • • • • •

Camellia sinensis (green tea) Eugenia jambolana Lam NAS preparation Echinacea purpurea (purple coneflower) Sambucus nigra L. (elderberries) others

Infectious bursal disease

• Ocimum sanctum and Argemone mexicana • combined extracts of rhizoma Dryopteridis crassirhizomatis and Fructus mume (RDCFM) • others

Swine flu

• • • • •

Porcine circovirus (PCV) related diseases

• sickle-leaved hare’s ear

Porcine epidemic diarrhea (PED)

• horny goat weed

Porcine reproductive and respiratory syndrome (PRRS)

• tea seed

Foot and mouth disease (FMD)

• kombucha • honey

Bovine viral diarrhea

• basil

Peste des petits ruminants

• goat weed

2

3

Swine

Ruminant

liquorice giloy neem ginger garlic

Mononegavirales (Yusoff and Tan, 2001). The virus infects more than 50% of the bird order and is one of the most common viral diseases of avians. The virulence of the virus is categorized into three pathotypes: the lentogenic strain (used as vaccine strain) that causes asymptomatic infection; the mesogenic strain that causes respiratory infection with moderate mortality; and the velogenic strain that causes gastrointestinal lesions (viscerotropic) or neurological infection resulting in 100% mortality. Vaccination of NDV is practiced in the commercial poultry industry in many countries especially in South East Asia, as the virus is endemic in these countries. Nevertheless, the backyard flocks have not been vaccinated against NDV, leading to the sporadic outbreaks consecutively over the years. Several medicinal plants have been used by farmers/owners in treating diseased birds as discussed in the following sections.

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Aloe species Although various species of aloe have been used against avian diseases including NDV, scientific analysis of these aloe species in the treatment of NDV is still poor. In 2002, Waihenya et al. (2002) evaluated the efficacy of the Aloe secundiflora crude extract on experimentally NDV-infected chicken. Four-month-old chickens, free of NDV antibodies, were used. Results showed that there were no significant differences in the final mortality rates between the treated and untreated chickens. Apparently, the survival of the infected chicken depended mainly on the antibody levels, on which the aloe has no significant effect. The aloe, however, could be a potential candidate on the management rather than the treatment of NDV. Abd-Alla et al. (2012) used various parts of Aloe hijazensis against hemagglutinating viruses such as NDV, avian influenza virus type-1, and egg-drop syndrome virus (EDSV) in specific-pathogen-free (SPF) chicken embryos. They reported that the flowers and leaves of A. hijazensis showed relatively higher antiviral activity than other parts of the plant. Azadirachta indica (neem) Neem, scientifically known as Azadirachta indica, has been shown to demonstrate a wide variety of therapeutic effects including antimicrobial activities (Kumar and Navaratnam, 2013; Gupta et al., 2017). The use of this plant in treating infections of various viruses such as poliovirus, bovine herpesvirus type-1, duck plague virus, and herpes simplex virus type-I has been reported [reviewed Kumar and Nayaratnam, 2013]. In a study by Helmy et al. (2007), the authors showed that neem extract from various parts of the plant exhibited antiviral activities with an IC50 of 4e8 mg/500 EID50 in embryonated chicken SPF eggs. However, detailed mechanisms of action were not known. In 2014, Gupta et al. investigated the immunological aspect of NDV-infected cells treated with neem leaves in vitro and in vivo. They found that neem leaf extract significantly reduced the NDV-stimulated splenocyte proliferation in mice to the level comparable to the uninfected control. This suggested that the extract demonstrated anti-NDV activity. Commiphora swynnertonii (Burtt) Commiphora swynnertonii is a tropical tree that is widely distributed in Asia and Africa. The plant has been shown to demonstrate antifungal, antiectoparasite, and antiviral activities. Bakari et al. (2012) investigated the antiviral properties of this plant against NDV. Various parts of the plant were extracted using DMSO and co-incubated with NDV prior to injecting into the embryonated eggs. At concentrations of 250 and 500 mg/mL, the survivability of the embryo has greatly improved and the hemagglutinin (HA) titer of these embryonated eggs were substantially reduced. The authors further investigated the efficacy of resinous extracts against NDV in chicken (Bakari et al., 2013). The extracts, at various concentrations, were given to the chicken before the challenge (prophylactic effect) or after the challenge (therapeutic effect). Results showed significant reduction in clinical symptoms or severity as well as in antibody titers in the chicken resulting to lower mortality rate. These findings indicate that the resinous extract has strong antiviral activity against NDV in chicken.

Poultry

119

Avian influenza virus Avian influenza virus (AIV) belongs to the family of Orthomyxoviridae. The virus has negative, single-stranded, eight segmented RNAs encoding for at least 11 viral proteins. The virus is divided into subtypes based on the HA and neuraminidase (NA) proteins on the virus surface responsible for the attachment and release of the virus, respectively (Abdelwhab and Hafez, 2012). Birds are the natural host for all the subtypes of AIV. Reassortment of the HA and NA with other subtypes could result in catastrophic pandemics in humans such as H1N1, H2N2, and H3N2 (Abdelwhab and Hafez, 2012). The virus is classified into highly pathogenic avian influenza virus (HPAIV) and low pathogenic AIV (LPAIV) based on its pathogenicity in poultry. Vaccination against AIV has not been very successful as multiple subtypes are co-circulating (i.e., H5, H7, and H9); hence, vaccination against multiple HA subtypes are required. In addition, immune pressure leading to the increased evolution rate of the virus is also one of the factors rendering the effectiveness of the vaccine. Treatments such as M2 blocker and neuraminidase inhibitors have been used to disrupt the lifecycle of the virus during infection. Alternatively, medicinal plants could be used to overcome the infection. Camellia sinensis (green tea) Catechins are the major phenolic compounds in green tea that exhibit antiviral and antimicrobial activities. In 2005, Song et al. (2005) showed that the epigallocatechin gallate (EGCG), epicatechin gallate (ECG), and epigallocatechin (EGC) could inhibit various stages of AIV replication. EGCG and ECG effectively inhibit the attachment of virus by interacting with the hemagglutination protein. Kim et al. (2013) reported similar results where they showed that EGCG did not affect the binding of the protein to receptors, but inhibited the hemifusion event between virus and the cellular membrane. In addition, catechins were found to suppress the RNA synthesis by inhibiting the endonuclease activity of AIV RNA polymerase (Song et al., 2005; Kuzuhara et al., 2009). Catechins also inhibit the release of the virus by interacting with the neuraminidase protein (Song et al., 2005). The authors proposed that the 3-galloyl group of the catechin skeleton might play an important role on the antiviral activity. Lee et al. (2012b) investigated the potential of antiinfluenza virus activity of green tea by-products against AIV infection in chicken. Antiviral effects, in a dose-dependent manner, were observed when the chickens were fed with 10 g/kg of feed. Significant reduction in the number of SPF chickens that shed AIV in cecal tonsil after the virus challenge was also observed. The authors suggested that green tea byproduct extract inhibits AIV effectively in the chicken intestines. Eugenia jambolana Lam Eugenia jambolana is a large evergreen tree that is widely distributed throughout India, Sri Lanka-Malaya, and Australia. Various parts of the tree such as the bark and leaves have been reported to be used against chronic diarrhea, dysentery, sore throat, as well as antibacterial and antiviral infections (Sood et al., 2012). They tested the antiviral activity of the leaves and bark crude extracts on HPAIV H5N1. The extracts demonstrated 100% inhibition of cytopathic effect (CPE) of the virus on MadineDarby Canine Kidney (MDCK) cells, further confirmed with up to 99% reduction in virus production in embryonated eggs.

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NAS preparation NAS preparation is a Chinese medicine prepared by the Yunnan eco-agricultural research institute consisting of Hedyotic diffusa, a common stonecrop herb, Abrotani herba, Folium isatidis, etc. (Shang et al., 2010). They infected 28-day-old chickens with H9N2 followed by a fourday treatment with different concentrations of NAS 48-hour postinfection. They showed that the chickens were free from AIV on the seventh day postinfection (five days after the first treatment), whereas the virus is still detectable in the positive control and the adamantanamine control group nine days postinfection. This finding suggested that NAS could be a potential drug candidate to control H9N2 subtype of AIV in chickens. Echinacea purpurea (purple coneflower) Echinacea contains compounds such as caffeic acid derivatives, alkylamides, and polysaccharides that have been shown to exhibit antimicrobial, antioxidant and immunomodulatory activities in vitro and in vivo (Hudson, 2012). The antiviral activity of this compound against AIV in embryonated chicken eggs was investigated by Karimi et al. (2014). They found that AIV was significantly neutralized by the extract when mixed prior to inoculation into the chicken embryos. Similar results were obtained when they performed qRT-PCR of the viral gene to determine the virus titer. However, the antiviral activity was not effective when the virus has entered the cells suggesting the extract exerts its activity by inhibiting the receptor-binding activity of the virus. Sambucus nigra L. (elderberries) Sambucus nigra L., or commonly known as black elderberries, exhibits antimicrobial, antioxidant, antiinflammatory effect in vitro and in vivo (Krawitz et al., 2011). Elderberries are rich in phenolic acids, flavonoids, catechins, and proanthocyanidins, of which some flavonoids have been shown to significantly block influenza virions in vitro (Roschek et al., 2009). Like the findings of Echinacea, Karimi et al. (2014) found that the compound inhibited the virus penetration when the extract and virus were mixed prior to inoculation but no effect was observed after the virus has entered the cells. Molecular detection of the virus such as qRT-PCR of the viral gene showed that the virus titer was significantly reduced.

Infectious bursal disease virus (IBDV) Infectious bursal disease virus (IBDV) is a member of the family Birnaviridae and genus Avibirnavirus with a nonenveloped double stranded RNA virion. Infection of the virus in three- to six-week-old chickens lead to the lymphoid depletion in the bursa of Fabricius resulting in immunosuppressive disease. The virus is categorized into three groups based on its virulence, i.e., mild strain, virulent strain (30%e60% mortality rate), and very virulent (more than 70% mortality rate). Like other poultry diseases, chickens are exposed and susceptible to IBDV infection, which causes substantial economic losses in the poultry industry. Vaccination against IBDV has been practiced worldwide. However, without proper biosecurity and effective vaccination program, the disease will still recur in the farm (Bagust, 2008). Currently, there is no effective treatment for IBDV infection. Researchers are constantly searching for medicinal plants alternatives in hope to obtain a therapeutic compound to inhibit the replication of the virus.

Swine

121

Ocimum sanctum and Argemone mexicana Ocimum sanctum and Argemone mexicana extracts have been reported to exhibit antiviral activities. Varshney et al. (2013) used the leave extracts of these plants to determine their antiviral effects in vitro and in vivo. At various concentrations, the extracts successfully reduce or inhibit the CPE of IBDV- and NDV-infected chick embryo fibroblasts (CEF). In addition, lower HA titers were also detected compared to that of the untreated control. The extracts were fed to chicken for 21 days at 250 mg/kg followed by IBDV and NDV challenge orally on day 22. Overall, the treated groups showed better protection with reduced clinical symptoms compared to untreated. Combined extracts of rhizoma Dryopteridis crassirhizomatis and Fructus mume (RDCFM) Rhizoma Dryopteridis crassirhizomatis (RDC) and Fructus mume (FM), separately, have been shown to exhibit antiviral properties previously (Lee et al., 2008; Yingsakmongkon et al., 2008). The combinatorial therapeutic effects of RDCFM on SPF chickens infected with IBDV were evaluated by Ou et al. (2013). The authors showed that the survival rate of the IBDV-challenged chicken (102.5 EID50) increased from 66.7% to 75% when treated with various concentrations of RDCFM compared to 50% of untreated control. Quantification of IBDV using real time RT-PCR showed that the virus loads in the bursa of Fabricius were significantly lowered in the treated group compared to the untreated group, which is also correlated to the IBDV antibody level. Other medicinal plants Chinese herb medicine consists of dipotassium glycyrrhizinate and ligustrazine hydrochloride was found to inhibit IBDV by inhibiting virus replication and/or inactivating virus directly in CEF cells (Sun et al., 2013). Alkaloids from the fruit pulp of Cucumis metuliferus, at concentration above 6.25 mg/mL were also found to inhibit the CPE of the virus on CEF cells (Anyanwu et al., 2017). Root extract of Withania somnifera (Ashwagandha plant) were tested to determine its anti-IBDV properties. Results showed up to 99.9% decrease in virus titer when IBDV-infected CEF was treated with W. somnifera at 25 mg (Pant et al., 2012). In addition, the leaves of M. oleifera Lam (MOL), dried fruits of Phyllanthus emblicus Linn (PEL), roots of Glycyrrhiza glabra Linn (GGL), and the leaves of E. jambolana Lam (EJL), at various concentrations, significantly inhibit IBDV in Vero cells (Ahmad et al., 2014).

Swine World pig meat production has nearly doubled over the last 20 years (Godfray et al., 2010). However, following such development, a wide range of diseases associated with reduced efficiency of food conversion, growth rates and significantly increased mortality rate, and continuous medication requirement in pigs have been reported in various countries. To make it worse, many established pig production systems are currently operating based on traditional farrow-to-finish systems, which involved mixing of age groups and minimal management opportunity, thus, increasing the risk of new diseases outbreaks. Among the

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examples of devastating outbreaks include the porcine respiratory disease complex in the United States and several other viruses such as the influenza virus, porcine reproductive and respiratory syndrome virus (PRRSV), and coronavirus in Asian countries. Diseases were normally controlled by the combination of vaccination programs and continuous use of antibiotics in feed or water. Unfortunately, such an approach has been unsuccessful and often leads to high production costs and endemic emergence of antibiotic resistant organisms. Traditionally, natural herbals have been used for relieving and curing many symptoms arising from viral infection including cold, flu, and other virus-related diseases (Samy et al., 2008). Though the effectiveness of these treatments is unproved, scientific research demonstrated that herbal extracts exert significant antiviral properties and could potentially be used as an alternative treatment for virus-based diseases in the farming industries.

Swine flu Swine influenza virus (SIV) is a respiratory disease of swine caused by a type A influenza virus. Swine flu is an emerging viral infection that is a present global public health problem. In pigs, influenza infection causes fever, lethargy, sneezing, coughing, breathing difficulties, and decreased appetite (Shah and Krishnamurthy, 2013). Liquorice Liquorice (Glycyrrhizaglabra) has been widely used in traditional medicine to lower cholesterol levels, heal respiratory tract disorders, and to boost immunity level (Samy et al., 2008). Scientists have been rediscovering the health benefits of liquorice. Liquorice derives its flavor mainly from a sweet-tasting compound called anethole and glycyrrhizinic acid, an antiviral compound significantly sweeter than sugar (Singh et al., 2003). Powdered liquorice root is an effective expectorant to promote the secretions of sputum by the air passages and has been used to treat cough since ancient times. The roots of the plant contain many phenolic and active compounds particularly glycyrrhizinic acid, which exhibits antiviral, antiinflammatory, antioxidant, and immune-modulating properties (Kapil and Sharma, 1997). These properties allow it to be an important supplement for flu prevention. Animal studies have revealed that liquorice is capable of stopping virus replication (Nose et al., 1998; Curreli et al., 2005). One study found that liquorice root protects cells from the infection of influenza A virus, and in already infected cells, causing a drastic reduction in the number infected cells (Wagner and Jurcic, 2002). Giloy (Guduchi) Tinosporacordifolia, also called Guduchi, is an herbaceous vine of the family Menispermaceae, indigenous to the tropical areas of India, Myanmar, and Sri Lanka. Ayurvedic Medicine stated that Guduchi is able to stimulate the body’s immune system by maintaining optimal levels of white blood cells such as macrophages. The plant is also used in dyspepsia and various types of fever. Traditional medicine suggested that mixing one foot long of the Giloy herb and seven leaves of Tulsicould effectively prevent infection of swine flu (Shah and Krishnamurthy, 2013). Though the recipe was not scientifically established, compound and

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elemental analysis indicated that Guduchi contains various diterpene compounds, including tinosporone, tinosporic acid, syringen, berberine, giloin as well as polysaccharides, such as arabinogalactan polysaccharide (Subhose et al., 2005; Sanchez-Lamar et al., 1999). These compounds possess adaptogenic and immunomodulating properties. Studies showed that Guduchi extract could cause a significant increase in IgG antibodies in serum, along with macrophage activation (Fortunatov, 1952), besides promotions of humoral immunity and stimulation of cell mediated immunity (Winston and Maimes, 2007). The plant has immense potential for use against novel H1N1 flu since it is a potent immunostimulant. Neem Azadirachta indica (neem) is a tree in the mahogany family Meliaceae. Neem tree extracts has been used in the Ayurvedic tradition for thousands of years to maintain health and overall well-being. The roots, bark, gum, leaves, fruit, seed kernels, and seed oil are all used in various therapeutic preparations. Neem leaves contain a wide range of flavonoids such as quercetin as well as nimbosterol and limonoids, includingazadirachtin, nimbin, and nimbidin, which are often used as antiviral agent in natural products (Choudhary et al., 2013; Sahoo, 2015). An in vitro assay indicated that dose and concentration played an important role in neem extract’s antiviral property (Shah and Krishnamurthy, 2013). A. indica has traditionally been used as an antiviral, and animal and laboratory research has shown promising results. While researchers have still not pinpointed the exact mode of action of neem phytoconstituents, there is some evidence to show that they interfere with viral reproduction, thus minimizing the impact of viral infections (Sahoo, 2015). Thus, neem can serve as a source of promising antiviral drugs. Ginger Zingiber officinale (ginger, Fig. 7.1) belongs to the family Zingiberaceae. Z. officinalis is one of the natural remedies for swine flu prevention. Traditional medicine practitioners often used ginger to boost the body’s immunity level, relief gastrointestinal illness, cure cough and flu, antinausea, antiinflammatory, and also aid digestion. The characteristic odor and

FIGURE 7.1

and flu releive.

Ginger or known as Zingiber officinale is believed to enhance immune system and help in digestion

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flavor of the ginger root comes from the mixture of zingerone, shogaols and gingerols, and volatile oils. Ginger contains gingerol, a pungent ingredient of ginger volatile oil with sulphur-containing compounds (allicin, alliin, and ajoene), and enzymes (allinase, peroxidase, and myrosinase), which exhibited antibiotic properties (Shah and Krishnamurthy, 2013). The allicins have fibrinolytic activity that reduces platelet aggregation by inhibiting prostaglandin E2. Compounds in ginger also increase levels of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase, which stimulate inflammatory reactions triggered by viral infections. Bioactive components analysis of ginger suggested that various antiinfluenza agents are presented in ginger including TNF-a, an antiinfluenza cytokine (Chopra and Nayar, 1956). Further animal trial showed similar findings, where gingerols from ginger extract is able to increase the motility of the gastrointestinal tract and have strong antimicrobial properties (Shah and Krishnamurthy, 2013). Garlic Alium sativum, lahsan (Hindi), or garlic (English), belongs to the family Alliaceae. A. sativum has been used throughout widely both for culinary and medicinal purposes (Fig. 7.2). Furthermore, garlic is a powerful natural antibiotic. Garlic has natural antiviral, antibacterial and immune-boosting properties. Traditional medicine used garlic to treat fungal, parasitic and viral infections, including influenza viruses for the past several decades (Hornung et al., 1994). Kim et al. (2005) investigated the antiviral properties of garlic toward human cytomegalovirus (HCMV) using tissue culture technique, plaque reduction and early antigen assay. A dose-dependent inhibitory effect of garlic extract (GE) was observed when GE was applied simultaneously with HCMV (Kim et al., 2005). In addition, past in vitro research also indicated antiviral effect of garlic against parainfluenza virus type 3 and human rhinovirus type 2 (Wang et al., 2006).

FIGURE 7.2 and fungal.

Garlic or Alium sativum traditionally used to treat microbial infection such as virus, bacteria, parasite

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Porcine circovirus (PCV) Porcine circovirus is the smallest known animal virus, belonging to the genus Circovirus in the Circoviridae family (Chen et al., 2012). PCV2 was demonstrated to be a causative agent of porcine circovirus-associated disease (PCVAD), which includes porcine multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex (PRDC), congenital tremor (CT), and reproductive failure (Lv et al., 2013; Duan et al., 2014). Sickle-leaved hare’s ear Bupleurum falcatum, also known as sickle-leaved hare’s ear, is a species of flowering plant in the Apiaceae family. B. falcatum has been used in Chinese medicine for over 2000 years as a “liver tonic.” B. falcatum is commonly prescribed by both Chinese and Japanese traditional medicine doctors for inflammatory and infectious diseases. Despite the precise mechanism of action remains unclear, B. falcatum has been found to possess antiinflammatory and antiviral properties (Lee et al., 2012a). Recent study by Yang et al. (2017) reported that several major triterpenoid saponins were identified in B. falcatum extract, including saikosaponin A (SSA) and saikosaponin D (SSD). These active components are reported to impart immunomodulatory, antiinflammatory, antibacterial, antiviral, and anticancer effects (Na-Bangchang and Karbwang, 2014). Furthermore, it has been shown that SSD could exhibit an antiproliferative effect in activated T-lymphocyte, via suppression of NF-kB, NF-AT, and AP-1 signaling (Tundis et al., 2009). Many studies have recently focused on the use of Chinese herbal medicines to treat or prevent PCV2-induced health disorders (Sun et al., 2016). Existing data showed that B. falcatum could reduce PCV2-induced pathological effects, which can cause an imbalance in a variety of protein levels and cell numbers through modulating the content of immunoglobulin as well as hemoglobin (HGB) (Darwich et al., 2003; Pejsak et al., 2011; Sipos et al., 2004).

Porcine epidemic diarrhea virus (PEDV) Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, dehydration, vomiting and high mortality in the piglets (Debouck et al., 1981). Most newborn piglets infected by PEDV would die and affected pigs normally exhibit severe symptoms, like massive diarrhea and dehydration, resulting in serious economic losses to the swine industry (Turgeon et al., 1980; Knowles and Reuter, 2012). Horny goat weed Epimedium koreanum, also known as horny goat weed or Nakai, is a genus of flowering plants in family Berberidaceae. Traditionally, E. koreanum has been used as an aphrodisiac, and to treat hypotensives and neurasthenia. E. koreanum contains a lot of flavonoids including icariin, icariside II, epimedin, epimedosides, hyperoside, qercetin, and chlorogenic acid. A recent report showed icariin in E. koreanum stimulates angiogenesis (Chung et al., 2008). Other researcher reported that flavonoids and icariin of E. koreanum Nakai improved the development of osteoblast (Zhang et al., 2008). Also, icariside II was found

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to induce apoptosis in human prostate cancer cells (Lee et al., 2009). But, the antiviral effect of E. koreanum was not reported until recently (Cho et al., 2012). Cho et al. (2012) demonstrated that water extract of E. koreanum exerts a potent antiviral activity on PED virus through both in vitro and in vivo animal models. Despite the underlying mechanism of E. koreanum remained unknown; test result suggested that E. koreanum exerts antiviral effect through modulating immune responses such as macrophage and lymphocyte stimulation. Porcine reproductive and respiratory syndrome virus (PRRSV) PRRSV is one of the major swine pathogens. This virus has caused significant economic losses to the swine industry (Neumann et al., 2005). This single-stranded RNA virus can cause reproductive failures in pregnant sows, respiratory disorder in piglets, immune suppression, and various secondary infections (Rossow, 1998).

Tea seed Besides being widely utilized in culinary, traditionally, Chinese communities often used tea seed extract to treat burn injuries. Previous studies demonstrated that tea seed saponins (TS), extracted from tea seeds, possess antifungal activities (Kuo et al., 2010), antiallergic (Matsuda et al., 2010), as well as antimicrobial and cardio-protective effects (Liao et al., 2009). Hayashi et al. (2000)reported that TS could inactivate human type A and B influenza virus, yet the underlying antiviral mechanisms remain unknown. A recent study investigated the inhibitory effects of TS on PRRSV and its underlying mechanisms of action using an in vitro model system (Li et al., 2015). PRRSV infection always results in persistent transmission and secondary infection. Li et al. (2015) demonstrates that TS can effectively inhibit PRRSV replication through multiple ways, including directly inactivating the virus or blocking the virus entry into the cells, and indirectly through the modulation of Poly (A)-binding protein (PABP). Such unique properties of TS might reduce the opportunity of development of drug-resistant strains.

Ruminants Ruminants, or four-stomached animals such as cow, goat, sheep, and buffalo, differ in their digestion physiology compared to other livestock (McAnally and Phillipson, 1944). Livestock provides a huge impact on the agricultural industry as a source of protein from meat and milk in large-scale or small-scale production. However, ruminants faced a major threat due to viral infections particularly because of several factors such as stress and poor immune response. Viral infection in ruminants may affect the animal production and performance, which may contribute to low economic returns. There are several ways to overcome viral infections in ruminants such as vaccination, drugs and herbs or traditional medicine (Lin et al., 2014). Nowadays, traditional medicine becoming one of the choice as it has shown to be safer, cheaper and efficient (Jassim and Naji, 2003). In this part, common herbs that have proven to show positive effects such toward common viral diseases in ruminants such as

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foot and mouth disease (FMD), bovine viral diarrhea (BDV), and peste des petits ruminants (PPR) will be discussed.

Foot and mouth disease (FMD) Foot and mouth disease is caused by Aphthovirus which belongs under Picornaviridae family. This disease is highly contagious and considered a fatal that affects all species of ruminants and swine (Grubman and Berry, 2004). This virus is highly infectious in nature and may be spread via aerosols, inanimate objects, and close contacts (Bayry and Kavery, 2001). The affected animals usually have blister-like lesions in the oral cavity and hoof area that lead to inappetence and lameness. Most of the affected animals show a drop in milk production, and the meat is banned from exportation. Among the herbs that can be used to prevent the disease are listed in the next sections. Kombucha Kombucha is a popular Chinese traditional medicine prepared from a culture of yeast and acetobacter, fermented with black tea and Chinese herbal extracts (green tea, chrysanthemum, liquorice, and Grosvenor momordica). This concoction has been suggested as an efficient probiotic (Greenwalt et al., 2000). It is acidic in nature, ranging between pH 2.5 to 4.6 and as FMD virus is sensitive to acidic condition or low pH, this mixture has shown to be effective in preventing FMD (Teoh et al., 2004). Based on an in vitro study in BHK21 cells, the kombucha herb can inhibit the growth of FMD virus. On the other hand, in vivo study showed it was capable of killing FMD virus with no toxicity effects on mice (Fu et al., 2014). In addition, spraying cattle in an FMD outbreak zone with kombucha protected against infection in a large-scale field trial (Fu et al., 2015). Honey In Kenya, an outbreak of FMD affecting 57.2% of the cattle was treated with honey, finger millet, and 97% sodium (Gakuya et al., 2011). The lesions of the ulcers and blister improved in three days following the topical application. Honey has been in use for treatment of infected wound for more than 2000 years, even before bacteria were discovered. Honey has been reported to have inhibitory effect to around 60 species of bacteria (Molan, 1992). Honey has antibacterial properties due to the production of hydrogen peroxide, which is formed and released slowly by glucose enzymes when the honey is diluted.

Bovine viral diarrhea Bovine viral diarrhea (BVD) is a disease of ruminants caused by pestivirus, which is originated from the family Flaviviridae. There are two biotypes of the virus depending on the ability to cause CPE (Ridpath, 2010). Pestivirus causing BVD normally spreads by close contacts among cattle populations and also vertical transmission that means transfer of virus from cow to fetus is highly possible. The affected fetus may lead to stillbirths, abortions or persistent infection in the calf after being born. Some of the cattle may have mucosal lesion especially in the intestinal tract leading to profuse diarrhea and anorexia and may progress to death (Thiel et al., 1996). Basil is one of the herbs that have been shown experimentally to reduce and treat BVD.

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Basil Basil, or Ocimum basilicum L. derived from the Lamiaceae family, is used as a cooking herb (Sartoratto et al., 2004). Despite its culinary usage, basil is used as traditional medicine of choice to treat respiratory and gastrointestinal tract disorders (Chiang et al., 2005). Basil has been demonstrated in many studies to have antivirus, antibacterial, and antifungal properties (Pozzatti et al., 2008; Suppakul et al., 2003). The effects of basil and its essential oils, known as monoterpenes, were tested against BVD virus at different time points of infection. It was demonstrated that the compound works directly on the particle of the virus based on the tremendous reduction of the virus in plaque assay (Kubica et al., 2014).

Peste des petits ruminants Peste des petits ruminants (PPR) mainly infect small ruminants such as sheep and goat. The disease is caused by morbillivirus, an RNA virus. The manifestation of the disease is associated with gastrointestinal tract (GIT) and respiratory system. The clinical signs are ranging from pyrexia (fever), stomatitis (inflammation of the oral cavity) and respiratory symptoms including ocular and nasal discharge and GIT signs include diarrhea and ulcers (Parida et al., 2015). Goat weed Goat weed or Ageratum conyzoides Linn in the form of extract metabolites or oil have antiinflammatory, analgesic, antipyretic, and antidepressant effects (Shekhar and Anju, 2012). It has been used in the treatment of PPR in combination with PPR vaccine and showed prominent results (Saliu et al., 2008). In addition, metabolites and extracts of goat weed dissolved in ethanol showed spasmolytic effects and protect gastric lining from ulcers.

Conclusion The overview of the usage of traditional herbs as antiviral agents in livestock is provided in this chapter. Based on the research findings presented, in addition to being a safer and economical alternative to drugs, herbal constituents showed positive responses in animals, which suggest that it may suppress viral replication and reduce the clinical signs of viral diseases in different productive animals. Although the usage of herbs have shown profound effects in reducing viral infections, total elimination is dependent on good management practice and biosecurity of the farms.

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C H A P T E R

8 Functional ingredients derived from aromatic plants Sonia A. Socaci1, 2, Anca C. Farca¸s1, Maria Tofana1 1

Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania; 2Institute of Life Sciences, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania O U T L I N E

Introduction

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Uses and applications

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Essential oils Chemical composition of essential oils Biological activities of essential oils Antibacterial and antifungal activities of essential oils Antioxidant activity of essential oils

134 135 136

Conclusions

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Acknowledgments

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References

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Further reading

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136 139

Introduction Medicinal and aromatic plants have always played an important role in human’s history, being used in gastronomy, traditional medicine, religious rituals, and other applications. there is still a growing interest toward these plants mainly due to the consumers’ increasing awareness and demand for both food and nonfood related products that have beneficial impact on their health. in order to meet the consumers new demands and to increase market competitiveness, the food industry, is focused on finding natural compounds as alternatives to synthetic preservatives and antioxidants used in food preservation. On the other hand, in

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00008-X

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Copyright © 2020 Elsevier Inc. All rights reserved.

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the animal production sector, the prophylactic use of antibiotics in animal feed for growth improvement and decreased mortality due to clinical diseases, has raised concerns about the transmission and proliferation of resistant bacteria via the food chain, leading to the ban of antibiotic growth promoters in the feed of livestock within the European Union, since 2006. Recent guidelines published by the Food Drug Association are exerting pressure for a ban in the United States in the near future (Brenes and Roura, 2010; Bento et al., 2013). As a consequence, new food and feed additives based on natural compounds from aromatic plants, including purified compounds, plant extracts or essential oils have been developed and their efficacy assessed and compared with the already existing ones. The main advantages of these plant derived additives include the fact that they are generally recognized as safe, is more difficult for bacteria to develop resistance to multi-component essential oils or plant extracts than to common antibiotics (single molecular entity) and they have many functional activities with positive impact on both food and feed quality, and human and animal health (Calo et al., 2015). Thus, scientific research regarding the chemical composition of medicinal and aromatic plants, especially the identification of bioactive compounds that could be exploited as functional ingredients in the development of innovative products, has shown an upsurge in the last decades. The purpose of this paper is to summarize the main biological activities of essential oils and extracts from aromatic plants in correlation with their functional constituents and the possible applications regarding their use in the development of products with benefits for human and animal health.

Essential oils All plants have the ability to produce volatile compounds, but in the majority of cases these molecules are present only in traces. Around 10% of the plant species can synthetize, secrete, and store in the cytoplasm of certain secretory cells a small amount of odoriferous essence. These plants are known as “aromatic plants” or “essential oil-bearing plants”. Plants from Lamiaceae (rosemary, sage, lavender, basil, etc.), Myrtaceae (eucalyptus, clove, etc.), and Rutaceae (citrus) families are among the richest in volatile aroma compound production (Ba¸ser and Buchbauer, 2010). Essential oils are secondary metabolites produced by aromatic plants having functional roles such as: protective agents, repellants for herbivores, pheromones responsible for attracting insects in order to facilitate the pollination and allelopathic communication between plants (Rozza et al., 2012). Essential oils are viscous lipophilic liquids that concentrate the characteristic scent of the aromatic plants, having a very high composition variability, in both qualitative and quantitative terms due to environmental and genetic factors such as plant cultivar, vegetative stage, time of harvest, geographical origin, plant part used, and extraction method (Brenes and Roura, 2010; Socaci, 2017). According to European Pharmacopoeia (fifth edition, 2004), essential oils may be obtained from different parts of plants by steam or water distillation or by mechanical processes (only in the case of Citrus). Among the parts of aromatic plants used for the extraction of essential oils are: flowers (lavender, geranium, chamomille, ylang-ylang, hop, clove), leaves

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FIGURE 8.1 The main extraction techniques for essential oils and plant extracts.

(mint, thyme, sage, rosemary), rhizomes (ginger), seeds (coriander), fruits (fennel, anise, citrus epicarp), bark, and wood (cinnamon, sandalwood, rosewood) (Dhifi et al., 2016). Extracts obtained by classical extraction techniques, such as solvent extraction with different organic solvents, or by more modern ones like supercritical fluid extraction, microwave-assisted extraction, ultrasound assisted extraction, and ionic-liquids extraction, are not considered essential oils, although these extracts possess similar aroma profiles with the raw material (Ba¸ser and Buchbauer, 2010). Moreover, depending on the solvent used in the extraction process, other bioactive compounds (such as polyphenols, carotenoids, etc.) can be retrieved from the plant material. These extracts are often used in the flavor and fragrance industry. In the search of new sources of functional ingredients with beneficial health properties, plant extracts are gaining more attention from pharmaceutical and food industry. The main extraction techniques for essential oils and plant extracts are presented in Fig. 8.1.

Chemical composition of essential oils All plants produce two main classes of metabolites: primary and secondary. The primary class include chemicals that constitute the basic building blocks of life: proteins, carbohydrates, lipids and nucleic acids. Secondary metabolites are those that occur in some species and not others and they are usually classified into terpenoids, shikimates, polyketides, and alkaloids. Essential oils are complex mixtures of tens or even hundreds of volatile and semivolatile compounds, generally of low molecular weight (under 300) with a vapor pressure (at atmospheric pressure and at room temperature) high enough to be partly found in vapor state. The main components are the terpenoids followed by the shikimates and polyketides (Ba¸ser and Buchbauer, 2010). Using gas chromatography and mass spectrometry, thousands of compounds have been identified so far in essential oils, including monoterpenes (limonene, myrcene, a-pinene, b-pinene), sesquiterpenes (humulene, farnesene, cadinene) as well as functional derivatives of alcohols (linalool, geraniol, menthol, a-bisabolol), ketones (menthone, camphor, verbenone, a-thujone, 6-methyl-5-hepten-2-one), aldehydes (geranial, hexanal, citronellal, cinnamaldehyde), esters (g-terpinyl acetate, linalyl acetate, bornyl acetate), phenols (thymol, eugenol, carvacrol), lactones, acids, aliphatic hydrocarbons and rarely

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even nitrogen- or sulfur-containing compounds (Brenes and Roura, 2010; Nazzaro et al., 2013; Dhifi et al., 2016). The complexity of essential oils’ composition is responsible for their multiple biological activities (e.g., antibacterial, antifungal, antioxidant and antiinflammatory, analgesics, sedatives, spasmolytic, local anesthetics) but also makes it difficult to identify all the constituents and to explain or attribute their biological activities to a certain compound (Brenes and Roura, 2010; Bento et al., 2013; Nazzaro et al., 2013; Dhifi et al., 2016; Games et al., 2016).

Biological activities of essential oils The mechanism of action of essential oils has not been yet fully elucidated. Studies conducted over the last decades have demonstrated that the compounds present in the essential oils may act on more than one target in the cell, such as, in the permeabilization or disruption of the cytoplasmic membrane, allowing the passage of nonspecific compounds or the release of cytoplasmic contents, or they may inhibit the ATPase enzyme responsible for energy generation of the cell, leading to cell death (Zhang et al., 2016; Gouvea et al., 2017). Moreover, due to the variety of chemical structures and properties of essential oil constituents, it is fair to assume that the biological activities of essential oils cannot be attributed to a single compound or to a specific mechanism. Thus, in the case of antibacterial activity for example, it is difficult to predict the susceptibility of a certain bacterial strain to the action of essential oils. (Bento et al., 2013). The biological activities of some of the most studied essential oils are summarized in Table 8.1. Antibacterial and antifungal activities of essential oils The antibacterial and antifungal activities of essential oils were the subject of many scientific studies and demonstrated by both in vitro and in vivo experiments (Ouwehand et al., 2010; Bento et al., 2013; Nazzaro et al., 2013; Radaelli et al., 2016). The lipophilic nature of the essential oils allows them to change into lipids of the cell membrane of bacteria, disrupting the structure, and making it more permeable, enabling the passage of nonspecific compounds and causing the leakage of ions and other cellular molecules which can finally lead to loss of viability or even cell death (Dhifi et al., 2016; Gouvea et al., 2017). Generally, the Gram-positive bacteria are more susceptible to the action of essential oils than Gramnegative bacteria. This susceptibility is due to the differences in the structure of bacterial cell walls. Cell walls of Gram-negative bacteria are more complex and possess an additional outer membrane. The outer membrane of Gram-negative bacteria is almost impermeable to hydrophobic molecules while the small hydrophilic molecules can pass through via abundant porin proteins. This is one of the reasons why Gram-negative bacteria are more resistant to essential oils and other natural extracts with antimicrobial activity (Nazzaro et al., 2013; Diao et al., 2014; Zhang et al., 2016). Many essential oils were tested to demonstrate their capacity to inactivate or inhibit the development of different microorganisms. Recently, Radaelli et al. (2016), conducted a study regarding the antimicrobial activity of essential oils extracted from Ocimum basilicum L. (basil), Rosmarinus officinalis L. (rosemary), Origanum majorana L. (marjoram), Mentha
piperita L. var. Piperita (peppermint), Thymus vulgaris L. (thyme) and Pimpinella anisum L. (anise) against

TABLE 8.1

Botanical sources, extraction method and biological activities of essential oils.

Botanical origin

Extraction method

Biological activity

References

Origanum rotundifolium/ vulgare

Hydrodistillation

Antibacterial agents against plant pathogenic bacteria, antifungal and antiparasitic properties; Active antimicrobial agent in packaging films and food preservation; Protective ability against oil oxidation in food systems

Martucci et al. (2015), Adame-Gallegos et al. (2016), Gormez et al. (2016), Ghadermazi et al. (2017), Mateo et al. (2017), Granata et al. (2018)

Antibacterial agents against plant pathogenic bacteria, antifungal and antiparasitic properties; Active antimicrobial agent in packaging films and food preservation, and in biomedical applications

Martucci et al. (2015), Adame-Gallegos et al. (2016), Gormez et al. (2016), Predoi et al. (2018)

Lavandula Hydrodistillation officinalis/angustifolia

Strong antibacterial and antifungal properties; Moderate in vitro anticancer activity

Desam et al. (2017), Abdel-Hameed et al. (2018)

Rosmarinus officinalis L.

Hydrodistillation; Microwave-assisted hydrodistillation

Antioxidant and antimicrobial activity and hepatoprotective potential

Karakaia et al. (2014) Raskovic et al. (2014)

Coriandrum sativum L.

Microwave-assisted hydrodistillation; hydrodistillation

Antioxidant and antimicrobial activity

Sourmaghi et al. (2015)

Salvia officinalis L.

Hydrodistillation

Protective ability against oil oxidation in food systems

Ghadermazi et al. (2017), Pop et al. (2016)

Cinnamomum zeylanicum

Hydrodistillation

Antifungal and anti-mycotoxigenic activity

Manso et al. (2015), Mateo et al. (2017)

Thymus capitatus

Water/steam distillation

Active antimicrobial agent in polymer-based nanocapsules as with application in food preservation

Teixeira et al. (2014), Granata et al. (2018)

Eucalyptus citriodora/gillii

Hydrodistillation

Potent fungitoxic, antiaflatoxin and nonphytotoxic properties; Strong antioxidant and antimicrobial properties

Hassine et al. (2012), Sebei et al. (2015), Lu et al. (2016), Pandey et al. (2016)

Syzygium aromaticum

Water/steam distillation

Innovative food packaging systems as with application in food preservation

Teixeira et al. (2014), Ghadermazi et al. (2017)

Citrus aurantifolia/ limon/sinensis/ latifolia

Hydrodistillation steam distillation

Antifungal and food preservative activities against a wide range of microbial pathogens; antimutagenic and strong antioxidant activity

Pandey et al. (2016), Hsouna et al. (2017), Toscano-Garibay et al. (2017)

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Hydrodistillation; Microwave-assisted hydrodistillation; Solvent free microwave extraction

Essential oils

Mentha longifolia L./piperita L.

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C. perfringens strain A (one of the main causative agents of food-borne diseases). Their results showed that all tested essential oils had bactericidal or bacteriostatic activity at their minimum inhibitory concentration, making them suitable alternatives to chemical preservatives and antibiotics. Nevertheless, the chemical structure, the presence of different functional groups, the concentration, the possible synergetic effects between the essential oil constituents, are influencing their antimicrobial activity. Thus, compounds from terpenes’ group do not possess high antimicrobial activity. Limonene, a-pinene, b-pinene, g-terpinene d-3carene, (þ)-sabinene or a-terpinene showed a very low or no antimicrobial activity against 25 of bacteria strains, including Aeromonas hydrophila, Bacillus subtilis, Clostridium sporogenes, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Lactobacillus plantarum, Leuconostoc cremoris, Proteus vulgaris, Staphylococcus aureus, Salmonella pullorum, Yersinia enterocolitica (Dorman and Deans, 2000). Instead, the antimicrobial activity of terpenoids (terpenes with added oxygen molecules or removed methyl groups) is depended on their functional group (Chouhan et al., 2017). For example, p-cymene (monoterpene) has less antimicrobial activity compared to its oxygenated derivatives carvacrol and thymol, but also can enhance the inhibitory effects of carvacrol when used together (Rattanachaikunsopon et al., 2010; Marchese et al., 2017). On the other hand, it seems that, in the case of carvacrol and thymol, the location of the functional group (OH) in the molecule does not influence the antimicrobial activity. According to Ultee et al. (2002), carvacrol and its isomer thymol have similar antimicrobial activity against B. cereus, S. aureus, and P. aeruginosa. The importance of the hydroxyl group bound to a benzene ring is sustained by the fact that menthol which lack the system of delocalized electrons (double bonds) and, consequently, the inability of the hydroxyl group to release its proton, has a low antimicrobial activity. The same was observed for carvacrol methyl ester which does not have the ability to release a proton and therefore is not considered an antimicrobial. Another class of functional constituents of essential oils studied for their antimicrobial activity are the phenylpropanoids. These compounds contain a six-carbon aromatic phenol group and a three-carbon propene tail from cinnamic acid, the most known being eugenol, isoeugenol, vanillin, safrole and cinnamaldehyde. As in the case of phenolic terpenoids, the antimicrobial activity of phenylpropanoids is dependent on the free hydroxyl groups but also on the type and number of substitutions on the aromatic ring. Generally, isoeugenol is more effective against yeasts, molds and bacteria than eugenol but both exhibit higher antimicrobial activity against Gram-negative bacteria than Gram-positive bacteria. Also, usually, cinnamaldehyde is less powerful than eugenol (Nazzaro et al., 2013; Chouhan et al., 2017). Nevertheless, the research conducted by Sharma et al. (2016) regarding the major attributes of phenylpropanoids from Cinnamonum spp. showed that cinnamaldehyde had good antibacterial activity (zone of inhibition 32e42 mm) against Bacillus cereus (MTCC 6840), Streptococcus mutans (MTCC 497), Proteus vulgaris (MTCC 7299), Salmonella typhi (MTCC 3917) and Bordetella bronchiseptica (MTCC 6838), while eugenol produced moderate activity at 80 mM/disc. Their mechanisms of action are different and antimicrobial potential depends on the tested microbial strains. While cinnamaldehyde acts like a growth inhibitor of Escherichia coli and Salmonella typhimurium without disintegrating the outer membrane or depleting the intracellular ATP pool, eugenol causes deterioration of the cell wall, lysis of cells,

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prevention of enzyme action in Enterobacter aerogenes and changes in the fatty acid profile of different bacteria (Nazzaro et al., 2013; Vergis et al., 2015). Antioxidant activity of essential oils As in the case of antimicrobial activity, the antioxidant potential of an essential oil is related to its composition. The presence of phenolics and secondary metabolites with conjugated double bonds contribute to their antioxidative potential (Dhifi et al., 2016). Phenolic constituents such as thymol, eugenol, carvacrol present in essential oils from clove, nutmeg, cinnamon, oregano, thyme, parsley, basil and others, have strong free radicals scavenging capacity, making the correspondent essential oils good antioxidant agents (Vergis et al., 2015). Other nonphenolic constituents, such as b-caryophyllene, terpinen-4-ol, a-terpinene, linanlool, 1,8-cineol, geranial/neral, menthone, citronellal, etc. may also contribute to the antioxidant activity of the essential oils (Franz et al., 2010). Used in feed additives, essential oils can interfere with the lipid metabolism in the animals, improving the oxidative stability of meat obtain from broilers, hen or turkeys (Botsoglou et al., 2002, 2005; Papageorgiou et al., 2003; Giannenas et al., 2005; Govaris et al., 2005). Essential oils most used in animal nutrition due to their antioxidative and antibacterial properties are those of oregano, thyme, rosemary and sage (Zeng et al., 2015). Supplementation of chickens’ diet with oregano essential oil (100 mg/kg) may lead to an increase in antioxidative status of broiler meat, probably due to the presence of antioxidant compounds in the essential oil, that may enter the circulatory system and retain in the muscle and/or other tissues (Botsoglou et al., 2002). The same research group perform a study on 6300 one-day old Cobb-500 chicken, assessing the effect on of diet supplementation with oregano and/or a-tocopheryl acetate. Feed supplementation with oregano (5 g/kg) delayed lipid oxidation in breast and thigh meat but was less effective compared with that exhibited by a-tocopheryl acetate supplementation. An additive effect was noticed when the combination of oregano (5 g/kg) and a-tocopheryl acetate supplementation was used (Giannenas et al., 2005). The effect of supplementing the diet of food-producing animals with essential oils is reflected also on the product quality, leading to improved dietary value, better oxidative stability and longer shelf-life of fat, meat and egg (Botsoglou et al., 2005; Govaris et al., 2005). However, the efficacy of essential oils to delay the lipid oxidation in meat it seems to depend on the type of meat. In the case of pork meat, no improvement of carcass and meat quality attributes were noticed when in the animals’ feed were supplemented with different concentration of oregano essential oil (Simitzis et al., 2010). The authors suggested that the lack of effect of dietary administrated oregano essential oil may be attributed to the fact that its components didn’t penetrate into the cell phospholipids membranes in pig muscles. Nonetheless, the differences in fatty acids composition between poultry and pork meat are worth considered. Poultry meat has a lowfat content but compared with pork meat it has a relative higher content of polyunsaturated fatty acids (60% vs. 17%, of total fat content), making it more susceptible to oxidation. Therefore, the dietary essential oil supplementation may trigger a more observable response on its oxidative stability (Zeng et al., 2015). Due to their antioxidant activity, essential oils, beside their importance in food industry, they play a major role in medicine in the prevention of chronic diseases caused by oxidative

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stress, such as heart diseases, brain dysfunction, immune system decline. Sharma et al. (2016) assessed in vitro the antioxidant, anti-proliferative and antibacterial activities of two phenylpropanoids: eugenol and cinnamaldehyde. Eugenol exhibited high free radical scavenging capacity with an IC50 value of 0.495 mM/mL. Cinnamaldehyde demonstrated considerable metal ion chelating capacity as well as antiproliferative potential against breast (T47D) and lung (NCI-H322) cancer cell lines.

Uses and applications Essential oils can be used as feed additives in livestock improving feed efficiency and animal production. As growth promoters their efficacy depends on factors such as dietary form, nutrient density and composition, essential oil composition and quality, dosage, age of animals, growth performance level. In poultry, they are also used for their antimicrobial, anticoccidial and antioxidant activities and carcass hygiene. Coccidiosis is a common parasitic disease caused by protozoa of the genus Eimeria leading to malnutrition and performance depression. The use of essential oils in poultry feed may result in a decrease of coccidian oocyst excretion and alleviation of intestinal lesions, thus reducing the need for conventional coccidiostats. Still, the action mechanism remains to be elucidated, one hypothesis is that the essential oils increase the mucus secretion and the creation of a thick layer of mucus on glandular stomach and wall of jejunum, suggesting villi-related protective properties of certain essential oil compounds (Franz et al., 2010; Giannenas et al., 2005; Zhai et al., 2018). Salmonellosis is one of the most frequently reported human zoonotic disease in the EU (Bento et al., 2013). Hence, the interest of the poultry industry in finding appropriate “green” and natural solutions to reduce the incidence of Salmonella contaminated broiler carcasses. Alali et al. (2013) assessed, among other parameters, the effect of an essential oil blend (carvacrol, thymol, eucalyptol, lemon) administered in drinking water on the Salmonella enterica serovar Heidelberg fecal shedding and colonization in broiler birds. Even though the addition of essential oil did not significantly reduce Salmonella Heidelberg colonization in ceca or fecal shedding in broilers, it may control Salmonella Heidelberg contamination in crops of broilers and hence may reduce the potential for cross-contamination of the carcass when the birds are processed. In another in vivo study (Bento et al., 2013), supplementation of diets with a blend of thymol and cinnamaldehyde had a positive impact on gut microbiota, growth performance and welfare in monogastric animals as well as in maintaining the feed stability acting as repellent to flour beetles. Moreover, the tested blend improved the food safety by lowering the incidence of the horizontal transmission of Salmonella infection in the farm. The combination between thymol and cinnamaldehyde showed selective antimicrobial properties, effectively inhibiting yeast, fungi and bacteria such as Salmonella and E. coli, while sparing the ones that beneficially modulate the gut microbiota. There are already available on the market feeds for poultry and swine that are fortified with essential oils blends and that were developed based on the existing scientific data from peer review studies and trials.

