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BOTANICAL RESEARCH AND PRACTICES

MEDICINAL PLANTS ANTIOXIDANT PROPERTIES, TRADITIONAL USES AND CONSERVATION STRATEGIES

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BOTANICAL RESEARCH AND PRACTICES

MEDICINAL PLANTS ANTIOXIDANT PROPERTIES, TRADITIONAL USES AND CONSERVATION STRATEGIES

DAVID ALEXANDRE MICAEL PEREIRA, PH.D. EDITOR

New York


Copyright © 2014 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data Medicinal plants : antioxidant properties, traditional uses and conservation strategies / editor: David Alexandre Micael Pereira. p. cm. Includes index. ISBN: (eBook)

1. Medicinal plants--Utilization. 2. Antioxidants. I. Pereira, David Alexandre Micael. QK99.A1M428 2013 581.6'34--dc23 2013035640

Published by Nova Science Publishers, Inc. † New York


Contents Preface Chapter 1

Chapter 2

vii Phenolic Compounds and Antioxidant Capacity of Medicinal Plants: A Review Sandra C. Gouveia, Vítor Spínola and Paula C. Castilho Potential Antioxidant Benefits of Commonly Used Fruits and Vegetables around the World Lourdes Rodríguez-Fragoso, Ulises Osuna-Martínez, Ana Isabel Gonzaga-Morales and Jorge Reyes-Esparza

1

41

Chapter 3

Hydroponic Production of Medicinal Plants Rita Maggini, Claudia Kiferle and Alberto Pardossi

Chapter 4

Flavonoids as Antioxidant Therapy for Metabolic Disorders B. S. Lakshmi, K. N. Sangeetha and K. Shilpa

117

Chapter 5

Use of Antioxidants to Control Obesity and Promote Weight Loss Vandana Gulati, Pankaj Gulati and Enzo A. Palombo

143

Chapter 6

Application of Antioxidant Plants as Anti-Hemolytic Agents João C. Fernandes and David M. Pereira

165

Chapter 7

Health Attributes, Antioxidant Properties and Phytochemical Composition of Traditional Medicinal Plants from Eastern Anatolia Izabela Konczak, Abdullah Dalar and Konrad A. Konczak-Islam

Chapter 8

Hetherotheca inuloides (Mexican Arnica) a Potent Antioxidant Effect as Neuro and Hepato-Protective Liliana Carmona-Aparicio, Noemí Cárdenas-Rodríguez, Bernardino Huerta-Gertrudis, José Luis Rodríguez-Chávez and Elvia Coballase-Urrutia


91

183

227

vi

Contents

Chapter 9

Meconopsis: Traditional Uses, Chemistry and Pharmacology Haifeng Wu, Xiaopo Zhang, Yan Zhou, Xiaofeng Zhang, Yao Li, Jingyi Zhang, Lisheng Ding, Junshan Yang and Xudong Xu

Chapter 10

A Case Study of Indigenous Medicinal Plants: Antioxidant Properties, Traditional Uses and Conservation Strategies Henry Lowe and Joseph Bryant

Index

243

259 267


Preface Nowadays, natural products and in particular medicinal plants, play an important role in human health and therapeutics. Across the world, several different cultures employ medicinal plants for the treatment of a wide range of pathological conditions. In this book, the authors address the antioxidant properties of several medicinal plants, as well as their traditional uses and conservation strategies. This is, without a doubt, a wonderful opportunity to have a closer insight into the chemistry, biological properties, conservation and traditional use of medicinal plants used around the world. Chapter 1 - Plants have been used for medicinal purposes since the origin of human civilization and their uses were described by the great civilizations of the ancient Chinese, Indian and Mediterranean. Nowadays, they continue to be the source of new medicines either by providing lead molecules or as natural herbal products (teas, tinctures, powders, poultices, infusions as well as other formulations). 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 preparations. Compounds produced by plants are divided in two groups: primary and secondary metabolites. Primary metabolites are compounds that possess fundamental roles in plant development steps such as phytosterols, acyl lipids, nucleotides, amino acids and organic acids. Secondary plant metabolites are structurally diverse and many are distributed among a limited number of plant species. Some of these compounds were found to have a key role in the protection of plants in several ways. Moreover, there are increasing evidences that modest long-term intakes of some specific classes of these compounds can favorable reduce and/or prevent the incidence of cancers and many chronic diseases such as cardiovascular disease, neurodegenerative disease, type II diabetes and hypertension, as well as the ageing process. Plant secondary metabolites can be grouped, based on their biosynthetic formation, into four groups: phenolic compounds, terpenoids, alkaloids and sulphur-containing compounds. Phenolic compounds are of great interest mainly due to their bioactive functions involved in human health-related issues. Oxidative stress and human health, namely in the pathogenesis of various diseases and disorders are related in different ways. Under stress, the human body will produce more harmful species, such as reactive oxygen species (ROS) than enzymatic antioxidants and nonenzymatic antioxidants, inducing cell damage. This effect is increased when there are not enough antioxidants to quench these harmful radicals.


viii

David Alexandre Micael Pereira

During a large period, artificial antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (Pg) and tert-butyl hydroquinone (TBHQ) were used as additives in foods and beverages. However, their use is now restricted since they are associated with high levels of cytotoxicity and carcinogenic effects. Therefore, there is a major need to find natural compounds with antioxidant properties and low toxicity associated. An antioxidant can be defined as a compound that inhibits or significantly delays the oxidation of substrates even if the compound is present in lower concentration than the oxidized substrate. Phenolic compounds and aromatic amines are free-radical scavengers and also present reducing properties. In this paper, the authors present an overview on phenolic compounds and their relation with antioxidant capacity of medicinal plants. Methods for extraction, detection and quantification of phenolic compounds and antioxidant capacity assays are revised and examples of important medicinal plants are presented. Chapter 2 - Reactive oxygen species (ROS) play a crucial role in human health. At low, regulated levels, ROS are involved in many vital physiological processes. They have a role in various signaling cascades, such as response to growth factor stimulation and control of inflammatory responses. They participate in the regulation of many cellular processes, including differentiation, proliferation, growth, apoptosis, cytoskeletal regulation, migration, and contraction. However, ROS also play an important role in a wide range of pathologies and many implicated diseases that are leading causes of death. It is common knowledge that plant-derived foods contain hundreds of active antioxidant compounds, including ascorbic acid, tocopherols, carotenoids, and a wide range of phytochemicals such as polyphenols. Many in vitro and animal studies have shown that a large range of dietary antioxidants, taken as extracts or as food components, have beneficial effects because they modulate oxidative stress and protect against oxidative damage and its complications. Dietary polyphenols have received a lot of attention from nutritionists, food scientists and consumers due to the role they play in human health. Polyphenols can induce antioxidant enzymes such as glutathione peroxidase, catalase and superoxide dismutase, which respectively decompose hydroperoxides, hydrogen peroxide and superoxide anions, also inhibiting the expression of enzymes such as xanthine oxidase. Medicinal plants are traditionally used in folk medicine as natural healing remedies with therapeutic effects such as the prevention of cardiovascular diseases, inflammation disorders, or reducing the risk of cancer. The antioxidant properties of medicinal plants depend on the plant, its variety, environmental conditions, climatic and seasonal variations, geographical regions of growth, degree of ripeness, growing practices, and many other factors such as post-harvest treatment and processing. The present chapter is limited to commonly consumed fruits and vegetables with significant nutritional and antioxidant beneficial effects in folk medicine. Here, the authors discuss the phytochemistry and antioxidant pharmacological properties of the following plant species: apple, berries, cranberry, grape, grapefruit, mango, orange, papaya, pomegranate, tangerine, avocado, broccoli, cactus, cauliflower, carrot, pepper, spinach, tomato, and watercress. The present chapter evidences the authors’ knowledge of the therapeutic properties of the antioxidant qualities of some fruits and vegetables is limited and seeks to provide an overall clear view of the antioxidant role of common fruits and vegetables, along with their health and diseasereduction benefits.


Preface

ix

Chapter 3 - Medicinal plants are specifically used for their contents of bioactive compounds, which are products of plant secondary metabolism with proven beneficial effects on human health. These substances are known to play a key role in the mechanisms of plant adaptation to the environment; they generally exhibit antioxidant properties and often act as defense molecules that are synthesized by plants in response to stress conditions. In the last decades, the interest by pharmaceutical companies towards the production of bioactive compounds from medicinal plants has considerably increased, especially in developed countries, in consideration of the consumers’ sensibility towards naturally sourced remedies. As a consequence, the traditional harvesting from the wild has become inadequate to sustain the market demand, and medicinal plants are increasingly cultivated on a commercial scale. On the other hand, the market requirement for standardized plant material cannot be fully satisfied by field crops, which are highly susceptible to year-to-year variability. Greenhouse hydroponics can contribute to overcome the drawbacks of conventional field cultivation, as it ensures a fast plant growth and allows both to control the growing environment and to change the composition of the nutrient solution that is fed to the plants. The application of a stress condition through a proper manipulation of the nutrient solution can stimulate secondary metabolism and promote the synthesis and accumulation of bioactive substances in plant tissues. This chapter presents some fundamental issues concerning the hydroponic production of raw plant material for the extraction of bioactive compounds. Literature data are reported on recent research concerning the hydroponic growing of medicinal plants, both under optimal conditions or under stress conditions to stimulate the production of secondary metabolites. Finally, basil is presented as a case study for the application of the hydroponic technique to the production of plant material for the extraction of rosmarinic acid, a bioactive secondary metabolite of well-known antioxidant activity. The present chapter points out the opportunities offered by the hydroponic growing of medicinal plants for the agro-industrial production of bioactive compounds. On the other hand, it also underlines the lack of information concerning the specific growing needs of the individual medicinal species. Despite the fact that at present a lot of molecules of pharmaceutical interest can be obtained from hydroponically-grown medicinal plants, suitable growing protocols are still required. Chapter 4 - Metabolic disorders, including diabetes and obesity, have been strongly associated with oxidative stress, due to a disproportionate release of free radicals, during the metabolism of excessive glucose and free fatty acids. Enhanced production of reactive oxygen species (ROS) and perturbed antioxidant defenses determine the chemical changes in virtually all cellular components resulting in their damage. ROS is generated through several mechanisms including oxidative phosphorylation, glucose auto-oxidation, advanced glycation end product (AGE) formation, activation of protein kinase C (PKC), nitric oxide synthase (NOS) and aldose reductase pathway among others. They also act as secondary messengers in the regulation of several intracellular signaling pathways. The most promising strategy to mitigate the effect of ROS induced oxidative damage is through the use of antioxidant molecules. Antioxidants, usually phytochemicals and micronutrients called as quenchers act either directly by free radical scavenging mechanisms or indirectly by enhancing the antioxidant status (enzymatic and non-enzymatic). As diabetes and obesity conditions initiate generation of free radicals, compounds that can manage these conditions


x


serve to be effective against these diseases and their complications. In this perspective, therapeutic intervention with the ability to reduce oxidative stress can impede or delay the onset of the metabolic disorder. Thus, agents possessing dual effect such as antidiabetic/anti-obesity and antioxidant activity are greatly in demand. The therapeutic effect of phytochemicals found in natural products to combat oxidative stress is gaining significance as they are recognized to be safe with a wide range of biological and pharmacological activities. Dietary components from plants such as polyphenols (flavonoids), terpenes and tannins are ubiquitous in nature and can effectively scavenge reactive oxygen and nitrogen species, thus, modulating the genes associated with metabolism and stress defense. This chapter discusses the sources of flavonoids, their potential antioxidant properties and the mechanism through which they exert their pharmacological effects in diabetes and obesity. Chapter 5 - The prevalence of overweight and obese individuals is increasing at an alarming rate across the globe. Obesity has become one of the most important avoidable risk factors for morbidity and mortality. The associated risks with obesity are cancer, diabetes and heart diseases. According to the World Health Organization, obesity is defined as abnormal or excessive fat accumulation that may impair health. In 2008, more than 1.4 billion adults were overweight and more than half a billion were obese. At least 2.8 million people die each year as a result of being overweight or obese. A person is considered obese if they possess a body mass index (BMI; a ratio of height to weight) greater than 30 whereas a healthy BMI should be 18.5 to 24.9. Obesity is the leading cause of death which can be prevented by diet and lifestyle modifications. Although the exact link between obesity and its associated risks is not clear, it is known that increased production of reactive oxygen species (ROS) is associated with cellular damage, including oxidation of cell membranes and proteins in conjunction with disturbances of cellular redox homeostasis. Free radicals are known to be involved in a number of human pathologies including atherosclerosis, cancer and hypertension. Studies have shown that obesity promotes increased plasma lipid peroxidation. Obesity also increases the mechanical and metabolic loads on the myocardium, thus increasing myocardial oxygen consumption. Therefore, antioxidants are capable of reversing these pathways and, in fact, can be helpful in preventing the deleterious effects caused by reactive oxygen species. However, antioxidants do not reduce obesity per se. Antioxidants are widely present in the plant kingdom and are known to prevent various disorders. Flavonoids, especially flavones, flavonols, flavanones, flavanols (catechins), anthocyanins, isoflavones and chalcones, are considered effective antioxidants associated with other pharmacological properties such as anti-cancer, anti-diabetic, anti-mutagenic, antithrombotic, anti-inflammatory and anti-HIV activities. Many studies have indicated that phenolic compounds such as o-coumaric acid, EGCG, esculetin, genistein, procyanidin, pycnogenol, rutin, and tea catechins, carnitine, CoQ10, choline, inositol and various herbs are effective in reducing obesity and promoting weight loss. This review will focus on recent examples of antioxidant nutrients, traditional medicines and foods that have been validated by scientific evaluation for controlling obesity or promoting weight loss. Chapter 6 - The use of medicinal plants represents the oldest and most common form of medication. Among the hundreds of studies published in the last two decades on medicinal plants research, the quest for new antioxidant drugs has a been pivotal. Some of those plants with antioxidant activity, as well as their bioactive components, have been in some cases further analyzed for a hypothetical anti-hemolytic potential. Although oxidative stress is not the primary etiology of diseases such as hemolytic anemias, it is believed to aggravate them.


Preface

xi

Therefore, the use of natural antioxidants, either as additives or as pharmaceutical supplements, may prevent or at least slow down free radical reactions that are responsible for provoking damage to essential red blood cell molecules. In this Chapter, the authors review the current knowledge regarding the use of medicinal plants as anti-hemolytic agents. Particular emphasis in the compounds responsible for this activity, as well as the mechanism of action is given. Chapter 7 - Anatolia - the westernmost protrusion of Asia, is at the forefront of the world richest sources of plant species. The mountainous and strongly fragmentized area is a home to over 11,000 plant species, of which 30% are endemic. Many of the world’s contemporary staple foods originated here. The extensive daily use of local plants for foods and medicine in Eastern Anatolia continues today and traditionally used plants outnumber the conventional sources of plant-based foods. Endemic plants are utilized daily in preparation of main meals, in salads and as herbal teas. They are used internally (e.g. herbal tea) and externally (e.g. poultice, decoction, ointment) to cure a number of ailments. This chapter presents the most frequently used traditional plants from the Eastern Anatolia and describes their uses, phytochemical compositions and antioxidant capacities. Their applications in ethnopharmacology in light of scientifically proven physiological activities are discussed. Chapter 8 - Hetherotheca inuloides (Mexican arnica) is a plant used in traditional medicine in different parts of the world; it is used in various presentations (tablets, beverages, ointments) for therapeutic purposes due to its anti-inflammatory, antimicrobial, analgesic, and antioxidant effects. As an antioxidant, it has attracted considerable interest because of the involvement of oxidative stress in various diseases affecting systemic and central levels. In particular, the focus of this chapter is to describe the evidence that demonstrates the ability of Mexican arnica to be used as a potent antioxidant, and how it can help protect the liver and brain, in experimental models affecting these organs. Chapter 9 - As the second-largest genus in the family Papaveraceae, Meconopsis comprises about 57 species among which 32 species are distributed in Qinghai-Tibet Plateau. The plants of Meconopsis have been prescribed as popular Tibetan medicine for the treatment of tuberculosis and hepatitis. The chemical constituents have been examined and the isolation of alkaloids, flavonoids and essential oils has been reported. Pharmacological activities include hepatoprotection and analgesic effects. The phytochemical and pharmacological studies on medicinal plants of Meconopsis genus have been reviewed in this chapter. Chapter 10 - Jamaica’s flora has a rich source of medicinal plants, with over 2900 species of identified flowering plants; 1788 plants have been identified to contain two or more bioactive compounds, of these 51 possessing anti-oxidant properties. Herbal medicine has been the source for many pharmaceutical and nutraceutical products based on well-researched and developed ethno-medicinal practices worldwide. It provides an alternative method for the management of various diseases, such as cancer, diabetes, hypertension among others, which are all alleviated by antioxidant compounds. Anti-oxidant activity is often times found in those plants that are edible and as such is able to alleviate oxidative stress when eaten. The Petiveria alliacea (guinea hen weed), the green coffee beans decoction (Coffee arabica), infusion, Hibiscus sabdariffa (Jamaican sorrel), the Eupatorium odoratum (jack in the bush) and Momordica charantia (cerasee) are among the common plants used on the island to promote oxidative relief.


xii


These plants are often administered in many different ways, including decoctions, macerations, infusions, tinctures or by cooking. Due to their usefulness as medicinal plants tissue culture has been used as a part of the conservation strategies that have been employed in preserving and maintaining the island’s flora.


In: Medicinal Plants Editor: David Alexandre Micael Pereira

ISBN: 978-1-62948-219-4 © 2014 Nova Science Publishers, Inc.

Chapter 1

Phenolic Compounds and Antioxidant Capacity of Medicinal Plants: A Review Sandra C. Gouveia, Vítor Spínola and Paula C. Castilho Centro de Química da Madeira, Departamento de Química, Universidade da Madeira, Campus Universitário da Penteada, Funchal, Portugal

Abstract Plants have been used for medicinal purposes since the origin of human civilization and their uses were described by the great civilizations of the ancient Chinese, Indian and Mediterranean. Nowadays, they continue to be the source of new medicines either by providing lead molecules or as natural herbal products (teas, tinctures, powders, poultices, infusions as well as other formulations). 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 preparations. Compounds produced by plants are divided in two groups: primary and secondary metabolites. Primary metabolites are compounds that possess fundamental roles in plant development steps such as phytosterols, acyl lipids, nucleotides, amino acids and organic acids. Secondary plant metabolites are structurally diverse and many are distributed among a limited number of plant species. Some of these compounds were found to have a key role in the protection of plants in several ways. Moreover, there are increasing evidences that modest long-term intakes of some specific classes of these compounds can favorable reduce and/or prevent the incidence of cancers and many chronic diseases such as cardiovascular disease, neurodegenerative disease, type II diabetes and hypertension, as well as the ageing process. Plant secondary metabolites can be grouped, based on their biosynthetic formation, into four groups: phenolic compounds, terpenoids, alkaloids and sulphur-containing compounds. Phenolic compounds are of great interest mainly due to their bioactive functions involved in human health-related issues.


2

Sandra C. Gouveia, Vítor Spínola and Paula C. Castilho Oxidative stress and human health, namely in the pathogenesis of various diseases and disorders are related in different ways. Under stress, the human body will produce more harmful species, such as reactive oxygen species (ROS) than enzymatic antioxidants and non-enzymatic antioxidants, inducing cell damage. This effect is increased when there are not enough antioxidants to quench these harmful radicals. During a large period, artificial antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (Pg) and tert-butyl hydroquinone (TBHQ) were used as additives in foods and beverages. However, their use is now restricted since they are associated with high levels of cytotoxicity and carcinogenic effects. Therefore, there is a major need to find natural compounds with antioxidant properties and low toxicity associated. An antioxidant can be defined as a compound that inhibits or significantly delays the oxidation of substrates even if the compound is present in lower concentration than the oxidized substrate. Phenolic compounds and aromatic amines are free-radical scavengers and also present reducing properties. In this paper, we present an overview on phenolic compounds and their relation with antioxidant capacity of medicinal plants. Methods for extraction, detection and quantification of phenolic compounds and antioxidant capacity assays are revised and examples of important medicinal plants are presented.

Keywords: Phenolic compounds, Antioxidant, Medicinal plants

Introduction 1. Medicinal Plants The use of plants in medicine is reported since the origin of human civilizations (Phillipson, 2001). Medicinal plants can be used in the form of crude drugs such as teas, tinctures, powders, poultices and infusions, as well as other formulations (Balunas and Kinghorn, 2005). The consumption of herbal products in the more affluent countries has increased in the past decades. In Europe, Germany is the country with the highest share of the herbal medicines market and it was reported that the sales of herbal medicinal products (HMPs) in 1997 were US$ 1.8 billion (Phillipson, 2007). Herbal treatments are still the most popular form of traditional medicine, and are highly lucrative in the international marketplace. Annual revenues in Western Europe reached US$ 5 billion in 2003-2004. In China, sales of products totaled US$ 14 billion in 2005. Herbal medicine revenue in Brazil was US$ 160 million in 2007. (cf. Traditional medicine Fact sheet N°134 December 2008, in: http://www.who.int/ mediacentre/factsheets/fs134/en/; accessed on 21st March 2011). The public access to these herbal medicinal products (HMP) led to the need of up-to-date monographs and to the use of standardized materials to prevent adverse effects including drug interactions in patients taking other over-the-counter or prescription medicines. The United States of America (USA) and Europe (EU) have been doing a great effort to regulate and license the commercialization of medicinal herbs to those patients who request to be treated with these products (Gurib-Fakim, 2006).


Phenolic Compounds and Antioxidant Capacity of Medicinal Plants

3

At the international level, the WHO has developed a strategy to review traditional medicines which includes a program to develop monographs for herbal ingredients. According to a WHO Fact Sheet published in 2008 (cf. above), in some Asian and African countries, 80% of the population depend on traditional medicine for primary health care. In many developed countries, 70% to 80% of the population has used some form of alternative or complementary medicine (e.g. phytotherapy or acupuncture) (Phillipson, 2001). In EU, the European Scientific Cooperative on Phytotherapy (ESCOP, formed in 1989) has as its main goal to advance the scientific status of phytomedicine and to assist with the harmonization of their regulatory status at the European level. ESCOP produces state-of-theart reviews of the therapeutic use of herbal medicinal products based on leading expertise across Europe. In the USA, the Food and Drug Administration (FDA) has responsibility for both food and drug products (Gurib-Fakim, 2006). In June 2008, following a public consultation, the FDA Scientific Committee published a guidance document for the safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements. In the EU, the EFSA Scientific Cooperation (ESCO) Working Group was created to advice on the adequacy of the proposed approach for the safety assessment of botanicals preparations. Standardization is a system that guarantees a minimum level of active components in the extract and is becoming increasingly important as a means of ensuring a consistent supply of high-quality phyto-pharmaceutical products. It can be defined as the establishment of reproducible pharmaceutical quality by comparing a product with established standard compounds and by defining minimum amounts of one or several compounds. In the field of phyto-medicines, standardization only applies to extracts. Standards for active ingredients to be used in medicinal products may be found in monographs and/or pharmacopeas (GuribFakim, 2006). Standardization permits comparison of the clinical effectiveness, pharmacological effects and side effects of a series of products (for example, against a placebo). Standardized products provide more security and increase the level of trust people have in herbal drugs. In summary, traditional medicine is the combination of knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures that are used to maintain health, as well as to prevent, diagnose, improve or treat physical and mental illnesses. 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 preparations. Pharmacognosy has provided information on pure natural compounds and foods with health benefits (Phillipson, 2007) and seeks the search for new drugs from natural sources combining different fields such as phytochemistry, microbial chemistry, biosynthesis, biotransformations, organic and analytical chemistry, among others. Wherever we are, trends to the rational use of medicinal plants dominate the measures being implemented by health and food authorities. At its essence, the traditional medicine must be supported by the isolation, characterization, synergy determination and validation of mode of action of active substances, and the knowledge of new biological active compounds is still a challenge since only a small percentage of plants over the world have been fully studied in a scientific manner.


4

Sandra C. Gouveia, Vítor Spínola and Paula C. Castilho

2. Phytochemicals in Plants Compounds produced by plants are divided in two groups: primary and secondary metabolites. Primary metabolites are compounds that have fundamental roles in plant development steps (photosynthesis, respiration and growth). Phytosterols, acyl lipids, nucleotides, sugars, amino acids and organic acids are examples of primary metabolites. Secondary plant metabolites are structurally diverse and numerous but are distributed among a very limited number of plant species. They represent an expression of the individuality of species, although in some cases they can be found in high concentrations. Some of these compounds were found to have a key role in the protection of plants in several ways (Crozier et al., 2006). In the lifecycle of the plant, they can assist reproduction by attracting pollinators, act as deterrents against herbivores and/or provide protection against harmful sun radiation. Some also have a role in human wellbeing. Modest long-term intakes of some specific classes of these compounds can favorable reduce and/or prevent the incidence of cancers and many chronic diseases such as cardiovascular disease, neurodegenerative disease, type II diabetes and hypertension, as well as the ageing process (Katalinic et al., 2010). Plant secondary metabolites can be grouped, based on their biosynthetic formation, into four groups: phenolic compounds, terpenoids, alkaloids and sulphur-containing compounds. Phenolic compounds are the most investigated, due to their bioactive functions involved in human health-related issues.

2.1. Phenolic Compounds Phenolic compounds are a class of low and medium molecular weight secondary metabolites biosynthesized both during normal plant development and in response to stress conditions, such as infection, wounding and UV radiation (Naczk and Shahidi, 2006). The chemical structure of phenolic compounds is characterized by the presence of at least one aromatic ring with one or more hydroxyl group attached. They are classified based on the number and arrangement of the carbon atoms of the basic structure (Table 1) and can be found in the free form or conjugated to sugar and organic acids residues. They can also be classified into different groups as a function of their number of phenol rings, as well as in the structural elements that bind these rings to one another. Distinctions are thus made between flavonoids, phenolic acids (e.g. hydroxybenzoic and hydroxycinnamic acids), stilbenes, lignins, lignans and proanthocyanidins. There are more than 5000 phenolic compounds described (Pyrzynska and Biesaga, 2009). 2.1.1. Flavonoids The term “flavonoid” is generally used to describe an extensive collection of natural products that include a C6-C3-C6 carbon framework or, more specifically, phenylbenzopyran functionality. Depending on the position of the linkage of the aromatic ring to the benzopyran (chroman) moiety, this group of natural products may be divided into three classes (Figure 1): the flavonoids (2-phenylbenzopyrans) 1, isoflavonoids (3-benzopyrans) 2, and the



5

neoflavonoids (4-benzopyrans) 3. These groups usually share a common chalcone precursor and therefore are biogenetically and structurally related (Marais et al., 2006). Table 1. Classification of phenolic compounds. Adapted from (Crozier et al., 2006) Number of carbons

Skeleton

Classification

Example

7

C6-C1

Phenolic acids

Gallic acid

8

C6-C2

Acetophenones

Gallacetophenone

8

C6-C2

Phenylacetic acid

p-Hydroxyphenyl-acetic acid

9

C6-C3

Hydroxycinnamic acids

p-Coumaric acid

9

C6-C3

Coumarins

Esculetin

10

C6-C4

Naphthoquinones

Juglone

13

C6-C1-C6

Xanthones

Mangiferin

14

C6-C2-C6

Stilbenes

Resveratol

15

C6-C3-C6

Flavonoids

Naringenin

Basic structure

The basic flavonoid skeleton is planar and may occur in several modified forms corresponding to additional hydroxylation, methylation and/or glycosylation. It is also possible to have aromatic and aliphatic acids, sulfate, prenyl, methylenedioxyl or isoprenyl groups attach to the flavonoid structure and its glycosides. The water solubility of flavonoids increases with the presence of glycoside and hydroxyl groups; however, methyl groups and isopentyl units turn flavonoids lipophilic.


6


The basics structures of the main classes of flavonoids are presented in Figure 2: Flavones, flavonols, flavan-3-ols, isoflavones, flavanones and anthocyanidins are the most abundant and dihydroflavonols, flavan-3,4-diols, coumarins, chalcones, dihydrochalcones and aurones are much less present in the common components of the human diet (Cuyckens and Claeys, 2004).

Figure 1. Types of flavonoids (Marais et al., 2006).

A. Flavonols Flavonols are characterized by an unsaturated 3-C chain with a double bond between C-2 and C-3 and by the presence of a hydroxyl group in position 3 (Figure 2). Conjugation commonly occurs at the 3- and 7-positions of the A-ring although substitutions at the 5, 7, 4’, 3’ and 5’ positions of the carbon ring have also been reported. Flavonols are the most abundant flavonoids and more than 450 flavonol aglycones are known; however, the number of flavonol conjugates is much higher due to the great number of glycosides moieties combinations. Quercetin, kaempferol and isorhamentin are the most common flavonol-type flavonoids found in fruits and vegetables (Prasain et al., 2004). B. Flavones Flavones are structurally similar to flavonols with a double bond between C-2 and C-3 but they lack hydroxylation at position 3. These compounds also present a variety of substitutions like hydroxylation, methylation, O- and C-alkylation, and glycosylation (Manach et al., 2004). The most common conjugated flavones are 7-O-glycosides and the Cglycosylation occurs mainly at C-8 and C-6 positions (Figure 2) (Cuyckens and Claeys, 2004). However, these types of flavonoids only appear in a few families of plants. Apigenin and luteolin are the major flavones found in the human diet, in grains, leafy vegetables and herbs (Zhang et al., 2010). C. Isoflavones Isoflavones possess the B-ring linked at the C-3 rather than the C-2 position (as in flavones) (Figure 2). They have structural similarities to estrogens but they are not steroids and normally have hydroxyl groups in C-7 and C-4’ positions in a configuration analogous to that of the hydroxyls in the estradiol molecule (Manach et al., 2004). O-glycosylation occurs with sugar groups linked preferentially to the 7-position of A-ring. This confers pseudo hormonal properties to these compounds, including the ability to bind to estrogens receptors. As such, they are consequently classified as phytoestrogens.



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Isoflavones are widely distributed in the plant kingdom but are found at high levels only in plants of the Leguminosae family, such as soybeans and their processed products (Naczk and Shahidi, 2006). Regarding the type of substitution on carbons C-5 and C-6, three main isoflavones aglycones are known: daidzein, genistein and glycitein (Luthria et al., 2007). These three aglycones can also occur in their acetyl, malonyl and hexoside forms.

Figure 2. Basic structures of the main classes of flavonoid. Common O- and C-glycosylation positions are indicated with an arrow (Cuyckens and Claeys, 2004).

D. Flavanones This type of flavonoid is characterized by the absence of a double bond between the C-2 and C-3 carbons of the B-ring, which gives an asymmetric carbon (C-2) as a chiral center (Figure 2). A large number of flavanones have the C-ring attached to the B-ring at C-2


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position with an α-configuration (Crozier et al., 2006). Hydroxylation, glycosylation and methylation are normal types of substitution for flavanones aglycones on the 7-position. These compounds are present in edible species and are found in tomatoes and certain aromatic plants such as mint, but are very rare in fruits with exception of the Citrus genus, where they can be found in high concentrations (Peterson et al., 2006). The main aglycones of flavanones are naringenin in grapefruit, hesperetin in oranges, and eriodictyol in lemons. The typical bitter taste of grapefruit is related to the glycosylation with neohesperidose of the flavanone aglycones, at the C-7 position: naringenin-7-O-neohesperidose. Rutinose conjugated flavanones are tasteless, like hesperitin-7-O-rutinoside (hesperidin) and naringenin-7-O-rutinoside in oranges. E. Flavan-3-ols This is the most complex type of flavonoids, varying from the simple monomers (+)catechin and its isomer (-)-epicatechin, to the oligomeric and polymeric proanthocyanidins (condensed tannins). Flavan-3-ols, proanthocyanidins and flavanones are molecules of low polarity due to the saturated bond between the C-2 and C-3 carbons in the C-ring. The two asymmetrical carbons C-2 and C-3 produce four isomers for each level of B-ring hydroxylation. These compounds do not present glycosylated forms in foods but can be hydroxylated to form the gallocatechins and can be esterified with gallic acid. Catechins are found largely in green tea but also in fruits, vegetables, red wine and chocolate (Manach et al., 2004). F. Anthocyanidins Anthocyanidins, mainly their glycosides and acylglycosides derivatives denominated anthocyanins, are the most important pigments in plants and fruits (red, blue and purple colors). They are glycosides of polyhydroxy and polymethoxy derivatives of 2phenylbenzopyrylium or flavylium salts (Figure 3) (Kong et al., 2003).

Figure 3. The flavylium cation. R1 and R2 are H, OH, or OCH3; R3 is a glycosyl or H; and R4 is OH or a glycosyl.

Anthocyanidins are more unstable than anthocyanins. The most important anthocyanidins are cyanidin, pelargonidin, delphinidin, peonidin and malvidin which are found as their glycosides in plants (Guzmán et al., 2009). Anthocyanidins always present a sugar moiety at the C-3 position and frequently on C-7, C-3’ and C-5’. Conjugation with hydroxycinnamates and organic acids is also common. In certain products, such as matured red wines and ports, chemical and enzymatic



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transformations occur and increase the number of ‘anthocyanin-derived polyphenols’. This contributes to the increase of the total intake of dietary phenols (Crozier et al., 2006). 2.1.2. Non-Flavonoids The main non-flavonoid phenolic compounds found in nature are the C6–C1 hydroxybenzoates, most notably gallic acid, which is the precursor of hydrolysable tannins, the C6–C3 hydroxycinammates and their conjugated derivatives, and the polyphenolic C6– C2–C6 stilbenes. Phenolic acids consist of two subgroups, the hydroxybenzoic and hydroxycinnamic acids and exist primarily as conjugates and are rarely found in their acidic forms, often found bound to alcohols, sugars, polysaccharides, or organic acids through ester bonds (Ignat et al., 2011). A. Hydroxybenzoates Hydroxybenzoate compounds include p-hydroxybenzoic, gallic, protocatechuic, vanillic, gentisic and syringic acids (Table 2) (Parveen et al., 2008). These compounds may be found in plants in their soluble form conjugated with sugar groups or organic acids and, in some cases, bound to cell wall fractions (lignin). The principal hydroxybenzoate is gallic acid, biosynthesized from phenylalanine via 3dehydroshikimic acid. Gallic acid can be converted to ellagic acid, which is the base unit for a wide range of gallotannins, forming hydrolysable tannins (polymers of gallic and ellagic acids). The content of these compounds in edible plants is generally very low, with exception of certain red fruits, black radish and onions. For example, tea is an important source of gallic acid (4.5 g/Kg fresh wt) (Manach et al., 2004). Table 2. Chemical structures of the most common hydroxybenzoates

R1

R2

Compound

H

H

p-Hydroxybenzoic acid

OH

OH

Gallic acid

H

OH

Protocatechuic acid

H

OCH3

Vanillic acid

OCH3

OCH3

Syringic acid

B. Hydroxycinnamates This class of compounds presents a much higher quantity and diversity rather than hydroxybenzoates. Cinnamic acid is a C6–C3 phenolic acid that is converted to a wide range of hydroxycinnamates. These are products of the phenylpropanoid pathway and are generally designated as phenylpropanoids (Crozier et al., 2006).


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The most important hydroxycinnamates are caffeic, p-coumaric, ferulic acids and their derivatives (Table 3). Hydroxycinnamates usually occur in several conjugated forms such as esters of hydroxyacids like quinic, shikimic and tartaric acid, as well as their sugar derivatives. Table 3. Chemical structure of three common hydroxycinnamates

R1

Compound

OH

Caffeic acid

H

p-Coumaric acid

OCH3

Ferulic acid

Caffeic acid occurs mainly as esters of quinic acid and the whole group of related isomers is generally denominated as “chlorogenic acids”. The true chlorogenic acid is 5-Ocaffeoylquinic acid (Figure 4). The number of caffeoyl moieties, their location and relative isomer abundance is often characteristic of a species. Coffee is a major dietary source of chlorogenic acids with intakes estimated at 0.5-1 g/day (Clifford, 2000).

Figure 4. Chlorogenic acid, 5-O-Caffeoylquinic acid structure.

C. Stilbenes This group of phenolic compounds has a C6-C2-C6 structure and is known to act as phytoalexins, antibiotic compounds produced as part of a plant's defense system against disease (fungal, bacterial and viral pathogens attacks). They occur in diversified sources like grapes, blueberries, cranberries, hops, peanuts, strawberries, red currants and some other botanical sources (Lee and Rennaker, 2007). Resveratrol (3,5,4-trihydroxy-stilbene) is the most common stilbene and occurs as both cis and trans isomers (Figure 5). In plants tissues, it is present primarily as trans-resveratrol3-O-glucoside (piceid). Grapes, peanuts and their products are considered the most important dietary sources of resveratrol. Trans-resveratrol and its hexoside are also present in high amounts in Polygonum cuspidatum (Japanese knotweed) (Crozier et al., 2006).



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Figure 5. Structures of trans and cis-Resveratrol.

3. Functions and Biosynthesis of Phenolic Compounds Phenolic compounds play different roles in plant physiology, against attacks by pathogens, herbivores and UV radiation of the sun (flavonoids). This type of compounds is also associated to the plant morphology (color and mechanical support in the case of lignin), growth (nutrient uptake, protein synthesis and enzyme activity) and reproduction (flavones, flavonols and anthocyanidins colors may attract pollinators) (Stalikas, 2007). The biosynthesis of phenolic compounds, namely flavonoids, hydroxycinnamates and phenolic acids involves a complex network of routes based principally on the shikimate, phenylpropanoid and flavonoid pathways. Phenylalanine, produced in plants via the shikimate pathway, is a common precursor for most phenolic compounds in higher plants (Crozier et al., 2006).

3.1. Phenolics and Hydroxycinnamates According to Crozier et al. (2006) gallic acid appears to be formed primarily via the shikimic acid pathway from 3-dehydroshikimic acid (Figure 6) although there are alternative routes from hydroxybenzoic acids. Enzyme studies with extracts from oak leaves have shown that gallic acid is converted to β-glucogallin which, in turn, is converted via a series of position-specific galloylation steps to penta-O-galloyl-glucose. Penta-O-galloyl-glucose is a pivotal intermediate that is further galloylated resulting in the synthesis of gallotannins and ellagitannins, the hydrolysable tannins (Crozier et al., 2006). Another possibility is that 3-dehydroshikimic acid to be directed to L-phenylalanine and start the phenylpropanoid pathway (Salminen and Karonen, 2011). Consecutive enzyme reactions give cinnamic acid, p-coumaric acid and p-coumaroyl-CoA. Cinnamic acid can also be metabolized to benzoic acid and salicylic acid. p-Coumaric acid is also metabolized via a series of hydroxylation and methylation reactions forming caffeic, ferulic, 5-hydroxyferulic and sinapic acids (Gallego-Giraldo et al., 2011; Lallemand et al., 2012; Shadle et al., 2007). Initially, caffeic acid was considered as the immediate precursor of 5-O-caffeoylquinic acid. However, recent molecular biology studies (Hoffmann et al., 2004) indicate that the main route to 5-O-caffeoylquinic acid, and presumably related caffeoylquinic acids, is from p-coumaroyl-CoA via 5-O-p-coumaroylquinic acid (Figure 6). p-Coumaroyl-CoA is also a vital intermediate leading to the synthesis of flavonoids and stilbenes.


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3.1.1. Flavonoids The C6–C3–C6 flavonoid structure is the product of two separate biosynthesis pathways (Figure 7). The bridge and the B-ring represent a phenylpropanoid unit synthesized from pcoumaroyl-CoA. The six carbons of ring-A originate from the condensation of three acetate units via the malonic acid pathway) (Crozier et al., 2006).

Figure 6. Schematic of the main pathways and key enzymes involved in the biosynthesis of hydrolysable tannins, salicylic acid, hydroxycinnamates and 5-O-Caffeoylquinic acid. Enzyme abbreviations: PAL, phenylalanine ammonia-lyase; BA2H, benzoic acid 2-hydroxylase; C4H, cinnamate 4hydroxylase; COMT-1, caffeic/5-hydroxyferulic acid O-methyltransferase; 4CL, coumarate CoA ligase; F5H, ferulate 5-hydroxylase; GT, galloyltransferase; ACoAC, acetylCoA carboxylase. Adapted from Crozier et al.,2006.

The conjugation of these two parts in a reaction catalysed by chalcone synthase (CHS) results in naringenin-chalcone. Isoflavones are produced in a slightly modified pathway through isoliquiritigenin, which lacks a 2’-hydroxyl group. The stereospecific conversion of naringenin-chalcone to naringenin by chalcone isomerase (CHI) is the central point of the flavonoid biosynthetic pathway. From this point several side branches are formed resulting in the production of different classes of flavonoids such as isoflavones, flavanones, flavones, flavonols, flavan-3-ols and anthocyanins (Figure 8) (Crozier et al., 2006).



Figure 7. Biosynthetic origin of the flavonoid skeleton.

F3H

Figure 8. Schematic of the main pathways and enzymes involved in the biosynthesis of stilbenes and flavonoids. Enzyme abbreviations: SS, stilbene synthase; CHS, chalcone synthase; CHR, chalcone reductase; CHI, chacone isomerase; IFS, isoflavone synthase; FNS, flavones synthase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin 4-reductase; F3H, flavanone 3hydroxylase; F3’H, flavonol 3’-hydroxylase; LAR, leucocyanidin 4-reductase; LDOX, leucocyanidin deoxygenase; ANR, anthocyanidin reductase; EU, extension units; TU, terminal unit (Adapted from Crozier et al., 2006).


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4. Phenolic Compounds and Health Benefits Since ancient cultures, phenolic compounds have been used in different medicinal applications. For example, the willow bark was used in the centuries B.C. to ease aches and pains, reduce fevers and inflammation; the substance responsible for these properties is a phenolic compound – salicin (isolated by Henri Leroux in 1829) (Mahdi, 2010). Nowadays, the incidence of chronic and degenerative diseases (such as cardiovascular disease, type II diabetes and some types of cancer) can be significantly reduced by changing lifestyle, in particular the diet. In the past decades, several studies were performed in order to correlate the consumption of high levels of dietary phenolic compounds and flavonoids (mainly in fruits and vegetables) to the reduction of degenerative diseases (Fang et al., 2007; Stalikas, 2007). The mode of action of phenolic compounds was initially thought to be due to direct scavenging of free radicals (reactive oxygen species) (Fraga, 2009; Soto et al., 2011; Vermerris and Nicholson, 2006). However, several recent studies have proven that interactions between various phytochemicals with different modes of action can increase efficacy and minimize toxicity (Mertens-Talcott et al., 2003). In particular, certain flavonoids have been shown to interact in the cancer development stages of initiation and promotion/progression. Among these flavonoids there are chalcones, flavanones, flavonols, flavones and isoflavones (Tringali, 2000). The bioavailability of phenolic compounds in foods (fruits, teas, vegetables, grains, coffees, spices, etc.) allows humans to consume them on a daily basis. The estimated range of consumption is 25 mg to 1 g a day, depending on diet (Stalikas, 2007). Accumulated evidence on the absorption and bioavailability of phenolic compounds and flavonoids in humans reveals that most of these compounds are modified during absorption and the metabolites that reach the cells and tissues are chemically and/or functionally distinct from the dietary sources (Fraga, 2009). Flavonoid glycosides are poorly absorbed until they have undergone hydrolysis by bacterial enzymes in the intestine. The resulting aglycones can be absorbed. However, recent studies suggest that a fair degree of absorption of flavonol glycosides can also occur in the small intestine (Stalikas, 2007). Even if negative effects have not been comprehensively reported, some phenolic compounds can be harmful when consumed in large doses. The reported negative properties attributed to phenolic compounds are the capacity to precipitate proteins, form complexes with polysaccharides, affect lipid metabolism and interfere with the bioavailability of metal ions. In all the health benefits described and associated to phenolic compounds, the key factor is their chemical structures and the different mechanisms of actions that they can undergo. The effects of phenolic compounds on human health have been well established in the last decades. Important biological and pharmacological properties, such as anti-inflammatory, antimutagenic and anticarcinogenic activities have been associated to phenolic compounds (Fang et al., 2007; Stalikas, 2007). One of the most important biological properties of phenolic compounds is the antioxidant activity against reactive species involved in ageing and in chronic, autoimmune, inflammatory, coronary and degenerative diseases (Soto et al., 2011; Vermerris and Nicholson, 2006).



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5. Extraction and Recovery of Phenolic Compounds Analysis of phenolic compounds present in crude plant extracts is based in a three steps procedure. First, extraction of compounds, second, clean-up of the extracts to eliminate interferences and/or concentrate phenolic compounds and, finally, the analysis of the extract and characterization of the compounds (Vichapong et al., 2010). The extraction method must be selected according to the plant material and type of compounds to be studied. The main aim is to achieve complete extraction, avoiding chemical modification and/or destruction of the compounds. Solid-liquid and liquid-liquid extractions are the most used methods to recover phenolic compounds from plants. They are widely used due to their efficiency, wide-range of applicability and its easy operation (Stalikas, 2007). The solubility of the compounds plays a very important role since it is dependent on their chemical frame which may vary from simple to highly polymerized structures. Also, the interaction between phenolic compounds with other plant compounds may lead to the formation of insoluble complexes difficult to extract. The most common solvents for phenolic compounds extraction are ethanol, methanol, propanol, ethyl acetate, acetone, dimethylformamide and their combinations, often with different proportions of water (Ignat et al., 2011; Naczk and Shahidi, 2006). Recovery of phenolic compounds from agriculture activities and beverages industries, such as olive mill waste waters, citrus transformation and wine making, is frequently performed by liquid–liquid extraction (Ignat et al., 2011). Solid-liquid extraction is mainly used to recover food components such as sucrose, lipids, proteins and also phenolic compounds (Ignat et al., 2011). For all these reasons, it has been difficult to establish a universal method for phenolic compounds extraction. Several recent reviews have compared and discussed the various techniques for extraction and analysis of plant phenolics (Khoddami et al., 2013; Santana et al., 2009). There are other techniques beside solvent extraction, such as the shake-flask technique, Soxhlet, ultrasound, supercritical fluid (SFE), microwave-assisted (MAE), pressurized liquid (PLE) and solid-phase extraction (SPE) matrix solid phase disruption (MSPD) (Capriotti et al., 2010) as well as solid adsorption (Vichapong et al., 2010). Soxhlet extraction, using aqueous methanol or acetonitrile, is frequently used to isolate flavonoids from crude extracts. The main disadvantages are long extraction times that can range from 12 hours to 24 hours and possible deterioration of thermolabile compounds (Li et al., 2005). Ultrasound method relies on the particles being broken apart mechanically, which improves solvent access to interior components resulting in a higher efficiency (Schantz, 2006). SFE is based on the fact that, close to the critical point, the solvent changes its properties rapidly with only slight variations of pressure. The extracts obtained by SFE technique are free from compounds degradated due to high temperatures and oxygen exposure. The extracts are also free from chlorophylls and other non-polar compounds insoluble in supercritical fluids (Ignat et al., 2011). The most common used critical fluid is supercritical carbon dioxide


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(SC–CO2), due to its benign effect on the environment, low toxicity, non-flammability and compatibility with processed foodstuffs. Often organic modifiers (like methanol) must be added to CO2 to recover polar phenolic compounds in particular flavonoids (Stalikas, 2007). Solvents such as n-hexane, dichloromethane, chloroform, and other normally used in industrial processes are being replaced by supercritical fluids due to regulatory and environmental pressures on hydrocarbon and ozone-depleting emissions (Ignat et al., 2011). The MAE method uses microwave energy to heat the sample–solvent mixtures in sealed or open vessels. The extraction solvents used for MAE must absorb microwaves, although the use of solvent mixtures with and without dipole moments opens up a variety of potential solvent mixtures (Schantz, 2006). In PLE, the solid or semisolid sample is placed in a closed cell. Conventional solvents are used in this technique and they are added to the cell at the start of the heating cycle. The higher efficiency of this method is related to the fact that it uses organic solvent at high temperature and pressure to extract analytes. The extraction is performed in an inert atmosphere and protected from light. This is very convenient for the purposes of automation, shorter extraction time, lower solvent consumption and on-line coupling of the extraction and separation techniques (Vichapong et al., 2010). One drawback of PLE is that wet samples require a drying step prior to analysis when using a non-polar extraction solvent (Schantz, 2006). This type of extraction was presented for the isolation of catechin and epicatechin from tea leaves and grape seeds (Stalikas, 2007). SPE is one of the most effective and versatile, methods of sample extraction. Utilizing low cost, reduction of processing time, pre-packed, disposable cartridges, sample components of interest are separated from other species by applying the extract to an appropriate chosen solid sorbent and selectively eluting the desired components. Besides the extraction techniques presented above mechanical processes are occasionally applied to enhance molecular interaction: mechanical stirring, continuous rotation and vortexing (Stalikas, 2007).

6. Separation and Detection of Phenolic Compounds Due to the multiple possibilities of isomer formation, the exact identification of phenolic compounds present in crude plant extracts requires separation and isolation. There was a notable increase in the development of the methodologies of separation in the last decades, namely in chromatographic techniques.

6.1. Conventional Chromatography Paper, thin-layer and packed open columns chromatographic methods have been used for the separation and purification of complex matrixes such as plant extracts. Thin-layer chromatography (TLC) and open column chromatography (CC) are still used as separation tools for many phenolic compounds (anthocyanins, flavonols, condensed



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tannins and phenolic acids) (Naczk and Shahidi, 2006) since they have the advantage of being simple, inexpensive and relatively fast. The implementation of a modern standardized methodology led to an increasing acceptance and recognition of high-performance thin-layer chromatography (HPTLC) as a competitive analytical method. HPTLC has many advantages, such as lower costs, short analysis time, the possibility of multiple detection, and specific chemical modification on the same plate (Ignat et al., 2011). HPTLC is also appropriate for the preliminary screening of plant crude extracts before HPLC analysis (Marston, 2007). CC is most often employed for the preparative scale separation of components from a crude plant extract, either gravimetrically or aided by the application of low pressure inert gas (flash column) (Cseke, 2006 ). There are a large variety of stationary phases such as alumina, silica, silica-diatomaceous earth, diatomaceous earth, cellulose, polyamide, cyano, diol and amino silica stationary phases which, combined with different mixtures of solvents, allow for separation of different types of phenolic compounds (Tsao and Deng, 2004). A simple way to visualize certain phenolic compounds is by UV light (350–365 nm or 250–260 nm), since some phenolic compounds fluoresce under this type of radiation. Generally, quantification is not the main goal of TLC studies. However, densitometry was successfully used in several studies.

6.2. Gas Chromatography Gas chromatography (GC) coupled with mass spectrometry (MS), is a powerful tool in separation and analysis but the lack of volatility of the majority of phenolic compounds makes this technique labor intensive since, in most cases, derivatization is necessary. Prior to chromatography separation, phenolics are usually transformed into more volatile derivatives by methylation, conversion into trimethylsilyl (TMS) derivatives, or derivatization with N-(tert-butyldimethylsily)-N-methyltrifluoroacetamide. Usually, these compounds are hydrolyzed and converted into their derivatives, injected into a non-polar column (Stalikas, 2007). The application of electron impact ionization (EI) with a selected ion monitoring (SIM) method generates a simplified ion chromatogram of the ions of interest (Prasain et al., 2004). In conventional GC, it is very difficult to analyze flavonoid glycosides even after derivatization. Glycoside hesperidin has been analyzed by high-temperature-high-resolution (HT-HR) GC-MS, with columns that can withstand temperatures up to 400 ºC (dos Santos Pereira et al., 2004). Even so, the use of GC-MS as a routine technique for screening samples for target analytes or unknown phenolic compounds is not the most suited due to the limitations mentioned above.

6.3. Capillary Electrophoresis Capillary electrophoresis (CE) separation is based on the different electrophoretic mobilities in solution of charged species in an electric field in small-diameter capillaries


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(Prasain et al., 2004). For the separation of phenolic compounds, complex formation with tetraborate molecules may influence negatively the separation. There are two different modes in CE separations based on the used buffers: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). CZE is the simplest mode of CE and has been applied to separate phenolic compounds. Charged species are separated from each other in the capillary, whereas all neutral species migrate at the same speed. Most of the flavonoids are weak acids, so alkaline buffers are used to ensure that the phenolic moiety is charged for electrophoretic separation. β-glycosides of flavonoids can be separated by CE using a borate buffer, which form a charged complex with the cis-diol moiety of the sugar ring (Prasain et al., 2004). MEKC uses surfactants, like sodium dodecyl sulfate (SDS) which form highly organized spherical micelles at levels above their critical micellar concentrations in the buffer. In this technique one should distinguish between neutral and charged analytes. Neutral compounds are separated based on hydrophobicity, which affects the analyte partitioning between the aqueous (moving with the electro-osmotic flow) and the micellar phases (charged and migrating with a different velocity). For charged analytes the separation by MEKC is based on both the degree of ionization and the hydrophobicity (de Rijke et al., 2006). MEKC has been extensively applied to separate phenolic acids and flavonoids (Česla et al., 2010; Huang et al., 2005b; Risso et al., 2007). Detection is usually performed by UV, but electrochemical, fluorescence and MS detectors are also used (de Rijke et al., 2006; Stalikas, 2007). Mass spectrometry (MS) revealed to be an excellent detector due to its high sensitivity, universal detection and selectivity with the capability of providing structural information. CE has been coupled to a large diversity of MS systems (Nevado et al., 2010). Electrospray ionization (ESI) has been reported as the ionization interface with the highest efficiency to use coupled with CE. ESI allows the detection of multiple chargeable species of high molecular mass and permits that CE eluted matrix can be introduced into the mass spectrometer through an ESI interface without splitting (Nevado et al., 2010). The low flow rates of CE (< 1µL/min) are also an advantage when using CE coupled with MS instruments.

6.4. High-Speed Counter Current Chromatography (HSCCC) In counter-current chromatography (CCC) there is no solid column packing material involved. It is an all-liquid separation technique which relies on the partition of a sample between two immiscible solvents and separation is dependent on the partition coefficient (k) (Marston, 2007; Tsao and Deng, 2004). In the HSCCC the stationary phase is immobilized by a centrifugal force and pressure. The force provides vigorous mixing between the two immiscible liquid phases, and retention of a very large fraction of the stationary phase (Tsao and Deng, 2004). The centrifugal field allows for the use of a liquid stationary phase in an open tube. The phase density difference and the centrifugal field are the only parameters involved in the equilibrium between the two liquid phases. The role of the phases can be switched during a run, consisting in the dual-mode of this technique (Ignat et al., 2011).



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The main advantages are related to the absence of a solid stationary phase: a) no irreversible adsorption; b) total recovery of injected sample; c) tailing minimized; d) low risk of sample decomposition; e) low solvent consumption; f) it reflects the real distribution profile of the extract; and g) low-cost (once the initial investment in an instrument has been made, no expensive columns and absorbents are required and only common solvents are consumed) (Marston, 2007; Tsao and Deng, 2004). The application of HSCCC technique has been used recently for the separation of phenolic compounds. Ignat et al. (2011) recently published a review on this subject with several references to the use of HSCCC in the separation of phenolic compounds from plant extracts.

6.5. High-Performance Liquid Chromatography High performance liquid chromatography (HPLC) has been described as the most useful tool for the qualitative and quantitative separation of phenolic compounds (Vichapong et al., 2010). Kalili and Villiers (2011) published recently a review of the recent developments in separation of phenolic compounds by HPLC. Reverse-phase C18 columns are extensively used with a binary solvent system containing acidified water (acetic, formic or phosphoric acid) and a less polar organic solvent as acetonitrile or methanol, possibly acidified (Tsao and Deng, 2004). Changing the pH and/or ionic strength of the solution will allow all compounds of interest to elute, ideally in a sequential manner. The identification of the compounds is achieved by combining the retention time and various detectors such as ultraviolet/visible (UV-Vis), diode-array (DAD), fluorescence, mass spectrometry (MS), electrochemical colorimetric array detection and nuclear magnetic resonance (NMR) (Vermerris and Nicholson, 2006).

Detectors Ultraviolet Detection Phenolic compounds have absorptions bands in the UV or UV/Vis region due to their conjugated double bonds and at least one aromatic ring present in their structures. Hydroxybenzoates have maximum absorption bands between 200 and 290 nm with exception of gentisic acid, which has an absorbance that extends to 355 nm. The hydroxycinnamates, show absorption bands in the range 270 to 360 nm due to additional conjugation (Stalikas, 2007). Flavonoids have two characteristic UV absorptions bands. Band I with a maximum in the 300-550 nm range, arises from the B-ring, and band II with a maximum between 240-285 nm, from the A-ring (Merken and Beecher, 2000). These absorption maxima can experience shifts to higher wavelengths (bathochromic shift) due to conjugation to sugar esters, or to lower wavelengths (hypsochromic shifts) due to O-glycosilation (Määttä et al., 2003). Simultaneous separation of mixtures of phenolic compounds is commonly detected at 280 nm for identification and quantification purposes. For each specific group of phenolic compounds, there are specific wavelengths of maximum absorption: hydroxybenzoic acids,


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flavan-3-ols and proanthocyanidins are collected at 280 nm, hydroxycinnamic acids at 320 nm, flavonols at 360 nm, flavones at 340 nm, and anthocyanins at 520 nm (Rice-Evans et al., 1996). Fluorescence Detection The use of fluorescence detection in phenolic compounds is used only occasionally, because the number of natural occurring phenolic compounds capable of fluorescence is limited. To extend the use of this type of detection to a larger number of compounds, derivatization must be employed. For example, quercetin and kaempferol can form complexes with metal cations exhibiting intense fluorescence (Stalikas, 2007). Classes of flavonoids that show native fluorescence include the isoflavones, flavonoids with an OH group in the C3-position, catechins and methoxylated flavones (de Rijke et al., 2006). Using fluorescence detection combined with UV detection allows distinguishing between fluorescent and non-fluorescent co-eluting compounds, but the establishment of the correct excitation and emission wavelength is crucial for a good detection (Stalikas, 2007). Electrochemical Detection Electrochemical detection is based on the capability of compounds to be oxidized or reduced at low-voltage potentials. Amperometric and conventional coulometric electrochemical detection are generally not compatible with the gradient elution mode (Stalikas, 2007). The development of multi-electrode array detection allowed the detection of phenolic compounds separated with a gradient elution in a wide range of samples such as wine (Mahler et al., 1988), biological matrices (Bugianesi et al., 2000; Wittemer and Veit, 2003) and plant extracts. Mass Spectrometry Detection UV-Vis data are a very important analytical tool but they are not enough for the complete identification of the composition of a complex mixture. In the last two decades, the use of mass spectrometry has increased as it became an essential analytical technique. Mass spectrometry detectors coupled online to HPLC (HPLCMS) dominate the literature related to the analysis of phenolic compounds in natural products (Stalikas, 2007). Mass spectrometers use the difference in mass-to-charge ratio (m/z) of ionized molecules to differentiate them. This requires, first, that compounds under analysis have been charged (often by deprotonation or protonation) and transferred into the gas phase, and second that they are separated as a function of their m/z values. These two steps are achieved by the mass spectrometer source and analyzer, respectively. There are two main types of ionization: the ion-spray techniques and the ion-desorption techniques (Tsao and Deng, 2004). The ionization sources reported in the analysis of phenolic compounds are diverse: fast atom bombardment (FAB), electrospray ionization (ESI), atmospheric pressure ionization (API) including atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo-ionization (APPI), matrix-assisted laser desorption ionization (MALDI) and thermospray analysis (TSP).