Conclusions

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Lipid oxidation is a major problem encountered during meat processing and refrigeration storage affecting the quality of the product (e.g., loss of desirable color, odor and flavor) and shortening its shelf-life. Some studies have demonstrated that the addition of essential oils into animals’ diet could have positive impact on meat quality. Rivaroli et al. (2016) supplemented the diet of young bulls with different concentrations of an essential oils blend, including oregano, garlic, lemon, rosemary, thyme, eucalyptus and sweet orange. The essential oils had no effect on chemical and fatty acid composition, meat color, water holding capacity or texture, but the inclusion of 3.5 g/day into the animals’ diet lead to a decrease in lipid oxidation. In one study, the results obtained by de Oliveira Monteschio et al. (2017) showed also reduced lipid oxidation and reduced color losses in meat during shelf-life when essential oils were used in animal diets. The essential oils can also be used as food additives in different meat derived products. Nieto et al. (2013) showed that the addition of rosemary and oregano essential oils into pork patties had antioxidative effect on protein thiol loss during their chill storage. Serious illness or even death may be caused by outbreaks of food poisoning associated with consuming contaminated products with enterohaemorrhagic E. coli O157:H7. Due to their antioxidant and antimicrobial properties, functional compounds from essential oils could be used to improve the safety and the shelf-life of raw fruit juices. The addition in low concentration (0.5e1.25 mM) of carvacrol and p-cymene (individually or in combination) in unpasteurized apple juice were biocidal against both spoilage yeasts and E. coli O157:H7 when stored at chill temperatures (Kisko et al., 2005). The antimicrobial agents from essential oils can be used in antimicrobial packaging. This preservation method is an alternative to the modified atmosphere packaging or addition of synthetic preservatives in food products. In antimicrobial packaging, volatile (essential oils) or nonvolatile (phenolics extracts) antimicrobials and antioxidant agents are incorporated into packaging materials in order to decrease the growth rate and maximum growth population and/or extended lag phase of target microorganism or by the inactivation by contact. When added in the appropriate amount, beside the inhibition of food-borne pathogenic bacteria such as Listeria, Salmonella, Aeromonas, Clostridium botulinum, Enterobacter, Staphylococci, and their toxins, the essential oils can also improve the organoleptic properties of the food product (Vergis et al., 2015). Aromatic plants are a source of natural bioactive compounds that display a range of properties among which antitumoral, antibacterial, antifungal, antiinflammatory and antioxidant activities were the most studied. The use of essential oils or purified constituents for medicinal purposes has known an upsurge in the quest of finding natural and safe alternatives to drugs which present unwanted side effects. Due to their antiinflammatory and antioxidant properties, Games et al. (2016) have evaluated the potential of p-cymene, carvacrol and thymol in the treatment of chronic obstructive pulmonary disease. Their findings showed that these functional compounds can reduce lung emphysema and inflammation in mice. Other applications of essential oils or aromatic plant extracts are given in Table 8.2.

142

TABLE 8.2 Examples of applications of essential oils in food and nonfood products. Applications

References

Rosemary essential oil (215 mg/L)

Prevented the growth of Clostridium spp. in sheep’s cheese

Moro et al. (2015)

Ginger essential oil (0.4%)

Probiotic yogurt - largest counts of viable probiotic bacteria (8.01 cfu/g)

Yangilar and Yildiz (2018)

Cumin and clove essential oils (75e225 mL/g)

Inhibition of five pathogenic strains (Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, Yersinia enterocolitica, Campylobacter jejuni, Clostridium perfringens, Staphylococcus aureus and Toxoplasma gondi) in lean red meat pulp

Hernández-Ochoa et al. (2014)

Thyme essential oil (0%, 0.5%, 1%, and 2%)

Chitosan film with thyme essential oil for packaging ready-to-eat meat - reduced yeast populations, enhanced color preservation

Quesada et al. (2016)

Clove essential oil (4500 and 9000 mg/L)

Decreased the mesophilic count in watermelon juice during 7 days storage at 37 C

Siddiqua et al. (2014)

Lemon essential oil (0.08%, 0.12%, and 0.16%)

Total inhibition of the germination and outgrowth of Acinetobacter acidoterrestris in lemon juice concentrate, under refrigerated storage over 11 days

Maldonado et al. (2013)

Myrtus communis and Lavandula angustifolia essential oils

Repellent activity against Sitophilus zeamais Motsch. (Coleoptera Dryophthoridae)

Bertoli et al. (2012)

German chamomile solution (twice a day compress)

Anti-inflammatory and antipruritic effects in the treatment of peristomal skin lesions

Charousaei et al. (2011)

Pinus koraiensis essential oil

Significantly reduced proliferation and migration of HCT116 colorectal cancer cells

Cho et al. (2014)

Garlic extract (3%) and tea tree oil (0.2%)

Toothbrush disinfectants against Streptococcus mutans

Chandrdas et al. (2014)

Essential oils extracted from five Artemisia species (78.63, 15.73, 3.15, 0.63 and 0.13 nL/cm2)

Repellent activity against Tribolium castaneum (Coleoptera: Tenebrionidae)

Liang et al. (2017)

8. Functional ingredients derived from aromatic plants

Essential oils/aromatic plant extracts

References

143

Conclusions Available scientific literature on the composition, properties, and manifold applications of essential oils or their main bioactive compounds reinforce the functional plant secondary metabolites’ importance and utility. Nonetheless, a better understanding of the essential-oil mechanisms of action and metabolism in animals is still needed as well as the elucidation of their synergistic or antagonist effects when used in blends and their interactions with other compounds. Even though there are studies on the benefits that essential oils could bring to animals’ or humans’ health, the research in this area requires further in-depth analysis, considering that the proven in vitro effect does not always translate into the same in vivo effect.

Acknowledgments This work was supported by two grants of Ministry of Research and Innovation, CNCS - UEFISCDI, project number PN-III-P1-1.1-TE-2016-0973 and project number PN-III-P1-1.1-PD-2016-0869 within PNCDI III.

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Cho, S.M., Lee, E.O., Kim, S.H., Lee, H.J., 2014. Essential oil of Pinus koraiensis inhibits cell proliferation and migration via inhibition of p21-activated kinase 1 pathway in HCT116 colorectal cancer cells. BMC Complement Altern. Med. 14, 275. https://doi.org/10.1186/1472-6882-14-275. Chouhan, S., Sharma, K., Guleria, S., 2017. Antimicrobial activity of some essential oilsdpresent status and future perspectives. Medicines 4 (58). https://doi.org/10.3390/medicines4030058. Desam, N.R., Al-Rajab, A.J., Sharma, M., et al., 2017. Chemical composition, antibacterial and antifungal activities of Saudi Arabian Mentha longifolia L. essential oil. J. Coast. Life Med. 5 (10), 441e446. Dhifi, W., Bellili, S., Jazi, S., et al., 2016. Essential oils’ chemical characterization and investigation of some biological activities: a critical review. Medicines 3 (25). https://doi.org/10.3390/medicines3040025. Diao, W.R., Hu, Q.P., Zhang, H., Xu, J.G., 2014. Chemical composition, antibacterial activity and mechanism of action of essential oil from seeds of fennel (Foeniculum vulgare Mill.). Food Control 35, 109e116. Dormans, H.J.D., Deans, S.G., 2000. Antimicrobials agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88, 308e316. Franz, C., Baser, K.H.C., Windisch, W., 2010. Essential oils and aromatic plants in animal feeding e a European perspective. A review. Flavour Fragrance J. 25, 327e340. Games, E., Guerreiro, M., Santana, F.R., et al., 2016. Structurally related monoterpenes p-cymene, carvacrol and thymol isolated from essential oil from leaves of Lippia sidoides Cham. (Verbenaceae) protect mice against elastase-induced emphysema. Molecules 21, 1390. https://doi.org/10.3390/molecules21101390. Ghadermazi, R., Keramat, J., Goli, S.A.H., 2017. Antioxidant activity of clove (Eugenia caryophyllata Thunb), oregano (Oringanum vulgare L) and sage (Salvia officinalis L) essential oils in various model systems. Int. Food Res. J. 24 (4), 1628e1635. Giannenas, I.A., Florou-Paneri, P., Botsoglou, N.A., et al., 2005. Effect of supplementing feed with oregano and/or alpha-tocopheryl acetate on growth of broiler chickens and oxidative stability of meat. J. Anim. Feed Sci. 14, 521e535. Gormez, A., Bozari, S., Yanmis, D., et al., 2016. The use of essential oils of Origanum rotundifolium as antimicrobial agent against plant pathogenic bacteria. J. Essential Oil Bearing Plants 19 (3), 656e663. Gouvea, F. d. S., Rosenthal, A., da Rocha Ferreira, E.H., 2017. Plant extract and essential oils added as antimicrobials to cheeses: a review. Ciencia Rural Santa Maria 47 (08), e20160908. Govaris, A., Botsoglou, E., Florou-Paneri, P., et al., 2005. Dietary supplementation of oregano essential oil and a-tocopheryl acetate on microbial growth and lipid oxidation of Turkey breast fillets during storage. Int. J. Poult. Sci. 4, 969e975. Granata, G., Stracquadanio, S., Leonardi, M., et al., 2018. Essential oils encapsulated in polymer-based nanocapsules as potential candidates for application in food preservation. Food Chem. 269, 286e292. Hassine, D.B., Abderrabba, M., Yan Yvon, Y., et al., 2012. Chemical composition and in vitro evaluation of the antioxidant and antimicrobial activities of Eucalyptus gillii essential oil and extracts. Molecules 17 (8), 9540e9558. Hernández-Ochoa, L., Aguirre-Prieto, Y.B., Nevárez-Moorillón, G.V., 2014. Use of essential oils and extracts from spices in meat protection. J. Food Sci. Technol. 51 (5), 957e963. Hsouna, A.B., Halima, N.B., Smaoui, S., Naceur Hamdi, N., 2017. Citrus lemon essential oil: chemical composition, antioxidant and antimicrobial activities with its preservative effect against Listeria monocytogenes inoculated in minced beef meat. Lipids Health Dis. 16, 146. https://doi.org/10.1186/s12944-017-0487-5. Karakaya, S., Nehir El, S., Karagozlu, N., et al., 2014. Microwave-assisted hydrodistillation of essential oil from rosemary. J. Food Sci. Technol. 51 (6), 1056e1065. Kisko, G., Roller, S., 2005. Carvacrol and p-cymene inactivate Escherichia coli O157:H7 in apple juice. BMC Microbiol. 5 (36). https://doi.org/10.1186/1471-2180-5-36. Liang, J.Y., Gu, J., Zhu, J.N., et al., 2017. Repellent activity of essential oils extracted from five Artemisia species against Tribolium castaneum (Coleoptera: Tenebrionidae). Bol Latinoam Caribe Plant Med Aromat 16 (5), 520e528. Lu, H., Shao, X., Cao, J., et al., 2016. Antimicrobial activity of eucalyptus essential oil against Pseudomonas in vitro and potential application in refrigerated storage of pork meat. Int. J. Food Sci. Technol. 51, 994e1001. Maldonado, M.C., Aban, M.P., Navarro, A.R., 2013. Chemicals and lemon essential oil effect on Alicyclobacillus acidoterrestris viability. Braz. J. Microbiol. 44 (4), 1133e1137. Manso, S., Becerril, R., Nerín, C., Gómez-Lus, R., 2014. Influence of pH and temperature variations on vapor phase action of an antifungal food packaging against five mold strains. Food Control 47, 20e26.

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Socaci, S.A., 2017. Tehnici de extractie, separare s¸ i analiza cromatografica a uleiurilor volatile. AcademicPres, ClujNapoca. Sourmaghi, M.H.S., Kiaee, G., Golfakhrabadi, F., et al., 2015. Comparison of essential oil composition and antimicrobial activity of Coriandrum sativum L. extracted by hydrodistillation and microwave-assisted hydrodistillation. J. Food Sci. Technol. 52 (4), 2452e2457. Teixeira, B., Marques, A., Pires, C., et al., 2014. Characterization of fish protein films incorporated with essential oils of clove, garlic and origanum: physical, antioxidant and antibacterial properties. Food Sci. Technol. 59 (1), 533e539. Toscano-Garibay, J.D., Arriaga-Alba, M., Sánchez-Navarrete, J., et al., 2017. Antimutagenic and antioxidant activity of the essential oils of Citrus sinensis and Citrus latifolia. Sci. Rep. 7 (1). https://doi.org/10.1038/s41598-017-11818-5. Ultee, A., Bennik, M.H.J., Moezelaar, R., 2002. The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus. Appl. Environ. Microbiol. 68 (4), 1561e1568. Vergis, J., Gokulakrishnan, P., Agarwal, R.K., Kumar, A., 2015. Essential oils as natural food antimicrobial agents: a review. Crit. Rev. Food Sci. Nutr. 55, 1320e1323. Yangilar, F., Yildiz, P.O., 2018. Effects of using combined essential oils on quality parameters of bio-yogurt. J. Food Process. Preserv. 42. https://doi.org/10.1111/jfpp.13332. Zeng, Z., Zhang, S., Wang, H., Piao, X., 2015. Essential oil and aromatic plants as feed additives in non-ruminant nutrition: a review. J. Anim. Sci. Biotechnol. 6 (7). https://doi.org/10.1186/s40104-015-0004-5. Zhai, H., Liu, H., Shikui, W., et al., 2018. Potential of essential oils for poultry and pigs. Animal Nutrit. 4, 179e186. Zhang, Y., Liu, X., Wang, Y., et al., 2016. Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Food Control 59, 282e289.

Further reading http://animalnutrition.dupont.com/productsservices/essential-oil-feed-solutions/.

C H A P T E R

9 Toxic or harmful components of aromatic plants in animal nutrition Maria Grazia Cappai1, Sabine Aboling2 1

Department of Veterinary Medicine, University of Sassari, Sassari, Italy; 2Institute of Animal Nutrition, University of Veterinary Medicine, Hannover, Foundation, Hanover, Germany O U T L I N E

Aromatic plants: toxicological properties in view of their ecological function 147

Potential adverse principles/traits of aromatic plants in animal nutrition

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Sensationdselection behaviord co-existence as essentials of the planteanimal interaction

References

156

Further reading

158

150

Aromatic plants: toxicological properties in view of their ecological function Aromatic secondary plant metabolites (ASPM) are biologically active substances synthesized by the living plant during plant-specific metabolic activities. From a plant perspective, ASPMs basically work as protective organic compounds against herbivory by insects or mammals (Brodie, 2009). Essential oils, such as thujone produced by the genus Artemisia, are a typical example of the two sides of an aromatic compound. As a phenol molecule thujone itself would hinder the physiology of the plant’s cells, therefore it is located in the plant’s vacuole (Fig. 9.1). Thujone is a volatile compound, and it is supposed to be perceived before ingestion by olfactory organs. Thus, thujone does work as a preventive antipastoral trait, at least from the first physical contact with mouth or nose. When the herbivore is going to ingest the plant thujone will be perceived by uppermost postingestion organs, via taste receptors of the gustatory

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00009-1

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Copyright © 2020 Elsevier Inc. All rights reserved.

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9. Toxic or harmful components of aromatic plants in animal nutrition

FIGURE 9.1 Mentha arvensis (left) and Humulus lupulus (right) with glands of essential oils (red arrows [gray in print version]). Those glands are single cells with a large vacuole in which the phenols ale accumulated. Located at the surface of the plant they are easily to destroyed. Then the phenol is immediately released and can be perceived olfactory or gustatory or even retronasal (Czarnecki and Fontanini, 2018) by the animal.

sense, too. From an initial contact on, the animal is able to associate that taste with the genus Artemisia by its smell or even its shape. That is, after a first postingestion contact, thujone probably has changed its signal character from a postingestion effect to a preventive antipastoral effect. However, we do not exactly know how the recognition works in case of not volatile compounds, like phenols (Czerny et al., 2011). It is but conceivable that the herbivores either smell phenols as ASPM within the plant’s vacuole before a post-ingestion contact in some cases, or that the cuticle of the plant releases certain amounts of volatile terpenes such as sesquiterpenes which act presumably as preingestion signal substances for the animal, thus influencing its feeding behavior (Catanese et al., 2016). There are two kinds of learning mechanism that are essential for mutual survival of plant and herbivore: Conditioned taste aversion (CTA) and conditioned taste preference (CTP) (Provenza et al., 1994). Both strictly species-specific mechanisms mean that there are positive or negative postingestion effects after the ingestion of aromatic plants (Pfister, 1992). For example, horses as grazers would avoid Artemisia because phenols are capable of destroying the gut mucosa in the small intestine (Anderson et al., 1983). Anecdotical reports showed that domestic horses, commonly kept as leisure horses, avoid the plant species on the pasture and accept it only dried and in combination with other feeding stuffs. In contrast, wild feral horses do ingest Artemisia, though in small amounts exclusively (Davies et al., 2014). Goats, as intermediate feeders, would tolerate Artemisia to a much larger extent. Their ruminal microbiota is capable of degrading those compounds into fractions that neither impair the gut mucosa nor damage the liver. Interestingly, a long-term study shows (Aboling, 2014) that under free-choice conditions, the foraging strategy of goats reveals not only a seasonal

Sensationdselection behaviordco-existence as essentials of the planteanimal interaction

149

FIGURE 9.2 Five free-roaming goats feed under free-choice conditions on Artemisia compared to nonphenolic

grasses to a distinct extent for example during autumn. Data from Aboling, S., 2014. Long-term survey of natural diet of goats on plant-species level during the year under free-choice conditions in Middle Europe. Running project, still unpublished.

preference of Artemisia in late summer and in autumn, but compared to nonaromatic grasses, Artemisia is eaten both in much smaller amounts and in an irregular monthly pattern (Fig. 9.2). Also, wild herbivores, such as deer and hares, would eat the plant almost around the whole year, but as a sort of supplemental feeding plant (Aboling, 2003). Basically, an aromatic plant like Artemisia could either be in large amounts part of a natural diet (intermediate feeders, browsers) or serves as a supplement component in their feed (grazers). From the herbivore perspective, it could be speculated that some dietetic properties of ASPM in Artemisia under free-choice conditions could be exploited wisely by the animal. For instance, an evidence-based report on antiparasitic properties in ruminants is available in the literature (Yarnell, 2007). From an ecological point of view, ASPM may explain the successful interaction between spontaneous vegetation and herbivores: mutual tolerance, mutual existence. Almost all plant families are considered to contain ASPM (Table 9.1). In all cases, there are either antipastoral traits or postingestion effects associated with a species-specific ecological function. Indeed, from the plant side, ASPM are probably meant to be recognized by the herbivore at a first time and acknowledged as a kind of natural traffic light: Green: Enjoy! (ASPM are missing, no need to eat carefully); Yellow: Caution (ASPM are present, safe ingestion of feed is recommended); Red: Beware (ASPM are dominating: Avoid for your own sake). In reality, due to co-evolutive mechanisms, the plant-animal-interaction leads to a kind of sustainable co-living. As a parallel, the same properties can be exploited for therapeutic use. In view of being a kind of natural feeding brake, ASPM synthesis in plants may not appear to be simply as lethal as such, but probably the interaction between vegetal and animal beings is expressed into a complex chemical communication, as to whether eating some plant species very carefully or not. Such ecological coordination between plants and animals renders potentially feasible to administer ASPM, according to adequate amounts adequately defined to avoid intoxication and exploit beneficial effects.

150 TABLE 9.1

9. Toxic or harmful components of aromatic plants in animal nutrition

Examples of families with representatives of prominent species with discernible aromatic contents in the widest sense to show the broad systematic range of occurrence of ASPM.

Plant species

Family

Secondary plant metabolite

Therapeutic appliance

Rumex acetosa

Polygonaceae

Acetic acid

Promotion of saliva production

Trigonella foenum-graecum

Fabaceae

Steroid saponins

Increase of appetite

Nasturtium officinale

Brassicaceae

Phenyl-mustard oils

Deworming

Artemisia

Asteraceae

Thujone

Deworming

Allium sativum

Liliaceae

Allyl oils

Deworming

Mentha x piperita

Lamiaceae

Menthol oils

Curing of mycodermatoses

Sensationdselection behaviordco-existence as essentials of the planteanimal interaction Co-existence is meant to involve the ecological balance between the plant biomass and plant feeders. ASPM behave like chemical means to inform the animal about safe eating or avoidance of certain plant species over time. In reality, animals can make their choice and pick different plant species out of the spontaneous vegetation with astonishing competence to minimize or fully exclude the risk of intoxication. There are lots of examples of such kind of successful co-existence (Fig. 9.3). The acceptance or avoidance of plants species capable of ASPM production is strictly species-specific on both plant’s and animal’s side. For example, Senecio possesses

FIGURE 9.3 Examples of sensation and selection behavior. (1) Jacobson’s organ in a goat buck, capable of perceiving the lowest concentrations of aromatic molecules; (2) Negative selection by horses in case of Senecio jacobaea, an aromatic plant from the Asteraceae family due to its perceptible sesquiterpen-lactones. Such 100% avoidance saves the life of horses on natural pastures. (3) Goat with spots of resin due to phenolic compounds in Pinus sylvatica. For this animal, leaves and bark of Pinus are an important feeding source in winter. The browser shows no health problems from the intake due to the co-evolutive developed detoxification mechanisms of ASPM beginning in the saliva and ending up in the liver. (4) Negative selection from donkeys of a species of the Brassicaceae family, with volatile Phenyl-Mustard oils. The same is true for the interaction between plant and animal in case of Senecio and horse: Although there is no other feed left, the pasture animals select this plant negatively due to its toxic ASPM. Photographs from the authors.

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pyrrolizidine alkaloids and aromatic sesquiterpenes that work as antipastoral traits. The species is negatively selected by horses but eaten in large amounts by grazing sheep (Berendonk et al., 2010). In contrast to horses and cows, sheep display to be capable of detoxifying strategies, thanks to the mutual benefits from the microbiota in the forestomachs. In such a way, toxic pyrrols do not get to the liver because they are broken down to nontoxic compounds before leaving the rumen (Brügmann et al., 2006; Fig. 9.4). Normally, the oxygenases in the liver are the most important detoxification system for herbivores. This system works if the plant-animal interaction is intact, when the animal is both trained and able to select under ecologically normal conditions. The liver in a horse possesses mono-oxygenases that degrade pyrrolizidine into smaller compounds. These toxic pyrrols can impair the metabolism of the hepatocyte. However, in case of very small ingested amounts of Senecio over a long time or a larger amount on one time, no intoxication occurs in the horse because the liver cells cope with the toxicants and little by little regenerate, if the horse is healthy. In contrast, even though herbivores like sheep are capable of tolerating pyrrolizidine alkaloids to a certain extent, large Senecio intakes from scarcity of feeding sources in the pasture for instance, may exceed the capacity of metabolic pathways for detoxification from the liver. In the case of other more sensitive herbivores, like horses, incapable of successful endogenous detoxification systems against Senecio alkaloids, the consumption of such plant is absolutely

FIGURE 9.4 Pathway of toxic pyrrols in distinct animal species. Data from Brügmann, M., Niemann, U., Wiedenfeld, H., Geburek, F., Jünnemann, D.F., 2006. Fallbericht aus der Pathologie: Tod eines Norwegers. In: N. L. f. V. u. Lebensmittelsicherheit. (Case report from department of pathology of the Lower Saxony Institute of Food Safety: Death of a Norwegian Fjord Horse)

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rare and the horse would try to avoid it completely (cf. Fig. 3.2). As said, in contrast to horses and cows sheep can feed on Senecio to a larger extent without apparent harm. The ruminal microbiota accounts some bacterial strains that can produce still unknown but nontoxic metabolites via the degradation of pyrrolizidine alkaloids. Their metabolized products can reach the liver to be safely excreted through the bile via the digestive system. In the case of sheep, it is interesting to point out how they also show signs of suppressed feed intake. It seems like that they (like the goats do with Artemisia) accept Senecio up to a certain amount through a self-limitation. Indeed, in vitro experiments show that in case of a concentration of alkaloids in the rumen liquid of >1000 mg/mL the detoxification system of the microbiota cannot cope with it any more (Craig et al., 1992).

Potential adverse principles/traits of aromatic plants in animal nutrition The concept at the basis of toxicological sciences considers the adverse effects of xenobiotics, capable to interfere with biochemical pathways in the organism and cause damage (sometimes potentially lethal). The term xenobiotic derives from the Greek word xenos, in this case meaning simply foreign (external to the body), to be distinguished from endogenous compounds synthesized by the animal. In view of such definition, APSM are exogenous chemicals which the animal body is incapable to synthesize and, that way, does not recognize as own molecules. After their intake, the metabolic systems of the animal are elicited to react in different ways to minimize the interference within own biochemical processes (Egea et al., 2016). Different biochemical strategies were developed through the co-evolved existence, but some animal species still appear to be more susceptible than others toward some APSM, whereas other plants can be eaten safely up to certain amounts by other animal species. In general terms, grazers appear more sensitive than browsers toward APSM. As biologically active compounds described in the literature, some are reported to possess both positive (anthelminthic) and negative (impaired physiological functions) effects. On the other hand, they are means against herbivory which make their consumption by the animal a border line condition. In such a way, the question could be “to eat, or not to eat?” and should consider the potential effects from the interaction between xenobiotics and biochemical processes of the animal (Marks et al., 1988). In view of such biological activity, the aware ingestion should be based on the evaluation of risks and benefits of such moderate consumption in the selection of plant species composing the natural diet of animals. However, the complexity of co-living between aromatic plants and animals in nature renders this argument both fascinating and delicate (Brodie, 2009). Thus, given the species diversity of aromatic plants and their compounds produced on one side and the different feeding habits and species-specific metabolic peculiarities of herbivores and omnivores on the other side, it is here underlined the relevance of functional diversity in particular in the highly complex plant-vertebrate-interaction (Bradshaw et al., 2003). That way, universal biological concepts were and will always be scientifically derived, proved and disapproved. Indeed, in the light of the diversity of natural ecosystems shaped over time on plant/animal interactions, the delicate balance of their co-living may lead to reckon that such subtle equilibrium is based on a very highly refined mechanism of mutual aid for surviving in the wild and not really to be poisonous sic et simpliciter (Varga et al., 2016).

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The topic of poisonous plants in animal nutrition covers one of the most important aspects within the scientific community as well as in the field, for improving farming practices and broaden the knowledge of stakeholders on such aspects (Senn and Suter, 2003). Chiefly, veterinary aspects on animal welfare and health are concerned (Heiman and Greenway, 2016). Unfortunately, sometimes evidence-based reports end with animal necropsy to address the cause of death (Gonzalez-Medina et al., 2017). The push behind the deliberate ingestion of dangerous amounts of poisonous plants by the animal is the most difficult clue to address. However, these are rare cases reported in the practice (Andersen et al., 2010), unless when animals graze/browse in an unknown environment or under particularly extreme circumstances, like in case of scarcity in feeding source variety. In such circumstances and not surprisingly, fatal poisoning may happen. Under artificial or exceptional conditions, the consumption of large amounts of toxicants with the diet can be determined by different factors, other than animal experience of smell or taste associated with harmful effects of certain plants. For instance, in many Mediterranean areas sheep flocks are fed on natural pastures and sheep can graze safely avoiding poisoning plants, despite heavily present in the spontaneous vegetation. However, when soils are ploughed after the drought season, underground organs like roots can be brought to the surface. Usually, such kind of feed is not accessible for sheep, thus no process of co-evolution could have occurred. Not surprisingly, a case of fatal poisoning happened in a sheep flock when such roots were those of Thapsia garganica L. (Apiaceae, Drias plant, also known as “deadly carrot”). Obviously, the deadly carrots were palatable and fleshy, all the more since the feed was scarce after the drought. In an ecological context, this means that species-specific antipastoral traits as ASPM (thapsigargin and thapsigargicin) were either not perceptible by the sheep or the natural traffic light “red: Beware” failed because the critical level of selection was low in the hungry sheep. In the end, all animals ingested the roots resulting into a mortality rate of 100% (Cappai et al., 2013). From the point of view of plant-animal interaction it is irrelevant that thapsigargin and thapsigargicin are of medical interest as possible antitumoral substances against prostate carcinoma (Denmeade et al., 2003; Denmeade and Isaacs, 2005; Jakobsen et al., 2001; Janssen et al., 2006). Specific nutritional aspects related to aromatic plants and their usage in animal nutrition (extensively discusses in other chapters of this book) instead mean to promote and exploit the benefits from APSM for different purposes, based on their regulating role of different metabolic processes. The recent investigations are aiming more and more to elucidate on the beneficial potentials of APSM and essential oils in animal nutrition. As seen, strong emphasis is given in this chapter to biological diversity, which is the basis for understanding the subtle equilibrium of co-existence and trophic dynamics of plant/animal interactions. When speaking of aromatic plants in animal nutrition, a huge importance was given to volatile compounds synthesized by the plant. However, perception of such scents is different if man and other animal species are considered. For instance, we use essential oils in cosmetics or in recipes in our kitchen and not only for nutraceutical purposes. Animals may perceive aromatic plants in the environment in different ways, due to their anatomical differences of the olfactory function (Frazier, 1992; Shipley, 1999; Pain and Revell, 2009; Goz’dziewska-Harajczuk et al., 2015; Kohl and Dearing, 2017). In general, animal species can be classified according to the different efficiencies in perceiving and transducing the signal generated by molecules smelled from the environment. Very briefly, the signal collected by peripheral endings distributed in the olfactory mucosa of

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turbinates in the nasal cavities reaches the primary olfactory cortex of the brain through the first pair of cranial nerves. From the primary olfactory cortex the signal is transmitted to other regions of the forebrain, to let the individual aware of the sensation and coordinate the reaction to the olfactory stimulus. Such reaction includes cognitive pathways and creates a databank of information, through which molecules are associated to the scent of a given plant. In our case, such cognitive behavior let us apprehend the presence of an aromatic plant from the volatile compounds and the specific scent, even without the sight of the plant (Bortolami and Callegari, 1999). The density of peripheral endings in the olfactory epithelium as well as its extension vary according to species. Even more, the primary olfactory cortex of the brain is differently developed in the different animal species. In view of such anatomical differences, animals are conventionally classified as macrosmatic and microsmatic, in relation to the development of the primary olfactory cortex of the brain, possibly reflecting the olfactory function. It is accepted that carnivores are macrosmatic and such peculiarities is also used for other purposes (detector dogs can target scents of distant objects). In nature, they would use these characteristics for hunting prey. Herbivores display to use efficiently the vomero-nasal organ (Fig. 3.1) to detect volatile molecules, through which they are capable to perceive the scent of plants. At this regard, aromatic plants seem to support the animals in the feed selection (Aboling, 2014; Egea et al., 2016; Frazier, 1992; Iason and Palo, 1991; Marks et al., 1988; Molyneux and Ralphs, 1992; Rogosic et al., 2006). Omnivores, like swine (both domestic and wild), do possess a well-developed olfactory cortex which is essential for their need to smell the scent of underground roots and insects, to target places for digging, and even proverbial is the swine’s brilliant ability to smell truffles. It is interesting to pose in relation the different anatomo-physiological functions and the feeding behavior of animals with the huge variety of volatile compounds produced by the several species of aromatic plants (and parts of them). In this scenario, the complexity of the co-evolution of animals and plants may be approached. In general terms, a toxicant can exert its action if it reaches target organs or tissues in the animal body. To do so, such xenobiotic should be ingested, absorbed, distributed and finally get to the target. Throughout the process, the animal body may try to react to reduce the presence and minimize the risk of adverse effects from the toxicant. However, the way the animal body handles the presence of the toxicant depends on animal species-specific countermeasures, developed through the adaptation to the seasonal availability of feeding sources composing the natural diet. First mechanisms of defense described in the literature in the postingestion phase is based on the modification of saliva production (Cappai et al., 2014). This occurrence may be elicited by the fact that after the first bite of part of an aromatic plant, bitter taste or astringent sensation may be experienced in the mouth. Bitter taste or astringency are capable to stimulate saliva production, to wash away the cause that led to such sensation. Such a learning based on taste sensation may be considered as the first step of the process of feed selection behavior, which could be adopted to avoid future experiences. However, other times, the speciesspecific adaptation to certain feeding sources is successful and the animal continues to feed on such plant or parts of it. In fact, if the saliva is at first abundantly secreted in the attempt to dilute the concentration of the xenobiotic in the mouth, it is in the modification of saliva composition that may solve the problem of harmful compounds, over a certain amount.

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Binding compounds may create stable complexes with the aim reduce the active dose of the toxicant by decreasing the absorption in the gastro-intestinal tract. Since the possibility to vary the saliva composition is genetically encoded, it is easy to understand why this strategy is specie-specific (Cappai et al., 2014; Clauss et al., 2002, 2004; Fickel et al., 1998; Gehrke et al., 2002; Juntheikki, 1996). Other animal strategies to minimize the toxic effects may instead rely on the presence of detoxifying processes. The liver can be considered as the chief biochemical laboratory of the animal body. However, enzyme synthesis for detoxification of toxicants are again genetically encoded and therefore depend on animal species. That way, some toxicants may be successfully detoxified in some animal species, while in some others may not. However, the possibility to detoxify xenobiotics also depends on the chemical structure of the xenobiotic and therefore different extents of tolerance toward the ingestion of harmful substances may also depend on the origin of the toxicant. The presence of toxicants in the diet may bring to liver overloading, with degeneration processes, potentially ending in the necrosis of the organ in hyperacute cases. The hepatic biochemical activity exceeding the normal function due to the presence of toxicants can also be dose-dependent. That way, if the animal learned the postingestion effects of toxicants in the diet, the choice to self-regulate the daily intake may take into account the detoxifying efficiency of the liver and how it can cope with a given dose. In ruminants, the presence of a large microbial community in the prestomach can help the liver in the detoxifying process, as said. Some ruminal bacterial strains in sheep are in fact capable to metabolize heterocyclic aromatic rings, like pyrrols, toxic for the liver and the nervous system. In this way, active toxicant may reach the target organs into very small amounts because the biotransformation occurred prior to systemic distribution in the posthepatic circulation. Also, salivary secretions as well as secretions in descending tracts of the gut display the function to bind compounds exerting protein precipitating activities, like tannins. By forming stable complexes, such secretions are capable to subtract tannins to direct contact with intestinal mucosa, meanwhile limiting intestinal absorption. Plant containing various amounts and a certain composition of toxicants may therefore not be readily accepted as feed by vertebrate herbivores. Only under certain circumstances, the selection of plants or parts containing APSM may enter in very small amounts in the natural diet of browsers and grazers. It should not be neglected that aromatic plants, too, contain valuable nutrients, to which the animal may turn to in case of necessity, but with caution. For example, phenols did warn horses against ingesting toxic seedlings of sycamore maple (Acer pseudoplatanus), however, those seedlings possess an attractive protein content of 497 g/mg DM (Aboling et al., 2019). The threshold of phenolic contents to be perceived at preingestion as repellent by the horses was 56.5 mg/g DM and did occur via olfactory senses in older seedlings, only. Thus, lower contents of phenols failed to be recognized resulting into fatal ingestion of seedlings. According to the postingestion experience, wise selection and own species-specific detoxification strategies may let animals intake safe amounts of aromatic plants, without reaching the toxic dose (selecting a plant when dry would limit the availability of some water soluble toxic compounds). As already mentioned, CTA and CTP (Provenza et al., 1994) play a key role in feed selection and involve positive or negative postingestion effects after the ingestion of aromatic plants. There are many studies in goats and sheep that learn to associate ASPM on the one hand and to balance the ingestion of those plants with the ingestion of protein-rich ones (Ralphs, 1992; Provenza et al. 1993, 1994; Provenza, 2004).

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In the modern feed industry, research and development is aiming to achieve the safe use of such natural, biologically active compounds to exploit for beneficial effects. However, the need to test each xenobiotic to assess its safe use in animal nutrition needs to be detailed to avoid any harmful condition. At present, no general rules can be laid down, given the diversity of APSM in the plant kingdom. In addition, the biological diversity of animal species and respective genetically encoded metabolic processes in synergy with the microbial community of the gut can lead to different extents of coping ability with the xenobiotics in the diet and potential toxic effects.

References Aboling, S., Scharmann, F., Bunzel, D., Kulling, S., 2019. Negative Selection of Sycamore Maple Seedlings (Acer pseudoplatanus) in Pasture Horses is Mediated Through Phenolic Compounds. Manuscript in preparation. Aboling, S., 2003. Flora und Äsung auf Wildäckern der Feldflur in Niedersachsen. (German). Z. Jagdwiss. 49, 161. Aboling, S., 2014. Long-term Survey of Natural Diet of Goats on Plant-Species Level During the Year Under FreeChoice Conditions in Middle Europe. Laufendes Projekt, noch unveröffentlicht. Andersen, K.B., Skaanild, M.T., Bertelsen, M.F., Brimer, L., 2010. Yew intoxication in brown bears; a novel approach to diagnosis. Eur. J. Wildl. Res. 56, 915e921. Anderson, G.A., Mount, M.E., Vrins, A.A., Ziemer, E.L., 1983. Fatal acorn poisoning in a horse: pathologic findings and diagnostic considerations Quercus. J. Am. Vet. Med. Assoc. 1105. Berendonk, C., Cerff, D., Hünting, K., Wiedenfeld, H., Becerra, J., Kuschak, M., 2010. Pyrrolizidine alkaloid level in Senecio jacobaea and Senecio erraticus - the effect of plant organ and forage conservation. In: Schnyder, H., Isselstein, J., Taube, F., Auerswald, K., Schellberg, J., Wachendorf, M., Herrmann, A., Gierus, M., Wrage, N., Hopkins, A. (Eds.), Grassland in a Changing World. Proceedings of the 23rd General Meeting of the European Grassland Federation, Kiel, Germany, 29th August - 2nd September 2010. Mecke Druck und Verlag, Duderstadt; Germany. Bortolami, R., Callegari, E., 1999. Neuroanatotmia ed Estesiologia Degli Animali Domestici. Edagricole, Italy. Bradshaw, R.H.W., Hannon, G.E., Lister, A.M., 2003. A long-term perspective on ungulateevegetation interactions. For. Ecol. Manag. 181, 267e280. Brodie, E.D., 2009. Toxins and venoms. Curr. Biol. 19. Brügmann, M., Niemann, U., Wiedenfeld, H., Geburek, F., Jünnemann, D.F., 2006. Fallbericht aus der Pathologie: Tod eines Norwegers. In: N. L. f. V. u. Lebensmittelsicherheit. Cappai, M.G., Garau, G., Aboling, S., Kamphues, J., Pinna, W., 2013. Fatal poisoning of sheep from roots of Drias plants (Thapsia garganica L.): a seasonal risk associated with soil management practices in a Mediterranean area. Large Animal Rev. 19, 292e294. Cappai, M.G., Wolf, P., Dimauro, C., Pinna, W., Kamphues, J., 2014. The bilateral parotidomegaly (hypertrophy) induced by acorn consumption in pigs is dependent on individual’s age but not on intake duration. Livest. Sci. 167 (1), 263e268. Catanese, F., Fernández, P., Villalba, J.J., Distel, R.A., 2016. The physiological consequences of ingesting a toxic plant (Diplotaxis tenuifolia) influence subsequent foraging decisions by sheep (Ovis aries). Physiol. Behav. 167, 238e247. Clauss, M., Gehrke, J., Fickel, J., Lechner-Doll, M., Flach, E.J., Dierenfeld, E.S., Hatt, J.M., 2002. Induction of salivary tannin-binding proteins in captive black rhinoceros (Diceros bicornis). In: Proceedings of the Joint Nutrition Symposium, p. 131. Clauss, M., Gehrke, J., Hatt, J.M., Dierenfeld, E.S., Flach, E.J., Hermes, R., Castell, J., Streike, J., Fickel, J., 2004. Tanninbinding salivary proteins in three captive rhinoceros species. Comp. Biochem. Physiol. A 140, 67e72. Craig, A.M., Latham, C.J., Blythe, L.L., Schmotzer, W.B., O’Connor, O.A., 1992. Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions. Appl. Environ. Microbiol. 58, 2730e2736. Czarnecki, L., Fontanini, A., 2018. Gustation and olfaction: the importance of place and time. Curr. Biol. 29, 18e20. Czerny, M., Brueckner, R., Kirchhoff, E., Schmitt, R., Buettner, A., 2011. The influence of molecular structure on odor qualities and odor detection thresholds of volatile alkylated phenols. Chem. Senses 36, 539e553.

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Davies, K.W., Collins, G., Boyd, C.S., 2014. Effects of feral free-roaming horses on semi-arid rangeland ecosystems: an example from the sagebrush steppe. Ecosphere 5, 127. Denmeade, S.R., Isaacs, J.T., 2005. The SERCA pump as a therapeutic target. Cancer Biol. Ther. 4, 69e77. Denmeade, S.R., Jakobsen, C.M., Janssen, S., Khan, S.R., Garrett, E.S., Lilja, H., Christensen, S.B., Isaacs, J.T., 2003. Prostate-specific antigen-activated thapsigargin prodrug as targeted therapy for prostate cancer. J. Natl. Cancer Inst. 95, 990e1000. Egea, A.V., Allegretti, L.I., Paez Lama, S.A., Grilli, D., Fucili, M., Guevara, J.C., Villalba, J.J., 2016. Diet mixing and condensed tannins help explain foraging preferences by Creole goats facing the physical and chemical diversity of native woody plants in the central Monte desert (Argentina). Anim. Feed Sci. Technol. 215, 47e57. Fickel, J., Göritz, F., Joest, B.A., Hildebrandt, T., Hofmann, R.R., Breves, G., 1998. Analysis of parotid and mixed saliva in roe deer. J. Comp. Physiol. 168, 257e264. Frazier, J.L., 1992. How animals perceive secondary plant compounds. In: Rosenthal, G., Berenbaum, M.R. (Eds.), Herbivores. Their Interactions with Secondary Plant Metabolites. Academic Press, Inc, San Diego, pp. 89e134. Gehrke, J., Fickel, J., Lechner-Doll, M., Lason, K., Clauss, M., 2002. Salivary tannin-binding proteins are not affected by mid-term feeding history in captive roe deer (Capreolus capreolus). In: Proceedings of the Joint Nutrition Symposium, p. 132. Gonzalez-Medina, S., Piercy, R.J., Ireland, J.L., Newton, J.R., Votion, D.M., 2017. Equine atypical myopathy in the UK: epidemiological characteristics of cases reported from 2011 to 2015 and factors associated with survival. Equine Vet. J. 49, 746e752. Go zdziewska-Harajczuk, K., Kleckowska-Nawrot, J., Janeczek, M., Zawadzki, M., 2015. Morphology of the lingual and buccal papillae in alpaca (Vicugna pacos) - light and scanning electron microscopy. Anat. Histol. Embryol. 44, 345e360. Heiman, M.L., Greenway, F.L., 2016. Review: a healthy gastrointestinal microbiome is dependent on dietary diversity. Mol. Metabol. 5, 317e320. Iason, G.R., Palo, R.T., 1991. Effects of birch phenolics on a grazing and a browsing mammal: a comparison of hares. J. Chem. Ecol. 17, 1733e1743. Jakobsen, C.M., Denmeade, S.R., Isaacs, J.T., Gady, A., Olsen, C.E., Christensen, S.B., 2001. Design, synthesis, and pharmacological evaluation of Thapsigargin analogues for targeting apoptosis to prostatic cancer cells. J. Med. Chem. 44, 4696e4703. Janssen, S., Rosen, D.M., Ricklis, R.M., Dionne, C.A., Lilja, H., Christensen, S.B., Isaacs, J.T., Denmeade, S.R., 2006. Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (hK2)-activated thapsigargin prodrug. Prostate 66, 358e368. Juntheikki, M.R., 1996. Comparison of tannin-binding proteins in saliva of Scandinavian and North American moose (Alces alces). Biochem. Syst. Ecol. 24, 595e601. Kohl, K.D., Dearing, M.D., 2017. Intestinal lymphatic transport: an overlooked pathway for understanding absorption of plant secondary compounds in vertebrate herbivores. J. Chem. Ecol. 43, 290e294. Marks, D.L., Swain, T., Goldstein, S., Richard, A., Leighton, M., 1988. Chemical correlates of rhesus monkey food choice: the influence of hydrolyzable tannins. J. Chem. Ecol. 14, 213e235. Molyneux, R.J., Ralphs, M.H., 1992. Plant toxins and palatability to herbivores. J. Range Manag. 13e18. Pain, S., Revell, D.K., 2009. Fodder Quality Specifications: Identifying Predictors of Preference between Hays. Kingston, ACT, Australia. Pfister, J.A., C, C.D., Provenza, F.D., 1992. Behavioral toxicology of livestock ingesting plant toxins. J. Range Manag. 45, 30e36. Provenza, F.D., Lynch, J.J., Nolan, J.V., 1993. The relative importance of mother and toxicosis in the selection of foods by lambs. J. Chem. Ecol. 19, 313e323. Provenza, F.D., Lynch, J.J., Burritt, E.A., Scott, C.B., 1994. How goats learn to distinguish between novel foods that differ in postingestive consequences. J. Chem. Ecol. 20, 609e624. Provenza, F.D., 2004. Behavioural mechanisms influencing use of plants with secondary metabolites. In: SandovalCastro, C.A., Hovell, F.D., Torres-Acosta, J., Ayala-Burgos, A. (Eds.), Herbivores - the Assessment of Intake, Digestibility and the Roles of Secondary Compounds. BSAS Publication Nottingham: University Press, pp. 183e195. Ralphs, M.H., 1992. Continued food aversion: training livestock to avoid eating poisonous plants. J. Range Manag. 45, 46e51.

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Rogosic, J., Estell, R.E., Skobic, D., Martinovic, A., Maric, S., 2006. Role of species diversity and secondary compound complementarity on diet selection of Mediterranean shrubs by goats. J. Chem. Ecol. 32 (6), 1279e1287. Senn, J., Suter, W., 2003. Ungulate browsing on silver fir (Abies alba) in the Swiss Alps: beliefs in search of supporting data. For. Ecol. Manag. 181, 151e164. Shipley, L.A., 1999. Grazers and browsers: how digestive morphology affects diet selection. Grazing behavior of livestock and wildlife. Idaho Forest Bulletin 70. Varga, A., Molnár, Z., Biró, M., Demeter, L., Gellény, K., Miókovics, E., Molnár, Á., Molnár, K., Ujházy, N., Ulicsni, V., Babai, D., 2016. Changing year-round habitat use of extensively grazing cattle, sheep and pigs in East-Central Europe between 1940 and 2014: consequences for conservation and policy. Agric. Ecosyst. Environ. 234, 142e153. Yarnell, E., 2007. Plant chemistry in veterinary medicine: medicinal constituents and their mechanisms of action A2 Wynn, S.G. In: Fougère, B.J. (Ed.), Veterinary Herbal Medicine. Mosby, Saint Louis, pp. 159e182. Chapter 11.

Further reading Appel, H.M., 1993. Phenolics in ecological interactions: the importance of oxidation. J. Chem. Ecol. 19, 1521e1552. Appel, H.M., Govenor, H.L., D’Ascenzo, M., Siska, E., Schultz, J.C., 2001. Limitations of folin assays of foliar phenolics in ecological studies. J. Chem. Ecol. 27, 761e778. Custódio, L., Patarra, J., Alberício, F., Neng, N. d. R., Nogueira, J.M.F., Romano, A., 2015. Phenolic composition, antioxidant potential and in vitro inhibitory activity of leaves and acorns of Quercus suber on key enzymes relevant for hyperglycemia and Alzheimer’s disease. Ind. Crops Prod. 64, 45e51. Dani, C., Oliboni, L.S., Agostini, F., Funchal, C., Serafini, L., Henriques, J.A., Salvador, M., 2010. Phenolic content of grapevine leaves (Vitis labrusca var. Bordo) and its neuroprotective effect against peroxide damage. Toxicol. Vitro 24, 148e153. Haslam, E., 1988. Plant polyphenols (syn. vegetable tannins) and chemical defenseda reappraisal. J. Chem. Ecol. 14, 1789e1805. Scharmann, F., Bunzel, D., Kulling, S., Aboling, S., 2019. Phenolic Profiles of Acer Pseudoplatanus as Affected by Plant Developmental Stage in the Light of Equine Atypical Myopathy. Stumpf, P.K., Conn, E.E., 1981. Secondary Plant Products. New York Academic Press, New York. Sunnerheim, K., Palo, R.T., Theander, O., Knutsson, P.-G., 1988. Chemical defense in birch. Platyphylloside: a phenol from Betula pendula inhibiting digestibility. J. Chem. Ecol. 14, 549e560.

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10 Application of aromatic plants and their extracts in diets of broiler chickens Li-Zhi Jin1, Yueming Dersjant-Li2, Ilias Giannenas3 1

Meritech and Huazhong Agricultural University Cooperative Innovation Center, Wuhan, P. R. China; Guangzhou Meritech Bioengineering Co., Ltd., Guangzhou, P. R. China; 2Consultant in Animal Nutrition, Nijkerk, The Netherlands; 3Laboratory of Nutrition, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece O U T L I N E Introduction

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Antioxidant and antiinflammatory actions Antimicrobial activity Effects on intestinal function Energy sparing effect

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Future implementations and conclusions 176 References

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Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00010-8

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animals and mostly in pigs and poultry. For this reason, they were given the name antibiotic growth promoters (AGPs). The main modes of actions of AGPs are based on their suppressing effects against is that the growth of the gastrointestinal microflora, making more nutrients available to the livestock animals. In poultry, it is extensively documented that AGPs can improve performance rates and feed utilization, decrease mortality in both clinical and subclinical diseases, and increase profitability. Development of bacterial resistance to AGPs in animals, however, has led to a growing public concern. In Europe, AGPs in animal feed have been banned since 2006, and worldwide a likely ban of AGPs in animal feed is on debate. Meanwhile, nutritional and management strategies have been developed to maintain optimum levels of growth performance and healthiness of animals fed AGP-free diets. In poultry, there is an increased use of alternative feed additives, among them, extracts of plants called phytogenics or phytobiotics, containing mostly the essential oils (EOs) of the plant material (Alcicek et al., 2003; Windisch et al., 2008; Christaki et al., 2012; Yang et al., 2015). An advantage of using phytobiotics is that they are residue free, generally considered as safe (GRAS); they are frequently used in the food industry as natural antimicrobials that have been proved to be an efficient alternative to AGPs (Brenes and Roura, 2010; Toghyani et al., 2010; Christaki et al., 2012; Giannenas et al., 2018a, 2018b, 2018c). The prefix “phyto” refers to plants, thus phytobiotics, also called as phytogenic feed additives or botanicals, are derived from aerial parts from cultivated or self-grown plants and bare EOs and other components such as flavonoids and glycosides. The EOs are oily liquids obtained from plant material that consists of terpenoid compounds with aromatic rings and exhibit relatively low-boiling temperature point. The EOs represent the very essence of order and flavor of the phenolic compounds of “herbs and spices,” typically extracted by distillation methods and named after the plant from which they are extracted. The effects of aromatic medicinal plants, their extracts and EOs have been reported in many literature studies, including their potent antimicrobial and antioxidant properties and their capacity to enhance digestive process. Also, to showcase their full multitude potential, other effects such as stimulation of the immune response, their antiinflammatory properties and their potential on growth promotion that is comparable to antibiotics should be also mentioned (Christaki et al., 2012; Giannenas et al., 2018a, 2018c). An extract is a substance made by extracting parts of plant materials, mostly by using a solvent such as ethanol or water. Extracts may be sold as solutions, pure compounds, or dry substances in form of powder. The aromatic principles of several plants, spices, nuts, herbs, fruits, etc., and flowers are marketed as extracts, or as EOs. The aim of this chapter is to review recent evidence based on experimental studies on the effect of phytobiotic products containing EOs and plant extracts on growth performance and feed utilization efficiency in broiler chickens. The mechanisms of action of those phytogenic products and their compounds will be also discussed. Because of the fact that oregano or origanum (Origanum vulgare L.) EO has high concentration of active substances and strongest antimicrobial activities, and has been extensively studied worldwide, thus this review has focused mainly on trial studies with oregano plant or EO or the related products.

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Mode of actions of aromatic medicinal plants, spices, or herbs, their extracts or essential oils in poultry Literature studies reported the potential of aromatic plants and their extracts such as EOs in possession of antimicrobial properties, digestion stimulation and antioxidant functions (Shi et al., 2012).

Palatability and feed intake The inclusion of herbal feed additives may positively or negatively influence the organoleptic characteristics of the diet such as aroma and taste. Feed palatability can be regarded as an important factor that determines overall performance. Nevertheless, several studies have showed that herbal feed additives did not affect feed intake compared to the experimental groups that were not fed with those additives either as EOs in healthy chickens (Botsoglou et al., 2002; Tiihonen et al., 2010) or ground herbs (Florou-Paneri et al., 2005; Giannenas et al., 2005). Similarly, recently published reviews reported that feed intake in chicks was unchanged by dietary inclusion of plant extracts and EOs (Brenes and Roura, 2010; Franz et al., 2010; Hashemi and Davoodi, 2011; Hippenstiel et al., 2011; Bozkurt et al., 2014). According to the findings of Amad et al. (2011) it was tasted that daily feed intake in chickens was not affected by increasing the dietary level of a mixture containing the EOs of anise, thyme, and oregano in comparison to chickens fed an unsupplemented diet. On the other hand, in a choice feed experiment oregano oil decreased feed intake in chickens (Symeon et al., 2010). However, feed intake was increased in coccidian challenged chickens by EO of oregano (Giannenas et al., 2003), ground herb of oregano (Giannenas et al., 2004), ground herb of Olympus tea (Florou-Paneri et al., 2004) and mixtures of herbal extracts containing active components of extracts from different plants such as Echinacea angustifolia, Cinchona succirubra, Ribes nigrum, and Agrimonia eupatoria (Christaki et al., 2004). Also, a carvacrol based feed additive enhanced feed intake in chicken challenged with Campylobacter (Arsi et al., 2014; Kelly et al., 2017) but commercial diets with plant extracts of Eremophila glabra, Acacia decurrens, and lemon myrtle containing their secondary compounds such as cineole, a-terpineol, and terpinene-4-ol did not affect food intake in Campylobacter challenged chickens (Kurekci et al., 2014). A possible explanation for the decreased feed consumption is that EOs possess an irritating odor and a pungent taste, which decreases food palatability. The pungency of the phytogenic compounds may decrease the feed intake of the animals according to the extensive review of (Brenes and Roura, 2010). In agreement to this hypothesis, Cabuk et al. (2006) reported that a dietary mix of plant extracts plants such as oregano, sage, laurel, fennel, citrus, myrtle drastically decreased feed intake in chicks from young broiler breeders by graded inclusion of this blend of plant EOs. Results of some other pertinent trials concluded that feeding pigs with extracts and EOs from caraway and fennel or from oregano and thyme also presented a dose-related detrimental effect on palatability (Jugl-Chizzola et al., 2006). In contrast to pigs, information of poultry concerning feed preference was rather scarce. It has been reported that poultry as birds might not be as sensitive as pigs to flavor or taste. Pigs are mammals and have different olfactory acuity, and Roura et al. (2008) also reported that birds are

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more tolerant to exposure of adequate levels of plant EOs than mammals. In a recent study (Giannenas et al., 2018b), a polyherbal feed additive was fed to broiler chickens positively affecting feed intake. It has been claimed that broiler chickens can show superior performance when fed with herbal feed additives whether they increase feed consumption; enhance digestion and consequently nutrient absorption. Thus, dietary herbal extracts can favor an improved status of health and structure of the gastrointestinal tract in order to be effectively resilient against bacterial or viral systemic or local challenges or a combination of most of those factors formerly presented (Van Der Aar et al., 2017).

Growth Performance Aromatic medicinal plants and their extracts are mostly claimed to be beneficially effective on the flavor and palatability of feed, thus enhancing the production performance (Windisch et al., 2008). Especially, during the last two decades, plenty of phytobased feed additives have been investigated. It has been mostly stated that dietary supplementation with herbal products to diets has growth promoting effect on broiler chickens. Thus, Yakhkeshi et al. (2011) studied the effects of different natural growth promoters in broiler chickens to compare the results with the groups feeding the diets with/without antibiotics. It was found that feeding the birds with the diets containing natural feed additives instead of antibiotics alleviated the negative effects of removing antibiotics from the diet of commercial poultry. Even under a detailed investigation on the literature, it is hard to find negative effects of plant extracts on poultry performance. Plant extracts and herbal EOs are regarded as growth enhancers in poultry diets however, their effectiveness is largely inconstant, due to some factors, such as the tested EOs, age of chickens, composition of experimental diets, and environment of the experimental conditions (Zhang et al., 2014). The earlier-mentioned variability on the effectiveness of the dietary usage of aromatic plants and their EOs as growth promoters will be discussed in detail in the current review. There are many different types of EOs known worldwide (about 3,000 EOs), of which about 300 exhibit remarkable commercial interest and are used mainly in aromatic, flavoring, and fragrance markets (Brenes and Roura, 2010). Among these EOs, extracts of oregano, rosemary, thymol, cinnamon, and coriander have been tested in poultry (Windisch et al., 2008). These EOs vary in composition and level of active substances due to many reasons (Brenes and Roura, 2010). As reviewed in the literature, the growth performance in poultry varies with different EO sources and dosage levels (Lee et al., 2004; Brenes and Roura, 2010; Giannenas et al., 2018c). As a result, sometimes, inconsistent results that were reported by several researchers in vivo could be related to experimental design, formulation of basal diets, and it could also be largely attributed to dosage of the additive, composition and the concentrations of the included active substances. In addition, the concentration of the bioactive compounds related to production (extraction) methods, and the origin of the plant materials used, as well as the cultivation parameters. Therefore, an EOs from the same plant species which are used in different trials may differ in composition and activities. For example, the main active substances that have antimicrobial functions in oregano EOs are carvacrol and thymol, which content may differ as largely as 5%e85% (Burt, 2004). Moreover, it is known that there are 60 plants with the same name of “oregano” in Europe (Lawrence

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and Reynolds, 1984; Giannenas et al., 2018a). Among them, Greek oregano (Origanum vulgare L. synonym Origanum heracleoticum ssp hirtum) has the highest content of the antimicrobial active substance carvacrol. Antibiotics have been previously used as effective tools to support animal health, growth performance as well as feed to gain ratio, especially in pig and poultry. Several different mechanisms have been proposed to clarify AGP-mediated growth enhancement, such as inhibition of subclinical diseases, reduction of metabolic cost of the challenged immune system, reduction of growth-depressing metabolites, such as ammonia, other nitrogenous compounds and bile degradation products formed by bacteria, lower fermentation cost, and enhancement of uptake and use of nutrients, as the intestinal wall in AGP-fed animals may be less thick (Allen et al., 2013; Giannenas et al., 2018a, 2018b, 2018c). All these points leverage the common hypothesis that the intestinal microbiota depress animal growth, either directly or indirectly, and that the major mechanism of AGP is based on its antimicrobial effects on intestinal microbiota and is emphasized according to its bactericidal activity. It is very common in germ-free animals a completely lack of effectiveness of antimicrobials as growth promoters. Also, germ-free animals after inoculation with bacteria fail to reach optimum growth performance. These seem to be the strongest arguments for the above stated hypothesis (Dibner and Richards, 2005; Vondruskova et al., 2010). Accordingly, herbal feed additives may be used as comparable operating tools to leverage animals’ health and performance. These plant-based feed additives have a major advantage over synthetic AGPs since they originate naturally from medicinal aromatic plants and they are routinely practiced as cooking ingredients in traditional recipes (De Smet, 2002; Varel, 2002). Recently, the World Health Organization (WHO) declared that the use of antimicrobials as growth feed additives in farm animals is a public health issue and that an urgent global synchronized planned action is necessary to reduce or completely eliminate the use of these compounds in animal nutrition, because several antimicrobial drugs that are used in productive animals are also used to cure important human infections, at the same time (WHO, 2014). An increasing pressure is now arising from the medical and veterinary communities, the regulatory agencies and the consumers to reduce or eliminate in-feed antibiotics of food-producing animals (Dibner and Richards, 2005; Sun et al., 2005; Gaucher et al., 2015).

Feed utilization In the search of the literature regarding the use of aromatic plants and their extracts in diets of broiler chickens, in contrast to the limited effects on feed intake, improvements in weight gain and feed conversion ratio dominate the observations. One mechanism that might explain this phenomenon is the stimulation of digestive enzyme secretions. The positive effects of EOs on digestive enzyme secretion from pancreas and intestinal mucosal have been constantly presented in several experiments with broiler chickens (Jamroz et al., 2006; Jang et al., 2007; Basmacioðlu Malayoðlu et al., 2010). These effects were confirmed by the increased digestibility of nutrients. However, these findings were not always synthesized and translated into equivalent improvement in growth performance (Lee et al., 2003;

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Botsoglou et al., 2004; Hernandez et al., 2004; Garcia et al., 2007; Amad et al., 2011). It is noteworthy that there is an inadequate description of the environmental conditions under which these trials were conducted. Poor hygienic conditions may depress growth performance of chickens, in that case, plant extracts and EOs may favorably affect the growth performance of broilers. Herbal extracts, plant EOs or their pure compounds have been broadly studied in the last decades as alternative growth performance enhancers. It was very early a pioneer study by Vogt et al. (1989) that herbs and spices were used as feed additives to support growth in broiler chickens. This might be possibly explained through their beneficial influences on animal metabolism and their ability to enhance digestion (Hernandez et al., 2004), to stimulate immune response (Gessner et al., 2013), to mediate inflammatory potential (Acamovic and Brooker, 2005; Gessner et al., 2013, 2016) and afford strong antibacterial, antioxidant and antimicrobial or antiparasitic properties (Christaki et al., 2012; Giannenas et al., 2013). Aromatic and medicinal plant extracts have long been regarded as a huge source of bioactive compounds with medicinal value, since they contain a variety of secondary metabolites, such as phenolic compounds, terpenes, steroids, and polyketides (Franz et al., 2010). It has been mentioned that various secondary metabolites phenolic compounds seem to have the highest potency (Gessner et al., 2016). It has been investigated that diet supplementation of poultry with different herbal extracts improved body weight gain, improved carcass quality, and reduced mortality and morbidity, however several published studies did not state noteworthy effects (Giannenas et al., 2003; Lee et al., 2003, 2004; Windisch et al., 2008; Christaki et al., 2012). Literature studies showed that dietary EOs could improve nutrients digestion (Lee et al., 2003; Basmacioðlu Malayoðlu et al., 2010). Some authors tested the effect of active substance in EOs, e.g., thymol and cinnamaldehyde at level of 100 ppm in the diets on enzyme activities in female broiler chickens (Lee et al., 2003, 2004). It was noticed that dietary EO affected enzyme activity. An increase in lipase by 29% and proteinase activity (trypsin activity increased by 18%) in the gastro intestinal tract was found by dietary addition of thymol in 21-day-old chickens. Betancourt et al. (2010) reported that addition of 200 ppm EO of Oregano majorana in the diet improved apparent ileal digestibility (AID) for energy and fat compared to unsupplemented diet and was comparable to the feed supplemented with antibiotics in broilers. The higher AID of fat was associated with higher body weight at 21 days. The EOs from Oregano vulgare h. at the same inclusion level, however, did not alter digestibility of fat. This may be related to the concentration of active substance in the EOs. Basmacioðlu Malayoðlu et al. (2010) showed that inclusion of oregano EO at 250 and 500 mg/kg to a wheat-soybean meal-based diet, substantially enhanced chymotrypsin activity in the gastrointestinal tract and improved digestion of crude protein and consequently digestibility values in broilers. This occurred with or without exogenous enzymes in diets. Similarly, Jang et al. (2007) found that total and specific activities of trypsin, the total activities of a-amylase and maltase were fortified in the intestinal chime of growing broilers that were given diets supplemented with 50 mg oregano EO/kg compared with chickens given unsupplemented diets.

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According to several authors (Lee et al., 2004; Jamroz et al., 2006; Basmacioðlu Malayoðlu et al., 2010; Bozkurt et al., 2014; Paraskeuas et al., 2016, 2017) it has been exhibited herbal EOs can stimulate lipase and proteinase activities, which in some cases may lead to increase digestible energy and metabolizable energy. The enzyme stimulation effect may be explained by stimulation of bile salt secretion. Lately, it was reported that dietary addition of protease and plant EOs in diets for broiler chicks improved growth performance (Giannenas et al., 2014a, 2014b). It was found that diet supplementation with protease and herbal extracts and, reduced the concentrations of excreta ammonia emission and increased nitrogen retention. Additionally, diet supplementation with a combination of a protease and EOs supported retention of nitrogen and reduced ammonia emissions in excreta by exhibiting a substantial synergy (Park and Kim, 2018). It seems to be that exploitation of aromatic and medicinal plants or herbs, spices their extracts or EOs are parts of a global project with synergies either based on the different herbs or among the traditional knowledge of different areas of the world.

Antioxidant and antiinflammatory actions The antioxidant activity of aromatic plants and their extracts in diets of broiler chickens as phytobiotics is another biological property of great interest. A huge volume of published studies in literature provide evidence of the ability of herbal extracts to deactivate free radicals and act as critical preventing factors against some chronic diseases such as neurogenerative disorders or hepatic failures (Kamatou and Viljoen, 2009). Antioxidant activity of the aromatic plants or herbs, their extracts or EOs is based on their ability of donating hydrogens or electrons and also delocalizing the unpaired electron within the phenolic aroma ring of their structure are the main mechanisms of protecting other biological molecules against oxidation, that previous researchers have been suggested (Fernandez-Panchon et al., 2008; Giannenas et al., 2013). According to Cuppett and Hall (1998), Brenes and Roura (2010) and Chirstaki et al. (2012) it has been extensively reported that a wide range of herbs and their extracts have potential antioxidant functions, especially those products derived from the Labiatae family such as oregano, thyme, basil, mint, rosemary, sage, savory, marjoram, hyssop, and lavender, due to their phenolic terpenoid compounds, e.g., carvacrol, thymol, rosmarinic acid, rosmarol, menthol, and eygenol. On the other hand, there is some evidence suggesting that the antioxidant properties of phytobiotics is not only caused by their phenolic substances, but also their nonphenolic compounds, such as glycosides (Milos et al., 2000). Cherian et al. (2013) phrased out that a dietary extract of Artemisia annua fed to broiler chickens showed an efficient antioxidant activity by reducing thiobarbituric acid reactive substances (TBARS) either in breast or in thigh meat. They suggested that the reduction in TBARS value could be due to individual or combined antioxidant properties of polyphenolic compounds or vitamin E in Artemisia annua. Placha et al. (2014) have demonstrated that supplementing the diet of broiler chickens with thymol can reduce the oxidation of fatty acids indicated by the lower malondialdehyde level in duodenal mucosa. Franz et al. (2010) have suggested that phytobiotics can beneficially affect some enzymes with antioxidant

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activity, such as superoxide dismutase, catalase, oxidase, glutathione peroxidase and, consequently affecting lipid metabolism in animals. Researchers have investigated the potential effect of extracts or EOs mostly from the Labiatae and the Asteraceae plant families containing phenolic compounds on improving the antioxidative capacity of poultry meat (Botsoglou et al., 2002, 2004; Young et al., 2003; Florou-Paneri et al., 2005; Giannenas et al., 2018c). Mohammadi Gheisar et al. (2015, 2017) have supplemented the diet of meat-type birds with a blend of extracts of medicinal aromatic plants containing thyme and reported that the TBARS values due to oxidation process in breast meat was substantially decreased due to the blend of extracts of medicinal aromatic plants. Antioxidant protection of dietary aromatic plants and their extracts is significant for two reasons. The first one is that it can promote health and performance of chickens. Another important consideration of the poultry industry, besides performance enhancement, is the quality of the poultry meat, especially during storage, guaranteeing a long shelf life. Lipid oxidation is a leading cause of quality deterioration in meat or muscle tissues and is well correlated with heme iron content and polyunsaturated fatty acids in meat (Rhee et al., 1996), and it is measured as rancidity. Dietary supplementation with plant-based antioxidants has been proved as an efficient tool to improve antioxidative capacity of broilers (Florou-Paneri et al., 2005; Christaki et al., 2012; Giannenas et al., 2016c). For this reason, aromatic medicinal plants, herbs, spices, and their extracts are examined as natural sources of antioxidants in order to protect meat from oxidation and prolong its storage time. Another important aspect in terms of antioxidant efficacy that has been further investigated are the synergism that is revealed when blends of aromatic medicinal plants, their extracts or EOs are simultaneously used in chicken nutrition. Very lately, it has been reported that a polyherbal feed additive containing extracts from the plants Allium sativum, Mentha piperata, Eucalayptus globulus, Cinnamomum camphora, Trigonella foenum graecum, Cichorium intybus, Trychyspermum ammi, Eruca sativa, and Zingiber officinale prevented the development of lipid oxidation in breast and thigh chicken meat kept under refrigerating storage for 4 days (Giannenas et al., 2018b). In the same study, the total phenolic (TP) content of the control diet was 0.4 mg/L gallic acid equivalents, the phenolic content of the extract was 15 mg/L gallic acid equivalents, whereas the content of the chicken breast and thigh meat in TP was augmented from control diet from 4.0 mg/L gallic acid equivalents, to 6.0 TP, respectively, when chickens fed 500 mg per kg of the herbal mixture. Similarly, another recent study (Ramos et al., 2017) showed that diet supplementation for 42 days with a Mexican oregano extract at the level of 100 mg per kg. The composition of this oregano oil extract was almost 31% thymol and 10% carvacrol. This extract after dietary supplementation for 6 weeks increased by 552% thymol content and 648% the carvacrol content in breast meat. These authors analyzed thymol and carvacrol by gas chromatography-mass spectrometry GS-MS. This modern and accurate analytical methodology can be used to identify and quantify the traceability of herbal phenolic constituents in meat or other animal tissues. The efficacy of herbal extracts as antioxidant feed additives needs to be evaluated and correlated with their phenolic content. In analogous very recent studies, the in vitro antioxidant activity of the tea tree EO was evaluated (Zhang et al., 2018). The antioxidant potential of this EO was evaluated by different methods such as TBARS and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, and the hydroxyl

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radical scavenging activity method. The tea tree EO was effective to lower DPPH with an EC50 (concentration for 50% of maximal effect) of 48.35 mg/mL and, at the same time, it reduced drastically lipid oxidative reactions with an IC50 (50% inhibitory concentration) of 135.9 mg/ mL. Moreover, it completely eliminated hydroxyl radical substances with an EC50 of 43.71 mg/mL. These findings showed that tea tree oil can serve as a very powerful natural based tool in reduction of oxidative stress in chickens. However, in vivo findings are not always as satisfactory as those in vitro studies. In this case, other divergent reactions may occur and in vivo experimental and large-scale studies are needed to guarantee their efficacy. Aromatic and medicinal plants or herbs, their extracts or EOs can have another important activity such as antiinflammatory or immune modulatory properties. In previous in vivo and in vitro studies, quercetin boosted neutrophil function and nuclear cell cytolytic activity and may improve innate immune function in poultry (Hodek et al., 2002; Saeed et al., 2017). Plant extracts including quercetin, have been reported to reduce inflammatory immune function by moderating the production of pro- and antiinflammatory molecules from cells of the innate and adaptive immune system (e.g., macrophages and T cells) and also, exhibit antiinflammatory activity on various cell types in animal and poultry species (Chirumbolo, 2010; Hager-Theodorides et al., 2014). Also, a dietary mixture of plant compounds with organic acids and probiotics modulated CD8þ to CD4þ blood lymphocytes subpopulations, although no main immune-stimulatory effect was found as the disease challenge of the animals was low (Giannenas et al., 2016a). Antioxidant compounds from the herbal extracts have been ingested by the feed, absorbed in the gastrointestinal tract and protected meat lipids from oxidative process and oxidation end products such as aldehydes formation in both breast and thigh meat samples. This antioxidant protection was considerably higher compared to the unsupplemented groups of chickens. Despite the fast metabolism of thymol and carvacrol in the anterior parts of the digestive tract, abundant quantities were found deposited in chicken breast (Ramos et al., 2017). Bioavailability and pharmacological mechanism of action of the herbal phenolic compounds are the underlying factors that determine these antioxidant health benefits (Fernandez-Panchon et al., 2008; Sedaghat and Karimi Torshizi, 2017). The antioxidant effect of the polyherbal dietary supplements is in agreement with previous results in chickens that were administered diets with 500 or 1000 ppm of an extract with 5% oregano and 0.5% sage EOs (Giannenas et al., 2016b, 2016c, 2016d). Another herbal feed additive containing menthol and anethole as major bioactive compounds improved growth performance of broiler chickens at the inclusion level of 100 mg/kg diet (Paraskeuas et al., 2017). It must be highlighted that the inclusion of the same additive at the level of 150 mg per kg of diet showed a considerably greater improvement in plasma total antioxidant capacity and a deeper reduction in meat cholesterol, proving that effectiveness and robustness of the impact of herbal feed additives are related to their inclusion level and composition. Plants such as herbs and spices (whole plants, leaves, or seeds, mainly used as feedstuffs) and their extracts (considered as additives) are being increasingly used in animal nutrition as appetizers, digestive and physiological stimulants, colorants, and antioxidants, and for the prevention and treatment of certain diseases or chronic pathological conditions in animals. Interest from the scientific community has been shifted to the identification of new efficient dietary nonantibiotic alternatives for chickens to reassure performance and establish a healthy and functional intestinal tract; herbal extracts should prove whether they are the

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reliable alternative in agrofood systems (Vogt et al., 1989; Giannenas et al., 2003; Christaki et al., 2012).

Antimicrobial activity Another basic and well-accepted mechanism that can explain the growth promoting effect of aromatic plants and their extracts in diets of broiler chickens as phytobiotics is the stabilization of ecosystem of gut microflora due to their potent antimicrobial activity. The balance of intestinal microflora on a favorable composition can lead to improved feed utilization and less exposure to growth-depressing disorders associated with digestion and metabolism (Williams and Losa, 2001; Franz et al., 2010; Bento et al., 2013; Kurekci et al., 2014; O’Bryan et al., 2015). The antimicrobial activity of EOs has been extensively investigated in several in vitro assays that showcased carvacrol and thymol as strong antimicrobials against pathogenic bacteria such as Escherichia coli and Salmonella typhimurium, both of which are potential risk factors of enteric infections (Franz et al., 2010; Hippenstiel et al., 2011; Bassole and Juliani, 2012). Carvacrol, thymol, and eugenol-exerted potent antimicrobial activity, have similar structure and exerted in combination, a further synergistically bacteriostatic and bactericidal activity, even in lower concentrations (Bassole and Juliani, 2012). Therefore, it is necessary to unravel the synergistic mechanism to optimize their formulation. Different in vitro methods as well as different pathogens exist for ranking the antimicrobial capacity of EO components, which could vary in a great extent. EOs used in poultry diets either individually or in combination or solely pure compounds have shown strong inhibitory effect on Clostridium perfringens and E. coli in the hindgut and ameliorated intestinal lesions and weight loss than the challenged control birds (Mitsch et al., 2004; Jamroz et al., 2006; Jerzsele et al., 2012). It is well known that the mechanism of antibacterial activity of herbal constituents is linked to their hydrophobicity, which disrupts the permeability of cell membranes and cell homeostasis with the consequence of loss of cellular components, influx of other substances, or even cell death (Windisch et al., 2008; Brenes and Roura, 2010; Solorzano-Santos and Miranda-Novales, 2012; O’ Bryan et al., 2015). Plant EOs and their components are mostly hydrophobic. This property enables the herbal compounds’ partition into the lipid layers in the bacterial cell wall and mitochondria, disturbing the structures and rendering them more permeable. It is of noted interest that gram-negative bacteria are more tolerant to the actions of EO than gram-positive bacteria due to their hydrophilic components in the outer membrane (Brenes and Roura, 2010; Giannenas et al., 2013; Seow et al., 2014). The antimicrobial properties of different plants derived compounds against several food pathogens, including Campylobacter jejuni, have been greatly demonstrated in vitro (Friedman et al., 2002; Koollanoor Johny et al., 2010). However, among the few studies reporting the results of in vivo trials, plant-derived compounds mostly failed to have a noticeable impact in reducing Campylobacter colonization in broilers (Hermans et al., 2011; Arsi et al., 2014; Kurekci et al., 2014; Guyard-Nicodeme et al., 2016). It has been hypothesized that these compounds could be absorbed or degraded before they reach the ceca (Hermans et al., 2011; Arsi et al., 2014) or that the ceca could protect Campylobacter from their antimicrobial activity (Arsi et al., 2014). The antimicrobial properties of EOs of aromatic plants and their components have been reviewed extensively (Lee et al., 2004; Brenes and Roura, 2010; Yang et al., 2015). Cowan

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et al. (1999) reported that 60% of EO derivatives examined to date were inhibitory to fungi while 30% inhibited bacteria. Usually, the EOs contain high level of phenolic components, including carvacrol, thymol and eugenol that own the strongest antibacterial properties against food-borne pathogens. Both the phenolics carvacrol and thymol are able to disintegrate the outer membrane of gram-negative bacteria, releasing lipopolysaccharides and increasing the permeability of the cytoplasmic membrane to ATP and depolarize the cytoplasmic membrane (Xu et al., 2008). Helander et al. (1998) investigated the antimicrobial mechanism of the two isomeric phenols, carvacrol and thymol, and the phenylpropanoid, cinnamaldehyde, on E. coli and S. typhimurium. It was observed that both carvacrol and thymol disintegrated the outer bacterial membranes in a similar manner, thus intracellular material from cells is transferred to the external medium due to membrane disruption. On the other hand, cinnamaldehyde did not affect the membrane, but showed potent inhibitory antibacterial activity. It was suggested that the phenylpropanoid cinnamadlehyde penetrates the bacterial membrane and thus can reach and affect the inner part of the cell. In agreement to the previous findings, it has also been hypothesized that whole EOs have greater antibacterial activity than their major components (e.g., solely carvacrol) and this suggests that the minor components in EOs are also critical to the activity and may have a synergistic effect (Brenes and Roura, 2010). The two major components of oregano EO, carvacrol and thymol, were found to give a synergistic effect when tested against S. aureus and P. aeruginosa (Lambert et al., 2001). Synergic effect between carvacrol and its biological precursor p-cymene has been observed. Although, p-cymene is a weak antibacterial substance, it can swell bacterial cell membranes to a larger degree compared to carvacrol. In this way, p-cymene may enable carvacrol to be more easily to transport into the cell so that a synergistic effect is achieved. However, a bacteriostatic or a bactericidal effect of plant extracts, EOs and herbal compounds is lower than antibiotics (Giannenas et al., 2003, 2004; Florou-Paneri et al., 2004; Christaki et al., 2004). In that case, the effective levels of those compounds are regarded to be considerably higher than the cost-effective levels in animal production. However, it is acknowledged that the industry uses feed additives to optimize the animal performance and health, principally in relation to feed input costs. It was observed that oregano EO has the strongest inhibition activities to E. Coli, Salmonella typhymurium, and staphylococcus aureus (Hammer et al., 1999; Burt, 2004; Mathlouthi et al., 2012), and fungus (Kocic-Tanackov et al., 2012). The high antimicrobial activity of oregano EO has also been reported in experiments with chickens. Roofchaee et al. (2011) examined the effects of dietary oregano EO on broiler caecum microbiota and serum antioxidant activity of broiler chickens. It was observed that populations of Lactobacilli remained unaffected; the populations of caecal coliforms were considerably decreased in chicken fed diets with 300 or 600 mg oregano EO per kg compared to chickens fed the nonsupplemented diets. Antioxidant activity of serum was higher in oregano EO supplementation group. It was concluded that oregano EO displayed potent antibacterial effects against cecal E. coli. In growing broiler chickens, Jang et al. (2007) reported that dietary addition of oregano EO at 50 mg/kg reduced Escherichia coli counts in digesta of both ileum and caecum when compared to control chicken; whereas Lactobacilli counts were unaffected. Moreover, dietary EO had similar effect on inhibition of E coli compared to the diet containing antibiotics. Additionally, diet inclusion of a combination of a protease and EO synergistically increased ileal Lactobacilli counts and reduced ileal E. coli counts in broilers (Giannenas et al., 2014a, 2014b;

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Park and Kim, 2018). Also, in two very recent studies it was shown that polyherbal feed additives with high phenolic content can present synergy among their combined constituents and as a result they promote growth and affect intestinal microbiota (Giannenas et al., 2018b, 2018c). Similarly, the antibacterial activity of the herbal dietary supplements is evidenced by alterations in intestinal microbiota either in small intestine or caecum (Tiihonen et al., 2010; Giannenas et al., 2016c, 2017; Skoufos et al., 2016; Tzora et al., 2017). The European ban on the nontherapeutic use of AGPs, as well as the limits on the use of other drugs have increased digestive disorders, morbidity and mortality in broiler chickens (Hao et al., 2014). Withdrawal of AGPs (and despite their use in low doses in feed) has resulted in a numerous increase in the prevalence of several pathogens such as Escherichia coli O157, Clostridium spp., Salmonella spp., Campylobacter spp., in the intestine of animals, arose the contamination of food and environment, and hence, enhanced the opportunities for humans to be infected by these pathogens (Danmap, 2011). Populations of Campylobacter in broiler chickens fed without antimicrobials was three-fold elevated than in broiler chickens that did not receive any approved antimicrobial drug (Heuer et al., 2001). This rather unexpected situation has increased the search for potent antimicrobial alternatives. In the postantibiotic era, applying dietary alternatives to antibiotics to support growth performance of broiler chickens has already become an ethos in order to enhance their productivity, sustain the optimum health status, and reduce mortality.

Effects on intestinal function The use of aromatic plants or herbs, their extracts, or EOs in poultry is also related to their effect on intestinal morphology and functionality. In addition to the findings of some studies that have shown enhancement of the digestive enzyme activity and absorption capacity, it has been demonstrated that those compounds may be able to support gut tissue morphology, and reactions of the gut-associated lymphatic system, as well as to stimulate intestinal mucus production which may further contribute to the relief from pathogen pressure through inhibiting adherence to the mucosa (Tsirtsikos et al., 2012). The gastrointestinal ecosystem despite its complicated establishment, is normally well organized and in active homeostasis. It is mainly composed of the intestinal cells in epithelium and endothelium, mucosal medium in regulation with immune system and intestinal microbiota, which is mainly composed of commensal and beneficial bacteria as well as bacterial pathogens sometimes. In recent experiments (Giannenas et al., 2016c, 2017, 2018c; Skoufos et al., 2016; Tzora et al., 2017) intestinal morphometry was influenced in broiler chickens fed diets supplemented with aromatic plants or herbs, their extracts or EOs. An increase in villus height and number of goblet cells in the chickens fed the herbal products compared to the control group was found. The villus height is an important indicator of the digestive function of broilers since it is directly associated with the absorptive capacity of the intestine mucous membrane. It has been reported that lower villus height leads to lower absorptive capability of the small intestine (Yamauchi et al., 2006; Laudadio et al., 2012). The presence of toxins even in low levels can negatively impact intestinal functionality or morphology in a severe way. Consequently, this can negative affect growth performance of chickens, although, the bird organism can resist a chronic period those associated definite negative impacts (Ghareeb et al., 2015).

Mode of actions of aromatic medicinal plants, spices, or herbs, their extracts or essential oils in poultry

171

According to (Wang et al., 2015) chickens challenged by lipopolysaccharides (LPS) from gram-negative bacteria, in necropsy were found to have drastically lower villus height of the small intestine compared to chickens that were not challenged with bacteria LPS. Furthermore, goblet cells can secrete sticky protein which has a protective effect on intestinal epithelium and on intestinal functionality (Kim and Ho, 2010; Antoni et al., 2014). The addition of a mixture of herbal EOs in broiler diets (containing at least 15 mg carvacrol per kg) notably increased goblet cells count and villus length (Reisinger et al., 2011). Similarly, a combination of oregano with attapulgite clay and organic acids had positive effects on intestinal morphometry and functionality with greater villus height, increased numbers of goblet cells and more intense nuclear intestinal cell functionality (Tzora et al., 2017). However, whether the change on intestinal wall and cells is related to an improvement of digestibility or impairment in immune function is still necessary to be fully ascertained. Since most research findings are readily available only for commercial products containing blends of herbal substances, there is still a need of using a systematic approach to explain the efficacy and the mode of action for each type of aromatic plant or herb, their extracts or EOs, as well as, the dose of active substances. Although the hypotheses are supported by credible experimental evidence, a full understanding of the mechanisms is still lacking. This is possibly one main reason that has limited the efforts in developing new alternatives including the products containing herbal compounds. Any change that could disturb the homeostasis would alter intestinal functionality and thus undermine gut health and even growth and welfare of broiler chickens (Giannenas, 2008; Giannenas et al., 2013). As described above in details, aromatic medicinal plants or herbs, spices, their extracts or EOs may have multiple functions as whole, including antimicrobial and antioxidant activities as well as digestion- and immune-enhancing properties (Brenes and Roura, 2010; Kostadinovic et al., 2015). To define the specific effect and target site (either animal host or its intestinal microbiota) of individual compounds remains critical and will facilitate their further application in feed (Christaki et al., 2012; Giannenas et al., 2018a).

Energy sparing effect Feed is the main cost in livestock production and energy contributes up to 75% of feed cost in poultry production. Grain and oils are the important energy sources in animal feed. After 2008, grain and oil prices have increased rapidly. Thus, lowering energy levels in the diet is economically attractive, but often leads to reduced growth performance. As several trials have shown, the effectiveness of oregano EO on improvement of intestinal health and nutrient digestion, an idea of dietary use of aromatic medicinal plants, herbs, spices, their extracts, and EOs in order to provide an energy saving effect was introduced. It can be expected that oregano EO can potentially compensate for a reduced energy level in the diet, while maintaining production performance. This hypothesis has been tested in experimental and field studies (Paraskeuas et al., 2016). It is generally accepted that when antibiotics growth promoters are used in poultry diets, feed utilization efficiency can be improved on average by 2%e5%. When feeds are formulated without AGPs, a higher energy level may be required. As mentioned earlier, the phenolic substances in EOs have an antimicrobial mode of action on the intestinal level,

172

10. Application of aromatic plants and their extracts in diets of broiler chickens

due to their disruption of bacterial cell walls. In addition, plant EOs have effect on the rate of enterocyte turnover and cellular exfoliation, thereby removing epithelial cells that are infected with pathogenic bacteria (Jin, 2010; Van Eerden and Star, 2010). Due to these functions, it can be expected that EOs can have a similar energy saving effect as AGPs in poultry. Some trial studies investigating the effect of oregano (Origanum vulgare L.) EO are reviewed below. A trial study in broilers was carried out to determine the effect of oregano essential oil (OEO) derived from Origanum vulgare L. (Phytogen, Meritech) on growth, feed utilization efficiency in broiler chickens. Diets with OEO were formulated to contain 3% less energy (Van Eerden and Star, 2010; Van Eerden et al., 2012). In that study, the OEO used was a commercial product, based on oregano EO that consists of main isomers of carvacrol and thymol (these two active substance comprising almost 81%e82% of the total oil) and more than 30 other substances including p-cymene and g-terpinene. The experiment used a 2  4 factorial design, with OEO at two levels (0 and 150 mg/kg) and dietary energy at four levels. The level of 150 mg/kg OEO was used in trial studies. Reduction of the fat content was used to decrease apparent metabolizable energy (AME) content in the diets while maintaining dietary crude protein levels. In the starter period (day 0e15), all diets had the same energy level. During the grower phase (day 15e30) and the finisher phase (day 30e36), the energy treatment was implemented with reduction of 30, 60, and 90 kcal/kg of diet, respectively. The nutrient composition was according to Dutch standards to satisfy nutrient requirements of broiler chickens. The broilers were supplied with a wheat/soybean meal-based diet during the entire experiment. Diets and water were available for ad libitum intake. Starter and grower diets contained a chemical and an ionophoric coccidiostat, respectively. The diets did not contain phytase or enzymes suitable for nonstarch polysaccharides. All diets were fed as pellets. In this study, during the first 15 days starter phase, test diets contained either 0 or 150 mg/kg

1.3

FCR

g (Feed intake / Weight gain)

750

650

550

1.2 0 mg /kg

150 mg / kg

Phytogen ® supplementaon Weight Gain

Feed intake

FCR

FIGURE 10.1 Weight gain (WG, g), feed intake (FI, g) and feed conversion ratio (FCR, g FI/g WG) during the starter period (0e15 days) in broiler chickens dietary supplemented with an oregano essential oil under the commercial product Phytogen (Meritech, China).

Mode of actions of aromatic medicinal plants, spices, or herbs, their extracts or essential oils in poultry

173

0.22

ECE

0.215

0.21

0 150

0.205

0.2 2940

2960

2980 3000 3020 3040 Dietary AME content, kcal/kg

3060

The effect of dietary apparent metabolisable energy (AME) content and oregano essential oil under the commercial product Phytogen (Meritech, China) on energy conversion efficiency (ECE, weight gain, g/energy intake, kcal) during the grower phase (15e30 days).

FIGURE 10.2

TABLE 10.1

Effect of oregano essential oil (OEO)* on intestinal health, growth performance, and feed utilization efficiency in chickens (mean  SD).

Treatments

Control

OEO (L0.7% AME)

OEO (L1% AME)

OEO (L2% AME)

Wet litter, %

2.43  0.07a

2.14  0.02c

2.20  0.09bc

2.26  0.10b

Soft dropping, %

3.38  0.08a

2.98  0.07b

3.03  0.09b

3.06  0.12b

WG, g/bird

3249  14a

3298  27.6c

3283  19.5bc

3273  17.5b

FCR, (g FI/g WG)

1.78  0.01a

1.61  0.02c

1.64  0.01b

1.65  0.02b

Feed cost (U, RMB#)

3621

3579

3577

3550

a

b

b

1.40b

Intestinal health parameters

Performance parameters

Average profit (U/bird)

0.78

1.52

1.36

Values with no superscript in common differ significantly (P < 0.05). * OEO is oregano essential oil derived from Origanum vulgare L.under the commercial product Phytogen (Meritech, China). # The renminbi or yuan is referred to the Chinese currency. a,b,c

OEO respectively but dietary AME content was maintained at the same level. Addition of OEO to the diet resulted in notably increased body weight and markedly lower feed to gain ratio. However, no major differences were observed in-feed intake (Fig. 10.1). The results showed 2.4% and 1.1% increase in growth rate and feed efficiency respectively during starter phase, indicating an energy saving effect in OEO supplemented groups. As reduction in dietary AME content resulted in an increased feed intake, the energy conversion efficiency (ECE, weight gain, g/energy intake, kcal) are calculated based on the original data and presented in Fig. 10.2. As shown in Fig. 10.2, without OEO supplementation, the ECE

174 TABLE 10.2

10. Application of aromatic plants and their extracts in diets of broiler chickens

The effect of dietary apparent metabolizable energy content and oregano essential oil (OEO)* supplementation on feed intake (FI, g), weight gain (WG, g) and feed conversion ratio (FCR, g FI/g WG) during the grower phase (15e30 days). FI, g

OEO, mg/kg

0

WG, g

150

Dif, %

0

150

FCR, g/g Dif, %

0

150

Dif, %

AME, kcal/kg 3050

2005

2007

100.1

1262

1281

101.5

1.590

1.567

98.6

3020

2035

2017

99.1

1285

1268

98.7

1.584

1.593

100.6

2990

2055

2018

98.2

1256

1269

101.0

1.637

1.590

97.1

2960

2066

2083

100.8

1273

1301

102.2

1.625

1.602

98.6

Mean

2040

2031

99.6

1269

1280

100.9

1.609

1.588

98.7

* OEO is oregano essential oil derived from Origanum vulgare L.under the commercial product Phytogen (Meritech, China).

maintained at more or less the same level at different dietary AME content, while with OEO supplementation, there was a linear increase in ECE with reducing dietary AME content. As shown in Table 10.1, reducing dietary energy levels with addition of OEO resulted in healthier intestinal status, this is related to the noted increase in average weight gain. Chickens fed OEO supplemented diets had lower feed consumption but greater weight gain and enhanced feed utilization efficiency. This indicated that at reduced dietary AME content, chicken fed OEO diet had lower energy requirement for maintenance and thus resulting in an energy saving effect. On the other word, the results indicated that supplementation of OEO enables the reduction in dietary AME content in grower phase while maintaining growth performance, maintaining or improving energy utilization efficiency in broiler growers. In TABLE 10.3

Economic benefit of using oregano essential oil (OEO)* at 150 mg/kg of diet in birds. Control

OS-0.7% energy

OS-1% energy

OS-2% energy

210

210

210

210

204

206

205

205

Purchasing costs (U, RMB )

420

420

420

420

Feed costs (U, RMB)

3621

3579

3577

3550

Final total weight, kg

1673.8

1690.5

1685.2

1682.5

Brutal income (U, RMB)

4514.7

4626.4

4590.6

4572.8

Other costs (U, RMB)

315

315

315

315

Initial no of birds Final no of birds #

y

Average profit (U/bird)

a

0.78

b

1.52

b

1.36

1.40b

Values with no superscript in common differ significantly (P < 0.05). * OEO is oregano essential oil derived from Origanum vulgare L. under the commercial product Phytogen (Meritech, China). y Based on final number of birds; values in the same row not sharing a common superscript are significantly different (P < .05). # The renminbi or yuan is referred to as the Chinese currency. a,b

Mode of actions of aromatic medicinal plants, spices, or herbs, their extracts or essential oils in poultry

175

y = 0.0101x + 1.585 R² = 0.7911

1.63 1.62 1.61 1.6 FCR

0 1.59 150 1.58 Linear (0 g/kg) 1.57 Linear (150 g/kg) 1.56 1.55 control

-30

-60

-90

Energy, kcal

FIGURE 10.3 The effect of dietary energy level and oregano essential oil under the commercial product Phytogen (Meritech, China) supplementation on feed conversion ratio (FCR, g FI/g WG) during the overall period (0e36 days).

the grower phase, production characteristics during the grower period are presented in Table 10.2. With reduction of AME content in treatment diets, there was a remarkably linear increase in-feed intake. Body weights were not affected by dietary AME content. Feed conversion ratios (FCRs) were particularly increased with decreasing dietary AME content. Supplementation of OEO decreased FCR in a positive trend. Thus, addition of OEO tended to result in a lower feed conversion ratio compared to control group. The overall performance 0e36 days, treatment diets contained reduced dietary AME content, therefore, it can be expected that the overall growth performance may not be influenced by dietary treatments (Table 10.3). It was noticed that only feed intake is considerably impacted by dietary energy levels. However, feed conversion ratio clearly increased with reduction of dietary AME content when OEO was not included in the diet, while with OEO inclusion, FCR was not affected by dietary AME content (Fig. 10.3). This implied that feed can be formulated at lower AME level with supplementation of OEO in grower and finisher phases, without adverse impacts on growth performance and feed utilization efficiency. Mortality varied between the groups however, analysis failed to show major differences between the different dietary treatments in any period, regardless of dietary AME content or supplementation of OEO. However, OEO supplementation reduced the overall mortality by 27%. On average, the mortality is 3.8% and 5.2% respectively in OEO group and control group. The experimental findings of the current research study clearly demonstrated that supplementation of OEO substantially decreased the incidences of wet litter and soft droppings, improved FCR and production performance. Reduction of dietary AME content with addition of OEO resulted in improved intestinal health status, this is related to the notable increase in average weight gain. Feed utilization efficiency is improved with OEO supplementation at reduced dietary AME content compared to control, however, the optimal

176

10. Application of aromatic plants and their extracts in diets of broiler chickens

feed utilization efficiency was observed with 0.7% energy reduction and 150 mg/kg OEO inclusion. Reducing energy content in the diets with inclusion of OEO reduced feed costs (Table 10.3). The profit per bird was lowest in control group, markedly elevated in OEO supplementation groups. The diet with 0.7% energy content reduction and 150 mg/kg OEO inclusion resulted in the best economical results. Hu et al. (2011) also carried out two experiments in boilers to determine the effect of OEO on growth performance and economic benefit. The first experiment took place in an experimental farm. One-day-old male chickens were divided into a control group (47,700 chickens) and an experimental group (42,000 chickens). Control group was fed standard commercial diets, whereas the experimental group was fed diets containing 200, 150, 100 mg/kg OEO, in phase 1 (1e14 d), phase 2 (15e35 d), and phase 3 (36e43 d), respectively. Results showed that weight gain was maintained at the same level for all treatments but feed conversion ratio in the oregano group was reduced by 4.3%. Brutal profit increased by 1U RMB (or US$0.14) per chicken in the test group. The second trial was done in a commercial farm, with similar experimental design, using 9,100 and 34,700 chickens in the test and control groups, respectively. Similar results were observed as in the first experiment; a substantial reduction in FCR (by 5.5%) in the oregano supplemented group was noted, whereas growth performance was not affected. Brutal profit increased by 0.54 U RMB (US$0.08) per chicken in test group. These field studies showed that feed can be formulated at reduced energy levels when OEO is used at 150 mg/kg, which can result in lower feed cost and economic benefit. Based on these trial studies, it can be deduced that OEO feed additives that containing high level of active substances and used at proper dosage, are effective alternatives for AGPs in poultry feed. Supplementation of OEO allows a reduction on energy levels by 1%e2% in the feed formulation, while maintaining growth performance and feed utilization efficiency. This will lead to reduced feed costs and increased economic benefit in poultry farms. Among phytogenic feed additives, literature studies showed that EO from oregano is a robust operating tool to support growth and enhance efficient use of feed in broiler chickens (Table 10.4). When dietary energy content is reduced by lowering fat inclusion, more carbohydrates may be used in the diets. In addition, due to the increased feed intake at low dietary energy content, more protein and carbohydrates will be consumed. This will increase the fermentation substances and stimulate the growth of pathogen bacteria in the intestine. It can be expected that oregano OEO supplementation suppresses the growth of pathogen bacteria and maintains the populations of the beneficial lactic acid bacteria, thus improving intestinal health and increasing feed utilization efficiency. This may explain the higher energy-saving effect of oregano OEO at reduced dietary energy levels (Jin, 2010; Van Der Aar et al., 2017).