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In the ESI technique, highly charged droplets are formed and ions are ejected by an ion evaporation process. An electric field is generated at the tip of a sprayer by applying a high voltage, with a close proximity of a counter electrode. Ions of one polarity are preferentially drawn into the drops by the electric field as they are separated from the bulk liquid. The electrospray stability has been improved and contamination of the source minimized by switching from the off axis sprayer geometry to an orthogonal sampling position (Nevado et al., 2010). This technique is typically performed either in the infusion mode or in combination with HPLC or capillary electrophoresis. In the infusion mode, the sample is introduced into a continuous liquid stream via an injection valve. In the APCI technique, the ions are formed at atmospheric pressure. A sample solution flows through a heated tube where it is volatilized and sprayed into a corona discharge with the aid of nitrogen nebulization. Ions are produced in the discharge and extracted into the mass spectrometer. One advantage of ESI source is a better S/N, due to the reduced number of ions in the spectral range of < 300 amu originating from the matrix and spraying solvent (Prasain et al., 2004). APCI, ESI, FAB and MALDI can operate in both positive (PI) and negative ionization mode (NI) (Tsao and Deng, 2004). There are different types of analyzers used in mass spectrometry and those reported for the study of phenolic compounds are: quadrupole (Q), magnetic sector, ion-trap (IT), time-offlight (TOF), and Fourier-transform ion cyclotron resonance (FT-ICR) that differ, among other factors, by the available mass range and resolution. Quadrupole analyzer is one of the most used in mass spectrometers since it is easy to handle, with a small size and relatively low cost. The main advantage of IT is the possibility to perform MSn experiments to obtain structural information, which is largely applied to phenolic compounds. TOF gives access to a theoretically unlimited mass range and is thus well suited for analysis of high molecular weight polymers, and also provides high resolution with accurate mass determination as low as 10 ppm (Xing et al., 2007). FT-ICR provides the highest mass resolution and most accurate mass determination, making it theoretically possible to assign molecular formula unambiguously for smaller molecules. Further information on the molecular structures of unknowns can be obtained by tandem mass spectrometry (MS/MS or MSn) experiments. This consists in isolating specific ions for fragmentation in a first stage of mass analysis and then inducing their dissociation by collision with inert gas molecules (argon or helium) to analyze the fragments thereby yielded in the second stage of mass analysis (Fulcrand et al., 2008).

7. MSn Identification and Structural Characterization of Phenolic Compounds The complete and unequivocal identification of each phenolic compound found in a plant extract can only be performed using NMR spectroscopy isolated and/or combined with other


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analytical techniques. This fact comes from the existence for a wide range number of phenolic compounds with positional isomers or chiral carbons. A large number of phenolic compounds have been studied directly or extracted from plants and characterized by 13C and 1H-NMR experiments. There are recent studies using HPLC for separation of components from crude extracts and the eluent is split between MS and NMR (March and Brodbelt, 2008), for simultaneous HPLC-MS and HPLC-NMR analysis. Nevertheless, the use of HPLC coupled to mass spectrometry, mostly ESI-MS has been widely used for structural identification of phenolic compounds present in several natural samples (Ablajan et al., 2006; Cuyckens and Claeys, 2004; Fabre et al., 2001; de Rijke et al., 2003; Ye et al., 2007). FAB was also used for identification of phenolic compounds after HPLC separation (Edenharder et al., 2001; Sano et al., 1999). A review on the application of MS techniques for the determination of flavonoids in biological samples was reported by Praisan et al. (2004).

7.1. Flavonoids Cuyckens and Claeys (2004) found that in the structure analysis of flavonoids by HPLC/ESI-MS/UV-DAD, the negative-ion mode is more sensitive and the fragmentation behavior is different, giving additional and complementary information, then the positive mode. Depending on the structure, flavonoid O-glycosides undergo collision-induced cleavage of the O-glycosidic bonds producing the free deprotonated aglycone. In order to help the analysis of mass fragmentation of flavonoid compounds, either as free aglycones and/or O-glycosilated aglycones. Ma et al. (1997) proposed a nomenclature for the main fragment ions obtained (Figure 9) (Cuyckens and Claeys, 2004). , , In the negative mode for free aglycones, the A and B labels correspond to ions containing intact A- and B-rings, respectively, in which i and j indicate the C-ring bonds that have been broken. For conjugated aglycones, Y is used to refer to the aglycone fragment , , [M–H–glycoside]-. When positive mode is used, the ions are denotade A and B , respectively.

Figure 9. Ion nomenclature used for flavonoid glycosides (illustrated on apigenin 7-O-rutinoside). Adaptaded from(Cuyckens and Claeys, 2004).



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The most useful fragmentations for the identification of flavonoid aglycones are those , that require cleavage of two C-C bond of the C-ring, resulting in structurally informative A ,

and B ions. These ions are obtained by specific retro Diels-Alder (RDA) reactions and give information on the number and type of substituent in the A- and B-rings (Cuyckens and Claeys, 2004). RDA reactions occur in six-membered cyclic structures containing a double bond and involve the relocation of three pairs of electrons in the cyclic ring. As a result, the cleavage of two -bonds and the formation of two π-bonds take place; for example, cyclohexene will fragment into butadiene and ethylene (de Rijke et al., 2006). The MSn analysis and main fragment ions of several flavonoid aglycones in the negative mode were reported by Fabre et al. (2001). The RDA C-ring cleavage of the 1,3 bonds giving , A and , B fragment ions appears as the main fragments in the negative ion mode, as it is also true for the positive mode. The , B ion is the major peak and it is characteristic for isoflavones (daidzein and genistein) (de Rijke et al., 2006). , A and , B fragments are reported at low intensity for some members of the main types of flavonoids. 3’,4’-dihydroxyflavonol (quercetin and fisetin) give characteristic 1,2 C-ring cleavage with , A ions as more abundant, rather than the , B fragment ions; this type of cleavage is not observed for other flavonols (Fabre et al., 2001). , A ions have also been detected from the fragmentation of two isoflavones (formononetin and biochanin A) (Aramendia et al., 1995; de Rijke et al., 2003). The number of hydroxyl groups in the B-ring is clearly observed in the fragmentation pattern. Flavonols with two or more hydroxyl groups in the B-ring display , A and , B . In some cases, a direct cleavage of the bond between the B- and C-rings, resulting in an [M-Bring-H]- fragment ion, can be observed (Cuyckens and Claeys, 2004). In addition to RDA reaction fragment ions, loss of small groups, such as H2O (18 Da), CO (28 Da), CO2 (44 Da) and C2H2O (42 Da), are commonly detected in negative and positive ion mode. These fragments are helpful in the identification of those specific functional groups. Compounds presenting methoxyl groups have a typical loss of 15 Da resulting in a [M − H − CH ]. radical ion (Cuyckens and Claeys, 2004). Flavonoids are found in nature often conjugated with sugar units. Glucose is the most commonly found sugar moiety followed by galactose, rhamnose, xylose and arabinose. Fragment ions from glyconjugate flavonoids are labelled based on the nomenclature introduced by Domon and Costello (Cuyckens and Claeys, 2004) represented in Figure 9. Y represents the diglycoside unit, with fragments that contain the aglycone part being denominated Y1 (loss of one sugar unit) and Y0 (loss of two sugar units); the relative sugar fragments are labeled B1 and B0. Ions formed due to the cleavage of the sugar ring, and which contain the aglycone, are designated , X , where j is the number of the interglycosidic bonds broken, counting from the aglycone; the superscripts k and l indicate the interglycosidic bonds, with the glycosidic bond linking the glycose part to the aglycone being numbered 0 (de Rijke et al., 2006). O-glycosides, C-glycosides and O,C-glycosides can be distinguished based on their MSn fragmentation pattern.


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Flavonoid O-glycosides can suffer both a collision-induced homolytic and heterolytic cleavage of the O-glycosidic bond producing deprotonated radical aglycone [Y − H]. and deprotonated aglycone ion, Y (Hvattum and Ekeberg, 2003). The radical aglycone ions are very common for deprotonated flavonol 3-O-glycosides. The nature and position of the glycoside group on the flavonol structure plays an important role on the formation of radical aglycone ions. Hvattum and Ekeberg (2003) verified that the product ion spectrum of kaempferol-7-O-neohesperidoside showed only a minor radical aglycone product ion, as opposed to kaempferol-3-O-rutinoside. The homolytic to heterolytic cleavage ratio increases with the increasing number of OH groups in the B-ring. There are minor differences between positional isomers:

,

B products

,

are more easily formed for 7-O-glycosides, whereas A fragments are more abundant for 4’O-glycosides (Cuyckens and Claeys, 2005). Flavonoid C-glycosides have the sugar moiety linked directly to the flavonoid aglycone via an acid-resistant C-C bond. Tandem MSn analysis in combination with CID allows for the characterization of this type of compounds both in negative and positive ion modes. The major fragment ions observed are related to the cross-ring cleavages of the sugar residue (Figure 10) and the loss of water molecules (Figure 11) (Cuyckens and Claeys, 2004).

Figure 10. Characteristic product ions formed by cross-ring cleavages in a pentose and hexose residue (Cuyckens and Claeys, 2004).

Figure 11. Loss of water observed for 6-C-glycosyl flavonoids involving the hydroxyl group at the 2’’position of the sugar residue and the hydroxyl group at the 5-or 7-position of the aglycone (Cuyckens and Claeys, 2004).



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The known C-glycosilation positions are the C-6 and/or C-8 of the flavonoid nucleus. Thus, the main goal is to differentiate 6-C- and 8-C-glycosyl flavonoids. The loss of a water molecule is observed, in positive and negative ion modes, and it is more pronounced for 6-Cthan 8-C-glycosyl compounds. In di-C-glycosides, sugar residues of different mass can be located, since the C6-sugar residue shows more extensive fragmentation than the C8-sugar residue. Very few flavonoid glycosides are commercially available as standards, so their quantitative analysis is seldom performed. Usually, plant extracts are subject to hydrolysis of those glycosides and the released aglycones are identified and quantified. In addition to glycosilation, several flavonoids have been described containing an acyl group linked to the sugar part. These acyl groups can be observed in mass spectrometry experiments, based on typical neutral losses. The most common acyl groups naturally occurring in flavonoids are acetyl, malonyl, benzoyl, galloyl, coumaroyl, feruloyl and sinapoyl (Cuyckens and Claeys, 2004). The exact linkage position of acyl groups to sugar units is difficult to define through ESI/MSn data, but they appear to be mainly linked at the 6-position of a hexose moiety which is confirmed when a , X fragment is present in the spectrum.

7.2. Non –flavonoids Ionization of hydroxybenzoic and hydroxycinnamic acids can be performed either in the negative (deprotonation, [M-H]-) or positive (protonation, [M+H]+) ion mode. Tandem mass spectrometry in the negative-ion mode of deprotonated phenolic acids produce a common loss of 44 Da by elimination of a carboxyl group from the deprotonated molecular ions, [M-H-CO2]-. As mentioned before, chlorogenic acids (CQA) are a family of esters formed between some trans-cinnamic acids (caffeic, coumaric, ferulic and tartaric) and (-)-quinic acid. This class of compounds is found in high levels in coffee, where esterification occurs at positions 3, 4 and 5 of the quinic acid structure. In addition to coffee, there are other plants rich in CQA and substitution at position C-1 has been reported in some Asteraceae, such as arnica and artichoke (Clifford et al., 2005). Despite that this class of compounds is widely distributed in nature, only few commercial standards are available, therefore accurate identification of individual compounds in complex samples is quite difficult. The application of tandem MSn fragmentation of the different isomers makes it possible to discriminate each one. Clifford and co-workers (Clifford, 2003, 2005) studied exhaustively these compounds by HPLC-ESI/MSn and presented a hierarchical key for the identification of caffeoylquinic acid (mono, di and tri-isomers), coumaroylquinic acids and feruloylquinic acids. For dicaffeoylquinic acids, the caffeoyl group is more or less easily removed, depending on which position of quinic acid it is connected, in the following order: 1~5 > 3 > 4. The discrimination between the 1-CQA and 5-CQA is easy to establish on HPLC using a reverse phase column since 5-CQA is more hydrophobic and therefore elutes later (Clifford et al., 2005). This key was also used for the identification of a large number of hydroxycinnamate esters of quinic, tartaric and shikimic acid in several species of Asteraceae (Clifford et al., 2005).


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These authors found cis and trans hydroxycinnamate moieties in CQAs and were able to distinguish them based on their fragmentation patterns, relative retention times and UV irradiation response.

8. Antioxidant Capacity and Phenolic Compounds 8.1. Antioxidant Capacity The relation between oxidative stress and human health, namely in the pathogenesis of various diseases and disorders has become a serious issue and as such has attracted much attention in the scientific community. Under stress, the human body will produce more harmful species, such as reactive oxygen species (ROS) (superoxide anion radicals, hydroxyl radicals and hydrogen peroxide), than enzymatic antioxidants (superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase) and non-enzymatic antioxidants (ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione, carotenoids, and flavonoids), inducing cell damage (Krishnaiah et al., 2010). This effect is increased when there are not enough antioxidants to quench these harmful radicals. Thus, human diet should be enriched with antioxidant compounds that can be of either artificial or natural origin. Foodstuffs themselves are prone to oxidation. During a large period, artificial antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (Pg) and tert-butyl hydroquinone (TBHQ) were used as additives in foods and beverages. However, their use is now restricted since they are associated with high levels of cytotoxicity and carcinogenic effects. Therefore, there is a major need to find natural compounds with antioxidant properties and low toxicity associated. An antioxidant can be defined as a compound (molecule, ion or a stable radical) that inhibits or significantly delays the oxidation of substrates even if the compound is present in lower concentration than the oxidized substrate (Matkowski, 2008). The antioxidants can be divided into preventing antioxidants, scavenging antioxidants, and repair and de novo antioxidants and they have their established roles in the defense network in vivo (Niki, 2010). The first line of defense is performed by the preventing antioxidants by suppressing the formation of reactive oxygen and nitrogen species (ROS/RNS). The scavenging antioxidants remove active species quickly before they attack biologically essential molecules. For example, carotenoids scavenge singlet oxygen either physically or chemically. Phenolic compounds and aromatic amines are free-radical scavengers. This type of mechanism is the second line of defense in vivo. The third line of defense is composed by various enzymes which repair damage, remove waste by-products and reconstitute the lost function. In addition, the adaptation mechanism functions as the fourth defense line, in which appropriate antioxidants are generated at the right time and transferred to the right position in the right concentration (Niki, 2010).



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8.1.1. Factors That Determine Radical Scavenging Capacity (RSC) The radical scavenging capacity reactions are determined by the redox property and/or ionization potential of the antioxidant. The impact of these two mechanisms is dependent on the environment. The RSC of a compound is determined by several factors: 1) the chemical reactivity toward free radicals and stoichiometric number, that is, rate of radical scavenging and number of radical molecules scavenged, 2) rate of antioxidant-derived radical, 3) interaction with other antioxidants, 4) concentration and mobility at the environment, 5) absorption, distribution, retention, and metabolism (Niki, 2010).

8.2. Methods to Determine Antioxidant Capacity In Vitro Many in vitro models have been applied for the evaluation of the antioxidant capacity of pure compounds and complex mixtures such as plants, food and biological samples. Still, the comparison and correlation of the results obtained from these distinct methods has to be formed carefully due to the different operating conditions and mechanisms of reaction. The term “antioxidant capacity” presents different meanings depending on the type of experiment and operator. It can refer to the capacity of a compound to scavenge free radicals or the capacity of a compound to resist to oxidation (Niki, 2010).

(A) Competitive scheme

(B) Non-Competitive scheme

Figure 12. Representation of competitive (A) and non-competitive (B) approaches for in vitro determination of antioxidant capacity.

The several types of antioxidant compounds are usually defined by their structure and mechanism of action and can be grouped in different ways according to the different authors. The most common and widely used methods to establish the antioxidant activity are colorimetric ones, based in competitive and non-competitive mechanisms followed by


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UV/Visible spectrophotometry (Figure 12). In the non-competitive methods, the antioxidants react with reactive species without the presence of any other competing target molecule. This type of assay involves two components in the initial reaction mixture: the antioxidant sample and the reactive species. In the competitive assays, the target species is a compound with high probability to be attacked in vivo by reactive species. As such, the antioxidant sample competes for those reactive species (radicals or non-radicals) (Magalhães et al., 2008). In close observation of the chemical reactions involved in the antioxidant process, the antioxidant capacities assays can be divided into two groups: hydrogen atom transfer (HAT) and single electron transfer (ET). HAT-based assays determine the capability of an antioxidant to scavenge free radicals by hydrogen transfers. HAT-assays are composed of a synthetic free radical generator, an oxidizable molecular probe and an antioxidant. The majority of HAT assays are kinetics based and involve a competitive reaction in which antioxidant and probe compete for peroxyl radicals thermally generated through the decomposition of azo compounds. R2N2 → 2 R . + N2 R. + O2 → ROO. The HAT mechanisms of antioxidant action in which the hydrogen atom (H) of a phenol (Ar-OH) is transferred to an ROO. radical can be summarized by the reaction (Apak et al., 2007): ROO. + AH/ArOH . → ROOH + A. /ArO. where the aryloxy radical (ArO.) formed from the reaction of antioxidant phenol with peroxyl radical is stabilized by resonance. The AH and ArOH species are the protected biomolecules and antioxidants, respectively. Effective phenolic antioxidants need to react faster than biomolecules with free radicals to protect the latter from oxidation. ET−based assays measure the reducing capacity of an antioxidant. In the reaction mixture there are antioxidants, oxidant and the probe. The probe is an oxidant that abstracts an electron from the antioxidant, causing the color changes of the probe (Huang et al., 2005a). The degree of color change (either an increase or decrease of absorbance at a given wavelength) is correlated to the concentration of antioxidants in the sample. The ET mechanism of antioxidant action is based on the reactions: (Apak et al., 2007) ROO. + AH/ArOH . → ROO . + AH . /AROH

.

AH . /ArOH . + H O ↔ A. /ArO. + H O ROO. + H O . ↔ ROOH + H O where the reactions are relatively slower than those of HAT– based assays, and are solvent and pH dependent. The aryloxy radical (ArO.) is subsequently oxidized to the corresponding



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quinone (Ar=O). The more stabilized the aryloxy radical is, the easier will be the oxidation from ArOH to Ar=O due to reduced redox potential. The following are examples of the most frequently in vitro systems for the evaluation of antioxidant capacity. 8.2.1. DPPH Method The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging assay was first described by Blois in 1958 and has been modified according to specific experimental conditions (Krishnaiah et al., 2010). Deep violet color

Yellow-white color

Figure 13. (a) DPPH radical structure and (b) structure of the reduced radical.

DPPH (Figure 13) is one of a few commercially and stable free radicals. It has a UV-Vis absorption maximum at 515 nm, which is responsible for its characteristic purple color. When mixed with an antioxidant/reducing sample, the DPPH radical Figure 13 (a) is reduced to the corresponding pale yellow hydrazine Figure 13 (b) (Magalhães et al., 2008). The reaction is usually performed in an organic solvent (methanol or ethanol) and the decrease of the absorbance at 515 nm is registered until a steady state is reached. The reaction mechanism was first assumed as being a HAT process but it is now known that the electron transfer (ET) reaction occurs faster than the hydrogen atom abstraction which is a very slow mechanism in strong hydrogen-bond accepting solvents (Foti et al., 2004). The results are generally expressed as the efficient concentration (EC50) which corresponds to the amount of antioxidant necessary to decrease in 50% the initial DPPH radical concentration. However, this calculation is dependent on the specific conditions used in the assay, mainly the DPPH initial concentration. Therefore, the construction of a calibration curve of a strong standard antioxidant compound like Trolox or ascorbic acid allows for the interpolation of the values of absorbance variation and the results are expressed as equivalent concentration (Magalhães et al., 2008). The DPPH assay presents some disadvantages such as the fact that the radical is more suitable for small scavenging molecules and big antioxidant molecules have a slow or inexistent activity towards DPPH. The reaction of DPPH with some compounds such as eugenol was found to be reversible, which can lead to false low antioxidant capacity of samples (Huang et al., 2005a).


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The absorbance variation can also be affected by compounds such as carotenoids that absorb at the working wavelength and also by the turbidity of the sample. 8.2.2. ABTS Method or Trolox Equivalent Antioxidant Capacity Method The ABTS method was first describe by Rice-Evans and modified by Miller in 1994 (Rice-Evans et al., 1996). In this method, it is necessary to generate the radical cation chromophore 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS•+) which has a blue/green colour and absorption maxima at wavelengths of 414, 645, 734 and 815 nm (Magalhães et al., 2008). A widely form used to produce the ABTS radical cation is via the chemical reaction of ABTS and potassium persulfate (Figure 14) which is stable for 2 days (Pinchuk et al., 2012). It is preferable to perform the detection at a wavelength of 734 nm, since the interference from other absorbing components and to sample turbidity will be reduced (Arnao, 2000). An important difference between ABTS and DPPH assay it that the ABTS radical cation can be solubilized in both aqueous and organic media, which allows measuring the contribution of hydrophilic and lipophilic compounds from samples (Arnao, 2000). In the same way of the DPPH decolorization assay, the decrease in absorbance is measured until a steady state is achieved. Reaction times in the range 1 to 30 minute have been reported. Trolox is the most common positive control used and the sample’s antioxidant capacity is expressed in terms of Trolox-equivalent.

Figure 14. Reaction of the generation of ABTS radical cation.

8.2.3. Ferric Reducing Antioxidant Power (FRAP) The FRAP assay measures the reducing power of a sample and it was first introduced by Benzie and Strain (Tsao et al., 2003). A sample with reducing power will reduce the yellow ferric 2,4,6- tripyridyl-s-triazine complex [Fe(III)-(TPTZ)2]3+ to the blue ferrous complex [Fe(II)-(TPTZ)2]2+ in acidic medium (Figure 15). The reaction is followed by an increase of absorbance at 593 nm and the variation in absorbance is related to a Fe(II) standard solution.



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The reaction time is typically 4 minutes (Magalhães et al., 2008) but for polyphenols the reaction occurs more slowly (> 30 minutes) until a plateau is usually observed. A drawback of this method is that any compound with a redox potential lower than that of the redox pair Fe(III)/Fe(II) can theoretically reduce Fe(III) to Fe(II) inducing a false FRAP value (Magalhães et al., 2008). Moreover, compounds with antioxidant properties that act as hydrogen transfers (such as thiols) will not react in FRAP assay. The low pH value used may induce protein precipitation when this method is applied to milk or plasma samples. This method has also been modified for the 96-well microplate reader, yielding better reproducibility and higher sample throughput (Tsao et al., 2003).

Figure 15. Structures of the two triazine complexs.

8.2.4. Oxygen Radical Absorbance Capacity (ORAC) Assay The oxygen radical absorbance capacity (ORAC) is one of the most used methods to measure the ROO˙ scavenging capacity. The intensity of fluorescence loss of a probe such as beta-phycoerythrin or fluorescein is measured over time under reproducible and constant flux of peroxyl radicals (Magalhães et al., 2008; Niki, 2010). When a sample with chain-breaking compounds is present in the reaction medium, the decay of fluorescence is reduced. The use of phycoerythrin presents some disadvantages like large lot-to-lot variability, photobleaching after exposure to excitation light, interaction with polyphenols by nonspecific protein binding and loss of fluorescence even without added radical generator (Magalhães et al., 2008). The use of fluorescein has overcome these limitations and the products generated from the reaction of fluorescein with peroxyl radicals have been characterized and are consistent with a HAT reaction mechanism (Tsao et al., 2003). The quantification of the antioxidant capacity is measured by the area-under-the-curve (AUC) technique with and without the antioxidant sample. The reaction is followed for large periods normally higher than 30 minutes. The ORAC values are expressed in reference to a calibration curve generally using Trolox as the positive control. Since there is a large number of lipophilic antioxidants, the ORAC assay has been modified in order to measure lipophilic and hydrophilic compounds. The best conditions were found to be a solution of 50% acetone: 50% water containing 7 % of randomly methylated βcyclodextrin as a water solubility enhancer (Huang et al., 2002).


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8.2.5. Total Radical-Trapping (TRAP) Assay The total radical-trapping antioxidant parameter assay was first described by Wayner in 1985 for the evaluation of the antioxidant capacity of human plasma (Magalhães et al., 2008). This method is based on the thermally decomposition of an azo-compound, producing a peroxyl radical flow at a constant temperature-dependent rate. The measurement of the time period in which oxygen uptake was inhibited by plasma is an indirect way to evaluate the antioxidant capacity. The target is the human plasma, while the oxygen consumed in the oxidation of plasma material is the probe molecule to follow the action of antioxidants (Magalhães et al., 2008). One of the main disadvantages of this method comes from the use of an oxygen electrode as detector, since it may not be stable over the necessary period. This issue can be eliminated by the use of R-phycoerythrin as the fluorescent target/probe which leads to the reaction being fluorimetric monitored. 8.2.6. Folin-Ciocalteu Method This method is widely used to measure the total phenolic content in different samples. The Folin-Ciocalteu reagent is a mixture of sodium tungstate, sodium molybdate, concentrated hydrochloric acid, phosphoric acid and water, to which lithium sulfate is added to give the intense yellow color. The exact chemical nature of this solution is not known, but it is believed to contain phosphomolybdic/phosphotungstic acid complexes (Huang et al., 2005a). The chemistry of this assay is based on the transfer of electrons in alkaline medium from phenolic compounds and other reducing species to molybdenum, forming blue complexes that can be detected at 750 - 765 nm (Magalhães et al., 2008). This method is non-specific to phenolic compounds because other non-phenolic compounds with reducing properties (ascorbic acid, Cu(I), Fe(II), sulfur dioxide, aromatic amines, etc.) can also react (Magalhães et al., 2008). However, a high correlation between the results obtained by this method and those obtained by other ET-based assays (FRAP, DPPH, ABTS, etc.) have been reported and consists on the main advantage of the Folin-Ciocalteu method. Stevanato, Fabris, and Momo (2004) proposed a method to assess the total phenolic content in tea using a polyphenol oxidase as being more specific than the Folin-Ciocalteu method. Nevertheless, the original FC method has become a routine assay in studying antioxidants, since it is simple and reproducible in most of the cases. (Huang et al., 2005a) Normally, gallic acid is used as reference compound and results are expressed as gallic acid equivalents. 8.2.7. β-Carotene-Linoleic Acid Bleaching Assay The evaluation of the antioxidant activity by the β-Carotene assay is based on the fact that the free radical linoleic acid attacks the highly unsaturated β-Carotene, and the presence of compounds with antioxidant properties delay the β-Carotene oxidation by neutralizing the free radicals in the medium (Gursoy et al., 2009).



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Readings are taken at a wavelength of 490 nm immediately after the beginning of reaction and at 15 minutes time intervals for 200-300 minutes. The inhibition power is normally expressed as percentage of inhibition (Siddhuraju and Becker, 2003).

9. Medicinal Plants with High Antioxidant Capacity and Phenolic Compounds The literature on medicinal plants phenolics and their bioactivity is huge. There are several comprehensive reviews focused on the antioxidant properties of different medicinal plant and the mechanisms of action of the various types of phenolic compounds. Huang and co-workers’ (Huang et al., 2009) review covers the most recent literature to summarize structural categories and molecular anticancer mechanisms of phenolic compounds from medicinal herbs and dietary plants. They conclude that various bioactivities of phenolic compounds, such as antioxidant, anticarcinogenic, or anti-mutagenic and antiinflammatory effects are responsible for their chemopreventive properties and also contribute to their inducing apoptosis by arresting cell cycle, regulating carcinogen metabolism and ontogenesis expression, inhibiting DNA binding and cell adhesion, migration, proliferation or differentiation, and blocking signaling pathways. They also emphasize that more information about the health benefits and the possible risks of dietary supplement or herbal medicines is needed to ensure their efficacy and safety. Stagos et al. (2012) review the chemopreventive properties plant polyphenols against HCC and discuss the molecular mechanisms accounting for this activity. Vanden Berghe (2012) discusses the possible epigenetic contributions of dietary polyphenols in cancer chemoprevention. Since epigenetic marks (epimutations) are reversible in contrast to genetic defects, chemopreventive nutritional polyphenols (soy, genistein, resveratrol, catechin, curcumin) are currently evaluated for their ability to reverse adverse epigenetic marks in cancer (stem) cells to attenuate tumorigenesis-progression, prevent metastasis or sensitize for drug sensitivity. The role of antioxidant versus pro-oxidant effects of green tea polyphenols in cancer prevention is discussed by endless researchers and a large number of reviews is available. Kim (2010) reviewed the naturally-occurring neuroprotective phenolics and their underlying mechanisms of neuroprotective actions. The neuroprotective activities of diverse polyphenol groups are potentially due to their capacity to modulate several cellular responses, especially reduction of oxidative stresses, anti-inflammation, and subsequent neuronal cell survival. Zhu et al. (2006) showed that hydroxycinnamic acid derivatives (HCA) were able to inhibit the cross-linking of protein induced by riboflavin mediated photo-oxidation. They found that HCA were also able to strongly protect lysozyme from gamma rays irradiation and to protect proteins against oxidation by scavenging oxidizing species and repairing the damaged protein. Feng et al. (2010) demonstrated that hydroxycinnamic acids have a significant activity in the prevention and treatment of lung cancer through antiproliferation, regulation of tumor cell division cycle, promotion of apoptosis, antioxidative effects, immune regulation, antimutagenic effects, stimulation of macrophage phagocytic activity and induction of gene expression and production of macrophage-related cytokines.


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Our research group studied the in vitro antioxidant activity of several Asteraceae plants endemic from Madeira Island, Portugal (Castilho et al., 2012; Gouveia and Castilho, 2010, 2011, 2012a, 2012b). Asteraceae studies usual focus on their contents in sesquitepenes lactones as bioactive compounds. However, we found that these plants show a high ratio hydrocynnamates/flavonoids and a very high antioxidant activity.

Conclusion There is a growing interest in substances exhibiting antioxidant properties, which can be obtained as food components or as specific preventive pharmaceuticals. The antioxidative and pharmacological properties of medicinal plants are usually related to the presence of phenolic compounds, especially phenolic acids and flavonoids, since polyphenols are known for their ability to prevent oxidative decay and provide a defense against the oxidative stress of free radicals for the plant itself. Phenolic compounds are a much diversified group of phytochemicals that are widely distributed in plants, and they may play an important role in preventing diseases such as obesity, coronary heart disease, colon cancer, gastrointestinal disorders and can also reduce the risk of diabetes. The biological properties of polyphenols and their health benefits have intensified research efforts to discover and use methods for the extraction, separation and identification of these compounds from natural sources. The standardization of these methods is highly desirable in order to make comparison of data an easier task for the scientific community and to the final consumer.

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Chapter 2

Potential Antioxidant Benefits of Commonly Used Fruits and Vegetables around the World Lourdes Rodríguez-Fragoso1, Ulises Osuna-Martínez2, Ana Isabel Gonzaga-Morales1 and Jorge Reyes-Esparza1 1

Universidad Autónoma del Estado de Morelos, Facultad de Farmacia, Cuernavaca, México 2 Universidad Autónoma de Sinaloa, Facultad de Ciencias Químico Biológicas, Cualiacán, México

Abstract Reactive oxygen species (ROS) play a crucial role in human health. At low, regulated levels, ROS are involved in many vital physiological processes. They have a role in various signaling cascades, such as response to growth factor stimulation and control of inflammatory responses. They participate in the regulation of many cellular processes, including differentiation, proliferation, growth, apoptosis, cytoskeletal regulation, migration, and contraction. However, ROS also play an important role in a wide range of pathologies and many implicated diseases that are leading causes of death. It is common knowledge that plant-derived foods contain hundreds of active antioxidant compounds, including ascorbic acid, tocopherols, carotenoids, and a wide range of phytochemicals such as polyphenols. Many in vitro and animal studies have shown that a large range of dietary antioxidants, taken as extracts or as food components, have beneficial effects because they modulate oxidative stress and protect against oxidative damage and its complications. Dietary polyphenols have received a lot of attention from nutritionists, food scientists and consumers due to the role they play in human health. Polyphenols can induce antioxidant enzymes such as glutathione peroxidase, catalase and superoxide dismutase, which respectively decompose hydroperoxides, hydrogen peroxide and superoxide anions, also inhibiting the expression of enzymes such as xanthine oxidase. Medicinal plants are traditionally used in folk medicine as natural healing 

Corresponding author: [email protected].


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L. Rodríguez-Fragoso, Ul. Osuna-Martínez, Ana Isabel Gonzaga-Morales et al. remedies with therapeutic effects such as the prevention of cardiovascular diseases, inflammation disorders, or reducing the risk of cancer. The antioxidant properties of medicinal plants depend on the plant, its variety, environmental conditions, climatic and seasonal variations, geographical regions of growth, degree of ripeness, growing practices, and many other factors such as post-harvest treatment and processing. The present chapter is limited to commonly consumed fruits and vegetables with significant nutritional and antioxidant beneficial effects in folk medicine. Here, we discuss the phytochemistry and antioxidant pharmacological properties of the following plant species: apple, berries, cranberry, grape, grapefruit, mango, orange, papaya, pomegranate, tangerine, avocado, broccoli, cactus, cauliflower, carrot, pepper, spinach, tomato, and watercress. The present chapter evidences our knowledge of the therapeutic properties of the antioxidant qualities of some fruits and vegetables is limited and seeks to provide an overall clear view of the antioxidant role of common fruits and vegetables, along with their health and disease-reduction benefits.

Introduction Over the past decades, we have discovered that reactive oxygen species (ROS) exert a multiplicity of biological effects across a wide spectrum that ranges from physiological regulatory functions to damaging alterations involved in the pathogenesis of an increasing number of diseases. All ROS types, including superoxide anions and hydrogen peroxide, have unpaired valence electrons or unstable bonds. At high concentrations, ROS react readily with proteins, lipids, carbohydrates, and nucleic acids, often inducing irreversible functional alterations or even complete destruction. When ROS were initially established as a biomedical concept it was thought they had exclusively toxic effects and were associated with pathologies. It is now clear that organisms have also developed methods for utilizing ROS in critical physiological processes (D’Autreaux and Toledano, 2007). Nowadays, people are advised to increase their intake of fresh fruit and vegetables based on the presumed benefits of the antioxidant content of plant substances. It is common knowledge that plant-derived foods contain hundreds of active antioxidant compounds, including ascorbic acid, tocopherols, carotenoids, and a wide range of phytochemicals such as polyphenols. Although there have been numerous studies on ROS scavenging involving fresh food products, few studies have focused on whether or not compounds in the diet can modulate ROS levels. Epidemiologic evidence suggests that regular consumption of fruits and vegetables may reduce the risk of some diseases, including cancer. Clinical pharmacologic interest in the efficacy and safety of the phytochemicals present in fruits and vegetables has grown during recent years due to the realization that many people selfmedicate using these agents. Here we review some of the antioxidant properties of the most widely used varieties to give medical practitioners an overview of the possible pharmacological and physiological effects of the common fruits and vegetables used by their patients. The present chapter is limited to species such as apple, berries cranberry, grape, grapefruit, mango, orange, papaya, pomegranate, tangerine, avocado, broccoli, cactus, cauliflower, carrot, pepper, spinach, tomato, and watercress. Based on our experience, these are the most frequently sought fruits and vegetables by people with health problems. Many have no consistently used, popular name in English; in other cases, the English name may


Potential Antioxidant Benefits of Commonly Used Fruits and Vegetables …

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refer to two or more botanically distinct species. It is not possible at this point to list all the fruits and vegetables commonly used by healthy and non-healthy people. Presently, there are only a few studies that combine a phytochemical and detailed analysis of the antioxidant properties of given fruits and vegetables (Heinrich, 2003), or studies that systematically explore their benefits, especially in regards to their antioxidant properties.

Reactive Oxygen Species: Source and Defense ROS are oxygen-containing molecules that are highly reactive in redox reactions. ROS are primarily produced intracellularly by two metabolic sources: the mitochondrial electrontransport chain, and oxygen-metabolizing enzymatic reactions such as xanthine oxidases, the cytochrome P450 system, NADPH oxidases, myeloperoxidase, and nitric oxide synthase (Bartosz, 2009). ROS levels are also dependent on oxygen concentrations. Most eukaryotic organisms require oxygen to survive. Oxygen is the terminal electron acceptor during energy production. It accepts an additional electron to create superoxide, a more reactive form of oxygen. Superoxide can be converted to hydrogen peroxide (H2O2) spontaneously. ROS were traditionally thought of as toxic by-products of living in an aerobic environment because they are known to damage cellular macromolecules, which could subsequently, lead to cell death. However, in recent years, several studies have shown that ROS can function as signaling molecules that regulate numerous cellular processes, including proliferation (Sauer et al., 2001). The second-messenger properties of ROS are believed to activate signaling pathways by activating tyrosine kinases, tyrosine phosphatases, MAP kinases, or ion channels. Furthermore, interactions between specific receptor-ligands are also known to generate ROS. This dual function of ROS as signaling molecules or toxins could result from differences in concentration, pulse duration, and subcellular localization (Menon and Goswami, 2007). Despite the constant generation of free radicals and oxidant species, living organisms have not only adapted to an unfriendly coexistence with these potentially toxic species, but have also developed mechanisms that use them advantageously (Bakkenist and Kastan, 2004). The arsenal of cellular defenses to control the magnitude of ROS generation is extensive and includes enzymatic (superoxide anion dismutases, catalases, glutathione peroxidase (GPx), peroxiredoxins, glutaredoxins, thioredoxins, sulfiredoxins) and nonenzymatic antioxidants (vitamins A, C, and E, glutathione (GSH), urate, bilirrubin). The coordinated action of antioxidant enzymes ensures efficient ROS removal. For example, the superoxide dismutases (SOD) catalyze the dismutation of superoxide anion into hydrogen peroxide, which, in turn, is converted into water and oxygen by GSH peroxidases and catalase. Considering the reactivity and site localization where free radicals and ROS are generated within cells, enzymatic antioxidant defenses are compartmentalized to neutralize these species more efficiently (Droge, 2002). For instance, SOD is localized in the cytosol (Cu/Zn SOD) or in the mitochondria, thus handling different pools of superoxide anion generated extra- or intramitochondrially. In addition, extracellular SOD (ecSOD) is predominantly found in the extracellular matrix and is known to regulate endothelial cells by preventing nitric oxide (NO) from reacting with superoxide anion. GPx, catalase, and peroxiredoxins control the fate of hydrogen peroxide produced from superoxide anion. Like ecSOD, GPx has also been found in plasma. GPx-1, a selenoprotein, is found in the cytosol


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L. Rodríguez-Fragoso, Ul. Osuna-Martínez, Ana Isabel Gonzaga-Morales et al.

and mitochondria of all cell types, whereas distinct peroxiredoxins can be located in the cytosol (Prx) or mitochondria (Prx-III). In addition to these efficient ROS scavenging enzymatic systems, there are also critical nonenzymatic antioxidants, some of which collaborate with enzymatic partners such as GSH. This critical antioxidant is a tripeptide (L-g-glutamyl-L-cysteinylglycine) that owes its antioxidant function to the sulfhydryl group of cysteine. It is synthesized in the cytosol of all cells from its constituents (amino acids, glutamate, cysteine, glycine) and is then compartmentalized in various suborganelles, where it plays a critical role in the detoxification of hydrogen peroxide produced from superoxide anion. In addition to its redox-modulating effects, GSH is a versatile antioxidant because of its function as a cofactor for GPx and glutathione reductase (GSR) in the so-called GSH redox cycle. The natural defense against ROS consists of antioxidant enzymes and antioxidant scavengers. The nuclear transcription factor Nrf2 is the master regulator of the gene expression of antioxidant enzymes. Three superoxide dismutases differing in their subcellular location catalyze the reaction of superoxide into oxygen and hydrogen peroxide. Thioredoxins, which also consist of several isoforms differing in subcellular localization, enable the reduction of oxidized proteins by cysteine thiol-disulfide exchange. Glutathione GPx reduces lipid hydroperoxides to alcohols and reduces hydrogen peroxide to water; GSH synthetase is responsible for synthesis of the major cellular antioxidant glutathione and therefore also plays an important role in ROS detoxification. Peroxiredoxins control cytokineinduced peroxide levels, thereby affecting signal transduction. Antioxidant scavengers are predominantly of dietary origin. These biomolecules include tocopherol (vitamin E), ascorbic acid (vitamin C), carotenoids, uric acid, and polyphenols (Hybertson et al., 2011). Most of these molecules are present in fruits and vegetables in our diet and play an important role in maintaining health because they act as free radical scavengers and antioxidants.

Impact of ROS in Health and Disease Although ROS have been classically known for their damaging effects, there is increasing evidence of their use in regulating and maintaining normal processes in living organisms (Assim et al., 2012). Therefore, the term “redox regulation” seems to better describe the redox status and its consequences. Both ROS and the protective antioxidant systems have to work in coordination to reach a state of redox homeostasis. There is evidence of the roles played by ROS in several physiological processes, such as maintaining vascular diameter and normal vascular cell function; participating with the tumor-relevant transcription factor and hypoxia-inducible factor (HIF) in sensing oxygen availability and initiating responses appropriate for cell survival; mounting an effective immune response (Tschopp, 2011); acting as possible signaling molecules in regulating skeletal muscle glucose uptake (Sandstrom et al., 2006); acting as a necessary cofactor for thyroperoxidase, the enzyme participating in a final step of thyroid hormone production (Erdamar et al., 2008); having a role in neuronal apoptosis during brain development, as well as in cognitive function (Massaad and Klann, 2011); modulating the aging process (Salminen and Kaarniranta, 2012), and regulating gene stability and transcription by affecting chromatin stability (Rajendran et al., 2011). In addition, muscular exercise renders us more resistant to oxidative damage.



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Cumulative evidence found over the years clearly supports the idea that ROS and oxidants are important factors in many different pathological processes (Brieger et al., 2012). The foundation for this pathophysiological role derives from the reactivity of these species to different cellular components such as lipids or DNA and, more specifically, proteins, because of the presence of cysteine residues. In most cases, as shown in cell-free or in in vitro cell systems, ROS and oxidant generation can disturb the functions of these vital cellular constituents, resulting in cell dysfunction or death. As a consequence, ROS contribute to a wide range of pathologies and many of the implicated diseases are leading causes of death. Cancers, cardiovascular diseases (CVD), and neurological diseases all show robust evidence for ROS involvement. ROS may not only contribute to cancer development through oncogenic mutations, but also via dysregulation, as in renal cell carcinoma (Perera and Bardeesy, 2011). Elevated levels of the HIF-1α contribute to tumor growth, angiogenesis, and metastasis. Thus, the simultaneous loss of a tumor suppressor and generation of ROS leads to a major alteration of the post-translational processing of the HIF-1α (Sarsour et al., 2009). ROS are also involved in a large number of CVD and the causal mechanisms are complex. In vascular smooth muscle cells from large arteries, NADPH oxidase (NOX) 1 is required for migration, hypertrophy, and proliferation, NOX4 mediates differentiation, and NOX1 and 2 are implicated in hypertension (Streeter et al., 2012). ROS have a role in neurological disease progression, primarily through the expression of NOX enzymes in microglia cells. While low ROS concentrations are required for brain function, high ROS concentrations contribute to diseases due to neurotoxicity (Sorce and Krause, 2009). ROS also have an important pathological role in the pathogenesis of some neurodegenerative disorders: Parkinson’s disease, Alzheimer´s disease, and amyotrophic lateral sclerosis (Terashvili et al., 2006). ROS have been implicated in several psychiatric diseases, including depression and autism. The most thoroughly studied example is schizophrenia, which illustrates yet more complicated and interesting roles for ROS (Sorse and Krause, 2009). On the other hand, many age-associated diseases of the eye, such as cataract and retinal degeneration, are thought to involve oxidative stress. Similarly, ageassociated hearing loss is thought to be a ROS-mediated disease (Bánfi et al., 2004). Regarding the function of ROS in metabolic disease and chronic inflammation, we could say that the prevalent metabolic state is the one described by the term “glucolipotoxicity,” in which excess extracellular glucose and fatty acids (FAs) exert various damaging effects. Excess glucose increases oxidative stress through several biochemical mechanisms, including glyceraldehydes autoxidation, protein kinase C activation, glycation, methyl glyoxal and sorbitol production, the hexosamine pathway, and oxidative phosphorylation (Robertson, 2004). It is increasingly acknowledged that diabetic complications are also strongly linked to a state of oxidative stress. Diabetic retinopathy, a major worldwide cause of blindness among adults, has been the focus of intensive research that demonstrates oxidative stress plays a vital role in its pathogenesis (Zhang et al., 2001). Obesity is also associated with a state of chronic inflammation in the adipose tissues as well as in other organs, where tissue-infiltrating monocytes/macrophages increase in number and in activity. Several active mediators, chemotactic molecules, cytokines, and adipokines, augment the chronic inflammatory state and result in the excessive production of ROS, causing systemic oxidative stress (Chen and Tinnett, 2008). Recently, it was also demonstrated that ROS induce the assembly and


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activation of inflammasomes and inhibit mitochondrial autophagy; both processes are related to aging and age-related diseases (Zhou et al., 2011). Taken together, the results of recently conducted research on molecular, subcellular organelles and cellular mechanisms involved in mediating ROS action offer promising avenues and propose novel, potentially therapeutic agents for ROS-linked diseases. There is no doubt that redox regulators, related active mediators, cellular organelle functions, and surrounding environments are all tied together in intricate networks affecting the whole body, metabolism, state of health and disease, and even lifespan.

Food as a Natural Source of Antioxidants Fruits and vegetables are known to be important components in a healthy diet, since they have low energy density and are sources of micronutrients, fiber, and other components with functional properties known as phytochemicals (Figure 1 and 2).

Figure 1. Phytochemicals polyphenols present in fruit and vegetables.



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Figure 2. Others phytochemicals non-polyphenols present in fruit and vegetables.

Several antioxidant supplementation strategies have been tested in humans based on the assumption that they will increase degradation of ROS and thereby reduce ROS-associated diseases. Notable treatments have included SOD and mimetics, peroxidase and mimetics, Vitamins A, C, and E, coenzyme Q10, β-carotene, and bioflavoids. Clinical studies using antioxidant food supplements have been largely disappointing. In fact, the long-term health consequences of many supplements are dubious. An example of this was the Iowa Women’s Health Study, where vitamin and mineral supplements were assessed for their relationship to total mortality in more than 30,000 elderly women. The use of food supplements, including those with antioxidant activity (multivitamins, vitamin B6), was associated with increased risk of total mortality (Mursu et al., 2011). The only supplements found to decrease mortality risk were calcium and vitamin D, certainly through mechanisms unrelated to oxidative stress. Thus, there is an apparent contradiction: on the one hand there is ample evidence of the role played by ROS in various diseases and antioxidant-rich food is generally associated with good health, but antioxidant supplements do not prevent disease and are even associated with a poor health outcome. Reportedly, the tissue concentrations that can be achieved with antioxidants might be far below the levels required to counteract a ROS-generating system. Antioxidants preferentially localize to subcellular compartments based on solubility. This is obviously not a problem with antioxidants of dietary origin (1 glass of red wine provides more than 1000 different compounds with antioxidant capacity). However, this is a limiting factor for single molecule supplements. Antioxidant food supplements (e.g., vitamin C) may also have pro-oxidant activity under certain circumstances, typically upon interaction with ROS. On the other hand, antioxidants scavenge ROS after their production. They are incapable of preventing oxidation of molecules that have a very high affinity for ROS, such as nitric oxide, which has an extremely rapid rate of reaction with superoxide. Antioxidants are most effective in


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combating low levels of ROS generation, but have a limited capacity to reduce high levels (Stanner et al., 2004). It is generally accepted that the beneficial effects of medicinal plants can be obtained from active constituents present in the whole plant, parts of the plant (e.g., flowers, fruits, roots or leaves), or plant materials or combinations thereof, whether in crude or processed state (De Smet, 2002). The concept of several active principle ingredients acting in a synergistic manner in natural remedies may be somewhat unusual to pharmaceutical scientists who are more accustomed to monotherapy using specific therapeutic agents. Consuming a diet rich in such plant foods will provide a wealth of phytochemicals, nonnutritive substances in plants that possess health-protective effects. Nuts, whole grains, fruits, and vegetables contain an abundance of phenolic compounds, terpenoids, pigments, and other natural antioxidants (including vitamins A, C, and E) that have been associated with protection from and/or treatment of chronic diseases such as heart disease, cancer, diabetes, and hypertension, as well as other medical conditions. Increased fruit and vegetable consumption can also help displace food that is high in saturated fats, sugar or salt. Low fruit and vegetable intake is among the top 10 risk factors contributing to mortality. The World Health Organization’s (WHO) study group on diet, nutrition and prevention of communicable diseases have recommend daily consumption of at least 400 g (14 oz) of fruits and vegetables (WHO, 2003). The US Department of Agriculture’s Food Guide Pyramid recommends that adults consume 5 to 9 servings of fruits and vegetables a day. The regular consumption of foods that are naturally high in antioxidants (fruits, vegetables, and whole grains) is associated with substantial health benefits. It has also been proposed that the additive and synergistic effects of phytochemicals in fruits and vegetables are responsible for their antioxidants activities, and that the benefits of plant-based diets are in part attributable to the complex mixture of phytochemicals present in whole foods (Liu, 2003) (See Table 1 and 2). Table 1. Fruits and their most common phytochemicals Fruit Apple Malus domestica

Commonly used parts Fruit pulp and peel

Cranberry Vaccinium macrocarpon

Fruits

Berries Rubus coreanus Rubus idaeus Rubus fruticosus Rubus leucodermis Grapes Vitis vinifera

Fruits

Fruits and seeds

Bioactive compounds Flavonoids, polyphenols, carotenoids, catechin, epicatechin, procyanidin, coumaric acid, chlorogenic acid, gallic acid, procyanidins, phloridzin, phloretin glycosides, caffeic acid, and chlorogenic acid (Golding et al., 2001). Phenolics compounds: phenolic acids Flavonoids: anthocyanidins and proanthocyanidins. Flavonols. Aglycones; myricetin, quercetin and kaempferol (Singh et al, 2009) Phenolics compounds, tannins, phenolic acids, organic acids, triterpenoids, flavonoids, gallotannin, ellagitannin, and anthocyanins (Yoon et al., 2003; Cho et al., 2005). Vitamins A, C, E and folic acid, calcium, and selenium (Tian et al, 2005). ß-carotene, lutein, phenolics, flavonoids and anthocyanins (Vislocky and Fernandez, 2010), carotenoids (Bunea, 2012), anthocyanins, catechins, resveratrol, phenolic acids, and procyanidins. (Kammerer et al., 2004).


Potential Antioxidant Benefits of Commonly Used Fruits and Vegetables … Grapefruit Citrus paradisi Mango Mangifera indica

Fruit pulp and peel Fruit pulp, peel and seeds

Orange Citrus sinensis

Fruit pulp, peel

Papaya Carica papaya

Fruit pulp, leaves and seeds

Pomegranate Punica granatum

Fruits

Tangerine Citrus tangerina Citrus reticulata Citrus deliciosa

Fruit pulp and peel

Vitamin C, carotenoids, phenolic compounds, flavonoids and furanocoumarins (Hanley and others, 2011). Phenolic compounds (Gallic acid, p-coumaric acid, ellagic acid, and protocatechuic acid), terpenoids, carotenoids, and ascorbic acid (Kim et al, 2011), flavonoids (quercetin and glycosylated xanthones such as mangiferin) (Berardini and others, 2005), vitamin E and vitamin C (Ajila et al., 2007). Vitamin C, flavonoids (hesperidin and naringenin), carotenoids (Benavente-García and Castillo 2008) and nthocyanins (Tarozzi et al., 2006). Proteolytic enzymes (papain, hymopapain), alkaloids (carpain, carpasemine), sulfurous compounds (benzyl isothiocyanate), flavonoids (Mahmood et al., 2005). Tannins, triterpenes, anthocyanins (Mahmood et al, 2005). Vitamin C, ferulic acid, p-coumaric acid, caffeic acid, carotenoids (lycopene, b-cryptoxanthin, and b-carotene) (Ajlia et al., 2010). Ellagitannins, polyphenols, tannins, anthocyanins, vitamin C, vitamin E, coenzyme Q10, and lipoic acid (Viuda-Martos et al., 2010). β-cryptoxanthin esters (Breithaupt and Bramedi, 2001), β-carotene (Holden et al., 1999), vitamins C, B1, B2 and B3, lutein and zeaxanthin, diosmin, polymethoxylated flavones (tangeretin, sinestein and nobiletin) (Gattuso et al., 2007).

Table 2. Vegetables and their most common phytochemicals Vegetable Avocado Persea americana

Commonly used parts Seeds and peel

Broccoli Brassica oleracea

Flowers and stem

Cauliflower Brassica oleracea Cactus Opuntia ficusindica

Flowers and stem

Carrot Daucus carota

Root

Pepper Capsicum annuum

Fruit

Spinach Spinacia oleracea

Leaves

Tomate Lycopersicum esculentum Watercress Nasturtium officinale

Fruit

Fruit and cladodes

Leaves

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Bioactive compounds Vitamin E, ascorbic acid, monounsaturated fatty acids and sterols (alkanols, aliphatic acetogenins, terpenoid) glycosides, flavonoids (rutin, catechin, and querceti) and coumarin (Deuester, 2001). Aliphatics glucosinolate and glucoraphanin (Cartea et al, 2010). Flavonoids (kaempferol, quercetin, isorhamnetin, sinapic, ferulic, caffeic and p-coumaric acids) (Velasco et al., 2011). Glucosinolates (thioglycosides), S-methyl cysteine sulfoxide, and isothiocyanate sulforaphane (Rodríguez-Hernández et al., 2012). Pectin, mucilage, aromadendrin, taxifolin, dihydroquercetin, isorhamnetin, vitexin, kaempferol, quercetin, derivatives of pyrone, ascorbic acid, betalains, betacyanins and flavonoids (Stintzing and Carle, 2005). Carotenoids, vitamins C and E, and phenolics (p-coumaric, chlorogenic, and caffeic acid), α- and β-carotene, quercetin, myrecetin, panaxynol, anthocyanidins (Surles et al, 2004). Vitamins C, A, and E, phenols, flavonoids, carotenoids and capsaicinoids (dihydrocapsaicin and capsaicin) (Watanabe and others, 2001. Materska and Perucka, 2005). Flavonoid glycosides, glucuronides and acylated di-and triglycosides of methylated and methylene dioxide derivatives of 6-oxygenated flavonols (Bergquist et al, 2005). Carotenoids, phytofluene, phytoene, neurosporene, γ -carotene, lycopene, phytoene, phytofluene, quercetin, polyphenols and kaempferol (Rao and Agarwal, 1999) Isothiocyanates, flavonoids (quercetin and hydroxycinnamic acids) (Gill et al, 2007). β-carotene, lutein and glucosinolates (Getahun and Chung, 1999).


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L. Rodríguez-Fragoso, Ul. Osuna-Martínez, Ana Isabel Gonzaga-Morales et al. Table 3. Experimental and clinical evidences of antioxidant effects of fruits

Fruit Apple

Cranberry

Berries

Grapes

Grapefruit Mango

Orange

Papaya

Pomegranate

Tangerine

Evidences. Antioxidant activity in vitro (Eberhardt et al., 2000). Reduction oxidative damage and effectively reduction the presence of tert-butylhydroperoxide induced ROS in vitro (Schaefer et al., 2006b). Favorable effects on antioxidant enzymes in liver including SOD, GSHPx in vivo (Décordé et al., 2008). Antioxidant activity in humans (Zhang K et al., 2004; Milbury et al., 2010; Yan et al., 2002; Duthie et al., 2006) Improvement in antioxidant status, protection from oxidation in a dose-dependent manner in atherosclerosis in humans (Steinberg, 2009). Protection membranes of living organism against the oxidative damage (Wojnicz et al., 2012). Reduction oxidized molecules of phosphatidylcholine liposome in an autooxidation process (Wolfe and Liu, 2007). Antioxidant activity (Seeram et al, 2006; Zikri et al, 2009). Reduction oxidative damage in women with metabolic syndrome (Basu et al., 2009) Prevention of radical scavenging and inhibition of lipid peroxidation. Regulation the expression of proinflammatory molecules in the ROS- and MMP-2-mediated pathways for antiulcer action . (Kim et al., 2011). Antioxidant activity, protecting low-density lipoprotein (LDL) against oxidation in vitro systems, preventing spleen cells from DNA damage induced by hydrogen peroxide (H2O2), and reducing oxidative stress in PC12 cells induced by addition of Fe2+ and t-butyl hydroperoxide (Dai et al., 2008). Prevent lipid oxidation, inhibit the production of reactive oxygen species (ROS), reduction of oxidative stress and improve glutathione/ oxidized glutathione in humans (Kar et al., 2009). Radical scavengers and chelating agents help to reduce physiological reactive oxygen species (Apostolou et al., 2013). Protection cells against the oxidative damage caused by free radicals (Hanley et al, 2011). Antioxidant and radical-scavenging properties (Guimarães et al., 2010). Antioxidant activity in vitro, in vivo (Ajila et al., 2007; Bischoff, 2008; Pardo-Andreu et al., 2006). Protection against oxidative damage in cells by ROS in vitro. Inhibition the oxidative hemolysis of erythrocytes induced by H2O2 (Alija and Prasada, 2008). Suppression ROS generation in vitro (Seeram, 2008). Reduction oxidative DNA damage and prevent meal-induced oxidative and inflammatory stress in circulating blood mononuclear cells. Reduction of reactive oxygen species (ROS) generation in vitro results (Milenkovic et al, 2011). Free radical scavenging property in vitro, in vivo (Arscott et al., 2010; Mehdipour et al, 2006). Protection against H2O2-induced oxidative DNA damage in rat pheochromocytoma tumor cells (Aruoma et al., 2006). Augmented intracellular GSH and catalase levels in SH-SY5Y neuronal cells treated with H2O2 insults and improvement the oxidant inhibitory effect of H2O2 on the assayed antioxidant enzymes (SOD, CAT and GPX) (Guizani et al., 2011). Antioxidative properties in vitro (Aviram et al, 2008). Reduction LDL oxidation and macrophage oxidative status in clinical trials (Aviram et al, 2004). Reduction oxidative stress. Decrease of protein and DNA damage, by the decline on GSH and GSSG levels without change of the GSH/ GSSG ratio, and by the decrease in antioxidant endogenous enzymes (GPx, CAT, SOD and GST) in vivo (Faria et al., 2007). Reduction lipid peroxidation and to scavenge free radicals (Dherani et al., 2008). Antioxidant activity (Puttongsiri and Haruenkit, 2010).