Future implementations and conclusions It is considered one of the main challenges in broiler industry to find effective natural infeed alternatives to antibiotics. As far as the efficacy and safety of herbal compounds is concerned, a systematic and comprehensive investigation is necessary due to their complex and variable composition. Furthermore, experimental trials in chickens fed diets supplemented

177

Future implementations and conclusions

TABLE 10.4

Summary of literature data mostly based on the effect of oregano essential oil (OEO; Origanum vulgare L.) solely or OEO in combination with other feed additives or polyherbal compounds on FCR*.

Broiler strain

Test period, d

Improved FCR, %

References

Cobb

35

2.0

Saini et al. (2003)

Cobb

45

4.9

Saini et al. (2003)

Arbor Acres

42

4.0

Saini et al. (2003)

Arbor Acres

42

7.6

Jin et al. (2012)

Cobb-500

38

4.2

Botsoglou et al. (2002)

Cobb-500

42

2.6

Giannenas et al. (2003)

Ross

41

1.5

Waldenstedt (2003)

Cobb-500

35

3.5

Giannenas et al. (2004)

Cobb-500

42

4.7

Giannenas et al. (2004)

Three-yellow chicken

49

14.8

Jin et al. (2012)

Arbor Acres

35

4.1

Jin et al. (2012)

Ross 307

42

4.6

Jin et al. (2012)

Arbor Acres

44

4.3

Hu et al. (2011)

Ross

42

1.9

Tiihonen et al. (2010)

Cobb-500

35

13.3

Tsinas et al. (2011)

Ross 308

42

10.1

Giannenas et al. (2014b)

Ross 308

42

1.6

Bozkurt et al. (2014)

Ross 308

42

1.5

Giannenas et al. (2016a)

Ross 308

42

7.3

Giannenas et al. (2016c)

Ross 308

44

1.7

Skoufos et al. (2016)

Ross 308

42

8.9

Tzora et al. (2017)

Ross 308

42

9.2

Giannenas et al. (2018b)

Ross 308

42

5.5

Giannenas et al. (2018c)

FCR, feed conversion ratio (g feed intake/g weight gain). Modified from Jin et al. (2012) and further developed for years 2011e18.

with herbal compounds may display potential side effects and other interactions with common feed ingredients (Cheng et al., 2014; Yang et al., 2015). In spite of the fact that plant extracts and herbal constituents are usually included in the GRAS category of food and feed additives by the Food and Drug Administration (FDA) of the United States or regarded as appetizers by the European Food Safety Authorities (EFSA), an extensive and comprehensive evaluation on potential toxicity is required to assure the safe use aromatic plants, herbal

178

10. Application of aromatic plants and their extracts in diets of broiler chickens

extracts, and EOs in broiler feeds as alternatives to pharmaceutical antimicrobials growth promoters (Christaki et al., 2012; Giannenas et al., 2013). A plethora of published studies showed that feed additives containing EOs from aromatic plants or herbal extracts are already marketed for use in animal production worldwide. EOs and plant extracts not only have a similar antimicrobial mode of action as AGPs but they possess other important bioactive properties, as well. Besides their antimicrobial function, the active compounds in plant extracts or EOs can further stimulate digestion, have antioxidant effect, stimulate immune function, these all together make it a promising alternative to AGPs in poultry feed. However, the effectiveness of a plant extract product or EO depends on its content of active components and the activities of these components. Another crucial aspect in broiler nutrition is the dietary energy content, which is the primary cost factor in poultry production. With the high feed costs, it is important to formulate diets with the lowest cost but maintaining growth performance and feed utilization efficiency in order to increase economic profit. This is an on-going challenge for all poultry nutritionists. After the withdrawal of feed AGPs, more attention has been paid to the nutritional management to improve intestinal health of chickens. There are commercially available feed additives in the market which may be used as alternative for AGPs. It is often reviewed that the trial results on using these additives are inconsistent, that may be due to the feed composition, experimental design, and environmental condition or absence of stress factors in poultry (Gong et al., 2014; Yang et al., 2015). There seem to be three main reasons associated with the inconsistency: (1) variations in the composition of the tested compounds due to plant growing locations, manufacturing methods, and the storage conditions; (2) variations in the dosages, especially in cases that may not be efficacious; (3) varied conditions during the trials such as environment (temperature, humidity, and air quality), genetics, age of chicks, feed composition and health status, as well as sanitary status and farm biosecurity systems. The practice of rearing chickens without using AGPs has been implemented in the European Union countries since 2006 and likewise, several countries in Asia and America are expected to follow in the coming era. The withdrawal of antibiotics from feed has implemented a new ethos in animal nutrition. In order to cater performance risks and higher incidences of intestinal diseases and mortality plant extracts as alternatives should be explored in a multitude of ways. This may appear in countries where antibiotic ban will be established. The cost-effectiveness in substituting antibiotics with alternatives is the most challenging one. This remains crucial for assuring long-term sustainable animal production. Herbal feed additives and plant compounds have a large variety of bioactive ingredients and thus represent one of the most promising categories of feed additives, as alternatives to AGPs. However, their application in food-animal production and especially in broiler industry is extensive, but inconclusive, largely owing to their inconsistent efficacy and lack of elucidation on the knowledge of the responsible mechanism of action. A more detailed understanding to figure out more on the effects of aromatic plants or herbs, their extracts, or EOs on intestinal function and microflora is necessary.

References

179

The clearance of mechanism of action of the dietary effects of aromatic medicinal plants, herbs, spices, their botanical extracts, or EOs will permit nutritionists and poultry scientists to make the optimum use of those substances for increased profitability and assured sustainability of poultry production. Finally, the possible risks in the use of aromatic plants or herbs, their extracts, or EOs in animal production and in human health need to be evaluated, even though those compounds are coming from nature and exist in plants in small quantities. Further investigations on a large scale with specific stress conditions or disease models that challenged chickens either with coccidia, such as Eimeria spp, or bacteria strains, such as Escherichia coli, Clostridium perfringens, Mycoplasma, or Salmonella spp, would provide more extensive and detailed knowledge on the effectiveness of aromatic medicinal plants, herbs, spices, their botanical extracts, or EOs as efficient feed additives.

References Acamovic, T., Brooker, J.D., 2005. Biochemistry of plant secondary metabolites and their effects in animals. Proc. Nutr. Soc. 64, 403e412. Alcicek, A., Bozkurt, M., Cabuk, M., 2003. The effect of an essential oil combination derived from selected herbs growing wild in Turkey on broiler performance. S. Afr. J. Anim. Sci. 33, 89e94. Allen, H.K., Levine, U.Y., Looft, T., Bandrick, M., Casey, T.A., 2013. Treatment, promotion, commotion: antibiotic alternatives in food producing animals. Trends Microbiol. 21, 114e119. Amad, A.A., Manner, K., Wendler, K.R., Neumann, K., Zentek, J., 2011. Effects of a phytogenic feed additive on growth performance and ileal nutrient digestibility in broiler chickens. Poultry Sci. 90, 2811e2816. Antoni, L., Nuding, S., Wehkamp, J., Stange, E.F., 2014. Intestinal barrier in inflammatory bowel disease. World J. Gastroenterol. 20, 1165e1179. Arsi, K., Donoghue, A.M., Venkitanarayanan, K., Kollanoor-Johny, A., Fanatico, A.C., Blore, P.J., Donoghue, D.J., 2014. The efficacy of the natural plant extracts, thymol and carvacrol against campylobacter colonization in broiler chickens. J. Food Saf. 34, 321e325. Basmacioðlu Malayoðlu, H., Baysal, S., Misirlioðlu, Z., Polat, M., Yilmaz, H., Turan, N., 2010. Effects of oregano essential oil with or without feed enzymes on growth performance, digestive enzyme, nutrient digestibility, lipid metabolism and immune response of broilers fed on wheat-soybean meal diets. Br. Poult. Sci. 51, 67e80. Bassole, I.H., Juliani, H.R., 2012. Essential oils in combination and their antimicrobial properties. Molecules 17, 3989e4006. Bento, M.H.L., Ouwehand, A.C., Tiihonen, K., Lahtinen, S., Nurminen, P., Saarinen, M.T., Schulze, H., Mygind, T., Fishcher, J., 2013. Essential oils and their use in animal feeds for monogastric animals-Effects on feed quality, gut microbiota, growth performance and food safety: a review. Vet. Med. 58, 449e458. Betancourt, L., Ariza-Nieto, C., Afanador-Téllez, G., 2010. Effects of feeding oregano essential oil to broilers on ileal digestibility and performance under high altitude conditions. Poultry Sci. 89, 384. Botsoglou, N.A., Christaki, E., Florou-Paneri, P., Giannenas, I., Papageorgiou, G., Spais, A.B., 2004. The effect of a mixture of herbal essential oils or a-tocopheryl acetate on performance parameters and oxidation of body lipid in broilers. S. Afr. J. Anim. Sci. 34, 52e61. Botsoglou, N.A., Florou-Paneri, P., Christaki, E., Fletouris, D.J., Spais, A.B., 2002. Effect of dietary oregano essential oil on performance of chickens and on iron-induced lipid oxidation of breast, thigh and abdominal fat tissues. Br. Poult. Sci. 43, 223e230. Bozkurt, M., Aysul, N., Kucukyilmaz, K., Aypak, S., Ege, G., Catli, A., Aksit, H., Coven, F., Seyrek, K., Cinar, M., 2014. Efficacy of in-feed preparations of an anticoccidial, multienzyme, prebiotic, probiotic, and herbal essential oil mixture in healthy and Eimeria spp.-infected broilers. Poultry Sci. 93, 389e399. Brenes, A., Roura, E., 2010. Essential oils in poultry nutrition: main effects and modes of action. Anim. Feed Sci. Technol. 158, 1e14.

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C H A P T E R

11 Application of aromatic plants and their extracts in the diets of laying hens David Harrington, Heidi Hall, David Wilde, Wendy Wakeman Anpario plc, Nottinghamshire, United Kingdom O U T L I N E Introduction

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Aromatic plants in layer well-being Gut microbiota modulation Stress Behavior Temperature stress Oxidative stress Immune regulation Skeletal development

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Feed conversion ratio (FCR) and feed intake Egg production including egg weight

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Egg characteristics Antioxidant stability Egg composition Egg shell Sensory evaluation

195 195 197 197 198

Conclusion

198

References

199

Introduction Through the application of quantitative genetics and sophisticated breeding techniques the modern laying hen is a highly productive animal. The goal of the primary breeders is to produce a layer genotype that is able to continue to produce increased number of

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00011-X

187

Copyright © 2020 Elsevier Inc. All rights reserved.

188

11. Application of aromatic plants and their extracts in the diets of laying hens

saleable eggs with increase persistency of lay, the “long life layer” with a 100-week production cycle (Bain et al., 2016). Accompanying this increased persistence is the requirement to maintain shell quality, skeletal integrity, and hen livability until the end of lay (Preisinger, 2018). Nutrition, biosecurity, and management are key factors in laying hen production to ensure optimal laying production, particularly with an increasing global focus on reducing the use of antibiotics and improving bird welfare. Increasingly strict targets for antibiotic use in the poultry industry limit the type and range of antibiotics that can be used. In the laying hen, the requirement for removal of eggs from the food chain to meet with strict antibiotic withdrawal periods has significant economic cost, hence the use of antibiotics has always been limited. With this increased pressure to remove medications that were once allowed, the potential for synergy between aromatic plants or even essential oils and antibiotics could prolong the efficacy of what is rapidly becoming a limited range of permissible antibiotics (Moussaoui and Alaoui, 2016; Chouhan et al., 2017). No withdrawal period is necessary when using aromatic plants which further strengthens their suitability as an alternative for antimicrobial management where necessary. Bird welfare, among other factors, has focused on housing and the replacement of existing cages with alternatives such as enriched/colony cages or more extensive systems that allow birds’ access to the floor and do not use cages. While these systems have a positive effect upon bird welfare, the opportunity for disease or parasitism is increased (Lay et al., 2011). An example is the increasing global incidence of the poultry red mite, Dermanyssus gallinae, where heavy infestations can lead to reduced hen productivity and even death. While essential oils have been used topically as mite repellents, scientific confirmation of their use in vivo hens are lacking despite anecdotal reports of their efficacy (Sparagano et al., 2014). Increased access to the floor or outdoor environment in alternative systems increases the exposure of birds to endoparasites such as helminths, histomonads, and Eimeria. The antiparasitic properties of plants are well characterized in vivo (Dhama et al., 2015) hence they are already used in laying hen production. Aromatic plants produce and exude aromatic substances (odorous volatile substances) and their extracts can include essential oil, gum exudate, balsam, and natural resin derived from any part of the plant. Many aromatic plants are species of the Lauraceae, Umbelliferae, Myrtaceae, and Lamiaceae families which includes cinnamon, fennel, eucalyptus, and oregano, although this grouping is not exclusive. Examples from other plant families include Nigella sativa in the Ranunculaceae family whose seeds produce an aromatic oil containing high levels of p-cymene, thymoquinone, and thymol (Kazemi, 2014) or essential oil from the flowers of Crocus sativus from the Iridaceae family that is high in safranal (Kanakis et al., 2004) which has been shown to have anticonvulsant properties (Hosseinzadeh and Talebzadeh, 2005). The dried stigma from C. sativus is known as saffron and is rich in crocin, a strong antioxidant (Botsoglou et al., 2005a). There have been a handful of reviews on plant use in laying hens (Steiner, 2009; Bozkurt et al., 2014). This chapter will revisit the use of aromatic plants in laying hen diets, particularly the Lauraceae, Umbelliferae, Myrtaceae, and Lamiaceae with a focus on their role in modulating the gut microbiota, managing stress and other physiological functions.

Aromatic plants in layer well-being

189

Aromatic plants in layer well-being Gut microbiota modulation The antimicrobial properties of extracts of aromatic plants are well documented in vitro (Roldán et al., 2010). The research on their use in vivo has been broad but their effect on the chicken microbiota and its characterization has received considerable focus, particularly in broilers for conditions that disrupt enteric health, such as the pathology Necrotic Enteritis caused by proliferating Clostridium perfringens (Diaz Carrasco et al., 2016). However, the layer microbiota is less well characterized and appears to differ from broilers in colonization pattern (Kers et al., 2018). Consequently, it is valuable to understand the effect of aromatic plants on the layer microbiota; they may well behave differently to the broiler. A handful of studies have looked at the impact of aromatic plants on the layer intestinal microbiota. A positive effect of oregano oil presented in microcapsules (100 mg/kg feed) was shown to increase numbers of Lactobacilli and Bifidobacteria while lowering levels of Escherichia coli and Salmonella in hens over a seven-week period (He et al., 2017). Bölükbasi and Erhan (2007) saw no difference in coliform counts following the feeding of thyme for 12 weeks to Lohman hens although the authors did observe a decrease in E. coli counts in birds fed 0.1 or 0.5% thyme. Following induced molting, a significant change in the cecal microbiota occurs, Clostridium perfringens and coliform counts increase while Lactobacillus spp. decreases (Bozkurt et al., 2016). However, the administration of a commercial product based on oregano essential oil (24 mg/kg) did not significantly alter the postmolt cecal microbiota of hens over the period 82e106 weeks, despite a trend in decreasing numbers of coliforms and increasing Lactobacillus spp (Bozkurt et al., 2016). Salmonella enteritidis and S. typhimurium are major food borne pathogens causing enteric illness in humans. Table eggs can be a source of infection and can become contaminated via either vertical or horizontal transmission. Upadhyaya et al. (2015) presented data indicating that the administration of trans-cinnamaldehyde (1.5%volume/weight) in the feed of Salmonella-infected hens over a 66-day period led to a 44% and 36% reduction in the number of Salmonella-positive egg shells and yolks respectively versus controls while also reducing the proportion of Salmonella-positive tissue samples (caeca, liver and oviduct). Hens fed a commercial product containing eugenol from (250 ppm) for three weeks prior to infection with S. enteritidis were Salmonella negative in caeca, liver, spleen and ovary 30 days postinoculation (Ordóñez et al., 2008). Arpásová et al. (2013) reported lower coliform, enterococci, lactobacilli, yeasts, and fungi counts in egg samples taken from 17-week-old hens fed thyme for 23 weeks. Cecal enteritis associated with intestinal spirochaetes of the genus Brachyspira is a disease complex mainly seen in laying hens and broiler breeders. Research work by Verlinden et al. (2013) indicated that among other compounds, carvacrol, thymol, linalool, nerolidol, eugenol, piperine, capsaicin, and cinnamaldehyde derived from aromatic plants demonstrated the lowest minimum inhibitory concentrations (MIC) against Brachyspira intermedia. When birds infected with B. intermedia were given feed supplemented with transcinnamaldehyde (500 mg/kg feed), the enumeration of Brachyspira was significantly decreased compare to control hens.

190

11. Application of aromatic plants and their extracts in the diets of laying hens

Various aromatic extracts influence the immune system including eugenol which has been shown to stimulate the inner mucus layer, a key mucosal barrier to microbes, and increase different bacterial families within the Clostridiales (Wlodarska et al., 2015). A working hypothesis is that eugenol modules the gut microbiota which leads to a thickening of the mucus layer which in turn confers a resistance to colonization by pathogenic bacteria (Wlodarska et al., 2015). While eugenol has been shown to stimulate the mucus layer to confer protection to the host, it is highly likely other aromatic plant derivatives or compounds could have a similar mode of action.

Stress Stress is multifactorial and examples of stressors can include nutritional change, handling, compromised welfare and temperature extremes. The production of corticosterone in response to a stressor suppresses follicular development and increases energy intake. Available energy is increased by decreasing the availability of the circulating yolk precursor and the prevention of yolk deposition in follicles leading to lower egg production (Mumma et al., 2006; Wang et al., 2017). Behavior Stress can also alter the performance of normal behaviors of the hen potentially leading to behavioral pathologies such as feather pecking or increased fear responses (Jones, 1996). Thymol has been reported to significantly lower fear-associated behaviors in quail when fed an equivalent 80/mg/day/bird over 100 days (Lábaque et al., 2013). Thymol acts as a positive allosteric modulator of GABA receptors (Garcia et al., 2006), which when activated, depress neuronal activity. Sedatives such as diazepam or the anesthetic propofol (an analog phenolic compound of thymol) work in a similar way (Riss et al., 2008; Bhandari and Kabra, 2014); at lower doses, propofol has also been shown to have anxyolitic properties (Kurt et al., 2003). Similarly, 200 mg/kg thyme fed to broilers increased feeding behavior (and performance) while lowering fear response (Ramadan, 2013), the reduction in fear response most likely allowing for increased time for feeding behavior. Dietary changes can initiate feather pecking that has the potential to lead to long-term injurious feather pecking, although the use of oregano in feed could potentially ameliorate the impact of diet change by “masking” the change (Dixon and Nicol, 2008). Banu et al. (2016) demonstrated a positive effect of 50% peppermint solution (20 mL/bird) on reducing brooding behavior as determined by the number of production days lost due to the hen attempting to incubate the egg. In backyard hens, peppermint decreased the time the hen was off the egg by 27 days. Temperature stress Temperature variance outside of the comfortable range of poultry, particularly excess heat, can result in compromised metabolic and immune function as well as disruption to the endocrine system, failure of gut integrity and tissue damage (Nidamanuri et al., 2017). Furthermore, reproductive organs can be adversely affected leading to reduced oviduct size and impaired follicle development. Cinnamon (40 mg/kg) added to the diet of

Aromatic plants in layer well-being

191

Lohmann LSL-Lite hens kept under cold stress conditions (8.8  3 C) over 8 weeks was observed to increase egg production 16% while cinnamon in combination with supplemental levels of zinc increased egg production 19% in comparison to unsupplemented controls (Torki et al., 2015). Similarly, Akbari et al. (2016) demonstrated that laying hens reared under low temperature conditions (6.8  3 C) and fed a combination of peppermint and thyme essential oil had improved performance (increased egg production and egg mass and reduced FCR) and egg quality versus controls. Conversely, hens under moderate heat stress (24 C) fed a mixture of essential oils from oregano, laurel, sage, fennel seed, myrtle, and citrus peel showed no improvement in production performance nor egg quality, although shell quality was improved over a 16-week period (Bozkurt et al., 2012a). Similarly, the inclusion of fennel essential oil in the diet of laying hens under high heat stress (34 C) did not influence performance parameters versus hens kept at 24 C (Gharaghani et al., 2015) and nor did an essential oil mixture (oregano, laurel, sage, myrtle, fennel, and citrus peel) in hens kept at 28 C (Özek et al., 2011). However, the same mixture of herbs used by Bozkurt et al. (2012a) was shown previously to significantly increase egg production and egg weight versus controls when birds were kept under conditions on average 30 C during a 20-week period (Çabuk et al., 2006). Oxidative stress Underpinning these physiological stress responses is oxidative stress, an imbalance in the endogenous antioxidant mechanism and prooxidants in the cell leading to excess reactive oxygen species and cellular damage. Environmental stress, such excess heat, increases the rate of oxidative stress in stressed birds (Nidamanuri et al., 2017). Essential oils from plant families such Lamiaceae have well documented antioxidant activity in vitro and in vivo in broilers (Matkowski and Piotrowska, 2006). Supplementation of hen diets with combinations of thyme, mint, rosemary and dill (200 mg/kg), rosemary and thyme (0.9%) or thyme (9 g/kg) or rosemary (6 g/kg) resulted in a significant reduction in serum and liver malondialdehyde (MDA) concentrations, reduced serum glutathione (GSH) activity or increased serum super oxide dismutase (SOD) (Alagawany and El-Hack, 2015; Alagawany et al., 2017; El-Hack et al., 2015; Mousavi et al., 2017). Similarly, Torki et al. (2018) reported a synergistic effect between chicory, rosemary, and dill on egg yolk oxidative capacity in hens fed the herbal mixture, increasing yolk glutathione peroxidase. Interestingly, in recent work by Chen et al. (2018) the administration of eucalyptus leaves at 0.8 g/kg did not significantly affect the hens total antioxidant capacity under normal conditions, although meat drip loss was 3% lower than controls. However, when hens were exposed to ethanol-induced oxidative damage, serum antioxidant status was significantly improved versus controls, resulting in lower liver damage. While undoubtedly aromatic plants and their extracts influence the antioxidant capacity of the hen, inferences on the effect upon performance are harder to draw. Evaluating serum and egg yolk antioxidant status, 50% of the evaluations presented in Table 11.1 showed no effect of antioxidant status in egg yolk or serum on performance. While antioxidant status in egg has been shown to reflect antioxidant status in serum (Sahin et al., 2010), the late-laying hen deposits antioxidants preferentially in the egg rather than the body (Loetscher et al., 2014). This could be a confounding factor in determining

192

TABLE 11.1 Effect seen of aromatic plant or extract on egg production, serum, and egg antioxidant capacity. Duration (weeks)b

Supplement

Presentation

Inclusion

Egg production

Serum MDA

Egg yolk MDA

References

43

10

Echinacea

Powder

5 g/kg

[

-

NC

Jahanian et al. (2015)

9

Eucalyptus

Filtrate

0.8 g/kg

NC

NC

Y

Chen et al. (2018)

37.5 28

12.9

Oregano

Dry herb

1%

[

-

Y

Radwan et al. (2008)

82

25

Oregano

Essential oil

24 mg/kg

[

Y

-

Bozkurt et al. (2016)

28

12.9

Rosemary

Dry herb

1%

[

-

Y

Radwan Nadia et al. (2008)

36

16

Rosemary

Powder

0.90%

[

Y

-

Alagawany et al. (2017)

28

12.9

Thyme

Dry herb

1%

[

-

Y

Radwan Nadia et al. (2008)

36

16

Thyme,

Powder

0.90%

[

Y

-

Alagawany et al. (2017)

28

12.9

Turmeric

Dry herb

1%

[

-

Y

Radwan Nadia et al. (2008)

42

4.3

Fennel

Not stated

20 g/kg

NC

-

NC

Gharaghani et al. (2015)

32

8.5

Oregano

Essential oil

100 mg/kg

NC

-

Y

Florou-Paneri et al. (2005)

32

8

Oregano

Dry herb

5 g/kg

NC

-

Y

Botsoglu et al. (2005b)

32

8

Rosemary

Dry herb

5 g/kg

NC

-

Y

Botsoglu et al. (2005b) c

78

4

Sage

Dried leaves

2.50%

NC

Y

NC

Loetscher et al. (2014)

42

6

Skullcap

Ethanol extract

0.5%

NC

-

Y

An et al. (2010)

36

16

Thyme

Powder

9 g/kg

NC

Y

e

El-Hack and Alagawany (2015)

NC, no change detected. a At initiation of test. b Length of test. c Lyophilized yolk.

11. Application of aromatic plants and their extracts in the diets of laying hens

Bird age (weeks)a

Aromatic plants in layer well-being

193

the level of aromatic plant required to stimulate a positive effect of antioxidant status on performance. The role of aromatic plants to modulate stress responses (whether behavioral or environmental) and the associated antioxidant status of the bird warrant further investigation. Increased understanding of the relationship between aromatic plants, antioxidant status in the hen and egg and the effect on stress will assist in their effective application and maximize their impact on production.

Immune regulation There are limited studies on the influence of aromatic plants on the layer immune system and while parallels can be drawn from work in broilers, their immune response does differ. Broilers tend toward a strong short-term humoral response (immunoglobulin M) while immunity in layers is biased toward a long-term humoral response (immunoglobulin Y) in combination with a strong cellular response (Koenen et al., 2002). Hens fed rosemary or thyme (0.9%) had higher circulating levels of IgY and IgM than control birds while rosemary appeared to stimulate the highest levels of circulating antibodies (Alagawany et al., 2017). A similar observation was made by Radwan Nadia et al. (2008), 1% rosemary, oregano or thyme herbs increased serum antibody titers. Antibody titers in response to vaccination with Infectious Bronchitis virus, Newcastle Disease virus, or Infectious Bursal Disease virus did not change significantly relative to control birds in hens kept on feed supplemented with a mixture of essential oils from oregano, laurel, sage, myrtle, fennel, and citrus peel from 52 to 68 weeks of age or in another study, hens from 36 to 51 weeks of age (Özek et al., 2011; Bozkurt et al., 2012a). However, it may well be that when birds are challenged with the associated viruses that a difference in the level of responding antibody titer might be observed. Similarly, aromatic plant extracts appear to have minimal influence on cellular immunity. Blood cell counts appear largely unaffected by the addition of eucalyptus, thyme or oregano although higher levels of eucalyptus can significantly alter heterophil:lymphocyte ratios (El-Motaal et al., 2008; Ghasemi et al., 2010; Bozkurt et al., 2016). Fennel essential oil (300 mg/kg) was reported to increase monocyte counts in hens (Nasiroleslami and Torki, 2010) while a mixture of garlic and thyme powder (0.2%) elevated lymphocyte counts when fed to 32-week-old hens for 6 weeks (Ghasemi et al., 2010). Hens fed eucalyptus powder (2e3/kg) had significantly higher wattle swelling on injection with phytohemagglutinin compared to control hens over the course of 24e72 h, indicating an elevated cell-mediated response (El-Motaal et al., 2008). Intestinal health is a key factor in immune regulation in the host and aromatic plants are known to influence the gut microbiota hence this could be the mode of action to influence immune markers. The long production life of the hen and an intensive vaccination program from day-old suggest that aromatic plants may have a role in supporting the immune system; it is a question of identifying the appropriate compounds.

Skeletal development Calcium for egg shell formation is obtained via the diet and mobilization from the medullary bone, for example the keel bone. Pathology of the keel bone such as fractures or

194

11. Application of aromatic plants and their extracts in the diets of laying hens

deformities have been reported as high as 83% in some laying flocks (Käppeli et al., 2011.) although incidence of pathology can vary greatly with housing type and age of hens. An imbalance in calcium utilization and mobilization from medullary and structural bone can lead to skeletal weakness resulting in bone fractures, compromising bird welfare as well as leading to poor egg shell quality and low egg production (Whitehead, 2004). Feed supplemented with fennel has been observed to increase serum calcium and phosphorus levels in hens fed calcium deficient diets. Furthermore, those same birds also had increased calcium and phosphorus mineralization of the tibia and increased bone strength (Hadavi et al., 2017). Olgun (2016) also demonstrated a positive effect of herbs on biomechanical properties of bone and increased calcium content. A mixture of herbal essential oils (garlic, oregano, and rosemary), oleoresins (rosemary and sage) and dry extract (Echinacea) fed to hens from 26 to 70 weeks of age increased the relative retention of calcium (calcium retained as a percentage  of calcium intake) but not phosphorus (Swiatkiewicz et al., 2018b). Despite the absence of any difference in bone mineralization, bone breaking strength, and yielding load were improved versus control birds. Conversely, in hens force molted at 82 weeks of age, biomechanical analysis of bones 14 weeks later did not show any difference in bone mineralization or breaking strength in comparison to controls (Bozkurt et al., 2016). Evidence would suggest that aromatic plants could have an influence on skeletal development in the laying hen, and perhaps pullet, but further study is necessary to identify if there is a causal relationship between plant and skeletal development.

Performance Feed conversion ratio (FCR) and feed intake Laying hens are fed to a target bodyweight according to genotype to achieve the most efficient egg production. Consequently, bodyweight gain in laying is not the most important zootechnical parameter versus FCR or feed intake, although eucalyptus leaf (3 g/kg) or a mixture of essential oils (oregano, laurel, sage, fennel seed, myrtle and citrus peel) has been shown to have a tendency to promote bodyweight gain (El-Motaal et al., 2008; Bozkurt et al., 2012a). When offered a choice, 21.2% of free-range laying hens with access to pasture preferentially foraged in basil over 5.2%, 9.9%, and 5.5% in oregano, parsley, and dill, respectively although no performance data were presented (Kosmidou et al., 2006). Clearly hens can differentiate and select forage but under commercial rearing and laying conditions, no choice in plant is offered. Dried peppermint leaves (20 g/kg feed) fed to 64-week-old hens for 12 weeks resulted in a 9% reduction in FCR (Abdel-Wareth and Lohakare, 2014) while a filtrate of ground peppermint leaves increased the bodyweight in hens over a 12-week period (Banu et al., 2016). A combination of peppermint and thyme essential oils fed to younger hens, 42-week-old, at 100 mg/kg diet led to a 7% decrease in FCR over an eight week period (Akbari et al., 2016). Improvements in FCR have also been reported using oregano, thyme, Echinacea, eucalyptus and rosemary or herbal mixtures (Çabuk et al., 2006; El-Motaal et al., 2008; Radwan Nadia et al., 2008; Abdel-Wareth et al., 2013; Abdel-Wareth, 2015; Jahanian et al., 2015; Bozkurt et al., 2016; Algawany et al., 2017; He et al., 2017). However,

Egg characteristics

195

a number of studies using the same herbs, albeit in some instances different presentations, or others have shown no effect upon FCR or feed intake (Table 11.2). Indeed, Bölükbasi and Erhan (2007) reported a 3% reduction in feed intake in 24-week-old hens fed 1% thyme.

Egg production including egg weight From reported studies, it would appear that the results of using aromatic plants in laying  hen diets are contradictory. Swiatkiewicz et al. (2018a) observed a 3.6% increase in egg production over a 46-week period in hens that were fed a mixture of herbal essential oils (garlic, oregano, and rosemary), oleoresins (rosemary and sage) and dry extract (Echinacea). Other researchers have reported similarly improved production parameters including egg production and egg weight using herbs (Table 11.2). Thyme and its extracts has been shown to increase egg production parameters on a number of occasions (Bölükbasi and Erhan, 2007; Abdel-Wareth, 2015; El-Hack et al., 2015). In contrast, Radwan Nadia et al. (2008), Arpásová et al. (2013), Ding et al. (2017) or Roth-Maier et al. (2005) reported no change in egg production, egg weight or egg mass when hens were fed peppermint, thyme, oregano, rosemary, turmeric or Echinacea, although age of administration, study duration, and herb presentation varied between studies. The difficulty to identify a causal relationship between aromatic plant and performance reflects the inconsistency in the available literature and highlights the needs for a standardized experimental design where possible. Only then, with a degree of experimental rigor, can plants begin to be evaluated for their efficacy on a reliable, reproducible platform. The duration of the evaluation period can also have a role to play in the detection of plant effects on performance. When study outcomes are ordered according to a positive effect on egg production or not (Table 11.2), the average duration of study demonstrating an increase in egg production is 13 weeks (range 7e22 weeks), versus 9 weeks (range 4e16 weeks) for studies where no change was demonstrated; average bird age at test did not differ. While this observation is based on a handful of evaluations (22), it would suggest that to demonstrate an effect of an aromatic additive on performance, the evaluation period needs to be as long as possible. In the current selection of studies, seven weeks was the minimum test period to observe a positive effect on egg production.

Egg characteristics Antioxidant stability The undesirable changes in the egg as a consequence of oxidation include off odor, color change, and flavor. Therefore, it is no surprise aromatic plants as natural antioxidants receive much attention. Dried sage (2.5%) fed to 78-week-old hens over four weeks significantly increased total tocopherol in egg yolk 2.3-fold versus control eggs from birds fed a basal diet containing 40 IU vitamin E (Loetscher et al., 2014). Oregano, Echinacea, rosemary, and skullcap supplemented in layer diets have all been shown to lower markers of lipid oxidation in fresh egg yolks or prolonged storage (Botsoglou et al., 2005b; Florou-Paneri et al., 2005; Radwan Nadia et al., 2008; An et al., 2010; Jahanian et al., 2015; Algawany et al., 2017; Batista

TABLE 11.2

Performance of hens fed aromatic plants or extracts. Egg production FCR

Feed Egg intake weight Source

Supplement

Inclusion

Presentation

7

30

Oregano

100 mg/g

Oil/microcapsule [

Y

NC

[

He et al. (2017)

8

46

Eucalyptus

3 g/kg

Powder

[

Y

Y

NC

El-Motaal et al. (2008)

10

43

Echinacea

5 g/kg

Powder

[

NC

NC

[

Jahanian et al. (2015)

12

64

Peppermint

20 g/kg

Dry herb

[

Y

[

NC

Abdel-Wareth and Lohakare (2014)

12.9

28

Oregano

1%

Dry herb

[

Y

NC

[

Radwan Nadia et al. (2008)

12.9

28

Rosemary

1%

Dry herb

[

Y

NC

[

Radwan Nadia et al. (2008)

12.9

28

Thyme

1%

Dry herb

[

Y

NC

[

Radwan Nadia et al. (2008)

16

36

Rosemary

0.90%

Powder

[

Y

NC

NC

Alagawany et al. (2017)

16

36

Thyme

1%

Powder

[

Y

NC

NC

Alagawany et al. (2017)

22

36

Commercial mixturec

24 g/kg

Essential oil

[

NC

NC

[

Bozkurt et al. (2012b)

4

78

Sage

2.50%

Dried leaves

NC

NC

NC

NC

Loetscher et al. (2014)

4.3

42

Peppermint

100 mg/kg

Essential oil

NC

NC

[

[

Akbari et al. (2016)

6

42

Skullcap

1%

Ethanol extract

NC

NC

NC

[

An et al. (2010)

8

32

Oregano

5 g/kg

Dry herb

NC

NC

NC

NC

Botsoglu et al. (2005b)

8

32

Rosemary

5 g/kg

Dry herb

NC

NC

NC

NC

Botsoglu et al. (2005b)

8.5

32

Oregano

100 mg/kg

Essential oil

NC

NC

NC

NC

Florou-Paneri et al. (2005)

9

37.5

Eucalyptus

0.8 g/kg

Filtrate

NC

Y

NC

NC

Chen et al. (2018)

12

54

Commercial mixtured

150 mg/kg

Powder

NC

NC

NC

NC

Ding et al. (2017)

12

26

Fennel

40 mg/kg

Alcohol extract

NC

NC

NC

[

Valkili and Heravi (2016)

12

26

Thyme

40 mg/kg

Alcohol extract

NC

NC

NC

[

Valkili and Heravi (2016)

12

24

Thyme

1%

Powder

NC

[

Y

NC

Bölükbasi et al. (2007)

16

36

Thyme

9 g/kg

Powder

NC

NC

NC

[

El-Hack and Alagawany (2015)

NC, no change detected. a Length of test. b At initiation of test. c Oregano, laurel, sage, fennel seed, myrtle and citrus peel, 24 g Essential oil. d Encapsulated thymol (13.5%) and cinnamaldehyde (4.5%).

11. Application of aromatic plants and their extracts in the diets of laying hens

Bird age (weeks)b

196

Duration (weeks)a

Egg characteristics

197

et al., 2017). Extracts of eucalyptus leaves fed to hens over 63 days at a dose rate of 0.8 g/kg were shown by Chen et al. (2018) to lower MDA levels in the egg yolk by 11.32% versus controls. Conversely, Galobart et al. (2001) saw no improvement in markers of lipid oxidation in eggs after feeding hens 1000 mg of a commercial rosemary powder while Torki et al. (2018) reported no change in GSH activity in egg yolks from birds experiencing heat stress. A hypothesis for the improved antioxidant status in eggs using natural antioxidants such as aromatic plants is a vitamin E sparing effect in the gut (Loetscher et al., 2014).

Egg composition A number of authors have presented data on the influence of egg composition by aromatic plants, including parameters such as protein, cholesterol and glucose but few studies appear to demonstrate any significant effect. Devi et al. (2017) reported a decrease in yolk cholesterol for the supplementation of feed with pudina leaf powder. More studies demonstrate an influence of aromatics on serum cholesterol for example, thyme, Echinacea or rosemary (El-Hack and Alagawany, 2015; Jahanian et al., 2015; Alagawany et al., 2017). Peppermint or thyme has been shown to have an effect on protein concentration (Abdel-Wareth and Lohakare, 2014; ElHack and Alagawany, 2015) but the authors do not relate this to egg composition. Conversely, Vakili and Heravi (2016) reported no significant difference in cholesterol concentration of yolk in eggs from birds fed thymol versus control birds (208.2 and 227.2 mg/yolk, respectively) yet an effect was seen in serum cholesterol levels (153.3 and 102.5 mg/dL for control and supplemented birds respectively). Compounds such as thymol and carvacrol influence lipid digestion via stimulation of digestive enzymes and antioxidant activity, thereby suggesting a possible mode of action for aromatic plants on serum and egg lipids such as cholesterol (Hashemipour et al., 2013). Haugh unit, the principal indicator of internal albumen quality, yolk score and yolk percentage all appear to follow a similarly inconsistent pattern to that observed with protein, cholesterol etc. Some studies report aromatics such as rosemary or thyme/thymol increasing Haugh unit (Abdel-Wareth, 2015; Alagawany et al., 2017), Echinacea increasing yolk color (Jahanian et al., 2015) or oregano increasing yolk index (Radwan Nadia et al., 2008). However, other studies report no effect on egg composition with the same herb or others such eucalyptus, pudina (Botsoglou et al., 2005b; Florou-Paneri et al., 2005; El-Motaal et al., 2008; Torki et al., 2015; Bozkurt et al., 2016; Devi et al., 2017).

Egg shell Although eggs tend to increase in size over the production cycle, shell weight does not increase proportionally, hence the shell tends to become thinner leading to more fragile eggs and increased risk of rejected eggs. In molted hens (82e106 weeks), a commercial oregano product did not significantly influence egg shell thickness nor percentage (Bozkurt et al., 2016). Similarly, neither dried oregano or rosemary had an effect on shell parameters after feeding to 28-week-old hens for 90 days (Radwan Nadia et al., 2008), while the use of thyme powder, thymol, cinnamaldehyde, Echinacea, eucalyptus and oregano in hens ranging from 24 to 54 weeks of age were reported to have no effect (Bölükbasi and Erhan, 2007; Ding et al., 2017; Jahanian et al., 2015; El-Motaal et al., 2008; He et al., 2017). However, data are

198

11. Application of aromatic plants and their extracts in the diets of laying hens

conflicting. Supplementation of layer feed with rosemary or thyme powder (0.9%) fed to hens from 36 to 52 weeks resulted in a significant increase in shell percentage and shell thickness versus un-supplemented controls (Alagawany et al., 2017). El-Hack and Alagawany (2015) observed that 9 g/kg thyme powder resulted in a 17% increase in shell percentage and 20% increase in shell thickness versus controls. While Abdel-Wareth and Lohakare (2014) saw an increase in shell thickness and percentage following the administration of peppermint leaves (20 g/kg) to 64-week-old hens over 12 weeks, Akbari et al. (2016) reported no such effect after feeding peppermint essential oil to 42-week-old hens over an 8-week period, albeit under conditions of cold stress. Fennel extract has also been associated with a reduction in cracked eggs when fed to 36-week-old hens over an 8-week period (Hadavi et al., 2017).

Sensory evaluation The sensory quality of the egg is important for sale of final product and consumer acceptance. Following feed supplementation with essential oils, compounds such as thymol are detectable at low levels in plasma and tissues with absorption rates ranging between 0.7% in plasma to 0.027% in liver and muscle to (Haselmeyer et al., 2015). Plant compounds fed to hens are readily detectable in the egg (Plagemann et al., 2011), although absorption rates from feed to egg yolk appears to be low, perillaldehyde > citral > geraniol > linalool > eugenol > terpineol > carvacrol

Kim et al. (1995)

a

Disk diffusion method

S. typhimurium

Citronellal > citral > geraniol > perillaldehyde > linalool > eugenol > terpineol > carvacrol

Ait-Ouazzou et al. (2011)

Disk diffusion method

S. enteritidis

Carvacrol > terpineol > linalool

Ait-Ouazzou et al. (2011)

Disk diffusion method

E. coli O157:H7

Carvacrol > terpineol > linalool

Frideman et al. (2002)

Microdilution þ agar culture

E. coli

Carvacrol, cinnamaldehyde > thymol > eugenol > geraniol

Frideman et al. (2002)

Microdilution þ agar culture

S. enterica

Cinnamaldehyde > thymol > carvacrol > eugenol > geraniol

Frideman et al. (2002)

Microdilution þ agar culture

C. jejuni

Cinnamaldehyde > carvacrol > eugenol > thymol > geraniol

Si et al. (2006)

b

Microdilution þ optical density

E. coli K88

Thymol, carvacrol > cinnamon oil > clove oil > eugenol

Si et al. (2006)

b

Microdilution þ optical density

E. coli O157:H7

Cinnamon oil > thymol > geraniol, clove oil, carvacrol > eugenol

Si et al. (2006)

b

Microdilution þ optical density

S. typhimurium DT 104

Cinnamon oil > carvacrol > thymol > clove oil

Van Zyl et al., (2006)

c

Microdilution p-iodonitrotetrazolium violet

S. aureus ATCC 25923

Carvacrol > geraniol > linalool > citronellal > eugenol

Van Zyl et al., (2006)

c

Microdilution t p-iodonitrotetrazolium violet

B. cereus ATCC 11778

Eugenol > carvacrol > geraniol > linalool > citronellal

Van Zyl et al., (2006)

c

Microdilution t p-iodonitrotetrazolium violet

E. coli ATCC 11775

Eugenol > carvacrol > geraniol > linalool > citronellal

Michiels et al. (2009)

d

Simulated stomach

Total anaerobic bacteria

Carvacrol > thymol > eugenol > trans-cinnamaldehyde

Michiels et al. (2009)

Simulated stomach

Coliform bacteria

Trans-cinnamaldehyde > carvacrol > thymol > eugenol

Michiels et al. (2009)

Simulated stomach

E. coli

Trans-cinnamaldehyde > carvacrol > thymol > eugenol

a

The ranking was based on 5% concentration. The ranking was based on minimum bactericidal concentrations. c The ranking was based on the concentration that resulted in complete growth inhibition of 107 cfu/mL. d The ranking was based on the concentration that gives a reduction of 0.5 log10 cfu/mL compared to control. Modified from Zhai H., Liu H., Wang S., Wu J., and Kluenter AM. Potential of essential oils for poultry and pigs. Anim. Nutri. 4, 2018, 179-186. b

232 TABLE 13.3

13. Application of plant essential oils in pig diets

Effects of essential oils and aromatic plants on the microflora in swine. Dose, g/kg

Species

Measured responses

References

Herbal extracts

7500

Weaned pigs

Reduced coliform bacteria counts in fecal; less diverse of microbiota in ileal digesta base on PCR-DGGE

Namkung et al.

EO blend

50e150

Weaned pigs

Increased Lactobacillus and decreased E. coli counts in feces

Li et al.

EO blend

1000

Weaned pigs

Increased Lactobacillus counts

Zhang et al.

Chinese medicinal herbs

1000/3000

Weaned pigs

Increased Lactobacilli counts in ileum and decreased Coliform counts in colon

Huang et al.

EO blend

100

Weaned pigs

Reduced E. coli and total aerobic bacteria in the rectum; increased Lactobacilli to E. coli ratio in colon

Li et al.

Phytogenic additive

50e150

Weaned pigs

Microbial counts in feces (aerobes, gram negatives, anaerobes and lactobacilli) didn’t change

Muhl and Liebert

Modified from Zeng, Z., Zhang, S., Wang, H., Piao, X. 2015. Essential oil and aromatic plants as feed additives in non[HYPHEN]ruminant nutrition: a review. J Anim. Sci Biotechnol, 6, 7.

piglets (Xu et al., 2014). Dietary supplementation with 100 mg/kg carvacrolethymol (1:1) increased populations of Lactobacillus and decreased populations of E. coli and decreased the intestinal oxidative stress (Wei et al., 2017). In addition, OEs protects against H2O2induced IPEC-J2 cell damage by inducing Nrf2 and related antioxidant enzymes (Zou et al., 2016). OEs also exerted a protective effect against diquat-induced oxidative injury in intestine of rats (Wei et al., 2015). The antioxidant activity of plant extracts is highly correlated with their chemical compositions (Teissedre and Waterhouse, 2000). The presence of phenolic OH groups in thymol, carvacrol, and other plant extracts act as hydrogen donors to the peroxy radicals produced during the first step in lipid oxidation, thus retarding the hydroxyl peroxide formation (Djeridane et al., 2006). Improved barrier function of the intestine During weaning, piglets suffer social, environmental and dietary stress, all of which contribute to a decrease in their performance and welfare. Numerous studies have shown that dysfunction of the intestine, which has important immunological, metabolic and barrier functions, has been demonstrated to play a crucial role in weaning-induced growth check (Campbell et al., 2013; Wijtten et al., 2011). Recent studies have indicated that the intestinal oxidative stress, intestinal inflammation and intestinal flora disorder can induce the decreased abundance of tight junction proteins including zonula occludens (ZO-1) and occludin and thus undermine the integrity of the intestinal barrier (Suzuki et al., 2011; Xu et al., 2014; Zhu et al., 2012). Dietary supplementation with 100 mg/kg carvacrolethymol

Application of EOs in boars

233

(1:1) had an increased population of Lactobacillus genus but reduced populations of Enterococcus genus and E. coli in the jejunum and decreased mRNA levels of TNF-a (Wei et al., 2017).

Application of EOs in sows During 2011e2013, several studies showed that pregnant sows had elevated oxidative stress during late gestation and lactation which was responsible for impaired milk production, reproductive performance, and finally longevity of sows (Berchieri-Ronchi et al., 2011; Lapointe, 2014; Zhao, 2011; Zhao et al., 2013). Accumulated evidence suggests that excessive ROS affect the insulin signaling cascade, which leads to insulin resistance (Rains and Jain, 2011; Vinayagamoorthi et al., 2008). Insulin resistance during peripartal period was shown to have a negative effect on lactation feed intake of sows (Mosnier et al., 2010; Weldon et al., 1994). Thus, dietary antioxidant concentrations need to be reevaluated for their sufficiency in sow diets, especially to prevent excessive oxidative stress during gestation and lactation. There is an increased systemic oxidative stress during late gestation and early lactation of sows. This is not conducive to the reproductive potential of sows. OEs have a strong antioxidant effect which can improve GSH-Px activity, reduce plasma MDA levels, and improve plasma total antioxidant capacity during late gestation and early lactation of sows. Therefore, removal of excess ROS, reduce damage to proteins, DNA, and cell membrane lipids which provide a better environment for embryo development. The EOs supplementation to sows’ diet improved performance of their piglets, which may be attributed to the reduced oxidative stress (Tan et al., 2015). TNF-a can inhibit insulin signaling downstream through multiple pathways and play an important role in insulin resistance. OEs can improve insulin resistance in sows by lowering plasma TNF-a and improve sows’ lactation intake. In addition, EOs diet also increased the sows’ counts of fecal Lactobacillus while reducing Escherichia coli and Enterococcus.

Application of EOs in boars The ROS in sperm is produced by the oxidation of NADPH by nicotinamide adenine nucleotide oxidase (NOX). NOX in sperm is distributed on the head and mitochondria of sperm. Mitochondria are the main site of sperm ROS production. In addition to the production of normal sperm, the ROS in semen can also be produced by white blood cells, immature sperm, and bacteria. Physiological levels of ROS play an important role in sperm capacitation, maturation, zona pellucida and cell signal transduction. Sperm is in an oxidative stress state when the level of ROS inside and outside the sperm exceeds the ability of the sperm to clear itself. Excessive ROS will play a toxic effect on sperm. ROS is expressed as attacking sperm PUFA, sperm nuclear DNA and sperm proteins. Therefore, impaired sperm membrane and mitochondrial membrane affects its functionality. OEs can alleviate the damage of ethanolinduced oxidative stress on mouse sperm production, sperm motility, and serum testosterone. It can effectively protect the damage of male testis induced by deltamethrin and improve the semen quality of male rats. The effect of OEs on the quality of boar semen is

234

13. Application of plant essential oils in pig diets

relatively small. Diet supplemented with 500 mg/kg EOs can improving boar semen antioxidant capacity, inhibit sperm lipid peroxidation to promote sperm motility. Attention should also be paid to the potential negative effects induced by EOs on semen quality. Essential oil from C. citratus reduced sperm motility, membrane functionality and integrity and mitochondrial membrane potential even in concentrations as low as 0.001%. However, the effect of OEs EOs on the quality of boar semen needs further research. Studying EOs on boar semen quality requires a focus on the types of EOs, dosages of diet supplementation, interaction with fat content, its fatty acid composition and boar breeds.

Effect of oregano oil against transportation stress Pig transport is an indispensable part in pork production process. When pigs are transported to the slaughterhouse, the pigs are subjected to a variety of stresses, including increased risk of reduced animal welfare, damage to the carcass quality of the pig, resulting in inferior meat quality and even mortality. During transportation, ROS levels can increase dramatically and any imbalance between production of these molecules and their safe disposal can lead to oxidative stress. Free radical oxidation is the main mechanism of food quality degradation, especially in meat products. It causes undesirable changes in taste, color, texture and nutritional value and can lead to the production of toxic compounds in meat, reducing consumer’s acceptability. Therefore, the oxidative state of pigs during slaughter is critical to meat quality. The addition of antioxidants to preslaughter fattening pigs is considered to be an effective means to improve the quality of fattening pork due to transport stress. In our previous study, pigs fed OEO showed reduced live weight contraction and higher hot carcass weights and dressing percentages after transportation than pigs fed a control diet (Zhang et al., 2015; Zou et al., 2016a,b). In addition, OEO were superior to vitamin E in increasing antioxidant enzyme activity, thereby reducing transportation-induced oxidative stress and improving meat quality. In addition to oxidative stress, increased intestinal permeability in fattening pigs during transport is reported to be closely related to meat quality and carcass traits. Therefore, the protective effect on the intestinal barrier of transport-stressed finishing pigs may be the site of action to alleviate the negative effects of transport stress in finishing pigs. In our previous study, we found that the villi were scattered and seriously desquamated in the jejunum of transport stress pigs. Interestingly, OEO was superior to Vit E in decreasing the stress response, thereby reducing transportation-induced intestinal injury and improving meat quality (Zou et al., 2017). These results indicate OEO can act as an efficient dietary supplement to alleviate transport stress in finishing pigs.

Conclusions EOs are naturally occurring phytochemicals which have various applications and have long been known and used throughout the world for treatment of many diseases. EOs have positive effects on digestion, gut microbial community, antioxidant effects, barrier function of the intestine, growth performance and welfare. These characteristics could be a useful

References

235

alternative to AGPs in animal diets. EOs can increase the performance of swine and growingfinishing pigs, alleviate transport stress in finishing pigs, and increase reproductive performance of boars and sows.

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Further reading

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Wijtten, P.J., van der Meulen, J., Verstegen, M.W., 2011. Intestinal barrier function and absorption in pigs after weaning: a review. Br. J. Nutr. 105, 967e981. Xu, C., Yang, S., Zhu, L., Cai, X., Sheng, Y., Zhu, S., Xu, J., 2014. Regulation of N-acetyl cysteine on gut redox status and major microbiota in weaned piglets. J. Anim. Sci. 92, 1504e1511. Yan, L., Meng, Q.W., Kim, I.H., 2011. The effect of an herb extract mixture on growth performance, nutrient digestibility, blood characteristics and fecal noxious gas content in growing pigs. Livest. Sci. 141, 143e147. Zeng, Z., Zhang, S., Wang, H., Piao, X., 2015. Essential oil and aromatic plants as feed additives in non-ruminant nutrition: a review. J Anim. Sci Biotechnol 6, 7. Zeng, Z., Xu, X., Zhang, Q., Li, P., Zhao, P., Li, Q., Liu, J., Piao, X., 2015. Effects of essential oil supplementation of a low-energy diet on performance, intestinal morphology and microflora, immune properties and antioxidant activities in weaned pigs. Anim. Sci. J. 86, 279e285. Zhai, H., Liu, H., Wang, S., Wu, J., Kluenter, A.M., 2018. Potential of essential oils for poultry and pigs. Anim. Nutri. 4, 179e186. Zhang, S., Jung, J.H., Kim, H.S., Kim, B.Y., Kim, I.H., 2012. Influences of phytoncide supplementation on growth performance, nutrient digestibility, blood profiles, diarrhea scores and fecal microflora shedding in weaningpigs, Asian-Australas. J. Anim. Sci. 25, 1309e1315. Zhang, T., Zhou, Y.F., Zou, Y., et al., 2015. Effects of dietary oregano essential oil supplementation on the stress response, antioxidative capacity, and HSPs mRNA expression of transported pigs[J]. Livest. Sci. 180, 143e149. Zhao, Y., 2011. Oxidative Stress Status and Reproductive Performance of Sows. Zhao, Y., Flowers, W., Saraiva, A., Yeum, K.-J., Kim, S., 2013. Effect of social ranks and gestation housing systems on oxidative stress status, reproductive performance, and immune status of sows. J. Anim. Sci. 91, 5848e5858. Zhu, L., Zhao, K., Chen, X., Xu, J., 2012. Impact of weaning and an antioxidant blend on intestinal barrier function and antioxidant status in pigs. J. Anim. Sci. 90, 2581e2589. Zou, Y., Wang, J., Peng, J., Wei, H., 2016a. Oregano essential oil induces SOD1 and GSH expression through Nrf2 activation and alleviates hydrogen peroxide-induced oxidative damage in IPEC-J2 cells. Oxid. Med. Cell. Longev. 2016. Zou, Y., Xiang, Q., Wang, J., et al., 2016b. Effects of oregano essential oil or quercetin supplementation on body weight loss, carcass characteristics, meat quality and antioxidant status in finishing pigs under transport stress [J]. Livest. Sci. 192, 33e38. Zou, Y., Hu, X.M., Zhang, T., et al., 2017. Effects of dietary oregano essential oil and vitamin E supplementation on meat quality, stress response and intestinal morphology in pigs following transport stress[J]. J. Vet. Med. Sci. 79 (2), 328e335.

Further reading Kaushik, M., Kumar, H.D., 2011. Studies on the effects of essential-oil-based feed additives on performance, ileal nutrient digestibility, and selected bacterial groups in the gastrointestinal tract of piglets. J. Anim. Sci. 89, 2106e2112. Li, P., Piao, X., Ru, Y., Han, X., Xue, L., Zhang, H., 2012a. Effects of adding essential oil to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health. Asian-Australas. J. Anim. Sci. 25, 1617e1626. Li, S.Y., Ru, Y.J., Liu, M., Xu, B., Péron, A., Shi, X.G., 2012b. The effect of essential oils on performance, immunity and gut microbial population in weaner pigs. Livest. Sci. 145, 119e123. Manzanilla, E.G., Perez, J.F., Martin, M., Kamel, C., Baucells, F., Gasa, J., 2004. Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. J. Anim. Sci. 82, 3210e3218. Naumann, C.R., Mikele, C.-W., Qureshi, W.A., Shaib, Y.H., 2006. Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs. J. Anim. Sci. 84, 2743e2751. Schöne, F., Vetter, A., Hartung, H., Bergmann, H., Biertümpfel, A., Richter, G., Müller, S., Breitschuh, G., 2010. Effects of essential oils from fennel (Foeniculi aetheroleum) and caraway (Carvi aetheroleum) in pigs. J. Anim. Physiol. Anim. Nutr. 90, 500e510. Zhang, Y., Gong, J., Yu, H., Guo, Q., Defelice, C., Hernandez, M., Yin, Y., Wang, Q., 2014. Alginate-whey protein dry powder optimized for target delivery of essential oils to the intestine of chickens. Poultry Sci. 93, 2514e2525.

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C H A P T E R

14 Application of aromatic plants and their extracts in aquaculture Ángel Hernández-Contreras, María Dolores Hernández IMIDA - Aquaculture, San Pedro del Pinatar, Region of Murcia, Spain O U T L I N E Introduction Applications of aromatic plants and their extracts in aquaculture Improved health and immunostimulation Nutrition and stimulation of growth and appetite Antiparasitic activity

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Latest properties discovered and possible uses 252

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Current regulatory status and future perspectives

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References

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Introduction Global aquaculture production continues to grow year after year, with a total of 80.0 million tons of different aquatic species produced in 2016. Of these, most are fin fish (54.1 million tons), followed by mollusks (17.1 million tons) and crustaceans (7.9 million tons). Altogether in 2016, they accounted for the first time for over half the fish consumed in the world (FAO, 2018). Despite the rapid growth of aquaculture, infectious diseases currently compromise the profitability of this activity. In pond fish aquaculture, 60% of the production is lost as the result of infectious diseases (Raman, 2017), while in the tropical marine shrimp sector, the fastest growing in the world, around 40% of the production is lost due to viral diseases

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00014-5

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Copyright © 2020 Elsevier Inc. All rights reserved.

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(>$3 billion) (Stentiford et al., 2012). To address these weaknesses, synthetic growth promoters and veterinary antibiotics have been used for more than 60 years to improve profitability in animal production, but they compromise food safety through the potential accumulation of residues in the animals and the environment (Gonzalez Ronquillo and Angeles Hernández, 2017). If no significant changes are made in the policies regulating their use worldwide, it is expected that their administration to animals for human consumption will increase by two-thirds by 2030, primarily in emerging economies (Laxminarayan et al., 2015). For all these reasons, studies are being conducted on the main groups of aquaculture organisms, using functional foods that promote growth, feed conversion, health or a combination of all of the above (Akhter et al., 2015). This trend is linked to greater social concern about healthy fish production and the prohibition of antibiotics as growth promoters in aquaculture (Guerreiro et al., 2017) after the emergence of resistant pathogens in aquatic ecosystems, which have even played a part in infectious disease outbreaks in humans in recent decades (Balcazar et al., 2006). Different strategies have been tested for the production of functional foods that are environmentally friendly and also improve food safety. Among them, the introduction of additives such as probiotics, prebiotics, synbiotics, and phytobiotics has been examined (perhaps to a lesser extent in the latter case, but with promising results) (Van Hai, 2015). The use of these plant-derived additives has been studied in great detail (Citarasu, 2010), with a growing interest in aromatic plants and their essential oils (Preedy, 2016; Sutili et al., 2017). Although there is no precise definition of aromatic plants, they are plants that produce and secrete aromatic substances, generally present in oils and commonly used in perfumery, cooking and sectors such as the food and pharmaceutical industries. Many of these aromatic plants belong to the Lauraceae, Umbelliferae, Myrtaceae, and Labiatae families (“Medicinal & Aromatic Plants,” n.d.). Aromatic plants have large amounts of active compounds, the vast majority of which are fat soluble and integrated in essential oils, and some of which are water-soluble. These essential oils have as main components terpenes and other molecules derived from them, such as terpenoids, aldehydes, ketones, acids, phenols, lactones, ethers, and esters (Tongnuanchan and Benjakul, 2014). The use of aromatic plants as substitutes for antibiotics and other synthetic substances is an eco-friendly strategy with great possibilities for success. The beneficial properties are often similar or superior to those of synthetic substances, and have been demonstrated through a large number of scientific studies (Reverter et al., 2014; Sutili et al., 2017). Moreover, their use can also have economic and marketing advantages when it comes to addressing the current demands of society in terms of food safety and for ensuring the eco-friendly production of animals that provides for their well-being. Natural immunostimulants are gaining interest as a substitute for the veterinary drugs, antibiotics and vaccines currently used in aquaculture. Unlike vaccines, immunostimulants improve the innate or nonspecific immune response to any pathogen (Galina et al., 2009). This holistic preventive strategy may be more successful, since infectious outbreaks are often associated with the physical condition of the fish, with most of the pathogens being opportunistic in nature. Aromatic plants and their extracts may be useful in this regard by improving the immune status and physical condition of the fish through their antistress, growth-promoting, appetite and immunostimulating properties (Reverter et al., 2014).

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Applications of aromatic plants and their extracts in aquaculture Improved health and immunostimulation The fight against emerging infectious diseases in aquaculture continues to be crucial to ensure the profitability, sustainability and continuity of the sector, since outbreaks cost millions of dollars annually around the world. Most of the infectious outbreaks come from wild hosts in adjacent waters and take advantage of the compromised immune system of the fish as the result of confinement, stress, and the genetic conditions of nonwild individuals. These outbreaks, which have occurred repeatedly since the start of aquaculture production, usually appear a few years after the introduction of each new species (Lafferty et al., 2015). In this scenario, and taking into account the growing reluctance to use veterinary drugs and other synthetic substances (Chakraborty and Hancz, 2011), aromatic plants and their extracts appear as promising alternatives to antibiotics and other synthetic medicines used in aquaculture. After an exponential growth in specific studies on fish health and immunology in relation to the use of aromatic plants and their active principles, there is already a wide range of aromatic plants whose immunostimulating and health-promoting properties have been demonstrated in fish (Table 14.1). The extracts and essential oils of aromatic plants contain many active compounds, some of which are relatively common in various aromatic plant genera and have attracted great interest for their immunostimulating and healthpromoting properties. The first of these, and perhaps the one that has been used the longest in the immunology of aquatic species, is allicin and its derivatives, which are present in garlic and onions (Fig. 14.1). As early as the 1980s and 1990s, the use of garlic was studied as a deworming treatment in fish (Boxaspen and Holm, 1991; Peña et al., 1988) with good results, having been used in previous decades, along with other natural compounds such as lime and onion, all over the world (Martins et al., 2002). Subsequent studies explored the use of this aromatic plant in aquaculture in both fresh form and as a dry extract or essential oil, revealing certain immunostimulating properties that generated great interest within the food additive industry. Researchers initially studied its effect on some general aspects of their physiology, growth, hematology, and apparent health (Diab et al., 2008; Metwally, 2009; Shalaby et al., 2006), and it was not until later that the immunostimulating effect of garlic was analyzed in a broad sense, delving deeper into specific aspects of the innate immune system. Sahu et al. (2007) studied the effect of increasing doses of dried, pulverized garlic (0.1%, 0.5% and 1%) added to the diet of Rohu (Labeo rohita) on the immune status and survival in a challenge with Aeromonas hidrophila. Subsequent studies analyzed the protective effect of garlic (Nya and Austin, 2011, 2009) or its bioactive compound, allicin (Nya et al., 2010), against the same pathogen in rainbow trout (Oncorhynchus mykiss). These studies addressed specific mechanisms of the innate immune system, such as respiratory explosion and phagocytic, lysozyme, antiprotease, and bactericidal activities, which were for the most part stimulated. Ndong and Fall (2011) also studied the effect of incorporating garlic in the diet on specific aspects of the immune system, in this case in hybrid tilapia (Oreochromis niloticus
Oreochromis aureus). They observed an increase in total white blood cells (WBC) count, respiratory explosion, phagocytic activity and index, and lysozyme activity with a dose of 0.5 g/kg, while the dose of 1 g/kg produced no changes. Later, Talpur and Ikhwanuddin (2012) included oven-dried

TABLE 14.1

Outstanding studies of aromatic plants or their extracts added to the feed of aquaculture species.

Aromatic plant

Fish

Type of extract

% in feed (w/w)

Growth promoter

Garlic

Oreochromis niloticus

natural/oil/powder

4/0.025/3.2

þ

Garlic

Oreochromis niloticus

powder

1e4

þ

þ

Shalaby et al. (2006)

Garlic

Oreochromis niloticus

crushed

1e2

þ

þ

Aly et al. (2008)

Garlic

Labeo rohita

powder

0.1e1

þ

Sahu et al. (2007)

Garlic

Oncorhynchus mykiss

oven dried/crushed

0.5e1

þ

Nya and Austin (2009)

Garlic (allicin)

Oncorhynchus mykiss

liquid pure allicin

0.5e1

þ

Nya et al. (2010)

Garlic

Oncorhynchus mykiss

crushed

0.5e1

þ

Nya and Austin (2011)

Garlic

Oreochromis niloticus x O. aureus

meal

0.5e1

e

þ

Ndong and Fall (2011)

Garlic

Lates calcarifer

oven dried/crushed

0.5e2

þ

þ

Talpur and Ikhwanuddin (2012)

Garlic/Ginger/ Turmeric/Fenugreek

Macrobrachium rosenbergii

powder

1

þ

Poongodi et al. (2012)

Garlic

Huso huso

garlic oil

50e200 (ppm)

þ

Tangestani et al. (2011)

Garlic (allicin)

Oncorhynchus mykiss

stabilized allicin

0.5e2

þ

Breyer et al. (2015)

þ

Garlic

Litopenaeus vannamei

powder

2e6

þ

Oregano

Ictalurus punctatus

essential oil

0.05

þ

Tymol-carvacrol

Oncorhynchus mykiss

powder

Tymol-carvacrol

Huso huso

Tymol/Carvacrol

Oncorhynchus mykiss

Immuno stimulant

References Metwally (2009)

Labrador et al. (2016) þ

Zheng et al. (2009)

0.1e0.3

þ

Ahmadifar et al. (2011)

powder

0.1e0.3

þ

Ahmadifar et al. (2014)

additive

0.1

þ

Giannenas et al. (2012)

þ

Carvacrol

Oncorhynchus mykiss

pure

1e5

þ

Yilmaz et al. (2015)

Oregano

Oncorhynchus mykiss

Essential oil

0.125e3 (mL/kg)

þ

Diler et al. (2016)

Zataria multiflora

Cyprinus carpio

Essential oil

30e120 (ppm)

þ

Soltani et al. (2010)

Carvacrol-thymolanethol-limonene

Ictalurus punctatus

additive

200 (g/ton)

þ

Peterson et al. (2015)

Summer savory

Pterophyllum scalare

Essential oil

0.1e0.4

þ

Ghafari Farsani et al. (2018)

Rosemary

Oreochromis sp.

Dried/ethyl acetate extract

15/4

þ

Abutbul et al. (2004)

Rosemary

Oreochromis sp.

Powder

4e16

þ

Zilberg et al. (2010)

Rosemary

Oreochromis mossambicus

Powder

1

þ

Yilmaz et al. (2013b)

Peppermint

Lates calcarifer

Powder

0.1e0.5

þ

þ

Talpur (2014)

Peppermint

Salmo trutta caspius

Ethanol extract

1e3

þ

þ

Adel et al. (2015b)

Peppermint

Rutilus frisii kutum

Ethanol extract

1e3

þ

þ

Adel et al. (2015a)

Fenugreek (seeds)

Sparus aurata

Crushed

1e10

þ

þ

Awad et al. (2015)

Fenugreek (seeds)

Sparus aurata

Crushed

þ

þ

Bahi et al. (2017)

Black cumin

Oncorhynchus mykiss

Methanolic extract

0.01e0.05

þ

Celik Altunoglu et al. (2017)

Saint John’s wort, Lemon balm, Rosemary

Salmo salar

Dry extract

0.6

þ

Reyes-Cerpa et al. (2018)

þ

þ

244

14. Application of aromatic plants and their extracts in aquaculture

Stereochemical structure of the most representative bioactive constituents from garlic (Allium sativum L.): alliin, allicin, allyl sulfide, (E)-ajoene, (Z)-ajoene and 1,2- vinyldithiin (Martins et al., 2016).

FIGURE 14.1

garlic in the diet of Asian sea bass (Lates calcarifer) at significantly higher doses of 5, 10, 15, and 20 g/kg. They observed improved survival in a challenge with Vibrio harvey, with significant differences at a dose of 10 g/kg. In general, the inclusion of garlic in the diet, at doses of 10 g/kg or higher, increased the values of phagocytic activity and index, superoxide anion production, lysozyme activity, bactericidal activity in serum and antiprotease activity. This is in contrast with the efficiency of the low doses used by the authors mentioned above, although several of them do not provide information about how it was added to the feed and whether there was a pretreatment, such as drying or heating. Studies have also been performed on the immunostimulating activity of garlic essential oil, although to a much lesser extent, probably due to the availability of other faster and cheaper formats. Tangestani et al. (2011) added to the diet of beluga sturgeon (Huso huso) increasing doses of essential oil of garlic and noted an improvement in the immune system as measured by several parameters, such as blood clotting, hemoglobin, and lymphocyte and eosinophil counts. Metwally (2009) also used essential oil of garlic without analyzing immunological aspects, although they noted an improvement in the activities of antioxidant enzymes in blood serum. Both the doses and the type of extract or essential oil used have varied considerably in the different studies, but it should be noted that harmful effects have been observed at high doses of stabilized allicin extract in rainbow trout (Breyer et al., 2015). While at low doses (0.5% and 1%),

Applications of aromatic plants and their extracts in aquaculture

245

survival increased when challenged with Aeromonas salmonicida, a potential inflammatory effect was observed at high doses (2%) in renal tissue, indicating a possible ineffectiveness and detrimental effect on health. The differences in the efficient dose observed by different authors may be closely related to the way the garlic is processed before it is added to the feed, as this could affect the enzymatic reaction, catalyzed by alliinase, that converts alliin into allicin (the main bioactive compound, along with the other sulfur derivatives). Crushing or chopping garlic before adding it to food or heating it is already known to be directly related to triggering this reaction and the resulting bioactive compounds (Song and Milner, 2001). This leads us to believe that it would be necessary to determine the abundance of each bioactive compound that is ultimately ingested by the fish in each study in order to obtain optimal dosages and draw clear conclusions. The immunostimulating effect of garlic has also been studied in crustaceans of interest to aquaculture, with positive results such as an increased survival in white leg shrimp (Litopenaeus vannamei) (Labrador et al., 2016) or greater expression of genes related to the immune system in kuruma shrimp (Marsupenaeus japonicus) (Tanekhy and Fall 2015). Other compounds from aromatic plants widely studied and known to have immunoregulatory properties are the monoterpene phenols thymol and carvacrol (Fig. 14.2), present in aromatic plants of the Lamiaceae family like thyme, oregano, or satureja, but also found in other families, such as Chenopodiaceae, Plantaginaceae, Umbelliferae, Verbenaceae, etc. (Kirimer et al., 1995). Their study as immunostimulants in fish has mainly focused on the essential oils of aromatic plants with a high concentration of thymol and carvacrol. Zheng et al. (2009) added additives of thymol, carvacrol, the combination of both and an essential oil of oregano (Origanum heracleoticum L.) to the diets of catfish (Ictalurus punctatus). After eight weeks of administering the diet, an improvement was observed in the activity of enzymes related to the immune system, such as lysozyme, superoxide dismutase, and catalase. In addition, following a challenge with Aeromonas hydrophila, both the combination of thymol

FIGURE 14.2

et al., 2013).

Pathway for the biosynthesis of thymol and carvacrol from L-terpinene and p-cymene (Rowshan

246

14. Application of aromatic plants and their extracts in aquaculture

and carvacrol and the essential oil of oregano reduced mortality, with essential oil of oregano being the most effective. The use of commercial concentrates with thymol and carvacrol has also been studied with regard to aspects related to the immune system. The lymphocyte blood count was found to increase with these concentrates of thymol-carvacrol in both rainbow trout and beluga sturgeon (Ahmadifar et al., 2014, 2011). Commercial extracts of these compounds were also used in the study by Giannenas et al. (2012), in this case in the form of an additive rich in carvacrol and another in thymol. They analyzed parameters related to the immune status of rainbow trout and found higher levels of lysozyme, total complement concentrations and catalase in fish fed both additives, but in particular in those consuming the carvacrol-rich additive. Subsequently, Yilmaz et al. (2015) studied the effect of several doses of pure carvacrol in the feed administered to rainbow trout, noting a general improvement in parameters related to the nonspecific immune system (lysozyme and myeloperoxidase activity). However, the results did not reveal a clear dose-dependent effect and did not allow for establishing an optimal dosage. Diler et al. (2016) also observed beneficial effects on health after adding increasing doses (0.125 mL/kg to 3 mL/kg) of essential oil of oregano (Origanum onites L.), rich in carvacrol, to the diet of rainbow trout for 90 days. Lysozyme activity in blood serum increased significantly with a dose of 3 mL/kg. Furthermore, following a bacterial challenge with Lactococcus garviae, mortality was reduced with all treatments, particularly with that of 3 mL/kg, in which all specimens survived. Other less common plants, such as “Shirazi thyme” or Iranian thyme (Zataria multiflora), the essential oil of which consists mainly of carvacrol and thymol, have also been tested as immunostimulants in fish (Soltani et al., 2010), specifically in common carp (Ciprinus carpio). After only eight days of administration and a challenge with Aeromonas hidrophila, it was possible to observe differences in the antibody titer, total WBC count, and bactericidal activity in blood serum. This essential oil has also been used in combination with others (Soltani et al., 2014). These authors tested the effect of Shirazi thyme and rosemary essential oils on the fish pathogen Streptococcus iniae, establishing their minimum inhibitory concentration (MIC). In addition, they found that at subinhibitory doses, these essential oils were able to reduce the expression of the SagA gene, the main gene encoding the streptolysin S (SLS) virulence factor. Based on these results, these essential oils have been proposed as a viable strategy for controlling streptococcosis through the inhibition of growth and suppression of SLS production. The combination of aromatic compounds such as carvacrol, thymol, anethole and limonene has been tested as an additive included in the diet of catfish (Peterson et al., 2015), and it has also been shown to have an immunostimulating effect at various levels. After a 14-day challenge with Edwarsiella tarda, mannose-binding lectin (MBL) levels were 15 times higher in fish fed an extract of aromatic compounds, and their survival was 43% greater than that of the control group. The essential oil of savory, rich in thymol and carvacrol, has also been shown to have an immunostimulating effect. Ghafari Farsani et al. (2018) found an increase in the level of lysozyme in blood serum with a dose of 200 mg/kg of savory essential oil, while immunoglobulin levels increased with doses of 200 and 400 mg/kg. Rosemary, and their active compounds (Fig. 14.3), is another of the aromatic plants most commonly studied as feed additive, due to its proven antidiabetic, hepatoprotective, choleretic, and antiadipogenic properties in mammals (Hernández et al., 2015). As for its use in aquaculture (Abutbul et al., 2004), obtained several rosemary extracts to be used against Streptococcus iniae in tilapia (Oreochromis sp.), with the ethyl acetate extract being the most

Applications of aromatic plants and their extracts in aquaculture

247

Chemical structure of (A) carnosic acid, (B) carnosol, (C) ursolic acid, and (D) rosmarinic acid (Seow and Lau, 2017).

FIGURE 14.3

effective in the in vitro tests. Using both this extract and a powder extract made from dried rosemary leaves, a five-day fish food experiment was conducted followed by a challenge with Streptococcus iniae (16 total days of feeding). Both formats proved to be effective, with a significant reduction in mortality as compared to the control, and no significant differences as compared to the group treated with oxytetracyclin at the end of the experiment. Zilberg et al. (2010) performed a dosage-response study with a similar dose to that used by Abutbul et al. (2004) and other lower doses, and found an optimal dose of 8% in a challenge with Streptococcus iniae and 16% in a challenge with Streptococcus agalactiae. The immunostimulating effect of rosemary extract in powder form has also been compared to that of other aromatic plants added to the diet of tilapia (Oreochromis mossambicus) (Yilmaz et al., 2013b). Following a challenge with Streptococcus iniae, mortality was significantly reduced with all of the aromatic plants used, although this reduction was greater with thyme and fenugreek than with rosemary. Another aromatic plant that belatedly captured the attention of researchers for its use in aquaculture is peppermint (Mentha piperita) (Fig. 14.4). Although its immunostimulating effect had already been studied in combination with other plants (Abasali and Mohamad, 2010), the first study in which it was added by itself to fish feed was conducted by Talpur (2014). They observed a protective effect in Asian sea bass against infection by Vibrio harveyi, in addition to an improvement of many blood parameters related to the immune system. The

248

FIGURE 14.4

14. Application of aromatic plants and their extracts in aquaculture

Chemical structure of the major constituents of mentha oil of different species (Kumar et al., 2011).

immunostimulating effect of this aromatic plant has also been demonstrated in two species from the Caspian Sea, Salmo trutta caspius and Rutilus frisii kutum (Adel et al., 2015a, 2015b). In both studies, dried peppermint powder was added to the fish diets at levels of 1%, 2%, and 3%, with similar effects observed in each case: a dose-dependent improvement of immunological parameters of the blood and mucus, and a decrease in lymphocyte counts, although other white blood cells remained unaltered. The addition of this aromatic plant has also recently been studied, this time in the form of essential oil, in the tambaqui (Colossoma macropomum), at dosages between 0.5% and 1.5%. However, the results obtained were unclear, as even though anthelmintic effects were observed against gill monogeneans, lysozyme activity and respiratory explosion were not increased, nor was the population of blood leukocytes. There are also studies addressing the immunostimulating properties of seeds from aromatic plants such as fenugreek and cumin (Awad et al., 2013; Bahi et al., 2017; Celik Altunoglu et al., 2017; Dorucu et al., 2009; Guardiola et al., 2017; Yilmaz et al., 2013a, 2013b), showing generally good results as immunostimulants, in addition to their nutritional characteristics. As can be seen, the number of studies on the use of aromatic plants as immunostimulants is exponentially increasing, and it is difficult to cover them all, but among the main plants

Applications of aromatic plants and their extracts in aquaculture

249

used, we can mention garlic, plants rich in carvacrol and thymol, rosemary, peppermint and fenugreek and cumin seeds, among others. The future use of these plants and compounds as immunostimulants may not lie in a single species, but rather in the combination of several of them (Reyes-Cerpa et al., 2018). However, in order to be able to define dosages and formats, it is important to include in all works an exhaustive analysis of the bioactive compounds in the extract used.

Nutrition and stimulation of growth and appetite The use of aromatic plants, and particularly their essential oils, in animal nutrition has great potential, possibly serving as a substitute for antibiotic growth promoters, due to their positive effects on digestion, intestinal microbiome, growth and health (Sutili et al., 2017). Even though antibiotic growth promoters (AGPs) appear to have little efficiency in optimized production systems, and in spite of the fact that their use has been greatly reduced in industrialized countries, their administration continues to increase in low-income countries, where the production systems are also more closely linked to the environment (Laxminarayan et al., 2015). In order to increase food safety in these countries and reduce the impact on the environment, it would be very useful to adopt green strategies, such as replacing AGPs with extracts and essential oils of aromatic plants, which also have other properties that perhaps play a greater role in the profitability of production and animal welfare. One of the aromatic plants most commonly studied as a growth promoter in fish and crustaceans is garlic. Shalaby et al. (2006) observed an increase in consumption, SGR and weight gain in Nile tilapia (Oreochromis niloticus) fed diets containing 10, 20, 30, and 40 g/kg of garlic for 12 weeks. Aly et al. (2008) also observed an improvement in the growth of Nile tilapia when administered diets with 10 and 20 g/kg of garlic. They also found that administration during a single month of the diet containing 20 g/kg of garlic produced significant changes that were maintained for another seven months in which they were fed a basal diet. This growth-promoting effect was also seen in juvenile specimens of European sea bass (Dicentrarcus labrax) with garlic and onion, which is also rich in the bioactive compound allicin. The optimal doses were 30 g/kg for garlic and 10 g/kg for onion. In Asian sea bass, an important growth promotion effect was seen for garlic, as well as positive effects on immunological and survival aspects (Talpur and Ikhwanuddin, 2012). Fish showed better growth and feed conversion rates with diets containing garlic, and this improvement was directly proportional to the dose (5e20 g/kg). Garlic has also been occasionally tested in crustaceans, and shown to be effective in Macrobrachium rosenbergii (Poongodi et al., 2012) and Litopenaeus vannamei (Javadzadeh et al., 2012). Other aromatic plants that have been tested in fish growth studies are the ones with thymol- and carvacrol-rich oils, such as thyme and oregano, and although they are less well known, plants from the Lippia sp. or Satureja sp. genus, among others. Zheng et al. (2009) observed a significant growth increase in catfish fed diets with carvacrol and carvacrol þ thymol additives, along with natural essential oil of oregano, with the latter showing the greatest effect. In the case of thymol administered by itself, there were no significant differences in growth. Afterward, Ahmadifar et al. (2011) supplemented rainbow trout diets with a commercial concentrate of thymol and carvacrol at greater doses (1, 2 and 3 g/kg)

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14. Application of aromatic plants and their extracts in aquaculture

and, curiously enough, they only observed a significant increase with the highest dose. This same additive also showed a growth-promoting effect in beluga sturgeon, starting at doses of 2 g/kg (Ahmadifar et al., 2014). These same aromatic compounds, tested separately in different additives in the diet of rainbow trout, showed no effect whatsoever on growth, but they did improve the feed conversion rate (Giannenas et al., 2012). A significant growth increase was observed with the use of oregano essential oil in the diets of rainbow trout at doses of 0.125, 1.5, 2.5, and 3.0 mL/kg, however, this effect was not dose-dependent (Diler et al., 2016). Another plant commonly studied, particularly for its immunostimulating properties, is peppermint. The use of this plant, dried and pulverized, at doses of 1, 2, 3, 4, and 5 g/kg, produced an improvement in the SGR and the FCR proportional to the dose (Talpur, 2014); in this case, the improvement was greater than that observed previously by these same authors for the addition of garlic (Talpur and Ikhwanuddin, 2012). In the case of the fish Rutilus frisii kutum, peppermint was capable of improving growth rate when added to the feed at a 3% dose, but lower doses did not have this effect (Adel et al., 2015b). The incorporation of fenugreek seeds in the diet of fish has also aroused relative interest in their properties, including its ability to stimulate growth. In the case of Nile tilapia, a dose of 1% of meal made from the seeds of fenugreek significantly improved growth (Abdel et al., 2009). A potent growth promotion effect was seen as well in gilthead seabream (Sparus aurata) that was directly proportional to the dose of fenugreek (1%e10%) in the diet (Awad et al., 2015); and it also improved growth and other performance parameters when mixed with probiotics in gilthead seabream diets (Bahi et al., 2017). It is worth noting that many works not discussed in this section have used extracts or oils of aromatic plants and found beneficial effects in other aspects related to health and wellbeing, but not to growth.

Antiparasitic activity Both saltwater and freshwater fish produced in aquaculture suffer infestations of ectoparasites that cause health problems in fish and great economic losses. The main ectoparasites in fish are monogeneans living in the skin, gills and even the eyes of the fish (Reverter et al., 2014; Vanhove et al., 2018). As the main treatment for these parasites is the use of chemotherapy baths that ultimately lead to the release of a number of these substances into the environment, extracts and essential oils of aromatic plants have attracted the interest of researchers for their anthelmintic properties (Table 14.2). As mentioned earlier, garlic was proven to be an anthelmintic (Peña et al., 1988) and effective against sea lice (Boxaspen and Holm, 1991) decades ago, but has only recently been tested in greater depth against monogeneans in fish of interest to aquaculture. Garlic extract rich in allicin administered in the feed has been shown to be very effective as a preventive treatment, successfully reducing Neobenedenia sp. infections by 70% when administered for 30 days (Militz et al., 2013a). In addition, it is also effective as a bath therapy for fish infested with this same parasite. In this way, it is effective against eggs and larvae, but relatively ineffective against juveniles (Militz et al., 2013b), so it may be advisable to use it in the diet as a preventive treatment if there is risk of infestation.

TABLE 14.2

Outstanding studies addressing therapeutic and protective uses of aromatic plants and their extracts against fish parasites.

Aromatic plant

Fish

Type of extract

Administration

Length of treatment

Parasite

References

Garlic

Cyprinus carpio

Minced/hexane/ aqueous

Bath

3 days

Capillaria sp.

Peña et al. (1988)

Garlic

Lates calcarifer

Aqueous extract

Oral

10e30 days

Neobenedenia sp.

Militz et al., 2013a,b

Garlic

Lates calcarifer

Aqueous extract

Bath

1h

Neobenedenia sp.

Militz et al., 2013a,b

Tea tree

Gasterosteus aculeatus

Essential oil

Bath

48 h

Gyrodactylus spp.

Steverding et al. (2005)

Eugenol

Colosssoma macropomum

1:20 (eugenol: ethanol)

Bath

5e15 min

Monogeneans

de Lima Boijink et al. (2015)

Lippia alba

Colosssoma macropomum

Essential oil

Bath

30 min

Monogeneans

Soares et al. (2016)

Lippia origanoides

Colosssoma macropomum

Essential oil

Bath

30e60 min

Monogeneans

Soares et al., 2017a

Lippia sidoides

Colosssoma macropomum

Essential oil

Bath

15e60 min

Monogeneans

Soares et al., 2017b

Clove basil

Colossoma macropomum

Essential oil

Bath

15 min

Monogeneans

de Lima Boijink et al. (2016)

Pepper, rosemary, peppermint

Oreochromis niloticus

Essential oil

Bath

3
10 min (24 h between treatments)

Monogeneans

de Oliveira Hashimoto et al. (2016)

Peppermint

Arapaima gigas

Essential oil

Bath

4h

Dawestrema spp.

Malheiros et al., 2016

Rosemary

Cyprinus carpio

Ethanol/aqueous

Bath

30e60 min

Dactylogyrus minutus

Zoral et al. (2017)

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14. Application of aromatic plants and their extracts in aquaculture

The essential oils of other aromatic plants have also recently been tested as anthelmintics. The essential oil of the Australian tea tree (Melaleuca alternifolia) has been used in baths against the parasite Gyrodactylus spp., reducing its prevalence in a dose-dependent manner when applied at between 1 and 30 ppm (Steverding et al., 2005). Eugenol, the main active compound in clove essential oil, used externally against monogeneans in tambaqui, was capable of reducing the prevalence of the parasite by 81%. It is worth noting that this reduction was not seen immediately, but rather a week after the treatment (de Lima Boijink et al., 2015). Tambaqui has also been used to test the anthelmintic effectiveness of other essential oils, such as those of several species of the genus Lippia sp. (Soares et al., 2017a, 2017b, 2016) or clove basil (Ocimum gratissimum) (de Lima Boijink et al., 2016). Ethanolic and aqueous extracts of rosemary have been demonstrated to be anthelmintics in carp, showing good effectiveness as a preventive therapy added to the feed and in therapeutic baths. The aqueous extract was less toxic to fish and, even though it showed a slightly lower effectiveness, it can be a useful, safe treatment to control monogenean parasites in fish (Zoral et al., 2017). Peppermint has also been reported to have antiparasitic effects, in addition to the immunostimulating properties commented on earlier. In Nile tilapia, a therapeutic bath with essential oil of peppermint at 40 mg/L was able to reduce the prevalence of some tilapia monogenean parasites by 70%, also improving hematological parameters (de Oliveira Hashimoto et al., 2016); however, this same concentration was not effective against the monogenean Dawestrema spp. in Arapaima gigas, and it also produced necrosis in the fish gills at higher doses (160 mg/L). This highlights the need to establish optimal, safe doses of each of these extracts and essential oils in each species of interest to aquaculture and for each pathogen. Once the toxicity limits and the optimum treatments for each disease have been established, this type of natural treatments can represent the definitive strategy to leave behind the use of treatments that contaminate fish for human consumption and the environment.

Latest properties discovered and possible uses In addition to the use of aromatic plant extracts and essential oils in nutrition and health, there is a wide variety of other uses that are gaining the interest of researchers, leveraging the benefits for food safety, availability and acceptance by consumers. One of them is the current use of essential oils and extracts from aromatic plants as anesthetics. Nowadays, the main anesthetics used in aquaculture are synthetic tricaine methanesulfonate (MS-222) and clove oil, which is of natural origin and whose main component is the aromatic compound eugenol. Until recently, however, no studies had been conducted on the anesthetic effect of other essential oils that are also rich in aromatic compounds, in spite of their great market availability and proven safety for humans. In the last decade, there have been several studies in this regard, mainly using plants from the families Lamiaceae, Verbenaceae, Lauraceae, and Myrtaceae. These experiences have mainly focused on South American fish and carp, while few studies of this type have been carried out on salmonids and Mediterranean fish (Hoseini et al., 2018). These new natural anesthetics have been

Latest properties discovered and possible uses

253

described as being less effective than clove essential oil and eugenol, but recently thymol has been reported to be slightly more effective in inducing anesthesia in carp, and it also has fewer secondary effects in terms of stress and tissue damage (Yousefi et al., 2018). In light of the effectiveness of the new extracts, oils and compounds obtained from aromatic plants, it would be interesting to explore this environmentally friendly strategy that provides a good level of food safety in other high-value species, such as salmonids and Mediterranean fish. Another novel aspect of extracts and essential oils from aromatic plants is the method used to incorporate them into feed, striving for optimal conservation, release and effect in the animals. Due to the low oxidative stability of many compounds and essential oils from aromatic plants, microencapsulation is becoming an increasingly attractive avenue to add essential oils to dry feed. This prevents interaction with other feed components and the release of undesirable odors, while at the same time protecting the active compounds from light, heat, moisture and oxidation. In addition, microencapsulation usually involves transforming the compound into an additive in powder form that can be homogenized in water and therefore used in different feed formats for fish (Sutili et al., 2017). Another type of use with great potential is the incorporation of oils or extracts in live prey for larvae. The larval phases of the different species of interest for aquaculture are usually the most difficult to manage, especially in the case of new species. The use of additives that improve the development and the immune system for better survival, which is normally compromised (Tovar-Ramı;rez et al., 2004), is very useful at this stage. Curiously enough, there have hardly been any experiences in this regard with white leg shrimp, but good results have been obtained when enriching brine shrimp with garlic extract at a dose of 200 mg/kg (Javadzadeh et al., 2012). One of the most novel focuses in terms of using the essential oils of aromatic plants is their use as functional additives in fish feed with a high proportion of vegetable ingredients. The harmful effect on marine fish of the inclusion of high percentages of soy meal is well known and is primarily related to the inflammation it produces in the intestine (soybean mealinduced enteritis). Even though safe inclusion limits have been established, such an available and economically viable replacement source of fish protein cannot be ruled out. For this reason, some authors have focused their studies on reducing the harmful effects of soy through the use of extracts and essential oils of aromatic plants. Similarly to the strategy followed with prebiotics and probiotics as additives in diets with low fish meal and oil content (Torrecillas et al., 2018, 2015), several authors have taken advantage of the immunomodulating and antiinflammatory effects of aromatic plant extracts and essential oils (Hernández et al., 2016) to reduce the secondary effects of including soy meal in the diet. Wang et al. (2016) used commercial compounds containing thymol and carvacrol in diets for Japanese sea bass (Lateolabrax japonicus), in which fish meal was partially replaced by soy meal. This compound added to a diet with 50% replacement reduced enteritis-related parameters, such as tissue disruption, lamina propria widening or reduction of intestinal folds. In gilthead seabream, a combination of essential oils of anise (Pimpinella anisum), sweet orange (Citrus sinensis) and oregano has been tested in diets that also had a high vegetable protein content (Rodrigues et al., 2018). The addition of these essential oils improved protein and fat retention, and also minimized fecal nitrogen loss.

́

254

14. Application of aromatic plants and their extracts in aquaculture

Current regulatory status and future perspectives One piece of evidence demonstrating the progress being made in the introduction of aromatic plant derivatives in animal feed and aquaculture is the inclusion of several of them on the list of food additives (EC, 2003), which is continuously updated by the European Union. With regard to the use of plants for curative and health purposes in fish, Commission Regulation (EC) No. 710/2009 (EC, 2009) concerning organic aquaculture permits the use of plant extracts without anesthetic effects as a veterinary treatment for fish. The use of allopathic medicines must be limited to a maximum of two treatments per year, or to once a year when the production cycle lasts less than 1 year. If these limits are exceeded, the fish cannot be sold as organic food. It should be further noted that the use of plant extracts for disinfection and cleaning of systems is allowed in farms producing organic products. In the case of the United States, it is the U.S. Food and Drug Administration and the U.S. Environmental Protection Agency that are in charge of regulating the use of plant extracts in aquaculture. In this sense, only garlic and onion have been considered by the FDA as new animal medications with low regulatory priority, and their use is permitted in salmonids for the treatment of infestations of helminths and sea lice (PPM number 1240.4200 2011). In spite of the great advances made in research on extracts and essential oils of aromatic plants and their use in aquaculture, existing regulations are advancing slowly, which may be limiting their use in the aquaculture industry. The use of bioactive plant compounds should be proposed in aquaculture, primarily as additives or immunostimulants, as opposed to medicines, since their registration as veterinary medications is a process that takes a long time and involves a greater economic cost (Bulfon et al., 2015). The success of implementing aromatic plants and their extracts in aquaculture can depend to a great extent on the success of organic aquaculture in which its use is practically a must. This type of aquaculture is experiencing great growth in Europe, and countries like Germany, France and Switzerland have particularly strong consumer markets (Ankamah-Yeboah et al., 2017). Furthermore, the regulation concerning organic aquaculture (EC, 2009) established that organic production also had to be based on organic juvenile fish starting in 2016. Given that the use of antibiotics would only be permitted within some very strict limits and the fish at this stage are very vulnerable to pathogens, this jeopardizes the viability of juvenile production. It is here where the use of immunostimulating compounds, health promoters and antistress compounds of a natural origin, such as extracts and essential oils of aromatic plants, play a crucial role. Recent studies on the market conditions of organic aquaculture in Europe (Ankamah-Yeboah et al., 2017), have evidenced the diversification of organic aquaculture products and the expansion of distribution channels, such as supermarkets, catering services, restaurants and online operators. In addition, the price that consumers are willing to pay is higher in organic aquaculture than in capture fishery with eco-labels. This review chapter highlights that marketing campaigns should place more emphasis on aspects such as absence on using synthetic additives and antibiotics, and animal health and well-being, as opposed to environmental aspects. To maintain safety standards and productivity, the speedy implementation of natural additives and medicines is crucial; among these, aromatic plants are one of the best candidates, given their proven effectiveness in fish.

References

255

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de Lima Boijink, C., Queiroz, C.A., Chagas, E.C., Chaves, F.C.M., Inoue, L.A.K.A., 2016. Anesthetic and anthelminthic effects of clove basil (Ocimum gratissimum) essential oil for tambaqui (Colossoma macropomum). Aquaculture 457, 24e28. de Oliveira Hashimoto, G.S., Neto, F.M., Ruiz, M.L., Acchile, M., Chagas, E.C., Chaves, F.C.M., Martins, M.L., 2016. Essential oils of Lippia sidoides and Mentha piperita against monogenean parasites and their influence on the hematology of Nile tilapia. Aquaculture 450, 182e186. Diab, A.S., Aly, S.M., John, G., Abde-Hadi, Y., Mohammed, M.F., 2008. Effect of garlic, black seed and Biogen as immunostimulants on the growth and survival of Nile tilapia, Oreochromis niloticus (Teleostei: cichlidae), and their response to artificial infection with Pseudomonas fluorescens. Afr. J. Aquat. Sci. 33 (1), 63e68. Diler, O., Gormez, O., Diler, I., Metin, S., 2016. Effect of oregano (Origanum onites L.) essential oil on growth, lysozyme and antioxidant activity and resistance against Lactococcus garvieae in rainbow trout, Oncorhynchus mykiss (Walbaum). Aquacult. Nutr. 23 (4), 844e851. Dorucu, M., Colak, S.O., Ispir, U., Altinterim, B., Celayir, Y., Colak, S.O., 2009. The effect of black cumin seeds, nigella sativa, on the immune response of rainbow trout, Oncorhynchus mykiss. Mediterr. Aquac. J. 2 (1), 27e33. EC, 2009. Commission Regulation (EC) No 710/2009 of 5 August 2009 amending Regulation (EC) No 889/2008 laying down detailed rules for the implementation of Council Regulation (EC) No 834/2007, as regards laying down detailed rules on organic aquaculture animal and seaweed production. OJ L 204, 15e34. EC, 2003. Regulation (EC) No 1831/2003 of the european parliament and of the council of 22 september 2003 on additives for use in animal nutrition. OJ L 268, 29e43. FAO, 2018. The State of World Fisheries and Aquaculture 2018, the State of World Fisheries and Aquaculture 2018 Meeting the Sustainable Development Goals. FAO, Rome, Italy. Galina, J., Yin, G., Ardó, L., Jeney, Z., 2009. The use of immunostimulating herbs in fish. An overview of research. Fish Physiol. Biochem. 35 (4), 669e676. Ghafari Farsani, H., Gerami, M.H., Farsani, M.N., Rashidiyan, G., Mehdipour, N., Ghanad, M., Faggio, C., 2018. Effect of different levels of essential oils (Satureja hortensis) in diet on improvement growth, blood biochemical and immunity of Angelfish (Pterophyllum scalare Schultze, 1823). Nat. Prod. Res. 1e6. Giannenas, I., Triantafillou, E., Stavrakakis, S., Margaroni, M., Mavridis, S., Steiner, T., Karagouni, E., 2012. Assessment of dietary supplementation with carvacrol or thymol containing feed additives on performance, intestinal microbiota and antioxidant status of rainbow trout (Oncorhynchus mykiss). Aquaculture 350e353, 26e32. Gonzalez Ronquillo, M., Angeles Hernandez, J.C., 2017. Antibiotic and synthetic growth promoters in animal diets: review of impact and analytical methods. Food Control 72, 255e267. Guardiola, F.A., Bahi, A., Bakhrouf, A., Esteban, M.A., 2017. Effects of dietary supplementation with fenugreek seeds, alone or in combination with probiotics, on gilthead seabream (Sparus aurata L.) skin mucosal immunity. Fish Shellfish Immunol. 65, 169e178. Guerreiro, I., Oliva-Teles, A., Enes, P., 2017. Prebiotics as functional ingredients: focus on Mediterranean fish aquaculture. Rev. Aquacult. 1e33. Hernández, A., García García, B., Caballero, M.J., Hernández, M.D., 2016. The inclusion of thyme essential oil in the feed of gilthead seabream (Sparus aurata) promotes changes in the frequency of lymphocyte aggregates in gutassociated lymphoid tissue. Aquacult. Res. 47 (10), 3341e3345. Hernández, A., García García, B., Caballero, M.J., Hernández, M.D., 2015. Preliminary insights into the incorporation of rosemary extract (Rosmarinus officinalis L.) in fish feed: influence on performance and physiology of gilthead seabream (Sparus aurata). Fish Physiol. Biochem. 41 (4), 1065e1074. Hoseini, S.M., Taheri Mirghaed, A., Yousefi, M., 2018. Application of herbal anaesthetics in aquaculture. Rev. Aquacult. Javadzadeh, M., Salarzadeh, A.R., Yahyavi, M., Hafezieh, M., Darvishpour, H., 2012. Effect of garlic extract on growth and survival rate in Litopenaeus vannamei post larvae. Iran. J. Fish. Sci. 21 (1), 39e46. Kirimer, N., Baser, K.H.C., Tümen, G., 1995. Carvacrol-rich plants in Turkey. Chem. Nat. Compd. 31 (1), 37e41. Kumar, P., Mishra, S., Malik, A., Satya, S., 2011. Insecticidal properties of Mentha species: a review. Ind. Crops Prod. 34 (1), 802e817. Labrador, J.R.P., Guiñares, R.C., Hontiveros, G.J.S., 2016. Effect of garlic powder-supplemented diets on the growth and survival of Pacific white leg shrimp (Litopenaeus vannamei). Cogent Food Agric. 2 (1).