Clearly, no single antioxidant can replace the natural combination of the thousands of phytochemicals that exist in whole foods. Given the history of the diverse intake of plant foods by mankind, it is sensible to encourage an assorted diet. The exact amounts of fruits and vegetables needed each day to minimize disease risk are not known and will require a great deal of additional research. However, data on the antioxidant benefits of fruits and vegetables suggest that it is not premature to advise increased intake of a variety of colorful fruits and



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vegetables. The safety of consuming concentrated extracts of fruits and vegetables that contain very high levels of phytochemicals is unknown and unwarranted at this time. However, the protective benefit of a phytochemical-rich diet is best obtained from frequent consumption of fruits, vegetables, and whole grain products (See Table 3 and 4). Table 4. Experimental and clinical evidences of antioxidant effects of vegetables Vegetable Avocado

Broccoli

Cauliflower Cactus

Carrot

Pepper Spinach Tomato

Evidences. Free radical-scavenging property, increases activities of SOD, CAT, GPx, and GST enzymes in vivo (Evan, 1998; Mahadeva et al., 2011) Antioxidant activity in vivo (Pahua-Ramos et al, 2012). Antioxidant activity (Herr and Buchler, 2010). Induction the expression of antioxidant enzymes such as glutathione reductase (GSSG-red) and NAD(P)H:quinine reductase (NQO1) in vivo (Guerrero-Beltrán et al., 2012). Antioxidant activity and inhibition of HMG-CoA reductase activity in vitro (Park et al., 1997). Scavenging activities against oxygen radicals in cell-free systems (Lim et al., 2008). Delays the pro-oxidative effects of proteins, DNA and lipids (Feugang et al., 2006). Protection erythrocytes against lipid oxidation induced in vitro by organic hydroperoxide (Butera et al., 2002). Antioxidant activity in vitro (Shebaby et al., 2012). Reduction oxidative DNA damage, increases levels of plasma antioxidants, and reduction inflammation (Hu et al., 2004). Decreases lipid peroxidation in humans (Potter et al, 2011). Antioxidant activity in humans (Collera-Zuñiga et al., 2005). Free radical scavenging activity in vitro (Kim et al., 2007). Antioxidants and free-radical scavenging in vitro (Aritomi et al., 1986). Induction of SOD in vivo (Schirrmacher et al., 2010). Antioxidant and decreases lipid peroxidation in vivo (Visioli et al., 2003). Free radical scavenger and potent inhibitor of lipid peroxidation in vivo (Periago et al., 2009).

Fruits and Vegetables: Examples with Clinical Relevance Apple (Malus domestica) Apple is one of those fruits that can play a role in decreasing the risk of chronic diseases due to its fiber content and chemical components such as flavonoids, polyphenols and carotenoids (Hyson, 2011). The concentration of phytochemicals in the apple peel varies greatly from that in the apple flesh. Some of the best-studied antioxidant compounds in apples include quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-rhamnoside, catechin, epicatechin, procyanidin, cyanidin-3-galactoside, coumaric acid, chlorogenic acid, gallic acid, and phloridzin (Guyot et al., 2003). It has been previously shown that peeled and unpeeled apples had high antioxidant activity and inhibited the growth of human cancer cells in vitro. Vitamin C was responsible for less than 0.4% of the antioxidant activity, indicating that other elements, such as phenolics, were the main contributors. The antioxidant and antiproliferative activities of unpeeled apples were greater than those of peeled apples (Eberhardt et al., 2000). It is also known that the concentration of total phenolic compounds is much greater in the peel than in the flesh (Escarpa and González, 1998). The flesh contains catechins, procyanidins,


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phloridzin, phloretin glycosides, caffeic acid, and chlorogenic acid, among others; the peel possesses all of these compounds and has additional flavonoids not found in the flesh, such as quercetin glycosides. Of the catechins, only (+)-catechin and (-)-epicatechin are present in appreciable amounts, with epicatechin being approximately twice as concentrated as catechin in the peels (Golding et al., 2001). Schaefer et al. crushed and extracted juice from cider and table apples harvested in Germany to prepare several polyphenolic mixtures. They found that all extracts significantly reduced oxidative damage and effectively reduced the presence of tert-Butyl hydroperoxide induced ROS. Although there were observed differences in effectiveness and specificity between each extract preparation, the effective range was comparable to quantities of phytochemicals found in apple juice (Schaefer et al., 2006b). Interestingly, it has been found that prolonged exposure to apple products resulted in even greater antioxidant capacity for some compounds, suggesting that metabolic products formed over a period of time may have differing antioxidant capacities from those of the parent phytochemicals and, in some cases, improved potential (Hyson, 2011). A study conducted in Turkey included 15 elderly participants (mean age 72 yrs; 8 female, 7 male) who ate fresh apples at a daily dose of 2 g/kg for 1 month. Pre- and post study values were compared to assess antioxidant activity in the participants’ erythrocytes and plasma. It was found that apple consumption increased antioxidant enzymes, including SOD and GPx, in erythrocytes and overall antioxidant potential in plasma. The upregulation of these enzymes suggests that regular apple consumption might promote a favorable milieu to reduce oxidation (Avci et al., 2007). Another study in hamsters evaluated the effects of adding daily apples and apple juice (pressed from fresh apples) to an atherogenic diet on lipids, oxidative markers, and early aortic lesions. Hamsters were provided with apples to an approximate human intake of 600 g/d (~2.5 large apples) or 500 mL of juice/d. Both products reduced the percentage of aortic surface area covered by foam cells (aortic fatty streak lesion area) by 48% in the apple group and 60% in the apple juice group compared to controls. Favorable effects on antioxidant enzymes in liver including SOD, GSHPx, and general markers of oxidation (hepatic TBARS) were significantly reduced (Décordé et al., 2008). It has been found on in vitro studies that intervention with either organic or conventionally grown apples (1 kg) did not affect antioxidant capacity on low-density lipoprotein (LDL) lag time tests in peripheral blood human lymphocytes. There was no effect on endogenous DNA strand breaks and Fpg-sensitive sites in peripheral blood lymphocytes, but apples strongly decreased oxidative DNA damage recognized by Endo III and increased the capacity to protect DNA against damage induced by iron chloride, indicating that both organically and conventionally grown apples have antigenotoxic potential (Briviba et al., 2007). As for the properties of individual compounds known to be present in apples, individual phytochemicals, including rutin, chlorogenic acid, and caffeic acid were all effective, with some reconstituted mixtures being more successful than the original in terms of antioxidant capacity and reducing DNA damage. The most effective compounds on all antioxidative parameters included quercetin and phloretin (Schaefer et al., 2006a). A number of in vitro studies have demonstrated that apples, apple extracts, and apple polyphenols possess high antioxidant capacity in vitro, including inhibition of copper induced LDL oxidation (Pearson et al., 1999).



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There are inconsistencies in the correlation between in vitro outcomes and in vivo antioxidant activity mediated by apple. This variability might be partially attributed to the many types of apples and apple components studied, in addition to varied reaction conditions including pH, concentration, types of ROS, and other study conditions.

Berries (Rubus coreanus, Rubus idaeus, Rubus fruticosus, Rubus leucodermis) Berries biological properties have been largely attributed to high levels of various phenolic compounds, as well as the interactive synergies among their natural phytochemical components (e.g., ellagic acid, quercetin, gallic acid, anthocyanidins, cyanidins, pelargonidins, catechins, kaempferol and salicylic acid). Berries also contain vitamins A, C, E and folic acid, calcium, and selenium (Tian et al., 2005). They have been shown to have a positive impact on several chronic conditions, including obesity, cardiovascular and neurodegenerative diseases, and cancer (Brownmiller et al., 2008). The major phenolic compounds present in blackberry (Rubus fruticosus L.) fruits are anthocyanins and ellagitannins (ETs). Sanguiin H-6 and lambertianin C are considered the two major ETs of strawberries (Fragaria x ananassa Duch.), blackberries (Rubus fruticosus), and raspberries (Rubus idaeus L.), and they have been reported as responsible for the high antioxidant capacity of these fruits (Beekwilder et al., 2005). Blackberry juices in two different preparations, with water and defatted milk, were administered to subjects in order to evaluate the possible effects of food matrix on plasma antioxidant capacity and enzymatic and non-enzymatic antioxidants. The consumption of blackberry juices led to an increase in plasma and urine antioxidant capacities, which could indicate the in vivo antioxidant potential. In plasma, this effect was more strongly associated with an increase in the ascorbate level and not with the polyphenols present in blackberry fruits. However, the antioxidant capacity in the urine was associated with anthocyanins and urate levels. An increase in plasma catalase activity (CAT) concurrent with plasma anthocyanin levels was therefore observed (Hassimoto et al., 2006). Korean raspberry (Rubus coreanus Miquel, RCM) has been used as a traditional remedy for several diseases (But et al., 1997). Scientists have studied the active components in raspberry and found it contains various antioxidants such as polyphenols, tannins, phenolic acids, organic acids, triterpenoids, flavonoids, gallotannin, ellagitannin, and anthocyanins (Yoon et al., 2003; Cho et al., 2005). Several studies have shown that a RCM extract has higher electron donation ability and prevents LDL oxidation in in vitro studies. Previous studies have shown that weeks of Korean raspberry supplementation did not reduce lipid peroxidation in healthy males who did not smoke or drink alcohol, suggesting that oxidative stress levels were relatively low in those subjects. In contrast, strawberry supplementation significantly reduced oxidative damage and lowered total cholesterol and LDL-cholesterol levels in women with metabolic syndrome or hyperlipidemia (Basu et al., 2009; Basu et al., 2010). These results suggest that the endogenous antioxidant defense system and antioxidant intake from the diet could adequately prevent oxidative stress in healthy subjects, but extra antioxidants do not reduce oxidative stress.


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Previous data have shown that anthocyanins from R. coreanus cause the reversal of naproxen-induced gastric epithelial cell damage through the prevention of radical scavenging and inhibition of lipid peroxidation. Furthermore, anthocyanins have an antiulcer effect due to their regulation of matrix metalloproteinase-2 (MMP-2) activity. Also, anthocyanins can regulate the expression of proinflammatory molecules in the ROS- and MMP-2-mediated pathways for antiulcer action (Kim et al., 2011). Raspberry constituents also have antioxidant and anti-inflammatory properties and inhibit cancer cell growth (Seeram et al., 2001). Black raspberries have a selective effect on the growth and apoptosis of highly tumorigenic rat esophageal epithelial cells in vitro; this may be due to preferential uptake and retention of its component anthocyanidins, which may also be responsible for the greater inhibitory effects of freeze-dried whole berries on tumor cells in vivo (Zikri et al, 2009). On the other hand, in studies using a rat model of nitrosamineinduced esophageal squamous cell carcinoma, black raspberries induced a reduction of proliferation, inflammation and angiogenesis while stimulating apoptosis and differentiation of premalignant cells and tissues, resulting in reduced tumor development. Genes associated with these cellular functions were also protectively modulated by black raspberry diets (Stoner et al., 2007). All of these data provide sufficient evidence of the antioxidant properties of berries.

Cranberry (Vaccinium macrocarpon) American cranberry is a fruit used as a prophylactic agent against urinary tract infections. Cranberry contains a great amount of phenolics, including simple phenolic acids, flavonoids that include anthocyanidins and proanthocyanidins, and flavonols (Rossi et al., 2010). These phenolics vary according to the degree of unsaturation, oxidation of the three-carbon segment, and polymerization, which may influence their biological activity. Only a small percentage of the total flavonol content in cranberry or cranberry juice exists as aglycones such as free myricetin, quercetin and kaempferol. When one thinks of cranberries, the color red comes to mind: this is due to the presence of anthocyanidins, the health benefits of which have been extensively studied (Singh et al., 2009). Due to its high content of flavonoids and phenolic acids, cranberry ranks highly among the fruits for both its antioxidant qualities and quantity (Vinson et al., 2001). As far as cranberry antioxidants are concerned, they mostly come under the form of phenolic acids and flavonoids (Chen et al., 2001) at portions of 44 and 56% respectively, based on total dry weight of non-nutrient antioxidants. Polyphenol antioxidants have previously been found in human plasma after drinking cranberry juice (CJ) and reached a maximum of 10 µM. On the other hand, several free phenolic acids have been found in human plasma following CJ consumption. Benzoic, ferulic and sinapic acids were found in human plasma from 45 up to 270 min after consumption. Phenolic acids are also identified in plasma but are not present in significant quantities in CJ, suggesting they could be metabolites from other phenolics from cranberries (Zhang and Zuo, 2004). The presence of phenolic antioxidants in human plasma has been found in bioavailability studies in healthy volunteers (Zhang K et al., 2004), in patients with coronary artery disease (Milbury et al., 2010) and patients with metabolic syndrome. The results of these and other studies lead us to believe that the high antioxidant activity of cranberry, and its extracts plays



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a substantial role in cardiovascular disease protection, cancer, and neurotoxicity, for example (Yan et al., 2002; Duthie et al., 2006). Cranberry compounds can cause an improvement in antioxidant status, which might be beneficial in the case of chronic diseases. It has been shown that when CJ is spiked on human plasma, LDL and very-low-density lipoprotein can be protected from oxidation in a dose-dependent manner. This second effect is directly related to atherosclerosis (Steinberg, 1989). Intervention trials, with or without placebo control and ranging from 2 to 16 weeks have reported cranberries improve oxidative stress, postprandial glycemic response, dyslipidemia, and atherosclerotic markers in healthy volunteers (Ruel et al. 2008) as well as in patients with type 2 Diabetes Mellitus (Lee et al. 2008). In contrast to these significant findings, Duthie et al. did a 2-week study of healthy female volunteers and reported no substantial changes in blood, cellular antioxidant status or surrogate biomarkers of CVD and cancer risks following cranberry juice versus placebo intervention (Duthie et al., 2006). Extensive research both on identification of the phytochemical in cranberries and their bioactivity indicates, among other things, that anthocyanins and flavonoids are strong antioxidants with the potency to protect the membranes of living organisms against oxidative damage (Wojnicz et al., 2012). It is very likely that the components of cranberry extract undergo oxidation, reducing the oxidized molecules of the phosphatidylcholine liposome in an autooxidation process (Wolfe and Liu, 2007).

Grapes (Vitis vinifera) The grape is considered a source of unique and potentially useful natural medicinal products; it is also used in the manufacturing of various industrial products (Yadav et al., 2009). The beneficial effects of grape and relevant grape-derived food products are believed to be related to a variety of bioactive components such as epicatechin gallate, procyanidin oligomers and their gallates, resveratrol, β-carotene, lutein, flavonoids and anthocyanins (Vislocky and Fernandez, 2010). A major group is that of phenolic antioxidants, which typically include anthocyanins, catechins, resveratrol, phenolic acids, and procyanidins (Bunea, 2012). Most grape phenolic antioxidants are distributed in grape skins or seeds. For instance, resveratrols, antochyanins and catechins are concentrated in the skin, while procyanidins concentrate in the seeds (Kammerer et al., 2004). Grapes contain a wide range of chemical substances such as sugars, organic acids, mineral salts, vitamins, enzymes, and also phytochemicals responsible for the sensory characteristics of wines and their health-related properties. Phytochemicals in grapes are mostly phenolic compounds. The antioxidant activities of the individual phenolic compounds may depend on structural factors such as the number of phenolic hydroxyl or methoxyl groups, flavone hydroxyl, keto groups, free carboxylic groups, and other structural features. The antioxidant activities of grape phenolics have been demonstrated in various model systems including protecting LDL against oxidation brought about by Cu2+, oxygen-centered radical-generating 2,2’-azobis(2-methylpropionamidine) dihydrochloride (AAPH), or peroxynitrite generating 3-morpholinosydnonimine in vitro systems, preventing spleen cells from DNA damage induced by hydrogen peroxide (H2O2), and reducing oxidative stress in PC12 cells induced by addition of Fe2+ and t-butyl hydroperoxide (Apostolou et al., 2013).


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Some studies showed that dietary intake of grape antioxidants helps prevent lipid oxidation and inhibit the production of ROS. For instance, dietary supplementation of grape seed extract (600 mg/day) for 4 weeks in high-cholesterol human subjects produced a reduction of oxidative stress and improved GSH/oxidized glutathione (GSSG) and total antioxidant status in a double-blinded randomized crossover human trial (Kar et al., 2009). Another study also demonstrated that grape seed extract supplementation (2× 300 mg/day) improved plasma antioxidant capacity (Vinson et al., 2001). A number of studies have shown that, when combined with each other or with other antioxidants, plant polyphenols exhibit stronger antioxidant activity compared to their individual one (Dai et al., 2008). It has been observed that grape antioxidants could act as free radical scavengers and chelating agents, in order to reduce the presence of ROS. One research group demonstrated that grape extracts exhibited strong antioxidant activity and prevented ROS-induced DNA damage (Apostolou et al., 2013). In the past years, a growing body of epidemiological studies and randomized controlled human trials have associated the consumption of grapes, wine, and grape juice with a wide variety of health-promoting effects, particularly a reduction in the risk of CVD, type-2 diabetes, certain types of cancers, and other chronic complications (Mellen et al., 2010; Feringa et al., 2011). Another biologically active and well-characterized constituent of the grape is resveratrol, which is known for its various medicinal properties in the treatment of human diseases (Yadav et al., 2009). The skin of grapes has a significant amount of resveratrol, the source behind the beneficial effects of red wines on cancer prevention and against coronary heart disease. The anticancer effects of grape antioxidants have been demonstrated in in vitro and in vivo models. In animal studies, resveratrol prevented or delayed the development of skin, mammary, and prostate tumors, as well as esophageal, gastric, small intestinal, colonic, pancreatic, and hepatic tumorigenesis. Resveratrol has also been shown to possess in vitro cytotoxic effects against a wide variety of human tumor cells, including lymphoid and myeloid cancer cells as well as skin, breast, ovary, cervix, prostate, stomach, colon, pancreas, liver and thyroid carcinoma (Morré and Morré, 2006). Grape antioxidants have been shown to induce cell cycle arrest and apoptosis in cancer cells as well as prevent carcinogenesis and cancer progression in rodent models (Aggarwal et al., 2004). Many studies have demonstrated that the phenolic compounds present in grapes could reduce the incidence of serious chronic problems such as atherosclerosis and CVD due to their antioxidant abilities (Zhu et al., 2012). In addition, grape oil helps dissolve thrombi in arteries, reducing platelet aggregation and preventing heart attacks; it can prevent hypertension and aid in the normalization of injuries caused by poor circulation due to obesity and diabetes. It may be used to treat obesity, cellulite, and stretch marks, since it aids tissue elasticity, reduces swelling and edema, restores collagen, and improves peripheral circulation (Agostini et al., 2012). Grapes also decreased the levels of lipid peroxidation in the liver and concomitantly increased the levels of hepatic enzymatic and nonenzymatic antioxidants (Pari and Suresh, 2008). Additionally, studies have also shown that the red wine prepared from grapes ameliorates oxidative stress in the liver of alcohol-fed rats, and helps prevent fatty liver and hepatic fibrosis (Assuncáo et al., 2009).



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Grapefruit (Citrus paradise) Citrus fruits have many healthy properties, most of them due to their high content of nutrients such as vitamin C, carotenoids, and phenolic compounds. These phytochemicals have antioxidant capacities and may protect cells against the oxidative damage caused by free radicals. Grapefruit juice is rich in a number of phytochemicals, including flavonoids and furanocoumarins (Hanley et al., 2011). The peel of grapefruit has five isolated compounds: friedelin, β-sitosterol, 7(3’,7’,11’,14’-tetramethy)-pentadec-2’,6’,10’-trienyloxycoumarin, limonin and cordialin B (Meera and Kalidhar, 2008). Medical data suggest grapefruit juice reduces atherosclerotic plaque formation and inhibits breast cancer cell proliferation and mammary cell tumorigenesis (Kiani and Imam, 2007). Many people suffer from chronic metabolic diseases (including hypertension, hyperlipidemia, and CVD) and they receive calcium channel antagonist therapy and 3hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors. Consumption of grapefruit and grapefruit juice may result in cardiometabolic benefits, since trials have shown it can increase serum HDL-cholesterol concentrations (Silver et al., 2011). It has also been shown that grapefruit juice positively affects bone quality and normalizes osteoclasts and decreased bone loss (Deyhim et al., 2006). Removing free radicals by eating foods rich in antioxidants will potentially improve antioxidant status, lower oxidative stress, and may even reduce bone fracture risk (Deyhim et al., 2008). The improvement in the bone quality of grapefruit-fed rats observed in this study could have been partially attributed to slowed-down bone resorption, increased bone mineral deposition, increased calcium absorption, the maintenance of plasma antioxidant capacity and decreased urinary magnesium excretion. Citrus peel is the main waste fraction of citrus fruits and has been widely studied because of its numerous biologically active compounds, including natural antioxidants such as phenolic acids and flavonoids (Manthey and Grohmann, 2001). It has been reported that heat treatment may liberate some low molecular weight phenolic compounds and hence increase the antioxidant capacity of citrus peel (Seok-Moon, 2004). Peel polar fractions revealed the highest contents in phenolics, flavonoids, ascorbic acid, carotenoids and reducing sugars, which certainly contribute to the highest antioxidant potential found in these fractions (Guimarães et al., 2010). Overall it was found that the peels of fruits are major sources of different antioxidants and these by-products of the juice extraction industry could be used as natural antioxidants. Many authors have reported antioxidant and radical-scavenging properties of essential oils and in some cases, a direct food-related application as well.

Mango (Mangifera indica, Mangifera pajang) Mango is one of most widely consumed tropical fruits; it is rich in nutritive and nonnutritive compounds, including ascorbic acid, carotenoids, and polyphenols, and it has been found that these phytochemical compounds contribute to disease-risk reduction (Shah et al., 2010). Previous studies of mango fruit have shown that it contains various classes of polyphenols, terpenoids, carotenoids, and ascorbic acid (Kim et al., 2011). Preliminary phytochemical screening revealed the presence of flavonoids, including quercetin and glycosylated xanthones such as mangiferin (Berardini et al., 2005). It has been demonstrated


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that quercetin possess antioxidant, antitumor, antihypertensive, anti-atherosclerotic, and antiinflammatory properties (Bischoff, 2008). Peels and seeds are the major by-products generated during the processing of mango, amounting to 35-60% of the total fruit weight. The peel is a good source of phytochemicals such as polyphenols, carotenoids, vitamin E and vitamin C (Ajila et al., 2007) and exhibits good antioxidant properties. Phenolics have a potent antioxidant activity and are believed to have health-promoting properties that make the consumption of fruits and derived processed products from fruit pulp, peels, and seed kernels a very healthy habit (Selles et al., 2002). It has been reported that polyphenol content of mango peel is higher than that of pulp and that peel extract from M. pajang fruits is a rich source of polyphenols (Ajila and Prasada, 2008). The major phenolic compounds were gallic acid, p-coumaric acid, ellagic acid, mangiferin, and protocatechuic acid. All these phenolic compounds were shown to exhibit antioxidant properties (Abdulrahman et al., 2011). Due to their antioxidant properties, the phytochemicals present in mango peel may exhibit protection against oxidative damage in cells by ROS. It has been previously reported that mango peel extract isolated from two varieties of mangoes at two different stages of maturity could inhibit the oxidative hemolysis of erythrocytes induced by H2O2 under experimental conditions (Ajila and Prasada, 2008). Previous studies of aqueous stem bark extract from a selected species of mango that was used in pharmaceutical formulations and as a food supplement in Cuba under the brand name of Vimang, report potent in vitro and in vivo antioxidant and anti-inflammatory activity, as well as prevention of age-associated oxidative stress (Pardo-Andreu et al., 2010). The significant cytotoxic activities of stem bark mango extract have been tested against the MCF 7, MDA-MB-435 and MDA-N breast cancer cell lines, as well as against the SW-620 colon cancer cell line and the 786-0 renal cancer cell line (Shah et al., 2010). Mangiferin also mediates the down-regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), suppresses NF-κB activation induced by inflammatory agents, including tumor nuclear factor alpha (TNF-α), increases intracellular GSH levels, and potentiates chemotherapeutic agent-mediated cell death. All these data suggest a potential role in combination cancer therapy (Knodler et al., 2008). It has been reported that mango contains compounds that have antioxidant activity, growth-arresting activity, and anti-tumor promotion activity. Whether these anticancer properties are maintained after digestion, absorption, and metabolism is unknown, although anticancer activity has been related to antioxidant activity in many studies (Percival et al., 2006).

Orange (Citrus sinensis) Citrus is one of the world’s most popular fruit crops; in addition to providing an ample supply of vitamin C, folic acid, potassium, and pectin, it contains a host of flavonoids, ascorbic acid and carotenoids that can potentially protect health. Its role in the prevention of several diseases has already been reported (Benavente-García and Castillo, 2008). Studies on the bioactive compounds and antioxidant activity of citrus have mainly focused on the fruits (peel, pulp and juice) and polar fractions (Gorinstein et al., 2001). It has been found that the peel is a major source of different antioxidants and this by-product of the juice extraction



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industry could be used as a natural antioxidant. A comprehensive study conducted on 21 Mauritian Citrus species demonstrated, with established correlations, that polyphenolic-rich extracts exhibited important antioxidant propensities in various test systems (Ramful et al., 2010a). Fruit juice of a new pigmented citrus hybrid named Omo-31 and those of its parents clementine cv. Oroval (Citrus clementina Hort. ex Tan.) and Moro orange [Citrus sinensis (L.) Osbeck] were analyzed during fruit maturation to determine juice yield, total soluble solids, total acidity, and potentially beneficial components such as vitamin C, flavanones, anthocyanins, and phenolic acids. At maturity stage, the amount of anthocyanins, flavanones, and hydroxycinnamic acids in Omo-31 was found to be notably higher than in its parents. Such high level of antioxidant substances makes this new fruit important for its nutritional benefits (Rapisarda et al., 2003). It has been found that organic red oranges have a higher phytochemical content (i.e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and bioactivity than integrated red oranges (Tarozzi et al., 2006). Recent results clearly show that Mauritian Citrus fruit extracts represent an important source of antioxidants, with a novel antioxidative role at the adipose tissue level and beneficial effects of antioxidant citrus extracts in a model of obesity-linked metabolic disorder (Ramful et al., 2010b). Orange juice antagonizes oxidative and inflammatory stress, an effect at least partially attributed to the presence of hesperidin and naringenin, which are two flavonoids that suppress ROS generation in vitro (Seeram, 2008). It was recently found that seven days consumption of red orange juice ameliorated endothelial function and reduced inflammation in non-diabetic subjects with increased cardiovascular risk (Buscemi et al., 2012). Other clinical studies in healthy subjects have shown that orange juice consumption reduced oxidative DNA damage and may prevent meal-induced oxidative and inflammatory stress in circulating blood mononuclear cells. The reduction of ROS generation and NF-κB binding following orange juice intake might be due to its flavonoid content, as suggested by in vitro results (Milenkovic et al., 2011).

Papaya (Carica papaya) Papaya fruit and seeds are widely used in medicine to prevent lipid peroxidation because of their high antioxidant contents, and are considered a preventive treatment against atherosclerosis and coronary heart diseases. Extracts from different papaya tissues have been shown to be bioactive. Carica papaya L. leaves and seeds are known to contain proteolytic enzymes (papain, hymopapain), alkaloids (carpain, carpasemine), sulfurous compounds (benzyl isothiocyanate), flavonoids, triterpenes, organic acids and oils (Mahmood et al., 2005). Papaya was reported to have in vitro free radical scavenging properties and effectively improve antioxidant defense while significantly decreasing the risk of age-related macular degeneration in human clinical trials (Arscott et al., 2010). It was also reported to protect against H2O2-induced oxidative DNA damage in rat pheochromocytoma tumor cells (Aruoma et al., 2006). Papaya fruit skin contains different bioactive compounds, such as ferulic acid, pcoumaric acid, caffeic acid, carotenoids (mostly lycopene, β-cryptoxanthin, and β-carotene) and vitamin C, which can collectively protect human cells from oxidative stress. Papaya epicarp extracts were effective in promoting wound-healing processes and cellular skin


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development; these remarkable effects were attributed to its photochemical and antioxidant activities (Ajlia et al., 2010). Studies about the toxicological and antioxidant potential of dried C. papaya juice in vitro and in vivo indicated its safety and antioxidative stress potential, which was found to be comparable to the standard antioxidant compound alpha-tocopherol (Mehdipour et al., 2006). It was recently found that papaya epicarp extracts augmented intracellular GSH and catalase levels in SH-SY5Y neuronal cells treated with H2O2 insults. Papaya epicarp extract can significantly ameliorate the oxidant inhibitory effect of H2O2 on the assayed antioxidant enzymes (SOD, CAT and GPx) (Guizani et al., 2011). Another compound found in papaya is licopene. This has been linked to antioxidant, antiproliferative (growth inhibition, cell cycle arrest, apoptosis), antiangiogenesis, antiinflammatory, and immunomodulation in prostate, lung, breast, gastric, liver, pancreas, colorectal, head, neck and skin cancer. Synergistic interaction with genistein, adriamycin, and cisplatin was also observed. Different targets include cyclin D1, Bcl-2, Bcl-xL, AKT, BAD, NF-κB, MMP-9, Sp-1, and IGF-BP3 (Amin et al., 2009). Fermented papaya preparation is a natural health food that has been commercially sold in Japan for 2 years. It is made by yeast fermentation of Carica papaya Linn. This fermented papaya preparation has been shown to increase SOD activity in the cortex and hippocampus in iron-induced epileptic foci of rats. These results suggest that the preparation has antioxidant actions and may serve as prophylactic food against age related and neurological diseases associated with free radicals (Imao et al., 1998).

Pomegranate (Punica granatum) Pomegranate is commonly eaten around the world and has been used in folk medicine across many cultures, especially in the Middle East, for a wide variety of therapeutic purposes (Shabtay et al., 2008). Pomegranate preparations contain very high levels of antioxidants compared to those of any other fruit or vegetable, including the amount of flavonoids and polyphenols. The juice is known to be a rich source of antioxidants given its polyphenol, tannins, and anthocyanin content, including vitamin C, vitamin E, coenzyme Q10, and lipoic acid. Anthocyanins are the most important group present in the arils or juice; they even give the fruit or juice its color (Viuda-Martos et al., 2010). It has been suggested that free radical scavenging and antioxidant activities of extracts from various parts of P. granatum play an important role in the prevention of free radical-related disease, including aging, wounds and ulcers (Harmam, 2001). Leaf extracts have shown free radical scavenging activity and antioxidant effects in vitro (Al-Muammar and Khan, 2012). The antioxidant activity is related to the diverse phenolic compounds present in the pomegranate, including the punicalagin isomer, ellagic acid, and anthocyanins (de Nigris et al., 2007). These compounds are known for their free radicalscavenging properties and inhibit lipid oxidation in vitro (Noda et al., 2002). Tzulker et al. suggested punicalagin is one of the major phytochemicals that contribute to the total antioxidant capacity of pomegranate juice, with anthocyanin playing a minor role (Tzulker et al., 2007).



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These antioxidants have been shown to protect against cholesterol oxidation and have anti-aging effects (de Nigris et al., 2006). Aviram et al., analyzed the action mechanism of pomegranate fruit parts (i.e., the peel, arils, seeds, and flower) in vivo and in vitro. All extracts were shown to possess antioxidative properties in vitro and pomegranate flower extract consumption resulted in lower serum lipids and glucose levels by 18% to 25% (Aviram et al., 2008). The flavonoids of pomegranate peel methanolic extract have also been shown to reduce lipid peroxyde and nitric oxide levels, and also scavenged the free radicals in brain tissue when they were coadministered with AlCl3 (Abdel, 2012). Ellagitanins, namely punicalagin, also have remarkable pharmacological activities, including anti-inflammatory, hepatoprotective and antigenotoxic ones (Lin et al., 2001). In a limited study of hypertensive patients, consumption of pomegranate juice for two weeks reduced systolic blood pressure by inhibiting the serum angiotensin-converting enzyme (Aviram and Dornfeld, 2001). Preliminary laboratory research and clinical trials showed that the juice of the pomegranate may be effective in reducing heart disease risk factors such as LDL oxidation, macrophage oxidative status, and foam cell formation (Aviram et al., 2004). The effect of pomegranate juice on cholesterol accumulation in macrophages, on cellular oxidation stress, and on cholesterol biosynthesis in a J774.A1 macrophage-like cell line has also been studied. Cells treated with pomegranate juice (polyphenol 75 mmol/L) showed a 40% decrease in the degradation of oxidized LDL, a decrease of 50% in the rate of macrophage cholesterol synthesis and a decrease of oxidative stress (Fuhrman et al., 2005). On the other hand, Rosenblat et al., also reported that the consumption of pomegranate juice by diabetic patients led to a decrease in oxidative stress in the patients’ serum and the macrophage uptake of oxidized LDL (Rosenblat et al., 2006). Recent research has shown that pomegranate extracts selectively inhibit the growth of breast, prostate, colon and lung cancer cells in culture. In preclinical animal studies, oral consumption of pomegranate extract inhibited growth of lung, skin, colon, and prostate tumors. An initial phase II clinical trial of pomegranate juice in patients with prostate cancer reported significant prolongation of prostate specific antigen doubling time (Mustafa et al., 2009). The use of pomegranate juice for 4 weeks in animals with hepatic oxidative stress showed a state of reduced oxidative stress. This was supported by the decrease of protein and DNA damage, the decline of GSH and GSSG levels without change of the GSH/GSSG ratio, and a decrease in antioxidant endogenous enzymes like GPx, CAT, SOD and glutathione Stranferase (GST) (Faria et al., 2007). The high antioxidant activities of the phytochemicals found in the pomegranate have led to the development of dietary supplements that contain biologically active polyphenols and ellagitannins. The overall antioxidant activity of pomegranate juice has been previously reported to exceed that of other red-purple fruits, red wine, and green tea (Rosenblat et al., 2006).

Tangerine (Citrus deliciosa, Citrus reticulate, Citrus tangerine) Tangerines, the smallest species in the economically important family of citrus fruits, contain high amounts of β-cryptoxanthin esters (Breithaupt and Bramedi, 2001). Additionally, tangerines are a source for β-carotene, lutein, and zeaxanthin (Holden et al., 1999). They also contain some potassium, magnesium, vitamins C, B1, B2 and B3, lutein and zeaxanthin. Like


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all citrus oils, tangerine oil has limonene as its major constituent, along with alpha-pinene, myrcene, gamma-terpinene, citronellal, linalool, nerol, neryl acetate, geranyl acetate, geraniol, thymol, and carvone (Lyle, 2006). Diosmin is one of the main components of citrus fruits. Polymethoxylated flavones, such as tangeretin and nobiletin, exist exclusively in the citrus genus and are especially common in the peels of tangerine (Gattuso et al., 2007). β-carotene and β-cryptoxanthin have pro-vitamin A activity as well as biological actions such as the ability to reduce lipid peroxidation and scavenge free radicals that may be important in maintaining health and preventing serious diseases such as cancer, pulmonary disorders, and cataracts (Dherani et al., 2008). It has been found that hand-pressed tangerine juice of C. reticulata Blanco cv. Sainampueng grown in northern Thailand is an excellent source of the polymethoxylated flavones tangeretin, nobiletin, and sinensetin. These peeled fruits had higher quantities of the flavanone glycosides narirutin, hesperidin, and didymin, but only small amounts of polymethoxylated flavones compared to the juice. An analysis of carotenoids and antioxidants in juice samples confirmed β-cryptoxanthin as the predominant carotenoid in these tangerines and revealed significantly higher levels of R-tocopherol in organic tangerine juice than that produced using conventional agrochemical-based and agrochemical safe grown fruits (Stuetz et al., 2010). Consumption of hand-pressed tangerine juice with high concentrations of tangeritin and nobiletin as well as a high content of flavanone glycosides, antioxidants, and β-cryptoxanthin might prove effective in reducing hypercholesterolemia, incidence of atherosclerosis and cardiovascular disease, and vitamin A deficiency. Previous studies on hamsters fed supplements with mixtures of 1% polymethoxylated flavonoids that contained tangeretin and nobiletin showed lower plasma concentrations of both triglycerides and cholesterol and reduced hepatic triglycerides (Kurowska and Manthey, 2004). Coating fruits, including citrics, to extend shelf life and improve glossiness has long been common method. Coating and storage temperature may lead to other chemical changes in fruits; it was reported that bioactive compounds, such as total polyphenol, vitamin C and hydroxycinnamicacids in blood oranges increased during storage at low temperatures (Rapisarda et al., 2008). Changes in ascorbic acid, total polyphenol, phenolic acids and antioxidant activity in juice extracted from coated Kiew Wan tangerine during storage at 4, 12 and 20 °C have been observed. The results of that study indicated that changes in the levels of ascorbic acid, total polyphenol, phenolic acids and antioxidant activity in coated tangerines were affected by storage period, regardless of temperatures. The ascorbic acid content decreased during the storage period, irrespective of temperature. As for the phenolic acids found (caffeic, p-coumaric, sinapic and ferulic acid), the level of each one increased during the early stage of storage and declined slightly at the end. Storage of coated tangerine at 4, 12 and 20°C did not affect the antioxidant components (Puttongsiri and Haruenkit, 2010). Phenolic acids also contributed to antioxidant activity in coated tangerines. Tangerines are sources of important nutrients for human health and the antioxidant activity in tangerine is associated with more than a single compound.

Avocado (Persea americana) Avocado is a good source of bioactive compounds, including vitamin E, ascorbic acid, carotenoids and soluble phenolics (Deuester, 2001). These compound classes may be divided



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into alkanols (also sometimes termed “aliphatic acetogenins”), terpenoid glycosides, various furan ring-containing derivatives, flavonoids, and coumarin (Corral-Aguayo et al., 2008). Growing data on the health benefits of avocadoes have led to increased consumption and further research (Whiley and Schaffer, 2002). Phytochemicals extracted from avocado can selectively induce several biological functions (Plaza et al., 2009). Avocado has traditionally been used due to its hypotensive, anti-inflammatory, and immune-enhancing effect (Adeyemi et al., 2002). Furthermore, avocado juice made from ripe fruit is very popular due to its numerous health benefits (Mahadeva et al., 2011). Ascorbic acid and GSH are the two major low molecular weight antioxidants that prevent oxidative damage in fruit. It has been shown that the GSH content increased in early harvested fruits and decreased in late harvested ones during storage. A similar trend was observed in ascorbic acid changes (Kevers et al., 2007). The mechanisms regulating the pool sizes of the two components are not yet fully understood, but high levels of GSH, may be associated with the high levels of ascorbic acid in the fruit (Noctor and Foyer, 1998). The reason for these phenomena may be linked to the fact that the ascorbic acid and total phenolic contents, along with antioxidant activity, are influenced by the harvest date of the avocado fruits during storage. Several beneficial medicinal properties of compounds present in the avocado seed and peel have been reported and are related to elevated levels of phenolic compounds (64% in seed, 23% in peel, and 13% in pulp). In addition, the seeds and peels of avocado also contribute 57% and 38% of the antioxidant capacities of the entire fruit, respectively (Wang et al., 2010). Rutin, catechin, and quercetin are widespread in nature and may act as powerful antioxidants in avocado (Terpinc et al., 2012). However, seeds contain the strongest antioxidant properties and highest phenol and procyanidin content compared to the pulp and edible portions (Wang et al., 2012). The phytochemicals present in P. americana fruit extract may contribute to the free radical-scavenging property of the extract (Mahadeva et al., 2011). Enzymatic antioxidants are also involved in the detoxification of free radicals and peroxides formed during the course of oxidative stress. Oral treatment with P. americana fruit extract in STZ-induced diabetic rats resulted in increased activities of SOD, CAT, GPx, and GST enzymes. This may be attributed to the free radical scavenging and anti-hyperglycaemic activities of P. Americana fruit extract (Evan, 1998; Mahadeva et al., 2011). It has been shown that oral treatment with avocado extract significantly decreased blood glucose levels and increased the insulin level in STZ-induced diabetic rats. The anti-hyperglycaemic effect of avocado fruits may be due to their stimulatory effect on remnant β-cells, allowing them to secrete more insulin, or to a favorable effect on regenerated β-cells, preventing the formation of glycosylated haemoglobin, and reducing liver peroxides (Mahadeva et al., 2011). Avocado seeds contain elevated levels of phenolic compounds and exhibit antioxidant properties. Avocado seeds reduced total cholesterol and LDL-cholesterol levels, as well as the prediction of the atherogenic index. Therefore, the antioxidant activity of phenolic compounds and dietary fiber in avocado seeds may be responsible for their hypocholesterolemic activity in a hyperlipidemic model of mice (Papua-Ramos et al., 2012). Recent studies indicate that phytochemicals extracted with 50% methanol from avocado fruits aid the proliferation of human lymphocyte cells and decrease chromosomal aberrations induced by cyclophosphamide (Paul et al., 2011). All of these data seem to indicate that the beneficial effects of P. americana are due to its antioxidants properties.


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Broccoli and cauliflower (Brassica oleracea) These leafy vegetables are unique among the common cruciferous vegetables that contain high levels of the aliphatics glucosinolate and glucoraphanin (Carteaet al., 2010). Glucosinolates are a class of organic compounds that contain sulfur and nitrogen and are derived from glucose and an amino acid. They are abundant in Brassica vegetables and believed to be the bioactive compounds responsible for many of the biological effects attributed to these greens, such as antioxidant properties, enzyme modulation, apoptosis and cell cycle controlling activities (Herr and Buchler, 2010). Flavonoids in Brassica foods are complex, containing up to five sugar residues, and these may be further substituted with hydroxycinnamic residues (Vallejo et al., 2004). Acylated flavonoids were detected in the extract and their UV spectra, characterized by a high maximum absorption of 330 nm and a little maximum between 255 and 268 nm, suggesting that the flavonoid-glycoside molecules were linked to hydroxycinnamic acid derivatives, in which sinapic, ferulic, caffeic and pcoumaric acids were the most abundant (Velasco et al., 2011). There is a wide variety of glucosinolates, but all share a β-thioglucoside N-hydroxysulfate common structure, containing a β-D-glucopyranosyl moiety and a variable side-chain derived from methionine, tryptophan or phenylalanine. Upon cellular disruption, glucosinolates are hydrolysed to various bioactive breakdown products by the endogenous enzyme myrosinase MYR). Isothiocyanates and indoleglucosinolate metabolites (in particular indol-3-carbinol) are the two major groups of autolytic breakdown products of glucosinolates. Both of them exhibit protective activities against different types of cancer (Velasco et al., 2011). Several epidemiological studies in Asia, the United States and Europe suggest that the consumption of vegetables from the Brassicaceae family, e.g., broccoli and cauliflower, may have protective effects against various types of cancers (Juge et al., 2007). Indole-3-carbinol is the main hydrolysis product of the glucosinolate glucobrassicin and can provide significant protection against cancer in animal models with a variety of chemical carcinogens, as well as in cultured human cancer cells. Initial clinical trials in women have shown that indole-3carbinol may prove a promising agent against cervical and breast cancers. Also, 3,3'diindolylmethane, an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been shown to exert anticancer effects in both in vivo and in vitro models (Choi et al., 2009). Their overall protective effects have been generally attributed to different biological activities, among them the modulation of phase-I (inhibition) and phase-II (induction) via xenobiotics metabolizing enzymes. It has been shown that Brassicacea extract enhanced GST and UDP-glucuronosyltransferase activities only after the breakdown of the glucosinolates by the intestinal or exogenous MYR, thus pointing to the fundamental role of isothiocynates, rather than glucosinolates, in up-regulating these xenobiotics metabolizing enzymes (Paolini and Nestle, 2003). Broccoli (Brassica oleracea var. Italica) has a high content of bioactive compounds, including glucosinolates, glycosylated flavonoids, and vitamin C. It also has anthocyanins from among the colored flavonoids, which are multifunctional food components given their antioxidant activity (Moreno et al., 2010). Brassicaceae are also able to attenuate oxidative stress, influencing the redox status of the cell by affecting GSH levels and inducing expression of antioxidant enzymes such as GSR and NAD(P)H:quininereductase (NQO1) in rat liver, kidney, and cardiovascular tissues (Guerrero-Beltrán et al., 2012).



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Cauliflower has glucosinolates (thioglucosides) and S-methyl cysteine sulfoxide. These compounds, which are derived in plant tissue by amino acid biosynthesis, show quite different toxicological effects and appear to possess anticarcinogenic properties. The percentage of isothiocyanate sulforaphane present in these vegetables may vary depending on conditions of hydrolysis, food handling, and preparation procedures (Rodríguez-Hernández et al., 2012). Biological activities of kale extract have been demonstrated in in vitro systems; they include antioxidant activity, inhibitory effect of abnormal cell growth, and inhibition of HMG-CoA reductase activity (Park et al., 1997). Kale juice supplementation has led to changes in serum antioxidant biokarmers, such as increased levels of Se and the activity of GPx, which imply the improvement of the serum antioxidant defense system (Kim et al., 2008). The hypocholesterolemic effect of kale extract on cholesterol metabolism through HMGCoA inhibition and bile acid synthesis has been studied in vitro systems (Park et al., 1997). Kale juice supplementation (150 mL/day for 12 weeks) resulted in substantial improvements in serum lipid profiles, especially with respect to HDL and LDL-cholesterol levels, the ratio of HDL- to LDL-cholesterol, and the antioxidant status of hypercholesterolemic men (Kim et al., 2008). On the other hand, Kim et al. compared changes in serum variables and net differences with respect to smoking status to evaluate if there was a difference in response to kale juice supplementation. However, whereas levels and GSH-Px activities responded more to kale juice supplementation in non-smokers, serum Se responded more among smokers. This depletion may be primarily due to the highly oxidant effect of tobacco smoke, which could lower the levels of antioxidant nutrients and increase oxidative stress (Kim et al., 2008). However, the cited study demonstrated that kale juice supplementation favorably influences serum lipid profiles and antioxidant systems.

Cactus (Opuntia ficus-indica) Cactus O. ficus-indica is widely distributed in Mexico and in all the American hemispheres, as well as in Africa and the Mediterranean basin (Acevedo et al., 1985). The fruit and cladode of Opuntia ficus yield high values of important nutrients such as minerals, vitamins and other antioxidants, it is utilized to treat several disorders; in addition, a recent study (Zourgui et al., 2008) showed the potential antigenotoxic activities of cactus cladodes against a single dose of mycotoxinzearalenone, a potent estrogenic metabolite. These data have made cactus pear fruits and cladodes perfect candidates for cytoprotective research. In Mexico, the O. ficus-indica (nopal) has been employed since pre-Columbian times as an important dietary and economic element (Betancourt-Domínguez et al., 2006). In Sicilian folk medicine it is used for treating gastric ulcers (Galati et al., 2001). O. ficus-indica prickly pads are an important source of nutritional elements like pectin, mucilage and minerals. The presence of total phenolic compounds (free and conjugated) with concentrations of 80-90 mg/100 g dried weight include aromadendrin, taxifolin or dihydroquercetin, isorhamnetin, vitexin, kaempferol, quercetin, and derivatives like myricitin, orientin and some derivatives of pyrone (Stintzing and Carle, 2005). O. ficus-indica fruit juice contains a rich variety of natural antioxidants, many phenol compounds, ascorbic acid, betalains, betacyanins, and a flavonoid fraction that consists mainly of rutin and isorhamnetin derivatives (Alimi et al., 2012). A number of studies have


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revealed a positive correlation between a diet rich in plant-based foods and reduced risk of diseases associated with oxidative stress such as cancer and cardiovascular and neurodegenerative diseases. O. ficus-indica fruit extract reportedly protected erythrocytes against lipid oxidation induced in vitro by organic hydroperoxide (Butera et al., 2002). Scavenging activity was restored in a dose dependent manner to near normal level in ethanolfed rats given prickly pear juice, and restoration of GSH levels was also observed (Alimi et al., 2012). The normalization of scavenging activity by prickly pear juice supplement could be due to the natural antioxidants, which could modulate the intrinsic imbalance between oxidant species and the antioxidant defense system. These fruits have shown several effects, including antiulcerogenic, antioxidant, anticancer and hepatoprotective activities (Kuti, 2004; Zou et al., 2005). Ascorbic acid is an important antioxidant and its content in cactus pear fruits is considerably higher than average ascorbic acid contents among some common fruits such as plums (7 mg/100 g fresh fruit), nectarines (10 mg/100 g fresh fruit) or peaches (9 mg/100 g fresh fruit) (Fernández-López et al., 2010). All the Opuntia species tested had significant amounts of flavonoids, with quercetin followed by isorhamnetin, luteolin and kaempferol. These compounds are more efficient antioxidants than vitamins, since flavonoids, and phenolic compounds in general, are able to delay the pro-oxidative effects on proteins, DNA and lipids through the generation of stable radicals (Feugang et al., 2006). On the other hand, it has been demonstrated that red-skinned cactus pear fruits contain taurine (7.7–11.2 mg/100 g fresh fruit) at the same level of Sicilian cultivars of O. ficus-indica but at a lower concentration than that reported for American and African cultivars (Tesoriere et al., 2005). It has been observed that O. ficus-indica phenolic compounds have antioxidant, anticancer, anti-inflammatory, analgesic, antiulcerogenic, hypoglycemic, hypolipidemic and hypocholesterolemic properties (Guevara-Arauza, 2009). Hepatoprotection may be related to the flavonoid fraction of the juice, but other compounds, such as vitamin C and betalains, could synergistically counteract many degenerative processes by means of their antioxidant activity (Galati et al., 2005). A previous study showed that O. ficus-indica glycoprotein had scavenging activities against oxygen radicals in cell-free systems, as well as cytoprotective and anti-apoptotic acitivities in oxygen radical-induced NIH/3T3 cells (Lim et al., 2008). O. ficus-indica glycoprotein did not have any cytotoxic effect and instead protected liver cells due to its scavenging activity against G/GO-induced radical production (Oh and Lim, 2006). These results show that O. ficus-indica glycoprotein exerts antioxidant and cytoprotective effects in vitro, either directly or indirectly. Anti-atherogenic effects have also been reported. The administration of O. ficus-indica glycoprotein decreased NO amounts in hyperlipidemic mice, probably via its antioxidant effects and by reducing lipid peroxidation (Oh et al., 2006). The oxidative damage caused by aflatoxin is considered the main mechanism leading to subsequent hepatoxicity. The pre and post-administration of cactus cladode extract with aflatoxin B significantly reduced this oxidative effect, which dropped to control level (Brahmi et al., 2011). The protective effects of cactus cladode extract to prevent and protect against oxidative damage is certainly associated to the presence of several antioxidants such as ascorbic acid, vitamin E, carotenoids, reduced GSH, flavonoids and phenolic acids actually detected in fruits and vegetables of different varieties of cactus (Shin et al., 2006).



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Carrot (Daucus carota) Carrots are widely consumed as food. Their active components, which include fibers, carotenoids, vitamins C and E, and phenolics such as p-coumaric, chlorogenic, and caffeic acid, have been amply studied (Surleset al., 2004). Anthocyanidins are the main antioxidants in purple-yellow and purple-orange carrots; chlorogenic acid is a major antioxidant in all carrots. Carotenoids do not contribute to total antioxidant capacity, but correlate with the antioxidant capacity of hydrophobic extracts. Purple-yellow carrots have the highest antioxidant capacity, followed by purple-orange carrots; the other carrots do not significantly differ (Potter et al., 2011). The chemical composition of the D. carota oil extracted from different parts of the plant consists mainly of phenylpropanoids, monoterpenes, sesquiterpenes, phenols and polyphenols, which include flavonoids (Maxia et al., 2009). A study on the dichloromethanemethanol extracts of the flower of wild carrot from Turkey revealed significant antioxidant properties and activities against several human cancer cell lines (Shebaby et al., 2012). The anticancer activity of D. carota oil extract could be partly explained by the presence of several sesquiterpenes and phenylpropanoids, or might be attributed to major/minor unknown compounds acting in synergy. Oral intake of carrot juice also displays other beneficial physiological effects, including reduced oxidative DNA damage, increased levels of plasma antioxidants, and reduced inflammation (Hu et al., 2004). Carrot juice significantly increased total plasma antioxidant capacity and decreased plasma malondialdehyde production. The decreased lipid peroxidation evident from drinking carrot juice is associated with increased antioxidant status independent of inflammatory markers, hormones, or increased cholesterol and triglyceride concentrations; it may also protect the cardiovascular system by increasing total antioxidant status and decreasing lipid peroxidation independent of any of the cardiovascular risk markers (Potter et al., 2011). The carotenoids present in D. carota L. are also antioxidants capable of reducing the risk of chronic diseases like cardiovascular ailments and cancer (Mech-Nowak et al., 2012). It was observed that the plasma antioxidant capacity of volunteers measured after 2 weeks of intervention with carrots had no effect on their antioxidant status despite an increase in plasma carotenoid concentration (Stracke et al., 2009). A possible explanation for the lack of effect on antioxidant status and LDL oxidation was that 2 weeks of intervention with blanched carrots might not be enough to enhance antioxidative status and protect LDL against oxidation. The biofortification of carrots has resulted in increased concentrations of bioactive compounds, namely carotenoids and polyphenols. The intake of biofortified carrot enhanced liver antioxidant capacity and vitamin A status in Mongolian gerbils. Liver antioxidant capacities in gerbils fed white carrots and supplemented with oil or vitamin A were lower, suggesting that the bioactive compounds in colored carrots, such as α-carotene, -carotene, lycopene and anthocyanins, may have enhanced liver antioxidant capacity either by acting directly as an antioxidant or indirectly by sparing -tocopherol, which was higher in gerbils fed colored carrots than in those fed white carrots and supplemented with vitamin A (Mills et al., 2008). The enhancement of liver antioxidant capacity observed in gerbils consuming biofortified carrots was likely due to the combined bioactivities of multiple compounds rather than the


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individual activities of carotenoids, anthocyanins, or phenolic acids, illustrating the synergistic benefits associated with the intake of whole foods.

Peppers (Capsicum annuum) Red pepper is used as a spice for enhancing the palatability of food and as a counterirritant in stomach medicines in many countries (Watanabe et al., 2001). The pungent principle of red pepper comes from a group of compounds called capsaicinoids, which possess a variety of biological properties and give it its spicy flavor. Two major capsaicinoids, dihydrocapsaicin and capsaicin are responsible for up to 90% of the total pungency of pepper fruits. In traditional medicine, C. annuum is used as a stimulant and, externally, as a rubefacient. It is also used to treat scarlet fever, putrid sore throat, hoarseness, dyspepsia, yellow fever, piles and snakebite (Ishtiaq et al., 2007). Capsaicin (8-methyl-Nvanillyl-6-nonenamide) is a principal component of Capsicum fruits and is known to have antioxidant properties; it has therefore been associated with potent antimutagenic and anticarcinogenic activities (Materska and Perucka, 2005). It is well known that vegetables undergo physical, structural, chemical and nutritional changes during hot-air drying, and these can affect attributes like texture, color, flavor and nutritional value. While both fresh and dried peppers exhibit a comparable phenol content of 127.5 and 116.7 mg/g extract respectively, a drastic reduction was observed after the frying process with a value of 15mg/g extract. A similar decrease was observed with C. annuum var. Senise cultivar, where the estimated total phenol content was 224.5 and 24.5 mg/g extract in fresh and fried peppers, respectively (Loizzo et al., 2013). Tundis et al. observed that the C. annuum var. acuminatum small has the highest observed quercetin content (56.0 μg/g dw). This value is 10 times higher than C. annuum var. cerasiferum (5.6 μg/g dw). Capsicum annuum var. acuminatum small also had the highest concentrations of luteolin and kaempferol. The best free radical scavenging activity was exerted by Capsicum annuum var. acuminatum (IC50 of 153.0 μg/ml). A comparative study, made by the same author showed that the best hypoglycaemic activity was exerted by C. annuum var. cerasiferum, with an IC value of 256.8 μg/ml and 356.8 μg/ml against α-amylase and α-glucosidase, respectively, in the lipophilic fraction. This fraction showed phytol, vitamin E, β-sitosterol, campesterol and certain FAs, methyl andethyl esters, as major constituents (Tundis et al., 2011). Previous studies have shown that fresh C. annuum var. Roggiano and Senise cultivar were capable of scavenging both 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis(3ethylbenzothiazoline-6-sulphonic acid solution radicals in a concentration dependent-manner. However, the drying and frying process drastically reduced the ability of samples to scavenge the 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid solution radicals (Loizzo et al., 2013). The results clearly evidenced the drying process allows for the retention of those phytochemicals able to exert their bioactivity, but frying drastically reduces the phytochemical content, especially of phenols, and consequently reduces both antioxidant activity and inhibition of carbohydrate-hydrolyzing enzymes. It has been shown that the most abundant flavonoid identified in both pepper cultivars, apigenin, inhibits α-glucosidase at 43% at 200 μM (Tadera et al., 2006). Administration of this flavone for 10 consecutive days in alloxan-treated diabetic animals increased levels of serum insulin while hardly inhibiting the



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glycation of plasma proteins (Liu et al., 2012). It was also found that pepper leaves not only possess antioxidant activity but also antiproliferative activity against HCT116 human colorectal carcinoma and MKN 45 gastric adenocarcinoma cell (Jeon et al., 2008). Several studies of red paprika have offered biological evidence of antitumor-promoting activity, reduction or prevention of chronic diseases such as cardiovascular disease, improvement of HDL-cholesterol, and hepatic gene regulation (Maokaa et al., 2004; Aizawa and Inakuma 2009). These studies show that the carotenoids in red paprika play a key role in these beneficial effects; capsanthin and capsorubin in particular, which are unique to red paprika, exert antioxidative and anti-tumor activities (Kim and Hwang, 2009). Paprika leaves also display potent biological actions such as free radical scavenging, and antimicrobial and tyrosinase inhibitory activities in various solvent fractions (Kim et al., 2007). Regarding C. annuum L., var. special, Kim et al. reported that, even though paprika leaves possesses phytochemicals such as lutein, γ-tocopherol, and other phenolic compounds, red paprika showed the strongest antioxidant activity. The antioxidant activity of paprika leaves appeared to be considerable when compared with β-carotene, which might be due to the combined activities of several phytochemicals, especially lutein and γ-tocopherol (Kim et al., 2011). Red hot peppers (C. annuum Tepin and Capsicum chinese Habanero) prevent Fe2+induced lipid peroxidation, probably because of their higher total phenol content and Fe chelating ability (Oboh, et al. 2007). Additionally, it has been proved that colored peppers (C. annuum) exhibit radical-scavenging activity (Chuah, et al. 2008). All varieties of red hot dried peppers, both extractable polyphenols and hydrolyzable polyphenol extracts, showed a high antioxidant capacity per g of dry matter. Hervert-Hernández et al. showed that arbol and chipotle varieties presented the highest values of antioxidant activities, followed by morita and guajillo (Hervert-Hernández et al., 2010). The antioxidant activity of pepper fruits may be mainly attributed to ascorbic acid, carotenoids, and capsaicinoids. In addition to the carotenoid composition of red dried hot peppers, a number of authors have identified capsanthin as the main carotenoid in several varieties of red peppers (Collera-Zuñiga et al., 2005). Capsaicin could also prevent the oxidation of oleic acid at cooking temperatures, as well as the formation of lipid hydroperoxides from the autoxidation of linoleic acid. Carotenoids have been found to play an important role in preventing oxidative damage, which is caused by free radicals in age related diseases such as cancer, and ageing itself (Tundis et al., 2011). These data provide basic evidence of peppers’ beneficial antioxidant properties.