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Lafferty, K.D., Harvell, C.D., Conrad, J.M., Friedman, C.S., Kent, M.L., Kuris, A.M., Powell, E.N., Rondeau, D., Saksida, S.M., 2015. Infectious diseases affect marine fisheries and aquaculture economics. Ann. Rev. Mar. Sci. 7, 471e496. Laxminarayan, R., Boeckel, T. Van, Teillant, A., 2015. The Economic Costs of Withdrawing Antimicrobial Growth Promoters from the Livestock Sector. OECD. Malheiros, D.F., Maciel, P.O., Videira, M.N., Tavares-Dias, M., 2016. Toxicity of the essential oil of Mentha piperita in Arapaima gigas (pirarucu) and antiparasitic effects on Dawestrema spp. (Monogenea). Aquaculture 455, 81e86. Martins, N., Petropoulos, S., Ferreira, I.C., 2016. Chemical composition and bioactive compounds of garlic (Allium sativum L.) as affected by pre-and post-harvest conditions: a review. Food Chem. 211, 41e50. Martins, M.L., Moraes, F.R., Miyazaki, D.M.Y., Brum, C.D., Onaka, E.M., Fenerick, J.J., Bozzo, F.R., 2002. Alternative treatment for Anacanthorus penilabiatus (Monogenea: dactylogyridae) infection in cultivated pacu, Piaractus mesopotamicus (Osteichthyes: characidae) in Brazil and its haematological effects. Parasite 9 (2), 175e180. Medicinal & Aromatic Plants [WWW Document], n.d. URL https://www.omicsonline.org/medicinal-aromaticplants.php. Metwally, M.A.A., 2009. Effects of garlic (Allium sativum) on some antioxidant activities in tilapia nilotica (Oreochromis niloticus). World J. Fish Mar. Sci. 1 (1), 56e64. Militz, T.A., Southgate, P.C., Carton, A.G., Hutson, K.S., 2013a. Dietary supplementation of garlic (Allium sativum) to prevent monogenean infection in aquaculture. Aquaculture 408e409, 95e99. Militz, T.A., Southgate, P.C., Carton, A.G., Hutson, K.S., 2013b. Efficacy of garlic (Allium sativum) extract applied as a therapeutic immersion treatment for Neobenedenia sp. management in aquaculture. J. Fish Dis. 37 (5), 451e461. Ndong, D., Fall, J., 2011. The effect of garlic (Allium sativum) on growth and immune responses of hybrid tilapia (Oreochromis niloticus x Oreochromis aureus). J. Clin. Immunol. Immunopathol. 3 (1), 1e9. Nya, E.J., Austin, B., 2011. Development of immunity in rainbow trout (Oncorhynchus mykiss, Walbaum) to Aeromonas hydrophila after the dietary application of garlic. Fish Shellfish Immunol. 30 (3), 845e850. Nya, E.J., Austin, B., 2009. Use of garlic, Allium sativum, to control Aeromonas hydrophila infection in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 32 (11), 963e970. Nya, E.J., Dawood, Z., Austin, B., 2010. The garlic component, allicin, prevents disease caused by Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 33 (4), 293e300. Peña, N., Auró, A., Sumano, H., 1988. A comparative trial of garlic, its extract and ammonium-potassium tartrate as anthelmintics in carp. J. Ethnopharmacol. 24 (2e3), 199e203. Peterson, B.C., Peatman, E., Ourth, D.D., Waldbieser, G.C., 2015. Effects of a phytogenic feed additive on growth performance, susceptibility of channel catfish to Edwardsiella ictaluri and levels of mannose binding lectin. Fish Shellfish Immunol. 44 (1), 21e25. Poongodi, R., Saravana Bhavan, P., Muralisankar, T., Radhakrishnan, S., 2012. Growth promoting potential of garlic, ginger, turmeric and fenugreek on the freshwater prawn Macrobrachium rosenbergii. Int. J. Pharma Bio Sci. 3 (4), 914e926. Preedy, V.R., 2016. Essential Oils in Food Preservation, Flavor and Safety. Academic Press. Raman, R.P., 2017. Applicability, feasibility and efficacy of phytotherapy in aquatic animal health management. Am. J. Plant Sci. 8 (2), 257. Reverter, M., Bontemps, N., Lecchini, D., Banaigs, B., Sasal, P., 2014. Use of plant extracts in fish aquaculture as an alternative to chemotherapy: current status and future perspectives. Aquaculture 433, 50e61. Reyes-Cerpa, S., Vallejos-Vidal, E., Gonzalez-Bown, M.J., Morales-Reyes, J., Pérez-Stuardo, D., Vargas, D., Imarai, M., Cifuentes, V., Spencer, E., Sandino, A.M., Reyes-López, F.E., 2018. Effect of yeast (Xanthophyllomyces dendrorhous) and plant (Saint John’s wort, lemon balm, and rosemary) extract based functional diets on antioxidant and immune status of Atlantic salmon (Salmo salar) subjected to crowding stress. Fish Shellfish Immunol. 74, 250e259. Rodrigues, V., Colen, R., Ribeiro, L., Santos, G., Gonçalves, R.A., Dias, J., 2018. Effect of dietary essential oils supplementation on growth performance, nutrient utilization, and protein digestibility of juvenile gilthead seabream fed a low-fishmeal diet. J. World Aquacult. Soc. 49 (4), 676e685. Rowshan, V., Bahmanzadegan, A., Saharkhiz, M.J., 2013. Influence of storage conditions on the essential oil composition of Thymus daenensis Celak. Ind. Crops Prod. 49, 97e101. Sahu, S., Das, B.K., Mishra, B.K., Pradhan, J., Sarangi, N., 2007. Effect of Allium sativum on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila. J. Appl. Ichthyol. 23 (1), 80e86.

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Seow, C.L., Lau, A.J., 2017. Differential activation of pregnane X receptor by carnosic acid, carnosol, ursolic acid, and rosmarinic acid. Pharmacol. Res. 120, 23e33. Shalaby, A.M., Khattab, Y.A., Abdel Rahman, A.M., 2006. Effects of Garlic (Allium sativum) and chloramphenicol on growth performance, physiological parameters and survival of Nile tilapia (Oreochromis niloticus). J. Venom. Anim. Toxins Incl. Trop. Dis. 12 (2), 172e201. Soares, B.V., Cardoso, A.C.F., Campos, R.R., Gonçalves, B.B., Santos, G.G., Chaves, F.C.M., Chagas, E.C., TavaresDias, M., 2017a. Antiparasitic, physiological and histological effects of the essential oil of Lippia origanoides (Verbenaceae) in native freshwater fish Colossoma macropomum. Aquaculture 469, 72e78. Soares, B.V., Neves, L.R., Ferreira, D.O., Oliveira, M.S.B., Chaves, F.C.M., Chagas, E.C., Gonçalves, R.A., TavaresDias, M., 2017b. Antiparasitic activity, histopathology and physiology of Colossoma macropomum (tambaqui) exposed to the essential oil of Lippia sidoides (Verbenaceae). Vet. Parasitol. 234, 49e56. Soares, B.V., Neves, L.R., Oliveira, M.S.B., Chaves, F.C.M., Dias, M.K.R., Chagas, E.C., Tavares-Dias, M., 2016. Antiparasitic activity of the essential oil of Lippia alba on ectoparasites of Colossoma macropomum (tambaqui) and its physiological and histopathological effects. Aquaculture 452, 107e114. Soltani, M., Ghodratnama, M., Ebrahimzadeh-Mosavi, H.A., Nikbakht-Brujeni, G., Mohamadian, S., Ghasemian, M., 2014. Shirazi thyme (Zataria multiflora Boiss) and Rosemary (Rosmarinus officinalis) essential oils repress expression of sagA, a streptolysin S-related gene in Streptococcus iniae. Aquaculture 430, 248e252. Soltani, M., Sheikhzadeh, N., Ebrahimzadeh-Mousavi, H.A., Zargar, A., 2010. Effects of Zataria multiflora essential oil on innate immune responses of common carp (Cyprinus carpio). J. Fish. Aquat. Sci. 5 (3), 191e199. Song, K., Milner, J.A., 2001. The influence of heating on the anticancer properties of garlic. J. Nutr. 131 (3), 1054Se1057S. Stentiford, G.D., Neil, D.M., Peeler, E.J., Shields, J.D., Small, H.J., Flegel, T.W., Vlak, J.M., Jones, B., Morado, F., Moss, S., Lotz, J., Bartholomay, L., Behringer, D.C., Hauton, C., Lightner, D.V., 2012. Disease will limit future food supply from the global crustacean fishery and aquaculture sectors. J. Invertebr. Pathol. 110 (2), 141e157. Steverding, D., Morgan, E., Tkaczynski, P., Walder, F., Tinsley, R., 2005. Effect of Australian tea tree oil on Gyrodactylus spp. infection of the three-spined stickleback Gasterosteus aculeatus. Dis. Aquat. Org. 66 (1), 29e32. Sutili, F.J., Gatlin, D.M., Heinzmann, B.M., Baldisserotto, B., 2017. Plant essential oils as fish diet additives: benefits on fish health and stability in feed. Rev. Aquacult. 10 (3), 716e726. Talpur, A.D., 2014. Mentha piperita (Peppermint) as feed additive enhanced growth performance, survival, immune response and disease resistance of Asian seabass, Lates calcarifer (Bloch) against Vibrio harveyi infection. Aquaculture 420e 421, 71e78. Talpur, A.D., Ikhwanuddin, M., 2012. Dietary effects of garlic (Allium sativum) on haemato-immunological parameters, survival, growth, and disease resistance against Vibrio harveyi infection in Asian sea bass, Lates calcarifer (Bloch). Aquaculture 364e 365, 6e12. Tanekhy, M., Fall, J., 2015. Expression of innate immunity genes in kuruma shrimp Marsupenaeus japonicus after in vivo stimulation with garlic extract (allicin). Vet. Med. 60 (1), 39e47. Tangestani, R., Doughikollaee, E.A., Ebrahimi, E., Zare, P., 2011. Effects of garlic essential oil as an immunostimulant on hematological indices of juvenile beluga (Huso huso). J. Vet. Res. 66 (3), 209e279. Tongnuanchan, P., Benjakul, S., 2014. Essential oils: extraction, bioactivities, and their uses for food preservation. J. Food Sci. 79 (7), R1231eR1249. Torrecillas, S., Montero, D., Caballero, M.J., Pittman, K.A., Custódio, M., Campo, A., Sweetman, J., Izquierdo, M., 2015. Dietary mannan oligosaccharides: counteracting the side effects of soybean meal oil inclusion on European sea bass (Dicentrarchus labrax) gut health and skin Mucosa mucus production? Front. Immunol. 6, 397. Torrecillas, S., Rivero-Ramírez, F., Izquierdo, M.S., Caballero, M.J., Makol, A., Suarez-Bregua, P., FernándezMontero, A., Rotllant, J., Montero, D., 2018. Feeding European sea bass (Dicentrarchus labrax) juveniles with a functional synbiotic additive (mannan oligosaccharides and Pediococcus acidilactici): an effective tool to reduce low fishmeal and fish oil gut health effects? Fish Shellfish Immunol. 81, 10e20. Tovar-Ramırez, D., Zambonino Infante, J., Cahu, C., Gatesoupe, F., Vázquez-Juárez, R., 2004. Influence of dietary live yeast on European sea bass (Dicentrarchus labrax) larval development. Aquaculture 234, 415e427. Van Hai, N., 2015. The use of medicinal plants as immunostimulants in aquaculture: a review. Aquaculture 446, 88e96.

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Vanhove, M.P.M., Briscoe, A.G., Jorissen, M.W.P., Littlewood, D.T.J., Huyse, T., 2018. The first next-generation sequencing approach to the mitochondrial phylogeny of African monogenean parasites (Platyhelminthes: Gyrodactylidae and Dactylogyridae). BMC Genomics 19 (1), 520. Wang, J., Tao, Q., Wang, Z., Mai, K., Xu, W., Zhang, Y., Ai, Q., 2016. Effects of fish meal replacement by soybean meal with supplementation of functional compound additives on intestinal morphology and microbiome of Japanese seabass (Lateolabrax japonicus). Aquacult. Res. 48 (5), 2186e2197. Yilmaz, E., Ergün, S., Yilmaz, S., 2015. Influence of carvacrol on the growth performance, hematological, non-specific immune and serum biochemistry parameters in rainbow trout (Oncorhynchus mykiss). Food Nutr. Sci. 6, 523e531. Yilmaz, S., Ergun, S., Celik, E.S., 2013a. Effect of dietary herbal supplements on some physiological conditions of Sea Bass Dicentrarchus labrax. J. Aquat. Anim. Health 25 (2), 98e103. Yilmaz, S., Ergün, S., Soytas, N., 2013b. Herbal supplements are useful for preventing streptococcal disease during first-feeding of tilapia fry, Oreochromis mossambicus. Isr. J. Aquacult. Bamidgeh 65. Yousefi, M., Hoseini, S.M., Vatnikov, Y.A., Nikishov, A.A., Kulikov, E.V., 2018. Thymol as a new anesthetic in common carp (Cyprinus carpio): efficacy and physiological effects in comparison with eugenol. Aquaculture 495, 376e383. Zheng, Z.L., Tan, J.Y.W., Liu, H.Y., Zhou, X.H., Xiang, X., Wang, K.Y., 2009. Evaluation of oregano essential oil (Origanum heracleoticum L.) on growth, antioxidant effect and resistance against Aeromonas hydrophila in channel catfish (Ictalurus punctatus). Aquaculture 292 (3e4), 214e218. Zilberg, D., Tal, A., Froyman, N., Abutbul, S., Dudai, N., Golan-Goldhirsh, A., 2010. Dried leaves of Rosmarinus officinalis as a treatment for streptococcosis in tilapia. J. Fish Dis. 33 (4), 361e369. Zoral, M.A., Futami, K., Endo, M., Maita, M., Katagiri, T., 2017. Anthelmintic activity of Rosmarinus officinalis against Dactylogyrus minutus (Monogenea) infections in Cyprinus carpio. Vet. Parasitol. 247, 1e6.

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C H A P T E R

15 Application of aromatic plants and their extracts in dairy animals Mariangela Caroprese, Maria Giovanna Ciliberti, Marzia Albenzio University of Foggia, Department of the Sciences of Agriculture, Food, and Environment, Foggia, Italy O U T L I N E Introduction

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Extraction methods of essential oils and use of abioc in animal nutrition 262 Aromatic plants and their extracts as modifiers of rumen fermentation

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Aromatics plant antimicrobial activities: effect on ruminant immune system 266

Aromatics plant antioxidant activities in dairy animals 268 Aromatic plants and their extracts as enhancer udder health

269

Conclusions and Future Direction

272

References

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Introduction In the last ten years, the European Commission’s overall strategy pointed out to tackle the emergency of bacteria resistant to antibiotics, due to both overexploitation and misuse of antibiotics. From January 2006 the European Commission has banned the use of antibiotics as growth promoters in animal feed. This prohibition has represented the final step in the phasing out of antibiotics used for nonmedicinal purposes and the first step when

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00015-7

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Copyright © 2020 Elsevier Inc. All rights reserved.

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15. Application of aromatic plants and their extracts in dairy animals

considering the public health. Only from January 1, 2017 the use of low doses of antibiotics in animal feed to promote growth and improve feed efficiency was effectively eliminated in the United States, as a result of new Food and Drug Administration (FDA) Veterinary Feed Directive. Consequently, farmers and stakeholders of the animal product supply and marketing chains are constantly searching for efficacious, safe, and cost-effective substances, which could help reduce the use of antibiotics (Simitzis, 2017). In this context, aromatic plants, including herbs and spices, with their secondary metabolites, i.e., phytochemicals or their essential oils (EOs), known as phytonutrients, have emerged as alternative growth promoters and antimicrobials improving the quality characteristics of animal products (Simitzis and Deligeorgis, 2011). Plant-derived phytochemicals are also named phytobiotics because are self-defense molecules of plants against animals and infectious organisms (Deans and Ritchie, 1987). Phytochemicals such as polyphenols, quinines, flavonols/flavonoids, alkaloids, polypeptides or their oxygen-substituted derivatives represents some of the main aromatic plant-derived bioactive compounds (ABIOC) deriving from aromatic plants (rosemary, oregano, sage, thymus, peppermint, and garlic), originated from the Mediterranean area (Perumalla and Hettiarachchy, 2011; Negi, 2012; Cowan, 1999). Moreover, ABIOC includes EO as well plant volatile aromatic compounds, which are a mixture of different chemical substances, between 20 and 60 components for each EO, containing terpenoids, alcohols, aldehydes, hydrocarbons, ketones, esters, and ethers (Dorman and Deans, 2000). The main components of EO are terpenoids, categorized as terpenes, among which monoterpenes are the most representative molecules portioning the 90% of many EO (limonene, thymol, carvacrol, linalool, carvone, geranyl acetate). Another class of molecules characterizing EO is that of phenylpropanoids, derived from phenylalanine (Hart et al., 2008), that in some cases are more abundant than terpenes, as in the case of cinnamon, clove, anisa (Patra, 2011). Anyway, ABIOC are usually residue free and generally recognized as safe (GRAS) (Windisch et al., 2009; Brenes and Roura, 2010; Varel, 2002). In this chapter the latest statements on the effects of ABIOC on dairy animals in terms of alterations in rumen fermentation, immune system, antioxidant activity, and udder health will be presented and discussed as depicted in Fig. 15.1. In general, the role of ABIOC, as alternative to synthetic antimicrobials, on the single animal compartments will demonstrate how their synergistic action could exert outcomes in terms of enhancement of milk production and improvement of milk quality and safety in dairy animals.

Extraction methods of essential oils and use of abioc in animal nutrition Different methods can be used to extract EO: steam distillation is the common used method for commercial production of EO; other methods include hydrodistillation, organic solvent extraction, microwave assisted extraction, supercritical CO2 extraction, ultrasonic extraction, and solvent-free extraction (Okoh et al., 2010). The first patent for solvent-free microwave extraction (SFME) of volatile natural substances was in 2003. The inventors (Chemat et al., 2003, EPD 03001183.7) produced a method of extraction of EO consisting of a microwave oven with a microwave chamber for receiving the biological material and a condensation chamber. The use of the term solvent-less or solvent-free extraction is linked to a number of advantages such as fast action, cleanliness, green method, low energy output and has been

Extraction methods of essential oils and use of abioc in animal nutrition

263

FIGURE 15.1 Schematic image of ABIOC effects on dairy animals on rumen, on mammary gland and udder health, on immune system, and on antioxidant status of dairy animals as summarized in this chapter.

found more effective than traditional extraction methods (Aslam et al., 2018). Moreover, the EO produced by microwave extraction method, and no solvent-free, need specific purification to be considered safe for human health. To this regard many opportunities and modifications can be possible in term of purification of EO by applying in combination with other extraction technique (Aslam et al., 2018). The incorporation of ABIOC in the diet of livestock can ensure the optimization of animal performance and increase nutrient availability. There is sufficient scientific evidence that herbs and EO are able to improve performance in piglets and poultry. On the contrary, very little information is available about the effects of using ABIOC as feed additives on feed intake and palatability in ruminant diet (Greathead, 2003; Rochfort et al., 2008). In growing lambs, negative effects of camphor and a-pinene, sprayed on alfalfa pellets, on intakes were observed, whereas no discernible effects of limonene, cis-jasmone, and b-caryophyllene on consumption were detected (Estell et al., 1998). However, the knowledge about the specific chemical interactions of ABIOC with feed intakes and the consequent alterations in feeding management is missing. In ruminant studies phytonutrients were found to be potential rumen modifiers by their antimicrobial activities against bacteria, protozoa, and fungi (Calsamiglia et al., 2007; Patra, 2012; Oh and Hristov, 2016). Therefore, particular attention has been devoted to their action on the reduction of methane production by shifting rumen fermentation toward propionate. However, many papers focusing on rumen fermentation shift induced by phytonutrients are in vitro batch or continuous culture studies, and

264

15. Application of aromatic plants and their extracts in dairy animals

this experimental condition is far from the in vivo application, particularly in dairy cows (Benchaar et al., 2008a). Nevertheless, on cattle some phenolic phytonutrients, among which cinnamaldehyde (CIN), eugenol (EUG), and capsicum (CAP) was tested in in vivo experiments, confirming a decrease of methane production by decreasing ruminal content of acetate and increasing of propionate (Cardozo et al., 2006; Yang et al., 2010). In ruminant nutrition, ABIOC are largely used as antioxidant and antiinflammatory supplements (Jungbauer and Medjakovic, 2012), and also as rumen fermentation modulators (Khiaosa-ard and Zebeli, 2013). The importance of studying the interconnections existing among metabolic traits, production traits (feed efficiency and milk production variables), and host-microbiome interactions (rumen microbiome and association of fermentation rate with microbial diversity), as hypothesized by Shären et al. (2018), should be essential to plan feed integration with ABIOC in order to guarantee a proper health status of dairy cows.

Aromatic plants and their extracts as modifiers of rumen fermentation In ruminants, the digestive tract is characterized by microflora colonizing the forestomachs in a true symbiosis with the host (Santra and Karim, 2003; Paul et al., 2010). The development of obligate and facultative anaerobes is naturally created by rumen environment, which receives a constant inflow of nutrients and fluids. Feed is continuously mixed being constantly supported by rumen contractions, and exposing feed to mechanical changes. All the undegraded and nonutilized feed ingredients, as well as a substantial biomass of microbial and infusorian cells, moves to the more distal compartments of the digestive tract: the reticulum and the abomasum. Both volatile fatty acids (VFA) and ammonia resulting from protein degradation are absorbed in the rumen contributing to the slight variations on its pH that is normally about 6e7 pH with a constant osmotic pressure and a temperature which is about the optimum range for microbial life processes: 38e40
S. Rumen fermentation can be modified through maximization or minimization of biochemical events in forestomachs. An integrative approach can be applied when a successful intervention is desirable, considering that any intervention could lead to specific long-term consequences and a chain of interrelated effects. The possibility to modify the rumen microbes in the forestomachs of ruminants is an essential point that can be optimized through diet structure, biotechnological products and probiotics, enzymatic preparations, dietary protein, dietary fat, etc. The rumen microbiome ecosystem is essential for the utilization of low-quality feed and the production of highquality protein in ruminants, and is characterized by resistance, resilience, and functional redundancy (Edwards et al., 2008; Weimer, 2015). The different populations of rumen microbes can be classified according to their ability to digest several substrates or specific substrates (Weimer, 2015), thus being named as cellulolytic, proteolytic, lipolytic, or amino-acid-fermenting microbes (Firkins and Yu, 2006). The main criticism of ruminant livestock production is its large environmental impact being responsible for 16%e25% of the global greenhouse gases emission and of about 33% to global anthropogenic methane emissions. Methane emission from ruminants principally derives from enteric fermentation (87% from rumen, and 13% from large intestine), and partially from their manure (Torrent and Johnson, 1994). Nutritional interventions based on ABIOC, rich in saponins, tannins, and EO can enhance rumen fermentation in order to reduce methane emission. The mitigation

Aromatic plants and their extracts as modifiers of rumen fermentation

265

of methane emission is direct on methanogenesis, and/or methanogens, protozoa, feed ingestion, and fermentation processes (Cobellis et al., 2016). In vitro experiments evaluating the effect of ABIOC on rumen fermentation displayed a wide range of results principally driven by the concentration of ABIOC tested and their synergistic action. In an experiment on in vitro rumen fermentation, a reduction of ammonia and total VFA concentrations was found in beef cattle when high concentrations of a number of EO were tested (3000 and 5000 mg/L of many of the oils), whereas a poor effect was observed if testing low concentrations. However, on the contrary, a dose-dependent effect was demonstrated for EUG, which increased ammonia concentration when tested at the lowest level (i.e., 0.3 and 3 mg/L), had no effect when tested at intermediate level (30 mg/L), and significantly decreased ammonia at high levels (300, 3000, and 5000 mg/L) (Cardozo et al., 2005). Rumen microbial populations detoxify EO by chemically reducing them into inert alcohols; this mechanism has been hypothesized to explain the absence of significant effects of EO, when tested doses lower than bactericidal level (Hart et al., 2008; Chizzola et al., 2004). A very exhaustive critical review on the effects of EO as rumen modifiers in ruminant nutrition has been recently published by Cobellis et al. (2016), suggesting the use of EO as promising natural substances to mitigate rumen methane and ammonia production, improve rumen fermentations, and reduce environmental impact of ruminant production. However, in order to satisfy a number of questions remained unanswered, such as the exact determination of EO active compounds, their effective doses, mode of action, effect on organoleptic characteristics of animal products, and costebenefit ratio before using EO, or more generally ABIOC, as additives in modulating ruminant nutrition at farm level (Cobellis et al., 2016), novel experiments have been performed in the last two years. Among these, Kolling et al. (2018) evaluated the effects of green tea and oregano extracts and their association as feed additives on performance and methane emissions of dairy cows between 28 and 87 days of lactation. Both oregano and green tea extract, when supplemented alone, reduced methane metrics without affecting productivity. The association of green tea and oregano extracts did not exert any additional reduction of methane emission and did not affect milk yield and DMI in comparison with single treatment. However, their association ameliorated the total-tract apparent digestibility, molar proportions of ruminal acetate and butyrate, and fatty acid profile in milk (Kolling et al., 2018). Lately, a blend of ABIOC deriving from mint, thyme, rosemary, and clove (Digestarom Dairy, BIOMIN Holding GmbH, Getzersdorf, Austria) was suggested as potential feed additive in cows to alleviate negative consequences of feeding highconcentrate, responsible for subacute ruminal acidosis (SARA) (Humer et al., 2018). This digestive disorder consists in an intermittent decline of ruminal pH, causing ruminal microbial imbalances, also called dysbiosis (Tajima et al., 2000; Khafipour et al., 2009). The importance of studying dysbiosis during SARA in cows is stressed by the production of microbe-derived toxic compounds, such as lipopolysaccharides (LPS) and biogenic amines (BA), that, in consequence of increased permeability of the rumen mucosa during SARA (Aschenbach and Gäbel, 2000), can translocate into systemic circulation, leading to systemic inflammation and metabolic derailments. Particularly, SARA challenge in cattle can result in displaced abomasum, laminitis, fatty liver, and plasma mineral disturbances (Zebeli and Matzler-Zebeli, 2012). However, the supplementation with ABIOC (Digestarom Dairy) showed a potent contrasting action against the increase of concentration of LPS, and BA in dry cows. Moreover, during the administration of concentrate-rich diet, which can cause

266 TABLE 15.1

15. Application of aromatic plants and their extracts in dairy animals

Influence of ABIOC (Digestarom Dairy) as feed additive supplementation on the concentration of ruminal LPS, biogenic amines (BA), and short-chain fatty acid (SCFA) profile in dairy cows subjected to two intermittent SARA feeding phases. SARA1a

SARA2a

CON

ABIOCb

CON

ABIOCb

151.353

85.893

154.074

194.513

Pyrrolidine

16.7

10.3

9.75

8.17

Histamine

21.6

7.42

6.40

2.18

Spermine

1.97

0.902

0.438

1.39

Propionate

23.8

21.0

27.5

25.1

Buryrate

13.0

16.0

11.8

13.9

Valerate

5.4

4.1

4.4

4.1

Caproate

1.7

1.2

0.8

0.9

Iso-valerate

0.8

1.2

1.1

1.3

LPS, endotoxin units/mL

Adapted by Humer et al. (2018)

Biogenic amines (BA), mg/kg

% of total SCFA in rumen Adapted by Neubauer et al. (2018)

SARA1 ¼ 65% concentrate for 1 wk; SARA2 ¼ 65% concentrate for 2 wk. ABIOC ¼ Digestarm Dairy (BIOMIN Holding GmbH, Getzersdorf, Austria)

a

b

both the reduction of ruminal pH and of rumen bacteria diversity, the supplementation with ABIOC (Digestarom Dairy) can increase ruminal activity, alleviating ruminal pH depression, reducing starching fermenting bacteria and increasing butyrate levels. Data on changes in LPS, BA, and % of short-chain fatty acid (SCFA) in rumen was presented in Table 15.1. Overall the supplementation with ABIOC (Digestarom Dairy) demonstrated its ability to alleviate the negative effects of concentrate-rich diet on ruminal microbial populations and reduce the production of toxic molecules in the rumen (Humer et al., 2018; Kröger et al., 2017; Neubauer et al., 2018). However, because of the high variability of the compounds and the concentrations tested, the results of the state of art are inconsistent and sometime contradictory. This lead the need of further in vivo research to better clarify the effects of ABIOC on voluntary intakes, rumen fermentation, and methane emission (Cobellis et al., 2016).

Aromatics plant antimicrobial activities: effect on ruminant immune system The cytoplasmic membrane of bacterial cells can be destroyed directly or by damaging the membrane protein causing an increase of its permeability. The mode of action of EO against bacterial cells was supposed by Burt (2004) based on their hydrophobic nature, and affinity with lipids. In particular, EO can activate cell lysis through cytoplasmic coagulation, or can modify the membrane permeability of bacterial cells for their lipophilicity (Burt, 2004).

Aromatics plant antimicrobial activities: effect on ruminant immune system

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Therefore, Gram-positive bacteria result more susceptible than Gram-negative bacteria to EO antimicrobial action due to hydrophobic nature of Gram-positive cytoplasmic membrane (Chao et al., 2000). Interestingly, toxicity of EO toward mammalian cells is inversely related to the lipophilicity of EO components, whereas toward bacteria and fungi is directly related to EO lipophilicity (Pauli, 2007, 2008). However, the evaluation of EO antimicrobial activity lead to contradictory results in some cases, probably due to a natural variability in their composition and to a different susceptibility of microorganisms to their action. The scarcity of information regarding EO production, storage, and age, as well as the different methods used for microbiological testing can be claimed to explain the different results obtained regarding EO antimicrobial activity (Pauli and Schilcher, 2010). The therapeutic uses of EO and/or single ABIOC in human medicine and care were deeply investigated in books on the argument (Hüsnü Can Baser and Buchbauer, 2010). Very few information is available on the effects of EO, or more generally of ABIOC, in veterinary medicine, and in particular on ruminant immune responses. However, in dairy cow ABIOC exerts a role in the modulation of the adaptive immune system, as also reported in monogastric species, specifically activating and inducing the expansion of CD4þ cells (Oh et al., 2013). Recent experiments tested different ABIOC administrated in the diet of dairy cows in a rumen-protected form or in bypass ruminal formulation in order to avoid the degradation of ABIOC by rumen microorganisms. Therefore, the abomasal administration (2 g/d per head) of capsicum from Capsicum frutescens L. and Capsicum anum L. var. concoides, increased the relative blood lymphocyte percentages. In addition, the blood proportion of total CD4þ cells and total CD4þ cells coexpressing the activation status signal and CD25 increased after the abomasal administration of curcumin from Curcuma longs L., and garlic. An apparent pro- or antiinflammatory effect of the abomasal administration of garlic, curcumin, and capsicum was excluded due to the absence of effects on proliferation and cytokine production. On the contrary, dairy cows challenged with LPS showed a modulation of acute phase response in terms of decreased blood cortisol, haptoglobin, and TBARS concentration after the administration of rumen-protected Capsicum oleoresin, from Capsicum frutescens L. and Capsicum anum L. var. concoides (Oh et al., 2017a). Finally, dietary supplementation of a granular form of increasing amounts of Capsicum oleoresin in dairy cows resulted in increased number of peripheral blood neutrophils, eosinophils, and neutrophils to lymphocytes ratio (Oh et al., 2015). Moreover, an increase in neutrophil phagocytosis was observed, suggesting that Capsicum oleoresin may affect directly their activity. It was hypothesized that the mechanism of action of ABIOC on immune cell activity can be exerted indirectly by the release of neuronal peptides or directly by a Transient Receptor Potential (TRP) cation channel of the subfamily Vanolloid member 1 (TRPV1) (Zimmerman et al., 1992; Heiner et al., 2003). The TRP is a cation channel family of receptors expressed in several cells such as immune cells, neurons and in other tissues, whose role is to detect changes in cell environment induced by temperature, pain, osmolarity, and other stimuli (Vriens et al., 2008; Voets et al., 2005). In general, ABIOC can bind to a family of TRP channels expressed on immune cells acting as secondary transducers of cell activation or ion transporters. The activation of TRP channels causes an increase of Ca2þ influx into intracellular compartment, activating transcription factors, such as nuclear factor (NF)-kB and nuclear factor of activated T-cells (NF-AT), to migrate into the nucleus of the cells (Berridge et al., 2000). Moreover, the influx of Ca2þ, promoted by TRP channels, causes an increase of internal store of Ca2þ in the endoplasmic reticulum (Gees et al., 2010). In

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particular, EUG capsaicin, and allicin activate TRPV1, allicin and cinnamaldehyde activate TRPA1, and EUG activates TRPV3. A representative scheme of ABIOC action on immune cells through TRP channel was reported in a review of Oh et al. (2017b).

Aromatics plant antioxidant activities in dairy animals A large body of evidence shows that ABIOC, besides their antimicrobial activities and antiinflammatory, have a strong antioxidant activity, often linked to their phenolic compounds. In particular, the antioxidant role of phenolic compounds is related to the increase of endogenous antioxidants such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) (Masella et al., 2005). Numerous glutathione-regulating genes contain response elements called xenobiotic response elements (XREs) and antioxidant response elements (AREs). It was demonstrated that ABIOC phenolic compounds can upregulate genes and transcription factors, including aryl hydrocarbon receptor (AhR), that is a ligand-activated transcription factor that binds to XREs, and nuclear factor E2 related factor (Nrf2), that binds AREs (Seymour et al., 2013). Carnosic acid, one of the major phenolic constituent of rosemary leaves, exhibits antioxidant activity even stronger than some synthetic antioxidants; in addition, clove, rosemary, cinnamon, and turmeric were found to have important antioxidant properties (Charles, 2013). Moreover, an important synergistic effect in ABIOC, in terms of antioxidant activity, was suggested (Aftab and Vieira, 2010), that can be used to prevent ruminant free-radical induced disorders, often related to acute phase response at the onset of infection (Cray et al., 2009). A preliminary experiment of Hashemzadeh-Cigari et al. (2014) showed that quaternary mixtures of herbs, represented by cinnamon bark, turmeric root, rosemary leaves, and clove, exploited a synergistic antioxidant activity (20%e29%) as compared to the activity exhibited by the single herb. Results from this preliminary study encourage the hypothesis that the combination of herbs synergistically potentiates the single response of each herb, leading to increased antioxidant activity on the whole. In sheep, in a vivo study, four herbs (rosemary, marigold, citrus, and grape) exerted strong antioxidant capacity (Gladine et al., 2007). Lately, the association of single rosemary, marigold, citrus, grape, and vitamin E, reduced oxidative stress in lactating cows when fed a diet rich in n-3 FA (Gobert et al., 2009). In recent studies in goats, juniper oil, containing 89.7% a-pinene (0.4e2 mL/kg of DM), increased activity of SOD (Yesilbag et al., 2016); whereas, a blend of herb powders (Woodfordia fruticosa, Solanum nigrum, and Trigonella foenum-graecum) containing hydrolysable tannins, steroidal saponins, and glycoalkaloids exhibited antioxidant action by increasing the activity of blood GSH peroxidase, CAT, and glutathione S-transferase (Choubey et al., 2016). Very few studies are available on the effects of ABIOC on oxidative stress in dairy cows. The abomasal infusion of Curcuma and Capsicum, and the dietary supplementation of Capsicum in dairy cows had no effects on the blood oxidative stress markers. However, when cows were challenged with LPS the supplementation of rumen-protected Capsicum decreased the blood concentrations of TBARS (Oh et al., 2017a). The abomasal infusion of garlic oil (2 g/d per head) increased blood concentrations of 8-isoprostane, a byproduct of lipid oxidation, suggesting that excessive dose of garlic oil can expose dairy cows to an increment of oxidative stress (Oh et al., 2013, 2015). Therefore, an optimization of doses and also the study and identification of synergistic interactions among ABIOC in

Aromatic plants and their extracts as enhancer udder health

269

animal feeding is required, to reduce costs of production and strengthen the antioxidant actions of ABIOC. In addition, ABIOC supplementation might be more effective when animals are under physiological or environmental stress conditions, or both (Gobert et al., 2009; Yang et al., 2010). Indeed, in transition cows chestnut tannins (mainly hydrolysable tannins) decreased an oxidative stress marker, malondialdehyde, and increased endogenous antioxidant enzymes such as SOD and GSH peroxidase in the blood and liver (Liu et al., 2013). Moreover, nutrition supplementation with excessive amounts of antioxidant micronutrients such as Vitamin E, Se, Zn, and Cu in ruminants was found to improve resistance against mammary infection, and, as a consequence, to enhance udder health (Politis, 2012; Scaletti et al., 2003). Previous findings further demonstrated that ABIOC could be potent healthenhancing supplements in ruminants in order to alleviate stressful condition activated by inflammatory responses, among which mastitis represent the main critical disease for the impairment of animal productivity.

Aromatic plants and their extracts as enhancer udder health Global milk production has been growing by 1.9% per annum over the last five years, with organic dairy products featured approximately 6% of the entire market in US dairy market in 2010. The prohibition of using synthetic antibiotics to treat mastitis has grown the interest to natural alternatives, such as aloe vera in combination with vitamin supplements (Pol and Ruegg, 2007). However, the treatment of mastitis is critical for organic dairy farmers to avoid the increase of udder problems and consequently the decrease the milk quality, even if no alternatives to synthetic antibiotics have been approved by the Food and Drug Administration (FDA) (Zwald et al., 2004). An appropriate scientific evaluation is essential to guarantee the effectiveness and safeness of the alternatives to antibiotics, and to improve milk quality and udder health. High somatic cell counts (SCC) of milk is indicative of poor udder health status and consequently of scarce milk quality, representing a major concern of modern dairy herds (Schukken et al., 2003). Cows experiencing high SCC produce less milk, causing significant economic losses to the dairy industry, due to around 70% of the costs associated with temporary or permanent decreases in milk production (Zhao and Lacasse, 2008). In cows with moderate or high SCC, a mix of 60% rosemary, 18% cinnamon bark, 18% turmeric, and 4% clove bud, administrated as a supplementation in the diet, was tested on feed intake, performance, udder health, ruminal fermentation, and milk plasma metabolites (Hashemzadeh-Cigari et al., 2014). Supplemented cows, both with moderate or high SCC, showed a reduction of the SCC and an enhancement of productive performance, thus improving the overall mammary health status. Moreover, supplemented cows also showed positive effects on feed intake and propionate formation in the rumen, resulting in enhanced milk production. Overall, this feeding strategy was particularly effective for the treatment of cows experiencing high SCC, in order to counteract the economic losses related to subclinical mastitis and its damage to mammary tissue with an enhancement of the productive performance. There is a scarcity of data concerning the effects of ABIOC on milk production, and the existing results are also conflictual. Nevertheless, dairy farmers market includes the phytoceuticals in animal diet. Feeding dairy cows with increasing doses (80, 120 g/cow/d) of a

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microencapsulated EO blend, caused an increase of milk energy content, without affecting milk yield, milk component yields or milk energy output (Spanghero et al., 2009). In addition, microencapsulated EO blend did not exert a difference in feed intake or body weight changes (Spanghero et al., 2009). Moreover, supplementations to dairy cows based on CIN, EUG, and CAP did not affect DMI, milk yield, milk composition, in terms of milk fat, true protein, lactose and fatty acid profile (Oh et al., 2018). In addition, the supplementation of dairy cows with CIN and EUG and their blend, at 15% and 28% respectively, did not result in an increase of DMI or milk yield and composition (Benchaar et al., 2008b, 2012; 2015; Benchaar, 2016; Tager and Krause, 2011; Tekippe et al., 2013). In other studies, CAP supplementation increased milk yield or feed efficiency of dairy cows (Oh et al., 2015, 2017a; Stelwagen et al., 2016), and the blend of CIN, EUG, and CAP, caused an increase of milk production in cows (Oguey and Wall, 2016). Only a slight effect on milk fatty acids composition was found in dairy cows supplemented with CIN and CAP (Benchaar and Chouinard, 2009; Oh et al., 2015). When garlic or garlic compounds were used to reduce enteric methane emissions, very little effects on milk yield, and DMI were found; however, a strong influence on the sensory and rheological characteristic of milk and cheese was registered (Rossi et al., 2018). Focusing on the study of natural alternatives to antibiotics on mammary gland, a crucial point for intramammary (IMM) treatment of mastitis is the selection of nonirritating substances, being the mammary gland highly susceptible to irritation. Numerous negative effects of ABIOC have been found, such as nausea and respiratory arrest for the oral administration of EO of thyme, mild gastrointestinal effects, and bleeding for garlic (Bent, 2008). EO can be toxic even in relatively small quantities. No specific common safety risks are associated with use of oregano oil products; however, typical daily doses of oregano EO for humans contain between 165 and 195 mg of carvacrol (Kinder et al., 2015). Human study reported that, after receiving topical oregano oil, the highest concentration of carvacrol detectable in plasma is 0.003 mg/mL (Mason et al., 2017). Topical administration of aromatic plants and their extracts can lead to the passage of ABIOC into bloodstream; however, no residues in milk deriving from ABIOC are allowed by the US Food and Drug Administration (US Food and Administration, 1994). Studies aiming at the evaluation of the efficacy of ABIOC treatments, both topical and oral, should be carried out during dry period to prevent inflammatory conditions during lactation. Dry period, the state of transition occurring from lactation to colostrogenesis, is indeed the most susceptible period for invading pathogens and consequently inflammation of the mammary gland in dairy cows (Oliver and Sordillo, 1988). Besides the scarcity of scientific prereviewed clinal studies on alternatives’ effectiveness (Ruegg, 2009), a positive effect of essential oils for improvement of milk quality in dairy cattle was found in the report of Karreman (2007). Plant-derived oils rich in thymol and carvacrol (Phyto-Mast) represent one of the approved IMM product for organic production by the Ohio Ecological Food and Farm Association, mainly used for improving milk quality. The in vitro antibacterial activity of Phyto-Mast, plants-derived oils, and their combination against common mastitis-causing pathogens cultured on milk was studied in Mullen et al. (2014a). In Table 15.2 is shown the in vitro growth of bacteria Staphylococcus aureus, Staphylococcus chromogenes, and Streptococcus uberis in milk when testing different concentration (vol/vol) of EO (%) as reported in Mullen et al. (2014a).

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Aromatic plants and their extracts as enhancer udder health

TABLE 15.2

Effect of EO derived from aromatic plant on growth of mastitis-causing microorganism in milk in vitro. Growth of mastitis-causing microorganism at different concentration of treatment (%, vol/vol)

Treatmenta

Common name

Staphylococcus aureus

Staphylococcus chromogenes

Streptococcus uberis

Angelica dahuricae (4%)

Bai zhi

88  27

52  31

102  31

Angelica sinensis (4%)

Chinese angelica

139  34

42  24

143  52

Gaultheria procumbens (4%)

Wintergreen

108  32

65  43

159  58

Glycyrrhiza uralensis (4%)

Chinese licorice

52  31

51  25

138  67

Thymus vulgaris (1%)

Thyme

137  108

35  15

22

Thymus vulgaris (2%)

00

00

00

Thymus vulgaris (3%)

00

00

00

Phyto-Mastb

237  107

47  16

114  58

a Growth of bacteria are presented as growth relative to milk þ bacteria control, averaged over three replications and presented as means  standard error. b Phyto-Mast (Bovinity Health LLC, Narvon, PA) contains Angelica dahuricae, Angelica sinensis, Gaultheria procumbens, Glycyrrhiza uralensis and Thymus vulgaris essential oil and is an intramammary herbal preparation used for improving milk quality. Adapted by Mullen, K.A., Lee, A.R., Lyman, R.L., Mason, S.E., Washburn, S.P., Anderson, K.L., 2014a. Short communication: an in vitro assessment of the antibacterial activity of plant-derived oils. J. Dairy Sci. 97, 5587e5591.

The four different EO tested at 4% failed to evidence the antibacterial activity; conversely, EO of thyme exerted consistent antibacterial activity against three mastitis-causing pathogens both at 2% and 3%. An in vivo study of Mullen et al. (2014b), both Phyto-Mast and Cinnatube (New AgriTech Enterprice, Locke, NY), an IMM therapeutic infusion, were tested during dry period. Both the herbal treatments registered a similar infection rate compared to conventional antibiotic therapy, without affecting milk quality and production. The previous statements encourage further demonstration on the efficacy of EO or blend of them to treat IMM infection, and as a consequence, on enhancement of lactation. Other phytoceutical products including a garlic-based tincture, administered via the oral or intravulvar (IVU) route (Dr. Paul’s CEG Tincture, Arcadia, WI, USA), and an oreganobased topical product (Uddersol, Ralco, Marshall, MN, USA) are available in USA to control mastitis. Garlic has an antibacterial activity in vitro (Cowan, 1999), whereas oregano and thyme contain carvacrol and thymol, which are potent antimicrobial molecules (Helander et al., 1998). Besides IMM administration, also topical (TOP) and IVU products for mastitis control did not scientifically evaluated in terms of effectiveness of the treatment. A TOP mint-based liniment treatment on mammary glands following IMM challenge with

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staphylococci was evaluated, but no significant reduction in bacterial numbers compared with control was observed (Knight et al., 2000). Conclusively, both oral and local administration of ABIOC can be efficacious in the treatment of mammary infection; however, no official methods are available for the detection of residues from ABIOC in milk. Moreover, it is unknown the interfering role of ABIOC with respect to screening tests for the evaluation of antibiotic residue in milk. In this context, Mullen at al. (2017) found that the antibiotic detection by Charm SL Beta-Lactam test (SLBL, ROSA Pearl Reader, Charm Sciences, Lawrence, MA, USA) and the Delvotest P (DSM, Delft, The Netherlands) was not affected by ABIOC residues.

Conclusions and Future Direction The increasing of misuse or overuse of antibiotics causing antimicrobial resistance is the major concern about human health assurance. In the last ten years, scientists have promoted the initiative to study new molecules declared safe to be used as efficient alternatives to antibiotics. A wide range of bioactive compounds from plants including EO have been proven for their positive effects at multifunctional level and are considered the future feed supplements for ensuring health of lactating animals and safety productions. As summarized in this chapter, ABIOC could positively manipulate gut microbiota and rumen fermentation, inhibit pathogenic mastitis-causing bacterial growth, prevent oxidation, and, as a result, improved quality of livestock production. Moreover, ABIOC could be used as feed supplementation in order to balance the decreasing of ruminal pH typical of SARA in dry cows, boosting the reduction of inflammatory state, reducing the release of LPS in rumen, and, as a consequence, its diffusion in blood and the risk of systemic inflammation. However, even if the action of ABIOC is considered overall positive, supported by in vitro, and, more recently, by in vivo studies in dairy animals, their effectiveness in animal production has not yet been proven to be consistent and some critical aspects need to be addressed before their commercial application as feed additive. An example can be the study of the optimal dose of ABIOC in relation to their application; thus, even if they are generally recognized as safe (GRAS) for use in the food and feed industry, their use at high concentrations could induce cytotoxic effects on living cells. Moreover, a combination among different ABIOC should be addressed in order to evaluate their synergistic action and realize the best blend for each specific requirement in ruminant. Finally, very little is known about the absorption, distribution, metabolism and excretion of ABIOC in ruminants, and regarding their action on parameters of animal welfare and productivity, particularly on mammary gland secretion, or their specific molecular mechanisms on immune system during stressful condition. Further research is needed to clarify these investigations and establish ABIOC the regular application in animal production.

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Perumalla, A.V.S., Hettiarachchy, N.S., 2011. Green tea and grape seed extractsdpotential applications in food safety and quality. Food Res. Int. 44, 827e839. Pol, M., Ruegg, P.L., 2007. Treatment practices and quantification of microbial drug usage in conventional and organic dairy farms in Wisconsin. J. Dairy Sci. 90, 249e261. Politis, I., 2012. Reevaluation of vitamin E supplementation of dairy cows: bioavailability, animal health and milk quality. Animal 6, 1427e1434. Publication number: 20040187340. Rochfort, S., Parker, A.J., Dunshea, F.R., 2008. Plant bioactives for ruminant health and productivity. Phytochemistry 69 (2), 299e322. Rossi, G., Sciavon, S., Lomolino, G., Cipolat-Gotet, C., Simonetto, A., Bittante, G., Tagliapietra, F., 2018. Garlic (Allium sativum L.) fed to dairy cows does not modify the cheese-making properties of milk but affects the color, texture, and flavor of ripened cheese. J. Dairy Sci. 101, 2005e2015. Ruegg, P.L., 2009. Management of mastitis on organic and conventional dairy farms. J. Anim. Sci. 87, 4582e4591. Santra, A., Karim, A., 2003. Rumen manipulation to improve animal productivity. J. Anim. Sci. 16, 748e763. Scaletti, R.W., Trammell, D.S., Smith, B.A., Harmon, R.J., 2003. Role of dietary copper in enhancing resistance to Escherichia coli mastitis. J. Dairy Sci. 86, 1240e1249. Schären, M., Kersten, S., Meyer, U., Hummel, J., Breves, G., Dänicke, S., 2018. Interrelations between the rumen microbiota and production, behavioral, rumen fermentation, metabolic, and immunological attributes of dairy cows. J. Dairy Sci. 101, 4615e4637. Schukken, Y.H., Wilson, D.J., Welcome, F., Garrison-Tikofsky, L., Gonzalez, R.N., 2003. Monitoring udder health and milk quality using somatic cell counts. Vet. Res. 34, 579e596. Seymour, E.M., Bennink, M.R., Bolling, S.F., 2013. Diet-relevant phytochemical intake affects the cardiac AhR and nfr2 transcriptome and reduces heart failure in hypertensive rats. J. Nutr. Biochem. 24, 1580e1586. Simitzis, P.E., Deligeorgis, S.G., 2011. The effects of natural antioxidants dietary supplementation on the properties of farm animal products. In: Animal Feed: Types, Nutrition, Safety. Nova Science Publishers, New York, USA, pp. 155e168. Simitzis, P.E., 2017. Enrichment of animal diets with essential oilsda great perspective on improving animal performance and quality characteristics of the derived products. Medicines 4, 35. Spanghero, M., Robinson, P.H., Zanfi, C., Fabbro, E., 2009. Effect of increasing doses of a microencapsulated blend of essential oils on performance of lactating primiparous dairy cows. Anim. Feed Sci. Technol. 153, 153e157. Stelwagen, K., Wall, E.H., Bravo, D.M., 2016. Effect of rumen-protected capsicum on milk production in early lactating cows in a pasture-based system. J. Dairy Sci. 99(E-Suppl. 1), 664. Tager, L.R., Krause, K.M., 2011. Effects of essential oils on rumen fermentation, milk production, and feeding behaviour in lactating dairy cows. J. Dairy Sci. 94, 2455e2464. Tajima, K., Arai, S., Ogata, K., Nagamine, T., Matsui, H., Nakamura, M., Aminov, R.I., Yashimi, B., 2000. Rumen bacterial community transition during adaptation to high-grain diet. Anaerobe 6, 273e284. Tekippe, J.A., Tacoma, R., Hristov, A.N., Lee, C., Oh, J., Heyler, K.S., Cassidy, T.W., Varga, G.A., Bravo, D., 2013. Effects of essential oils on ruminal fermentation and lactation performance of dairy cows. J. Dairy Sci. 96, 7892e7903. Torrent, J., Johnson, D.E., 1994. Methane production in the large intestine of sheep. In: Aquilera, J.F. (Ed.), Energy Metabolism of Farm Animals. CSIC Publishing Services, Granada, Spain, pp. 391e394. EAAP Publication No. 76. US Food and Drug Administration, 1994. Animal Medicinal Drug Use Clarification Act of 1994. http://www.fda. gov/AnimalVeteri nary/GuidanceComplianceEnforcement/ActsRulesRegulations/ucm085377. Varel, V.H., 2002. Livestock manure odor abatement with plant-derived oils and nitrogen conservation with urease inhibitors: a review. J. Anim. Sci. 80, E1eE7. Voets, T., Talavera, K., Owsianik, G., Nilius, B., 2005. Sensing with TRP channels. Nat. Chem. Biol. 1, 85e89. Vriens, J., Nilius, B., Vennekens, R., 2008. Herbal compounds and toxin modulating TRP channels. Curr. Neuropharmacol. 6, 79e96. Weimer, P.J., 2015. Redundancy, resilience and host specificity of the ruminal microbiota: implications for engineering improved ruminal fermentations. Front. Microbiol. 6, 296. Windisch, W., Rohrer, E., Schedle, K., 2009. Phytogenic feed additives to young piglets and poultry: mechanisms and application. In: Steiner, T. (Ed.), Phytogenics in Animal Nutrition: Natural Concepts to Optimize Gut Health and Performance. Nottingham University Press, Nottingham, UK, pp. 19e38.

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C H A P T E R

16 The effects of aromatic plants and their extracts in food products Bojana Filipcev University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia O U T L I N E Introduction

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Application of aromatic herbs in food Effects in food Application in different food systems Cereal grains Bread and bakery products Biscuits

280 280 281 281 282 285

Products of animal origin (meat, fish, dairy products) 285 Fruits and vegetables 289 Use of EOs in active packaging systems 289 Conclusion

290

References

291

Introduction In the modern food industry, great interest persists for natural bioactive compounds due to growing consumer demands for functional food with evidenced health benefits and use of natural additives in processed food products. Many plant-occurring phytochemicals can be considered adequate for these purposes. There is a paucity of literature on investigations of culinary herbs and spices and their role as contributors of bioactive compounds with prohealth effects as well as their food protective properties (Opara and Chohan, 2014; Embuscado, 2015; Carvalho Costa et al., 2015; Shahidi and Ambigaipalan, 2015; Tapsell et al., 2006). Along with culinary herbs and spices, medicinal plants have also been proposed as promising sources of natural antimicrobial and antioxidant agents for the food industry

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00016-9

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Copyright © 2020 Elsevier Inc. All rights reserved.

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16. The effects of aromatic plants and their extracts in food products

(Ortega-Ramirez et al., 2014). The medicinal and food protective effects of aromatic plants are based on the action of their biologically active compounds. These compounds are constituents of essential oils and extracts of herbs and spices, and provide strong antioxidant and antimicrobial activities.

Application of aromatic herbs in food Effects in food Inclusion of aromatic plants and their derivatives into various food systems received considerable popularity despite all uncertainties that exist regarding their true potential and value-added effect and has become an extensively studied topic. Fig. 16.1 visualizes the most important value-added effects provided by inclusion of aromatic herbs into food formulations. The food industry may benefit mostly from the antioxidative and antimicrobial properties. Aromatic herbs and their derivatives are potential natural alternatives to synthetic antioxidants and preservatives. As being natural, they are especially interesting for use in minimally processed food, organic food, and functional food, i.e., by food producers that opt for “clear labels.” They can be added to food directly (in fresh or dried state) or its constituents may be extracted and added to food in the form of essential oils (EOs) or extracts. Direct addition of EOs to food is limited by the intense aroma (Hyldgaard et al., 2012). Various other strategies have been employed to overcome this problem, such as their encapsulation in edible and biodegradable polymers as parts of active packaging systems or their encapsulation into nanoemulsions in order to prevent interaction with food matrices (Hyldgaard et al., 2012).

FIGURE 16.1 The roles aromatic plants may provide in food systems, the associated active ingredients, and the presumed mechanisms of action.

Application of aromatic herbs in food

281

As antioxidants, aromatic herbs and EOs were found to exhibit an ability to prevent, minimize, or slow down the rate of lipid oxidation in various food systems (fats, oils, meat, biscuits) during storage under ambient conditions or refrigerated state (Embuscado, 2015; Jiang and Xiong, 2016; Hygreeva et al., 2014). As part of their basic action (oxidation retarding), natural antioxidants from herbs exerted additional ability to alleviate the formation of various cytotoxic compounds during thermal processing of food, for example, meat (Jiang and Xiong, 2016) and biscuits (Navarro and Morales, 2017). EOs obtained from aromatic and medicinal plants are considered the best alternatives to synthetic preservatives due to strong antimicrobial activities (Jayasena and Jo, 2013). As antimicrobials, EOs are prioritized over crude herbal material due to better storage stability, higher concentration of active substances, easier manipulation, no seasonal variation, and standardization (Tipsrisukond et al., 1998). Being complex mixtures of components, EOs exert multiple antimicrobial properties. In food preservation strategies, EOs are often applied as a part of a hurdle technology in which several preservation factors are combined to provide microbial stability of the product (Calo et al., 2015). Many EOs show synergistic antimicrobial activity when used in combination. It was shown that in food systems, higher dose of EOs is needed for effective antimicrobial action than that observed in in vitro assays (Calo et al., 2015; Bassolé and Juliani, 2012). The improvement of nutraceutic function of food is also an expected beneficial effect of addition of aromatic herbs to food and is closely associated to the known healthpromoting properties of aromatic herbs and their derivatives. But their real value and potential, in this sense, has not been fully confirmed as it depends on numerous other factors. An additional benefit of the food use of aromatic plants may be the reduced intake of salt (Carvalho Costa et al., 2015).

Application in different food systems Cereal grains There has been an increasing interest in testing the feasibility of EOs as storage insecticides due to growing concerns about human health, environmental persistence, and development of insect resistance related to continued application of current synthetic insecticides. Several plants and their derivatives exert pesticide properties and are traditionally used to protect the cereal grains against pest attack. Today, EOs are considered as promising alternatives to synthetic fumigants due to their antiinsect pest ability. The action of EOs on storage pests is variable (Ningombam et al., 2017) and spans from an acute toxicity, through a feeding inhibition and an impairment of the reproductive system, to a repellent action. In addition to storage pest toxicity, EOs have other desirable fumigant properties like high volatility and low mammalian toxicity (Shaaya et al., 1997). Due to high volatility, EOs can be easily removed by aeration. It is believed that insecticidal and repellent effects of EOs involve a neurotoxic mode of action (Kostyukovsky et al., 2002). Despite a large body of data documenting the bioactivity and insecticidal activity of botanical insecticides from numerous sources against storage pests, a small number of botanical

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16. The effects of aromatic plants and their extracts in food products

products are commercialized and used in practice. The reasons are numerous (Adarkwah et al., 2017): e natural or “bio insecticides” are less effective than synthetic insecticides due to slower action, absence of knock-down effect, and fast degradation when exposed to sunlight; e their use may be impractical if higher doses are required to achieve the antiinsect effect; e lack of standardization of natural products; e legislative difficulties related to registration and patenting of natural products; e higher price. Efficiency of herbal insecticides is usually investigated and confirmed under laboratory conditions. There is a lack of data on their action and effectiveness upon up-scaling under real field and storage conditions. Kłys et al. (2017) propose that further research work should focus on identifying and isolating the chemical compounds from plant materials, seeking for those with the maximal efficiency. They deem plant-derived repellents as important components in the integrated pest management system.

Bread and bakery products The importance of bread in a human diet is well known. As a commodity, mass-consumed on a daily basis, it has been recognized to have high potential to be a delivery vehicle for many deficient nutrients (Rosell, 2003). Improved nutrient profile and functionality in bread can be achieved by supplementing its formulation with new ingredients, rich in bioactive substances and antioxidants. Inclusion of aromatic herbs in bread formulation can contribute to improved mineral pattern and antioxidative activity (overviewed in Pestoric et al. (2017)). However, similarly to other food products, a compromise has to be made between the applied dose of herbal ingredient and sensory acceptability of the product (Pestoric et al., 2017). Considerably improved antioxidant activity of bread was reported upon supplementation of versatile spice and herbal ingredients: turmeric (powder, essential oil, and residue), coriander leaf (powder), green tea (powder), fennel seed (powder), ginger (powder), black tea (extract), mixture of green tea powder with oregano and tomato paste, etc. (Dziki et al., 2014). The applied doses varied between 0.5% and 8% (flour basis) and the majority of authors reported almost linear relation between bread antioxidativity and dose of herbal ingredient. The herbal ingredients usually contribute to increase in total phenolic compounds in the enriched bread. Green tea extract added to bread provided low-glycemic features such as significantly lower level of glucose released during simulated pancreatic digestion and rapidly digested starch (Goh et al., 2015). Besides antioxidativity and “pro-health” potential, supplementation of bread with herbal material affects other important attributes such as color, taste, odor, volume, and texture. The overall effect is very different and greatly varies from case to case, as it depends on the nature of applied herbs, supplementation level, interaction with other ingredients, etc. Reported changes in bread properties are summarized in Table 16.1. Dough properties are also affected by addition of herbal material, but the effects are not discussed here.

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Application of aromatic herbs in food

TABLE 16.1

Effect of aromatic herb addition on attributes of bread and bakery products.

Herbal ingredient

Applied dose a

Product attributes and observed effects

Reference

Couch grass, nettle, artichoke, kale, fenugreek, yellow tea extract

50 g/kg (f.b.) leaf or seed 10 g/kg (f.b.) extract

Triticale crisp bread (extruded) Texture n.e.b Color, variable (depending on a herb) Overall acceptance for couch grass, nettle, yel. tea bc Overall acceptance for artichoke, kale fenugreek ad

Makowska et al. (2017)

Black tea extract

50 mL infusion per 90 g flour Infusion: tea to water (v/v) proportion 0:5e5:0

Northern style Chinese steamed bread Crust darkness b Specific volume n.e. Crust and crumb hardness n.e. Sensory properties n.e.

Zhu et al. (2016)

Chameleon plant (Houttuynia cordata, Thunb)

0.1%e1.0% 0.1% 0.5%

Bread Antioxidant activity b Volume a Overall acceptability b

Park (2015)

Green tea and green coffee extracts

0.25%e1.0% (on weight of comp. substituting flour)

Fried fine yeasted-pastry (doughnuts) Antioxidant activity b Acrylamide formation a Hardness b Volume a Sensory properties n.e.

Budryn et al. (2013)

Green tea extract (95% total polyphenols)

0.5%e1.0% (f.b.)

Steamed bread Specific volume n.e.

Ananingsih et al. (2013)

Oregano

1%e4%

Bread Overall acceptability at 2% level n.e.

Dhillon et al. (2013)

Fennel seeds

3%e15% (f.b.) powder

Bread Antioxidant activity b Specific volume a Crust color (CIELab) a Crumb L* a a* b* b Crumb firmness b Sensory acceptability at 5%e7% Ue

Das et al. (2013)

Coriander leaf

1%e7% (f.b.) powder

White bread Specific volume a Crumb firmness b Crust color (CIELab) a Crumb L* a a* b* b Sensory acceptance at 3%e5% U

Das et al. (2012)

(Continued)

284 TABLE 16.1

16. The effects of aromatic plants and their extracts in food products

Effect of aromatic herb addition on attributes of bread and bakery products.dcont'd Product attributes and observed effects

Herbal ingredient

Applied dose

Ginger

0.0%, 3.0%, 4.5%, 6.0% Bread (f.b.) powder Antioxidant activity (TPC, RSA-DPPH) b Crumb firmness and gumminess b Crumb pore uniformity, geometry and fineness n.e. Color (L*, h0 b, C* Sensory acceptance at 3% U; at >3% a;

Balestra et al. (2011)

Korean turmeric

0%e8% (f.b.) powder

Bread Antioxidant activity and total phenolics b Specific volume a Crumb hardness b Crumb L* a a* b* b Aroma n.e. Taste a Texture n.e. Overall up to 4% U

Lim, SH, Ghafoor, Hwang and Park (2011)

“Vitalplant” herbal blend (leaves of mint and birch, fruits of parsley and caraway, alder buckthorn bark)

2% (f.b.) powder

Bread Specific volume b Crumb texture b Crumb elasticity, pore fineness b Taste U

 Simurina et al. (2008)

8% (f.b.) extract

Specific volume a Crumb texture a Crumb elasticity, pore fineness a Taste U

1%e2% (f.b.) powder

Bread Specific volume b Crumb elasticity, pore fineness, crust color b Overall sensory acceptability b

“Probavit” herbal blend (gentian root, wormwood, centaury, fruits of caraway, fennel, anise, leaves of mint and balm, chamomile flower, angelica root)

“Anemit” herbal blend Powder, applied levels Bread (leaves of nettle and not reported Specific volume a ironwort, fruits of Crumb elasticity, pore fineness, crust color a rosehip) Aroma, melting-in-the-mouth b f.b. flour basis. n.e. no-effect. c b increase in attribute intensity. d a decrease in attribute intensity. e U acceptable quality. a

b

Reference

Brkic et al. (2001)

Brkic et al. (2001)

Application of aromatic herbs in food

285

Biscuits Unlike bakery products, biscuits seem to be more convenient for supplementation with nontraditional ingredients since the development of biscuits’ structure and texture has been less compromised by addition of novel ingredients. The major limitation expected is related to the impact of aromatic herbs on sensory properties and acceptance. Similarly to other food commodities, fortification of biscuits is supposed to enhance products’ functionality (by increasing antioxidative activity and improving nutritional profile) and to increase shelf-life (by preventing or retarding oxidative rancidity). Table 16.2 lists the reported findings on the effects of incorporation of various aromatic herbs into biscuit formulation. One of the most important benefits of inclusion of herbs into biscuit formulation would be the delay of the onset of rancidity and replacement of use of synthetic antioxidants. Some promising results are reported in Table 16.2 for biscuit matrix. Izzreen and Noriham (2011) studied the effect of some Malaysian herbal aqueous extracts on the shelf-life of cakes and found that only the extract of tenggek burung (Melicope ptelefolia) had antioxidative activity comparable to synthetic preservatives BHA/BHT but it was not superior to them. In soda crackers, the addition of fine powders of marjoram and spearmint at 0.5% and 1% level produced an antioxidant effect (Bassiouny and Hassanien, 1990). Plant extracts of amla (Emblica officinalis), drumstick leaves (Moringa oleifera), and raisins (Vitis vinifera) provided more efficient control of lipid oxidation in biscuits during 6 weeks of storage in comparison to BHA. Ingredients rich in phenolic compounds may be a promising strategy to alleviate the formation of advanced lipoxidation endproducts (ALEs) based on the results of the study on the antiglycative capacity of olive leaf extract in wheat-flour biscuit model (Navarro and Morales, 2017). De Oliviera et al. (2009) recommended medicinal herbs guarana (Paullinea cupana) and catuaba (Anemopaegma mirandum) as good sources of fibers and minerals (copper, iron and zinc) in gluten-free biscuits with a relatively satisfactory potential of consumers’ preference.

Products of animal origin (meat, fish, dairy products) In meat and processed meat products, herbal extracts may be used to extend their shelf-life by preventing oxidative rancidity and microbial spoilage as well as by reducing the incidence of pathogenic microorganisms. Meat and meat products are highly susceptible to fat and protein oxidation due to the high ratio of polyunsaturated fatty acids, deficiency of endogenous antioxidants, high levels of prooxidants such as heme species, high salt concentrations, etc. (Kanner et al., 1988). The oxidative changes in meat are highly undesirable because they impair product quality and have negative implication on human health. The problem of oxidative decay in meat is conventionally solved by use of synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG) which are scrutinized due to potentially harmful side effects related to toxicity. The use of aromatic herbs as potential sources of natural antioxidants has been extensively investigated and results reviewed (Jiang and Xiong, 2016; Falowo et al., 2014; Shah et al., 2014; Hygreeva et al., 2014; Karre et al., 2013). Karre et al. (2013) concluded that pine bark extract, rosemary, oregano, and other spices are capable of performing the role of

TABLE 16.2 Effect of aromatic herb addition on biscuit attributes. Herbal ingredient

Biscuit functionality

References

Hardness a Fracturability n.e. (extracts except at 2%) a (powders) Color (a*, b*) bb Note: effects more expressed with extracts for hardness and color

Antioxidant activity (DPPH) b Lipid oxidation (malonaldehyde formation) a Note: effects more expressed with powdered herbs

Misan (2010); Pestoric et al. (2014)

Extracts of carrot, 1% (w/w) grape leaf and turmeric

Sensory properties a but Uc Spread factor b (except carrot) Texture a

Oxidative stability b Hefnawy et al. (2016) Note: better performance in comparison to synthetic antioxidants (BHA, TBHQ)

Green and yellow tea leaves (Camellia sinensis)

Sensory properties U (taste, crispness, overall acceptance a, after taste b for green tea)

Antioxidant activity (DPPH, ABTS, ORACFL, PCL) b Hemicellulose, insoluble fiber, proteins b Total phenolics b Lipid oxidation a Note: better performance for yellow tea

Gramza-Michałowska et al. (2016)

Appearance, color a Crispiness, overall acceptability b Appearance, color, taste, aroma, overall acceptability a

-

Eagappan et al. (2015)

“Vitalplant” herbal blend (leaves of mint and birch, fruits of parsley and caraway, alder buckthorn bark), powder and extract

Applied dose 2%,4%,6% powder or extract

5.5% powdered

Tribulus terrestris 10% (L) fruit extract powder 20%

Biscuit quality attributes a

Holly basil (Ocimum sanctum) and moringa leaves

Powder blend (1:1 w/w) at 4%, 8%, 12% (flour replacement level)

Appearance, color, texture, acceptance a Note:at lowest applied dose, product was acceptable.

Crude fiber b Ash b Carbohydrate a

Ariful Alam, Jahangir Alam, Abdul Hakim, Obidul Huq, & Golam Moktadir (2014)

Mixture of extracts (liquorice (Glycyrrhiza glabra) root, holly basil (Ocimum sanctum), anise (Pimpinella anisum))

Extract 1 (0.75% liquorice, 0.175% basil, 0.3% anise (f.b.d)) Extract 2 (0.37% liquorice, 0.25% anise, 0.25% basil (f.b.)

Sensory properties of wheat and wheat/barley biscuits U

-

Rattan et al. (2014)

Ayurvedic herbs (Shatavari, Ashwagandha, Yastimadhu)

Each herb 3% (f.b.), powder

Sensory properties U

-

Mehta (2013)

Guduchi (Tinospora cordiofolia)

Dry leaves powder (2.5%, 5.0%, 7.5% f.b.)

Spread ratio a Breaking strength b Dough water absorption b Dough stability a Dough hardness b Dough cohesiveness, springiness a Sensory properties at 5% U

Proteins b Dietary fibers b Fe b Ca b Antioxidant activity b b-carotene b

Sharma et al. (2013)

Mint (Mentha spicta L)

Powder (1%) Extract (500 mg) Pure menthol (100 ppm)

Acceptability for mint powderU Note: Powdered mint provided best texture properties, taste and mouthfeel.

-

Shivani et al. (2006)

b increase in attribute intensity. a decrease in attribute intensity. c U acceptable quality. d f.b. flour basis. b a

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antioxidants in meat and poultry. Similar findings were recapitulated by Shah et al. (2014); green tea, pine bark, rosemary, nettle, and cinnamon were found to have similar or superior antioxidant properties in comparison to some conventional antioxidants in meat processing. Hygreeva et al. (2014) summarized findings which confirmed more effective inhibition of lipid oxidation in cooked, fermented and irradiated meat products by herbal extracts. Results suggest that combination of different herbal extracts may exhibit better antioxidative activity due to synergistic action of compounds. Fallowo et al. (2014) emphasized the multifunctional role of natural antioxidants: natural antioxidants such as EOs and organic acids, simultaneously perform antimicrobial and preservative roles and lower the microbial spoilage of meat during processing and storage. Moreover, application of natural antioxidants in meat processing stabilizes cholesterol levels and reduces the incidence of harmful substances such cholesterol oxidation products, malondialdehyde and heterocyclic amines which forms during thermal treatment of meat (Falowo et al., 2014; Jiang and Xiong, 2016). Meat and meat products are highly perishable and susceptible to microbial spoilage due to high content of moisture and essential nutrients (Jayasena and Jo, 2013). In addition, meat and meat products have been reported as frequent vehicles of pathogenic bacteria such as Salmonella spp., Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Clostridium spp., Aeromonas, etc., which can result in outbreaks of foodborne diseases with serious consequences. Antimicrobial preservatives in meat are used to increase long-term stability and to increase safety by controlling pathogens (Hygreeva et al., 2014). Aromatic herbs and derivatives have enormous potential for use as natural preservatives in meat processing. Scientific literature provides plentiful examples on good antimicrobial efficacy of EOs of oregano, parsley, rosemary, clove, lemongrass, sage, and vanillin in various meat products (Hygreeva et al., 2014). Burt (2004) compared the antimicrobial activity of different EOs and found that it follows the order: oregano > clove > coriander > cinnamon > thyme > mint > rosemary > mustard > cilantro/sage. However, results are not straightforward as some studies report on low antimicrobial effects against pathogens in contaminated meat products (Grosso et al., 2008) while some studies recorded different antimicrobial efficacy of natural preservatives in different meat products. It was inferred that the activity of plant-based antimicrobials may be impaired by the high-fat content of meat products (Tajkarimi et al., 2010). Moreover, differences exist between the EOs activity in vitro and in food. The major limitations of the use of natural antimicrobials include several important factors: variable efficacy in food, applicability of low doses due to strong odor and high costs (Tajkarimi et al., 2010). Therefore, it has been suggested to combine them with already existing preservation technologies to ensure the required level of preservation action (Jayasena and Jo, 2013). The literature demonstrates successful application of EOs in prolonging the shelf-life of fish, alone or in combination with other preservation techniques (Alex and Kannan Eagappan, 2017; Lucera et al., 2012). EOs were found to efficiently extend the shelf-life and quality parameters of fish meat: thyme oil in refrigerated Nile tilapia fillets (Khalafalla et al., 2015); tea polyphenols and rosemary extracts in refrigerated air-packed crucian carp (Tingting et al., 2012); and icing from thyme, oregano, and clove in chilled anchovies (Bensid et al., 2014) are only some of the relevant examples. Dairy products are mainly susceptible to hydrolytic and oxidative rancidity and may suffer deterioration due to exposure to heat, light, oxygen, metal ions, enzymes, etc. Recent works confirmed that EOs can be efficient in preserving the microbial stability of various

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products (whey, cheese, yoghurt, milk) by reducing or elimination pathogenic bacteria (Teplá et al., 2016; Lucera et al., 2012). Moreover, EOs can serve as antioxidants in dairy products (Alenisan et al., 2017) and are especially valuable as natural preservatives in processing of organic dairy products (Yangilar&Yildiz, 2018; Asensio et al., 2015).

Fruits and vegetables Fruits and vegetables are perishable foods which undergo quality decay during storage and processing. Quality decay mainly involves dehydration, color changes, softening, surface pitting, microbial spoilage, chilling injuries during cold storage, etc. (Serrano et al., 2008). EOs of different plants have strong antifungal properties capable of suppressing fungal growth in fresh produce. Efficiency of various EOs (thyme oil, cinnamon oil, lemon oil, lemongrass oil, oregano oil, clove extract) against common spoilage fungi was confirmed in in vitro tests on many commercial fruits like mandarins, banana, strawberries, apricots, table grapes, orange, avocado, etc. (Sivakumar and Bautista-Ba nos, 2014). This allows the use of EOs by spraying or dipping to control postharvest decay in many fruits and vegetables (cherries, citruses, apples, peaches and cabbages) (Sivakumar and Bautista-Ba nos, 2014; Lucera et al., 2012). Moreover, EOs can be used as ingredients in biopesticides to fumigate fruits on mothertrees. Treatments with EOs have been more efficient when more EOs were combined (Sivakumar and Bautista-Ba nos, 2014) or when naturally resistant fruit cultivars were used. According to (Ayala-Zavala, Gonzáles-Aguilar, & Del-Toro-Sánchez, 2009), combined EOs have high potential to prevent decay of fresh-fruit cuts. These EO combinations may be commercially viable if well-optimized in terms of aroma notes and antimicrobial activity and would provide extra aroma to a fresh product, in addition to microbial safety. Herbal extracts can be used to control microbial enzymatic spoilage of unpasteurized fruit juices (Damjanovic-Vratnica, 2016). In vegetables, application of EOs in washing water showed promising results. The action of EOs may be augmented by chilling or decreasing pH of food (Tajkarimi et al., 2010). Some EOs demonstrated inhibitory effects against food-borne pathogens in different media (fresh lettuce, tomato, vegetable dishes) (Damjanovic-Vratnica, 2016). Due to GRAS status of EOs, they can be considered as safe treatments in food. Thymol and carvacrol were recommended by WHO for the control of postharvest fruit decay (Sivakumar and Bautista-Ba nos, 2014). Since 2013, thymol has been approved as natural pesticide by EU (Reg EU 568, 2013). However, organoleptic impact remains the major issue which may limit the applicability of EOs in some cases. In practice, the most viable solutions involve the use of EOs as a part of hurdle technology or in modified-atmosphere packaging (MAP) systems (Tajkarimi et al., 2010). Packaging of minimally processed fruits and vegetables in modified atmosphere is frequently applied in the marketplace. In such systems, EOs can be used as secondary preservatives to prevent the growth of psychrotolerant pathogens and spoilage bacteria (Holley and Patel, 2005).

Use of EOs in active packaging systems In addition to direct application in food, EOs can be indirectly used to extend product shelf-life by incorporating them into active food packaging (Ribeiro-Santos et al., 2017a).

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The preservation of foods occurs by diffusion of active compounds from EOs to the food (Ribeiro-Santos et al., 2017a). Active packaging materials are used in the form of films and coatings (Krochta, 2002). EOs can be contained in separate containers (sachets) or integrated into the packaging material (Ribeiro-Santos et al., 2017b). Depending on the composition of EOs and their interaction with the polymer matrix, EOs-incorporated films and coatings can yield antimicrobial and antioxidative properties (Atarés and Chiralt, 2016). Incorporation of EOs into carrier matrix may promote the antimicrobial capability of EOs in comparison to the capability of EOs directly applied on food (Atarés and Chiralt, 2016). Antioxidative capacity of EOs incorporated films depends on the intrinsic activity of EOs and film permeability to oxygen (Atarés and Chiralt, 2016). EOs incorporated into films and coatings have been applied in several foods (meat, fish, dairy, bakery, vegetables) and successfully increased the shelf-life and preserved the eating quality. Type of films, EOs, food and associated effects are listed in detail in Ribeiro-Santos et al. (2017b). Active films and coatings with built-in EOs demonstrate high efficiency in conjunction with modified atmosphere packaging, good hygiene and manufacturing practices (Hyun et al., 2015; Kykkidou et al., 2009). The side-effect of incorporation of EOs into the polymer network is the induction of microstructural changes in films and coatings. The microstructural changes greatly influence the mechanical, barrier and optical properties of matrices (causing typically some weakening, reduced water permeability and increased opaqueness) which limit their use (Atarés and Chiralt, 2016). The changes are variable and hard to predict because they depend on the specific interactions between the oil components and the polymer matrix which are associated with the nature of EOs, applied concentration and homogenization technique (Atarés and Chiralt, 2016). To avoid the effect of undesirable alterations in the properties of packaging films, a possible alternative is the use of encapsulation to increase the stability and efficiency of EOs in packaging materials (Ribeiro-Santos et al., 2017a). Encapsulated EOs can be integrated within different matrices to form an active food packaging (Ribeiro-Santos et al., 2017a). Ribeiro-Santos et al. (2017a) compiled the main conclusions of several studies on the antimicrobial efficiency of micro- and nanoencapsulated EOs. The majority of studies revealed improved performance of encapsulated EOs against the growth of tested microorganisms but more research is necessary to optimize the effectiveness of EOs in the active packaging materials. Moreover, the use of nanotechnology in food systems requires further extensive research addressing the issues of potential toxicity to humans.

Conclusion Undoubtedly, aromatic herbs and their derivatives have great potential for use in food and food packaging due to their outstanding antimicrobial and antioxidant properties. In addition, a number of aromatic herbs, extracts, oils, and oil compounds have a GRAS status and their application can be considered as safe treatments, not involving any regulatory related issues. As such, aromatic herbs and their derivatives may be natural solutions to replace synthetic additives and increase the shelf-life of products. The application of natural antioxidants and microbial preservatives in foods adds extra value and supports the commercial viability of the product. Furthermore, due to presence of numerous bioactive substances,

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the added health and nutritional benefit is an additional advantage of supplementing food with aromatic and medicinal herbs. Issues to be considered when applying aromatic herbs in food systems are related to the impacts on sensory properties and consumer perception as well as to the optimization of their effects in food. At this moment, direct replacement of synthetic additives with natural ones based on aromatic herbs is not rational because of lack of understanding of the mechanisms of their actions, interactions with other food components, susceptibility of microorganisms, etc. The most applicable scenario is the use of aromatic herbs as a part of a multihurdle system to preserve food stability.

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17 The effects of plant extracts on the immune system of livestock: the isoquinoline alkaloids model Valeria Artuso-Ponte, Anja Pastor, Manfred Andratsch Phytobiotics Futterzusatzstoffe GmbH Eltville, Germany O U T L I N E AGP removal and gut health

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The use of antimicrobials and the risk of antimicrobial resistance 296 The public health concern

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The role of the mucosal immune system 297 The role of NF-kB

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Causes and consequences of intestinal inflammation

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Plant metabolites with antiinflammatory properties 300 Nonnitrogen secondary metabolites

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Isoquinoline alkaloids

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Mode of action of isoquinoline alkaloids: inhibition of NF-kB activation

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Consequences of reducing inflammation with isoquinoline alkaloids 304 Isoquinoline alkaloids, and gut health and stress

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Regulating intestinal inflammation and the effects on animal performance

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Public health concerns about the use of antimicrobials as growth promoters (AGP) in food animals and the development of resistant bacteria have led to the ban of AGP in the European Union and to a more judicious use in many other countries. Removing AGP from the feed of

Feed Additives https://doi.org/10.1016/B978-0-12-814700-9.00017-0

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production animals has reduced the occurrence of antimicrobial resistance, but it has increased the incidence of enteric diseases, negatively affecting animal welfare and health. This chapter is aimed to define the role of intestinal inflammation in food producing animals and to describe plant extracts and other compounds with well-known antiinflammatory effect and their benefits on gut health and growth performance.

AGP removal and gut health Several reports have indicated that removing AGP from the feed significantly decreased the prevalence of antimicrobial resistance without affecting production parameters (Emborg et al., 2001). However, other studies have shown that morbidity and mortality significantly increased after AGP were removed, thus increasing the use of antimicrobials for treatment and prevention and increasing the production cost (McEwen and Fedorka-Cray, 2002). Additionally, it has been reported that intestinal health and welfare were negatively affected when subtherapeutic antimicrobials were removed. For example, enteric infections due to E. coli and Lawsonia intracellularis have been more prevalent after removing AGP, increasing dramatically the need for therapeutic antimicrobials and negatively affecting the health and the production performance of the animals. The removal of AGP has also increased the number of bacteria that potentially can be transmitted to humans, such as Salmonella spp. and Campylobacter spp. (Hao et al., 2014). It is known that a “healthy gut” is essential to good animal performance. However, there is not a clear definition of what is gut health. The term “gut health” involves physiological, physical, and immune components that interact with each other and ensure that the animal can perform at its maximum genetic capacity and cope with internal and external stressors (Kogut and Arsenault, 2016). “Gut health” is not a simple definition, instead it is a concept that involves the interaction between the microbiome, the intestinal barrier and the nutrients present in the feed, which prevent the occurrence of diseases and allow the appropriate digestion and absorption of nutrients that will be used by the animal to grow and perform at its genetic capacity.

The use of antimicrobials and the risk of antimicrobial resistance Antimicrobials have been widely used in food animals for decades to promote growth and improve feed efficiency, as well as to reduce morbidity and mortality rates (Kim et al., 2012). The exact mechanism of action of subtherapeutic antimicrobials to improve growth is not completely understood (Kim et al., 2012). Potential mechanisms include: (1) Reduction of bacteria that compete with the host animal for nutrients; (2) Enhancement of the immune system; and (3) Modulation of metabolic activity or shifting the balance to a more beneficial microbial population (Labro, 1998; McEwen and Fedorka-Cray, 2002; Kim et al., 2012; Looft et al., 2012). The extensive use of antimicrobials, especially at subtherapeutic levels, has been associated with the development of resistant bacteria (Phillips, 1998; Callaway et al., 2004; Doyle and Erickson, 2006). When antimicrobials are used, resistant bacteria can survive and multiply

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and eventually, they will maintain, accumulate and spread their resistance genes (Mathew et al., 2007). Antimicrobial resistance can be developed by any bacteria, including pathogens and commensal flora. Antimicrobial resistance is a complex process in which resistant bacteria can be transmitted between animals and humans through the consumption of contaminated food (Mathew et al., 2007), direct contact and through contact with a contaminated environment, animal waste, vectors and carriers (Box et al., 2005). Furthermore, antimicrobial resistance can be transmitted from commensal bacteria to zoonotic enteropathogens and vice versa (McEwen and Fedorka-Cray, 2002).

The public health concern Numerous countries have established surveillance programs to improve the early detection of antimicrobial resistance, as well as to preserve the power and lifespan of antimicrobials and to provide guidelines for development and use of new antimicrobials (Mathew et al., 2007). For example, in the United States the National Antimicrobial Resistance Monitoring System (NARMS) was created in 1996 to identify new antimicrobial-resistant bacteria and to provide reliable information about antimicrobial resistance to veterinarians and physicians. In production animals, antimicrobial resistance has been mainly related to the use of nontherapeutic antimicrobials used as growth promoters (Looft et al., 2012; Robbins et al., 2013). The use of AGP in food animals have also been associated with the development of antimicrobial resistance in humans and the increase in human illnesses due to resistant foodborne pathogens (Anderson et al., 2003). Several organizations have recommended discontinuing the use of AGP that are of the same classes of antimicrobials used in human medicine (Anderson et al., 2003). Since 1998, the European Union has banned the use of tylosin, spiramycin, bacitracin, and virginiamycin as growth promoters because of their similar structure to antimicrobials used in humans. Furthermore, the European Union has banned the use of all AGP since 2006 (European Commission, 2005). The potential negative consequences of the removal of AGP on gut health and growth performance have increased the interest for finding alternatives to maintain good animal performance, reduce the prevalence of bacterial infections and improve the quality of food products by promoting gut health (Windisch et al., 2008; Yakhkeshi et al., 2011; Morgan, 2017). In this chapter, we will focus on alternatives that influence the immune system, particularly those reducing intestinal inflammation.

The role of the mucosal immune system The intestine is the largest organ of the body in direct contact with food antigens, pathogens, toxins and commensal bacteria. The epithelium is the first line of defense and is composed by four differentiated cell types: enterocytes, mucus-producing cells (i.e., Goblet cells), cells that produce antimicrobial peptides (i.e., Paneth cells) and hormone-producing cells. Underlying the epithelium, the lamina propria comprises the immune cells, including

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macrophages and dendritic cells, which are responsible for the innate immune response. Dendritic cells are capable of sampling antigens from the intestinal lumen through their specialized cytoplasmic extensions intercalated between enterocytes and presenting the antigens to T and B cells in the lamina propria and the Payer patches. This interaction represents the close association between the innate and the adaptive immune response in the gut (Santaolalla et al., 2011). Inflammation is defined as the “response of vascularized tissues to infections and tissue damage that brings cells and molecules of host defense from the circulation to the sites where they are needed to eliminate the offending agent” (Kumar et al., 2015). Inflammation is the first nonspecific innate immune response to infection and tissue damage. Innate immune cells can recognize the damage and pathogen invasion through specialized receptors, known as pattern recognition receptors (PRRs) located on the cell surface and inside the cells (Mogensen, 2009). As in other parts of the body, inflammation in the intestine starts with the activation of PRRs, such as toll-like receptors (TLRs) present in the intestinal epithelial cells and immune cells of the lamina propria. It is well-known that the transcription factor NF-kB plays a key role in initiating intestinal inflammation and promoting the production of cytokines, chemokines and the recruitment of acute inflammatory cells (Berkes et al., 2003). Once PRRs are triggered, a signaling cascade is initiated to promote the recruitment of leukocytes to the damaged/infected area. The signaling cascade involves the production and release of several cytokines, chemokines and stimulatory molecules, which play an important role on the vascular system to increase the blood flow and the permeability of the blood vessels and to ensure the adhesion and migration of inflammatory cells to the site of inflammation and the subsequent activation (Newton and Dixit, 2012). Mediators of the inflammatory response include prostaglandins, histamine, nitric oxide (NO) and cytokines such as tumor necrosis factor (TNF) and interleukin 1 (IL-1). Activated leukocytes including dendritic cells, macrophages and neutrophils remove the cause of the injury and repair the damaged tissue. Furthermore, these activated leukocytes produce proinflammatory and antiinflammatory cytokines to amplify and regulate the inflammatory response and mediate the adaptive immune response. Additionally, cytokines promote the production of acute-phase proteins (APP) that will affect the immune response systemically (Newton and Dixit, 2012).

The role of NF-kB A common signaling occurrence of the PRRs is the activation of the transcription factor NF-kB. It is well-known that NF-kB plays a crucial role in the inflammatory response as its activation induces the transcription of proinflammatory genes encoding proinflammatory cytokines and chemokines (Tak and Firestein, 2001). Normally, NF-kB stays inactive in the cytoplasm of most cells due to its sequestration by inhibitory proteins such as the IkB family of proteins, mainly IkBa. The canonical activation of NF-kB involves the stimulation of PRRs by microbial molecules and proinflammatory cytokines such as IL-1 and TNF (Newton and Dixit, 2012). The activation of these receptors promotes the degradation of the IkBa inhibitory

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protein through phosphorylation by the IkB kinase (IKK) complex, resulting in the translocation of NF-kB to the nucleus and the initiation of the transcription of proinflammatory genes (Liu et al., 2017). Consequently, the production of proinflammatory cytokines, chemokines, and adhesion molecules is increased. Moreover, the activation of the NF-kB transcription factor is involved in regulating cell proliferation, apoptosis and cell differentiation (Liu et al., 2017). The release of cytokines and chemokines is responsible for local changes (i.e., heat, redness, pain, and edema) as well as systemic changes. Systemic consequences of inflammation include the synthesis and release of acute-phase proteins (APP) in the liver, including C-reactive protein, serum amyloid A, and haptoglobin, the activation of the nitric oxide synthase and other enzymes involved in gluconeogenesis and glycogenolysis in the liver, as well as insulin resistance. Additionally, behavioral changes are observed including anorexia and lethargy. Neuroendocrine changes induced by inflammation promote fever and the release of corticotropin and cortisol (Gabay and Kushner, 1999).

Causes and consequences of intestinal inflammation Enteric pathogens are well-known causes of intestinal barrier dysfunction (Berkes et al., 2003). Bacteria and their toxins can alter the integrity of the intestinal epithelium directly, by affecting the morphology and structure of the tight junction proteins, or indirectly, by causing intestinal inflammation. Pathogens and their toxins are not the only cause of intestinal inflammation. Feed ingredients and nonnutritional components of the feed can also trigger the immune system and initiate the cascade of inflammation. The complex process of inflammation is tightly regulated to ensure that the cause of the injury is removed, and the tissue is repaired. However, if this regulation fails or if the cause of inflammation persists, it can have deleterious effects to the animal’s health and performance. In fact, the dysregulation of NF-kB has been associated with chronic inflammatory conditions in humans, such as ulcerative colitis and Crohn’s disease (Berkes et al., 2003; Nanthakumar et al., 2011). Mounting an immune response and even maintaining an alert, responsive, but controlled immune system is a nutritionally demanding process that requires the redistribution of nutrients (Lochmiller and Deerenberg, 2003). In other words, mounting an immune response, even if it is mild, requires nutrients that could have been used for other physiological processes. This model of nutrient allocation during an immune response assumes the negotiation between growth, reproduction, thermoregulation, and immunity (Sheldon and Verhulst, 1996). The initial acute-phase of the immune response is characterized by a state of hypermetabolism and protein malnutrition (Lochmiller and Deerenberg, 2003). Proinflammatory cytokines, including IL-1, TNF, and IL-6 can induce anorexia. Consequently, the body must use its own reserves to supply the immune system with the necessary nutrients and energy. In the skeletal muscle, proteins are degraded (proteolysis), and the synthesis of proteins is decreased to rise the availability of amino acids, which will be used in the liver to synthesize glucose as a source of energy, a process called gluconeogenesis. At the same time, glycogen

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and lipids are also degraded (i.e., glycolysis and lipolysis, respectively) to generate more energy for the immune system (Lochmiller and Deerenberg, 2003). If inflammation persists, the animal will lose weight and body condition. Mercier et al. (2002) has estimated that protein synthesis in the muscle decreases by 23% under chronic intestinal inflammation. Moreover, protein synthesis in the liver and the intestine increases by 63% and 19% respectively, due to the synthesis of acute-phase proteins and the replacement of damaged intestinal cells (Mercier et al., 2002). In addition, intestinal inflammation is accompanied by malabsorption, which restricts the availability of energy and nutrients even more. Malabsorption may be due to the consequent bacterial overgrowth and the structural changes of the intestinal epithelium due to inflammation. It is known that intestinal inflammation negatively affects the intestinal barrier function by altering the tight junctions, leading to the “leaky gut” syndrome. In addition, fluids and electrolytes are secreted to the lumen, inducing diarrhea and contributing to the state of malabsorption (Berkes et al., 2003). The exact cost of inflammation to performance is difficult to predict. However, it has been estimated that a mild immune response in pigs reduces body weight gain and feed intake by 21% and 15% respectively (Spurlock et al., 1997). In chickens, body weight gain was reduced by 13%e18% (Klasing et al., 1987). Chronic inflammation negatively affects animal performance. Therefore, herbs and plant extracts which exert local antiinflammatory properties are of great interest to the animal industry. Regulating excessive intestinal inflammation has the potential to reduce the cost of the immune response, increase the availability of nutrients for growth and production, and decrease the occurrence of enteric diseases by maintaining a healthier intestinal barrier function.

Plant metabolites with antiinflammatory properties Plant extracts are substances or compounds that can be removed from the tissue of a plant, usually by the treatment with a solvent. One of the purposes is their use as a feed additive and individual desirable characteristic is the antiinflammatory property produced by secondary metabolites (SMs). Regarding their molecular structure, SMs can be classified in two distinct groups: with or without nitrogen. On one hand, there are alkaloids, nonprotein amino acids, glucosides, glucosinolates, amines, lectins, and peptides, on the other are terpenes, phenols, polyacetylens, carbohydrates, and organic acids (Wink, 2015). Secondary plant metabolites do not support the growth and the development of the plants. They play a significant role in the defense system and are required by the plant to survive in its environment. They pose numerous characteristics, such as antimicrobial, antioxidative, antifungal, antiviral, anticancerogenic, and antiinflammatory (Osbourn and Lanzotti, 2009).

Nonnitrogen secondary metabolites Polyphenols are a structural group of mainly natural, organic chemicals characterized by the presence of large multiples of phenol structural units. They play important roles in the distribution and abundance of plants, the effects of environmental factors upon the

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abundance of plants, and the interactions among and between plants and other organisms (Osbourn and Lanzotti, 2009). Flavonoids, the biggest class of polyphenols, are characterized by two aromatic rings that carry several phenolic hydroxyl or methoxyl groups. In addition, they often occur as glycosides and are stored in vacuoles (Kabera et al., 2014). Flavonoids can be divided into six subclasses: flavanols (e.g., catechin, gallocatechin), flavanones (e.g., hesperidine, naringenine), flavones (e.g., luteoline, apigenine), flavonols (e.g., quercetin, kaempferol), isoflavones (e.g., genistein, daidzein) and anthocyanidins (e.g., cyanidin, malvidin). For clarification, the term “tannin” by extension is widely applied to any large polyphenolic compound containing enough hydroxyls and other suitable groups (such as carboxyls) to form strong complexes with various macromolecules. The concentration of flavonoids in food and feed differs in quality and quantity particularly regarding the type and used part of plant, as well as cultivation, harvesting and processing conditions. Bioavailability is a pharmacological value describing the amount of a substance which reaches unchanged the systemic metabolism to be then available at potential target organs or tissues (Aktories et al., 2013). The bioavailability of a substance is primarily affected by absorption, distribution, metabolization and elimination and differs enormously between flavonoids. For example, the flavonoid aglyca (nonglycosylated molecule) is better absorbed in monogastrics than in ruminants (Beyer, 2015). As most of the plant flavonoids (with exception of flavanols) are glycosylated, the molecules need to be hydrolysated by enzymes before they become available. The systemic availability of flavonoids also depends on the absorption in the gut epithelium, the mucous and serous secretion intensity of their conjugates, and the secretion via the bile. Polyphenols are degraded and excreted as fast as possible. In ruminants, the availability of flavonoids is different in comparison to monogastric species due to the comprehensive degradation and modulation by the ruminal microbiota. The antiinflammatory effect of the flavonol quercetin has been investigated quite well in in vitro studies. Quercetin scavenges reactive oxygen and nitrogen species, targets prominent proinflammatory signaling pathways, such as NF-kB and MAPK (Fürst and Zündorf, 2014) and has an inhibiting effect on signaling pathway enzymes. Epigallocatechin-gallate (EGCG), the most prominent member of the family of green tea catechins, makes up the biggest amount in green tea product available on the feed and food market due to its molecule size. Antiinflammatory, antioxidant, antiinfective, and anticancer effects are well described. EGCG induces apoptosis by affecting regulatory proteins of the cell cycle and inhibition of NF-kB, inhibits growth factor-depending signaling, proteasome-dependent degradation, MAPK-pathway, and the expression of COX-2 (Fürst and Zündorf, 2014). Another group of substances with antiinflammatory properties are essential oils (EOs). EOs have a complex composition, containing from a dozen to several hundred components. Most components identified in essential oils includes terpenes (oxygenated or not), with monoterpenes and sesquiterpenes prevailing. Nevertheless, allyl- and propenylphenols (phenylpropanoids) are also important (Miguel, 2010). An essential oil is defined internationally as the product obtained by distillation, or by a suitable mechanical process without heating of a plant or some parts of it (Miguel, 2010). Essential oils are complex mixtures of hydrocarbons and their oxygenated derivatives.