Spinach (Spinacia oleracea) Spinacia oleracea L. is an annual plant (occasionally biennial), native to central and southwest. It is a dietary powerhouse, full of vitamins and minerals (Nayak et al., 2010). It is a rich source of chemoprotective substances such as folic acid, flavonoids, lutein, zeaxanthine, β-carotene and chlorophylls, which may contribute to the maintenance of the genetic material. An important antioxidant, it is usually consumed after boiling the fresh or frozen leaves (Schirrmacher et al., 2010). Freshly cut spinach leaves contain approximately 1g of total flavonoids per kilogram, and the occurrence of at least 10 flavonoid glycosides has


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been reported. These are glucuronides and acylated di- and triglycosides of methylated and methylene dioxide derivatives of 6-oxygenated flavonols (Bergquist et al., 2005). Extensive conjugation across the flavonoid structure and numerous hydroxyl groups enhance their antioxidative properties, allowing them to act as reducing agents, hydrogen- or electron-donating agents, or singlet oxygen scavengers (Aritomi et al., 1986). The antioxidant capacity of spinach flavonoids has been determined by the free-radical scavenging assay using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and was compared with that of Trolox, a synthetic analogue of vitamin E. The most active products were those derived from patuletin with a 3′,4′-dihydroxyl group. The incorporation of a feruloyl residue increased the freeradical scavenging activity. Boiling fresh-cut spinach leaves extracted approximately 50% of the total flavonoids and 60% of the vitamin C; a decrease in the total antioxidant activity was observed during storage of leaves (Gil et al., 1999). A pronounced antioxidant effect has been observed immediately after spinach consumption and this can be taken as an indication that this effect is at least partly attributable to direct scavenging effects and not to indirect mechanisms such as induction of antioxidant enzymes, which are seen only after extended time periods (Moser et al., 2011). The observed antioxidant effects of spinach intake are partly supported by the research of Schirrmacher et al., who found a slight induction of SOD and a modest reduction in the malondialdehyde levels in human plasma after a 10-day consumption trial (Schirrmacher et al., 2010). Preadministration of glycolipid extracts from spinach (20 mg/kg body weight) prevented villous atrophy, misaligned crypts, and increased inflammatory cytokines in rat jejunum treated with 5-FU (300 mg/kg body weight). Mono-galactosyl-diacylglycerol and digalactosyldiacylglycerol are primary components of the extracts, and have anti-oxidative and antiinflammatory effects. In Caco-2 cells, monogalactosyl-diacylglycerol and diglactosyldiacylglycerol inhibited the production of ROS induced by phorbol ester (Shiota et al., 2010). The effect of spinach product consumption on antioxidant activity in human blood has been tested in healthy volunteers. The spinach groups received 20 g/day/subject of whole-leaf minced, liquid, or liquefied spinach for 3 weeks and were compared with a control group that received a basic diet. The consumption of spinach resulted in greater erythrocytic GSR activity and lower erythrocytic catalase and serum α-tocopherol responses (Castenmiller et al., 1999). The beneficial effect conferred by the natural antioxidants present in spinach may be mediated through its antioxidative and/or anti-inflammatory properties.

Tomato (Lycopersicum esculentum) Tomatoes (Lycopersicon esculentum) and tomato-based products are a source of phytochemicals such as carotenoids (e.g., phytofluene, phytoene, neurosporene, γ-carotene, and ζ-carotene, flavonols (e.g., quercetin and kaempferol), phytosterols, and phenylpropanoids (Tan et al., 2010). Tomatoes and tomato sauces and puree are said to help reduce urinary tract disease symptoms and may have anticancer properties (Polívkováet al., 2010). Tomato consumption might be beneficial for reducing cardiovascular risks associated with type 2 Diabetes (Shidfar et al., 2011). Tomato plants produce two tomato monoterpene synthases, LeMTS1, and LeMTS2 (Van Schie et al., 2007). Tomato consumption has been



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associated with decreased risk of breast cancer (Zhang et al., 2009), head and neck cancers (Freedman et al., 2008), and might also offer strong protection against neurodegenerative diseases (Rao and Balachandran, 2002). It has been shown that the regular intake of tomato products for 3 weeks decreases lipid peroxidation markers associated with cardiovascular disease (Visioli et al., 2003). It has been reported that daily intake of a tomato drink (LycoMato), formulated with a lycopene, phytoene, phytofluene, and R-tocopherol oleoresin increases plasma and lymphocyte carotenoid concentrations while augmenting cellular antioxidant protection (Porrini et al., 2005). Tomato ripening involves the breakdown of chlorophylls and build-up of carotenoids, accompanied by a continuous increase in lycopene, the carotenoid responsible for the red color of ripe tomatoes. The ascorbic acid content was significantly higher in Ronaldo tomatoes than in Siena and Copo. The values ranged from 5.05 to 8.21 mg/100 g for green samples, from 5.99 to 8.26 mg/100 g for pink tomatoes, and from 7.91 to 15.41 mg/100 g for red tomatoes, thus displaying a significant increase during ripening (Shi and Maguer, 2000). Antioxidant activity was higher in red tomatoes of the Siena and Copo cultivars than in the Ronaldo variety, which may be due to the differences in the antioxidant compound content and their synergistic effect in measured antioxidant activity. The ferric reducing ability of both tomato extracts and hydrophilic tomato extracts increased significantly from green to red tomatoes in all three cultivars, since ripe tomatoes had higher antioxidant-compound content than unripe tomatoes (Periago et al., 2009). Red tomatoes exhibit a better antioxidant composition based on their higher lycopene, total phenolic, flavonoid and ascorbic acid contents. As a result of this antioxidant content, they display greater ferric reducing capacity but have reduced lipid oxidation inhibition activity. The antioxidant activity of tomatoes is most probably due to hydrophilic antioxidants, especially total phenols and flavonoids. Lycopene is a highly unsaturated hydrocarbon containing 11conjugated and 2 unconjugated double bonds. A sapolyene, it undergoes cis-transisomerization induced by light, thermal energy and chemical reactions. In human plasma, lycopene is present as an isomeric mixture, with 50% as cis isomers. Although comparative bioavailability values for lycopene from 67 different tomato products are unknown, lycopene from processed tomato products appears to be more bioavailable than that from raw tomatoes (Rao and Agarwal, 1999). Processed tomato products such as juice, ketchup, paste, sauce and soup are all good dietary sources of lycopene. Several studies have indicated that lycopene is an effective antioxidant and free radical scavenger (Mourvaki et al., 2005), and is also a potent inhibitor of lipid peroxidation and low-density lipoprotein oxidation in vivo (Periago et al., 2009). Lycopene is the most important carotenoid present in tomatoes and tomato products, and their dietary intake of the latter has been linked to a decreased risk of chronic illnesses such as cancer and cardiovascular disease (Riccioniet al., 2008; Waliszewski and Blasco, 2010). The protective effects of resveratrol against cardiovascular disease are due to its effects on the platelet aggregation inhibition activity and its strong antioxidant potential (Olas and Wachowics, 2005). Concentrations of total resveratrol in tomato skin ranged from 18.4 ng/g in the MicroTom variety, 2 orders of magnitude below those determined in the skin of seedless red grapes (2.78 mg/g), suggesting that this tomato variety is unlikely to contribute adequate amounts of resveratrol in a normal diet to produce the health benefits associated with this phytonutrient (Ragab et al., 2006).


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A number of studies have shown that flavonoids and hydroxycinnamic acids are the major phenolics in tomatoes (Fleuriet et al., 1985). Among the many tomato components (e.g., vitamin C and polyphenols) credited with healthful properties, carotenoids and lycopene, in particular, are being actively researched.

Watercress (Nasturtium officinale) The leaves of watercress (Nasturtium officinale) are also widely used as a home remedy (Launert, 1981). Watercress contains one of the highest concentrations of glucosinolates per gram weight of any vegetable as well as containing high concentrations of carotenoids such as lutein, and β-carotene, along with other important bioactive phytochemicals (Getahun and Chung 1999). These phytochemicals have also been associated with various anticarcinogenic properties, including antioxidant activities. Members of the Cruciferae family have also been shown to contain high amounts of phenolic compounds (Gill et al., 2007). Watercress supplementation was associated with reductions in basal DNA damage, in basal plus oxidative purine DNA damage, and in basal DNA damage in response to ex vivo hydrogen peroxide challenge. The mechanisms of antigenotoxic effects due to watercress supplementation are unknown, although this may be related to antioxidant status and changes in GST activity (Torbergsen and Collins, 2000). A diet high in watercress is associated in a number of epidemiological studies with a reduced cancer risk in a number of sites, including colon, lung, lymphatic system and possibly prostate, (Higdon et al., 2007; London et al., 2009). Beneficial changes after watercress intervention were associated with an increase in plasma lutein and β-carotene (100% and 33%, respectively). These results support the theory that consumption of watercress can be linked to a reduced risk of cancer via decreased damage to DNA and possible modulation of antioxidant status by increasing those phytochemicals (Gill et al., 2007). Phenethylisothiocyanate has been reported to have several anti-carcinogenic effects, including the inhibition of phase I enzymes and/or the activation of phase II enzymes (Canistro et al., 2004). It has been observed that, in the rat liver and colon, phenethylisothiocyanate (PEITC) leads to an induction of the total GST activity, such as the induction of the quinine reductase by 7-methylsulfinylheptyl-isothiocyanates and 8methylsulfinyloctyl-isothiocyanates (Rose et al., 2000). In vitro studies showed that watercress extract increased SOD2 activity while PEITC had no impact. This may be due to the potential influence of further bioactive watercress constituents such as quercetin glycosides or hydroxycinnamic acids (Buettner et al., 2006). Another study demonstrated that an extract from watercress modulated gene expression in human peripheral blood cells in vitro and that this was also reflected in a modulation of enzyme activity in vivo, particularly in individuals with the GSTM1*0 genotype (Hofmann et al., 2009). The level of ROS produced by polymorphonuclear leucocytes also increases, accompanied by a decrease in the activity of many tissue antioxidant enzymes. It has been observed that watercress reduces oxidative stress and enhances antioxidant capacity in hypercholesterolaemic rats. Watercress extract prevented the high-fat diet-induced elevation of malondialdehyde (MDA), significantly reduced MDA content in liver homogenates, and



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increased GSH level in liver. This suggests that watercress extract can either increase the biosynthesis of GSH or reduce the extent of oxidative stress leading to less GSH degradation; it may, in fact, have both of these effects (Yazdanparast et al., 2008).

Conclusion Exploring the healing powers of plants is an ancient phenomenon. Hippocrates (460-377 B.C), the father of medicine, said, “Let thy food be thy medicine and thy medicine be thy food.” Such an idea reflected the importance of dietary supplements for their therapeutic and preventive bioactive components, elevated margin of safety, and desired range of efficacy. Although traditional healers have long used plants to prevent or cure various conditions, nowadays there is an increased interest in the health benefits of foods and researchers have begun to look beyond the basic nutritional benefits of foodstuffs into disease prevention and health enhancing ingredients. This chapter brings together evidence of the beneficial influence of fruits, vegetables or their components (phytochemicals) given their potential antioxidant properties (Figure 3). Interestingly, most of the fruits and vegetables here examined contain a very similar phytochemical mix.

Figure 3. Potential antioxidant properties of phytochemicals present in fruit and vegetable.


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Currently, most of the fruits and vegetables produced worldwide are still consumed fresh. A very small quantity (1.5%) goes into the manufacturing of pickled products, fruit and vegetable drinks, purées, jellies, candy, juices, jam, and dried fruits. The demand for fruit and vegetable beverages has increased in many countries over the last few years. This may be attributed to changes in dietary habits, taste preferences, and the lifestyle of present-day consumers. It is well known that fruit and vegetable beverages have higher nutritional, medicinal, and calorific values compared to synthetic beverages. Moreover, owing to high acidity, astringency, bitterness, and such other factors in some of these foodstuffs, the preparation of processed products becomes limited despite having high nutritional value. Epidemiological studies suggest that vegetarianism is associated with reduced risks of cancer, cardiovascular and neurodegenerative disorders. This is consistent with the fact that the incidence of these disorders is lower among some populations where fruits and vegetables are the main elements in the human diet. Since diseases like cancer are multifactorial phenomena in which many normal cellular pathways become aberrant, it is highly unlikely that one agent could prove effective against such disorders. This chapter presented evidence of the potential antioxidant properties of fruits and vegetables and their role in regulating and maintaining normal processes in living organisms. The presence of multiple phytochemicals in these foodstuffs suggests that the combined bioactivities of multiple compounds result in the synergistic benefits associated with the intake of whole foods. There is no doubt regarding the protective benefits of phytochemical-rich foodstuffs, but these effects are most successfully obtained from frequent consumption of unprocessed natural fruits and vegetables. The concept of food as medicine needs to be propagated to ensure healthy feeding habits. However, more studies are required to acquire a better understanding of the mechanisms behind the potential health benefits of dietary phytochemicals.

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Chapter 3

Hydroponic Production of Medicinal Plants Rita Maggini*, Claudia Kiferle and Alberto Pardossi Department of Agriculture, Food and Environment, University of Pisa, Pisa, Italy

Abstract Medicinal plants are specifically used for their contents of bioactive compounds, which are products of plant secondary metabolism with proven beneficial effects on human health. These substances are known to play a key role in the mechanisms of plant adaptation to the environment; they generally exhibit antioxidant properties and often act as defense molecules that are synthesized by plants in response to stress conditions. In the last decades, the interest by pharmaceutical companies towards the production of bioactive compounds from medicinal plants has considerably increased, especially in developed countries, in consideration of the consumers’ sensibility towards naturally sourced remedies. As a consequence, the traditional harvesting from the wild has become inadequate to sustain the market demand, and medicinal plants are increasingly cultivated on a commercial scale. On the other hand, the market requirement for standardized plant material cannot be fully satisfied by field crops, which are highly susceptible to year-to-year variability. Greenhouse hydroponics can contribute to overcome the drawbacks of conventional field cultivation, as it ensures a fast plant growth and allows both to control the growing environment and to change the composition of the nutrient solution that is fed to the plants. The application of a stress condition through a proper manipulation of the nutrient solution can stimulate secondary metabolism and promote the synthesis and accumulation of bioactive substances in plant tissues. This chapter presents some fundamental issues concerning the hydroponic production of raw plant material for the extraction of bioactive compounds. Literature data are reported on recent research concerning the hydroponic growing of medicinal plants, both under optimal conditions or under stress conditions to stimulate the production of secondary metabolites. Finally, basil is presented as a case study for the application of the hydroponic technique to the production of plant material for the *

[email protected].


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Rita Maggini, Claudia Kiferle and Alberto Pardossi extraction of rosmarinic acid, a bioactive secondary metabolite of well-known antioxidant activity. The present chapter points out the opportunities offered by the hydroponic growing of medicinal plants for the agro-industrial production of bioactive compounds. On the other hand, it also underlines the lack of information concerning the specific growing needs of the individual medicinal species. Despite the fact that at present a lot of molecules of pharmaceutical interest can be obtained from hydroponically-grown medicinal plants, suitable growing protocols are still required.

Introduction Secondary Metabolism and Antioxidants Medicinal plants are specifically used for their contents of bioactive compounds, which are compounds of proven beneficial effects on human health. The molecules that constitute active principles for the human organism are produced in plants by the secondary metabolism. As secondary metabolites, these compounds are not directly involved in the fundamental functions that determine plant growth and development, such as photosynthesis, respiration or tissue formation. Rather, they are involved in the interactions between the plant and the environment where it lives, and play a central role in the mechanisms of plant adaptation to both abiotic and biotic stresses. Secondary metabolites accomplish a lot of different functions in plants. These molecules can have an attractive role towards insects or animals for plant reproduction through pollination or seed dispersion (for example, the colours of anthocyanins may attract pronube insects or birds). Alternatively, secondary metabolites (such as some substances belonging to the class of naphto- or benzoquinones) can be involved in allelopatic interactions, to inhibit germination or development of competing plants. Anyway, because of their key role in plant survival, a major function of secondary metabolites is defense. Because they are anchored to the soil, plants cannot escape the harmful action of a stress agent. Thus, they have developed effective biochemical pathways for the synthesis of organic molecules that can counteract the effects of a stress condition. These molecules are synthesized by plants either in response to a biotic stress (for example, against herbivores or pathogens), or to react against an abiotic stress (for example, UV radiation or toxic substances). Under stress conditions, plant growth may be reduced, and this in turn may result in a new pattern of resource partitioning. According to the carbon/nitrogen (C/N) balance hypothesis (Bryant et al., 1983), stress conditions which limit growth more than photosynthesis cause excessive carbohydrates production, providing additional carbon skeletons for the synthesis of secondary metabolites. It is well known that stress conditions in plants also cause an increase in the production of reactive oxygen species (ROS), such as hydroxyl radical (HO•), superoxide radical (O2•-) or hydrogen peroxide (H2O2) (e.g., Ercal et al., 2001; Ksouri et al., 2007; Mehrizi et al., 2012). Oxidative imbalances in plants activate several protective mechanisms to eliminate or reduce ROS (Domìnguez-Valdivia et al., 2008; Wang et al., 2011), such as the enhancement of the activity of antioxidant enzymes like ascorbate peroxidase (APX), catalase (CAT) and


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superoxide dismutase (SOD), and the production of antioxidant compounds (Gill and Tuteja, 2010). That is why antioxidants generally act as defense molecules. Plant defence metabolites arise from the main secondary metabolic routes, the phenylpropanoid, the isoprenoid and the alkaloid pathways (Iriti and Faoro, 2009). Among defence molecules, phenolic compounds, which are synthesized through the phenylpropanoid metabolic pathway and play a key role in the scavenging of ROS, are one of the most numerous group of plant secondary metabolites, with more than 8000 structures currently known (Soobrattee et al., 2005), which are widely distributed throughout the plant kingdom. These include flavonoids, anthocyanins, tannins, caffeic acid derivatives and lignin, which are abundantly contained in plant tissues (Grace and Logan, 2000). The free radical scavenging and antioxidant activities of phenolic compounds depend on the number and the position of the hydroxyl groups that are linked to the aromatic ring (Soobrattee et al., 2005, Hinneburg et al. 2006). In particular, the molecules with hydroxyl groups in the ortho or para positions of a benzene ring are easily oxidized to the corresponding quinonic forms; the radical intermediate of this redox reaction is capable to stabilize the unpaired electron by delocalization. The occurrence of an environmental conditions that impairs the plant's aerobic or photosynthetic metabolism (such as high light intensity, adverse temperature, drought, osmotic imbalance or mineral disorders), causes inevitably enhanced generation of ROS (Ksouri et al., 2007). When the production of ROS prevails over the antioxidant power of cells, it results in oxidative stress. Cells under oxidative stress display various dysfunctions due to lesions caused by ROS to lipids, proteins and DNA. Plants with high levels of antioxidants have been reported to have a great resistance to this oxidative damage (e.g.: Foyer and Shigeoka, 2011; Landi et al., 2012). Antioxidant compounds are involved in the mechanisms of plant tolerance to stress conditions (Iriti and Faoro, 2009). For example, higher levels of phenolics have been reported in salt tolerant plant species compared to non-tolerant ones (Gill and Tuteja, 2010). Excess aluminum (Al) stress increased the concentration of flavonoids in Al-tolerant populations of Cunila galioides Benth, a naturally occurring medicinal and aromatic plant native of south Brazil (Mossi et al., 2011). In rosemary (Rosmarinus officinalis L.) copper (Cu) nutrition resulted effective in counteracting salt-induced oxidative damage (Mehrizi et al., 2012), as Cu reduced lipid peroxidation and membrane permeability, whereas it increased total phenol content of salt-stressed plants. Furthermore, it was suggested that the relevant anthocyanins level in the leaves of a red cultivar (Red Rubin) of basil (Ocimum basilicum L.) could significantly contribute to tolerance towards boron (B) toxicity (Landi et al., 2013). In addition to the usually strong antioxidant activity, secondary metabolites often have pharmacological properties, and can act as antiinflammatory, antibacterial, antiviral, antimicotic, anticancer, immunomodulating molecules. For instance, phenolic compounds have a large number of therapeutic applications, such as the prevention and treatment of cardiovascular, neurodegenerative, diabetes, cancer and inflammatory diseases. The medicinal actions of phenolics are mostly ascribed to their antioxidant capacity, chelation of redox active metal ions, modulation of gene expression and interaction with the cell signalling pathways (Soobrattee et al., 2005, Hinneburg et al. 2006). Houghon (2001) estimated that about 40% of the pharmaceutical products used in western countries were initially discovered from natural sources, and that 25% of these sources were represented by higher plants. Rates (2001) reported that about 25% of the drugs


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prescribed worldwide came from plants, 121 such active compounds being in current use. Moreover, more than 10% of the 252 drugs considered as essential by the World Health Organization are exclusively of plant origin and a significant number of synthetic drugs are obtained from natural precursors (Rates, 2001).

Market Requirements and Field Cultivation Because of the presence of bioactive molecules, a lot of plants are regarded as primary sources of important natural substances for food, cosmetic and pharmaceutical products. Plant-derived substances are present on the market mainly to satisfy the consumers’ preference towards natural products. The sensibility of the consumers towards naturally sourced products is particularly strong when dealing with natural remedies. The use of herbal medicines has increased in recent years due to their usually low prices, and also to the common misconception that herbs are safe and without side effects, being of natural origin (Rahimi et al., 2012). In the last decades the consumption of natural remedies has undergone a substantial increase, in particular in developed countries. In addition, several important drugs cannot yet be synthesized economically and are still obtained from plants (Rates, 2001). This has increased the interest by pharmaceutical companies towards the identification, development and production of active principles from medicinal plants. Medicinal plants are traditionally collected from the wild. Among the 50 thousand medicinal species in use, approximately two thirds are obtained from wild collection (Canter et al., 2005). Although what is sensed as natural is also sensed as safe, the plant material collected at the spontaneous state is often of poor quality or even poisonous (Atanassova et al., 2011; Prasad et al., 2012). This represents a serious problem for medical doctors, researchers and consumers, and has been the cause of a strong public healthcare concern and a regulatory demand for high quality, uniformity and safety of medicinal plant products (Stewart and Lovett-Doust, 2003; Atanassova et al., 2011). Regulatory legislation has been introduced in recent years in North America and in the European Union to discipline the safety and quality specifications of herbal preparations (Zheng et al., 2006b; Vlietinck et al., 2009). Quality and safety standards (e.g. maximum allowed concentrations of heavy metals) have been enacted and put into effect in many countries (Rahimi et al., 2012). Moreover, although the traditional collection from the wild is still a low-cost practice in many developing countries (Prasad et al., 2012), with the increasing popularity and rapid growth of the global market for herbal medicine, wild collection has become a danger for ecosystems and for the conservation of plant species. Special attention is required for species at risk of extinction. It has been reported that environmental destruction due to the harvesting of a wild licorice (Glycyrrhiza glabra Linn.), is becoming a serious problem (Sato et al., 2004). Arcostaphylos uva-ursa (bearberry) and Piper methysticum (kava) are two other widely used herbal medicines threatened by wild harvesting (Canter et al., 2005). Furthermore, Crosby and Cracker (2007) reported about the development of tissue culture techniques for the moringa genus, to ensure the maintenance of germplasm. As a consequence of these safety and environmental issues, currently medicinal plants are also cultivated on a commercial scale, although they remain minor crops. Despite the recent interest towards medicinal plants, which is also the result of the



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consumers' acceptance of new food and health products (Ehret et al., 2002), in Europe only 10% of commercially used medicinal species are cultivated (Canter et al., 2005). Recently Prasad et al. (2012) reported about the need for sustainable and viable production methods, while Vlietinck et al. (2009) pointed out the necessity of controlled cultivation to ensure the production of herbal substances of high quality. Medicinal plants cultivation is mainly controlled by pharmaceutical companies, and generally consists of an intensive cropping system for the production of high quantities of biomass at low cost. During the last years the market of organically-certified natural remedies has also increased in developed countries (Craker, 2007). This has encouraged the organic cultivation of medicinal plants, which is principally directed to the herbal market. Anyway, the main purpose of pharmaceutical industry is to purchase the required amount of raw material for the production of pharmaceutical preparations in a planned and regular way. The bioactive substances to be used for the commercial preparations are concentrated from the raw plant material by means of industrial extraction processes, and the whole production process is subjected to a strict quality control. Therefore, the first step is quality assessment of the starting material. In contrast with the need for high quality standards, a lot of cultivation areas are arranged in developing countries, usually far from the production units, to keep costs at a low level. As a consequence, the quality of the raw material that arrives at the extraction laboratories is often poor. In particular, the presence and concentration of the bioactive substances which have to be extracted may be not sufficient, depending not only on the selection of the plant species and variety, but also on the agronomic techniques (Letchamo et al., 2002). On the other hand, despite the market trend and the growing interest towards the cultivation of medicinal plants, the knowledge about the needs of a lot of medicinal species is still scarce. Some medicinal plants are reported to be difficult to grow in open field (e.g, Echinacea spp.; Li, 1998) and generally the agronomic techniques are not yet optimized (Briskin, 2000). Nevertheless, the market requires standardized plant material both in quantitative and in qualitative terms, that is the biomass production and the concentration of bioactive compounds in the tissues should be not only as high as possible, but also as constant as possible. Unfortunately, generally these requirements cannot be fulfilled by field crops, which in contrast undergo a marked year-to-year variability. This is due mainly to genetic and geographic factors, but also to the variations in environmental conditions that were experienced by the plants during growth and development (Brechner et al., 2007). All these factors affect both the biomass production and the synthesis of secondary metabolites, and often are responsible of discrepancies between the actual concentration of active principles in medicinal preparations and the concentrations indicated on the label. For instance, Brechner et al. (2007) reported that the components of Hypericum perforatum are often found to vary by a factor of two compared to the concentrations reported on labels for the prepared drug. Similar claims have been reported for Echinacea preparations (Mølgaard et al., 2003). Another problem connected to field cultivation is the incidence of biological, chemical or physical contamination of plant tissues. The plants at harvest could be spoiled by foreign material such as weeds, soil particles, soil pollutants and pathogens, which could interfere with post harvest handling and processing and could have a detrimental effect on the quality of the final product. The optimization of the cultural techniques, within the frame of either traditional or organic cultivation, should be a critical step to improve the quality of the raw material.


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However, conventional cultivation cannot remove the effects of the fluctuations in the environmental conditions, which strongly contribute to the variability of field crops.

Greenhouse Hydroponics In consideration of the market requirements for a standardized product with a high content of bioactive principles, several efforts are directed to the setup of suitable growing conditions for the stimulation of the plant secondary metabolism. The variability of the content of bioactive compounds is one of the major limitations in using plants as sources of these molecules for the pharmaceutical industry. On the other hand, open-field culture does not allow a strict control of the growing conditions and of the secondary metabolism. Therefore, the development of alternative systems for the production of medicinal plants could be an effective tool to overcome the drawbacks linked to field cultivation. Hydroponics is a growing system, where the nutrient elements that are normally found in the soil are dissolved in proper amounts in the irrigation water that is supplied to the plants. This system includes several techniques, which generally differ for the methods employed for the delivery of the nutrient solution to the culture. Hydroponics is also referred to as 'soilless culture', because the plants are cultivated in pure nutrient solution (water culture) or in artificial growing media (substrate culture) that replace the common agricultural soil (Pardossi et al., 2006). Hydroponics may fulfill both legal and industrial requirements for medicinal plants, as it offers several advantages over conventional field cultivation. For instance, the use of water and fertilizers is more efficient with hydroponics; the plants can be grown on a year-round basis; the quantity and quality of the production are highly predictable because do not depend on geographic area or pedoclimatic conditions; plant contamination is absent or minimal; the plant material is easy to be processed and extracted (e.g.: Mulabagal and Tsay, 2004; Pardossi et al., 2006; Raviv and Lieth, 2007). With hydroponics, the management of irrigation and fertilization associated to the effective control and optimization of the climatic conditions enables the standardization of the production process and enhances plant growth and development. Therefore, both a shorter growing cycle and a higher yield can be obtained in comparison with conventional cultivation. The limitation of the growing cycle offers the additional opportunity to set up more consecutive cultures within one year. Therefore, a considerable increase in total biomass production can be obtained if appropriate scheduling of planting and multiple harvesting scheme are adopted. A further major advantage of hydroponics is the possibility to deliberately expose the plants to stress factors that are known to elicit an increase in the concentrations of secondary metabolites (Brechner et al., 2007; Rahimi et al., 2012). With this technique, the management of important growing parameters such as climatic conditions or mineral nutrition represents the main tool for the regulation of secondary metabolism. In particular, a proper change of the composition of the nutrient solution could stimulate the secondary metabolism and favour the accumulation of bioactive compounds in the tissues (Briskin, 2000). In a similar way, in vitro culture systems such as tissue or cell culture, may represent a valid alternative to conventional agriculture for the production of plant metabolites (e.g. Kiferle et al., 2011). In vitro culture allows to regulate plant biosynthetic pathways in a



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strictly controlled and aseptic environment. Several strategies can be applied in artificial cultivation systems to stimulate the production of active substances. For instance, the use of elicitors (e.g. methyl jasmonate, salicylic acid and yeast extract) is known to enhance the accumulation of bioactive compounds in tissue cultures (Zhao et al., 2005). Anyway, although bioactive compounds from several species have been obtained by means of tissue culture (Matkowsky, 2008; Karuppusamy, 2009), this technique does not ensure such a high level of biomass production per unit area as hydroponics. In addition, in vitro culture is a complex technology that requires skilled operators and expensive structures (Ahloowalia and Prakash, 2002; Nair et al., 2013). Considerable effort has been devoted to the increase of the production efficiency and to the reduction of investment and running costs of in vitro systems, for example by partial mechanization of some cultural steps or by means of bioreactors (Ahloowalia and Savangikar, 2002; Levin and Tanny, 2002). Nevertheless, in 2002 it was estimated that in vitro production of any compound with a market price lower than US$ 1000 per kilogram was not economically sustainable (Rao and Ravishankar, 2002). At present, in vitro production of phytochemicals on a commercial scale is still limited only to very few high-value plant secondary metabolites (Weathers et al., 2010). One example is taxol, an important anticancer drug produced by Taxus spp., which accumulates in the bark of the yew tree. Due to the slow growth of yew trees and to the low bark concentration of taxol, an effective cell culture of Taxus was developed for taxol production (Zhong, 2002). Taxus spp. were also the subject of early studies on the hydroponic growing of medicinal plants. Wickremesinhe e Arteca (1994) reported about the growing of Taxus x media and Taxus cuspidata for the production of taxol. Greenhouse hydroponics involves lower running costs compared to those of in vitro cultivation (Montero et al., 2009); therefore, this technique could represent a cost-effective system to produce plant material for the extraction of pharmaceutical molecules. Hayden (2006) reported about the opportunities provided by soilless culture for the production of medicinal crops in controlled environments for improving quality, purity, consistency, bioactivity, and biomass production on a commercial scale. Anyway, the evaluation of the economic profitability of greenhouse hydroponics for the production of niche crops such as medicinal plants should take into account challenges as well as opportunities. In particular, the market could undergo either a rapid growth or a rapid decrease. Successful cultivation of medicinal plants on a commercial scale implies to overcome the difficulty of predicting which extracts will remain on the market (Canter et al., 2005). Along with production factors (such as disease and pest control, quality, production costs and yield) and the need for local infrastructure (such as warehousing and processing facilities), careful market research and consultation with buyers are essential issues for the production of specialty crops such as medicinal plants in greenhouses (Ehret et al., 2002). Among the different hydroponic systems, the floating raft system represents a low-cost technology that is suitable for growing leafy vegetables under greenhouse conditions. Miceli et al. (2003) indicated the floating system as the easiest and least expensive way to produce leafy vegetables when soil cultivation is no longer feasible. The floating system is a simple technique, where the plants are grown on polystyrene trays, which float on a layer of stagnant nutrient solution. This is about 30 cm deep to allow root growth and development, is aerated to avoid root zone hypoxia, and is regularly checked for pH and electrical conductivity to prevent nutrient imbalance. In the floating raft system, the plants are grown with their bare roots dipping directly into the nutrient solution.


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For its simplicity and low investment and running costs, this technique has found practical application in commercial production, and is typically employed for short cycle, high density, fresh-cut (that is minimally processed) leafy vegetables (Pardossi et al., 2006; Rodríguez-Hidalgo et al. 2010). Because the plants are grown in pure nutrient solution without the aid of a growing medium, the floating system offers additional specific advantages over field cultivation. For example, the plant density is generally much higher than in the soil, thus resulting in a high biomass production. Moreover, the absence of a growing medium is a particularly favourable condition for the harvesting of the root system. Along with the aboveground parts, the root tissues obtained from the floating system are not spoiled by substrate particles and can be easily removed from the nutrient solution without damage or loss of material.

Hydroponic Growing of Medicinal Plants At present, a lot of studies have been undertaken relating to the hydroponic growing of medicinal plants. Since the year 2000, about 185,000 works have been published concerning medicinal species and about 8,000 have been published concerning hydroponics (source: Scopus; www.scopus.com/home.url; accessed 13th June 2013); only 430 papers report about the hydroponic culture of medicinal plants. The viability and the advantages of this growing system for the production of secondary metabolites from medicinal plants have been demonstrated for a lot of species (e.g.; Dorais et al., 2001; Léonhart et al, 2002; Hyden, 2006; Azarmi et al., 2012). For example, Stewart and Lovett-Doust (2003) pointed out that greenhouse hydroponic cultivation under controlled environmental conditions in Calendula officinalis could ensure pesticide-free conditions, lacking environmental contaminants, resulting in superior product quality and consistency. Brechner et al. (2007) emphasized that growing Hypericum perforatum in controlled environments, such as the greenhouse or growth chamber, can remove wide variations of common variables such as temperature, insect and disease pressures, and water status. Recently, Prasad et al. (2012) reported that the hydroponic systems can be an effective platform for the production of clean and good quality Centella asiatica herb for the pharmaceutical companies. It was also observed that greenhouse hydroponics could help to overcome germination and establishment problems which may arise with the soil cultivation of medicinal species that are difficult to grow in open field (e.g.: Canter et al., 2005; Crosby and Cracker, 2007; Dall'Acqua et al., 2010). Tabatabaie et al. (2007) reported that hydroponics could be used for the production of both valerian (Valeriana officinalis var common) and lemon verbena (Lipia citriodora var. Verbena) under glasshouse. These authors employed different types of soilless culture for both species, and obtained the highest fresh biomass production using the floating raft system. The same technique was successfully applied also to the cultivation of Camptotheca acuminata, which is used for the production of the anticancer molecule camptothecine (Li and Liu, 2005). In general, hydroponics ensures a high biomass production, because the nutrient elements are readily available at the root zone and can be easily taken up by the plants. Therefore, higher production of plant material can be obtained with hydroponics (particularly, with water culture) compared to that of soil-grown crops (e.g.: Dorais et al., 2001; Letchamo et al., 2002).



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Generally, plant growth is also much faster in hydroponic culture than in open field. For example, Léonhart et al. (2002) reported that Tanacetum parthenium, Achillea millefolium, Taraxacum officinale and Calendula officinalis were all well adapted to greenhouse hydroponic growing conditions and provided abundant yield and high produce quality in a short time period. Dorais et al. (2001) evaluated the growth of several medicinal plants in a floating raft system and found that, after 50-120 days, both the root and the shoot dry weight of Achillea millefolium, Artemisia vulgaris, Inula helenium, Stellaria media, Taraxacum officinale and Valeriana officinalis were much higher in the floating system compared to those of field-grown plants. With the exception of Taraxacum officinale, in all the species under examination the rate of biomass accumulation was faster in the aboveground parts than in the roots; Artemisia vulgaris showed the fastest growth rate. This result was in agreement with those of a previous study carried out with 31 species belonging to the Asteraceae family and grown hydroponically (Almeida-Cortez et al., 1999). Among them, Artemisia vulgaris exhibited the fastest growth rate (0.226 g g-1 day-1). Similarly, Echinacea spp., which is traditionally cultivated two to four years in open field, provided high biomass yields in hydroponics in a much shorter time period (a few months only). For example, in Echinacea angustifolia the root yield harvested in nearly eight months from two consecutive hydroponic cultures was comparable with the yield reported in the literature for field cultivations lasting two years or more (Maggini et al., 2012). Moreover, the production yield in Echinacea purpurea was found to increase 2.3 times compared to the average soil cultivation in North America (Letchamo et al, 2002). All these studies evidenced that hydroponics could really offer the opportunity to shorten the growing cycle used in conventional field cultivation and increase at the same time the biomass production. Together with a higher biomass yield, a higher concentration of secondary metabolites has also been obtained with hydroponics for a lot of medicinal species. Among secondary metabolites, essential oils have been often found in higher amounts in plants grown hydroponically compared to those grown in open field. For some officinal plants such as Pelargonium roseum, Cymbopogon citratus, Ocimum gratissimum, Vetiveria zizanioides e Nepeta transcaucasica, the hydroponic system provided 5-6 times more essential oil than traditional cultivation. Moreover, hydroponically produced essential oil of Pelargonium roseum had a higher geraniol content and was therefore of better quality (Mairapetyan, 1999). Recently, Azarmi et al. (2012) indicated the floating system as an efficient method to produce large biomass of Aloysia citriodora L with high content of volatile oil. In addition to essential oils, other classes of secondary metabolites have been found at higher concentrations in hydroponically-grown than in soil-grown medicinal plants. Tadevosyan et al. (2005) reported about the hydroponic cultivation of Humulus (a species used in Armenian traditional medicine) as an efficient and prospective technique in the Ararat Valley conditions. Humulus plants grown in hydroponics contained higher concentrations of alkaloids, tannins and essential oil than those cultivated in the soil. The content of hypericin, hyperforin and pseudohypericin in the flower tissues of hydroponically-grown Hypericum perforatum was similar or higher than those previously reported for field-grown plants (Murch et al., 2002). It was found that, under outside hydroponic conditions, Celandine poppy (Chelidonium majus L.) presented higher contents of alkaloid, tannins and vitamin C, and lemon catmint (Nepeta cataria L. var. citriodora) contained remarkably higher concentrations of essential oil, tannins and vitamin C compared to field cultivated plants (Manukyan, 2005).


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In contrast with these results, some studies on the production of secondary metabolites in Echinacea angustifolia reported much lower root concentrations of caffeic acid derivatives (especially of the marker compound echinacoside) in hydroponically-grown plants (Zheng et al., 2006b; Maggini et al., 2010, 2012; Sabra et al., 2012) than in field-grown crops (Berti et al., 2002). This was probably the consequence of plant harvesting after only a few months of hydroponic cultivation, whereas field-grown Echinacea plants are commonly harvested after a few years (ontogenetic effect). Anyhow, all these studies indicated that, although a lot of medicinal species are easily adapted to greenhouse hydroponic conditions and have been successfully cultivated by this growing system, other species still require further work for the development of profitable growing protocols, based on the knowledge of their specific growing needs.

Manipulation of Growing Conditions Several studies have shown that in greenhouse hydroponic culture the accumulation of secondary metabolites of pharmaceutical interest can be stimulated by modifying the composition of the nutrient solution (e.g. Briskin, 2000; Maia et al., 2001; Zheng et al., 2006b; Montanari et al. 2008; Kiferle et al., 2013) or the climate inside the greenhouse, such as temperature (McChesney, 1999) or light conditions (Giorgi et al., 2007; Hou et al., 2010). Some authors reported that treating hydroponically-grown plants with growth regulators (Wikremesinhe and Arteca, 1996) or bio-stimulants (Parađiković et al., 2011) resulted in larger production of secondary metabolites. Plant mineral nutrition may affect both plant growth and secondary metabolism (Briskin, 2000; Zheng et al., 2006a). Nitrogen (N) is the most important nutrient for plants. As a consequence, N starvation is a primary cause for growth reduction, as it limits primary metabolism, thus reducing the production of biomass. However, N deficiency may have an opposite effect on secondary metabolism. The C/N balance hypothesis proposed by Bryant et al. (1983), assumes that the C/N ratio within the plant regulates the concentration of C-based secondary metabolites. A limitation of N supply which restricts growth more than photosynthesis, results in over-production of carbohydrates. These compounds are in part allocated to C-based secondary metabolites. At the same time, N deficiency enhances the formation of reactive oxygen species (Kováčik et al., 2007). For these reasons, phenolic compounds, which are a major group of C-based antioxidant molecules, play a central role in plants’ adaptation to N starvation. In chamomile (Matricaria chamomilla) an increase in the production of phenolics was observed in N-deficient plants (Kováčik et al., 2007). In several medicinal plants, lowering the concentration of N in the nutrient solution resulted in an increase of the content of bioactive secondary metabolites. For example, feeding licorice (Glycyrrhiza glabra Linn.) plants with dilute nutrient solution (approximately equivalent to a quarter unit of Hoagland solution) provided the highest glycyrrhizin content in root tissues and the highest plant growth (Sato et al., 2004). In Camptotheca acuminata, decreasing N concentration in the hydroponic nutrient solution increased the content of the secondary metabolite camptothecine (Li and Liu, 2005). In soilless culture, N is usually supplied as nitrate (NO3-; Pardossi et al., 2006). Some authors (e.g. Munoz et al., 2008; Massa et al., 2010) proposed to lower the NO3concentration in the nutrient solution to reduce the environmental impact of soilless culture



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associated with NO3- leaching. Decreasing the concentration of NO3- in the nutrient solution also reduces the accumulation of NO3- in leafy vegetables (Santamaria et al., 1998), which is potentially toxic to human health. Like N, phosphorus (P) is an essential nutrient for plants. Stewart and Lovett-Doust (2003) reported that Calendula officinalis showed promise as a medicinal greenhouse crop that requires low P levels for optimal production of inflorescence, which is the target tissue containing bioactive compounds. Moreover, due to the xerophytic characteristics of this species, the best results in terms of flower-head tissues production were obtained when relatively low ratios of P relative to N and potassium (K) were associated to intermittent watering regime. The authors suggested that discontinuous water and nutrient supply in hydroponic culture may be widely applicable to medicinal plants, since a lot of species share Calendula’s xerophytic characteristics. Nutrient solutions differing in concentrations and ratios of N, P, and K were reported to influence also the synthesis of various pharmaceutical compounds such as alkaloids, essential oils, tannins, and vitamin C in Chelidonium majus L. and Nepeta cataria L. (Manukyan, 2005). Sodium (Na+) and chloride (Cl-) are the most common non-nutrient ions dissolved in irrigation water. The induction of a salt stress by addition of sodium chloride (NaCl) to the nutrient solution determines a rise in the electrical conductivity and results in osmotic stress, as well as ion (Na+ or Cl-) cytotoxicity (Saleh and Maftoon, 2008; Silva et al., 2008; Munns and Tester, 2008; Dashti et al., 2010), and oxidative damage to macromolecules and cell structure (Neto et al., 2006; Eraslan et al., 2007). Depending on the species, salt stress may have different effects on the production of plant secondary metabolites. For example, salinity was reported to decrease the production of essential oils in Matricaria chamomilla (Razmjoo et al., 2008) and Melissa officinalis (Ozturk et al., 2004), and to have no significant effect on the content of echinacoside per plant in Echinacea angustifolia (Maggini et al., 2013). Mehrizi et al. (2012) observed that salinity induced oxidative stress in hydroponically-grown rosemary, resulting in lipid peroxidation and increase in cell membrane permeability to toxic ions, which in turn reduced plant growth. As a response to oxidative damage, the total phenolic content in medicinal plants was often reported to be influenced by salinity (Mehziri et al 2012¸ Navarro et al., 2006; Ksouri et al., 2007). A strong correlation between salt tolerance and antioxidant capacity was found in several plant species (Gill and Tuteja, 2010). In particular, higher levels of phenolics were reported in salt tolerant species compared to non tolerant ones. Together with NO3-, ammonium (NH4+) is a main source of N and is readily absorbed by plants. However, likewise excess Na+ or Cl-, excess NH4+ may have a toxic effect on plants, although the biochemical mechanisms of NH4+ toxicity remain to be further elucidated (Britto and Kronzucker, 2002). The concentrations at which the toxic effects are observed depend on plant species. Several studies have been conducted on the effect of NH4+ on the growth of some crop species (e.g.: Britto and Kronzucker, 2002; Savvas et al., 2006; Cárdenas-Navarro et al., 2006; Cao et al., 2011). One of the main effects of NH4+ toxicity is a lower root/shoot ratio (Kiferle et al., 2013), although the opposite was observed in some species (Britto and Kronzucker, 2002). On the other hand, the presence of NH4+ along with NO3- could have also favorable implications, as NH4+ may reduce NO3- absorption. In addition, in hydroponics NH4+ may help in managing the pH of the nutrient solution, as it controls the alkaline drift in pH determined by NO3- assimilation (Savvas, 2001). The pH of the nutrient solution is known to


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affect plant growth and metabolism, as reported for the hydroponic culture of Artemisia afra Jacq. (Koehorst et al. 2010). At the same time, NH4+ absorption may alter intracellular pH gradients, which affect a lot of metabolic pathways (Dixon and Paiva, 1995). Little information has been reported concerning the response of plant secondary metabolism to N form. Anyway, the use of nutrient solutions supplemented with both NH4+ and NO3- at different ratios was reported to affect the production of bioactive compounds in medicinal species grown in hydroponics. It was observed that the supply of 50% total N as NH4+ enhanced the accumulation of the alkaloids catharanthine and vinblastine in Catharanthus roseus (Guo et al., 2012). In contrast, the supply of a mixture of NH4+ and NO3- in Echinacea angustifolia decreased the concentration of some caffeic acid derivatives (Montanari et al., 2008). At the same time, a decrease was also observed in the activity of phenylalanine ammonia lyase, a key enzyme of the phenylpropanoid pathway involved in the biosynthesis of these secondary metabolites (Montanari et al., 2008). In sweet basil irrigated with a nutrient solution containing 10.0 mM NH4+, the total content of essential oil was markedly reduced as compared to the plants fed exclusively with NO3- (Adler et al., 1989). A scarce oxygen (O2) level in the root zone (hypoxia) is a further cause of metabolism imbalance. Although the effect of hypoxia on the secondary metabolism of medicinal plants has been scarcely investigated, in floating system this condition may occur in the stagnant nutrient solution, especially in warm season, as high temperatures may reduce O2 solubility while increasing root respiration (Gorbe and Calatayud, 2010). An adequate O2 level is necessary to ensure root functionality, whereas O2 deficiency reduces the uptake of both water and nutrients such as NO3- (Horchani et al., 2010; Ferrante et al., 2003). Moreover, O2 deficit enhances the formation of reactive oxygen species (Colmer and Voesenek, 2009). Anyway, a large part of the literature on the effects of hypoxia concerns plant growth with little attention paid to secondary metabolism. Growth reduction is considered one of the first adaptive plant responses to hypoxia, as this allows to conserve energy, inhibiting a wide range of ATP-consuming processes to decrease O2 demand (Geigenberger, 2003). The detrimental effect of low O2 in the root zone of plants grown in hydroponics was observed in several crop species (e.g.: Ferrante et al., 2003; Shi et al., 2007). Plant sensitivity to hypoxia conditions depends on plant species and may vary even among different cultivars of the same species. In some cultivars of Medicago sativa the growth of both roots and shoots was limited by waterlogging, while in other cultivars only root growth was severely restricted, whereas shoot biomass was unaffected (Smethurst and Shabala, 2003). Under root zone hypoxia conditions, a differential response between the root system and the aerial organs may be associated to ethylene entrapment in submerged plant tissues, as a consequence of the much lower gas diffusion rate in water than in air (Visser and Vosenek, 2004). Ethylene plays a key role in the mechanisms of plant adaptation to hypoxia, for instance by regulating the formation of adventitious roots and aerenchyma (Licausi, 2011). On the other hand, this hormone is known to inhibit root growth, even at low concentration (Abeles et al., 1992). In addition to a change in the composition of the nutrient solution, a proper modification of the growing conditions could also result effective in stimulating the secondary metabolism. For example, it was found that: low temperatures increased the accumulation of morphine in Papaver somniferum (McChesney, 1999); water stress increased the concentration of flavonolignans in primary blooms of Silybum marianum (L.) Gaertn. (Belitz and Sams,2007); low irradiance favored the accumulation of glycyrrhizic acid and liquiritin in the roots of



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Glycyrrhiza uralensis Fisch. (Hou et al., 2010). Several experiments demonstrated that supplemental lighting on medicinal plants grown hydroponically under greenhouse accumulated more bioactive molecules compared to field-grown crops (Pedneault et al., 2002; Brechner et al., 2007). In contrast, an opposite effect of supplemental lighting was reported on other medicinal species. For example, the concentration of phenolic compounds from Tarassacum officinale was 6.2 times higher in field-grown plants compared to those cultivated in hydroponic culture. In Inula helenium, sesquiterpene lactones were more concentrated in field-grown root compared to hydroponically-grown root and parthenolide was more concentrated in field-grown flowers and leaves than in the same organs of hydroponically-grown plants (Pedneault et al., 2002).

A Case Study: Basil Basil (Ocimum basilicum L.) is one of the most important species belonging to the genus Ocimum, in the Lamiaceae family. The genus Ocimum encompasses a huge number of medicinal species and varieties, characterized by a large variability in morphology and habitats, flavours, scents, and uses (Putievsky and Galambosi, 1999). A lot of them are mainly cultivated to be used for culinary preparations. This species includes a large number of varieties and cultivars with distinct morphological traits and chemotypes (Simon et al., 1999), which range from typical green-leaf varieties (Genovese, Lettuce leaf, Gigante) to purple-colored genotypes (Dark Opal, Red Rubin) or lemon-flavoured cultivars (Citriodorum). Basil is cultivated worldwide, and is also grown hydroponically (Miceli et al., 2003). Whereas some varieties are used as ornamental plants, basil is mainly used for food preparations (Makri and Kintzios, 2007). The fresh green leaves of some cultivars (sweet basil; for example Genovese) are commonly used for the preparation of the well-known Italian ‘pesto’ sauce, now largely diffused all over the world (Miele et al., 2001). Basil is also an important source of essential oils and of rosmarinic acid (Kiferle et al., 2011). The essential oils are extensively used in food and pharmaceutical industry, perfumery, cosmetics and herbal medicine (Makri and Kintzios, 2007; Hussain et al., 2008). The composition and concentration of the essential oils is largely variable in dependence of cultivars and growing conditions. However, linalool, chavicol and methyl-chavicol, eugenol and methyl-eugenol, estragole, methyl-cinnamate, have all been reported as the dominant volatile constituents (Lee et al., 2005; Makri and Kintzios, 2007; Klimánková et al., 2008). The relative content of each constituent can often enable to differentiate among distinct cultivars (Klimánková et al., 2008). Rosmarinic acid is one of the most abundant antioxidant phenolic compounds accumulated by basil (Jayasinghe et al., 2003; Li et al., 2007; Makri and Kintzios, 2007; Juliani et al., 2008; Lee and Scagel 2009). Rosmarinic acid is widely distributed in the plant kingdom, but represents a characteristic secondary metabolite of several medicinal plants (e.g. Salvia officinalis, Mentha x piperita, Thymus vulgaris, Melissa officinalis) in the Boraginaceae and Lamiaceae families (Petersen and Simmonds, 2003; Petersen et al., 2009). As a caffeic acid derivative, rosmarinic acid belongs to the class of phenylpropanoids (Kurkin, 2013). The molecule is formally obtained by esterification of the carboxylic group of


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caffeic acid with the alpha hydroxyl group of 3,4-dihydroxyphenyllactic acid. The pure compound was isolated for the first time in Rosmarinus officinalis by Scarpati and Oriente (1958), while the complete biosynthetic pathway from the precursors tyrosine and phenylalanine was fully elucidated 45 years later by Petersen and Simmonds (2003). Rosmarinic acid is a strong free radical scavenging agent. The antioxidant properties of this secondary metabolite are due to the presence of two couples of hydroxyl groups, each couple being located in the ortho positions of a benzene ring. A large number of additional biological activities have been described for rosmarinic acid: adstringent, anti-inflammatory, anti-mutagen, anti-bacterial and anti-viral properties have been attributed to this compound (Petersen and Simmonds, 2003; Juliani et al., 2008). Likewise the vast majority of plant secondary metabolites, rosmarinic acid accumulation for a given genotype is strongly affected by many factors, including growing and environmental conditions, phenological stage, plant organ (Del Baño et al., 2003; Juliani et al., 2008; Shiga et al., 2009).

Experiments at the University of Pisa This section reports a synthesis of the main results obtained in a series of experiments carried out at the University of Pisa (Italy) with the green-leaf basil cultivar Genovese grown in floating system (Kiferle et al., 2011, 2012, 2013). These experiments were aimed at studying the applicability of greenhouse hydroponics to the agro-industrial production of rosmarinic acid (hereafter indicated as RA). When grown hydroponically, basil plants showed a fast growth and leaf concentration of RA ranged from 4 to 29 mg/g DW (Kiferle et al., 2011). Roots also contained significant concentrations of RA. Although the shoot accounted for more than 90% total dry mass, in principle the whole plant could be processed for RA extraction, as the floating system facilitates also the harvesting of clean root tissues (Kiferle et al., 2011). All the determinations were conducted on non-dehydrated fresh or frozen (-80°C) samples, as desiccation at 70°C was found to reduce the content of RA in basil tissues up to 40% (Kiferle et al., 2011). Leaf RA concentrations reported in the literature for sweet basil varied from less than 0.1 mg/g DW (Sgherri et al., 2010) to nearly 100 mg/g DW (Javanmardi et al., 2002). This wide range is probably the consequence of differences in plant genotype and growing conditions, or in the method used for the determination of rosmarinic acid. In some experiments attempts were made to increase the production of RA in sweet basil while maintaining the same biomass production. In particular, the plants were exposed to a moderate NaCl salinity stress, to a moderate hypoxia condition or to a change in N nutrition. In order to study the effect of salinity, a control treatment and two different saline treatments were compared. In the latter treatments, proper amounts of NaCl were added to the control nutrient solution (Kiferle et al., 2012). Although the response to saline stress may change in different cultivars (Attia et al., 2011; Omer et al., 2008), the cultivar Genovese resulted moderately tolerant to a NaCl-induced salinity stress (Kiferle et al., 2012). In agreement with these results, other authors found that NaCl concentrations up to 50 mM did not affect the growth of sweet basil grown in water culture (Attia et al., 2009; Tarchoune et al., 2009, 2010). In contrast, Bernstein et al. (2010) reported that NaCl salinity reduced



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significantly root and shoot growth in hydroponically-grown sweet basil, especially at concentrations higher than 50 mM. The content of RA in leaf tissues resulted unaffected by NaCl salinity (Kiferle et al., 2012). This result was in disagreement with those observed under slightly different growing conditions by other authors (Tarchoune et al., 2009), which found that 50 mM NaCl markedly reduced the leaf concentration of RA (as well as those of caffeic and vanillic acids) in basil cultivar Genovese. In contrast, the root content of RA was found to increase significantly in both saline treatments (Kiferle et al., 2012). In another experiment, hypoxia conditions were easily induced in the culture by simply disconnecting the aeration of the nutrient solution. The cultivar Genovese appeared moderately tolerant to a moderate hypoxia stress. Hypoxia did not affect significantly shoot growth, while a marked reduction was observed in root dry weight (Kiferle et al., 2012). This result was in agreement with those of other studies (Drew, 1983; Incrocci et al., 2000; Shi et al., 2007). The root tissues were affected by hypoxia also for the accumulation of RA, whereas the leaf content of this metabolite was not modified by the oxygen level in the nutrient solution (Kiferle et al., 2012). In contrast to these findings, some authors observed an increase in the level of phenolic compounds in plants grown under root hypoxia, for instance in both shoots and roots of Hypericum brasiliense (Nacif de Abreu and Mazzafera, 2005) and in the stems of Eucalyptus marginata (Burgess et al., 1999). In the latter work, the increase in the concentration of phenolic compounds was also linked to the increased activity of some enzymes involved in their biosynthesis, such as phenylalanine ammonia lyase, 4-coumarate coenzyme A ligase and cinnamyl alcohol dehydrogenase. Two distinct types of experiments were carried out concerning the influence of N nutrition. In the first one, N was entirely supplied as NO3-, at the concentrations of 10, 5 or 0.5 mM (Kiferle et al., 2013). The former concentration is the standard level of NO3- that is considered as optimal, and similar concentrations are generally employed in hydroponic cultivation (Pardossi et al., 2006; Sonneveld and Voogt, 2009). In the second experiment, the total N content was kept constant at the optimal concentration value, and the NO3-/NH4+ ratio was modified (Kiferle et al., 2013). Overall, the growth parameters were higher at the optimal concentration of 10 mM, except the root dry matter, which increased at the lowest NO3- concentration. This was an expected result, as it is known that N deficiency inhibits shoot growth while stimulating root growth (Clarkson, 1985), because this adaptive mechanism enhances the plant’s ability to absorb nutritive ions from the growing medium. In a similar way, several growth parameters indicated that the supply of NH4+, alone or in mixture with NO3-, had a marked detrimental effect on plant growth (Kiferle et al., 2013). In contrast with these findings, in pot-grown sweet basil the biomass production resulted unaffected by the use of salt fertilizers containing NO3- or NH4+ (Tesi, 1995; Adler et al., 1989). The decrease in NO3- concentration increased the content of RA in the tissues (Kiferle et al., 2013). In agreement with this outcome, an increase in leaf RA content of sweet basil grown under limited N availability was reported by Nguyen and Niemeyer (2008). The concentration of RA was affected also by the N form. In leaf tissues, the presence of NH4+ ion had the undesired effect of decreasing the level of rosmarinic acid, even in the presence of NO3- (Kiferle et al., 2013). The following table (Table 1) summarizes the main effects that were determined in the leaf tissues of sweet basil cultivar Genovese by a change in the composition of the nutrient solution (Kiferle et al., 2012, 2013).


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Table 1. Effect of modifications of the nutrient solution on biomass production and leaf content of rosmarinic acid in sweet basil (Ocimum basilicum L.), cultivar Genovese. The symbols + and ¯ indicate higher or lower values than those obtained under standard growing conditions while the letters ‘ns’ indicate no significant change. DW: dry weight. See text for details

Dry biomass (g/plant) Rosmarinic acid (mg /g DW) a b

Salinitya (NaCl addition) ns ns

Low N levelb (N as NO3-) ns +

NH4+ additionb (constant total N) _ _

Hypoxiaa ns ns

Kiferle et al., 2012. Kiferle et al., 2013.

Overall, salinity or hypoxia did not have a significant effect either on the leaf biomass production or on the content of RA, which remained unchanged in leaf tissues. On the other hand, a clearly detrimental effect was observed for both growth and RA production in the presence of NH4+. Thus, the use of this ion in the nutrient solution should be avoided for hydroponic cultivation of sweet basil. The best results were provided by a decrease in the level of NO3- supplied to the plants, compared to the standard concentrations generally used in hydroponic culture (Pardossi et al., 2006; Sonneveld and Voogt, 2009). When plants were grown with a NO3- concentration of 5 mM, leaf and total RA content was significantly greater than at 10 mM, the typical concentration of hydroponic nutrient solutions. All these findings suggested the potential of greenhouse hydroponic culture of sweet basil for the agro-industrial production of RA, as a large amount of biomass with a high concentration of this antioxidant compound could be produced in a few weeks. The concentration of RA could be further increased by a proper change in the composition of the nutrient solution, specifically by a decrease in the NO3- level compared to the typical concentration of hydroponic nutrient solutions (10 mM or higher). The above reported results also have some important operative and environmental implications, as they suggest that poor quality (i.e. moderately saline) irrigation water can be used in water culture of sweet basil and that the aeration of nutrient solution is not a crucial factor for optimal plant growth and RA production of this species. Furthermore, the reduction of NO3- concentration in the culture solution results in lower environmental impact, as less N fertilisers are applied and the leaching of NO3- with nutrient solution discharge is limited. A further outcome of the experiments described in this section was that different basil genotypes accumulated different amounts of RA (Kiferle et al., 2011) and that, as a consequence, cultivar selection is recommended for production improvement.