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They have been used since ancient times, in many different traditional healing systems all over the world, because of their biological activities. If essential oils can scavenge some free radicals, they can also act as antiinflammatory agents, because one of the inflammatory responses is the oxidative burst that occurs in diverse cells (monocytes, neutrophils, eosinophils, and macrophages). Many preclinical studies have documented antimicrobial, antioxidant, antiinflammatory and anticancer activities of essential oils in several cell culture and animal models (Miguel, 2010). EOs and their compounds have proven in vitro efficacy as antimicrobial, antioxidant, immunomodulating and antiinflammatory agents (Miguel, 2010), which may be the basis of their protective effect against gut inflammatory diseases. Chamomile essential oil, for example, has been used for centuries as an antiinflammatory substance. The antiinflammatory activity of essential oils may be attributed not only to their antioxidant activities, but also to their interaction with signaling cascades involving cytokines and regulatory transcription factors. Also, an impact on the expression of proinflammatory genes is conceivable (Miguel, 2010). The benefits of EOs applications in animal’s diets depend on diverse parameters. These parameters can be, on one hand, the unstable composition and the different levels of inclusion in the diet, and on the other hand, variable animal genetic factors (Hüsnu Can Baser and Buchbauer, 2009).

Nitrogen secondary metabolites Alkaloids are nitrogen compounds of natural origin biosynthesized from amino acids. However, not all possess a basic character, which is to contain at least one cyclically bound nitrogen, a reacting alkaline and show biological activity. Alkaloids represent one of the biggest groups of natural products and often play a vital role in plant defense against herbivores and other interspecies defenses. These compounds have important ecological functions, providing protection against attack by herbivores and microbes and serving as attractants for pollinators and seed-dispersing agents. They contribute to competition and invasiveness (of what) by suppressing the growth of neighboring plant species, a characteristic which also is called allelopathy (Osbourn and Lanzotti, 2009). Several are phytoalexins, antimicrobial and often antioxidative substances that accumulate rapidly within areas of pathogen infection. Alkaloids are additional elements in flower pigments to attract pollinators, hormones and signal molecules. Finally, they are potential alternatives of the development of new pharmaceutical and crop protection products (Osbourn and Lanzotti, 2009). Besides morphine, codeine, capsaicin, piperin, and quinine, there are a series of examples of alkaloids with specific functions, which can be extended almost endlessly; adalin and propylein as a defense secretion of ladybirds; samandarin, a steroidal alkaloid secreted by the fire salamander; mescaline, a drug which occurs naturally in the peyote cactus; gramin, produced by barley and used as basic product of tryptophan synthesis; atropine which occurs naturally in a number of plants of the nightshade family and is mentioned in the WHO model list of essential medicines, sanguinarine, as well as chelerythrine, protopine and allocryptopine, produced by plume poppy, Macleaya cordata (Kosina et al., 2010).

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Macleaya cordata is described in numerous publications and is used in traditional Chinese medicine. It is well-known for its antimicrobial, antibacterial and antiinflammatory effects, anticancerous, involved in induction of apoptosis and finally, worldwide used as a feed additive (Zeng et al., 2013).

Isoquinoline alkaloids Sanguinarine is one of the pioneers and has already entered the field in the past century. In 1997 Madan M. Chaturvedi first described it as a potent inhibitor of the NF-kB activation, I-kB phosphorylation and degradation. The nuclear factor NF-kB is a pleiotropic transcription factor whose activation results in inflammation, viral replication, and growth modulation. NF-kB is almost found in all cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet irradiation, and bacterial or viral antigens. It plays a leading role in regulating the immune response to infection. Due to its role in pathogenesis, NF-kB is considered a key target for drug development. Sanguinarine and its relatives belong to the group of (benzyl) isoquinoline alkaloids. To the present, more than 3000 of these alkaloids are known. This type of alkaloid derives from two molecules of tyrosine. The central intermediate in their biosynthesis, reticuline, can undergo various rearrangements and modifications to produce the different structural classes of benzylisoquinolines (Zeng et al., 2013). Since then, several studies have demonstrated the antiinflammatory activity of alkaloids, involving inhibition or regulation of important inflammation mediators, such as the beforementioned NF-kB, COX-2, and iNOS (Alves de Almeida et al., 2017). Lopes Souto et al. (2011) reviewed published studies to evaluate the antiinflammatory effects of alkaloids and reported 40 of these compounds with significant activity. Alkaloids emerge as potential agents for intestinal inflammatory disorders. Depending on their type and molecular structure, they are absorbed to a greater or lesser extent. Regarding their toxicity, it is not a disadvantage, when they are badly absorbed. It does not influence their mode of action. Nrf2 is another transcription factor that regulates the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation (Vrba et al., 2012). The authors showed that sanguinarine may, under appropriate conditions, increase the capacity of the enzymatic antioxidant defense system via activation of the p38 MAPK/ Nrf2 pathway. Sanguinarine and its close relatives, protopine and chelerythrine, are well described in the literature. An overview on PubMed (July 2018) shows 796 publications for sanguinarine, and 2442 and 248 for protopine and chelerythrine, respectively. Berberine, the rising star on the night sky of isoquinoline alkaloids and its abilities are described in 4909 articles. Nevertheless, morphine shows more than 55,000 publications, and caffeine passed the 30,000 publications in 2016 and is now at 31,849. The interest in alkaloids remains high. The keyword “alkaloid” leads to 446,813 entries and the first publication dates 1873 where J. Thompson describes the use and abuse of Nux vomica and its alkaloids in the British Medical Journal. Isoquinoline alkaloids from the plant Macleaya cordata are very well-known antiinflammatory compounds and they have been used as feed additives in animals for decades

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(Kosina et al., 2010). In the following section of this chapter, the antiinflammatory properties of isoquinoline alkaloids will be described in detail, including the mode of action and the subsequent effects on the inflammatory cascade, as well as the benefits on gut health and performance.

Mode of action of isoquinoline alkaloids: inhibition of NF-kB activation Sanguinarine is capable of blocking “the tumor necrosis factor-induced phosphorylation and degradation of IkBa, an inhibitory subunit of NF-kB, and inhibits translocation of p65 subunit (of NF-kB) to the nucleus” (Chaturverdi et al., 1997). The result is that NF-kB remains inactivated in the cytoplasm and it cannot start the transcription of proinflammatory genes encoding proinflammatory cytokines in the nucleous. Consequently, less proinflammatory cytokines are synthesized and released, reducing the magnitude of the inflammatory response.

Consequences of reducing inflammation with isoquinoline alkaloids Cytokines are immunomodulating proteins which have their role in cell signaling. They are released by macrophages, T-lymphocytes, or fibroblasts, and include interleukins, lymphokines and cell signal molecules, such as interferon and tumor necrosis factor. Soler et al. (2016) investigated the effect of isoquinoline alkaloids on proinflammatory cytokine expression on a monoculture of enterocytes, using the intestinal cell line IPEC-J2. Acetylsalicylic acid, known for its antiinflammatory and growth promoting activities, served as positive control (Xu et al., 1990; Botting, 2010). The study used E. coli. to trigger inflammation. Results indicated that acetylsalicylic acid and isoquinoline alkaloids significantly decreased the relative abundance of proinflammatory cytokines (Fig. 17.1). However, no significant differences were found between isoquinoline alkaloids and acetylsalicylic acid for TNF-ɑ and IL1b. This study underlined that isoquinoline alkaloids are truly antiinflammatory compounds and reduce the expression of proinflammatory cytokines. Macrophages play a critical role in the first line defense against microbial infections. Macrophages are antigen-presenting cells and they can interact with appropriate T cell receptors or they can clear the infectious agent by itself. This process, known as phagocytosis, requires that macrophages produce radical oxygen species, lysozyme, proteolytic enzymes, or nitric oxide (NO). Cytokine-activated macrophages release NO in high concentrations. NO is believed to be involved in the regulation of apoptosis, as well as in the pathogenesis of inflammatory disorders of the gut, joints, and lungs (Sharma et al., 2007). The inducible nitric oxide synthase (iNOS) catalyzes the production of NO from L-Arginine. Therefore, iNOS can be measured to evaluate macrophage activity and the degree of inflammation. Khadem et al. (2014) investigated the effect of isoquinoline alkaloid supplementation on the expression of iNOS in broiler chickens. In this study, Ross 308 broiler chicks were used and supplemented with either oxytetracycline or isoquinoline alkaloids. At day 35, the jejunal gene expression of iNOS was evaluated. Results indicated that supplementation with isoquinoline alkaloid reduced the expression of iNOS in a dose-dependent manner. Additionally,

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The response of IPEC-J2 cells to a challenge with an enterotoxigenic E. coli strain in the presence or absence of antiinflammatory compounds. Control ¼ PBS; ASA ¼ acetylsalicylic acid; IQ ¼ isoquinoline alkaloids. Experiments were done in triplicate (n ¼ 3). Different letters indicate a significant difference (P < .05).

FIGURE 17.1

oxytetracycline also significantly reduced iNOS expression. The results for feed conversion reflected the in vitro resultsda dose-dependent effect was observed when isoquinoline alkaloids were supplemented. It can be assumed that a decrease in inflammation was mirrored in a better feed conversion. Furthermore, Pickler et al. (2013) showed that less immune cells associated with inflammation are expressed when isoquinoline alkaloids are used. In this study, broilers were challenged with Salmonella Enteritidis and changes in the intestinal morphology and the expression of immune cells in the intestinal mucosa and the peripheral blood were assessed. The use of isoquinoline alkaloids resulted in a significantly lower expression of goblet and T-lymphocyte CD3þ cells in the duodenum and jejunum, further supporting the antiinflammatory effect of isoquinoline alkaloids. The production of APPs is part of the unspecific immune response. Following tissue damage due to infection or injury, cytokines will be released and reach the liver. Here, in the presence of cortisol, they will promote the synthesis of so-called APP. During an acute inflammation, the plasma levels of acute-phase proteins will increase (positive APP, e.g., C-reactive protein, serum amyloid A, haptoglobin) or decrease (negative APPs, e.g., transferrin, albumin) many folds of their normal concentration. APP have a variety of functions, e.g., to inhibit the growth of the infectious agent, give negative feedback on the inflammatory response, or recruit more immune cells to the site of infection. Weaning is one of the most stressful events for pigs due to social, immunological, infectious, metabolic and nutritional changes. Stress is known to be associated with intestinal inflammation, resulting in an increased production of cytokines and APP, which negatively influences intestinal morphology, leading to a reduced absorptive capacity, a disruption of the intestinal barrier and therefore to a greater risk for bacterial infections (Pié et al., 2004; Al-Sadi et al., 2010). Kantas et al. (2015) investigated the effect of isoquinoline alkaloids on

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the growth performance and the level of APP in weaning piglets. In this study, over 800 weaned piglets were used. Results indicated that levels of haptoglobin and serum amyloid A were significantly reduced in isoquinoline-supplemented pigs after 7 and 14 days of supplementation in a dose-dependent manner. Additionally, feed conversion and average daily gain were significantly improved in supplemented pigs, suggesting that isoquinoline alkaloids can be used to reduce intestinal inflammation associated with weaning stress, thus resulting in better performance of postweaning pigs.

Isoquinoline alkaloids, and gut health and stress Tight junctions are protein junctional complexes between enterocytes. Their function is to prevent paracellular leakage of big molecules (including pathogens). Cytokines and some pathogens are known to interfere with tight junction proteins in a negative way, possibly leading to a disruption of the gut barrier function. A good review about how enteric pathogens may lead to a disruption of the intestinal barrier through alteration of the tight junctions is found in Awad et al. (2017). Liu et al. (2016) assessed the effect of dietary supplementation with isoquinoline alkaloids on tight junction proteins in growing pigs. This study found a higher expression of the tight junction proteins zonulin-1 (ZO-1) and claudin-1 in isoquinoline alkaloid-supplemented pigs, indicating a healthier and less permeable intestinal barrier (Fig. 17.2). Stress is very well-known to cause intestinal inflammation and intestinal barrier dysfunction. Cortisol is widely used as a marker of stress and it can be measured in saliva, which represents the active portion of the hormone. Artuso-Ponte et al. (2015) investigated the effect of isoquinoline alkaloids supplementation on salivary cortisol in pigs before and after transportation to the slaughterhouse and the correlation between stress and carcass contamination with Salmonella spp. The results of this study showed that salivary cortisol concentrations were not different between pigs of the negative control group and pigs supplemented with isoquinoline alkaloids at the beginning of the study and before the pigs were transported to the slaughterhouse. However, after transportation, only nonsupplemented pigs showed a significant increase in salivary cortisol compared to pretransport levels, while no significant

FIGURE 17.2 Relative protein abundance of tight junction proteins in the jejunal mucosa of pigs (n ¼ 6). Different letters indicate a significant difference (P < .05).

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differences were observed in the isoquinoline alkaloids fed pigs. Additionally, the cortisol concentrations after transportation were significantly higher in nonsupplemented compared to isoquinoline alkaloids-supplemented pigs. Moreover, this study also showed that contamination of the carcasses with Salmonella spp. was lower in isoquinoline alkaloidssupplemented pigs, which had lower levels of salivary cortisol. These results indicated that isoquinoline alkaloids can regulate stress response and can reduce the shedding of bacteria that can potentially contaminate the carcasses and increase the food safety risk. The results from this study were further confirmed by comparable data obtained in sows (farrowing stress; Suwannathada et al., 2015) and piglets (weaning stress; Artuso-Ponte et al., 2018).

Regulating intestinal inflammation and the effects on animal performance Isoquinoline alkaloids are antiinflammatory, therefore contributing to a physiological range of intestinal inflammation. Consequently, gut integrity is improved, leading to a better nutrient digestibility (Boroojeni et al., 2018). This is correlated with a better feed conversion and growth (Kantas et al., 2015; Khadem et al., 2014). For example, an analysis of more than 20 worldwide university studies indicated that the use of isoquinoline alkaloids in broiler chickens improved feed conversion ratio by 4.1% and 2.7% in challenged and unchallenged birds, respectively, while daily body weight gain was improved by 5.0% and 3.6%, respectively (Pastor and Kraieski, 2018) (Table 17.1). In conclusion, the presented research data shows that antiinflammatory plant extracts, such as isoquinoline alkaloids, are truly antiinflammatory compounds. Isoquinoline alkaloids TABLE 17.1

Effects of isoquinoline alkaloids on gut health.

Effect

Mode of action

References

Antiinflammatory

Inhibition of NF-kB

Chaturverdi et al. (1997)

Antiinflammatory

Lower cytokine expression

Soler et al. (2016)

Antiinflammatory

Reduced iNOS expression

Khadem et al. (2014)

Antiinflammatory

Minimized expression of goblet and T-lymphocyte CD3þ cells

Pickler et al. (2013)

Antiinflammatory

Decreased production of acute-phase proteins

Kantas et al. (2015)

Improved gut integrity

Higher expression of tight junction proteins

Liu et al. (2016)

Improved gut integrity

Better nutrient digestibility

Boroojeni et al. (2018)

Stress relief

Lower cortisol levels in saliva and serum

Artuso-Ponte et al. (2015); Artuso-Ponte et al. (2018); Suwannathada et al. (2015)

Improved performance

Improved feed conversion and growth

Kantas et al. (2015); Khadem et al. (2014); Pastor and Kraieski (2018)

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can positively influence gut health by affecting the inflammation cascade in several steps, starting with the inhibition of the NF-kB activation. Consequently, cytokine and APP production may be reduced, and cortisol levels minimized. Furthermore, it has been shown that the use antiinflammatory compounds can result in a better and sustainable animal production.

References Aktories, K., Förstermann, U., Hofmann, B.F., Starke, K., 2013. Allgemeine und spezielle Pharmakologie und Toxikologie. Elsevier, Urban & Fischer Verlag. Alves de Almeida, A.C., Meira de-Faria, F., Dunder, R.J., Bognoni Manzo, L.P., Monteiro Souza-Brito, A.R., Luiz-Ferreira, A., 2017. Recent trends in pharmacological activity of alkaloids in animal colitis: potential use for inflammatory bowel disease. Evid. Based Complement Altern. Med. 2017. Al-Sadi, R., Ye, D., Said, H.M., Ma, T.Y., 2010. IL-1ß-Induced increase in intestinal epithelial tight junction permeability is mediated by MEKK-1 activation of canonical NF-kB pathway. Am. J. Pathol. 177 (5), 2310e2322. Anderson, A.D., Nelson, J.M., Rossiter, S., Angulo, F.J., 2003. Public health consequences of use of antimicrobial agents in food animals in the United States. Microb. Drug Res. 9, 373e379. Artuso-Ponte, V., Moeller, S., Rajala-Schultz, P., Medardus, J.J., Munyalo, J., Lim, K., Gebreyes, W.A., 2015. Supplementation with quaternary benzo(c)phenanthridine alkaloids decreased salivary cortisol and Salmonella shedding in pigs after transportation to the slaughterhouse. Foodborne Pathog. Dis. 12 (11), 1e7. Artuso-Ponte, V., van Leeuwen, J., da Silva, C., 2018. Effect of isoquinoline alkaloids in combination with organic acids on stress response and incidence of diarrhea of post-weaning pigs. In: The 25th International Pig Veterinary Society Congress and International PRRS Symposium, 11.06.e14.06.2018, Chongqing, China. Awad, W.A., Hess, C., Hess, M., 2017. Enteric pathogens and their toxin-induced disruption of the intestinal barrier through alteration of tight junctions in chickens. Toxins 9 (29), 60e82. Berkes, J., Viswanathan, V.K., Savkovic, S.D., Hecht, G., 2003. Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation. Gut 52, 439e451. Beyer, B., 2015. Untersuchungen zur Bioverfügbarkeit von Catechinen und Quercetin beim Rind. Agrar- und Ernährungswissenschaftliche Fakultät. Christian-Albrechts-Universität zu Kiel. Dissertation). Boroojeni, F.G., Männer, K., Zentek, J., 2018. The impacts of Macleaya cordata extract and naringin inclusion in postweaning piglet diets on performance, nutrient digestibility and intestinal histomorphology. Arch. Anim. Nutr. 72 (3), 178e189. Botting, R.M., 2010. Vane’s discovery of the mechanism of action of aspirin changed our understanding of its clinical pharmacology. Pharmacol. Rep. 62, 518e525. Box, A.T.A., Mevius, D.J., Schellen, P., Verhoef, J., Fluit, A.C., 2005. Integrons in Escherichia coli from food-producing animals in the Netherlands. Microb. Drug Res. 11, 53e57. Callaway, T.R., Anderson, R.C., Edrington, T.S., Genovese, K.J., Harvey, R.B., Poole, T.L., Nisbet, D.J., 2004. Recent pre-harvest supplementation strategies to reduce carriage and shedding of zoonotic enteric bacterial pathogens in food animals. Anim. Health Res. Rev. 5, 35e47. Chaturvedi, M.M., Kumar, A., Darnay, B.G., Chainy, G.B.N., Agarwal, S., Aggarwal, B.B., 1997. Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-kB activation, IkBɑ phosphorylation, and degradation. J. Biol. Chem. 272 (48), 30129e30134. Doyle, M.P., Erickson, M.C., 2006. Reducing the carriage of foodborne pathogens in livestock and poultry. Poultry Sci. 85, 960e973. Emborg, H., Ersbøll, A.K., Heuer, O.E., Wegener, H.C., 2001. The effect of discontinuing the use of antimicrobial growth promoters on the productivity in the Danish broiler production. Prev. Vet. Med. 50, 53e70. European Commission, 2005. Ban on Antibiotics as Growth Promoters in Animal Feed Enters into Effect. HMSO. Fürst, R., Zündorf, I., 2014. Plant-derived ant-inflammatory compounds: hopes and disappointments regarding the translation of preclinical knowledge into clinical progress. Mediat. Inflamm. 2014. Gabay, C., Kushner, I., 1999. Acute-phase proteins and other systemic responses to inflammation. N. Engl. J. Med. 340 (6), 448e455. Hao, H., Haihong, H., Guyue, C., Zahid, I., Xiaohui, A., Hafiz, I.H., Lingli, H., Menghong, D., Yulian, W., Zhenli, L., Zonghui, Y., 2014. Benefits and risks of antimicrobial use in food-producing animals. Front. Microbiol. 5 (288), 1e11.

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C H A P T E R

18 Effects of phytobiotics in healthy or disease challenged animals Ioannis Skoufos1, Eleftherios Bonos1, Ioannis Anastasiou2, Anastasios Tsinas1, Athina Tzora1 1

Department of Agriculture, School of Agriculture, University of Ioannina, Arta, Greece; 2 Trinity Nutrition Ltd., Cavan, Ireland O U T L I N E

Introduction

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In vitro and in vivo antimicrobial and antiparasitic activities of aromatic and medicinal plants, herbs, their extracts and essential oils in poultry Coccidial infections Necrotic enteritis Campylobacter infection

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In vitro and in vivo antistress activities of aromatic and medicinal plants, herbs, their extracts and essential oils in ruminants 325 Effects of dietary phytobiotics in rabbits 327 Conclusions

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Introduction Animal production systems have relied upon antibiotics for healthy animal development, but this comes with the known outcome of selection of resistant bacteria (Alekshun and Levy, 2007) and resulting in an increase in the reservoir of resistance genes that may enter the food

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Copyright © 2020 Elsevier Inc. All rights reserved.

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chain and ultimately the human microbiome (Sommer et al., 2010). For example, in poultry, Escherichia coli is often a natural component of the chicken gut flora inhabiting their mucosal surfaces (Dho-Moulin and Fairbrother, 1999), but some pathogenic types such as avian pathogenic E. coli (APEC) are associated with localized or systemic diseases collectively named colibacillosis (Stordeur and Mainil, 2002). Because of APEC infections, the poultry industry suffers from the loss of chickens and birds, which affects food economy (Dho-Moulin and Fairbrother, 1999). These APEC infections were first controlled using antibacterial drugs, but unfortunately, the misuse of the antibiotics led to the rise of antibiotic-resistant strains instead (Nhung et al. 2017). Recently, the use of antibiotics as growth promoters (AGPs) has been curtailed by legislation in European Union (EU) and EU-associated countries since 2006 (Christaki et al., 2012; Gaucher et al., 2015). The reason of this prohibition was the widespread development of resistant bacterial strains to evolve over a short period and transfer resistance to other strains of bacteria populations. The major concern of the scientific community was the appearance that human cases of untreatable bacterial diseases with lethal ending were increasing dramatically, showing that pathogenic bacteria gained resistance to antibiotics greater than before (NRS, 1980; Gaucher et al., 2015). The basal scientific ground for the common use of antibiotics given in small amounts in animal diet was their halting effect on the growth of bacteria, prevention of disease outbreaks and enhancement of growth rate, particularly in intensively reared animals (NRS, 1980). Therefore, extensive effort has been devoted toward developing alternative products to antibiotics to favor intestinal health by research teams in academia, in the animal industry, as well as in EU funded projects and research collaborations and investment (Timbermont et al., 2011; European Commission, 2018). A vast source of natural antimicrobial compounds has been used by humanity and animals since antiquity (Christaki et al., 2012). Aromatic plants and extracts, herbs, medicinal plants were very familiar globally, however, there exists a large gap between indigenous herbal practices and contemporary medical sciences in philosophy, theories and their applications, especially in animal diet and health. It is predominantly the last three decades that animal nutritionists and veterinarians have begun to explore aromatic and medicinal plants and their extracts in this regard, as many plants produce secondary metabolites which have noteworthy antimicrobial properties against a wide range of microorganisms (Christaki et al., 2012). Herbal essential oils (EOs) and their active components have prompted researchers and academics to investigate their potential to improve production efficiency in poultry, pigs and ruminant farm animals. Herbal EOs are considered safe for human and animal consumption and are categorized as generally recognized as safe (GRAS) in the EU countries under the European Food Safety Authorities and in USA by Food and Drug Association (Windisch et al., 2008; Brenes and Roura, 2010). In the modern literature, there are published experimental evidences on the efficiency of phytobased products. In some extensive reviews about aromatic plants, extracts or EOs in animal nutrition, their mode of action was examined and experimental evidence on their use was presented in healthy or diseased animals (Windisch et al., 2008; Brenes and Roura, 2010; Bozkurt et al., 2013; Giannenas et al., 2018c). Research of plant or botanical products

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under both pharmacological and industrial protocols is necessary to establish their extensive use in animal industry. This chapter discusses recent developments in use of phytobased products, which are a relatively new class of feed additives, to potentially benefit poultry, swine, small and large ruminants, and rabbits either healthy or challenged by various bacterial microorganisms, parasites, or stressful conditions. Some representative intestinal diseases such as necrotic enteritis and coccidiosis in poultry will be described and alternative antimicrobial ways of control based on plant extracts will be discussed. A literature search on bacterial challenges in young piglets and heat stress in ruminants will be also presented. Dietary solutions based on aromatic and medicinal plants, herbal extracts and their EOs will consecutively be assessed along with their mechanisms of action. Further discussion will also explain possible reasons for varying responses, as well as, challenges and opportunities in the development of phytobased products, with emphasis on the marketplace and modernization.

Aromatic and medicinal plants, herbs, their extracts and essential oils as feed additives Aromatic and medicinal plants, herbs and spices, their extracts EOs have been used as alternative medicine based on ethnoveterinary knowledge (Chang, 2000). Aromatic medicinal plants, their EOs, or herbal extracts have been found to exhibit antimicrobial activity, antiparasitic activity, and possess antiviral and antioxidative properties (Fig.18.1; Christaki et al., 2012; Giannenas et al., 2018b). Aromatic, medicinal plants of botanical origin or herbs and spices and their extracts can be used in animal feeding after drying and grounding. Herbal extracts can be prepared by different extraction methods with various solvents, such as organic solvents (ethanol, methanol or toluene). Plant EOs are mixtures of fragrant, volatile compounds, extracted by steam water distillation (Oyen and Dung, 1999). The term “essential” originates in the theory of “quinta essential” of Paracelsus, who alleged that this “quintessence” was an important part of medicine preparation (Oyen and Dung, 1999). Since, the term “essential oil” is not scientifically defined according to modern knowledge, it was suggested to be used the term “volatile oil” instead, though even today the term “essential oil” is used more often (Hay and Waterman, 1993). Natural plant products may have a variable composition of individual compounds. For example, the percentage of the two main constituents of thyme EOs, i.e., thymol and carvacrol, can vary from as low as 3% to as high as 60% (Lawrence and Reynolds, 1984). Similar variation can be found also in the EO of oregano (Origanum vulgare ssp. hirtum), which is obtained by steam-distillation of the plant, that contains more than 30 bioactive components, most of which are phenolic substances with varying activities (Economou et al., 1991; Sivropoulou et al., 1996; Adam et al., 1998). Major components are carvacrol and thymol, which represent over 80% of the total oil (Adam et al., 1998), with carvacrol to be the main component in most cases, although sometimes it can be found only in traces (Sivropoulou et al., 1996). The concentration of the other major constituents such as the two monoterpene

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Antioxidant

Flavor enhancers

Antimicrobial

Beneficial effects of phytobiotics

Antiinflammatory

Antiparasitic

Anti-stress

FIGURE 18.1

Immuneenhancers

Effects of phytobiotics as feed additives.

hydrocarbons, g-terpinene and p-cymene, that often constitute about 5% and 7% of the total oil, respectively, also vary and the effect of oregano EO is often also variable because it depends on all the constituents working together (Adam et al., 1998). Cinnamaldehyde, found in large amounts in cinnamon EO totals to about 60%e75% (Duke, 1986). Due to this wide variation in composition, the biological effects of the EOs may vary (Schilcer, 1985; Janssen et al., 1989; Deans and Waterman, 1993). This diversity of EOs could urge future research to select pure compounds, i.e., thymol, cinnamaldehyde, beta-ionone and carvacrol, for evaluating their probable use to replace antibiotics in animal production. A major advantage of the use of aromatic plants and their extracts as feed additives is that they are considered to be safe products (Jones, 1996; Christaki et al., 2012). If they were unsafe, the use of herbs, spices and their extracts in human food would have to be minimized, as they would not probably affect only the aromatic characteristics of food (Jones, 2002). Despite this popularity of aromatic plants and their extracts, if they were to be used as alternative feed additives, in large scale in animal feeds, they should be well researched in terms of their mechanisms of action, toxicity and clinical effects. In should be noted that even today, there are much hyperbole associated with herbal products, for example being implicitly safe, absent of side effects or totally efficient; Of course these claims are not acceptable from a scientific and industrial point of view (Giannenas, 2008). Sound pharmacological research on aromatic medicinal plants and their extracts is necessary in the overall strategy to prevent or treat animals’ diseases.

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In vitro and in vivo antimicrobial and antiparasitic activities of aromatic and medicinal plants, herbs, their extracts and essential oils in poultry A recent study (Al-Mnaser and Woodward, 2017) focusing on assessing the effects of polyphenols in the form of oregano extract EOs presented very interesting data. Oregano polyphenols can be bacteriostatic (inhibitory or restrictor effect) at sub-lethal concentrations and bactericidal (killing effect) at or above the minimum inhibitory concentration (MIC) level on many microorganisms. Oregano extract suppressed antibiotic-resistant APEC strains, which are responsible for causing colibacillosis in poultry reared for human consumption. A total of 12 APEC strains isolated from infected birds have been characterized microbiologically and biochemically, using standard laboratory methods. The phytochemical, aqueous oregano, inhibited and killed the 12 APEC strains including those that were antibiotic resistant (MIC range 0.3e0.5 mg/mL), and inhibited their biofilm formation. The authors suggested that their future work will take into more in-depth dimensions in justifying the use of oregano extract in the poultry industry. The current data generated provides very promising evidence that oregano extract may be used in novel ways to control potentially pathogenic and antibiotic-resistant APEC strains and provide alternative strategies to the use of antibiotics. Oregano extract showed its bactericidal and antibiofilm activities against antibiotic-resistant APEC and at a very low concentration in comparison with antibiotics (Al-Mnaser and Woodward, 2017). This is possibly due to its antibacterial activities resorting to its phenolic content (Rhayour et al., 2003). A previous study (Williams, 1997) showed that phenols used as disinfectants exhibit oocysticide action versus E. tenella by in vivo and in vitro tests. In nature, several aromatic plants are rich sources of phenolic compounds and especially those of the Labiatae family (Vekiari et al., 1993). Among which oregano presents particular interest as the active ingredients in its EOs are mostly phenolic compounds (Vekiari et al., 1993). Also, other researchers found that thymol and carvacrol show important biological activities, as feed additives (Ibrir et al., 2001, 2009; Christaki et al., 2012). Apart from the volatile compounds in the EOs oregano contains also nonvolatile compounds that also exhibit biological activity (Vekiari et al., 1993). The antimicrobial actions of phenols, are targeting against the bacterial cell wall, influencing the cell wall structure. Phenols affect with the cytoplasmic membrane and increase its permeability for cations, like Hþ and Kþ (Sikkema et al., 1995; Ultee et al., 2002a, 2002b). A large dissipation of ion gradients causes injury to the cell processes and may lead to outflow of cellular constituents, loss of water equilibrium, change of the membrane polarity, inhibition of ATP synthesis, and death (Ultee et al., 2002a, 2002b). It has been proposed that the major phenolic compounds exert lipophilic properties, based on their chemical structure (Farag et al., 1989; Conner, 1993). Helander et al. (1998) who examined the antibacterial effect of two isomeric phenols, carvacrol and thymol, and the phenylpropanoid, cinnamaldehyde, on Escherichia coli O157 and Salmonella typhimurium, noted that both substances can cause damage to the bacterial cell membrane. It has been proposed that the lipophilicity regulates the ability of terpenoids and phenylpropanoids penetrate the cell membrane of the bacteria (Helander et al., 1998). It has also been suggested that chemical properties, such as the existence of functional groups (Farag et al., 1989), and aromaticity modify their activity

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(Bowles and Miller, 1993). Some substances, such as cinnamaldehyde contain sulfhydryl groups, which are indispensable for the fungal growth that can form charge transfer complexes with electron donors in the fungus cells, which can negate cell division and hinder with cell metabolism (Bang et al., 2000). Other examples indicated that cinnamaldehyde, originating from the cinnamon EO is a strong inhibitor of Clostridium perfringens and Bacteroides fragilis and a moderate inhibitor for Bifidobacterium longum and Lactobacillus acidophilus found in human feces (Lee and Ahn, 1998). The selective inhibition by cinnamaldehyde on pathogenic, intestinal bacteria may have an effect on the balance of the gastrointestinal microflora. The in vitro antimicrobial activities of EOs derived mainly from oregano, thyme, and cinnamon have been extensively examined (Deans and Ritchie, 1987; Meeker and Linke, 1988; Biondi et al., 1993; Juven et al., 1994; Sivropoulou et al., 1996; Hammer et al., 1999; Chen et al., 2008; Al-Kassie, 2009; Al-Mnaser and Woodward, 2017). Based on their in vitro antimicrobial activity, the EOs application as prophylactic and therapeutic agents in animal production is under examination. Some early studies showed that it was possible to rear broilers without growth promoters and coccidiostats by incorporating in their diet oregano EOs or ground oregano (Waldenstedt, 2003; Florou-Paneri et al., 2004; Giannenas et al., 2005). Other early field studies conducted by Köhler (1997) with commercial preparations of EOs of various plants reduced substantially colony forming units of C. perfringens. It should be further noticed that the comparison was made against chickens that received a diet containing zinc bacitracin at the level of 20 ppm. A slightly lower ileal adenosine triphosphate (ATP) concentration has been reported (Veldman and Enting, 1996), which is an indicator of microbial activity in broilers (Smits et al., 1997). In addition, a dietary mixture of capsicum, cinnamaldehyde and carvacrol resulted in lower populations of Escherichia coli and C. perfringens in ceca (Jamroz and Kamel, 2002). Alcicek et al. (2003) reported that a dietary EO mixture derived from six aromatic plants, including oregano, laurel, sage, myrtle, fennel and citrus increased chicken performance parameters. Another important mechanism that could explain the antimicrobial effect of phytobiotics is that they can interfere with or inhibit the bacterial quorum-sensing signals, i.e., the intercellular communication among bacterial cells (Szabo et al., 2010). This communication uses mediators such as homoserine lactones and can coordinate prokaryotes and eukaryotes gene expression (Williams, 2007). It has been claimed that quorum sensing exhibits a very important role in plant and animal diseases, caused by different bacterial, for example Agrobacterium vitis, Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas putida, and E. coli. Thus, by affecting quorum sensing, phytobiotics can limit bacterial pathogenicity and antibiotic resistance, reduce biofilm formation and lessen virulence of the infection (Ishida et al., 2008; Szabo et al., 2010). A number of EOs from plants such as geranium (Geranium robertianum L., Geraniaceae), rose (Rosa damascena L., Rosaceae), laventer (Lavandula angustifolia L., Labiatae), rosemary (Rosmarinus officinalis L., Lamiaceae), tea tree (Camellia sinensis) and lemon (Citrus lemon) that can limit bacterial growth, have been shown to block quorum-sensing mechanisms in vitro (Schelz et al., 2006; Szabo et al., 2010; Alvarez et al., 2012; Kerekes et al., 2013). Quorum sensing blocking by phytobiotics could be a new form of medicinal approach, especially against infections that are resistant to antibiotics. There seem to be additional mechanisms of action that could explain the health benefits of dietary phytobiotics in animal nutrition. For example, phytobiotic supplementation can

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improve gastrointestinal mucosa immunity and morphology, and prevent bacterial colonization (Kiczorowska et al., 2017). Morphological analysis of the gastrointestinal tract often shows elongation of villi and increased crypt depth, and increased activity of epithelial cells, along with release of proinflammatory cytokines (Kumar et al., 2014). Moreover, in many herbs there are substances with immunostimulatory properties, such as Echinacea (Echinacea Moench), liquorice (Glycyrrhiza L.), and garlic (Allium sativum L.). The active plant substances could augment the activity of lymphocytes, macrophages, and natural killer cells, supporting phagocytosis or stimulating interferon synthesis (Kiczorowska et al., 2017). According to Liu et al. (2014), this effect is possibly regulated by gene expression, including genes that regulate immune response. In another trial, anise seeds’ addition (10 g/kg) in broiler chicken diets, resulted higher antibody titer against avian influenza (Yazdi et al., 2014). Recently, a lot of pertinent studies with EO of different plants or combinations of EOs have been reported that populations of lactic acid bacteria remained unaffected, whereas that dietary addition of EO at 50 mg/kg resulted in a decreased E. coli population in ileo-cecal digesta (Jang et al., 2007). Also, populations of cecal E. coli were significantly lower in 300 and 600 mg dietary oregano oil supplementation per kg of feed, whereas Lactobacilli population was unaffected (Roofchaee et al., 2011). Additionally, diet supplementation of broilers with a combination of a protease and EO synergistically increased ileal Lactobacilli populations, and reduced ileal E. coli populations (Giannenas et al., 2014b; Park and Kim, 2018). Similarly, the antibacterial effect of the herbal dietary supplements is evidenced by alterations in intestinal microbiota either in small intestine or caecum (Tiihonen et al., 2010; Giannenas et al., 2016, 2018c; Skoufos et al., 2016; Tzora et al., 2017). Also, it was very lately reported that polyherbal feed additives can promote growth due to their effects on intestinal microbiota based on their high phenolic content and synergy among their combined constituents (Giannenas et al., 2018b, 2018c). Some intestinal diseases are an important concern to the broiler poultry industry because of lost productivity, increased mortality, reduced welfare of birds and the associated contamination of poultry products with pathogen bacteria and or their toxins for human consumption (Mitsch et al., 2004; Giannenas and Kyriazakis, 2009; Gaucher et al., 2015). In this line, major examples in broilers are coccidiosis, necrotic enteritis and campylobacter infection.

Coccidial infections Avian coccidiosis is the result of the intracellular growth and replication of protozoan parasites of the genus Eimeria (phylum Apicomplexa). This intestinal parasitosis is a foremost source of production and major economic losses for the poultry producer all over the world (Ekstrand et al., 1994; Wallach et al., 1995). There are seven species of Eimeria which are considered to be the main causative agents of chicken coccidiosis: E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and E. tenella (Shirley, 1986). These intestinal parasites are differentiated by biological differences, such as preferred intestinal site for intracellular infection, differences of life cycle stages and times, and immunological specificity. Some of these species show reproductive isolation. The coccidia E. acervulina, E. maxima and E. tenella are regarded as the most important for the chicken industry, based on their omnipresence in chicken flocks and strong innate pathogenicity (Crouch et al., 2003; McDougald, 2003).

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Coccidiosis that has been identified as a detrimental disease as early as 1920 remains one of the most significant problems of the poultry industry worldwide and results in large annual losses (Naidoo et al., 2008; Giannenas et al., 2013a). Today, the largest proportion of chickens reared in the world consist of standard broilers, which are the result of decades of breeding programs. The cost of coccidiosis for the chicken industry is the combination of feed or water medication costs, veterinary expertise costs, and production reduction. Commercially reared broilers are especially vulnerable to the disease, due to the intensity of farming, the use of deep litter and the avoidance of use of breeding broilers in cages. The main anticoccidials added to feeds are the polyether (ionophore) group of chemotherapeutics, sulphonamides, pyrimidine derivatives, triazinetriones, and the benzenacetonitriles (Naidoo et al., 2008; Kant et al., 2013). Until recently this availability of dietary anticoccidial medications, supported the large growth and low cost of production for the poultry industry. However, over the last 20 years the large use or even misuse of these drugs, gradually led to the development of resistant coccidian strains (Long, 1982; Chapman, 1997; Giannenas et al., 2014a). Today, there is evidence of coccidial resistance against all anticoccidial drugs (Chapman, 1997; Chapman et al., 2005; Bozkurt et al., 2013). Additionally, consumers are considerably worried about appearance and levels of drug residues in poultry products (McEvoy, 2001; Young and Craig, 2001), and they demand that broiler chickens should be raised without without drugs (Barton, 1999; Christaki et al., 2012). Presently, in Europe, there is pressure to gradually phase out the dietary use of ionophore antibiotics in animal production (Christaki et al., 2012). Certain anticoccidials have been prohibited, such as nicarbazin. Although a total ban on the use of coccidiostats in animal feeds has not taken effect in most countries, there is a growing effort for antibiotic free production in US and Canada, trying to stop the emerging problem of antimicrobial resistance (Gaucher et al., 2015). Since broiler chickens will be raised without antibiotics or anticoccidials the poultry experts should provide them a health protective umbrella that is wide enough to confront bacteria, coccidia, parasites, or stressful conditions. A widely used experimental model to artificially challenge poultry has been described in detail by Conway and McKenzieet (2007). Briefly, the model involves that birds will be orally infected with 105 sporulated oocysts of E. acervulina, 106 sporulated oocysts of E. maxima and 105 sporulated oocysts of E. tenella. However, at least for E. tenella a challenge on day 10 or 14 of age with a suspension containing 5 to 10  104 was shown to be accepted as disease model. This reliable experimental model is useful for the evaluation of the predisposing effects of putative risk factors and efficacy of preventive measures (Giannenas et al., 2003, 2013b, 2014a; Bozkurt et al., 2013). Otherwise, a combined inoculation with oocysts of various Eimerian species may provoke a disease and challenge the affected birds. In the last three decades several studies showed the in vitro and in vivo anticoccidial effects of phytobiotic plants and substances. In coccidian-challenged chickens, Allen et al. (1997) found that dried leaves of Artemisia annua can shield chickens against to E. tenella infection. It was found that Artemisia annua could be investigated as a potential source of anticoccidial compounds in poultry. Pure compounds found in of A. annua, i.e., artemisinin, 1,8-cineole and camphor at the levels of 17, 119 and 119 ppm, respectively, were added to chicken broiler feeds until the third week of age. At the second week of age, 50% of the chickens were inoculated with E. acervulina and E. tenella. A moderate prophylactic effect against the coccidia was found in the supplemented chicks, especially in those fed diets

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with artemisinin. In another trial, Youn and Noh (2001) showed that Sophora flavescens EOs were more effective than Artemisia annua to treat E. tenella parasitation in chickens. Earlier, Akhtar and Rifaat (1985) tested the anticoccidial potential of EOs of Melia azedarach L. (Bakain) in naturally parasitized chickens. Evans et al. (2001) also investigated whether a combination of EOs from clove (1.0%), thyme (0.1%), peppermint (0.1%), and lemon (0.1%) could limit the coccidia oocyte production and the population of C. perfringens in broiler chicks when artificially inoculated. Chicks that consumed the feed supplemented with an EO mixture displayed a lower oocyte excretion in comparison with those fed the basal feeds. Giannenas et al. (2003) in an inaugurate study, found that the EO of oregano displayed an anticoccidial effect versus E. tenella when supplemented into chicken feeds at 300 mg/kg. In another experiment, broiler chickens challenged with E. tenella, were administered feeds supplemented with ground oregano herb. The inoculations of the chickens with E. tenella was performed at 14 days of age by oral administration of a 2 mL suspension of 5  104 sporulated oocysts. Based on the performance parameters (weight gain and feed conversion ratio of parasitized vs. nonparasitized chicks) the dietary supplementation with ground oregano limited the negative effects of E. tenella parasitation. Also, the recorded values of the severity of bloody diarrhea, mortality, caecal lesion scores and coccidian oocyst output per gram of feces suggested that dietary supplementation of 5.0 and 7.5 g oregano per kg of feed showed the most pronounced effects against the infection with E. tenella (Giannenas et al., 2004). These findings supported the hypothesis that dietary oregano could act as an alternative to ionophore antibiotics for protection against coccidial infection in broiler chickens. Another study examined the effect of dietary supplementation with Olympus tea (Sideritis scardica) on the performance parameters of broiler chickens inoculated at 14 days of age with 6  104 sporulated oocysts of E. tenella (Florou-Paneri et al., 2004). Data based on evaluation of performance parameters along with the severity of bloody diarrhea, mortality rate, caecal lesion score and oocyst output showed that supplementation with Olympus tea at 10 g/kg feed, could lessen the negative effects of the coccidial infection. However, the exerted coccidiostatic effect against E. tenella was considerably lower than that exhibited by lasalocid. Similar results were also noticed in another work studied the effect of dietary supplementation with a commercial preparation of herbal extracts from the plants Agrimonia eupatoria, Ehcinacea angustifolia, Ribes nigrum and Cinchona succirubra (Christaki et al., 2004). Broiler chickens were inoculated with 6  104 sporulated oocysts of E. tenella at 14 days of age, and results indicated that the blend of plant extracts or herbal EOs exerted a positive effect on postinfection performance compared to the challenged birds. Additionally, other researchers also noticed the positive effect of dietary supplementation with a saponin-based herbal extract, which noticeably improved feed conversion efficiency compared to control birds and those that were fed a tannin-based extract after experimental infection with 4  104 sporulated oocysts of E. tenella and 4  106 sporulated oocysts of E. acervulina at 8 days of age (Muriel et al., 2005). Also, Oviedo-Rondon et al. (2006) noted a remarkable effect of the dietary use of a commercial blend of herbal extracts (thymol, eugenol, curcumin, and piperin from the plants Thymus vulgaris, Syzygium aromaticum, Cinnaminum zeylanicum and Pipper nigrum, respectively). Broiler chickens were experimentally infected with sporulated oocysts of E. tenella E. acervulina and E. maxima at 19 days of age, and half of the challenged birds were also vaccinated with the Advent coccidian vaccine at day of hatch. It has been declared that a mixture of EOs had a beneficial outcome on

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postinfection performance compared to the challenged birds and may serve as an alternative to antibiotic anticoccidial drugs, although, no benefits were observed for vaccinated broilers against coccidian that were given this extract. However, it must be noted that the chickens were given 100 ppm of the product containing 30% active substances and possibly the dosage was low. In another study, three coated plant extracts (RepaXol in two dietary formulations) were examined for their minimal lethal concentration against histomonads in vitro and their actions against histomonadal growth in cultures (Hauck and Hafez, 2008). After 48 hours, plant extracts killed the histomonads when added at 1.25e2.5 mL/mL. Coated plant extracts also limited the growth of microorganisms at the same concentrations. Dietary supplementation with Tulbaghia violacea exhibited a positive effect on the rate of oocyst shedding and promoted a similar food conversion ratio to toltrazuril (Naidoo et al., 2008). This finding showed that the use of antioxidant-containing plants may also support in the prevention of eimeriosis. However, the active plant constituents must be easily partitioned in lipids in order to penetrate intracellularly in large quantities to affect the protozoan parasites. This offers a strong incentive for future appraisal of the anticoccidial efficacy of plant extracts in larger studies. Similarly, Tsinas et al. (2011) examined the effect of dietary oregano EO supplementation on the performance of challenged broilers with E. acervulina and E. maxima oocysts at the second week of life. Dietary oregano oil in both levels of supplementation (30 and 60 mg/kg) reported to enhance weight gain and feed to gain ratio at the same level to the chickens that were protected by the approved anticoccidial salinomycin or the nonchallenged birds. The challenged control presented lower performance than those of the other groups for the 10 days postinfection. The authors after evaluating performance parameters along with the extent of bloody diarrhea, mortality, morbidity and survival rate, intestinal lesion score and shedding of oocysts in the faces, clearly pointed that oregano EOs exhibited an anticoccidial effect analogous to that exerted by salinomycin against E. acervulina and E. maxima. Similarly, in a trial with oregano ground plant, it appeared that oregano limited the negative effects of E. tenella infection on body weight and the feed efficiency, with the most effective supplementation levels of oregano found to be 5.0 and 7.5 g/kg of feed (Giannenas et al., 2004). It was hypothesized that the nonprotective role of oregano at 10 g/kg could be due to action of its phenolic constituents, which at elevated levels of supplementation might have toxic or negative effects on feed palatability. It is possible that phenolic compounds at high levels could exert an undesired activity on the bird enterocytes (Weber and De Bont, 1996). It should be taken into consideration that phenolic compounds, such as carvacrol and thymol may affect the upper layer of mature enterocytes of the intestinal mucosa that are already infected by the intracellular pathogens. Carvacrol has an hydrophobic character that could result in interaction with the cellular membranes (Sikkema et al., 1995). Increased levels of concentration of carvacrol, may cause the accumulation and interaction of this substance with the cellular membranes phospholipid belayer, affecting the membrane fluidity (Weber and De Bont, 1996; Giannenas et al., 2003). Based on the recently published literature, phytobiotics hold promise as alternative dietary methods to control chicken coccidiosis. However, additional investigation is required to identify their specific modes of action, most effective levels of administration and possible interactions when used in blends versus single components.

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Necrotic enteritis Necrotic enteritis (NE) of domestic broiler chickens is considered to be a worldwide disease, potentially fatal with flock mortality rates to range from 1% to 30% (Dahiya et al., 2006). The causative agent of NE, C. perfringens, types A or C is a nearly ubiquitous gram positive, spore forming, extremely prolific, toxigenic anaerobic bacteria (Van Immerseel et al., 2004; Timbermont et al., 2011; Chan et al., 2016; Tsiouris, 2016). This type of bacteria is common in soil, dust, feces, feed, farm litter and intestinal digesta. NE typically appears in broiler chickens at 2e6 weeks of age and appears with a sudden onset of diarrhea. Post mortem examination often shows intestinal mucosal necrosis and an overgrowth of C. perfringens in the small intestine (Timbermont et al., 2011; Tsiouris et al., 2015; Tsiouris, 2016). NE annual cost worldwide for the poultry producers is quite significant (Chan et al., 2016). Moreover, since it is not easy to diagnose mild or subclinical forms of NE, probably losses from the subclinical form of NS are even larger, due to the limited bird growth, reduced carcass weight, and worse feed utilization (Timbermont et al., 2011; Tsiouris et al., 2015; Tsiouris, 2016). Although large outbreaks of NS are infrequent, they can cause high mortality and severe economic losses. Generally, C. perfringens is often found in the intestinal digesta of healthy chicken at low populations (