Conclusion Greenhouse hydroponic technology is currently applied to the commercial-scale production of fresh or minimally-processed herbs (including basil) for the vegetables market. This well-known and commonly employed technology could be easily applied also to the production of biomass for the extraction of bioactive molecules.



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The production efficiency of this growing system could be further improved by accurate variety selection, as the content of bioactive compounds in medicinal plants is strongly dependent on the genotype. It is generally acknowledged that greenhouse hydroponic cultivation is a profitable system for medicinal plants production in terms of biomass yield and quality of the raw material, which is clean and easy to be harvested and processed. A low-cost greenhouse hydroponic system such as the floating raft system, which has found actual commercial application for the production of high density, short cycle leafy vegetables, may also result economically profitable, especially if the species to be grown are selected both for their economic value and bio-active properties. With greenhouse hydroponics, the cultural cycle can be sensibly shortened. While this is an evident advantage for biomass production, it may be a limiting factor for the synthesis and accumulation of sufficient amounts of bioactive substances in the tissues, as we found in Echinacea angustifolia (Maggini et al., 2012). On the other hand, we found that the floating raft system provided a suitable growing method for the agro-industrial production of RA from basil (Kiferle et al., 2011, 2012, 2013). The greenhouse hydroponic growing of this species may be considered as a model system for the production of plant material for the extraction of bioactive compounds, as a large amount of biomass with high concentration of bioactive compound could be produced in few weeks. In addition, proper manipulation of the characteristics of the nutrient solution (e.g. N concentration) may increase the production of the metabolite(s) of interest. In our experiments on basil, the determinations were conducted on fresh or frozen (80°C) samples, which contained much more RA than oven dried tissues (Kiferle et al., 2011). Medicinal plant material generally undergoes desiccation, as dried tissues are easier to handle and process. This common post-harvest practice prevents undesired microbial degradation and facilitates storage and transportation to the processing unit. In contrast, a special production scheme is required for the processing of fresh material. In particular, the greenhouses for the cultivation of medicinal plants should be located close to the processing units, a short-term storage should be planned before extraction and suitable cold rooms should be available. In order to evaluate the profitability of this scheme, which resembles the one for the industrial production of fresh-cut vegetables, the overall costs to obtain secondary metabolites from fresh or dry plant material should be compared. The literature data evidence that there is still a lack of information on the suitable growing practices for medicinal plants production in hydroponics, and suggest that specific cultural protocols should be developed for each species individually.

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Chapter 4

Flavonoids as Antioxidant Therapy for Metabolic Disorders B. S. Lakshmi1, K. N. Sangeetha2 and K. Shilpa1 1

2

Centre for Biotechnology, Anna University, Chennai, India Department of Biochemistry, University of Madras, Chennai, India

Abstract Metabolic disorders, including diabetes and obesity, have been strongly associated with oxidative stress, due to a disproportionate release of free radicals, during the metabolism of excessive glucose and free fatty acids. Enhanced production of reactive oxygen species (ROS) and perturbed antioxidant defenses determine the chemical changes in virtually all cellular components resulting in their damage. ROS is generated through several mechanisms including oxidative phosphorylation, glucose auto-oxidation, advanced glycation end product (AGE) formation, activation of protein kinase C (PKC), nitric oxide synthase (NOS) and aldose reductase pathway among others. They also act as secondary messengers in the regulation of several intracellular signaling pathways. The most promising strategy to mitigate the effect of ROS induced oxidative damage is through the use of antioxidant molecules. Antioxidants, usually phytochemicals and micronutrients called as quenchers act either directly by free radical scavenging mechanisms or indirectly by enhancing the antioxidant status (enzymatic and non-enzymatic). As diabetes and obesity conditions initiate generation of free radicals, compounds that can manage these conditions serve to be effective against these diseases and their complications. In this perspective, therapeutic intervention with the ability to reduce oxidative stress can impede or delay the onset of the metabolic disorder. Thus, agents possessing dual effect such as anti-diabetic/anti-obesity and antioxidant activity are greatly in demand. The therapeutic effect of phytochemicals found in natural products to combat oxidative stress is gaining significance as they are recognized to be safe with a wide range of biological and pharmacological activities. Dietary components from plants such as polyphenols (flavonoids), terpenes and tannins are ubiquitous in nature and can effectively scavenge reactive oxygen and nitrogen species, thus, modulating the genes associated with metabolism and stress defense. This chapter discusses the sources of flavonoids, their potential antioxidant properties and the mechanism through which they exert their pharmacological effects in diabetes and obesity.


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B. S. Lakshmi, K. N. Sangeetha and K. Shilpa

Introduction Since ancient civilizations, traditional medicine systems have used plants for the treatment of various diseases and disorders. Isolation of bioactive compounds from various medicinal plants began as early as the 19th century in the ancient Chinese, Indian and North African civilizations. Several bioactive molecules have been identified from plants in the past century, prominent among them being atropine and hyoscine from Solanaceae sp., codeine and morphine from opium poppy, digoxin from Digitalis leaves, quinine from Cinchona bark, vinblastine and vincristine from Catharanthus roseus and reserpine from Rauwolfia sp. Years of research work has led to the discovery of several bioactive principles for the treatment of diseases. Natural products and their derivatives or their derived pharmacophore contribute to more than 50% of all the medicines used in the world. It has also been observed that 67% of drugs used for the treatment of human cancers and 70% anti-bacterial, anti-fungal, antiparasitic, and anti-viral are naturally derived / inspired drugs [Gurib-Fakim, 2006].

Free Radicals and Oxidative Stress In recent years, there has been an increasing awareness among people towards understanding the role of free radicals on human health. Cells in our body use oxygen to generate energy and the process results in the production of free radicals by the mitochondria. Free radicals such as superoxide anion (•O2-), hydroxyl radical (•OH) and nitric oxide (NO•) are produced as by-products of several reactions occurring using oxygen and these free radicals are highly reactive. Free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known as pro-oxidants. ROS and RNS are produced as a result of cellular redox processes and have special affinity for lipids, proteins and nucleic acids. Free radicals at lower concentrations are essential for several physiological functions of cell including gene expression, cellular growth, defense against infection and as stimulating agents in biochemical processes. However at higher concentrations, free radicals can damage cell membranes and lipoproteins by lipid peroxidation, proteins by structural changes causing loss of activity and damage deoxyribonucleic acid (DNA) by DNA strand breaks leading to cell mutation. However, the body has certain antioxidant enzymes as special defense mechanisms which can be divided into two categories: enzymatic mechanisms and nonenzymatic mechanisms for neutralizing the effects of these pro-oxidants (Figure 1a). Any imbalance between the pro-oxidant system and the antioxidant defenses lead to a stressful environment defined as “oxidative stress”, which ultimately ends up in oxidative cell damage with prolonged stress (Figure 1b) [Maritim et al., 2003]. Oxidative stress plays a major role in the development of chronic and degenerative diseases such as cancer, arthritis, aging, diabetes, autoimmune disorders, cardiovascular and neurodegenerative diseases [Maritim et al., 2003]. Despite considerable progress in the field of drug discovery, there is an increase in the progression of these diseases globally, with metabolic disorders being the prime targets, due to their associated long term complications.


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Figure 1. Pro-oxidant and antioxidant status during a) physiological and b) oxidative stress condition.

Hence, there is an urgent need to meet the demands of the disease with a multi-modal therapeutic approach that includes multi-targeted action with lesser side effects. Although, development of modern medicine has resulted in the advent of modern pharmacotherapeutics, there is a need to look for new drugs to modify the course of complications associated with metabolic disorders. At present, the therapy for metabolic disorders relies mainly on approaches using synthetic agents. These synthetic therapies have limited efficacy, limited tolerability and significant mechanism-based side effects, thus desperately demanding for alternative approaches. Natural products with their structural and chemical diversity, biochemical specificity and molecular characteristics, are ideal for screening and identification of bioactive molecules for drug discovery process [Tiwari and Rao, 2002]. It has also been observed that certain cases of metabolic disorders respond well to natural remedies in comparison to conventional drugs.

Plants as an Alternate Therapy In many parts of the world, medicinal plants form the backbone of traditional medicine, and are the source for several secondary metabolites exhibiting bioactivity for the treatment of various diseases and metabolic disorders [Farnsworth, 1994]. Additionally, secondary metabolites have applications in food and cosmetic industry as additives, due to the preservative effects exhibited by the antioxidant and antimicrobial constituents [Škrovánková et al., 2012]. Till date, tropical rain forests remain a vast reservoir and continue to provide bioactive compounds used for the development of new drugs. At present, about 50 drugs have been discovered from tropical plants and the possibility of finding more bioactive compounds


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are enormous as only about 1% of tropical species have been explored for their bioactive potential. According to WHO survey, 80% of world population, primarily those of developing countries, rely on plant based medicines for the treatment of various diseases and disorders. Herbal remedies in traditional cultures have been developed through trial and error methods over several centuries, with the most important remedies being passed on verbally from one generation to another. Presently, since there exists a better understanding of human physiology, the bioactivities exhibited by plants in the treatment of various diseases and disorders can be understood better. The commonly used plants exhibiting antioxidant activity include neem, turmeric, ginger, rosemary, sage, oregano, marjoram, basil, thyme, mints, balm, cumin, fennel and caraway among others [Škrovánková et al., 2012]. These medicinal plants are a reservoir of secondary metabolites, which act either individually, additively or in synergy with several different plants in exhibiting their biological effects [Cragg and Newmann, 2005]. Among the various secondary metabolites produced by plants in response to stress, phenols form the largest group, ranging from simple structures with one aromatic ring to complex polymers such as tannins and lignins [Phillipson, 2001 and Gurib-Fakim, 2006]. The secondary metabolites such as flavonoids, stilbenes, tannins, coumarins, lignans and lignins exhibit various biological effects including antioxidant activity [Packer et al., 1999; Yu-Ling et al 2012] and are involved in the elimination of free radicals that are responsible for various chronic and degenerative diseases, including inflammation, stroke, diabetes mellitus and cancer.

Plants as Antioxidants Nearly all plants possess compounds that exhibit antioxidant activity as a defense mechanism. These plant antioxidants play a vital role in human health care by serving as reducing agents, free radical scavengers, complexes of pro-oxidant metals, and, quenchers of singlet oxygen formation. The most common natural antioxidants are polyphenols that include flavonoids (flavanols, isoflavones, flavones, catchins, flavanones), cinnamic acid derivatives, coumarins, tocopherols and poly functional organic acids.

Polyphenols Polyphenols are secondary metabolites of plants and are generally involved in defense against ultraviolet (UV) radiation or aggression by pathogens. In food, polyphenols contribute to the bitterness, astringency, color, flavor, odor and oxidative stability. They offer protection against development of cancers, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases. Plant phenolic compounds are formed from the common intermediate, phenylalanine or a close precursor shikimic acid [Ilja and Peter, 2005]. Polyphenols may be classified into different groups based on the number of phenol rings they contain and the structural elements that bind these rings to one another. The main classes



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include phenolic acids, flavonoids, stilbenes and lignans. This chapter will discuss about flavonoids and their role against metabolic diseases like diabetes and obesity.

Flavonoids Flavonoids are plant secondary metabolites that are best known for imparting characteristic red, blue, and purple pigments in various plant tissues including fruits, vegetables, grains, bark, roots, stems, flowers. More than 4000 structurally distinctive flavonoids have been identified from plants [Brahmachari, 2011] and many of them are been known to perform better than many well-known antioxidants, such as ascorbate (vitamin C) and α-tocopherol (vitamin E) based on in vitro antioxidant assays because of their strong capacity to donate electrons or hydrogen atoms [Hernańdez et al., 2009]. Human consumption of plant derived flavonoids is approximately 1 g per day [Kuhnau, 1976] with beverages like tea, coffee, red wine and beer containing large amounts of flavonoids and herbal remedies containing flavonoids being used around the world. Flavonoids act as copigment, contributing to the colour in plants and help in the pollination by attracting animals by their colours and also in the protection of plants from stress, such as damage caused by UV [Gurib-Fakim, 2006 and Cody et al., 1986]. Moderate to high amounts of flavonoids are present in tea, fruits (apples, blueberries), dark chocolate and red wine, whereas, broccoli or fruit juices (cranberry and orange) provide relatively low levels of flavonoid [Beecher, 2003]. Flavonoids are involved in an array of processes, including plant–pathogen interactions, pollination and seed development [Williams and Grayer, 2004; Winkel-Shirley, 2001]. Plants have been found to produce flavonoids in response to various biotic and abiotic stresses, such as wounding, drought, metal toxicity, nutrient deprivation, infection and are also observed to act as a deterrent for herbivores [Winkel-Shirley, 2001; Van Breusegem and Dat 2006; Cadenas 1995; Winkel-Shirley 2002; Dixon and Paiva, 1995 and Hernández et al., 2009]. Besides their role in protecting plants, their consumption by humans have been known to improve health by preventing degenerative diseases associated with oxidative stress, as flavonoids are known to act as scavengers of free radicals such as ROS [Rice-Evans et al., 1997 and Pourcel et al., 2007]. Flavonoids are polyphenolic compounds, formed by addition of malonyl CoA to the phenylpropanoid molecule coumaroyl CoA [Saxena et al., 2012]. The aromatic cycles of flavonoids undergo modifications like hydroxylations, methylations, glycosylations, acylations or prenylations, which account for the diversity of flavonoid class [Pourcel et al., 2007]. However, all flavonoids share a basic skeleton structure consisting of C6-C3-C6 with two aromatic C6 rings and a heterocyclic rings containing one oxygen atom. The molecular structures of flavonoids determine their capacity to act as antioxidants. Flavonoids are available in the form of flavonols, flavones, isoflavones, flavonones in major dietary sources such as tea, red wine, apple, tomato, orange, lemon, grape fruit, ginkgo, cherry, onion, parsley, soyabeans, neem, thyme and other legumes [Saxena et al, 2012]. In recent years, various investigations have been carried out to characterize the effect of plant derived secondary metabolites on free radicals scavenging. The genes that govern the biosynthesis of antioxidant flavonoids are present in liverworts and mosses and are mostly up-regulated as a consequence of severe stress [Agatia et al., 2012]. Increasing evidence of different physiological functions exhibited by flavonoids in


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response to stress and understanding how plants control the types and amounts of flavonoids that are produced in response, aid in the process of isolation of these bioactive flavonoids. Attempts to understand the role of flavonoids in stress protection, as well as in defining the mechanisms that control the amounts and varieties of flavonoids produced in plants, in response to diverse environmental cues still remains elusive [Chalker-Scott, 1999]. Flavonoids having small molecular weight are responsible for the tartness and bitterness of many fruits, whereas larger molecular weight flavonoids especially tannins are responsible for their astringency [Di Carlo et al., 1999]. Investigation of the molecular basis of flavonoid function in reducing stress, along with its contribution in the regulation of biochemical mechanisms and control of the types and amounts of flavonoids synthesized under different conditions, continues to be a high priority for research [Winkel-Shirley, 2002]. Research in the field of flavonoids had increased since the discovery of a new compound isolated from oranges which was believed to be a member of a new class of vitamins (designated as vitamin P), but later was identified to be a flavonoid (rutin). In response to this discovery, intensive research was undertaken to isolate the individual flavonoids and probe their mechanism of action [Nijveldt et al., 2001].

Biosynthesis of Flavonoids Flavonoids are biosynthesized via a combination of the shikimic acid and acylpolymalonate pathways, where cinnamic acid derivative (phenylpropane) acts as a starting compound in polyketide synthesis. Cinnamic acid, synthesized from shikimic acid, following basic substitutions such as hydroxylations and reductions, results in the formation of different classes of flavonoids [Di Carlo et al., 1999]. Flavonoids have a common structure of diphenyl propanes ([A] C6 - [B] C3 - [C] C6), consisting of two aromatic rings linked through three carbons [Gurib-Fakim, 2006] (Figure 2). The various classes of flavonoids are divided based on the connection of the B ring to the C ring; the level of oxidation of the C ring from the basic benzo-γ-pyrone structure, as well as the variation in the number and substitution pattern of the hydroxyl groups and the extent of glycosylation of the heterocyclic rings [Amić et al., 2003], they appear to occur as aglycones, glycosides and methylated derivatives. The flavonoids include flavones, flavonols, flavanols, flavonones, and anthocyanidins [Narayana et al., 2001].

Figure 2. General structure of a flavonoid.



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Subclasses of Flavonoids The subclasses of flavonoids and their sources are discussed in detail (Table 1).

Aglycone Aglycone is a flavonoid consisting of a benzene ring (A) condensed with a six membered ring (C), which in the 2-position carries a phenyl ring (B) as a substituent.

Flavones and Flavonols The flavonoid’s six-membered ring substituted with α-pyrone is classified as flavones and flavonols. Flavones are characterized by a planar structure because of a double bond in the central aromatic ring such as apigenin, luteolin, kaempferol and myricetin [Grotewold 2007]. Flavonols and flavones are the most widely distributed flavonoids, which include quercetin, kaempferol, myricetin, chrysin and apigenin [Winkel-Shirley, 2002]. The main dietary sources of flavonols and flavones include tea and onions [Grotewold, 2007]. Flavonols are the most ancient and widespread flavonoids and exhibit a wide range of potent physiological activity and are even synthesized in mosses and ferns [Winkel-Shirley, 2002]. Among the flavonols, quercetin is the most frequently occurring compound in foods like onions, apples, broccoli, and berries. [Nijveldt et al., 2001].

Flavanols and Flavonones Flavanols and flavonones are the class of flavonoids in which the six-membered ring is a dihydro derivative. Flavanols differ from flavonones with hydroxyl group in the third position and a C2-C3 double bond [Narayana et al., 2001]. The first group, flavanols are termed pycnogenols because they tend to form dimers by condensation of two identical compound; proanthocyanidines are examples of flavanol dimers. [Di Carlo et al., 1999]. Examples of flavanols include narigin, epicatechin and gallocatechin [Nijveldt et al., 2001]. The second group is the flavanones, mostly found in citrus fruits, for example naringenin and hesperidin [Grotewold, 2007].

Isoflavonoids Flavonoids have a second position of the benzenoid substitution, whereas isoflavonoids are the class where benzenoid substitution occurs at third position [Narayana et al., 2001] such as genistein, daidezin and biochanin A [Grotewold, 2007].



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Table 1. Natural sources of Flavonoids (Modified From Narayana et al., 2001; Nijveldt et al., 2001 and Beecher, 2003) Flavonoid Flavonols

Flavonones

Flavones

Flavanols

Flavan-3-o1s Isoflavones Anthocyanins

Examples Kaempferol Morin Rutin Myricetin Quercetin Quercetrin Myricitrin Spirenoside Galangin Robinin Kaempferide Fisetin Hesperitin Naringin Naringenin Eriodictyol Hesperidin Pinocembrin Likvirtin Rpoifolin Apigenin Tangeretin Flavone Baicalein Luteolin Chrysin Techtochrysin Diosmetin Diosmin Silibinin Silymarin Taxifolin Pinobanksin Catechin Genistein Daidzin Cyanidin Delphinidin Malvidin Pelargonidin Peonidin Petunidin

Rich food sources Nearly ubiquitous in foods such as tea, grape, cranberry, olive, onion, tomato etc

Citrus foods

Green leafy spices likeparsley

Teas, red grapes and red wines Tea, grapes Soybeans, soy foods and legumes Red, purple and blue Berries



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Anthocyanin Anthocyanins are closely related to flavonoids with an open C-ring. They are the class of compounds responsible for the red-blue pigments in plants. Anthocyanins have been found to occur as anthocyanins (glycosides) and anthocyanidins (aglycones), both have been found to be water-soluble [Gurib-Fakim, 2006; Di Carlo et al., 1999]. Anthocyanins are found mainly in fruits with red or blue color such as strawberries and other berries, grapes, wine, and tea and their examples include cyanidin and pelargonidin [Nijveldt et al., 2001].

Minor Flavonoids Dihydroflavones and dihydrochalcones have been considered as minor flavonoids because of their limited natural distribution [Di Carlo et al., 1999].

Location of Flavonoids in Plants Stress-responsive dihydroxy B-ring-substituted flavonoids have great potential to inhibit the generation of ROS. These flavonoids are located within or in the proximity of centres of ROS generation in severely stressed plants. Efficient mechanisms have been recently identified for the transport of flavonoids from the endoplasmic reticulum (ER)/ the site of their biosynthesis, to different cellular compartments. The mechanism underlying flavonoidmediated ROS reduction in plants is still unclear [Agatia et al., 2012].

Membrane Flavonoids A number of flavonoids with high in vitro antioxidant activity have been found to be hydrophobic since their biological function is associated with membranes. The solubility of each flavonoid ranges from moderately hydrophobic (luteolin and epigallocatechin) to strongly hydrophilic (flavonol glycosides and anthocyanins) [Hernańdez et al., 2009].

Flavonoids in Chloroplast Chloroplasts are considered a major source of intracellular hydrogen peroxide (H2O2) in photosynthetic plant tissues [Mehler, 1951], suggesting that glycosylated flavonoids play a role in the antioxidant machinery, acting by scavenging singlet oxygen and stabilizing the outer membrane [Zaprometov and Nikolaeva, 2003].

Vacuolar Flavonoids Anthocyanins and proanthocyanins have been found to accumulate in vacuoles, contributing to pigmentation and photoprotection. It is noted that vacuolar flavonoids can



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only exhibit their antioxidant potential by disruption of the physical barrier created by tonoplast, thereby creating contact with cytosolic oxidizing agents [Gould et al., 2002].

Nuclear Flavonoids Flavonoids such as flavonols, flavan-3-ols and chalcones have been detected in plant nuclei and nucleus of mesophyll cells. They contain dihydroxy B-ring substituted flavonoids, which inhibit ROS-generation by making complexes with Iron (Fe) and Copper (Cu) ions [Agatia et al., 2012]. It has been suggested that nuclear flavonoids protect DNA from oxidative damage caused due to ROS, however, no direct antioxidative action of nuclear flavonoids has been reported, instead, they indirectly protect DNA by screening UV radiation and chelating transition metals, consequently preventing the Fenton reaction [Melidou et al., 2005, Polster et al., 2006].

Extracellular Flavonoids Extracellular flavonoids indirectly protect cellular components from photooxidation by acting as UV-light screen [Jordan, 1996]. Flavonoids present in cuticles and epicuticular waxes serve as an antioxidant barrier against oxidizing pollutants, such as ozone (O3) and sulphur dioxide (SO2) [Toma´s-Barberań et al., 1988, Alcerito et al., 2002], however no experimental evidence is yet available.

Biological Activities of Flavonoids Flavonoids exhibit positive impact on human health by modulating many enzyme activities affecting several cellular systems [Di Carlo et al., 1999]. Flavonoids have been evidenced for a wide range of biological activities in humans, suggesting that these compounds exhibit numerous pharmacological activities such as anti-inflammatory, analgesic, anti-tumour, anti-HIV, anti-diarrhoeal, anti-fungal, anti-hepatotoxic, anti-lipolytic, antioxidant, vasodilator, anti-spasmodic , immunostimulant, anti-osteoporotic, anti-ulcerogenic, anti-viral and anti-microbial [Gurib-Fakim, 2006]. Flavonoids have also been found to inhibit the activity of several enzymes such as aldose- reductase and xanthine-oxidase [Di Carlo et al., 1999].

Diabetes and Oxidative Stress Glucose homeostasis represents the balance between intake (glucose absorption from the gut), tissue utilization (glycolysis, pentose phosphate pathway activity, tricarboxylic acid cycle activity, glycogen synthesis) and endogenous production of glucose (glycogenolysis and gluconeogenesis) [Shinji et al., 2009]. Glucose homeostasis is maintained by the highly coordinated interaction of three physiologic processes: insulin secretion, tissue glucose uptake and hepatic glucose production. Perturbation in glucose homeostasis leads to a chronic



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increase in blood glucose concentration, resulting in the heterogeneous metabolic disorder defined as diabetes. Over a period of time, diabetes develops into numerous other metabolic aberrations, resulting in diabetic complications, both vascular (cardiovascular complications, nephropathy, neuropathy, retinopathy and embryopathy) or a-vascular (cataract and glaucoma). The main reason attributed for this effect is that chronic supra-physiological glucose concentration generates free radicals, thus creating an oxidative stress which negatively affects a large number of organs and tissues [King and Loeken, 2004].

Diabetes - Induced Oxidative Stress There are multiple sources of oxidative stress in diabetes including non-enzymatic, enzymatic and mitochondrial pathways (Figure 3).

Figure 3. Hyperglycemia-induced oxidative stress.

Non-enzymatic Non-enzymatic sources of oxidative stress originate from the oxidative biochemistry of glucose. Glucose can undergo autoxidation and generate •OH radicals [Al-Rawi, 2012]. In addition, glucose reacts with proteins in a non-enzymatic manner leading to the development of Amadori products, followed by formation of AGEs with generation of ROS at multiple steps during the process. Once AGEs are formed, they bind to various receptors for AGEs (RAGE), thereby generating ROS [Alison Goldin et al., 2006]. It has also been proposed that carbonyl stress, rather than oxidative stress, involving both sugars and lipids would be the relevant source of oxidative stress in diabetes [Ferdinando et al., 2010]. Under hyperglycemic



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condition, there is an enhanced metabolism of glucose through the polyol (sorbitol) pathway, which also results in enhanced production of •O2-. Superoxides can activate several other damaging pathways in diabetes including accelerated formation of AGEs, polyol pathway, hexosamine pathway and PKC, all of which have been proven to be involved in micro- and macro vascular complications. Mitochondrial respiratory chain is another source of non-enzymatic generation of reactive species. During the oxidative phosphorylation process, electrons are transferred from electron carriers nicotinamide adenine dinucleotide (NADH) and reduced flavin adenine dinucleotide (FADH2), through four complexes in the inner mitochondrial membrane, to oxygen, generating adenosine triphosphate (ATP) in the process [Green et al., 2004]. Excessive levels of glucose leads to an overdrive of the electron transport chain in the mitochondria resulting in overproduction of superoxide anions, thereby creating oxidative stress. Nishikawa et al. (2000) have demonstrated that generation of excess pyruvate via accelerated glycolysis under hyperglycemic conditions floods the mitochondria and causes •O2- generation at the level of Complex II in the respiratory chain. Increased generation of ROS and especially •O2- precedes the activation of four major pathways involved in the development of diabetic complications. It has been postulated that mitochondrial •O2- is the crucial initiator that turns oxidative stress into diabetes by stimulating more ROS and RNS production via downstream activation of PKC, nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase and nuclear factor κB (NF-κB)-mediated cytokine production. Nevertheless, hyperglycemic condition activates production of ROS thereby stimulating the stress related signaling mechanisms such as NFκB, p38-mitogen activated protein kinase (MAPK) and signal transducer and activator of transcription- janus kinase (STAT-JAK) which reduces the expression of antioxidant enzymes by glycation of these proteins [Taniyama and Griendling, 2003].

Enzymatic Sources Enzymatic sources of ROS in diabetes includes nitric oxide synthase (NOS), NAD(P)H oxidase, xanthine oxidase and polyol pathway. All isoforms of NOS require five cofactors/prosthetic groups such as flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, tetrahydrobiopterin (BH4) and Ca2+-calmodulin. In the absence of its substrate L-arginine or its cofactors, NOS may produce •O2- instead of •NO which is referred to as the uncoupled state of NOS [Jeanette et al., 2005]. NAD(P)H oxidase (Nox), is a membrane associated enzyme that consists of five subunits and is a major source of •O2- production by the electron reduction of oxygen using electron donors like NAD(P)H or NADH. There is plausible evidence that PKC is stimulated in diabetes via multiple mechanisms such as polyol pathway and angiotensin II (Ang II), and activates Nox [Amiri et al., 2002]. Enhanced generation of ROS due to increased expression of Nox has been implicated in diabetes and its associated complications like atherosclerosis, hypertension, renal and neural diseases [Singh et al., 2009]. Xanthine oxidase catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid, producing superoxide in both the reactions. The enzyme is derived from xanthine dehydrogenase, and over expression of xanthine oxidase results in increased oxidative stress [Berry and Hare, 2004].



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Polyol pathway is involved in the conversion of glucose to sorbitol. During hyperglycemia, intracellular glucose level rises and glucose is converted to sorbitol by stimulation of enzyme aldose reductase and coenzyme NAD(P)H. Depending upon the severity of hyperglycemia, upto 30% of glucose can be diverted to polyol pathway. Disproportionate diversion of glucose to this pathway leads to production of sorbitol, ROS, AGE and deficiency of glutathione (GSH) [Ferdinando et al., 2010]. Intracellular accumulation of sorbitol is harmful as it causes cell damage, and also potently activates stresssensitive signaling pathways including p38-MAPK and c-jun N-terminal kinase (JNK) which have been proposed to play important role diabetic complications [Newsholme et al., 2007].

Obesity and Oxidative Stress Obesity is a chronic disease of multifactorial origin and can be defined as an increase in the accumulation of body fat. It is a state of chronic oxidative stress that arises due to clustering sources of abnormalities like hyperglycemia, hyperleptinemia, increased tissue lipid levels, inadequate antioxidant defenses, increased rates of free radical formation, enzymatic sources within the endothelium, and chronic inflammation [Vincent and Taylor, 2006]. This chronic oxidative stress may be the mechanism underlying the development of co-morbidities in obesity. High fat diet and adipoctyte secretory proteins play a major role in obesity induced oxidative stress. High fat diet: Chronic hyper-nutrition such as high fat high carbohydrate (HFHC) meals, high dietary saturated fatty acids (SFA) and trans-fatty acids leads to the accumulation of fat in the adipose tissue, inducing oxidative stress through multiple biochemical mechanisms such as superoxide generation from Nox, oxidative phosphorylation, glyceraldehyde autooxidation, PKC activation, polyol and hexosamine pathways [Isabella et al., 2013]. Adipocyte secretory proteins: Adipose tissue is a triglyceride (TG) storage tissue as well as a source for substances with endocrine, paracrine and autocrine action, called the adipokines or adipocytokines (plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNF-α), resistin, leptin, and adiponectin). Adipokines play a role in the homeostasis of various physiological processes. These adipokines secreted by the adipose tissues induce production of ROS, leading to oxidative stress [Fernández et al., 2011]. Hence, adipose tissue is considered as an independent factor for the generation of systemic oxidative stress. However, oxidative stress can be a consequence and also a trigger of obesity by increasing the pre-adipocyte proliferation, adipocyte differentiation and size of mature adipocytes [Sonoli et al., 2011; Barth et al., 2010 and Kluth et al., 2011]. The generated ROS controls body weight by exerting different effects on hypothalamic neurons that control satiety and hunger behaviour [Esposito et al., 2006]. Other factors that contribute to oxidative stress in obesity are abnormal post-prandial ROS generation [Chrysohoou et al., 2007], hyperleptinemia [Hartwich et al., 2007], chronic inflammation [Patel et al., 2007], tissue dysfunction [Steppan and Lazar, 2004], and low antioxidant defenses [Block et al., 2002 and Keaney et al., 2003].


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Mechanisms of Obesity - Induced Oxidative Stress There are several mechanisms by which obesity induces oxidative stress through ROS generation (Figure 4).

Figure 4. Obesity induced oxidative stress.

Fat cell accumulation: During the state of obesity, adipocytes are unable to function as an energy storage organ, due to excessive fat accumulation resulting in lipotoxicity. Intracellular triglycerides inhibit the adenosine nucleotide translocator (ANT), leading to ATP accumulation in mitochondria. The mitochondrial adenosine diphosphate (ADP) drop, reduces the speed of oxidative phosphorylation and mitochondrial uncoupling, promoting electron leakage and free radical release. Free radical release triggers oxidative stress leading to mitochondrial DNA damage, mitochondrial dysfunction along with ER stress characterized by impaired protein folding, lipid droplet creation and hepatic cholesterol accumulation. During ER stress, mis-folded proteins activate the unfolded protein response (UPR) that is responsible for ER biogenesis, protein folding and degradation of aberrantly packaged



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proteins. If UPR is prolonged, the persistent oxidative protein folding machinery causes ROS production with subsequent systemic release of free fatty acids and inflammatory mediators. Similarly, over-consumption of oxygen by the fat cells generates free radicals in the mitochondrial respiratory chain that is found coupled with oxidative phosphorylation in mitochondria [Isabella et al., 2013]. Hyperleptinemia: Mitochondrial and peroxisomal oxidation of fatty acids also induces ROS generation. Leptin is a hormone produced by adipose tissue, which regulates appetite and exerts protective effects against lipotoxicity in non-adipose tissues. Hyperleptinemia occurs during obesity and induces oxidative stress, mainly by increasing mitochondrial and peroxisomal fatty acid oxidation. Hyperleptinemia stimulates proliferation and activation of monocytes/macrophages alongwith production of interleukin 6 (IL-6) and TNF-α [Hartwich et al., 2007]. Antioxidant enzyme depletion: Upon increase of adipose tissue, the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), are significantly diminished. Finally, high ROS production and decrease in antioxidant capacity leads to various abnormalities, among which endothelial dysfunction is characterized by a reduction in the bioavailability of vasodilators, particularly NO, and an increase in endothelium-derived contractile factors causing atherosclerotic disease [Amirkhizi et al., 2007]. Lipid peroxidation: Lipid peroxidation during obesity is another major cause of oxidative stress. Fatty acid accumulation during obesity leads to an excessive generation of free radicals. The generated free radicals create a stressful environment and readily react with lipids in the cell membrane forming lipid peroxide that causes oxidative degradation of lipids. Lipid peroxidation leads to an elevation in the formation of end products such as malondialdehyde, hydroperoxides, 4-hydroxynonenal, isoprostanes and conjugated dienes. Lipid peroxidation is associated with several indices of adiposity and a low systemic antioxidant defense (antioxidant enzymes, tissue dietary antioxidants, GSH) [Isabella et al., 2013]. Similarly, ROS can stimulate oxidation of low-density lipoprotein (LDL) and oxidized LDL, which are not recognized by the LDL receptor, can be taken up by scavenger receptors in macrophages leading to foam cell formation and atherosclerotic plaques [Dorien et al., 2007]. Protein oxidation: Advanced oxidation protein products (AOPP) are recognized as markers of oxidative damage to proteins during oxidative stress. They are derived from oxidation-modified albumin, fibrinogen and lipoproteins. Oxidative stress is the main element in this modification and the most significant is the myeloperoxidase/ H2O2/ halide system. AOPP have their own particular biological proprieties, similar to those of AGEs, and also bind to the same receptor, i.e. RAGE. Physiologically, AOPP are formed during the whole life in small quantities and increase with age. Significant elevation of 8-hydroxydeoxyguanosine, an AOPP occurs during obesity induced oxidative stress [Piwowar, 2010].


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Antioxidants Antioxidants are substances that interact with and stabilize free radicals thus protecting the cells from damage. The antioxidants may be exogenous or endogenous in nature. The endogenous antioxidants can be classified as enzymatic and non-enzymatic. Enzymatic antioxidants include SOD, CAT, GPx, glutathione reductase (GRx), which prevent the transformation of ROS, thus converting them into stable molecules like water and molecular oxygen [Gupta and Sharma, 2006]. The non-enzymatic antioxidants can be divided into metabolic antioxidants and nutrient antioxidants. Metabolic antioxidants like lipoic acid, GSH, L-arginine, uric acid, bilirubin among others maintain the antioxidant equilibrium by primarily acting as cofactors for the antioxidant enzymes. Another major source of antioxidants, are the nutrient antioxidants belonging to exogenous antioxidants, and comprising of compounds that can be taken as supplements through foods such as vitamin E, vitamin C, carotenoids and trace elements like Selenium (Se), Copper (Cu), Zinc (Zn), Manganese (Mn) which play a crucial role by preventing lipid peroxidation damage [Ashok et al., 2012]. Antioxidants can be classified into first line of defense, second line of defense and third line of defense antioxidants [Gupta and Sharma, 2006]. First line of defense antioxidants includes SOD, CAT, GPx, GRx and some trace elements like Se, Cu, Zn and Mn. SOD acts by quenching superoxides and catalase functions by catalysing the conversion of H2O2 to water and molecular oxygen. Selenium and vitamin E efficiently scavenge the peroxides from cytosol and cell membrane. Copper exerts its activity with cytosolic SOD and Zn exhibits its activity through alcohol dehydrogenase, alkaline phosphatase and carbonic anhydrase. Second line of defense includes glutathione, uric acid, bilirubin, vitamin E, carotenoids, flavonoids, albumin and vitamin C. GSH is a good scavenger of superoxides, hydroxyl radicals and lipid peroxides. β-carotene is an excellent scavenger of singlet oxygen. Vitamin C directly interacts with superoxides and hydroxyl radicals. Vitamin E scavenges peroxyl radicals, the intermediates in lipid peroxidation and is responsible for protecting polyunsaturated fatty acids (PUFA) and low density lipoprotein (LDL) against lipid peroxidation. Flavonoids are phenolic compounds from plants that inhibits lipid peroxidation and lipoxygenases. Vitamin C and E helps to minimize the consequences of lipid peroxidation by binding transition metal ions like Cu and Fe, thereby inhibiting free radical stimulation. The third line of antioxidant enzymes includes lipases, proteases, transferases, DNA repair enzymes, methionine sulphoxide reductases (MSRA) among others. They are a complex group of enzymes that repair the damaged DNA, proteins, oxidized lipids and peroxides. They also play vital role in stopping the chain propagation of peroxyl lipid radicals.

Antioxidant Flavonoids with Anti-diabetic Activities Bio-flavonoids are well-known for their multi-directional biological activities. Their potential role in the treatment of diabetes has become the focus of investigation in recent times due to their remarkable health benefits. Flavonoids have been reported to exert anti-



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diabetic effects through their capacity to avoid glucose absorption or to improve glucose tolerance [Jung et al., 2006]. It has also been demonstrated that flavonoids can act as insulin secretagogues or insulin mimetics, probably by influencing the pleiotropic mechanisms to attenuate the diabetic complications. Flavonoid drug candidates have also been found to stimulate glucose uptake in peripheral tissues and regulate the activity of the rate-limiting enzymes involved in carbohydrate metabolism pathway [Matsui et al., 2006; Brahmachari, 2006; Brahmachari and Gorai, 2006a and 2006b; Brahmachari, 2009 and Qi et al., 2010].

Anti-hyperglycemic Effect in Diabetic Rats It has been demonstrated that various flavonoids including chrysin and its derivatives, prunin (naringenin 7-O- β -D-glucoside), silymarin, isoquercetrin and rutin showed significant anti-hyperglycemic effects on diabetic rats [Choi et al., 1991; Shin et al., 1999; Velussi et al., 1997; Hnatyszyn et al., 2002]. Similarly, isoorientin isolated from Cecropia obtusifolia [Andrade-Cetto and Wiedenfeld, 2001] and flavonoid glycosides from Phyllanthus fracternus [Hukeri and Kalyani, 1988] exhibit potent hypoglycemic activity in diabetic and alloxanised rats respectively. Coutareagenin (5-hydroxy-7-methoxy-4-(3,4-dihdroxyphenyl)2H-benzo-1- pyran-2-one), a neoflavonoid from the bark of Hintonia latiflora has been reported to exhibit promising anti-diabetic efficacy in menopausal diabetic women [Korec et al., 2000]. Kaempferol-3,7-O-(α)-dirhamnopyranoside (kaempferitrin) [De Sousa et al., 2004] and its structurally similar derivative Kaempferol- 3-neohesperidoside, a glycosylated flavonoid along with its Vanadium complex (kaempferol-3-neohesperidoside-VO(IV) complex) exhibits significant hypoglycemic effect in normal and alloxan-induced diabetic rats [Cazarolli et al., 2006 and Shukla et al., 2004]. Similarly, quercetin showed promising antidiabetic activity in streptozotocin (STZ)-diabetic rats when treated individually or when complexed with vanadium [Vessal et al., 2003]. Flavones such as 4',5-dihyroxy-6,7dimethoxyflavone-3-O-β-D-xylopyranoside (xylopyranoside), [1′′(R)-5,4′,1′′-trihydroxy-6,7(3′′,3′′- dimethylchromano) flavone and flavanone (2S)-4′-O-methyl-6- methyl-8prenylnaringenin were found to possess promising anti-hyperglycemic activity by decreasing glucose level of STZ- induced diabetic rats. Apigenin-6-C-β-L-fucopyranoside and apigenin6-C-(2′′-O- α -L-rhamnopyranosyl)- β -L-fucopyranoside were observed to stimulate glucoseinduced insulin secretion in hyperglycemic rats [Brahmachari, 2011].

Aldose Reductase Inhibitory Activity Aldose reductase is an enzyme that catalyzes NAD(P)H-dependent conversion of glucose to sorbitol in polyol pathway of glucose metabolism. Excessive conversion of glucose to sorbitol leads to several diabetic complications andinhibition of this enzyme aids in the protection of micro complications associated with diabetes. Several flavonoids known for their antioxidant potential have been found to be effective in the inhibition of aldose reductase. Myrciacitrins isolated from Myrcia multiflora [Matsuda et al., 2002], C-glucosidic flavone derivative named as isoaffineyin (5,7,4,3′,5′-pentahydroxyflavone-6-C-glucoside) from Manikara indica [Haraguichi et al., 2003], flavonol glycoside, quercetin 3-O-α-Larabinopyranosyl-β-D-glucopyranoside along with the known flavonoid glycosides such as



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kaempferol 3-O-β-D-glucopyranoside exerted promising inhibition of porcine lens aldose reductase activity [Kim et al., 2004]. Likewise, engeletin, astilbin (dihydroflavonol glycosides), isorhamnetin 3-O-β-D-glucoside also possesses significant inhibitory activity against rat lens aldose reductase (RLAR) in vitro [Wirasathien et al., 2007 and Lee et al., 2009]. Matsuda et al. (2002) demonstrated that the flavone constituents, 3′,4′dihydroxyflavone, 3′,4′,7-trihydroxyflavone, luteolin, luteolin 7-O- β -D-glucopyranoside and the flavonoid glycosides, quercitrin, guaijaverin and desmanthin-1 exhibited potential aldose reductase inhibitory activity.

Glycosidase Enzyme Inhibitory Activity The membrane-bound intestinal α-glucosidases hydrolyze oligosaccharides, trisaccharides and disaccharides to glucose and other monosaccharides in the small intestine. Hence, as a therapeutic measure for diabetes, α-glucosidase inhibitors are in demand to reduce the impact of carbohydrates on blood sugar. Certain flavonoids and its derivatives have been reported to exhibit α-glucosidase inhibitory activity. Hydroxy flavonoids such as 6hydroxyapigenin (scutellarein), 6-hydroxyapigenin-7-O-β-D-glucopyranoside,6-hydroxyluteolin-7-O- β -D-glucopyranoside, feruloylglucosides such as 6-hydroxyapigenin-7-O-(6-Oferuloyl)-β-D-glucopyranoside,6-hydroxyluteolin-7-O-(6-O-feruloyl)-β-D-glucopyranoside isolated from Origanum majorana and flavonoid 6-hydroxyluteolin and 5,6,7trihydroxyflavone (baicalein) from Scutellaria baicalensis showed rat intestinal α-glucosidase inhibitory activity [Brahmachari, 2011]. Similarly, it has been reported that prenylated flavonols isolated from the roots of Dorstenia psilurus were found to exhibit glycosidase enzyme inhibitory activity against α-glucosidase, β-glucosidase, and α-mannosidase [Kawabata et al., 2003; Harborne and Williams, 1971; Miyaichi et al., 1989; Ravn et al., 1990; Ulubelen et al., 1980; Ranganathan et al., 1980; Harborne, 1967 and Nishioka et al., 1998].

Insulin Signaling Genistein derivatives significantly stimulated the uptake of glucose through adenosine monophosphate-activated kinase (AMPK), glucose transporter protein 4 (GLUT4) and glucose transporter protein 1 (GLUT1) pathway and also exhibited inhibition of protein tyrosine phosphatase 1B (PTP1B) in L6 myotubes, thereby exhibiting promising anti-diabetic activity [Lee et al., 2009]. Flavonoid apigenin-6-C- β -L-fucopyranoside stimulated glycogen synthesis in rat soleus muscle through insulin signal transduction. Kaempferol-3neohesperidoside stimulated glycogen synthesis in rat soleus muscle through phosphatidylinositol-3-kinase (PI3K) - glycogen synthase kinase 3 (GSK 3) pathway and mitogen-activated protein kinase/extracellular signal regulated kinases (MEK) - protein phosphatase-1 (PP-1) pathway. Epigallocatechin 3-gallate enhances tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), MAPK, p70s6k, and PI3K activity, and reduces phosphoenolpyruvate carboxykinase gene expression through PI3K [Brahmachari, 2011].



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Glycation Inhibitors Glycation is the process of covalent bonding of a protein or lipid molecule with a sugar molecule without the controlling action of an enzyme. Glycation is the first step in the evolution of the sugar molecules through a complex series of very slow reactions in the body including Amadori reactions, Schiff base reactions, and Maillard reactions which lead to the formation of AGEs. Some AGEs are non-reactive whereas others are more reactive than the sugars they are derived from, by releasing highly oxidizing products such as H2O2, thereby impairing the functioning of biomolecules, and also been implicated in many age-related chronic diseases. Flavonoids have been found to be very effective in inhibiting the formation of advanced glycation products by acting as glycation inhibitors. Flavonoids such as astragalin, quercetin, 3-O- β -D-glucopyranoside (isoquercetin) from the leaves of Eucommia ulmoides were found to be acting as glycation inhibitors [Kim et al., 2004]. Luteolin 6-C-(6′′O-trans-caffeoylglucoside) from Phyllostachys nigra [Jung et al., 2007] and two flavan-3-ol derivatives from Actinidia arguta showed inhibitory efficacy against AGEs [Jang et al., 2009]. Also, few more AGE inhibitors such as the dihydroflavonol glycosides [Wirasathien et al., 2007], isoflavone C-glucosides and the 2,3-dioxygenated flavanone (erigeroflavanone) isolated from Pueraria lobata and Erigeron annuus have also been reported [Kim et al., 2006 and Yoo et al., 2008]. Lipid Peroxidation Inhibitors Flavonoids serve as potent inhibitors of lipid peroxidation process by scavenging free radicals, protecting LDL associated antioxidants, α-tocopherol and carotenoids from oxidation, regeneration of vitamin E from oxidized α-tocopherol, chelation of transition metal ions and protection of cells against oxidative damage by inhibiting xanthine oxidase, NAD(P)H oxidase or lipoxygenase. The flavonol catechin prevented plasma lipid peroxidation and also inhibited LDL oxidation induced by copper ions [Aviram and Fuhrman, 1998]. Flavonol, quercetin, rutin, luteolin also inhibited copper induced LDL oxidation more effectively than kaempferol by chelating copper ions. Likewise, other flavonoids that inhibit LDL oxidation are morin, fisetin, gossypetin and hydroxy cinnamic acid derived phenolic acids like caffeic, ferulic, p-coumaric acid and isoflavan glabridin. Flavonoids are suitable for protecting cell membrane from free radical induced oxidative damage, as they were both lipophilic and hydrophilic, resulting in reduced cell mediated oxidation by LDL [Rice-Evans and Packer, 2006]. Licochalcone B and D from Glycyrrhiza inflata inhibited superoxide production in xanthine/xanthine oxidase system. It also inhibited mitochondrial lipid peroxidation by Fe (III) ADP/NADH and protected red blood cells against oxidative haemolysis. Antioxidants isolated from licorice include isoflavans glabridin, hispaglabridin A,B, 4-O-methyl glabridin and two chalcones isoprenyl chalcone and isolipuritegenin. Glabridin inhibits 2,2'-azobis-2-methyl-propanimidamide,dihydrochloride (AAPH) and copper induced LDL oxidation, inhibited the formation of aldehydes, lipid peroxides and oxysterols. It also inhibited the consumption of β-carotene and lycopene in the presence of LDL oxidation but could not protect vitamin E from oxidation. It inhibited PKC required for p47 phosphorylation, the primary event in the inhibition of NAD(P)H oxidase induced


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macrophage mediated oxidation of LDL. Other flavonoids like epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin and gallic acid show an inhibition of LDL oxidation [Rice-Evans and Packer, 2006].

Pharmacokinetics of Flavonoids Limited data is available on the amount of flavonoids absorbed by human, however, studies conducted on animal models have shown that flavonoids bound to p-glycosides are non-absorbable, whereas only aglycones, without a sugar molecule can pass through the gut wall. It has been observed that only in the colon, hydrolysis of l3-glycosidic bonds occurs by micro-organisms which degrade dietary flavonoids. There are no reports of any enzymes capable of splitting the bond present or secreted into the gut. After absorption, flavonoids are metabolized primarily in liver [Hackett, 1986], nevertheless intestinal wall and kidney are considered as the secondary sites of metabolism. Overall, the pharmacokinetics depends on the origin of flavonoids. It has been observed that flavonoids in citrus fruits, are poorly metabolized by the intestinal microflora. Quercetin is not absorbed in human and rutin is poorly absorbed, whereas procyanido lignanes are readily absorbed in mice. Flavonoids which are metabolized by intestinal bacteria are converted to hormone-like compounds.The microorganisms in the colon hydrolyze glucuronides and sulphates, which then most probably enable absorption of the liberated aglycones. Flavonoids, once absorbed, influence many biological functions, making them beneficial in a variety of human disorders [Di Carlo et al., 1999].

Conclusion Metabolic disorders are an epidemic condition progressing rapidly in developing countries thus attracting concern. The main factor associated with metabolic disorders such as diabetes or obesity is the oxidative stress involving surplus release of free radicals combined with a disturbed antioxidant status. Since, the metabolic disorders are either a consequence or an initiator of oxidative stress, therapeutic strategies aimed to counter them should be multifunctional, possessing both antioxidant and anti-diabetic/ anti-obesity activities. In recent times, bioactive principle based treatment approaches have gained greater significance because of their remarkable beneficial effects especially in their multifunctional activities. Hence, therapeutic approaches using natural sources mainly from plants have increased. Among the several phytoactive constituents, polyphenols are the front runners of antioxidant activity. Flavonoids, are naturally occurring phenolic compounds with a broad range of biological activities such as anti-hyperglycemic, activators of insulin signaling and inhibition of intestinal α -glucosidase enzyme, aldose reductase activity, lipid peroxidation and glycation. As these bioflavonoids are multifunctional (antioxidant and anti-diabetic/antiobesity) and represent an unparalleled source of molecular diversity, their therapeutic role as drug candidates for the treatment of metabolic disorders could be defined in relation to the drug discovery process.



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Chapter 5

Use of Antioxidants to Control Obesity and Promote Weight Loss Vandana Gulati*, Pankaj Gulati and Enzo A. Palombo Environment and Biotechnology Centre, Faculty of Life and Social Sciences, Swinburne University of Technology, Hawthorn, Victoria, Australia

Abstract The prevalence of overweight and obese individuals is increasing at an alarming rate across the globe. Obesity has become one of the most important avoidable risk factors for morbidity and mortality. The associated risks with obesity are cancer, diabetes and heart diseases. According to the World Health Organization, obesity is defined as abnormal or excessive fat accumulation that may impair health. In 2008, more than 1.4 billion adults were overweight and more than half a billion were obese. At least 2.8 million people die each year as a result of being overweight or obese. A person is considered obese if they possess a body mass index (BMI; a ratio of height to weight) greater than 30 whereas a healthy BMI should be 18.5 to 24.9. Obesity is the leading cause of death which can be prevented by diet and lifestyle modifications. Although the exact link between obesity and its associated risks is not clear, it is known that increased production of reactive oxygen species (ROS) is associated with cellular damage, including oxidation of cell membranes and proteins in conjunction with disturbances of cellular redox homeostasis. Free radicals are known to be involved in a number of human pathologies including atherosclerosis, cancer and hypertension. Studies have shown that obesity promotes increased plasma lipid peroxidation. Obesity also increases the mechanical and metabolic loads on the myocardium, thus increasing myocardial oxygen consumption. Therefore, antioxidants are capable of reversing these pathways and, in fact, can be helpful in preventing the deleterious effects caused by reactive oxygen species. However, antioxidants do not reduce obesity per se. Antioxidants are widely present in the plant kingdom and are known to prevent various disorders. Flavonoids, especially flavones, flavonols, flavanones, flavanols (catechins), anthocyanins, isoflavones and chalcones, are considered effective antioxidants associated with other pharmacological properties such as anti-cancer, anti-diabetic, anti-mutagenic, *



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Vandana Gulati, Pankaj Gulati and Enzo A. Palombo anti-thrombotic, anti-inflammatory and anti-HIV activities. Many studies have indicated that phenolic compounds such as o-coumaric acid, EGCG, esculetin, genistein, procyanidin, pycnogenol, rutin, and tea catechins, carnitine, CoQ10, choline, inositol and various herbs are effective in reducing obesity and promoting weight loss. This review will focus on recent examples of antioxidant nutrients, traditional medicines and foods that have been validated by scientific evaluation for controlling obesity or promoting weight loss.

Keywords: Medicinal plants, antioxidant, anti-obesity and polyphenols

Introduction The word ‘obese’ originates from the Latin word obesus, which means to be ‘stout, fat or plump.’ Obesity develops when energy intake exceeds expenditure. The excess energy is stored in adipose tissues. The balance between energy intake and expenditure is influenced by a complex interplay of genetic, environmental and social factors. Progression to obesity may develop to a stage where some signals trigger cellular and biochemical events that lead to insulin resistance and metabolic syndrome (Achike et al. 2011). Body mass index (BMI) is a simple index of weight-for-height that is commonly used to classify overweightness and obesity in adults. Globally, overweightness and obesity are the fifth leading risk for death. According to World Health Organisation data, at least 2.8 million adults die each year as a result of being overweight or obese. In 2011, more than 40 million children under the age of five were overweight. Obesity was once considered a problem of high-income nations, but it is now on the rise in low- and middle-income countries. More than 30 million overweight children are living in developing countries and 10 million in developed countries (WHO 2013). A strong link has been found between increased oxidative stress in accumulated fat and the pathogenic mechanism of obesity and obesity-associated metabolic disorder. It has also been reported that obesity is a strong independent predictor of systemic oxidative stress which may be the source of several metabolic dysfunctions such as inflammation, hypertension, and impaired glucose intake in muscle and fat related to obesity. Therefore, dietary factors having potential antioxidant effect would offer an effective and beneficial strategy to reduce oxidative stress and its related complications (Gaillet et al. 2012). Excess body fat reduces the quality of life, increases healthcare-associated costs and increases the risk of death due to several associated disorders. Genetic predisposition, an inactive lifestyle and high caloric intake lead to obesity. Therefore, lifestyle changes affecting dietary habits and physical activity are essential to promote weight loss (Moro et al. 2000). Several reports indicate that low-carbohydrate diets are effective in producing rapid weight loss and beneficial metabolic changes. However, the lack of long-term studies and reports of some undesirable effects, such as increased levels of ketone bodies, high losses of body water, headache, constipation and, especially, difficulties in maintaining weight loss after the diet, make it difficult to recommend these diets as a healthy option for weight loss (Strychar 2006). A review of the scientific evidence stated that an ideal diet may be the one containing moderate protein content (30%), high monounsaturated and omega-3 fatty acids, lowglycemic index carbohydrates (40%), and adequate quantities of fibers, isoflavones, calcium


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and antioxidant minerals. Since adherence to healthy dietary patterns can be difficult, meal replacement and dietary supplements should be considered as effective strategies for weight loss, weight maintenance and treatment of metabolic syndrome (Abete et al. 2010). Adipocytes are the primary site of energy storage and these accumulate triacylglycerol that results from an energy imbalance (Figure 1). It has been reported that adipocyte dysfunction plays an important role in the development of obesity which occurs when adipocytes accumulate a large amount of fat and become enlarged. It is characterized at the cellular level by an increase in the number and size of adipocytes differentiated from preadipocytes in adipose tissue (Hsu et al. 2008).

Figure 1. Adipocyte life-cycle: Mesenchymal stem cells are the precursors of several different types of cells, including myoblasts, chondroblasts, osteoblasts and preadipocytes. Once preadipocytes are triggered to mature, they begin to change shape and undergo cell division known as clonal expansion, followed by initiation of the genetic program that allows them to synthesize and store triglycerides. Mature adipocytes can continue storing lipid when energy intake exceeds output, and they can mobilize and oxidize lipid when energy output exceeds input. Mature adipocytes can also undergo apoptotic cell death under certain conditions, modified from Rayalam et al (Rayalam et al. 2008).

Many studies have shown that polyphenolic antioxidants such as EGCG, genistein, esculetin, gallic acid, quercetin and naringenin suppress the adipocyte differentiation process and induce apoptosis in 3T3-L1 adipocytes through activation of AMP-activated protein kinase (Figure 2) (Lin et al. 2005; Rayalam et al. 2008).


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Vandana Gulati, Pankaj Gulati and Enzo A. Palombo

Figure 2. Effect of selected natural compounds on the different stages of the adipocyte life-cycle: Genistein, resveratrol and quercetin inhibit preadipocyte proliferation and EGCG, quercetin and c-lipoic acid suppress lipid accumulation in maturing preadipocytes. They also trigger lipolysis and induce apoptosis in mature adipocytes. EGCG induces apoptosis in both preadipocytes and mature adipocytes, and it can inhibit lipid accumulation in maturing preadipocytes. Quercetin also has multiple effects: it can inhibit preadipocyte proliferation, induce preadipocyte apoptosis and stimulate lipolysis in mature adipocytes. Ajoene + CLA are especially potent in inducing apoptosis in mature adipocytes, modified from Rayalam et al (Rayalam et al. 2008).

If reduction in caloric intake is properly maintained, it can be effective for both prevention and reversal of adiposity and its associated health consequences. Diets focused on particular macronutrient intakes such as very low fat diet, inclusion of antioxidants and diets rich in fibre would be effective. Randomized trials have shown that specific dietary factors impact numerous established and novel cardiovascular and obesity-related risk factors; some of these dietary factors may be involved in reversing adipocyte changes. Also, in rodent models of obesity, a diet high in marine omega-3 fatty acids reduced adipocyte hypertrophy (de Ferranti et al. 2008). Adipose tissue has the capacity to directly trigger endothelial dysfunction by secreting a variety of molecules, such as pro-inflammatory cytokines and leptin, which can mediate the release of C-reactive protein (CRP) from the liver and also impair endothelial function (Brook et al. 2004). Antioxidant enriched diets could be applied in nutritional therapy of obesity by increasing the health benefits related to weight loss and protection against a free radical attack, which will further decrease the risk of coronary heart disease. Fruits are often considered as healthy foods because they contain a variety of compounds with antioxidant capacity, such as vitamins C and E, carotenoids, flavonoids, and polyphenols, which may produce beneficial actions. The ability of two hypo-caloric diets with different fruit contents



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to improve antioxidant biomarkers related to lipid peroxidation in fifteen obese women was estimated and a significant decrease in Low-density lipoprotein (LDL) cholesterol levels in obese women was observed who followed the high fruit diet (Crujeiras et al. 2006).

Factors Affecting Obesity Genetic and Environmental Factors: 40% to 80% of the variance of BMI can be attributed to genetic factors. It is estimated that heredity accounts for factors relevant to energy balance such as body fat distribution, resting metabolic rate, energy expenditure after overeating, lipoprotein lipase activity and basal rates of lipolysis. Over 250 genetic markers have been described in association with obesity-related variables in humans (e.g., BMI, skin-fold thickness, waist-to-hip ratio, fat mass, and percent fat mass) (George et al. 2011). Endocrine and Metabolic Factors: Both endocrine and metabolic factors contribute to obesity. The hypothalamus may influence caloric balance due to actions on feeding through effects on the neuroendocrine system involved with appetite and behavior, through effects on energy expenditure and hormone secretion through effects on secretion of growth hormone, thyroid-related hormones, cortisol, insulin and sex steroids. Thus, decreased leptin/insulin activity in the central nervous system (CNS) may promote obesity through increased caloric balance and weight gain (Bays 2004). Psychological Factors: A few causative personality characteristics are related to obesity and research evidence strongly suggests that obesity is not a unitary syndrome. Instead, it appears to be the end result of a complex interaction within and between both physical and psychological factors (George et al. 2011). Food intake: Some people tend to eat more during periods of heavy exercise, during pregnancy or during depressive episodes and are unable to re-establish their former eating habits. The increase in obesity can usually be related to the type of food consumed, such as increased intake of sugar and fat (George et al. 2011). Circadian Rhythm: A number of studies have provided support for a link between the altered sleep/wake patterns associated with 24-hour lifestyle and obesity. Studies have also reported that obese patients are sleepier during the day and more likely to experience disturbed sleep at night compared with normal weight controls. Various disease states in humans result from alterations in circadian patterns of metabolism. Numerous environmental influences also promote adipocyte proliferation and differentiation. These studies suggest that the circadian clock within the adipocyte may also be a potential regulator of triglyceride metabolism and that impairment of this molecular mechanism may contribute towards adiposity (Bray et al. 2007). Energy expenditure and thermogenesis: Basal metabolic rate (BMR) in obese subjects is higher than in lean subjects, which is not surprising since obesity is associated with an increase in lean body mass. Obese patients tend to expend more energy during physical activity as they have a larger mass to move. On the other hand, many obese patients decrease their amount of physical activity. The energy expended on walking at 3 miles per hour is only 15.5 kJ/min (3.7 kcal/min) and, therefore, increasing exercise plays only a small part in losing weight (George et al. 2011).


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Vandana Gulati, Pankaj Gulati and Enzo A. Palombo Control of Appetite: Appetite is the desire to eat and this usually initiates food intake. Following a meal, cholecystokinin (CCK), bombesin, glucagons-like peptide 1 (GLP1), enterostatin and somatostatin are released from the small intestine and glucagons and insulin from the pancreas. All of these hormones have been implicated in the control of satiety and, therefore, are considered as the most promising antiobesity targets (Bays 2004).

The ideal anti-obesity drug would produce sustained weight loss with minimal side effects. Various anti-obesity drugs were launched into the market but withdrawn due to unacceptable side effects (Table 1). A recent review by Rodgers concluded that “the history of anti-obesity drug development is far from glorious, with transient magic bullets and only a handful of agents currently licensed for clinical use” (Rodgers et al. 2010). Table 1. Currently available anti-obesity drugs* Drug name

Mechanism of Action

Side effects

Dextroamphetamine

Appetite suppressant

Tachycardia and hypertension

Sibutramine

Noradrenaline and 5-HT uptake inhibitor

Increase in blood pressure and heart rate

Structure

NH2

N

Cl

Orlistat

Lipase inhibitor

O

Fecal urgency, oily stools, abdominal pain, flatus

H3C

O

(H2C)10 CH3

O

H3C

H H

H O H

CH3

NH O H

* Reference: Bays (2004).

The mechanisms that regulate energy balance have substantial built-in redundancy, overlap considerably with other physiological functions and are influenced by social and psychological factors which limit the effectiveness of pharmacological interventions. The drugs which target metabolic tissue pathways, such as adipocytes, liver and skeletal muscle, have shown potential in preclinical studies but none of them has yet reached clinical



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development. Recent improvements in the understanding of peptidergic signalling of hunger and satiety from the gastrointestinal tract mediated by ghrelin, cholecystokinin (CCK), peptide Y Y (PY Y) and glucagon-like peptide-1 (GLP-1), and of homeostatic mechanisms related to leptin and its upstream pathways in the hypothalamus, have opened up new possibilities (Rodgers et al. 2012). The pharmacological management of obesity is at an exciting crossroads. For agents that meet preliminary requirements for selectivity of action and potential safety profile, extensive real-world testing is required by regulators for efficacy in terms of weight loss as well as long-term benefits for prevention and treatment. Successful discovery and development of potent and safe drugs for the prevention and treatment of obesity will probably require polytherapeutic strategies (Halford et al. 2010). Current treatments for obesity have not been successful in maintaining long-term weight loss, demonstrating the urgent need for new insight into mechanisms that may lead to obesity and altered metabolism. Therefore, more emphasis should be given on nutrients and botanicals showing antioxidant effects and they may provide a safe and more effective remedy for obesity.

Nutritional Therapies in Weight Management Selenium (Se): Selenium is a cofactor for glutathione peroxidase which is required for the reduction of peroxides thus helps in reducing oxidative stress (Lubos et al. 2011). A randomized trial was conducted with 37 morbidly obese women. The participants consumed one Brazil nut, which is a good source of selenium (providing approximately 290 mg of Se a day), for 8 weeks. Blood tests showed that consumption of one Brazil nut daily effectively increased Se status and resulted in increased glutathione peroxidase activity in obese women (Cominetti et al. 2011). Calcium (Ca): Dietary Ca appears to play a pivotal role in the regulation of energy metabolism and obesity risk. Zemel and co-workers observed that patients in the highest quartile of adiposity were negatively associated with Ca and dairy product intake (Zemel 2004). A more recent nutritional intervention trial also demonstrated that higher low-fat dairy intake among overweight type-2 diabetic patients on isocaloric-restricted regimens enhanced the weight-loss process. The proposed mechanisms are primarily mediated by circulating calcitriol. The increased calcitriol produced in response to low-Ca diets stimulates adipocyte Ca influx and consequently, promotes adiposity, while higher Ca diets inhibit lipogenesis, stimulate lipolysis, lipid oxidation and thermogenesis and inhibit diet-induced obesity in mice. Moreover, a published meta-analysis concluded that dietary Ca has the potential to increase fecal fat excretion, which could be relevant for preventing weight (re)gain. However, some investigators did not find dietary Ca enrichment to have beneficial effects during a weight-loss process. Thus, the effect of Ca on weight loss continues to be unclear, indicating that more long-term studies are required (Abete et al. 2010). Another study also investigated the effect of Ca and vitamin D supplemented orange juice on weight loss and reduction of visceral adipose tissue in overweight and obese adults. Two parallel, double-blind, placebo-controlled trials were conducted with either regular or reduced-energy (lite) orange juice for 16-weeks with 171 participants. The treatment groups


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consumed three 240-mL glasses of Orange juice (regular or lite) fortified with 350 mg Ca and 100 IU vitamin D per serving, and the control groups consumed either unfortified regular or lite orange juice. Computed tomography scans of visceral adipose tissue and subcutaneous adipose tissue were performed. The results suggested that, in overweight and obese adults, a moderate reduction in energy intake and supplementation of Ca and vitamin D in juice beverages led to a beneficial reduction in intra-abdominal fat (Rosenblum et al. 2012). Magnesium (Mg): Mg is a cofactor for more than 300 enzymes involved in bioenergetics, protein phosphorylation, glutathione production and synthesis of cyclic adenosine monophosphate (cAMP). It also affects the structure and function of nucleic acids, cell membranes and ion channels. The Mg content of food is greatly reduced by processing. Strong epidemiologic and mechanistic data support a role for Mg deficiency in the genesis of insulin resistance and metabolic syndrome. Deficiency of this mineral contributes to the development of the metabolic syndrome such as obesity, diabetes, diabetic vascular complications, dyslipidemia, hypertension and insulin resistance. The mechanism of this effect in obese humans is multifactorial and involves reduced tyrosine kinase activity at the insulin receptor, modulation of intracellular Ca activity and increased circulating tumour necrosis factor (TNF)-α levels (Cave et al. 2008). Zinc (Zn): Zn is a potent antioxidant and has an important role in immune defence functions. The expression of multiple zinc transporter proteins in adipose tissue is altered in obesity, which varies from one region to another (subcutaneous to intra-abdominal adipose tissue). Zn status modulates obesity and metabolic syndrome. In a large clinical study, both low consumption of dietary Zn and low serum Zn levels were associated with an increased prevalence of diabetes, hypertension, hypercholesterolemia and coronary artery disease. Animal studies have demonstrated potential mechanisms and implied a plausible therapeutic role for Zn in obesity. In rats fed with high fructose diet along with zinc supplementation showed improved insulin sensitivity and antioxidant status. Also, due to its antioxidant action, it provides a protective effect against ischemia/reperfusion injury, which could be relevant for critically ill obese patients (Cave et al. 2008). Low Zn status had been observed in several obese and diabetic patients (Suliburska et al. 2012). Another study in 2009 suggested that 20 mg Zn given daily to 60 obese children in a randomized, blinded, crossover trial resulted in significant reductions in fasting plasma glucose levels and fasting insulin levels. The authors concluded that, in addition to lifestyle modifications, Zn supplementation might be a useful and safe additional intervention for improving cardio metabolic risk factors related to childhood obesity (Bruney 2011). Vitamin A: Retinoic acid decreases lipid accumulation, glycerol 3-phosphate dehydrogenase (GPDH) activity and peroxisome proliferator-activated receptor (PPAR)γ expression. In one study, feeding a high dose of retinol (129 mg/kg a day) to obese rats reduced adipose tissue mass and body weight. The anti-adipogenic effects are exerted through a number of mechanisms; retinoic acid blocks C/EBPβ (CCAAT-enhancer-binding protein) mediated induction of downstream genes, notably PPARγ, prevents entry of the preadipocytes into the growth-arrested phase and interacts with activators of PPARγ through retinoic acid receptors (Bonet et al. 2003). Vitamin D: The status of vitamin D is strongly associated with variation in subcutaneous and visceral adiposity. In mouse epididymal fat tissue cultures, 1,25(OH)2D3 markedly suppressed the expression of PPARγ and C/EBPβ and in 3T3-L1 preadipocytes, ≥0.5 nM



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1,25(OH)2D3 decreased viability and lipid accumulation and induced apoptosis (Andersen et al. 2010). Multivitamins and multiminerals: The effects of supplementation with multivitamin and multimineral on adiposity, energy expenditure and lipid profiles in obese Chinese women have been evaluated. In a 26-week, randomized, double-blind, placebo-controlled intervention study, 96 obese Chinese women (average BMI = 28 kg m2) participated. The trial was randomized into three groups, receiving either one tablet of multivitamin and mineral supplement (MMS), or Ca 162 mg or identical placebo daily during the study period. A total of 87 participants completed the study. After 26 weeks, results suggested that, in obese individuals, multivitamin and mineral supplementation could reduce body weight and fatness and improve serum lipid profiles which may be through increased energy expenditure and fat oxidation (Li et al. 2010). Arginine: Arginine is a non-essential amino acid mainly required for cell division, immune defence and removing ammonia from the body. It also regulates gene expression, mitochondrial biogenesis, brown adipose tissue (BAT) development and cellular signalling transduction pathways (Jobgen et al. 2009). A 21 day, randomized placebo-controlled trial on 33 hospitalized middle-aged, obese (BMI = 39.1 ± 0.5 kg/m2) participants with dietcontrolled Type-2 diabetes mellitus was conducted. Each patient received a low-calorie diet (1,000 kcal/day) and a regular exercise-training program (45 min twice a day for 5 days/week) during the study. They were randomized to 8.3 g arginine/day (approximately 80 mg/kg body weight per day) or placebo. Both groups of participants exhibited reductions in body weight, fat mass, waist circumference and circulating levels of glucose, fructosamine and insulin. Moreover, increases in antioxidant capacity and circulating levels of adiponectin were observed. Over the 3-week period of study, fat-free mass was maintained in the arginine group but reduced by 1.6 kg in the placebo group. Also, arginine supplementation to obese participants promoted fat reduction and spared lean body mass during weight loss (McKnight et al. 2010). Other studies with both animals and humans have also indicated that arginine supplementation may be a novel therapy for obesity and metabolic syndrome, acting via decreased plasma levels of glucose, homocysteine, fatty acids, dimethyl-arginines, triglycerides with concurrent improvement in insulin sensitivity. Alpha lipoic acid (ALA): Lipoic acid is an organo-sulfur compound with good antioxidant activity. 1127 obese and pre-obese people (445 men and 682 women, 18-60 years old) were screened in a study. According to the BMI, 53% were obese, and 43% were preobese. All were treated for 4 months with 800 mg/day of lipoic acid (ALA). In the pre-obese group, significant reductions of weight (8%, in both genders), BMI (2 points), blood pressure and abdominal circumference (female 6 cm, male 7 cm) were observed. In the obese group, significant reductions of weight (9%, in both gender), BMI (female 3 point, male 4 point), blood pressure and abdominal circumference (female 9 cm, male 11 cm) were seen (Carbonelli et al. 2010). Conjugated linoleic Acid (CLA): The potential mechanisms responsible for the antiobesity properties of CLA isomer in rodent models include decreased energy intake by suppressing appetite, increased energy expenditure, decreased lipogenesis and adipogenesis, increased lipolysis and apoptosis. Several studies have also shown that CLA regulates both leptin and adiponectin (Prieto-Hontoria et al. 2011).


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Omega-3 fatty acids: Omega-3 poly-unsaturated fatty acids (PUFAs), specifically the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) components, are very helpful in management of obesity as they reduce the inflammatory response through numerous distinct mechanisms. Studies have shown that they decrease activation and release of TNF-α, activate PPAR-γ and PPAR-α, decrease serum triglycerides levels and inflammatory prostaglandins and alter adiponectin levels. In a prospective randomized trial, participants with non-alcoholic fatty liver disease (NAFLD) who received 1 g/d of omega-3 PUFA for 12 months had significantly decreased serum levels of alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase and triglycerides compared with placebo controls (Cave et al. 2008).

Role of Plants and Bioactive Compounds in Weight Loss Grape seed extract: Grape seed extract from Vitis vinifera was administered to obese rats (induced by high fat diet) for 45 days and the findings suggested that grape seed extract was a safe anti-obesity and cardio protective agent. It may also play a role in inflammatory damaging conditions such as stroke (Charradi et al. 2011). Curcumin and resveratrol combination: Curcumin (Figure 3) exhibits multiple anticarcinogenic effects and its role in chemoprevention is being assessed in clinical trials. It has also been shown to be protective in rat models of diverse diseases such as atherosclerosis, ischemia reperfusion injury, cystic fibrosis and diabetes mellitus (Sharma et al. 2006). Curcumin, resveratrol and their combination (3% suspended solution) were found to significantly suppress increased body weight, showing anti-obesity action in rats fed with a high fat diet for eight weeks. Decreases in plasma glucose and insulin levels were observed. This combination also lowered fat accumulation, suppressed triacylglycerol, total cholesterol, free fatty acids and normalized the activity of antioxidant enzymes in the liver (Hussein et al. 2013). O

O

HO

OH OCH3

OCH3

Figure 3. Curcumin.

Resveratrol: Resveratrol is a naturally occurring phytoalexin derived from red wines and grape juice (Figure 4) induces cell apoptosis in 3T3-L1 adipocytes by increasing the expression of Sirt1, Cytochrome c, cleaved Caspase 9 and cleaved Caspase 3. A combined



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treatment of resveratrol, genistein and quercetin enhanced apoptosis in pre- and lipid-filled mature murine adipocytes. Further, a combination of resveratrol and genistein has a stronger effect on induction of adipocyte apoptosis (Zhang et al. 2012). OH

HO

OH

Figure 4. Resveratrol.

Guggulsterones: Guugulsterones (Figure 5) are steroidal compounds extracted from Commiphora myrrh. A trial was conducted with 58 adult obese participants given either 1.53.0 g/day Guggulu (Medohar) for 30 days or no drug. All participants were on a restricted diet (1200-1600 cals/day) and completed a brisk walk for 30 minutes daily. The mean weight reduction in participants was higher in the Guggulu group without any adverse effects (Bhatt et al. 1995). The effect of guggulsterone has been determined on apoptosis, adipogenesis and lipolysis of 3T3-L1 cells. Results showed that guggulsterone isomers exert anti-obesity effects by inducing apoptosis and lipolysis in mature adipocytes (Shah et al. 2012). H

O

O

Figure 5. Guggulsterone.

Hydroxycitric acid (HCA): The bioactive compound HCA, (Figure 6) isolated from the dried fruit rind of Garcinia cambogia, along with the micronutrient niacin-bound chromium provided safe weight loss in adults (Bruney 2011). HCA also inhibits adenosine triphosphate citrate lyase and has been used in the treatment of obesity. A double-blind, randomized, placebo-controlled study has investigated the effects of G. cambogia extract (containing 100 mg HCA/day) on visceral fat. The participants included were aged 20-65 years with a visceral fat area > 90 cm2. Forty-four participants were randomized at baseline and 39 completed the study. At 16 weeks, the Garcinia group had significantly reduced visceral, subcutaneous and


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total fat areas compared with the placebo group. No severe adverse or rebound effects were observed at any time in the test period. Therefore, this study suggested that Garcinia may be useful for the prevention and reduction of accumulation of visceral fat (Hayamizu et al. 2003).

O

HO

OH

O

O OH HO

OH

Figure 6. Hydroxycitric acid.

Phaseolus vulgaris: White kidney beans inhibit carbohydrate hydrolysing enzymes, resulting in flatulence. Based on this, products containing the French white bean, Phaseolus vulgaris, have been widely marketed as weight loss aids. A double-blind, placebo-controlled clinical study in 2007 included 60 slightly overweight people. The participants were given either placebo or a Phaseolus extract once daily 30 minutes prior to a main meal rich in carbohydrates. Over the next 30 days of the study, the results indicated that Phaseolus treatment led to a significantly greater reduction of body weight and improvement of lean/fat ratio as compared to placebo (Obiro et al. 2008). Hoodia pilifera: A novel compound from South African succulent plants of the Hoodia family has been reported to reduce appetite in animals. The compound, identified as P57, extracted from Hoodia pilifera and Hoodia gordonii was also tested clinically where it showed positive results in suppressing appetite and reduced body weight in obese individuals. An oral dose (6.25–50 mg/kg) resulted in decreased food consumption as compared to control (fenfluramine) and the reduction in food intake induced by Hoodia compounds was greater (Gooda Sahib et al. 2012). Caralluma fimbriata: Caralluma, an edible cactus used by tribal Indians to suppress hunger and increase endurance, has shown be anti-obesity activity. Fifty men and women ingested 1 gm of Caralluma extract per day for 60 days. Compared to placebo, the extract appeared to suppress appetite and significantly reduce waist circumference. Feeding of the extract also resulted in a decrease in body weight, BMI, hip circumference and body fat in overweight individuals (Gooda Sahib et al. 2012). Ephedra sinica: Ephedra extract induced significant decrease in serum cholesterol, triglyceride, glucose, fasting insulin and leptin levels in obese and overweight women (Hackman et al. 2006). Ephedra alkaloids are commonly combined with caffeine or botanical sources of caffeine (e.g., guarana, yerba maté) for weight loss. A recent meta-analysis of clinical trials showed a weight loss of 0.9 kg per month for Ephedra-containing supplements compared with placebo (Robert B. Saper 2004). Ephedra sinica reduced weight gain and epididymal fat accumulation, improved glucose intolerance, decreased triglycerides and increased high-density lipoprotein cholesterol in high fat diet fed mice. Gene expression



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analysis revealed that Ephedra sinica upregulated the expression of adiponectin and PPAR-α and downregulated the expression of TNF-α. Therefore, it may reduce obesity and hyperglycemia (Song et al. 2012). Lagerstroemia speciosa: Food containing 5% Lagerstroemia, also known as banaba water extract, was used to feed female obese mice and a significant reduction in body weight was observed as compared with control mice fed with a regular diet. Liver triglyceride content was also reduced by more than 40% in the banaba water extract-treated mice (Klein et al. 2007). Cissus quadrangularis: This plant helps in fighting obesity and symptoms of metabolic syndrome due to the presence of flavonoids and indanes, as well as phytosterols and ketosteroids which have shown promise as powerful and efficient antioxidants. They also appear efficient for lipase and amylase inhibition, thereby providing a mechanism for weight loss via reduced oxidative stress, dietary fat and carbohydrate blocking (Mishra et al. 2010). Irvingia gabonensis: This plant is commonly known as bush mango, dikanut or African mango. The high soluble fibre content lowers plasma cholesterol, triglycerides and glucose concentrations. The glycoproteins in the seeds seem to inhibit hydrolase enzymes by blocking the active sites or altering enzyme configuration. A combination of Irvingia gabonensis and Cissus quadrangularis was studied for 10 weeks in a randomized, double-blind, placebocontrolled trial on 72 obese or overweight participants. Capsules containing the placebo or active formulations were administered twice daily before meals without the involvement of major dietary changes or exercises. Anthropomorphic and serological measurements (body weight, body fat, waist size; total plasma cholesterol, LDL cholesterol and fasting blood glucose level) were taken at baseline and at 4, 8 and 10 weeks. The combination resulted in larger reductions in these measurements, suggesting it may be useful in the management of obesity and its related complications (Oben et al. 2008). Raspberry ketones (RK): These major aromatic compounds of raspberries (Figure 7) were tested to investigate their effect on obesity using in vivo experiments. Mice were fed a high fat diet including 0.5, 1, or 2% RK for 10 weeks. The results indicated that RK prevented the high fat diet-induced elevations in body weight and the weights of the liver and visceral adipose tissues; they also significantly increased norepinephrine-induced lipolysis associated with the translocation of hormone-sensitive lipase from the cytosol to lipid droplets in rat epididymal fat cells. Therefore, RK prevented and improved obesity and fatty liver. RK stimulated the metabolism of white and brown adipose tissues and inhibited small intestinal absorption of dietary fat by suppressing pancreatic lipase activity. As an agent effective in preventing both fat- and sugar-induced obesity, RK might exert its anti-obesity effect via an increase of norepinephrine-induced lipolysis in white adipocytes and by enhancement of thermogenesis in brown adipose tissue (Morimoto et al. 2005). Dioscorea nipponica: Methanol extract of Dioscorea nipponica showed potent inhibitory activity against pancreatic lipase enzyme due to the saponins dioscine and diosgenin present in this plant. When Sprague-Dawley rats were fed with high fat diet, both the saponin compounds were significantly able to suppress blood triacylglycerols, gained less body weight and adipose tissue compared to controls in an eight week experimental study (Kwon et al. 2003). Asparagus officinalis: Asparagus is consumed as a healthy and nutritious vegetable in many parts of the world. Due to the presence of various bioactive compounds such as flavonoids, steroidal saponins and polysaccharides, this plant possesses strong antioxidant,


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immunoprotective, anti-inflammatory, antifungal, hepatoprotective, antitumour, hypolipidaemic and hypoglycaemic activity (Gautam et al. 2004). Ethanolic and aqueous extracts of Asparagus officinalis significantly decreased body weight gain, serum total cholesterol and serum low-density lipoprotein cholesterol in hyperlipidaemic mice when administered at a daily dose of 200 mg/kg for eight weeks. Also, serum high-density lipoprotein cholesterol levels were evidently increased. Moreover, both extracts dramatically decreased the activities of alanine and aspartate transaminases in serum with improvements in superoxide dismutase and total antioxidant capacity. Therefore, Asparagus has strong hypolipidaemic and hepatoprotective actions and would be a helpful supplement for weight loss (Zhu et al. 2010).

O

HO Figure 7. Raspberry ketone.

Nigella sativa: In a randomized double blind clinical trial in fifty male obese subjects, extracts of Nigella sativa seeds showed significant reduction in body weight, waist circumference and triglycerides (Ranjbar et al. 2013). Favourable results were reported in another randomized double blind clinical trial using Nigella sativa seeds in capsules. Reductions in body–mass index, waist–hip ratio, blood pressure, fasting blood sugar and serum lipids were observed (Qidwai et al. 2009). Murraya koenigii: Murraya koenigii, commonly known as ‘Curry leaves’ is native to India and traditionally used as a spice for its characteristic flavour and aroma. The aromatic leaves are considered as a tonic, anthelmintic, analgesic, digestive, and appetizer, being widely used in Indian cookery for flavouring food stuffs. The leaves contain carbazole alkaloids, flavonoids, furanocoumarins, terpenoids and tannins and have shown strong hypolipidemic activity and have been indicated for the treatment of the mild form of diabetes. The dichloromethane and ethyl acetate extracts of Murraya koenigii leaves significantly reduced the body weight gain, plasma total cholesterol and triglyceride levels when given orally at a dose of 300 mg/kg/day to the high fat diet induced obese rats for two weeks. The observed antiobesity and antihyperlipidemic activities of these extracts are correlated with the carbazole alkaloids and a bioactive compound, mahanimbine (Birari et al. 2010). Crocus sativa: A randomized, placebo-controlled, double-blind study evaluated the efficacy of Satiereal (Inoreal Ltd, Plerin, France - a novel extract of saffron stigma) supplementation on body weight changes over an eight week period in healthy, mildly overweight women. Saffron reduced snacking and enhanced satiety through its moodimproving effect, and thus contributed to weight loss. The enrolled subjects consumed 1 capsule of Satiereal (containing 176.5 mg of extract) per day (n = 31) or a matching placebo (n = 29) with no restrictions on the caloric intake during the study. Satiereal caused a



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significantly greater body weight reduction than placebo and the mean snacking frequency was also significantly decreased. The study indicated that due to enhanced satiety effect, combination of an adequate diet with Satiereal supplementation might help people to achieve weight loss (Gout et al. 2010). Lycium barbarum: Two separate randomized, double-blind, placebo-controlled, small clinical studies were conducted in healthy human adults for fourteen days to investigate the effect of L. barbarum fruit juice on caloric expenditure and waist circumference. A standardized L. barbarum fruit juice, GoChi, was administered in three doses (30, 60, and 120 ml) and found to significantly decrease waist circumference and increase metabolic rate, relative to placebo-treated control subjects (Amagase et al. 2011). Tamarindus indicus: The antiobesity effect of aqueous extract of tamarind pulp was investigated in diet-induced obese Sprague–Dawley rats. The obesity was induced via high fat diet and tamarind extract at 5, 25, or 50 mg/kg was given orally for ten weeks. It was observed that the extract decreased the levels of plasma total cholesterol, low density lipoprotein (LDL), and triglyceride, and increased high-density lipoprotein (HDL), with the concomitant reduction of body weight. Moreover, it also decreased plasma leptin and fatty acid synthase (FAS) activity and enhanced the efficiency of the antioxidant defense system. Therefore, tamarind improved obesity-related parameters in blood, liver, and adipose tissue and suppressed obesity induced by a high fat diet, possibly by regulating lipid metabolism and lowering plasma leptin and FAS levels in rat model (Azman et al. 2012). Citrus aurantium: The study investigated the lipolytic effect of a citrus-based polyphenolic dietary supplement, SINETROL (1.4 g/day), in overweight subjects for twelve weeks. SINETROL is extracted by physical treatment (crushing of fruits, cold pressure of juice, extraction, centrifugation, filtration, spray drying) of specific varieties of red orange (Citrus sinensis L. Osbeck (Blood group), sweet orange (Citrus aurantium L. var. sinensis), bitter orange (Citrus aurantium L. var.amara), grapefruit (citrus paradise) and guarana (Paulinia cupanna). SINETROL stimulated lipolytic activity and significantly decreased body fat and body weight. The effects were linked to polyphenolic composition of this supplement and its resulting synergistic activity. SINETROL is a potent inhibitor of cAMPphosphodiesterase (PDE), suggesting its lipolytic effect is mediated by cAMP-PDE inhibition. Therefore, it may prevent obesity by decreasing BMI (Dallas et al. 2008). Glycyrrhiza glabra: Licorice (Glycyrrhiza glabra Linn) is a well-known medicinal plant and glabridin is an isoflavan isolated from licorice. The anti-obesity effect of glabridin and glabridin-rich supercritical fluid extract of licorice was investigated. Glabridin effectively inhibited adipogenesis in 3T3-L1 cells. The inhibitory effect resulted from inhibiting the induction of the transcriptional factors CCAAT enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma. Mice were fed with high fat diet containing 0, 0.1% and 0.25% licorice supercritical extract for eight weeks. The extract significantly reduced weight gain, white adipose tissue and fat cell size in a dose-dependent manner. In the liver, it effectively inhibited high fat diet-induced hepatic steatosis through downregulation of gluconeogenesis-related phosphoenolpyruvate carboxykinase and glucose 6-phosphatase and upregulation of the β-oxidation-related carnitine palmitoyl transferase. Therefore, the results suggested that glabridin and glabridin-rich licorice extract would be effective anti-obesity agents (Ahn et al. 2013). The other flavonoid compounds of licorice have also shown strong antioxidant and antiobesity activity (Birari et al. 2011).


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Capsicum annum: Fermented red pepper paste is made with glutinous rice, Meju (fermented soybean blocks), salt, and red pepper (Capsicum annuum L.) powder and is fermented for several months. It is one of the best known traditional foods in Korea has been recognised for its antiobesity activity by decreasing body weight and lipid levels rats fed with high fat diet (Rhee et al. 2003). It is also reported to inhibit lipid accumulation and stimulated lipolysis in 3T3-L1 adipocytes (Ahn et al. 2006). In another study, 28 female volunteers (BMI more than 23 kg/m2) aged 19 to 60 years were treated with fermented red pepper paste for twelve weeks. Marked cholesterol modulation was observed in the fermented red pepper paste-treated group with low levels of urinary hypoxanthine (Kim et al. 2010). Allium sativum: Garlic extracts exert anti-cancer, antioxidant, antimicrobial, antiobesity and anti-inflammatory effects (Banerjee et al. 2003; Kuda et al. 2004). The effect of thiacremonone, a sulfur compound isolated from garlic significantly inhibited 3T3-L1 differentiation via down-regulation of adipogenesis-related transcription factors and adipogenic markers without any cytotoxic effect. Thiacremonone resulted in AMPK activation, resulted in the suppression of intracellular lipid droplet levels, reduction of lipid synthesis and increases in fatty acid oxidation therefore it may be a promising compound for the treatment of obesity (Kim et al. 2012). Zingiber officinale: Two grams of powdered ginger dissolved in hot water induced significant increase in thermogenic effect in healthy overweight men and influenced feelings of satiety without any adverse effects (Mansour et al. 2012). In another study, the effect of ginger supplementation was investigated in obese men and a significant decrease in total cholesterol, body fat percentage, fat mass, waist circumference, waist-hip-ratio and significant increase for fat free mass after the ten week period was observed. However, mean BMI, HDL, LDL and triglyceride remained unchanged. Therefore, the results suggested that resistance training along with ginger supplementation may be an effective therapeutic strategy to induce favourable changes in lipid profiles and body composition in obese individuals (Atashak et al. 2011). Punica granatum: The anti-obesity effect of the pomegranate leaf extract was investigated in a mouse model of high fat diet-induced obesity and hyperlipidemia. The animals were treated with 400 or 800 mg/kg/day of pomegranate extract for five weeks. The extract-treated groups showed a significant decrease in body weight, energy intake, adipose tissue and serum, total cholesterol, triglycerides, glucose levels and high density lipoprotein after 5 weeks treatment. The extract inhibited the development of obesity and hyperlipidemia in high fat diet-induced obese mice by inhibiting the pancreatic lipase activity and suppressing energy intake. It may be a novel appetite suppressant that only affects obesity resulting from a high fat diet (Lei et al. 2007). Other plant compounds: Exotic fruits such as litchi, mangosteen, Acai, goji berries, pomegranate and avocado have been shown to decrease body weight, fat mass and adipocity in a number of studies which may be due to presence of various bioactive constituents (Devalaraja et al. 2011). Many studies have suggested that tea and tea polyphenols have beneficial effects on weight loss and prevention of obesity. These decrease lipid and carbohydrate absorption, increases lipid metabolism, inhibit de novo lipogenesis and increases carbohydrate utilization. The main polyphenols present in tea are catechins, (-)Epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin- 3-gallate (ECG) and (-)epigallocatechin-3-gallate (EGCG) (Grove et al. 2010).



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Conclusion Obesity continues to be a major health challenge in the modern world. If not controlled, it progresses to metabolic syndrome through a complex pathophysiological process. Available evidence indicates that obesity is a metabolic disease largely caused by lifestyle and genetic variants. It leads to the deposition of excess fat in adipose tissues and, under certain conditions that remain unclear, in the viscera. The excess fat deposits trigger low-grade inflammatory processes that recruit inflammatory cells, with the accompanying release of inflammatory cytokines (e.g. TNF-a, IL-6 etc.), thus triggering a cascade of pathophysiological processes that lead to complications such as hypertension, atherosclerosis, dyslipidaemia, insulin resistance and diabetes mellitus, which characterize metabolic syndrome. The popularity of alternative medicine is increasing in demand of natural health products as drugs have failed to give desirable long-term results. Various plants and natural products have been assessed for their potential anti-obesity effect. There is increasing evidence that plants can exert anti-obesity effects through various mechanisms such as antilipase and anti-adipogenesis effects, or by suppressing appetite. Dietary phytochemicals might be employed as anti-obesity agents because they may suppress growth of adipose tissue, inhibit differentiation of preadipocytes, stimulate lipolysis and induce apoptosis of existing adipocytes, thereby reducing adipose tissue mass. Medicinal plants are gaining more credibility in the scientific community. There are thousands of unexplored plants across the world, some of which have traditionally been used to maintain ideal body weight or as slimming agents, thus justifying the need for deeper research in this field.

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Chapter 6

Application of Antioxidant Plants as Anti-Hemolytic Agents 1

João C. Fernandes1,2,3 and David M. Pereira3,4

Laboratory of Pharmacology and Experimental Therapeutics, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal 2 Institute for Molecular and Cellular Biology, University of Porto, Porto, Portugal 3 Biochemistry Laboratory, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal 4 Institute for Traditional Medicine, Porto, Portugal

Abstract The use of medicinal plants represents the oldest and most common form of medication. Among the hundreds of studies published in the last two decades on medicinal plants research, the quest for new antioxidant drugs has a been pivotal. Some of those plants with antioxidant activity, as well as their bioactive components, have been in some cases further analyzed for a hypothetical anti-hemolytic potential. Although oxidative stress is not the primary etiology of diseases such as hemolytic anemias, it is believed to aggravate them. Therefore, the use of natural antioxidants, either as additives or as pharmaceutical supplements, may prevent or at least slow down free radical reactions that are responsible for provoking damage to essential red blood cell molecules. In this Chapter, we review the current knowledge regarding the use of medicinal plants as anti-hemolytic agents. Particular emphasis in the compounds responsible for this activity, as well as the mechanism of action is given.

Introduction Since ancient times, humans have been looking for drugs in nature in order to prevent or heal a wide number of diseases. In earlier stages, medicinal plants were used in an intuitive and empirical way. Over time, the fundaments for the precise use of certain plants in the


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treatment of specific diseases were being discovered and gradually the empiric use of those plant species was abandoned and started to be supported by evidences (Halberstein, 2005). In the last decades, the scientific community has been trying to identify the specific molecules in herbs that account for their health promotion benefits. This search has been focusing primarily on plant metabolites (most of them are powerful antioxidants), phytochemicals, vitamins and fiber content. Countless studies have been performed and published upon the role of specific plant metabolites in disease prevention or treatment, mainly in diseases originated by reactive oxygen species (ROS). Coronary heart diseases, ulcers, cancers and neurodegenerative diseases (e.g. Parkinson’s and Alzheimer’s), besides overall ageing, are a few examples of human diseases and health conditions that may be instigated by free radicals (Morrell, 2008; Sugamura & Keaney, 2011 ). ROS are metabolic byproducts of aerobic metabolism of cells resulting from normal physiological processes such as energy production, which inevitably leads to the generation of oxidative molecules: superoxide (O2-•), hydrogen peroxide (H2O2) or hydroxyl radical (•OH) (O’Brien et al., 2012). Transition metals like Fe2+ or Cu2+, although required for certain enzymatic functions, exacerbate oxidative damage by catalyzing the conversion of hydrogen peroxide into •OH, a highly reactive radical that will immediately react with any biological molecule, notably lipids (O’Brien et al., 2012). Another important free radical is nitric oxide (NO). This molecule, produced by the nitric oxide synthase (NOS) enzymes, exerts various physiologic functions as a neuromediator, regulator of immune functions, or vasodilator. However, it can react with superoxide to form peroxynitrite (ONOO-), an extremely potent cellular oxidant. Furthermore, environmental sources such as cigarette smoke, pollutants, ultraviolet light, ionizing radiation and xenobiotics also possess an important role on free radicals production (Beckman et al., 1990). To a certain extent, cells can protect themselves against oxidative damage (Figure 1). The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide to H2O2, which is then converted to H2O by gluthatione peroxidase (GPx) or to O2 + H2O by catalase (CAT) (Miwa et al., 2008). The reaction catalyzed by GPx requires gluthatione (GSH), which is converted to reduced gluthatione (GSSG). The concentrations of GSH and GSSG, as well as their ratio, reflect the redox state of the cell and are crucial for an efficient ROS detoxification. The detrimental effect of transition metals is minimized through the action of proteins like ferritin, transferrin, lactoferrin that can store Fe2+ ions, thus keeping their free cellular concentration as low as possible (Marí et al., 2009). Finally, cells are also protected by other non-enzymatic agents with endogenous and exogenous sources such as uric acid, bilirubin, Vitamin E, Vitamin C, carotenoids, polyphenols, among others (Marí et al., 2009). Among the various diseases that oxidative stress is believed to aggravate, hemolytic anemias are given particular attention throughout this chapter (Huang et al., 2005). Oxidative stress has been implicated in the beta-hemoglobinopathies (sickle cell anemia and thalassemia), glucose-6-phosphate dehydrogenase deficiency, unstable hemoglobin, hereditary spherocytosis, congenital dyserythropoietic anemias, paroxysmal nocturnal hemoglobinuria and aging of red blood cells (RBCs) (Halliwell & Gutteridge, 1984; Huang et al., 2005). Although oxidative stress is not the primary etiology of these diseases, such damage to erythroid cells plays a crucial role in hemolysis due to ineffective erythropoiesis in the bone marrow and short survival of RBCs in the circulation. Moreover, platelets and polymorphonuclear (PMN) white cells are also exposed to oxidative stress. As a result, some


Application of Antioxidant Plants as Anti-Hemolytic Agents

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patients develop thromboembolic phenomena and recurrent bacterial infections in addition to the chronic anemia (Halliwell & Gutteridge, 1984). There are numerous intracellular events that precede oxidant-induced cell hemolysis (Figure 2). These include oxidation of hemoglobin, generation of high-molecular weight proteins or protein ionic shifts, lipid peroxidation, changes in cell deformability, phospholipid flip-flop processes within the lipid bilayer leaflet of the membrane and carbonylation – an irreversible post-translational amino acid residue modification (Fibach & Rachmilewitz, 2008; Mazor et al., 2001). RBCs have well known enzymatic (CAT, SOD, peroxyredoxins and GPx) and non-enzymatic (GSSG and ascorbate) antioxidant systems, which prevent or slow protein oxidation, thus reducing ROS into less reactive intermediates. However, these endogenous defenses are not enough to protect RBCs whenever an unusual physical or chemical stress occurs, mainly due to the limited biosynthesis capacity of these cells and their poor repair mechanisms. Therefore, the circulating RBCs suffer and accumulate physical and chemical changes, which become more pronounced with cell aging, hence resulting in various pathologies as already described (Kruckeberg et al., 1987). The use of antioxidants may prevent or ameliorate free radical reactions in vivo, thereby prevent some of the damage to essential erythrocyte molecules presented above. There are numerous evidences suggesting that several plants, used either as a whole or their individual constituents, have substantial protective effects upon erythrocytes oxidative damage (Figure 3). Serafini et al. (2002) showed that the ingestion of tea produced a significant increase in human plasma antioxidant capacity in vivo, further suggesting that ingestion of medicinal plants with antioxidant capacity would strength the protection of RBCs membrane from free radical-induced oxidative damage, and ultimately from hemolysis. Therefore, several studies have been focusing on medicinal plants regarding their capacity to prevent RBC membrane damage and, eventually, hemolysis, which occurs mainly due to lipid oxidation of erythrocyte membrane mediated by ROS as the ones generated by H2O2 or by 2,2'-azobisisobutyramidinium chloride (AAPH) (Pandey & Rizvi, 2010). Aqueous extracts of sage (Salvia sp.), savory (Satureja montana L.), myrtle leaf (Myrtus communis L.), yarrow (Achillea millefolium L.), agrimony (Agrimonia eupatoria L.) and walnut-tree (Juglans regia L.) were reported by Gião et al. (2010) as to possess good capacity to inhibit H2O2-induced hemolysis in an in vitro system. This natural oxidant is regularly used in vitro to initiate radical formation in intact cells. Its main target in RBCs is hemoglobin, which is consequently converted to either met- or ferry-haemoglobin (its oxidized forms). In addition, exposure of RBCs to H2O2 also results in changes of RBC membrane proteins, as well as lipid peroxidation. As a consequence of these oxidative modifications, drastic changes in RBC shape and membrane structure may occur, which will eventually lead to lysis. The six extracts showed a hemolysis inhibition rate above 80%, at low concentrations [0.00050.005% (w/v)], with savory displaying the highest inhibition (>95%). In addition, thyme, weet-amber and eucalyptus also showed a relevant protection although higher concentrations were requiresd [0.025-0.5% (w/v)]. The authors hypothesize that these differences may be explained by the different profiles in phenolic content between the plants, thus suggesting that the higher anti-hemolytic activity is related with the amount of hydrogen donors and singlet oxygen quenchers. Heath (Calluna vulgaris L. Hull) also displayed a dual effect: at low


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concentrations is showed a protective effect, however at high concentrations [> 0.05% (w/v)] pro-hemolytic effect was found. Using the same oxidative agent, Manian et al. (2008) reported the high anti-hemolytic effect (about 70-80% hemolysis inhibition) of banyan tree (Ficus bengalensis L.) and Indian fig tree (Ficus racemosa L.) from both bark and root. Both plants inhibited the lipid peroxidation of RBC membranes and •OH scavenging capacity. Simon et al. (2000) demonstrated that rooibos (Aspalathus linearis R.Dahlgr.) is able to inhibit significantly the hemolysis induced by H2O2, whether as decoct (more than 50%) or as an hot water extract (above 70%), at concentrations around 1.5 mg/ mL. Coulibaly et al. (2011) concluded that goat weed (Scoparia dulcis L) may have therapeutic applications due to its antioxidant properties as it was able to prevent RBC lysis (56%-83%) at a concentration of 300 µg/mL. The extract with lower flavonol concentration exhibited the highest protection, thus suggesting that the protective role of this plant against H2O2-mediated oxidation is not determined by the flavonol content. Sangkitikomol (2012) also assessed the effect of 30 plant extracts on delaying erythrocyte hemolysis induced by AAPH. This oxidative agent can decompose to form carbon-centered radicals that can react with O2 to yield peroxyl radicals capable of removing hydrogen from membrane lipids and to stimulate lipid peroxidation. In general, all the extracts tested delayed the hemolytic process to some extent. Nevertheless, aleppo oak (Quercus infectoria Oliv), clove [Eugenia caryophyllus (Spreng.) Bullock & S. G. Harrison], rose (Rosa domescena Mill), mexican marigold (Tagetes erecta L.), and eugenia (Syzygium gratum Wight) exhibited higher protection, increasing the time required to achieve 50 % hemolysis above 210 min, against the 120 min seen for positive control. This capacity to protect RBCs was attributed by Sangkitikomol to the plant polyphenols content: according to the author, the polyphenols protect the structure and function of RBC membranes by interacting with the surface of the membrane through hydrogen bonding, which in turn impairs the access of oxidants to the cell. Furthermore, Sangkitikomol reported that the above-mentioned plants were also capable of inhibiting hemoglobin oxidation induced by Nacethylphenylhydrazine (APHZ) – source of free radical formation inside cytosol of RBCs, leading to oxidation of proteins, mainly hemoglobin. Quince (Cydonia oblonga Mill) leaf extracts were analyzed by Costa el al. (2009) and showed a low capacity to inhibit AAPHinduced hemolysis, lower than 40% at 50 µg/mL, mainly due to its major phenolic compound constituent, 5-O-caffeoylquinic acid. Asghar & Masood (2008) reported the capacity of milk thistle (Silybum marinum L.) to scavenge peroxyl radicals generated by AAPH. At concentrations in the 0.05-0.5 mg/ml range, hemolysis was inhibited by about 32.5-92.5%. Moreover, the authors suggest that the anti-hemolytic capacity of this plant may also be associated to its capacity to increase both SOD and GPx. The golden root (Rhodiola rosea L.) has also been reported as possessing anti-hemolytic capacity. Battistelli and co-workers have used hypochlorous acid as an oxidative agent, which is associated with oxidation of GSH and of -SH groups of membrane proteins, as well as with cross-linking of membrane proteins and extensive disruption of the membrane inducing lysis; 100 µL of golden root aqueous extract (41.6 mg/mL) was sufficient to reduce hemolysis in 50%; 200 µL completely inhibit hemolysis and RBC deformability (Battistelli et al., 2005). Atrooz (2013) analyzed two aromatic plants seeds, cumin (Cuminum cyminum L.) and caraway (Carum carvi L.), with proved medicinal properties and assessed their capacity to protect RBCs from FeSO4-induced



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hemolysis; both plants showed anti-hemolytic activity, however cumin possessed higher capacity – over 60% inhibition at 100 µM against 45% by caraway at the same concentration. Several other works have been published in the last years demonstrating the high capacity of other plant extracts, but mainly reporting the anti-hemolytic capacity of plant components in particular: Lam et al. (2007) isolated 6-gingerol and 3,3′,5-trihydroxy-4′-methoxystilbene 3-β-D-glucoside, phenolic compounds from ginger (Zingiber officinale Roscoe) and rhubarb (Rheum rhabarbarum L.), respectively. At 50 µM, 3,3′,5-trihydroxy-4′-methoxystilbene 3-βD-glucoside showed higher protection (65%) than trolox (40%) in AAPH-induced hemolysis in RBC. Differently, 6-gingerol only exerted some effect when at 100 µM. Overall, Lam et al. concluded that phenolic compound from rhubarb is almost two times more potent than trolox while 6-gingerol was only about half as potent as trolox in this assay. In addition, the authors also reported a strong inhibition against lipid peroxidation of RBC membranes in a tertbutylhydroperoxide (tBHP)/hemin oxidation system. Dai and co-workers (2006) isolated several flavonols and their glycosides, which are commonly presented in several plants and fruit trees – myricetin (eg. gum tree - Acacia nilotica; moringa - Moringa oleifera; Aloe vera - Aloe barbadensis), quercetin (Ginkgo biloba; St. John's wort - Hypericum perforatum; American Elderberry - Sambucus canadensis…), morin (Apple Guava - Psidium guajava; Osage Orange leaf - Maclura pomifera; Old Fustic - Maclura tinctoria…), kaempferol (Candle Bush - Cassia alata; Small-leaved Lime - Tilia cordata; Field Horsetail - Equisetum arvense…), rutin (Buckwheat - Fagopyrum esculentum; Rhubarb - Rheum rhabarbarum; Fava d'anta - Dimorphandra mollis…), quercetin galactopyranoside (American saw-wort Saussurea americana; Peepal - Ficus religiosa), quercetin rhamnopyranoside (Amur Maple Acer ginnala; Peepal - Ficus religiosa), and kaempferol glucopyranoside (Sohanjana Moringa oleifera; Aloe vera), and analyzed their effectiveness to protect RBCs from AAPHinduced hemolysis. The results demonstrated that all these flavonols (and their glycosides) are effective antioxidants, however the compounds bearing ortho-dihydroxyl functionality (myricetin, quercetin, rutin, quercetin galactopyranoside, quercetin rhamnopyranoside) possess remarkably higher activity than the ones bearing no such functionality (Dai et al., 2006). This is to be expected as the ortho-hydroxyl would make the oxidation intermediate, ortho-hydroxyl phenoxyl radical, more stable due to the intramolecular hydrogen bonding interaction. Green tea (Camellia sinensis L. Kuntze) is the most studied plant regarding the capacity to prevent erythrocyte lysis. It is an excellent source of polyphenol antioxidants including epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG) and gallic acid (GA) (Ma et al., 2000). Due to the high amount of studies on the antihemolytic effect by green tea, it is already understood that its effects are not exclusively due to interruption of free-radical chain reaction (by trapping the free radicals), but also due to its ability to enhance the activity of endogenous antioxidant enzymes such as CAT, SOD, and/or glutathione antioxidant enzyme systems. Furthermore, the capacity of green tea polyphenols to act as chelators of catalytic cations involved in initiation of free radicals or to function synergistically with α-tocopherol (vitamin E) and L-ascorbic acid (vitamin C), protects RBC membranes from oxidation (Basu et al., 2003). Ma et al. (2000) reported that all the polyphenols in green tea referred above were able to suppress erythrocyte hemolysis from AAPH-oxidation in the following sequence EGCG>EGC>ECG≈EC>GA.


Figure 1. ROS generation in RBC and its defenses.


Figure 2. Intracellular events that precede oxidant-induced cell hemolysis.


Table 1. Summary of plants previously tested for their anti-hemolytic capacity Common name

Botanical name

Part used

State

RBC

Activity*

Concentration

Ref

Agrimony

Agrimonia eupatoria L

Leaf

Aqueous extract

Human

+++

0.005%

(Gião et al., 2010)

Aleppo oak

Quercus infectoria Oliv

ND

Aqueous/methanol extract (80:20)

Human

++

<0.25%

(Sangkitikomol, 2012)

Avocado

Persea Americana L

Leaf

Aqueous extract

Human

++

0.1%


Silk bananas

Musa sapientum

Leaf

Methanol extract

Human

++

0.1%

(Sahaa et al., 2013)

Banyan tree

Ficus bengalensis L.

Aerial root

Acetone extract

Cow

++

0.05%

(Marian et al., 2008)

Methanol extract

Cow

++

0.05%

(Marian et al., 2008)

Aqueous extract

Quails

++

0.175%

(Simon et al., 2000)

Acetone extract

Human

++

100 µm

(Atrooz, 2013)

Black tea

Camellia sinensis L.

Leaf

Caraway

Carum carvi L.

Seed

Methanol extract

Human

++

100 µm

(Atrooz, 2013)

Ceylon ironwood

Mesua férrea L

Flower


Human

++

<0.25%


Chamberbitter

Phyllanthus urinaria L

Leaf


Human

++

<0.25%



Human

++

<0.25%


++

0.02%

(Rajeshwari et al., 2012)

Clove

Eugenia caryophyllusI Spreng

Buds

Human

Coriander

Coriandrum sativum

Leaf & Seed

Methanol extract

Cumin

Cuminum cyminum L

Seed

Acetone extract

Human

+

100 µm

(Atrooz, 2013)

Methanol extract

Human

++

100 µm

(Atrooz, 2013)

Aqueous extract

Human

++

0.05%


++

<0.25%


++

0.0384%

(Nabavi et al., 2012)

++

0.01%

(Sabuncuoğlu & Şöhretoğlu, 2012)

++

0,0025%

(He et al., 2008)

Eucalyptus

Eucalyptus globules L

Leaf

Eugenia

Syzygium gratum Wight

Leaf


Human

Gamak

Dorema aitchisonii Korovin

Aerial parts

Ethanol extract

Rat

Genarium

Geranium tuberosum L

Flower

Methanol extract

Ginkgo

Ginkgo biloba L

Leaf

Aqueous extract

Human Human

*Activity classification: (+) - < 40% hemolysis inhibition; (++) – 40% < hemolysis inhibition < 80%; (+++) < 80% hemolysis inhibition.


Common name

Botanical name

Part used

State Hexane Extract

Goat weed

Scoparia dulcis L

Plant

Chloroform extract Methanol extract

RBC Rat Rat Rat Human

Golden root

Rhodiola rósea L.

Root

Aqueous extract

Green tea

Camellia sinensis L.

Leaf

Aqueous extract

Heath

Calluna vulgaris L Hyssopus angustifolius

Leaf Flower Stem Leaf

Indian fig tree

Ficus racemosa L

Stem bark

Indian lotus

Nelumbo Mucifera Gaertn

Flower

Aqueous extract Methanol extract Methanol extract Methanol extract Acetone extract Methanol extract Aqueous/methanol extract (80:20)

Peppermint

Mentha piperita L

Aerial parts

Ethanol extract

Mexican marigold

Tagetes erecta L

Leaf


Seed

Aqueous extract

Seed

Aqueous extract

Hyssop

Milk thistle

Silybum marinum L

Myrtle Neem

Myrtus communis L Azadirachta indica A Juss

Leaf Leaf

Aqueous extract Aqueous/methanol extract (80:20)

Nilgiri barberry

Berberis tinctoria Lesch

Fruit

Methanol extract

Pale Cranesbill

Biebersteinia multifida DC

Roots

Aqueous extract

Activity*

Concentration

+++

0.03%

++

0.03%

+++

0.03%

Human Human Rat Human Rat Rat Rat Cow Cow Human Rat

+++ ++ +++ +++ ++ ++ ++ ++ +++ +++

100µl of 41.6mg/ml 200µl of 41.6mg/ml 0.054% 0.0025% 20µm 0.0005% 0.0065% 0,0024% 0.0026% 0.05% 0.05% 0.05%

++

0.084%

Human Human

++

<0.25%

+++

0.05

+

0.005

Human Human Cow

+++ ++

0.005% <0.25%

++

0.0071%

Rat

+

N.D.

Human

Human

++ +++



Ref (Coulibaly et al., 2011) (Coulibaly et al., 2011) (Coulibaly et al., 2011) (Battistelli et al., 2005) (Battistelli et al., 2005) (Schmitz et al., 2008) (Costa et al., 2009) (Zhang et al., 1997) (Gião et al., 2010) (Nabavi et al., 2012) (Nabavi et al., 2012) (Nabavi et al., 2012) (Marian et al., 2008) (Marian et al., 2008) (Sangkitikomol, 2012) (Ebrahimzadeh et al., 2010) (Sangkitikomol, 2012) (Asghar & Masood, 2008) (Asghar & Masood, 2008) (Gião et al., 2010) (Sangkitikomol, 2012) (Sasikumar et al., 2012) (Nabavi et al., 2010)

Table 1. (Continued) Common name Quince Raspberry

Botanical name Cydonia oblonga Mill Rubus idaeus L

Part used Leaf Leaf

Rooibos tea

Aspalathus linearis R.Dahlgr

Leaf

Rose Sage Savory Screwpine Sweet-amber Thyme Walnut-tree Yarrow

Rosa domescena Mill Salvia sp Satureja Montana L Pandanus amaryllifolius Roxb Hypericum androsaemum L Thymus vulgaris L Juglans regia L Achillea millefolium L

Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf

State Methanol extract Aqueous extract Powder Aqueous extract Aqueous/methanol extract (80:20) Aqueous extract Aqueous extract Aqueous/methanol extract (80:20) Aqueous extract Aqueous extract Aqueous extract Aqueous extract

Zombi pea

Vigna vexillata L

Seed

Acetone extract

RBC Human Human Quails Quails Human Human Human Human Human Human Human Human Human

Activity* + ++ ++ ++ ++ +++ +++ ++ +++ +++ +++ +++

Concentration 0.005% 0.025% 3.1 g/1 kg 0.15% <0.25% 0.005% 0.005% <0.25% 0.025% 0.0005% 0.005% 0.0005%

+++

0.05%



Ref (Costa et al., 2009) (Gião et al., 2010) (Simon et al., 2000) (Simon et al., 2000) (Sangkitikomol, 2012) (Gião et al., 2010) (Gião et al., 2010) (Sangkitikomol, 2012) (Gião et al., 2010) (Gião et al., 2010) (Gião et al., 2010) (Gião et al., 2010) (Sowndhararajan et al., 2010)


175

Figure 3. Optical microscopy images of RBCs population incubated with savory, with and without AAPH.

The authors suggested that green tea polyphenols could be capable of trapping the initiating radicals of AAPH and inhibit RBC membrane lipid peroxidation, hence protecting these cells from hemolysis. In another study performed by Zhang et al. (1997), it was demonstrated that EGCG and EGC possess a higher anti-hemolytic activity in vitro (above 85% inhibition at 20µM, over AAPH) than ECG or EC; nevertheless, all the epicatechin isomers, in combination or separately, demonstrated to be effective antioxidants both in vitro and in vivo protecting RBC membrane to oxidation. EGC and EC were the only compounds found in blood after the ingestion of green tea polyphenols extracts. Analysis to membrane lipid content revealed that all four epicatechin isomers significantly prevented loss of arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) compared to RBCs incubated with AAPH, suggesting that hemolysis was associated with a significant decrease in these polyunsaturated fatty acids. Sangkitikomol et al. (2012) reported that among the 30 plants and fruits tested in their research, C. sinensis presented one of the highest protection rates. Moreover, Schmitz et al. (2008) results indicated that in a concentration of 0.54 mg/mL, green tea extracts inhibited 81.6% of AAPH-induced hemolysis, thus demonstrating to


176


possess an excellent anti-hemolytic capacity. Costa et al. (2009) also reported the antioxidant capacity of green tea extracts upon AAPH radicals, by inhibiting hemolysis in more than 50% at a concentration of 25 µg/mL, further suggesting that polyphenols may be interacting with the membrane bilayer – decreasing its fluidity and therefore the diffusion of free radicals into the cell membranes and its consequent damaging effects. Basu et al. (2003) reported that the obese participants of their study ingesting green tea (4 cups of 8oz boiled tea per day or 2 capsules) for 8 weeks had an increase in blood glutathione (1783 to 2395 μg/g), hence suggesting that green tea may provide antioxidant protection to cells. In other study published by Coimbra et al. (2006), 30 human subjects had their RBCs membrane analyzed after 4 weeks drinking 1L of green tea daily. The authors found a significant reduction in the oxidative stress within the erythrocyte, as suggested by a significantly lower value of membrane binding hemoglobin (a parameter of oxidative stress within the RBCs) and by changes in band 3 profile towards a normal mean profile, namely an increase in the band 3 monomer (directly related with changes in RBC cytoskeleton and therefore in its shape). The results led the authors to conclude that drinking green tea has beneficial effects, such as reducing oxidative stress and, therefore, protecting the individual from oxidative stressderived diseases. Ginkgo biloba L. has also been subjected to several assays involving RBC hemolysis. The leaves of this tree have been used in Chinese medicine for thousands of years. Today it has become one of the most popular and commonly used herbal remedies due to its wide spectrum of beneficial effects on health. Liu et al. (2009) tested the anti-hemolytic potential of G. biloba leaf extracts and its main components – flavonoids (mainly quercetin, kaempferol, isorhamnetin and their glycosides) and terpenes (ginkgolides A, B, C and J as well as bilobalide). Both ginko extract and its flavonoids demonstrated to significantly inhibit hemolysis, while terpenes didn’t seem to have any effect. He et al. (2008) reported a dual effect of G. biloba leaf extract on the amyloid peptide (Aβ25-35)-induced hemolysis. In the presence of G. biloba leaf extract, the hemolytic rate was inhibited by 70% at a dose of approximately 25 μg/ml. Higher doses of G. biloba leaf extract increased the hemolytic rate significantly. In addition, He et al. also demonstrated that G. biloba leaf extract increased the hypotonic resistance of red blood cells (decreasing hemolysis rate), or decreased membrane fragility in the hypotonic environment. Furthermore, the author also reported an increase in GSH, as well as a protective effect over hemoglobin oxidation. Huang et al. (2009) published the results of their research involving 25 type-2 diabetes patients with retinopathy, taking 240 mg of dry extract of G. biloba leaves. Conclusions suggested that there was an improvement in RBC deformability as well as in viscoelasticity, which is compatible with lower RBC lysis, as well as a decrease in membrane lipid peroxidation. However, the pro-oxidant effects found for certain concentrations, as well as the lack of conclusive results in some research works, turns it necessary to have further studies on G. biloba extracts. Aloe vera L. has been used as a traditional medicine and as an ingredient in many products due to its vast reported antioxidant properties. Among A. vera constituents, barbaloin and its aglycone aloe-emodin are the major constituents of leaves (Patel et al., 2012). The content of barbaloin in the juice of aloe leaves was reported to be 15-40%. Mazzula et al. (2012) demonstrated the antioxidant activity of this plant by maintaining RBCs membrane integrity when exposed to AAPH; A. vera (25-100 µM) not only prevented RBCs lysis – avoiding proteolytic degradation of band-3 anion exchanger membrane protein, as it also prevented the consumption of the cytosolic antioxidantGSH by AAPH. Singh et al.



177

(2000) also reported an increase of several cytosolic enzymes involved in RBC defences against oxidative damage – SOD, GPx and CAT, when mice were fed with fresh leaf pulp extract of Aloe vera (30 µl and 60 µl/day/mice daily for 14 days). Furthermore, Lam et al. (2007) demonstrated that barbaloin prevented several RBC membrane changes, such as lipid peroxidation, protein crosslinking and sulfhydryl group oxidation induced by AAPH or by hemin (a trivalent ferric oxidant which also exists in vivo); moreover, compared with trolox, barbaloin showed to possess higher effectiveness protecting RBCs from AAPH-induced hemolysis (trolox IC50 = 59.2 µM; barbaloin IC50 = 31.7µM) (Dai et al., 2006). The protective effects upon RBCs have also been studied upon fruit trees and some vegetables, which in some cases do not fit the common designation of medicinal plants. Olive tree leaves have deserved in the last decade numerous studies upon its capacity to protect RBCs from oxidative stress (Garcia et al., 2013). The classes of phenols present in olives and olive tree leaves are phenolic alcohols (such as hydroxytyrosol), secoiridoids, flavonoids and lignans. Secoiridoids are the most representative class in olives with the glycoside oleuropein being present up to 14% of the dry weight. Paiva-Martins and colleagues have published a group of papers demonstrating that several olive polyphenols (hydroxytyrosol , oleuropein and its hydrolysis products) can significantly protect RBCs from oxidative damage in a dosedependent manner (from 3 to 80 µM) (Paiva-Martins et al., 2009; Paiva-Martins et al., 2010; Paiva-Martins et al., 2013). All the compounds showed capacity to significantly prevent hemolysis induced by AAPH and by H2O2, as well as prevent hemoglobin oxidation and changes on RBC protein membrane profile induced by the same oxidative agents. Extracts of onions (Allium cepa L.) have also been accounted for beneficial effects upon RBCs (Jaiswal et al., 2001). The layers of onions are a rich source of flavonoids, consisting mainly quercetin-3,4´-O-diglucoside and quercetin-4´-O-monoglucoside. Jaiswal & Rizvi (2001) reported a significant protection of oxidative stress by onion outer layers extract on RBCs subjected to tert-butyl hydroperoxide treatment (hemolysis inhibition around 40%), as well as an increase in GSH levels; the results are more pronounced for outer layers as compared to the inner layers due to variation in the quantities of quercetin according to layer position. Leelarungrayub et al. (2004) reported that shallot (Allium ascalonicum L.) possess the capacity to inhibit RBC hemolysis induced by H2O2 (around 80% by hexane-extract shallot and only about 20% by water-extract shallot, at 1.0 mg/mL); additionally, the hexane-extract shallot (200µg/mL) also reduced GSH oxidation and lipid peroxidation induced by AAPH. Pharmacological studies of garlic extracts and its components, such as S-allylcysteine, have revealed capacity to improve peripheral circulation, protect vascular endothelial cells from oxidant injury or reduce plasma lipids. For this reason, results attained with garlic led to the publication of a group of papers on its anti-hemolytic activity (Moriguchi et al.2001; Morihara et al.2007). Moriguchi et al. (2001) has reported that garlic extract prevents the decrease of erythrocyte deformability induced by lipid peroxidation in a dose-dependent manner; also, the authors stated that garlic significantly suppressed FeSO4-induced hemolysis, as well as significantly suppressed the hemolysis rate even without induction of peroxidation – this results led the author to suggest that garlic extracts may protect RBC membranes by mechanisms other than protection from lipid peroxidation, such as the stabilization of erythrocyte membranes. Morihara et al. (2007) demonstrated in his research that garlic extract [0.14-0.57% (w/v)] was able to reduce peroxynitrite-induced hemolysis in a concentrationdependent manner, as well as S-allylcysteine (1-10 mM dose-dependently) [56]. In summary, the investigation on garlic led the researchers to conclude that it improves microcirculation


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and rheological blood properties and conserves the structure and role of RBCs, not only through an antioxidant process but also via the glycolysis pathway and membrane stabilization of RBCs.

Conclusion Many common plants contain diverse therapeutic bioactive ingredients that may turn out to be valuable in the future as a primary or as supplemental therapies, when carefully applied. However, the use of several medicinal plants is often empiric and unreasonable due to lack of information. Therefore, research in this area should be carried on until the agents responsible for the activity are determined or, as the case may be, the most active fraction or extracts have been discovered, as well as perfectly defined the range of secure concentrations. Among the several therapeutic areas where medicinal plants may play an important role in the future, the use of some of the plants with antioxidant activity mentioned in this chapter might be of great matter as support therapy against certain medical blood conditions, such as sickle cell anemia, glucose-6-phosphate dehydrogenase deficiency, and congenital dyserythropoietic anemias, or even to retard red blood cells aging.

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Chapter 7

Health Attributes, Antioxidant Properties and Phytochemical Composition of Traditional Medicinal Plants from Eastern Anatolia Izabela Konczak,1,* Abdullah Dalar2 and Konrad A. Konczak-Islam3 1

2

CSIRO Animal, Food and Health Sciences, North Ryde, NSW, Australia YüzüncüYıl University, Faculty of Science, Department of Biology, Van, Turkey 3 New South Wales University, Kensington, NSW, Australia

Abstract Anatolia - the westernmost protrusion of Asia, is at the forefront of the world richest sources of plant species. The mountainous and strongly fragmentized area is a home to over 11,000 plant species, of which 30% are endemic. Many of the world’s contemporary staple foods originated here. The extensive daily use of local plants for foods and medicine in Eastern Anatolia continues today and traditionally used plants outnumber the conventional sources of plant-based foods. Endemic plants are utilized daily in preparation of main meals, in salads and as herbal teas. They are used internally (e.g. herbal tea) and externally (e.g. poultice, decoction, ointment) to cure a number of ailments. This chapter presents the most frequently used traditional plants from the Eastern Anatolia and describes their uses, phytochemical compositions and antioxidant capacities. Their applications in ethnopharmacology in light of scientifically proven physiological activities are discussed.

Keywords: Eastern Anatolia, traditional remedy, medicinal plant, ethnobotanical, antioxidant, enzyme-inhibitory activity

*

Corresponding Author address: Email: [email protected]; [email protected].


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Izabela Konczak, Abdullah Dalar and Konrad A. Konczak-Islam

Introduction Traditional medicinal plants, also known as herbal medicines, botanical medicines or phytomedicines, refer to medicinal products obtained from plant roots, stems, leaves, bark, seeds, fruits and flowers that can be used to promote general health and treat diseases. These herbal materials are used directly in a prescription formulation or processed into various ready-to-use products (Tang and Halliwell, 2010). Traditional medicinal products constitute multi-billion-dollars industries worldwide and are becoming increasingly popular as a cost effective alternative, or complementary, to conventional medicine. Traditional medicines have been and continue to be used in every country around the world in some capacity. In the developing world, 70-95% of the population depends on traditional medicine for primary health care needs (Robinson and Zhang, 2011). In the developed world the interest in natural pharmaceuticals steadily increases and plant-based pharmaceuticals are seen as supportive medicine or an alternative to conventional medicine with fewer side effects. However, to utilize the traditional knowledge in the development of plant based pharmaceuticals, the identity of active components, their efficacy and their safety has to be investigated. The use of herbal products as a medicine, based on a traditional knowledge generated over centuries, is still the dominant way of curing disease in the eastern part of Turkey. With regards to floristic diversity, Turkey is one of the richest countries in the temperate world. The Turkish flora is estimated to contain more than 11,000 specific and intraspecific taxa of higher plants of which about 3000 are endemic. This exceeds the total number of endemic species found in Europe. With this exceptional diversity Turkey is the centre of genetic resources of many cultivated forms of annual and perennial, herbaceous and woody plants (Ağaoğlu et al., 1997).

Ethnobotanical Medicine in Turkey – Historical Perspective The use of wild plants for medical purposes in Turkey is a long tradition. However, the number of studies capturing these uses are limited. Early studies presented a list of plants used in various regions of Turkey and their application to cure health disorders (Başar, 1972; Baytop, 1984; Cubukcu and Ozhatay, 1987). In other studies the local uses of plants were reported primarily as a part of floristic studies (Tonbul and Altan, 1989). In the 1990’s the research teams of E. Yesilada and E. Sezik of the Gazi University, Ankara, Turkey, in collaboration with M. Tabata’s team of Kyoto University, Japan, carried out the first, extensive, systematic surveys with the objective of recording the local knowledge of medicinal plant uses across Turkey. This invaluable contribution of both research centers resulted in a number of consecutive research papers listing traditional medicinal plants and methods of preparation and administration in the treatment of medical conditions in the following regions of Turkey: north-eastern Anatolia (Sezik et al., 1991), Kastamonu province (Sezik et al., 1992), Mediterranean region (Yesilada et al., 1993), Black Sea region (Fujita et al., 1995), Western Anatolia (Honda et al., 1996), Southern Anatolia (Taurus Mountains region) (Yesilada et al., 1995), Eastern Anatolia, Van and Bitlis provinces (Tabata et al., 1994), Eastern Anatolia, Erzurum, Erzı̇ ncan, Aǧri, Kars, Iǧdir provinces (Sezik


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et al., 1997), Central Anatolia (Sezik et al., 2001), and north-west Anatolia (Yesilada et al., 1999). The information obtained through these extensive field studies has been used to create the first comprehensive “Data Bank of Turkish Folk Medicine”, which describes plantoriginated remedies obtained from 1011 plants belonging to 107 plant families (Yesialda, 2001). A few years later the number of plant species used in Turkey as folk remedy has been estimated to be around 1500 (Yesilada, 2005). According to Yesilada (2001) the most frequently employed plant families in traditional Turkish ethnic medicine are the Lamiaceae, Rosaceae and Asteraceae families. The frequency of application of traditional medicinal species is presented in Figure 1.

Figure 1. The frequency of application of commonly used ethnobotanicals of Turkey in traditional remedies. (According to Yesilada, 2001).

The most commonly used medicinal herbs recorded by Yesilada (2001) were Plantago species (P. major and P. lanceolata), applied in 371 remedies, Rosa species (R. canina and R. montana; 354 remedies) and Urtica species (U. dioica and U. urens; 327 remedies). A vast number of remedies have been developed to treat similar health problems, for example 1149 remedies were recorded for dermatological problems, 601 for respiratory tract illnesses, 466


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for skeleto-muscular problems, 425 for urological ailments, 360 for parasitic and microbial infections, 278 for nervous system related conditions, 176 for endocrine and metabolic diseases, 174 for immune system and neoplastic diseases, 120 for genital system problems and 36 remedies for the haematopoietic and lymphoid systems (Yesilada, 2001). Frequently the same plant or plant parts were used individually or in combination in the preparation of different remedies, and the same plant was often used to treat various conditions in different parts of the country.

Eastern Anatolia – A Pot of Gold for Medicinal Plants Eastern Anatolia is the largest (approximately 170 000 km2) of seven geographical divisions of Turkey with 14 provinces, bordering Iraq, Iran, Nakhichevan, Armenia, and Georgia (Figure 2); it is included in the Irano-Turanian phytogeographical region, covered with deciduous-mixed forests and deciduous tree steppes (Altundag and Ozturk, 2011). The area is surrounded by coastal mountain ranges, which protects it from the moderating effect of sea breezes. Subsequently, the winters are cold and long, with snow lasting for several months. They are followed by very short and rainy springs, and hot and dry summers. The mountainous and strongly fragmentised area offers numerous microclimatic and ecological zones, which provided suitable conditions for the development of diverse flora (Tan, 1998; Özgőkçe and Özçelik, 2004). The most important floral biodiversity centers in Eastern Anatolia are Munzur and Anti Taurus Mountains, Elbistan-Darende, Kemaliye, Kesis Mountain, and Harput and Hazar Lake (Altundag and Ozturk, 2011).

Figure 2. Geographical location of the Eastern Anatolia region of Turkey (in black). Photograph licensed under the Creative Commonsa Attribution 3.0 Unported license by The Emirr. (http://commons.wikimedia.org/wiki/File:Latrans-Turkey_location_Eastern_Anatolia_ Region. svg).

The Eastern Anatolia region is regarded as one of the richest areas of plant biodiversity in Turkey with over 3,000 vascular plant taxa, of which nearly 800 are endemic. The local population has a comprehensive knowledge of traditional herbal medicines which are commonly used to prevent or to treat disease. This knowledge was developed over centuries,



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when Anatolia served as a passageway between the continents of Europe, Asia and Africa. A variety of flora, fauna and cultures owe their geographical spread to this passageway. Over many centuries a number of races and tribes originated from distant lands settled in Turkey, bringing knowledge of plants and their various uses. According to Yesilada (2005), a number of plant remedies used currently in Turkey were described on clay tablets that have survived from the Mesopotamian civilizations, i.e., Sumerians, Assyrians and Akkadians, and Hettites, and on papyrus documents from the Egyptians. This heritage, combined with the local flora, has contributed to an extensive use of plants in daily life as a food and medicine (Coşkun and Gençler Özkan, 2005). The knowledge of herbal medicines in this region is still mostly passed on orally (with the exception of a few codices) from one generation of religious healers (referred to as‘şeyh’) and local healers (‘hekim’) to the next. The utilization of local plants for medical purposes in Anatolia has a long history. According to Solecki (1976) human fossils from the Middle Paleolithic age (about 60,000 years ago) discovered in the Shanidar cave, Kurdish Alps, were surrounded by an extraordinary amounts of plant pollen, later identified as Achillea (milfoil or yarrow), Senecio (groundsel or ragwort), Centaurea solstitialis (St. Barnaby’s thistle), Centaurea cyanus (cornflower, blue bottle, or blue bonnet), Liliaceae (or Muscari type, grape hyacinth), Zphedra altissima (joint pine or woody horsetail) and Althaea (hollyhocks). The author emphasize the importance of this finding, citing it as evidence that the relationship between humans and plants in this area has developed over a prolonged period of time. Dioscorides Pedanius (circa 40-90 AD), originally of Anazarbus in southern Anatolia, and a surgeon with the army of the Roman Emperor Nero, wrote the book “Materia Medica”, recognised as the precursor to all modern pharmacopeias. In his book Pedanius described around 600 plants used for medical purposes by the Greeks, Romans and other ethnic groups. As established by Guenther (1934), the majority of the described plants had originated from the Anatolian peninsula. Most of these plants are still in use by the local inhabitants of Anatolia (Yesilada and Sezik, 2003) and it has been suggested that Materia Medica could be the oldest comprehensive document on Anatolian folk medicine (Yesilada, 2005). The continual use of the same medicinal plants for over two thousand years emphasizes the value of an empirical tradition based on trial and error. Ethnobotanical surveys undertaken by Tabata and collaborators in the 1990’s and data published by other authors have revealed that a large number of medicinal plants are found in the states of Agri, Ardahan, Bingöl, Bitlis, Elazig, Erzincan, Erzurum, Hakkari, Igdir, Kars, Malatya, Muú, Tunceli and Van. In the Van and Bitlis provinces alone 40 medicinal plants belonging to 19 families are commonly used (Tabata et al., 1994), while in the Erzurum, Erzı̇ ncan, Aǧri, Kars and Iǧdir provinces 87 plant species belonging to 38 families are used (Sezik et al., 1997). Recent study by Tetik and collaborators reported the use of 123 medicinal plants with 15 being recorded for the first time in the Malatya province (Tetik et al., 2013). In Konalga (Êzdînan-Martanis), an ancient village of Eastern Anatolia, 211 plant taxa are currently in use, with 87 used as food and 87 used for medical purposes (Mükemre, 2013). This chapter aims to capture the historical and traditional knowledge in light of the most recent research reports and findings, presenting an overview of traditional medicinal plants from Eastern Anatolia (especially from the Van province), their uses, known phytochemical compositions and antioxidant capacities. Their application in ethnopharmacology in light of scientifically proven physiological activities are also discussed.


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Collection, Preservation and Application of Traditional Medicine in Eastern Anatolia The majority of traditional medicinal plants until now are collected from the wild. Some endemic plants grow in hardly accessible mountainous areas and can only be collected on dedicated trips. Fresh plants are gathered during summers, dried in the dark at room temperature and stored for winters. Traditionally, drying enabled local people to use medicinal plants throughout the year. Today the herbs can also be purchased from local herbspice stores (aktars); fresh plants are available from spring to autumn, and dried during winters. Depending on the source, the whole plant or its individual parts, including leaf, shoot, stem, branch, bud, bark, flower, fruit, seed, stylus, root, bulb, tuber, cortex, latex, juice, root sap and gum are utilized for the preparation of herbal remedies. The simplest way of applying herbal remedies is a direct application of fresh or dried plant parts. Direct application includes an oral uptake and external use. According to Tabata and collaborators 45% of medicinal preparations of Eastern Anatolia are used directly (Tabata et al., 1994). Direct application of fresh plant material on the skin is the main external use of herbal remedies presented in this study. This includes placing an intact plant or its parts (e.g. whole leaf) on skin, or macerated prior to the use, and used as poultice spread on cloth over the skin. The remedy can be also made by boiling herbs in a small amount of water and making a decoction, which is applied on skin or used in a bath. Dry plant powder or macerated fresh plant material mixed with a carrier can also be used to prepare an ointment. Frequently the same plant can be applied externally in various ways: pounded and placed directly on skin, in a bath or as an ointment. For example, different part of Juglans regia (seed, leaf, fruit, bark and cortex) are eaten fresh, placed fresh on the skin, used to prepare an ointment, pounded and applied directly on skin, or used to prepare a poultice that is placed on a cloth and wrapped around the injured area (Kaval, 2011; Özgökçe and Özçelik, 2004; Tabata et al., 1994; Tuzlacı and Doğan, 2010). From 182 traditional medicinal plants from Eastern Anatolia described in this chapter 68 plants (37.4%) are used externally, with poultice being the most frequent way of application (14.8%), closely followed by application of a fresh plant (12.6%) and decoctions, which are used externally on skin (7.2%) or in bath (3.3%). Most of the species are used in more than one form of preparation (e.g. decoction or ointment) and often the same preparation is utilised in different ways (for instance, the decoction as drink, compress or bath). Ointments have the lowest application rate of 5.5%. Tabata and co-workers reported earlier that applications of ointment can be made with Vaseline, honey, butter, filtered yoghurt, alum, or even flour mixed with soap (Tabata et al., 1994). Honey and butter are the most frequent applied carriers of plant constituents. Rarely, juice or latex (extracted by squeezing plant parts) is used externally (< 1%). In one case, plant (Eryngium billardieri) juice is used for sniffing to quit smoking. The remedies are used externally to treat rheumatism, hemorrhoids, burns, abscesses, wounds, eczema, fungal infections, asthma or even cancerous uterus (Plantago lanceolata and Plantago maritime). Taking a medicated bath is especially practiced for the treatment of rheumatism and cold.



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In an earlier study by Tabata and collaborators liquid preparations (decoctions or infusions) were reported to be the second most common way of medicine application in the Eastern Anatolia with a frequency of 39% (Tabata et al., 1994). Recently Kaval (2011) reported that 43.3% of medicinal preparations are used as decoction and 16.6% are used as a fresh plant material. In the current study we have observed an increase of liquid preparations for internal use to 52.7%. Liquid preparations are used to treat a vast variety of conditions, from gastrointestinal problems, including abdominal pain, stomach ache, stomach cancer and constipation, through to treating diabetes, hypertension, hypercholesterolemia, hemorrhoids, kidney disorders, epilepsy and boosting the immune system. Dalar and Konczak (2013) reported that herbal infusions are especially common in the Eastern Anatolia and their consumption is an essential part of a lifestyle, with their primary function being to warm the body during long cold winters and cool summer’s days of the mountain climate. Traditionally as much as 10 to 15 cups of various herbal infusions were consumed per day, which made the herbal teas the main dietary beverage. Although currently the black tea of Camellia sinensis leaf is also used, consumption of herbal teas is still preferred due to strong beliefs in their medicinal properties. Herbal infusions are prepared using predominantly leaves, flowers, complete aerial parts and fruits. A quarter of the medicinal herbs (25.8%) identified in this study are consumed fresh. These plants are employed multi-contextually, as medicine and food, and frequently there is no clear border between their use as food and as medicine. For example, aerial parts of Malva neglecta are used for preparation of salad, fresh leaves are mixed with yogurt and used as an appetizer or they are cooked and included in main meals - as ingredients of soups and omelets (Dalar et al., 2012). Some herbs are used as flavouring agents in cakes, and occasionally in liqueurs and in desserts (as spices).

Traditional Medicinal Plants of Eastern Anatolia: Current Status The list of common examples of ethnobotanical medicines utilized daily in the Eastern Anatolia to cure common and minor ailments, including the plant parts used, ways of herbal remedy preparation, application and medical conditions treated, is presented in Table 1. The 182 plants listed represent 47 families. The most frequently represented plant family is the Asteraceae (formerly Compositaceae) with 36 species, followed by the Lamiaceae (formerly Labiatae) with 21 species, Apiaceae with 12 species, Malvaceae with 11 species and Rosaceae with 8 species. Plants representing these five families make up 48.9% of all the plant sources utilized as traditional medicine presented in this chapter. Half of the traditional medicinal plants listed (91 plants) are used to treat only one ailment, 41 plants (22.5%) are used for two different ailments, and there are 24 plants (13.2%) that are used to treat three ailments. The remaining 14.3% of plants are applied to cure four or more ailments, with the most versatile being Mentha longifolia L., Lamiaceae, used to treat 15, followed by Cichorium intybus L., Asteraceae and Malva neglecta Wallr., Malvaceae, both used for the treatment of 12 and Urtica dioica L., Urticaceae, used to treat 10 ailments.


Table 1. Traditional application of medicinal plants in Eastern Anatolia Family/Species name AMARANTHACEAE Amaranthus retroflexus L. ANACARDIACEAE Pistacia atlantica Desf.

Local names

Plant part

Therapeutic effect/ailments treated

Application

References

Horoz ibiği

LF

Sterility

Infusion

Özgökçe and Özçelik, 2004

Kizvan, nervend

LX, FR (immature) GM FR

Keratoma, wound healing, mouth diseases Stomach disorders Common colds, diuretic, urinary inflammations Burns, antiseptic, diarrhoea

Chewing

Tuzlacı and Doğan, 2010

Pistacia khinjuk Stocks Pistacia terebinthus L.

Gezan, Menegiç, çedene

Eaten fresh Decoction (int.)

Kaval, 2011 Özgökçe and Özçelik, 2004; Cakilcioglu et al., 2010 Tabata et al., 1994; Cakilcioglu et al., 2010; Cakilcioglu et al., 2011

Rhus coriaria L.

Sumak

FR+SD, FR(mature)

APIACEAE Anethum graveolens L.

Sıbıt

AE

Hypercholesterolemia

Anthriscus slyvestris (L.) Hoffm. Diplotaenia cachrydifolia Boiss. Eryngium billardieri Delar.

Gımıgımi Siyabu Tüsü, gelenk

Carminative Rheumatism, diabetes Antifungal, stomach ache, bronchitis, sinusitis, catarrh, wounds, quit smoking, abscesses,

Eryngium bornmuelleri Ferula orientalis L.

Tusi Heliz, çağşir, Kıngor,

FR RT RT, BR, WP, GM RT, AE RS, LX, WP

Ferula haussknechtii Wolff. ex. Rech f. Foeniculum vulgare R. Mill. Grammosciadium platycarpum Boiss. &Hausskn. Heracleum antasiaticum Manden Heracleum persicum Desf.

Kermeğ Rizyane Rizyane

RS SD AE

wound healing Abdominal pain in children Stomach ache

Hellis Soy

LF AE

Wound healing Diabetes

Johrenia dichotoma DC. subsp. sintenisii Bornm

Serzer

AE

Wound healing

Toothache, stoma cancer Wormy wounds, eczema, stomach ache, fungal infection, Immunostimulant,

Decoction (ext.), infusion (int.)

Eaten fresh, decoction Decoction (int.) Decoction (int.-ext.) Fresh (on wounds), decoction, eaten fresh (after peeling), juice (sniffered) Infusion (int.) Fresh (on wounds), decoction (int.), infusion (int.), poultice (ext.) Fresh (on wounds) Powder Decoction

Kaval, 2011 Altundag and Ozturk, 2011 Kaval, 2011 Kaval, 2011; Tabata et al., 1994; Sezik et al., 1997

Mükemre, 2013 Kaval, 2011; Tuzlacı and Doğan, 2010, Mükemre, 2013 Kaval, 2011 Kaval, 2011 Kaval, 2011

Crushed (ext.) Tetik et al., 2013 Eaten fresh, Mükemre, 2013 decoction (int.) Powder ( on wounds) Kaval, 2011


Family/Species name Prangos pabularia Lindl. ARACEAE Arum dentrucatum C.A Mey. Ex Schott var. vidensis. ARISTOLOCHIACEAE Aristolochia bottae Jaub. & Spach.

Local names Kerkule

Plant part RS

Therapeutic effect/ailments treated Wound healing

Application Fresh ( on wounds)

References Kaval, 2011

Karibel

RT

Diabetes

Decoction (int.)

Özgökçe and Özçelik, 2004; Altundag and Ozturk, 2011

Guhkıtık

RT, AE

Wound healing, erythematic,

Poultice (on wounds), ointment (poultice mixed with honey)

Kaval, 2011

Zilasur, zehir otu

BD, FR, LF BD

Scabies, fungal infection Scabies

Pounded (ext.), crushed (ext.) Pounded (ext.)

Özgökçe and Özçelik, 2004; Tuzlacı and Doğan, 2010 Tabata et al., 1994

FL, LF, AE FL, LF, WP

Stomach ache, carminative, expectorant, Infusion (int.) colds Diuretic, urinary antiseptic, Decoction (int.), antispasmodic, antianemic, menstrual pounded (ext.) disorders, stomach ache, colds, astringent

ASCLEPIDACEAE Vinetoxicum canescens (Wild.) Decne. subsp. canescens Vinetoxicum tmoleum Boiss. ASTERACEAE Achillea biebersteinii Afan.

Arı çiçeği, bovijan

Achillea millefolium L. subsp. millefolium

Civan perçemi, herezan

Achillea vermicularis Trin

Civan perçemi, bovijan Papatya Papatya Sarı papatya Sarı papatya Nuserk, top telli, kriz, loşlek

WP, AE AE AE FL FL LF, FL, RT

Swelling of stoma, colds, stomach disorders Common cold Common cold Diuretic, stomach ache Diabetes, throat diseases Haemorrhoids, inflammation, eye diseases, swelling of stomach ache, abscesses, sun stroke

Loşlek

LF

Sunstroke

Top telli,

LF, RT AE

Swelling of stomach, abscesses, Dyspnoea and diabetes

Anthemis austriaca Jacq. Anthemis cotula L. Anthemis nobilis L. Anthemis tinctoria L. var. tinctoria Arctium minus (Hill.) Bernh. subsp. pubens (Bab.) Arenes

Arctium minus (Hill.) Bernh. subsp. minus Arctium tomentosum Miller var. glabrum (Körnicke) Arenes Artemisia absinthium L.

Zilasur

Bevüjana kuvi

Tuzlacı and Doğan, 2010; Mükemre, 2013 Özgökçe and Özçelik, 2004; Cakilcioglu et al., 2010; Cakilcioglu et al., 2011; Altundag and Ozturk, 2011 Decoction (int.), Özgökçe and Özçelik, 2004; infusion (int.) Mükemre, 2013 Decoction Kaval, 2011 Decoction Kaval, 2011 Infusion (int.) Özgökçe and Özçelik, 2004 Decoction (int.) Cakilcioglu et al., 2010 Fresh (ext.), red parts Kaval, 2011; Özgökçe and pounded, juice, Özçelik, 2004; Tabata et al., boiled with milk 1994; Sezik et al., 1997 (ext.) Direct application Altundag and Ozturk, 2011 (ext.) Boiled with milk Özgökçe and Özçelik, 2004 (ext.) Decoction (int.) Kaval, 2011


Table 1. (Continued) Family/Species name Artemisia spicigera C.Koch.

Local names Giyabend

Plant part AE

Bellis perennis L. Centaurea karduchorum Boiss. Centaurea glastifolia L.

Koyun gözü, Güya brinok Tahlisk

FL WP AE

Centaurea iberica Trev. ex Spreng. Centaurea pterocaula Trautv. Centaurea virgata Lam.

Kelembeşk Şermnik Şaladir

LF LF WP

Centaurea solstitialis L. subsp. solstitialis Cichorium intybus L.

Belhok, zerik

AE

Kaniş, talişk, hindiba, kanej

LX, AE, RT

Circium sp. Cirsium pubigerum (Desf.) DC. var. spinosum Pet. Gundelia tournefortii L. var. tournefortii

Kilindor Kivariavi

RT RT

Kenger

Helianthus tuberosus L. Helichrysum armenium D.C. subsp. armenium Helichrysum plicatum DC. subsp. plicatum Inula helenium L. S ubsp. vanensis Grierson Inula helenium L. subsp. pseudohelenium Grierson

Therapeutic effect/ailments treated Rheumatism, headache, cough, stomach diseases Diarrhoea, diuretic, purgative Wound healing Prostate

Application Decoction (ext./int.)

References Kaval, 2011 Özgökçe and Özçelik, 2004 Mükemre, 2013 Kaval, 2011

Venom Diarrhoea Stomach ache, wound healing, antiallergic Malaria

Infusion (int.) Powdered (ext.) Decoction (then dry) (ext.) Eaten fresh Decoction (int.) Decoction (ext.), ash, + butter (ointment) Decoction (int.)

Abdominal pain, epilepsy, prostate, hypertension, haemorrhoids, kidney disorders, depurative, hypercholesterolemia, hepatic, asthma, stomach ache, wound healing Haemorrhoids, internal diseases Swelling of the body

Eaten Fresh (mixed with water), decoction (then dry), boiled long time (int.), infusion (int.) Decoction (int.) Poultice (ext.)

Kaval, 2011 Kaval, 2011 Tabata et al., 1994; Altundag and Ozturk, 2011 Tuzlacı and Doğan, 2010; Altundag and Ozturk, 2011 Kaval, 2011; Özgökçe and Özçelik, 2004; Tabata et al., 1994; Tetik et al., 2013; Altundag and Ozturk, 2011; Mükemre 2013 Tuzlacı and Doğan, 2010 Kaval, 2011

SH, SD, LX, RT, ST

Vitiligo, cold, catarrh, aphrodisiac, diarrhoea, diabetes, orexigenic

Sevka ağı Guyazerk

BB AE

Diabetes Kidney stones, cholesterol, diabetes

Included in meals, coffee substitute, , decoction (int.), direct application (ext.) Eaten fresh Decoction (int.)

Özgökçe and Özçelik, 2004; Tabata et al., 1994; Tetik et al., 2013; Cakilcioglu et al., 2011; Altundag and Ozturk, 2011 Kaval, 2011 Mükemre, 2013

Gülülga zer, herdemtaze İngüz, peniruk

AE, SH, FL

Kidney stones

AE

Haemorrhoids

Kaval, 2011; Özgökçe and Özçelik, 2004 Kaval, 2011

Andız

RT

Chest pain

Decoction, infusion (int.) Ointment ( mixed with Vaseline/cream) Infusion (int.)



Family/Species name Lactuca saligna L. Lactuca serriola L. Senecio vernalis Scorzonera latifolia (Fisch. & C.A. Mey.) DC.

Local names Tehlişka geva Kaju Gurutik, kanok, nermend

Plant part BD AE BR+FL, AE LX, RT, WP

Therapeutic effect/ailments treated Hypertension Haemorrhoids, sinusitis Flatulence Pain, sterility, headache

Scorzonera tomentosa L.

Arvent

LX

Wound healing

Tanacetum argyrophyllum (C.Koch) Tvzel. var. argyrophyllum Tanacetum balsamita L. subsp. balsamitoides (Schultz Bip.) Grierson. Tanacetum chilliophyllum (Fisch. & C.A.Mey) Schultz Bip. var. chilliophyllum Taraxacum montanum (C.A. Mey.) DC. BERBERIDACEAE Berberis crataegina DC.

Bevüjan

AE

Diabetes

Gıyakeçk

LF

Wound healing

Bevüjan

AE

Pıtot

Zirinç

Application Eaten fresh Infusion (int.) Infusion (int.) Poultice, gum (ext.)

References Kaval, 2011 Tetik et al., 2013 Tuzlacı and Doğan, 2010 Kaval, 2011; Özgökçe and Özçelik, 2004; Mükemre, 2013 Ointment (with butter Tuzlacı and Doğan, 2010 and wax) Decoction Kaval, 2011 Kaval, 2011

Diabetes

Fresh ( as poultice ) Decoction

LX

Eye redness, wound healing

Fresh on skin

Kaval, 2011

Diabetes, colds, haemorrhoids

Altundag and Ozturk, 2011

Jaundince in animals Orexigenic Epilepsy

Eaten fresh, decoction (int.) Int. (by licking) Eaten fresh Decoction (int.)

Ointment (with butter and Pinus sp woodext.) Ointment (with butter, ext.) Eaten fresh, decoction (ext.) Ointment (with butter, ext.) Infusion (int.)

Sezik et al., 1997

Berberis vulgaris L. Bunias orientalis L. Leontice leontopetalum L. subsp. ewersmannii (Bunge) Coode BORAGINACEAE Alkanna megacarpa D.C.

Karamuk Galatürpenk Kırkbaş otu

FR, RT RT ST RT

Havaciva

RT (bark)

Wound healing, burns

Alkanna orientalis (L.) Boiss. var. orientalis Anchusa azurea Mill. var. azurea

Havacıva otu, hovacov Mıjmejok

RT

Wound healing

FL

Stomach ache, asthma, colds

Echium italicum L.

Deve dilli, goriz

RT

Wound healing

Heliotropium circinatum Griseb.

Bostan otu

BR+FL

Cancer


Kaval, 2011

Özgökçe and Özçelik, 2004 Altundag and Ozturk, 2011 Özgökçe and Özçelik, 2004

Özgökçe and Özçelik, 2004; Tabata et al., 1994 Mükemre, 2013 Özgökçe and Özçelik, 2004; Tabata et al., 1994 Tuzlacı and Doğan, 2010

Table 1. (Continued) Family/Species name Nonea pulla (L.) DC.

Local names Güzrik, mıjmıj

Plant part LF, RT

Therapeutic effect/ailments treated Venom, antiseptic

Application Eaten fresh, poultice (ext.)

References Kaval, 2011; Altundag and Ozturk, 2011

Keselmehmut, ğulik

Heartache, kidney pain, stomach disorders, diarrhoea, wound healing Wound healing, burns, vulnerary

Decoction, powdered (ext.) Ointment (with butter,), pounded (ext.) Moistened, with sugar (ext.) Decoction (int.) Infusion (int.) Powder (ext.), infusion (int.)

Kaval, 2011; Sezik et al., 1997 Tabata et al., 1994; Altundag and Ozturk, 2011

BRASSICACEAE Alyssum pateri Nyár. subsp. pateri Asymnea rigidum Grosh.

Nujdan

AE, WP LX

Brassica oleracea L.

Lahana

LF

Abscesses

Capsella bursa-pastoris (L.) Medik Cardamine uliginosa Bieb. Lepidium latifolum L.

Munzur otu Nujdar

AE AE LF

Diabetes Kidney stones Wound healing, vulnerary

Mayasıl otu

LF

Haemorrhoids

Boiled (ext.)


RT

Sterility

Boiled (int.)


Abdulselam, daraling, RT, juri ruvi, tırye ruvi FR

Stomach disorders, constipation,hypertension

Decoction, fresh, powder (int.)

Kaval, 2011, Mükemre, 2013

Dikenli ardıç

FR

Rheumatism

Decoction (ext. bath)


Topalak

RT

Diuretic

Infusion (int.)


Puk, buki

LX

Antiseptic

Tabata et al., 1994

Gevrik

LX

Antiseptic for wounds and cuts

Directly on skin (ext.) As above

Keringan, hoşil, huşil

LX

Constipation

Eaten fresh

Tabata et al., 1994

CARYOPHYLACEAE Telephium imperati L. subsp. orientale (Boiss) Nyman CRASSULACEA Umblicus erectus DC CUCURBITACEAE Bryonia multiflora Boiss. & Heldr. CUPRESSACEAE Juniperus oxycedrus L. CYPERACEAE Cyperus rotundus DIPSACACEAE Scabiosa sulfurea Boiss. et Huet Cephalaria sparsipilosa Matthews EUPHORBIACEAE Euphorbia sp.


Tabata et al.,1994 Tuzlacı and Doğan, 2010 Tuzlacı and Doğan, 2010 Tabata et al., 1994; Özgökçe and Özçelik, 2004; Altundag and Ozturk, 2011

Sezik et al., 1997

Family/Species name Euphorbia denticulata Lam.

Local names Hekletis

Plant part LX

Therapeutic effect/ailments treated Abdominal pain, diarrhoea

Euphorbia heteradena Jaub.& Spach Euphorbia macrocarpa Boiss. & Buhse Euphorbia macroclada Boiss.

Sütleğen

LX

Huşil

Euphorbia virgata Waldst. & Kit Tan EQUISETACEAE Equisetum arvense L. E. fluviatile L. FABACEAE Glycyrrhiza glabra L. var. glandulifera (Waldst. & Kit.) Boiss. Medicago sativa L. subsp. sativa Trifolium repens L. var. giganteum Lag-Foss. Trigonella foenum-graecum L. GERANIACEAE Pelargonium quercetorum Agnew GLOBULARIACEAE Globularia trichosantha Fisch. & Mey. HYPERICACEAE Hypericum perforatum JUNCACEAE Juncus inflexus L.

References Kaval, 2011

Constipation

Application Eaten fresh (with water) Eaten fresh

LX

For wound healing

Fresh (ext.)

Kaval, 2011

Sütleğen, yılan otu

LX

Eczema, fungal infection


Şilomali

LX

Constipation

Directly on skin (ext.) Eaten fresh (with sugar)


Kaval, 2011

Giyagezık, at kuyruğu AE, WP Getgedok AE

Urinary problems, diuretic Hypertension, kidney pain

Decoction, Infusion (int.) Decoction

Kaval, 2011; Özgökçe and Özçelik, 2004 Kaval, 2011

Mekik kökü, meyan, sus, meyan kökü

RT

Diabetes, heartache, cough, bronchitis, stomach ache, diarrhoea, digestive

Decoction (int.)

Hespıst Sebelk nefel

WH AE

Astringent Stomach disorders

Poultice (ext.) Decoction

Kaval, 2011; Özgökçe and Özçelik, 2004; Sezik et al., 1997; Tetik et al., 2013 Kaval, 2011 Kaval, 2011

Pıltan

SH

Hypoglycaemic

Pounded, decoction (int.)


Tolk

RT

Intestinal worm in children

Decoction

Kaval, 2011

Ahu

LF

Parasites

Infusion (int.)


Binbir delikotu, kantaron

WP, FL

Stomach ache, sedative

Decoction (int.), infusion (int.)

Özgökçe and Özçelik, 2004; Cakilcioglu et al., 2011

Pizak

RT

Kidney stones

Decoction

Kaval, 2011


Table 1. (Continued) Family/Species name JUNGLADACEAE Juglans regia L.

Local names

Plant part


Application

References

Güz, ceviz

SD, LF, FR, BA, CO

Haemorrhoids, bleeding, abscesses, wounds, hypoglycaemic, fungal infection, eczema

Eaten fresh (int.), ointment, (mixed with honey), pounded, poultice wrapped in a cloth (ext.)

Kaval, 2011; Özgökçe and Özçelik, 2004; Tabata et al., 1994; Tuzlacı and Doğan, 2010

LABIATEA Mentha longifolia (L.) Hud. subsp. longifolia

Püng, tüylü nane, punk, yarpuz, pünk

AE, LF, WP

Decoction [int./ext. Tabata et al., 1994; Kaval, (bath)], infusion (int.) 2011; Özgökçe and Özçelik, 2004; Tuzlacı and Doğan, 2010; Sezik et al., 1997; Cakilcioglu et al., 2011

Mentha pulegium L.

Püjan, havşan, nane

Decoction (int.)

Origanum acutidens (Hand-mazz) letswaart Mentha spicata L.

Zemul

WP, FL BR+FL

Common cold, flu, catarrh, heart palpitations, asthma, abdominal pains, internal diseases, menstrual pain, stomach ache, cough, bronchitis, headache, haemorrhoids, sunstroke, antispasmodic Gall bladder disorders Abdominal pains

Decoction (int.)

Tabata et al., 1994; Özgökçe and Özçelik, 2004; Tuzlacı and Doğan, 2010

Pünk, pune, nane

WP, AE, LF

Rheumatism, antispasmodic, colds, flu

Mercanköşk Catır, kuş zemulu Cantir, cantirik

ST WP, LF, BR+FL WP

Sedative, stomach ache Wound healing, stomaache, epilepsy, cold Stomach ache

Decoction [(int./ext. (bath)] Eaten fresh Decoction (int.), infusion (int.) Decoction (int.)

Tabata et al., 1994; Cakilcioglu et al., 2011 Özgökçe and Özçelik, 2004; Özgökçe and Özçelik, 2004; Tuzlacı and Doğan, 2010 Tabata et al., 1994

Canter, kekik otu

WP

Wound healing

Tabata et al., 1994

Prunella vulgaris L. Phlomis armeniaca Wild.

Sosın Belgsesing Şalba, çalba

Abdominal pain Antipyretic, colds, asthma, bronchitis

Phlomis pungens Wild. Salvia candidissima Vahl. subsp. candidissima Salvia multicaulis Vahl.

Şalba Galabor

AE WP, FL WP LF

Powdered, with honey (int.) Decoction Infusion (int.)

Cough Cold

Infusion (int.) Infusion (int.)

Kaval, 2011 Altundag and Ozturk, 2011; Mükemre, 2013 Altundag and Ozturk, 2011 Tuzlacı and Doğan, 2010

Adaçayı

AE

Diabetes

Decoction (int.)

Cakilcioglu et al., 2010

Origanum majorana L. Origanum vulgare L. subsp. gracile (C. Koch) letswaart Origanum vulgare L. subsp. viride Origanum vulgare L. subsp. vulgare


Family/Species name Salvia nemorosa L.

Local names Gemdaş

Plant part WP

Therapeutic effect/ailments treated Bleeding, wounds

Application Pounded (ext.)

Salvia sclarea L.

Dağ çayı

BR+FL, LF

Respiratory system diseases, cold

Salvia verticillata L.

Hart, ada çayı

LF

Catarrh, common colds, nausea

Infusion (int.), with lemon juice) Decoction (int.)

Saturea hortensis L. Teucrium chamaedrys L. subsp. sinuatum (Cèlak) Rech. F. Teucrium polium L.

Anıh, dağ kekiği BR+FL Keselmehmut, derman AE, WP Keselmehmut, LF, beyaz ot, AE, merve, neman, ST bovijana şin Zahter WP Çatıra kuvi AE

Immunostimulant Rheumatism, stomach disorders, poison, cancer Stoma disorders, kidney pain, cancer, haemorrhoids, diarrhoea, internal diseases, bleeding

Included in foods Decoction (int.)

Antiseptic Dyspnoea, stomach disorders

Eaten fresh Decoction (int,.)

Halbet

SD

Hypoglycaemic

Astragalus brachycalyx

Geven, keven

SH, GM

Immunostimulant, cancer

Medicago minima (L.) Bart. LILIACEAE Allium cepa L.

Gurnik

FR

Cardiac diseases

Pounded or decoction Tabata et al., 1994 (int.) Crushed with honey Tuzlacı and Doğan, 2010 (int.) Infusion (int.) Tuzlacı and Doğan, 2010

Sirmuk

Digestive, urinary inflammations

Eaten fresh

Tetik et al., 2013

Dağ sarmısağı

RT, LF BB

Hypotensive , cancer, ringworm

Crushed (int.)


Corin Yara otu

LF RT

Anaemia Wound healing

Altundag and Ozturk, 2011 Tuzlacı and Doğan, 2010

Gulik

RT, LF (juice)

Diabetes, fungal infection, eczema

Gül falcı

BD

Rheumatism

Eaten fresh Crushed with Plantago major leaves (ext.) Decoction (int.), crushed (ext.) Eaten fresh

Thymbra spicata L. Thymus kotschyanus Boiss. & Hohen var. kotschyanus LEGUMINOSAE Trigonella foenum-graceum L.

Allium macrochaetum Boiss. subsp. tuncelianum Allium sintenisii Freyn Asphodeline tenuior Ledeb. subsp. Tenuiflora E. Tuzlacı var. tenuiflora Eremurus spectabilis Bieb. Merendera trigyna (Steven ex Adam) Stapf.

Decoction (int.), eaten fresh, pounded (ext.)


References Sezik et al., 1997; Altundag and Ozturk, 2011 Tuzlacı and Doğan, 2010 Tabata et al., 1994; Özgökçe and Özçelik, 2004; Tuzlacı and Doğan, 2010 Tuzlacı and Doğan, 2010 Kaval, 2011; Özgökçe and Özçelik, 2004 Tabata et al., 1994; Kaval, 2011; Özgökçe and Özçelik, 2004; Sezik et al., 1997 Mükemre, 2013 Özgökçe and Özçelik, 2004 Kaval, 2011

Tuzlacı and Doğan, 2010 Özgökçe and Özçelik, 2004;

Table 1. (Continued) Family/Species name MALVACEAE Alcea apterocarpa (Fenzl) Boiss.

Local names

Plant part


Application

Huri

Anti-inflammatory, skin disorders, kidney stones, stomach ache, cough

Decoction [(int.-ext. Altundag and Ozturk, 2011 (bath)], infusion (int.)

Alcea calvertii (Boiss.) Boiss.

Hıra çiçeği, hayro

Kidney stone

Alcea dissecta (Baker) Zoh.

Govik

RT, SH, WP RT, WP LF

Infusion (int.), decoction (ext.) Poultice (ext.)

Alcea excubita Iljin Alcea fasciculiflora Zohary

Amervans Hatmi

FL, LF RT

Expectorant, cold Kidney stone, abscesses

Alcea flavovirens (Boiss. & Buhse.) Iljin Alcea kurdica

Heru, hiro, hero

RT, WP, LF

Kidney pain, urinary problems

Infusion (int.) Decoction (int.), residue (ext.) Decoction (int.)

Hero

Kidney ache, kidney stone, asthma

Decoction (int.)

Alceae setosa Alef. Alcea hohenackeri (Boiss. & Huet ) Boiss. Althea officinalis Malva neglecta Wallr.

Hatmi Hero

RT, LF LF LF, RT WP LF, AE, WP

Tabata et al., 1994; Kaval, 2011 Mükemre, 2013

Expectorant, diuretic, emollient Kidney pain, urinary problems, stomach disorders Diuretic, antilithic Kidney and bladder stone, abscesses, wound healing, stomach disorders, abdominal pain, peptic ulcer, common colds, infertility, bronchitis, indigestion, vaginal candidiasis

Infusion (int.) Decoction (int.)

Özgökçe and Özçelik, 2004 Kaval, 2011

Infusion (int.) Poultice (ext.), decoction [(int.-ext. (bath)], infusion (int.)

Özgökçe and Özçelik, 2004 Tabata et al., 1994; Kaval, 2011; Özgökçe and Özçelik, 2004; Sezik et al., 1997; Tetik et al., 2013

PAEONIACEAE Paeonia mascula (L.) Miller subsp. arietina (Anders.) Cullen et Heywood PLANTAGINACEAE Plantago atrata Hoppe

Hatmi Tolik, dolik, berberu, korkut, Tolgabadinga, Tolgaküvi, ebegümeci, hiru

Injury, asthma

References

Özgökçe and Özçelik, 2004; Altundag and Ozturk, 2011 Tuzlacı and Doğan, 2010; Altundag and Ozturk, 2011 Tuzlacı and Doğan, 2010 Özgökçe and Özçelik, 2004

Gulorç, gülhorç

AE

Diabetes

Infusion (int.), decoction (int.)


Sinir otu

LF

Wound healing

Fresh (ext.)



Family/Species name Plantago lanceolata L.

Local names Plant part Giyamambel, LF giyabironug, sinirotu, belhevzar, mambel, ilan dili, zimanugmari Belhevzar, belgbirim, LF belghavz, belghevizar, boğa yaprağı, amin

Therapeutic effect/ailments treated Stoma disorders, diabetes, haemorrhoids, wound healing, abscesses, recovery of incisions, cancerous uterus

Yılan dili

LF

Cancer of uterus

Decoction (ext.)

References Kaval, 2011; Özgökçe and Özçelik, 2004; Tabata et al., 1994; Mükemre, 2013 Tabata et al., 1994; Kaval, 2011; Özgökçe and Özçelik, 2004; Tuzlacı and Doğan, 2010; Sezik et al., 1997 Özgökçe and Özçelik, 2004

Mısır

SL

Antilithic, diuretic, haemorrhoids

Infusion (int.)


Revas, ribes, ışgın, uşkun, revas, rimbez, rives

RT, SD, SH, LF, ST

Diabetes, ulcer, diarrhoea, anthelmintic, haemorrhoids, digestion problems, stomach disorders, expectorant

Powder (boiled in water), decoction (int.), eaten fresh

Rumex acetosella L. Rumex cripsus L.

Tirşok Kuzu kulağı

ST (juice), AE LF

Chewing fresh Pounded (ext.)

Rumex tuberosus L. subsp. horizontalis (Koch.) Rech. PORTULACACEAE Portulaca oleracea

Tırşoka kera

LF

Diabetes, stomach ache Haemorrhoids, anti-inflammatory, antiphlogistic, antirheumatic wound healing

Kaval, 2011; Özgökçe and Özçelik, 2004; Tabata et al., 1994; Tuzlacı and Doğan, 2010; Mükemre, 2013 Tuzlacı and Doğan, 2010 Özgökçe and Özçelik, 2004

Poultice (ext.)

Kaval, 2011

Pırpar, pimpirim, semiz otu, parpar, pirpirim

AE

Urethra infection, inflamed wounds, cardiac disorders, diabetes, constipation

Decoction (int.), crushed (ext.), infusion (int.)

Tuzlacı and Doğan, 2010; Tetik et al., 2013

Cüngk Çörekotu

LF SD

Rheumatism Diabetes, ulcer

Poultice (ext.) Decoction (int.)

Kaval, 2011 Özgökçe and Özçelik, 2004

Muhabbet çiçeği

RT

Stomach pains

Eaten fresh


Gıyabirin Helhelok

LF FR

Astringent Prostate

Poultice (ext.) Eaten fresh

Kaval, 2011 Kaval, 2011

P. major L. subsp. major

Plantago maritime L. POACEAE Zea mays L. POLYGONACEAE Rheum ribes L.

RANUNCULACEAE Ranunculus kotschyi Boiss. Nigella sativa RESEDACEA Reseda lutea L. var lutea ROSACEAE Alchemilla hessii. Rothm. Cerasus microcarpa (C.A. Mey.) Boiss. subsp. tortuosa (Boiss.& Hausskn.) Browicz

Wound healing, abscesses

Application Eaten fresh, decoction, poultice (ext.), fresh on skin (ext.) Poultice (fresh), powder (dry), fresh on skin (ext.)


Table 1. (Continued) Family/Species name Cerasus mahalep L. Crataegus monogyna Jacq. Pyrus syriaca Boiss. var. syriaca Rosa canina L.

Local names Mahlep Alıç Şekok Şilank, şilan, kuşburnu, domuzturpu Şilank

Plant part FR FR FR FR, RT, LF

Therapeutic effect/ailments treated Diabetes Sedative, antispasmodic Expectorant, stomach ache Common cold, cough, haemorrhoids, stomach ache, asthma, immunostimulant, bronchitis,

Application Infusion (int.) Eaten fresh Infusion (int.) Decoction (int.), stewed (int.), infusion (int.)

FR

Common cold and cough

Decoction

Düdırk, dırne, mımirk, tütürk

SD, FR

Tonsillitis, sore throat, strengthen of eyes, kidney stones

Eaten fresh, dry powder in water (gargle),

Tabata et al., 1994; Kaval, 2011

Babelisk Yoğurt otu, yanikotu

AE FL

Haemorrhoids All cancers, burns

Fresh (ext.) Powder (int.) (ext.)

Kaval, 2011 Tabata et al., 1994; Özgökçe and Özçelik, 2004; Altundag and Ozturk, 2011

RHAMNACEAE Paliurus spina-christi Mill.

Kenari

BR

Antifungal

Fresh (on wounds then burned)

Kaval, 2011

SALICACEAE Populus nigra L. Salix aegyptiaca L. Salix alba L.

Kavak Belgebi Belgibizeri, söğüt

LF LF LF

Rheumatism Toothache Toothache, rheumatism

Decoction (bath) Poultice Poultice, decoction (bath)

Tabata et al., 1994 Kaval, 2011 Kaval, 2011; Tabata et al., 1994

Masicerk

BR+FL

Arthralgia, haemorrhoids, rheumatism

Masicark

AE

Rheumatism

Tuzlacı and Doğan, 2010; Mükemre, 2013 Kaval, 2011

Süpürge out

LF

Inflammed wounds

Crushed (with wheat flour), infusion (int.) Steam of brewed water applied directly (ext.) Directly on skin (ext.)

Rosa heckeliana Tratt. subsp. vanheurckiana (Crĕp.) Ö. Nilsson Rubus caesius L.

RUBIACEAE Galium consanguineum Boiss. Galium verum L. subsp. glabrescens Ehrend

SCROPHULARIACEAE Verbascum cheiranthifolium Boiss. var. cheiranthifolium Verbascum speciosum Schrad.

Scrophularia libanotica Boiss, var. urartuensis R. Mill.


References Cakilcioglu et al., 2011 Özgökçe and Özçelik, 2004 Tuzlacı and Doğan, 2010 Tabata et al., 1994; Kaval, 2011; Özgökçe and Özçelik, 2004; Tuzlacı and Doğan, 2010; Sezik et al., 1997 Kaval, 2011


Family/Species name SOLONACEAE Hyoscyamus niger L.

Local names

Plant part


Application

References

Hırdal, hargel, harundol, ban out, batbat Dağdoğan

SD

Toothache, tooth cavity

Inhaled, directly on tooth (ext.)

RT+SD

Intoxication

Eaten fresh

Tütün

LF

Wound healing

Powder (ext.)

Tabata et al., 1994; Özgökçe and Özçelik, 2004; Sezik et al., 1997 Tabata et al., 1994; Özgökçe and Özçelik, 2004; Tabata et al., 1994

Tevri

BR, BK

Toothache, rheumatism, cough

Fresh, decoction powder (ext.)

Kaval, 2011

Derdoğan

FR

Diarrhoea

Eaten fresh


Gezınk, geznek, yığınç, dezink, gezınk, ısırgan otu, gezik, ğıjırtken, çinçar, dırık

SD, AE, WP, LF

Cancer, common cold, rheumatism, diarrhoea, kidney stone, joint pain, stomach ache, cough, diabetes, losing weight

Eaten fresh (mixed with honey), decoction (int.-ext.), fresh (ext.), poultice (ext.), infusion (int.)

Urtica urens L.

Isırgan otu, çinçar

LF, WP

Diarrhoea, diuretic, rheumatism

VIOLACEAE Viola odorata L.

Bınevşok

WH

Stomach disorders, kidney pain

Decoction (then dry), Kaval, 2011 eaten fresh

VALERIANACEAE Valeriana officinalis L. Valeriana alliarifolia Adams ZYGOPHYLLACEAE Tribulus terrestris L.

Kediotu

AE RT

Kidney stone Sedative, antispasmodic

Decoction (int.) Tea (int.)

Tuzlacı and Doğan, 2010 Özgökçe and Özçelik, 2004;

Kidney stone, diarrhoea

Decoction (int.)

Peganum harmala

Üzerlik

AE, FR RT, SD

Haemorrhoids, prostatitis, urinary incontinence, stomach ache

Decoction (int.), roasted (int.)

Kaval, 2011; Cakilcioglu et al., 2011 Tabata et al., 1994; Özgökçe and Özçelik, 2004, Sezik et al., 1997

Hyoscyamus reticulates L. Nicotiana tabacum L. THYMELAECEAE Daphne mucronata Royle ULMACEAE Celtis tournefortii Lam. URTICACEAE Urtica dioica L.

Sedıdan, pıtırak

Kaval, 2011; Özgökçe and Özçelik, 2004; Tabata et al., 1994; Tuzlacı and Doğan, 2010; Sezik et al., 1997; Cakilcioglu et al., 2011; Mükemre, 2013 Tea (int.), fresh (ext.) Tabata et al., 1994; Özgökçe and Özçelik, 2004; Sezik et al., 1997

AE: aerial part; BB: bulb; BA: bark; BR: branch; BD: bud; BK: bark; CO: cortex; GM: gum; FL: Flower; FR: fruit; LF: leaf; LX: latex; RT: root; RS: root sap; SD: seed; SH: shoot, SL: stylus; ST: stem; WH: whole plant. Ext: external, Int: internal.


Table 2. Selected ailments and their traditional remedies used in Eastern Anatolia Health condition Wounds, injuries

Stomach disorders

Abdominal pain Carminative /flatulence Constipation Haemorrhoids Diabetes

Rheumatism Diuretic Kidney pain Kidney stones Gallbladder disorders Urinary inflammation Cardiac disorders Eye problems Common cold

Traditional remedy Nicotiana tabacum, Scrophularia libanotica, Rumex tuberosus, Portulaca oleracea, Plantago major, Plantago atrata, Plantago lanceolata, Malva neglecta, Asphodeline tenuior, Salvia nemorosa, Origanum vulgare, Juglans regia, Euphorbia macrocarpa, Lepidium latifolum, Asymnea rigidum, Alyssum pateri, Echium italicum, Alkanna orientalis, Alkanna megacarpa, Taraxacum montanum, Tanacetum balsamita, Scorzonera tomentosa, Cichorium intybus, Centaurea karduchorum, Aristolochia bottae, Pistacia atlantica, Ferula orientalis, Ferula haussknechtii, Heracleum antasiaticum, Johrenia dichotoma, Prangos pabularia Pistacia khinjuk, Eryngium billardieri, Ferula orientalis, Grammosciadium platycarpum, Achillea biebersteinii, Achillea millefolium, Achillea vermicularis, Anthemis nobilis, Arctium minus (Hill.) Bernh. subsp. pubens, Arctium tomentosum, Artemisia spicigera, Centaurea virgata, Anchusa azurea Mill. var. azurea, Alyssum pateri Nyár. subsp. pateri, Bryonia multiflora, Glycyrrhiza glabra L. var. glandulifera, Trifolium repens, Hypericum perforatum, Origanum majorana, Teucrium chamaedrys, Teucrium polium, Thymus kotschyanus, Alcea apterocarpa, Alcea hohenackeri, Plantago lanceolata, Rheum ribes, Rumex acetosella, Reseda lutea L. var lutea, Pyrus syriaca, Rosa canina, Urtica dioica, Viola odorata, Peganum harmala Foeniculum vulgare, Cichorium intybus, Euphorbia denticulata, Origanum acutidens, Prunella vulgaris, Alcea hohenackeri, Mentha longifolia, Malva neglecta Anthriscus slyvestris, Achillea biebersteinii, Senecio vernalis Euphorbia sp, Euphorbia heteradena, Euphorbia virgata, Portulaca oleracea, Peganum harmala, Galium consanguineum, Rosa canina, Zea mays, Rheum ribes, Rumex cripsus, Plantago lanceolata, Teucrium polium, Mentha longifolia, Juglans regia, Telephium imperati , Circium sp., Inula helenium, Berberis crataegina, Berberis crataegina, Diplotaenia cachrydifolia Boiss, Heracleum persicum, Arum dentrucatum, Anthemis tinctoria L. var. tinctoria, Artemisia absinthium, Gundelia tournefortii L. var. tournefortii,., Helianthus tuberosus L., Helichrysum armenium D.C. subsp. armenium, Tanacetum argyrophyllum, Tanacetum chilliophyllum, Berberis crataegina, Capsella bursa-pastoris, Glycyrrhiza glabra L. var. glandulifera, Juglans regia, Salvia multicaulis, Trigonella foenum-graceum, Eremurus spectabilis Bieb., Paeonia mascula, Plantago lanceolata, Rheum ribes, Rumex acetosella, Portulaca oleracea, Nigella sativa., Cerasus mahalep, Urtica dioica Diplotaenia cachrydifolia, Artemisia spicigera, Juniperus oxycedrus, Mentha spicata, Teucrium chamaedrys, Merendera trigyna, Ranunculus kotschyi, Populus nigra, Salix alba, Verbascum cheiranthifolium, Verbascum speciosum, Daphne mucronata, Urtica urens, Urtica dioica Achillea millefolium , Anthemis nobilis , Bellis perennis, Cyperus rotundus, Cichorium intybus, Helichrysum armenium, Juncus inflexus, Alcea calvertii, Alcea fasciculiflora, Alcea flavovirens, Alcea kurdica, Alcea hohenackeri, Malva neglecta, Equisetum fluviatile, Teucrium polium, Valeriana officinalis, Tribulus terrestris, Allium cepa, Viola odorata Helichrysum armenium, Helichrysum plicatum, Cardamine uliginosa, Juncus inflexus, Alcea apterocarpa, Alcea calvertii, Alcea fasciculiflora, Alcea kurdica, Malva neglecta, Urtica dioica, Valeriana officinalis, Tribulus terrestris, Rubus caesius Mentha pulegium, Malva neglecta Pistacia terebinthus, Allium cepa Alyssum pateri, Glycyrrhiza glabra, Medicago minima, Portulaca oleracea, Arctium minus, Rubus caesius, Arctium minus, Rubus caesius, Taraxacum montanum (eye redness) Urtica dioica, Rosa canina, Rosa heckeliana, Malva neglecta, Salvia sclarea, Salvia verticillata, Phlomis armeniaca, Salvia candidissima, Origanum vulgare, Mentha spicata, Mentha longifolia, Achillea millefolium, Anthemis austriaca, Anthemis cotula, Anchusa azurea


Health condition Malaria Hypocholesterolemia Hypertension Burns Antiseptic Toothache Tonsilitis Parasites Venome poisoning

Traditional remedy Centaurea solstitialis L. subsp. solstitialis Anethum graveolens, Helichrysum armenium D.C. subsp. armenium, Cichorium intybus Equisetum fluviatile Rhus coriaria, Alkanna megacarpa, Asymnea rigidum, Galium verum Rhus coriaria L., Scabiosa sulfurea, Cephalaria sparsipilosa Daphne mucronata, Hyoscyamus niger, Salix alba, Salix aegyptiaca Rubus caesius Pelargonium quercetorum,Globularia trichosantha, Allium macrochaetum Centaurea iberica, Nonea pulla


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Table 2 presents the list of selected ailments and medicinal plants used in their treatment. This table shows that frequently multiple herbs have been identified to treat the same health disorder. For example, 32 herbs (17.6% of the total number) have been identified for the treatment of wounds and 33 herbs (18.1%) for the treatment of stomach disorders. The percentages of herbs used to treat these two common health conditions listed in this chapter are higher than those cited by Kaval: 10.1% for wound healing and 12.8% for stomach disorders (Kaval, 2011). Diabetes and cancer are two prevailing health conditions of the contemporary society. In Eastern Anatolia a high number of plant sources have also been identified for their treatment. Twenty five herbs (13.7%) are in use to alleviate the symptoms of diabetes. The percentage of herbs to treat diabetes presented in this listing is double that reported earlier (7.3%) by Kaval (2011). A relatively high number of plants (12 plants; 6.6%) are applied in cancer treatment. Eryngium bornmuelleri is used specifically to cure stomach cancer, while Plantago lanceolata and Plantago maritime are used to treat the cancer of uterus. Galium verum L. subsp. glabrescens Ehrend is used for the treatment of all types of cancers. These plants represent extremely valuable resources for future research aimed at identification of active constituents and possibly - development of new anti-diabetic or anti-cancer agents. Table 2 presents also 15 medicinal plants (8.2%) utilized for the treatment of kidney disorders, and additional 13 plants (7.1%) identified to cure specifically kidney stones. In the last group six plants belong to the Malvaceae family. Alternative uses of a few members of the same plant family to cure one ailment may indicate a presence of the same active components. Relatively high number of plants (14; 7.7%) are used for the treatments of rheumatism; the percentage of plants utilized to cure this condition is in agreement with earlier report by Kaval (2011). The application of herbal remedies from Eastern Anatolia listed in this chapter addresses common illnesses (e.g. abdominal pain or common cold) as well as specific conditions, such as gall bladder stones, tonsillitis, and stomach cancer. Two common chronic conditions related to the modern lifestyle are also treated; hypertension with Equisetum fluviatile, and hypocholesterolemia with Anethum graveolens, Helichrysum armenium D.C. subsp. Armenium, and Cichorium intybus. Natural medicines to treat both conditions are in high demand. Traditional medicinal plants presented in this chapter may provide valuable leads for identification of physiologically active natural compounds for pharmaceutical uses.

The Most Versatile Herbs: Phytochemical Composition and Pharmacological Activities The majority of original research papers and review publications dedicated to ethnobotanicals from Eastern Anatolia that have been published to date are aimed at providing information on the identity of the medicinal plants and their traditional uses. Physiological activities of plants and plant-originated products arise from the presence of secondary metabolites in the tissue used. Contemporary research indicates that these compounds significantly influence the way a plant responds to stress. For example,



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anthocyanins are implicated in tolerance to environmental stress, including drought, ultraviolet light (UV-B) and heavy metals, and also facilitate plant resistance to pathogens (bacteria and fungi) and herbivores (Gould, 2004). These compounds are often highly efficient antioxidants, which mitigate injuries by scavenging free radicals and reactive oxygen species (e.g. in case of photooxidative injury in leaves). Accordingly, plants growing at high elevation are exposed to an increased flux of solar ultraviolet-B radiation (Blumthaler and Ambach, 1990) and photooxidative injuries and subsequently produce more antioxidants to alleviate the harmful effects of free radicals. A high number of Eastern Anatolia’s herbal remedies are collected in mountainous areas. Out of the 182 plant species listed in Table 1, four plants have an outstanding frequency of application - they are used to cure 10 or more different health disorders. In order to clarify the reasons behind their efficacy, understood over generations of ‘trial and error’, their phytochemical composition is discussed. Mentha longlifolia L. (mint) is a very popular herb, represented by 20 to 30 species growing mostly in temperate regions. It is a fast growing, perennial plant, spreading through underground rootstock. The leaves are soft, lanceolate, and are formed in pairs along a square-shaped stem, which can reach 0.5 - 1 m in height. Small flowers, white to mauve, are gathered to form spikes at the tip of the stems. The herb is collected from the wild and can be cultivated for its essential oils. In the Eastern Anatolia the plant is used to treat common cold, flu, catarrh, heart palpitations, asthma, abdominal pains, internal diseases, menstrual pain, stomach ache, cough, bronchitis, headache, hemorrhoids and sunstroke. It is also used as an antispasmodic agent (Table 1). It is used internally, as a decoction and herbal infusion, and externally, as a decoction in a bath. Mentha is also used as a condiment in various foods such as beverages, ice creams, candies, cakes and in meats to adjust their taste and odor. M. longifolia represents the Lamiaceae family, which contains large amounts of phenolic acids and flavonoids in a variety of structural forms, including flavones, flavonols, flavonones, dihydroflavonols and chalcones (Motamed and Naghibi, 2010). Flavonoids exhibit pronounced antioxidant capacity, can act as reducing agents, hydrogen-donating antioxidants and singlet oxygen quenchers (Hvattum and Ekeberg, 2003). This quality contributes towards their physiological activities and numerous health benefits, including the reduction of oxidative stress, protection against coronary heart disease or cancer (Seelinger et al., 2008). According to Kosar and co-workers, Mentha sp. are rich sources of tannins, saponins, phenolic acids and flavonoids (Kosar et al., 2004). The total phenolic content of M. longifolia is 107.21±34.2 mg GAE (gallic acid equivalent)/g DW (dry weight) and flavonoid content is 42.47±7.97 mg QE (quercetin equivalent)/g DW. The contribution of flavonoids to the total phenolics is high: 39.61% (Raj et al., 2010). Orhan and collaborators identified flavonoids luteolin 7-O-glucoside, luteolin 7-O-rutinoside and luteolin 7-O-glucuronide in n-butanol extract of the aerial parts of M. longifolia (L.) Hudson subsp. longifolia from Eastern Anatolia (Orhan et al., 2012). The same authors reported pronounced antimutagenic activity of these flavonoids. Similarly, apigenin 7-O-glucoside isolated from M. longifolia exerted strong antimutagenic activities (Ames test) with inhibition rates ranging from 27.2% (S. typhimurium TA1535: 0.4 μM/plate) to 91.1% (S. typhimurium TA1537: 0.2 μM/plate) and inhibited yeast growth (yeast deletion assay) from 4% to 57.7% (Gulluce et al., 2013). Many parts of M. longifolia, including leaves, flowers and stems, are used in herbal teas or as additives, due to their aroma and flavor. The sources of this unique flavor are the


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essential oils - secondary metabolites that are highly enriched in compounds based on an isoprene structure. An important component of essential oils are terpenes, which occur as monoterpens, diterpenes, triterpenes, and tetraterpenes (C10, C20, C30, and C40), as well as hemiterpenes (C5) and sesquiterpenes (C15). Many terpens, such as menthol, present in M. longifolia and camphor, have medicinal value (Cowan, 1999). Gulluce and collaborators identified 45 compounds comprising the essential oil of M. longifolia ssp. longifolia from the north-eastern part of Anatolia (Gulluce et al., 2007). The dominating compounds of this mixture were: cis-piperitone epoxide (18.4%), pulegone (15.5%), piperitenone oxide (14.7%), menthone (7.9%), isomenthone (6.6%), thymol (6.6%), trans-piperitone epoxide (4.1%), carvone (4.9), β-caryophyllene (2.6%), camphor (1.6%), γ-muurolene (1.1%), and piperitenone (1.0%). The remaining 33 compounds (α-pinene, β-pinene, β-myrcene, 3octanol, α-terpinene, p-cymene, limonene, (Z)-β-ocimene, (E)-β-ocimene, γ-terpinene, cissabinene hydrate, linalool, pinocamphone, terpinen-4-ol, α-terpineol, dihydro carvone, bornyl acetate, sabinyl acetate, thymol acetate, β-bourbonene, nepetalactone, β-copaene, αcaryophyllene, bicyclogermacrene, β-bisabolene, γ-cadinene, d-cadinene, spathulenol, caryophyllene oxide, salvial-4(14)-en-1-one, β-atlantol, humulene epoxide II) were present at levels of less than 1%. Cichorium intybus L. (common chicory) of Asteracea family, known locally as hindiba or kanej, is an erect perennial herb 80-90 cm in height, predominantly with bright blue flowers, occasionally white or pink, a reddish leaf and a fleshy taproot up to 75 cm in length. Stems are stiff and have length from 20 to 100 cm. Basal leaves are shortly petiolate, oblanceolata. The flowers open early in the day and close soon after noon. C. intybus is an edible and a medicinal plant. Aerial parts are used for preparation of meals or salad and roots are used as a chewing gum. The herb is also used as an animal feed. All plant parts, including roots, leaves, flowers and aerial parts, are dried and ground into a fine powder for later use in a variety of herbal remedies. C. inytbus is a typical Mediterranean plant, indigenous to Europe, Western Asia, Egypt and North America. It is cultivated in Europe for their roots (C. intybus var. sativum), which are baked, ground, and used as a coffee substitute and an additive. It is also used in herbal mixtures in the symptomatic treatment of digestive disorders such as flatulence, slow digestion, belching, and epigastric distension as well as to promote renal and digestive elimination functions. C. intybus is one of the broadly used herbal medicines. This chapter reports its use for abdominal pain, epilepsy, prostate, hypertension, hemorrhoids, kidney disorders, hypercholesterolemia, hepatic, asthma, stomach ache, wound healing as well as antiseptic, depurative and cleansing agent. This medicinal plant is consumed fresh, and is used to prepare decoctions and infusions (Table 1). An interesting preparation has been developed for epilepsy treatment, whereby a decoction prepared by boiling the pounded roots for 2-3 hours is taken on an empty stomach in the morning for 3 consecutive days (Tabata et al., 1994). Nandagopal and Kumari (2007) named C. intybus a ‘multipurpose medicinal plant’ used as: antihepatotoxic, antiulcerogenic, antiinflammatory, appetizer, digestive, stomachic, liver tonic, cholagogue, cardiotonic, depurative, diuretic, emmenagogue, febrifuge, and alexeteric. It is useful in vitiated conditions of kapha and pitta, cephalalgia, heapatomegaly, inflammations, anorexia, dyspepsia, flatulence, colic, gout, burning sensation, allergic conditions of skin, jaundice, splenomegaly, hyperdipsia, skin diseases, leprosy, strangury, amenorrhoea, chronic and bilious fevers, ophthalmia, pharyngitis, vomiting; it is also used to treat AIDS, cancer, diabetes, dysmenorrhoea, impotence, insomnia, splenitis and tachicardia



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(Nandagopal and Kumari, 2007). Pharmacological investigation of the root extract of C. intybus revealed immunomodulatory and anticancer properties (Hazra et al., 2002). Animal studies have revealed that preparations from C. intybus roots lowered the serum and liver lipid concentration in rats (Kim and Shin, 1998). Further, it has been reported that chicory root extract decreased cholesterol absorption by 30% (p<0.05) in the jejunum and by 41% (p<0.05) in the perfused ileum, which is in agreement with its traditional uses to reduce hypercholesterolemia. Moreover, antihepatoxic effect (the ability to prevent liver damage) of C. intybus also has been reported (Kim, 2000; Ahmed et al., 2003). C. intybus accumulates two major groups of physiologically active phytochemicals: phenolic compounds and sesquiterpene lactones. The main phenolic compounds of leaf include monocaffeoyl tartaric acid, chlorogenic acid (5-O-caffeoylquinic acid), chicoric acid (dicaffeoyltartaric acid), cyanidin 3-O-glucoside, delphinidin 3-O-(6′′ malonyl) glucoside, cyanidin 3-O-(6′′ malonyl) glucoside, quercetin 3-O-glucuronide and luteolin 7-Oglucuronide (Innocenti et al., 2005). Laveli (2008) along with the above named phenolic compounds reported the presence of quercetin 3-O-glucoside, as well as tentatively identified an acylated derivative of quercetin 3-O-glucoside. Flower petals of C. intybus are a rich source of anthocyanins, containing delphinidin 3,5di-O-(6-O-malonyl-β-d-glucoside), delphinidin 3-O-(6-O-malonyl-β-d-glucoside)-5-O-β-dglucoside, delphinidin 3-O-β-d-glucoside-5-O-(6-O-malonyl-β-d-glucoside) and delphinidin 3,5-di-O-β-d-glucoside (Nørbæk et al., 2002). The sesquiterpene lactone chemistry of Cichorium species is dominated by lactucin-like guaianolides and their glycosides, present in roots and aerial parts. Kisiel and Zielinska (2001) reported that root and leaf samples of C. intybus contain sesquiterpenoid aglycones and glycosides. The leaves contain β-13-dihydrolactucin, jacquinelin, lactucin, 8desoxylactucin, lactucopicrin and its derivatives, crepidiaside B, and loliolide, while the root contains a mixture of methyl and ethyl esters of p-hydroxyphenylacetic acid, cichorioside B and sonchuside A, ixerisoside D and magnolialide. Moreover, root of the C. intybus plant produces a small number of germacrane- and eudesmane-type sesquiterpene lactones and their glycosides. This information is in agreement with Bais and Ravishankar (2001), who reported that the main sesquiterpene lactones of C. intybus are lactucin, 8 deoxylactucin, and lactucopicrin found in the roots and the heads of the plant and considered to be responsible for the bitter taste of chicory. The leaves and roots also contain trace amounts of other bitter sesquiterpene lactones such as guaianolides, lactupin, deoxylactupin, eudesmonolides and guanomanolides (Bais and Ravishankar, 2001). According to Varotto and collaborators numerous compounds such as inulin, bitter sesquiterpene lactones, coumarins, flavonoids and vitamins present in the tuberous root of C. intybus are of medical importance (Varotto et al., 2000). Malva neglecta Wallr. (common mallow) of Malvaceae family is an annual herb with orbicular, shallowly lobed leaves. Flowers are borne in fascicles of the leaf axils. In Turkey a number of Malva species are edible as leafy vegetables (known as tolık or ebegümeci) prepared in various forms. For example, they are stuffed with bulgur wheat or rice or are boiled and used as a side dish (Dalar et al., 2012). The plant is widespread in all regions of Turkey and, although regarded elsewhere as a weed, in Turkey it is used as food and folk medicine in a number of conditions, including kidney and bladder stones, abscesses, external wounds, stomach disorders, abdominal pain, peptic ulcer, common colds, infertility, bronchitis, indigestion and vaginal candidiasis (Table 1).


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Research data on the composition of phytochemicals in M. neglecta is limited. Dalar and collaborators reported that the level of total phenolic compounds in different plant parts ranged from 3.40.3 (root) to 17.40.3 (leaf) mg GAE /g DW. The leaf contained the highest level of phenolics and was followed by flower, fruit, stem and root (Dalar et al., 2012). The levels of total phenolics in M. neglecta compared favorably with 30 traditional Chinese medicinal plants evaluated by Wong and collaborators (Wong et al., 2006). The level of flavonoids ranged from 0.97±0.02 (root) to 7.21±0.28 (leaf) mg RE (rutin equivalent)/g DW, which, respectively, represented 29.1% (root) and 41.5% (leaf) of the total phenolics. The distribution of flavonoids in various plant organs followed the order: root


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followed by those for flavonoids and antioxidant capacities, which is in agreement with the finding that these two groups of phenolic compounds are the major contributors to the composition of phenolic compounds of M. neglecta. These results suggest that phenolic compounds, especially flavonoids and hydroxycinnamic acids, are the potential sources of medicinal properties of this plant. Leaves of M. neglecta are the primary source for the preparation of decoctions used in the traditional medicine of Eastern Anatolia. The exceptionally high ORAC value of the leaf extract supports their medicinal properties with suppression of oxidative stress as one of the potential mechanisms of action. Decoction of M. neglecta is commonly used in Eastern Anatolia to heal wounds (Table 1). Zare and collaborators investigated the effect of chloroform, ethanol and water extracts of M. neglecta and M. sylvestris on some bacterial and fungal contaminants of wound infections. Ethanol-based extracts exhibited the highest antibacterial activity, and were especially effective against Streptococcus pyogenes, followed by Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus vulgaris. Aqueous and chloroform extracts had more pronounced antifungal activity against Aspergillus niger, Aspergillus fumigatus and Candida albicans (Zare et al., 2012). Urtica dioica L. (nettle) of Urticaceae family is a perennial, herbaceous plant with opposite, sharply toothed leaves and white, stinging trichomes. The stalk contains sclerenchymatic fibers, which can be used in the textile industry (Pinelli et al., 2008). This herb grows in nitrogen-rich soils (nitrophilous herb) and is widely distributed throughout the temperate regions of the world. It has a long history of use as a traditional medicine and is utilized in cosmetics, textiles, and biodynamic agriculture. Moreover, U. dioica represents a very nutritious and easily digested food, high in minerals (especially iron), vitamin C and provitamin A (Allardice, 1993). The traditional medicinal uses of U. dioica in Eastern Anatolia include treatment of kidney and bladder stones, abscesses, external wounds, stomach disorders, abdominal pain, peptic ulcers, common colds, infertility, bronchitis, indigestion and vaginal candidiasis (Table 1). Seeds and aqueous extracts of the aerial parts of this herb have also been occasionally used in Turkey as herbal medicine by cancer patients (Akbay et al., 2003). The physiological activities of U. dioica were a subject of multiple studies. Riehemann and collaborators reported inhibition of proinflammatory transcription factor NFkB by a standardized extract of U. dioica leaves (Riehemann et al., 1999). In agreement, a clinical pilot study established that consumption of a stew made of U. dioica enhances the effectiveness of antirheumatic non-steroid anti-inflammatory drugs (NSAID; Chrubasik et al., 1997). Other reports described the uses of U. dioica to treat eczema, gout, anemia and allergic rhinitis (Bone and Mill, 2000; Fischer, 1997). U. dioica leaf extract inhibits platelet aggregation (El Haouari et al., 2006) and can therefore be used in the prevention and treatment of cardiovascular conditions. Earlier studies have shown that U. dioica leaf is a valuable source of vitamin C (Martınez-Para and Torija-Isasa, 1980) and minerals (Weiss, 1988). However the physiological activities of U. dioica can be assigned mostly to the presence of 3 groups of phytochemicals: phenolic compounds, carotenoids and fatty acids. Pinelli and collaborators reported the presence of three classes of phenolic compounds in the leaf and stalk of U. dioica: hydroxycinnamic acid derivatives (main compounds being chlorogenic acid and 2-Ocaffeoyl-malic acid); flavonols (rutin, quercetin p-coumaroyl-glucoside, kaempferol 3-Oglucoside, kaempferol 3-O-rutinoside, isorhamnetin 3-O-rutinoside) and anthocyanins


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[peonidin 3-O-rutinoside, rosinidin 3-O-rutinoside, peonidin 3-O-(6′′-O-p-coumaroylglucoside)]. Chlorogenic and 2-O-caffeoylmalic acid are the main phenolic compounds (76.5% of total phenolics) of the leaf. Stalk extracts also contain chlorogenic acid, 2-Ocaffeoyl-malic acid, p-coumaric acid, and a caffeic acid derivative as well as flavonols and anthocyanins (Pinelli et al., 2008). Contemporary research revealed pronounced health-enhancing properties of the numerous phenolic compounds listed above. Chlorogenic acid - the dominating phenolic compound of U. dioica - is the active anti-diabetic agent of Nerium indicum leaf, an Indian folk remedy for type II diabetes (Ishikawa et al., 2007). Flavonoids are especially widely researched due to their pleiotropic health beneficial effects; they frequently possess potent antioxidant capacities and a wide array of biochemical functions resulting in promotion of good health. For example, they are involved in immune function, including modulation of gene expression, enzymes activity and cholesterol and histamine metabolism. They reduce coronary heart disease risk, exhibit antimutagenic, anti-viral, antidiabetic, anti-obesity and chemopreventative properties (Pandey and Rizvi, 2009; Kroon and Williamson, 2005; Kim et al., 2010a; Kim et al., 2010b). Guil-Guerrero and collaborators conducted a comprehensive evaluation of carotenoids and fatty acids in different parts of U. dioica plant. They have reported the presence of 9 carotenoids in the leaves of U. dioica. For all leaf maturity levels, lutein, lutein isomers, βcarotene and β-carotene isomers were the major carotenoids. Neoxanthin, violaxanthin and lycopene were also important contributors in specific leaf maturity stages (Guil-Guerrero, et al., 2003). The same authors have established that U. dioica leaf is among the most suitable plant sources of fatty acids for human nutrition with a high n-3 to n-6 acids ratio of 3.51. The saturated fatty acids were found in all plant parts, with palmitic acid being the dominating compound (17.9% in mature leaves to 25.4% in seeds) and little amounts of stearic acid. The monounsaturated fatty acids were present in seeds: erucic acid (1.2%) and in root: oleic acid (8.7%), palmitoleic acid (2.6%, also present in stem at 0.5%) and gadoleic acid (1.2%). The dominating fatty acid in leaves was the α-linolenic acid, also present in seeds. Guil-Guerrero and collaborators have concluded that young leaves of U. dioica are a valuable source of fatty acids and carotenoids for human nutrition. Three of the four plants described above: M. longifolia, C. intybus and U. dioica accumulate diverse plant secondary metabolites: phenolic compounds and essential oils in M. longifolia, phenolic compounds and sesquiterpene lactones (terpens) in C. intybus and phenolic acids, carotenoids and fatty acids in U. dioica. The concurrent presence and high level of phytochemicals, representing various groups and resulting in exceptionally rich composition of plant secondary metabolites, is the common quality of these broadly applied, all-round traditional medicinal plants. This rich composition may be the key to their exceptional curing properties observed by many generations of traditional healers. The major secondary metabolites of M. neglecta identified to date are phenolic compounds, however further in-depth studies are needed to investigate the secondary metabolites of this plant.



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Antioxidant Capacities and Enzyme-inhibitory Activities of Selected Medicinal Plants from the Van Province Oxidative stress is an imbalance in the redox status of a cell between the production of free radicals - reactive oxygen/nitrogen species (ROS, RNS), and antioxidant defense mechanisms. Free radicals are highly unstable molecules, formed as a part of natural metabolism as well as originating from external sources like smoking, pollution, radiation or exposure to chemicals. Maintaining the oxidative stress for a prolonged period of time leads to the damage of cell components, including DNA, lipids and proteins, which may result in potential mutations and ultimately the formation of cancer, diabetes, hypertension, heart problems or other chronic conditions as well as accelerated ageing (Halliwell, 2007). Defense against oxidative stress is therefore an important factor in preventing the development of many diseases. Antioxidants have the ability to neutralize free radicals, by receiving or donating an electron, without becoming free radicals themselves as they are stable in both forms. However, when an antioxidant neutralizes a free radical it becomes inactive. Therefore our body requires a continuous delivery of antioxidants from food. Phytochemicals, especially dietary polyphenols, are potent antioxidants capable of scavenging and intercepting free radicals, thus preventing cellular molecule damage. Frequently, a high antioxidant capacity is an indication of a plant’s health-enhancing properties. Phenolic compounds i) scavenge radical species such as reactive oxygen/nitrogen species (ROS/RNS), ii) suppress ROS/RNS formation by inhibiting some enzymes or chelating trace metals involved in free radical production, and iii) upregulate or protect the antioxidant defense system (Dai and Mumper, 2010). The number of studies dedicated to antioxidant testing of traditional medicinal plants from the Van province of Eastern Anatolia are limited. Table 3 presents the available data on antioxidant activities of methanol extracts of some herbs, traditionally used for medical purposes by the local population, as well as antioxidant capacities of herbal teas prepared from selected herbs. Among the extracts, Verbascum cheiranthifolium exhibited superior antioxidant activity to all other plants, as evaluated in two reagent-based antioxidant capacity assays: ORAC and FRAP. The ORAC values ranged from 905.0±5.3 (stem) to 2262.6±8.5 (flower) µmol TE/g DW (Table 3). Flower extract exhibited the highest activity and was followed by leaf and stem (Dalar and Konczak, 2012). The ORAC values of V. cheiranthifolium were approximately 3-fold those of Centaurea karduchorum, 1.7 those of Eryngium bornmuelleri and 1.3 those of Dactylorhiza chuhensis. Interestingly, V. cheiranthifolium is among the most extensively used medical plants in the region. The exceptionally high ORAC values of V. cheiranthifolium leaf and flowers, were comparable to those of the commonly used herbs Jasminum grandiflorum flower (Spanish jasmine) (2330±64 µmol TE/g DW) and Rosa damascena (Damascus rose) (2382±62 µmol TE/g DW) (Dudonné et al., 2009). Among these selected plants from the Van province V. cheiranthifolium and Plantago lanceolata leaf exhibited comparable and the highest total reducing capacities (FRAP assay; 368.45.0 and 369.1±19.4 µmol Fe+2/g DW, respectively), which were approximately 1.5fold that of E. bornmuelleri and D. chuhensis leaves and 7-fold that of the C. karduchorum


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leaf. The FRAP values of V. cheiranthifolium and P. lanceolata (Table 3) were comparable to those of the long and widely used medicinal trees Juniperus communis (common juniper) (240±9 µmol Fe+2/g DW) and Cananga odorata (cananga tree) (370±2 µmol Fe+2/g DW) (Dudonné et al., 2009). The results of both assays applied in this study have clearly shown that among all plant parts, leaves exhibited the highest antioxidant capacity. The utilization of aerial parts, especially leaves and flowers, have been the dominating ways of preparation of traditional medicines in the Eastern Anatolia region of Turkey for centuries. The antioxidant capacities of the leaves and flowers of these herbs were found to be relatively high and comparable or superior to antioxidant capacities of numerous Chinese and Ayurvedic medicinal plants and commonly used medicinal herbs, which may suggest that antioxidant capacities of these plants may be relevant to their physiological activities (Dalar and Konczak, 2012). The most common application of these selected plants in the Van province is preparation of herbal teas. Lyophilised hydrophilic extracts representing six herbal infusions (M. neglecta, P. lanceolata, Salvia limbata, Phlomis armeniaca, Anchonium elichrysifolium, and V. cheiranthifolium) also exhibited potent oxygen radical scavenging abilities and high total reducing capacities (Table 3). The high ORAC and FRAP values of these herbal teas indicate that the evaluated plant sources comprise a rich mixture of phytochemicals, able to donate a hydrogen cation (ORAC assay) or a free electron (FRAP assay), and could therefore offer comprehensive protection from reactive oxygen species and oxidants, which may effectively reduce oxidative stress. The level of total phenolics in the lyophilized herbal infusions, as evaluated using the Folin-Ciocalteu method, varied from 27.9±0.4 mg GAE/g DW (M. neglecta) to 80.4±1.8 mg GAE/g DW (P. lanceolata) (Table 3). P. lanceolata herbal tea had the highest total phenolic content (Table 3), which was similar to that of commercially available black tea (Camellia sinensis) Yellow Label (84.9±8.03 mg GAE/g DW). V. cheiranthifolium had a similar level of total phenolics to that of black tea Cameron Highlands trade (60.6±5.4 mg GAE/g DW); P. armeniaca and S. limbata had higher or comparable phenolics levels to that of oregano (58.6±3.7 mg GAE/g DW), while A. elichrysifolium and M. neglecta had similar phenolics levels to rosemary (28.1±0.8 mg GAE/g DW) (Dalar and Konczak, 2013). In another study Şahin and collaborators (2004) have identified exceptionally rich source of phenolic compounds in Origanum vulgare L. subsp. vulgare. The same herb exhibited very high radical scavenging activity towards the di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) radicals (Table 3). The level of phenolic compounds of Heracleum persicum and Gundelia tournefortii was comparable to that of E. bornmuelleri leaf (Çoruh et al., 2007b). Antioxidant testing of selected plant sources conducted to date suggest that antioxidant capacities of these common herbs from the Van province of Eastern Anatolia are comparable to those of traditional medicinal plants used by other cultures, for example Chinese medicine (Dalar et al., 2012). Phenolic compounds, predominantly flavonoids and phenolic acids (Table 3, Table 4), were identified as the major sources of antioxidant capacities of hydrophilic extracts obtained from these plants.


Table 3. Antioxidant capacities of traditional medicinal plants from Eastern Anatolia Antioxidant test Extraction ORAC (Oxygen radical absorbance capacity) assay Malva neglecta Aqueous methanol plant parts (root, stem, leaf, flower, fruit, whole plant) Plantago lanceolata Aqueous methanol plant parts (root, stem, leaf, flower, fruit, whole plant) Cichorium inytbus Aqueous methanol plant parts (root, stem, leaf, flower, whole plant) Eryngium bornmuelleri Aqueous methanol plant parts (root, stem, leaf, flower) Centaurea karduchorum Aqueous methanol plant parts (root, stem, leaf, flower) Verbascum cheiranthifolium Aqueous methanol plant parts (stem, leaf, flower) Malva neglecta tea Lyophilized ethanol Plantago lanceolata tea Salvia limbata tea Phlomis armeniaca tea Anchonium elichrysifolium tea Verbascum cheiranthifolium tea FRAP (Ferric reducing antioxidant power)assay Malva neglecta Aqueous methanol plant parts (root, stem, leaf, flower, fruit, whole plant) Plantago lanceolata Aqueous methanol plant parts (root, stem, leaf, flower, fruit, whole plant) Cichorium inytbus Aqueous methanol plant parts (root, stem, leaf, flower, whole plant)

Results

Compounds identified

References

425.3±6.7 – 898.9±14.9 µmol Trolox eq./gDW

Phenolics

Dalar et al., 2012


Phenolics

Dalar et al., 2012


Phenolics

Dalar, unpublished result

406.6±3.1 – 1489.0±17.0 µmol Trolox eq./gDW 326.9±7.2 – 674.7±13.7 µmol Trolox eq./gDW 905.0±5.3 – 2262±8.5 µmol Trolox eq./gDW 1638.4±218.3 µmol Trolox eq./gDW

Phenolics

Dalar and Konczak, 2012 Dalar and Konczak, 2012 Dalar and Konczak, 2012 Dalar and Konczak, 2013

Phenolics Phenolics Phenolics

3343.9±215.2 µmol Trolox eq./gDW 3602.9±52.1 µmol Trolox eq./gDW 2978.9±74.6 µmol Trolox eq./gDW 2239.5±241.5 µmol Trolox eq./gDW 4265.9±132.9 µmol Trolox eq./gDW 39.2±1.2 – 190.3±6.7 µmol Fe2+/gDW

Phenolics

Dalar et al., 2012

130.4±11.5 – 369.1±19.4 µmol Fe2+/gDW

Phenolics

Dalar et al., 2012

90.1±9.5 – 251.6±9.7 µmol Fe2+/gDW

Phenolics



Table 3. (Continued) Antioxidant test Eryngium bornmuelleri plant parts (root, stem, leaf, flower) Centaurea karduchorum plant parts (root, stem, leaf, flower) Verbascum cheiranthifolium plant parts (stem, leaf, flower) Malva neglecta tea Plantago lanceolata tea Salvia limbata tea Phlomis armeniaca tea Anchonium elichrysifolium tea Verbascum cheiranthifolium tea Eryngium bornmuelleri leaf sequential fractions

Extraction Aqueous methanol

Results 55.2±5.9 – 250.3±2.2 µmol Fe2+/gDW

Compounds identified Phenolics

Aqueous methanol

42.1±1.6 – 67.7±1.4 µmol Fe2+/gDW

Phenolics

Aqueous methanol

223.8±3.6 – 368.4±5.0 µmol Fe2+/gDW

Phenolics

Lyophilized ethanol

390.8±13.5 µmol Fe2+/gDW

Phenolics

1130.8±48.2 µmol Fe2+/gDW 930.4±18.8 µmol Fe2+/gDW 853.0±8.9 µmol Fe2+/gDW 402.8±7.9 µmol Fe2+/gDW 1123.5±52.4 µmol Fe2+/gDW 435.4±18.5 – 909.1±37.5 µmol Fe2+/gDW

References Dalar and Konczak, 2012 Dalar and Konczak, 2012 Dalar and Konczak, 2012 Dalar and Konczak, 2013

Erygium bornmuelleri leaf

Ethanol Acetone Pure water Lyophilized ethanol

Centaurea karduchorum leaf

Lyophilized ethanol

305.9±13.7 µmol Trolox eq./gDW

Aqueous methanol

3.4±0.3 – 17.4±0.3 mg Gallic acid Eq./gDW

Phenolics

Dalar et al., 2012

Aqueous methanol


Phenolics

Dalar et al., 2012

Aqueous methanol


Phenolics


Aqueous methanol

3.6±0.3 – 25.8±0.2 mg Gallic acid Eq./gDW 4.4±0.1 – 7.4±0.1 mg Gallic acid Eq./gDW

Phenolic compounds

Dalar and Konczak, 2012 Dalar and Konczak, 2012

Total phenolics (Folin-Ciocalteu reducing) assay Malva neglecta plant parts (root, stem, leaf, flower, fruit, whole plant) Plantago lanceolata plant parts (root, stem, leaf, flower, fruit, whole plant) Cichorium inytbus plant parts (root, stem, leaf, flower, whole plant) Eryngium bornmuelleri plant parts (root, stem, leaf, flower) Centaurea karduchorum plant parts (root, stem, leaf, flower)

Aqueous methanol

813.6±39.9 µmol Trolox eq./gDW

Rutin, flavonoid glucosides, phenolic acids

Dalar et al., 2013

Flavonoid glucosides, chlorogenic acid Flavonod glucosides

Dalar, unpublished results Dalar, unpublished results

Phenolic compounds


Antioxidant test Verbascum cheiranthifolium plant parts (stem, leaf, flower) Malva neglecta tea

Extraction Aqueous methanol Lyophilized ethanol

Plantago lanceolata tea Salvia limbata tea Phlomis armeniaca tea Anchonium elichrysifolium tea Verbascum cheiranthifolium tea Eryngium bornmuelleri leaf sequential fractions

Results 20.2±0.6 – 33.1±0.4 mg Gallic acid Eq./gDW 27.9±0.4 mg Gallic acid Eq./gDW 80.4±1.8 mg Gallic acid Eq./gDW 56.0±0.9 mg Gallic acid Eq./gDW 60.2±3.5 mg Gallic acid Eq./gDW 31.2±0.2 mg Gallic acid Eq./gDW 67.0±3.3 mg Gallic acid Eq./gDW 44.4±1.7 – 91.9±4.4 mg Gallic acid Eq./gDW

Erygium bornmuelleri leaf

Ethanol Acetone Pure water Lyophilized ethanol

Centaurea karduchorum leaf

Lyophilized ethanol

26.9±1.1 mg Gallic acid Eq./gDW

Gundelia tournefortii L. seed and aerial parts

Lyophilized methanol

Heracleum persicum Prangos ferulacea Chaerophyllum macropodum Rheum ribes stem

Lyophilized methanol Lyophilized methanol Lyophilized methanol Lyophilized chloroform

Aerial parts: 64.4±4.8 mg Gallic acid Eq./gDW Seed: 105.1±8.7 mg Gallic acid Eq./gDW 65.1±6.4 mg Gallic acid Eq./gDW 34.0±7.0 mg Gallic acid Eq./gDW 59.6±2.8 mg Gallic acid Eq./gDW 22.68±1.10 mg Gallic acid Eq./gDW

Rheum ribes stem



Rheum ribes root

Lyophilized chloroform


Rheum ribes root



Mentha longifolia L. subsp. longifolia


45 mg Gallic acid Eq./gDW

Origanum vulgare subsp. vulgare


220 mg Gallic acid Eq./gDW


Compounds identified Phenolic compounds Phenolic compounds

References Dalar and Konczak, 2012 Dalar and Konczak, 2013


Dalar et al., 2013

Flavonoid glucosides, chlorogenic acid Flavonod glucosides

Dalar, unpublished results Dalar, unpublished results Çoruh et al., 2007a

Phenolic compounds

Phenolic compounds Phenolic compounds Phenolic compounds Phenolic compounds and essential oils Phenolic compounds and essential oils Phenolic compounds and essential oils Phenolic compounds and essential oils Phenolic compounds and essential oils Phenolic compounds and essential oils


Çoruh et al., 2007b Çoruh et al., 2007b Çoruh et al., 2007b Öztürk et al., 2007 Öztürk et al., 2007 Öztürk et al., 2007 Öztürk et al., 2007 Gulluce et al., 2007 Şahin et al., 2004

Table 3. (Continued) Antioxidant test Extraction Results DPPH (di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium free radical scavenging activity) assay Gundelia tournefortii L. seed and aerial Lyophilized methanol High radical scavenging activities parts Aerial parts- IC50:0.442 mg/ml Seed- IC50: 0.073 mg/ml Heracleum persicum Lyophilized methanol High radical scavenging activities IC50:0.438 mg/ml Prangos ferulacea Lyophilized methanol High radical scavenging activities IC50:0.242 mg/ml Chaerophyllum macropodum Lyophilized methanol High radical scavenging activities IC50:0.623 mg/ml Rheum ribes stem Lyophilized methanol High radical scavenging activities 87.07±0.54 % at 100 µg concentration Rheum ribes root Lyophilized methanol High radical scavenging activities 60.60±0.86 % at 100 µg concentration Antioxidant test Extraction Results Rheum ribes root Lyophilized methanol High radical scavenging activities 50.87±0.30 % at 100 µg concentration Mentha longifolia L. subsp. longifolia Lyophilized methanol High radical scavenging activities IC50: 57.4 µg/ml Origanum vulgare subsp. vulgare Lyophilized methanol High radical scavenging activities IC50: 9.9 µg/ml


References

Phenolic compounds

Çoruh et al., 2007

Phenolic compounds

Çoruh et al., 2007

Phenolic compounds

Çoruh et al., 2007

Phenolic compounds

Çoruh et al., 2007

Phenolic compounds and essential oils Phenolics compounds and essential oils Compounds identified Phenolic compounds and essential oils Phenolic compounds and essential oils Phenolic compounds and essential oils

Öztürk et al., 2007 Öztürk et al., 2007 References Öztürk et al., 2007 Gulluce et al., 2007 Şahin et al., 2004

Table 4. Enzyme inhibitory activities of traditional medicinal plants from Eastern Anatolia Enzyme inhibitory assay Alfa-amylase inhibitory activity Herbal Teas: Malva neglecta Plantago lanceolata, Salvia limbata, Phlomis armeniaca, Anchonium elichrysifolium, Verbascum cheiranthifolium

Extraction

Lyophilized ethanol

Enzyme inhibitory activity


References

Inhibition (%) at concentration 4 mg/ml 23.6±0.5 32.3±0.4 28.3±1.0 13.5±0.4 22.4±0.9 67.8±1.1

Phenolic compounds

Dalar and Konczak, 2013


Enzyme inhibitory assay Eryngium bornmuelleri leaf sequential fractions Alfa-glucosidase inhibitory activity Herbal Teas: Malva neglecta Plantago lanceolata, Salvia limbata, Phlomis armeniaca, Anchonium elichrysifolium, Verbascum cheiranthifolium Eryngium bornmuelleri leaf sequential fractions Malva neglecta whole plant Plantago lanceolata whole plant Cichorium intybus whole plant

Pancreatic lipase inhibitory activity Herbal Teas: Malva neglecta Plantago lanceolata, Salvia limbata, Phlomis armeniaca, Anchonium elichrysifolium, Verbascum cheiranthifolium Eryngium bornmuelleri leaf sequential fractions Malva neglecta whole plant Plantago lanceolata whole plant

Extraction Ethanol Acetone Pure water Lyophilized ethanol

Ethanol Acetone Pure water Lyophilized ethanol Lyophilized ethanol Lyophilized ethanol

Lyophilized ethanol

Ethanol Acetone Pure water Lyophilized ethanol Lyophilized ethanol

Enzyme inhibitory activity IC50*: 8.3±0.1 mg/ml IC50: 9.6±0.2 mg/ml IC50:19.4±0.4 mg/ml

Compounds identified Rutin, flavonoid glucosides, phenolic acids

References Dalar et al., 2013

Phenolic compounds


IC50: 13.83±0.17 IC50: 1.43±0.04 IC50: 3.28±0.23 IC50: 2.54±0.02 IC50: 12.53±0.28 IC50: 2.03±0.10 IC50: 8.5±0.1 mg/ml IC50: 10.4±0.3 mg/ml IC50:19.1±0.6 mg/ml IC50: 10.02±0.05


Dalar et al., 2013

Phenolic compounds

IC50: 1.68±0.17

Flavonoid glucosides

IC50: 6.04±0.05

Chicoric acid, caftaric acid, chlorogenic acid, flavonoid glucosides

Dalar, unpublished data Dalar, unpublished data Dalar, unpublished data

Phenolic compounds



Dalar et al., 2013

Phenolic compounds

Dalar, unpublished data Dalar, unpublished data

IC50: 10.21±0.08 IC50: 4.53±0.08 IC50: 6.72±0.09 IC50: 8.11±0.04 IC50: 8.56±0.10 IC50: 4.76±0.07 IC50: 5.9±0.1 mg/ml IC50: 5.0±0.1 mg/ml IC50: 6.5±0.2 mg/ml IC50: 12.96±0.38 IC50: 6.21±0.06

Flavonoid glucosides


Table 4. (Continued) Enzyme inhibitory assay Cichorium intybus whole plant

Extraction Lyophilized ethanol

Angiotensin converting enzyme (ACE) inhibitory activity Herbal Teas: Lyophilized Malva neglecta ethanol Plantago lanceolata, Salvia limbata, Phlomis armeniaca, Anchonium elichrysifolium, Verbascum cheiranthifolium

Enzyme inhibitory activity IC50: 3.97±0.20

Compounds identified Chicoric acid, caftaric acid, chlorogenic acid, flavonoid glucosides

References Dalar, unpublished data

Inhibition (%) at concentration 0.6 mg/ml

Phenolic compounds


34.0±2.0 21.0±2.0 35.0±2.0 27.0±2.0 21.0±2.0 23.0±4.0

*IC50 – half maximum inhibitory concentration.



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The same extracts commonly consumed in the Van province as herbal teas successfully suppressed the activities of key enzymes involved in metabolic syndrome: α-glucosidase, pancreatic lipase and the angiotensin I-converting enzyme (ACE) and, with the exception of V. cheiranthifolium, had a moderate to low inhibitory activity against α-amylase (Table 4). Natural α-amylase and α-glucosidase inhibitors from plant sources offer an attractive strategy to control postprandial hyperglycemia. These inhibitors in particular, which have a lower activity against α-amylase and a stronger activity against α-glucosidase, can be used as an effective therapy for postprandial hyperglycemia with minimal side effects (Kwon, et al., 2006), attributed to the simultaneous inhibition of both enzymes, which results in abnormal bacterial fermentation in the colon due to the presence of undigested carbohydrates, leading to abdominal distention, flatulence, meteorism and even diarrhea (Apostolidis et al., 2006). P. lanceolata, P. armeniaca and S. limbata exhibited weak inhibitory activities against αamylase and pronounced inhibitory activities against α-glucosidase, which may suggest their potential anti-diabetic properties. Moreover, P. lanceolata was the most potent inhibitor of pancreatic lipase; this result suggests that P. lanceolata may be considered as a potential candidate for further studies towards identification of anti-diabetic and anti-obesity activities (Dalar and Konczak, 2013).

Conclusion The uses of traditional medicinal plants are based on observations of their action and effectiveness arising from their application on humans over centuries. This chapter showed that herbs which are used to cure multiple health conditions, namely Mentha longifolia, Cichorium intybus and Urtica dioica, are characterized by a concomitant presence of phytochemicals representing various groups: phenolic compounds, carotenoids, fatty acids and terpenes. The rich composition of phytochemicals may hold the key to the efficacy of these commonly used traditional medicinal plants. The majority of Eastern Anatolian medicinal plants evaluated to date exhibited an antioxidant capacity comparable to medicinal plants utilized by other cultures, including traditional Chinese and Ayurvedic systems. This chapter presents a number of plant sources, selected by traditional wisdom, to cure chronic health conditions associated with the contemporary fast-paced lifestyle, including diabetes, obesity, hypertension or hypercholesterolemia. Among the listed medicinal plants are those used for the treatment of various types of cancer; Eryngium bornmuelleri, Anchonium elichrysifolium, Plantago lanceolata, Plantago maritime, Galium verum, Teucrium polium, and Heliotropium circinatum. Until now our knowledge of both the phytochemical composition and mechanism of physiological actions of these traditional medicinal plants is limited. In fact, the majority of these sources have not been subjected to any studies. Traditional medicinal plants presented in this chapter may provide valuable leads for the identification of physiologically active natural compounds for pharmaceutical uses. These plants, selected by generations of traditional healers, represent a vast and underutilized resource for the development of newer and more efficient pharmacological treatments, and a novel approach to curing the diseases of our age. Their value should not be underestimated.


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Chapter 8

Hetherotheca inuloides (Mexican Arnica) a Potent Antioxidant Effect as Neuro and Hepato-Protective Liliana Carmona-Aparicio, Noemí Cárdenas-Rodríguez, Bernardino Huerta-Gertrudis, José Luis Rodríguez-Chávez and Elvia Coballase-Urrutia* Laboratory of Neurochemistry, National Institute of Pediatrics, Chemistry Institute, UNAM, Mexico

Abstract Hetherotheca inuloides (Mexican arnica) is a plant used in traditional medicine in different parts of the world; it is used in various presentations (tablets, beverages, ointments) for therapeutic purposes due to its anti-inflammatory, antimicrobial, analgesic, and antioxidant effects. As an antioxidant, it has attracted considerable interest because of the involvement of oxidative stress in various diseases affecting systemic and central levels. In particular, the focus of this chapter is to describe the evidence that demonstrates the ability of Mexican arnica to be used as a potent antioxidant, and how it can help protect the liver and brain, in experimental models affecting these organs.

Keywords: Hetherotheca inuloides, oxidative stress, antioxidant, neuroprotective and hepatoprotective effects

Introduction The use of plants for medicinal purposes comes from the early history of civilization. The study of substances that make up these plants and their possible mechanisms in health * Corresponding Author address: Email:[email protected].


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L. Carmona-Aparicio, N. Cárdenas-Rodríguez, B. Huerta-Gertrudis et al.

benefits is an area of broad interest, together with the mechanisms underlying diseases that afflict humans. It has now been determined that major biochemical mechanisms associated to pathologies affecting the peripheral nervous system (such as atherosclerosis, ischemia reperfusion injury, diabetes, liver cirrhosis, cancer, epilepsy, Alzheimer's, Parkinson's, Huntington's, etc.), as well as in aging processes and inflammation, is the generation of free radicals (FR) and reactive oxygen species (ROS) (Segura et al., 2000; Halliwell and Whiteman, 2004; Matkowski and Piotrowska, 2006; Wang et al., 2006; Valko et al., 2007). These oxidizing species cause cumulative damage of molecules essential to body function, such as proteins, lipids and DNA (Szabó and Ohshima, 1997). However, the body has its own defense mechanisms to deal with the action of oxidizing species. In certain situations antioxidant defenses may be overwhelmed by excessive ROS generation. This imbalance between oxidants and antioxidants species is known as oxidative stress, which is associated with many diseases as the ones mentioned above (Basaga, 1990; Cárdenas-Rodríguez, 2006). Endogenous antioxidant systems such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and antioxidants with thiol groups, are the first defenses against FR. There also exist non-enzymatic systems, such as glutathione (GSH), vitamin E (tocopherol), vitamin C (ascorbic acid), bilirubin and uric acid that can be enhanced by incorporation into the diet (Matés and Sánchez-Jiménez 1999; Valko et al., 2007,). There are abundant scientific studies demonstrating that flavonoids, carotenoids, vitamin C and vitamin E, may act by capturing these ROS. While most reports are based on the determination of antioxidant capacity, in vitro assays and physico-chemical nature can be extrapolated to potential in vivo protection (Cotelle, 2001; Fang et al., 2002; Albano, 2006; Valko et al., 2007). In this chapter we will give detailed evidence on the neuro- and hepatoprotector effects of Hetheroteca inuloides (Mexican arnica).

Generalities of Medicinal Plants Plants are a rich source of bioactive phytochemicals, more than 5000 of them have been characterized in fruits, vegetables and grains. However, there is still a large number unmarked. It is necessary to continue studies to characterize the effect of these phytochemicals and get to know their potential applications in health benefits.Currently, the phytochemicals are mainly in carotenoids, phenols, alkaloids, nitrogen-containing compounds and thiol groups (-SH). There is evidence suggesting that they are either involved or are a major source for the prevention of chronic diseases (Liu, 2003 and 2004). The functions that are attributed to natural phytochemicals include antioxidant, immune system enhancer, anti-inflammatory, antimutagen, anticancerigen and antibacterial. There is evidence that might be helpful in diseases induced by oxidative stress caused by different harmful agents such as free radicals (FR) (Liu, 2003 and 2004; Matowski and Piotrowska, 2006). On the other hand, it is very important that users who consume or use the plants in therapy for various diseases, are aware of their potential and their possible adverse effects. In Mexico it is known that there are about 30,000 species of plants registered in 1997 by the National Indigenous Institute, with 3,000 documented uses in traditional medicine for


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people living in rural and urban communities. This is considered evidence of their effectiveness (Almaguer, 2001; Martínez, 1984 and 1989) .

Medicinal Plants and Oxidative Stress In the annals of medical history, few events have had such a profound impact as the result of knowing about the existence of FR and reactive oxygen species (ROS) and their influence on living organisms. The production of ROS is a natural process, unavoidable and constant, a continuous biological process, a consequence of life in an aerobic environment. All cells, regardless of their type, are permanently producing ROS (Valko, 2007). ROS production and oxidative stress are associated with tissue injury and many pathological processes, including atherosclerosis, diabetes, cancer, neurodegenerative diseases, liver diseases and normal aging (Halliwell and Gutteridge, 2006; Seifried et al., 2007; Valko, 2007). This has caused a lot of research, aimed to understand and regulate the production of FR and oxidative stress (Bayraktar et al., 2010; Goldibi and Laher, 2010). These oxidizing species cause cumulative damage in molecules essential to body function, such as proteins, lipids and DNA (Halliwell and Gutteride, 2006; Valko et al, 2007). However, the body has its own defense mechanisms to deal with the action of oxidizing species. In certain situations antioxidant defenses may be overwhelmed by excessive ROS generation. This imbalance between oxidants and antioxidants species known as oxidative stress, which is associated with many diseases as the ones mentioned above. Endogenous antioxidant systems such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and gluthione reductase (GR) are the first defenses against FR (Matés and Sánchez-Jiménez, 1999; Halliwell and Gutteridge, 2006; Valko et al, 2007). There also exist non-enzymatic systems, such as glutathione (GSH), vitamin E (tocopherol), vitamin C (ascorbic acid), bilirubin and uric acid that can be enhanced by incorporation into the diet (Fang et al., 2002; Albano, 2006; Valko et al, 2007). There are abundant scientific studies demonstrating that flavonoids, carotenoids, vitamin C and vitamin E, may act by capturing these ROS (Albano, 2006; Valko et al, 2007). While most reports are based on the determination of antioxidant capacity, in vitro assays and physico-chemical nature can be extrapolated to potential in vivo protection. In this chapter we will give detailed evidence on the neuro- and hepato-protector effects of Hetheroteca inuloides (Mexican arnica).

Heterotheca inuloides Generalities In Mexico plant groups of different species share the common folk name "arnica" derived from Arnica montana, european specie widely used in folk medicine in Europe. This group of medicinal plants known as “arnica” and A. montana have similar characteristics and uses.


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These plants are included in the genus Mentzelia, Thitonia, Trixis, Verbesina, Helenium, and Heterotheca (Villaseñor Ríos and Espinosa García, 1998). Four species are recognized in Heterotheca sect. Heterotheca, all of them are found in Mexico: H. grandiflora Nutt., H. subaxilaris Britt, Rusby, H. inuloides Cass (with three varieties), and H. leptoglosa DC. H. inuloides and H. leptoglosa are endemic to Mexico. The H. inuloides is also known as: arnica mexicana or Mexican arnica, acahua, acahualli, field arnica, mountain arnica, arnica the country, arnika (Purepecha), cuateteco, false arnica and tlályetl (Martínez, 1984). H. inuloides has elongated leaves and broad, its flowers are grouped and placed in a circle. It is usually found in cool and temperate climates. It can measure up to 1 m high, and is found in pine and oak forests. For its widespread medicinal use, it is often grown in home gardens and harvesting is done at the time of flowering.It is distributed in Chihuahua, Durango, Guerrero (Coyuca de Catalan), Jalisco Guadalajara, Michoacán (Zitacuaro, Tacuaro, Coalcoman Vazquez), Hidalgo (Gangueo), Morelia (Ocampo), Oaxaca, Puebla, Nuevo León, Zacatecas, San Luis Potosí, Veracruz, Colima, Tlaxcala, Puebla, Distrito Federal, Guanajuato, Morelos, Querétaro, Nayarit and Estado de México (Argueta et al., 1994; Villaseñor Ríos and Espinosa García, 1998; De Rzedowski and Rzedowski, 2001).

Medicinal Propierties In traditional medicine H. inuloides became very popular because it was attributed medicinal properties, plus it is readily available and inexpensive. The whole plant is used and consumed as decoct to ease the pain of lung, heart, muscle, rheumatism, stomach, kidney and ulcers. It is also used as compresses, dressing, as an ointment mixed with butter or poultices to heal wounds, bruises, pimples, rash, swollen bumps, sores (Villaseñor Ríos and Espinosa García, 1998; De Rzedowski and Rzedowski, 2001). The aerial parts (flowers, petal or stamens) in decoction is used to wash wounds, applied to bruises and welts. For inflammation and ovaries matrix compresses are applied. The foliage is used as an analgesic for chest pain, heartburn, gastritis, and internal and external traumas. The leaves are boiled to unswelland disinfect wounds and the water derived from this boiling is used to induce appetite. It has also been reported to be used for: bladder irritation, kidney cancer, nerves and eye bath in order to promote inflammatory states, but no one has defined the part of the plant used. In the market H. inuloides is found in different forms for example, homeopathic tinctures, teas and ointments, which are used for skin infections, fevers, bumps and sores (Villaseñor Ríos and Espinosa García, 1998; De Rzedowski and Rzedowski, 2001). In recent years, results of research on the characterization of various components of the flower and its biological effects have been reported.

Therapheutics Effects of Diferents Mexican Arnica Extracts From the plant, the following pure compounds have been characterized: luteolin, kaempferol, 3,7-dimethyl-kaempferol, kaempferol-3, 7,4'-trimethylether, 6-methoxykaempferol-3,7-dimethyl ether, quercetin, quercetin-3,3'-dimethylether, quercetin-3, 4'-



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dimethyl ether, quercetin-3 ,7-dimethyl, 3',4'-tetramethylether, quercetin-3,7,3'trimethylether, quercetin-3,7,4'-trimethylether among others. The structures of these compounds were established by methodologies including UV spectroscopy, nuclear magnetic resonance and mass spectra (Jerga et al., 1990). Sereveral of the plant constituents are polyacetylenes, cadinanes, triterpenes, sterols, sesquiterpenes and flavonoids with different biological properties (Table 1). Table 1. Different pharmacological properties of the metabolites derived from acetone and methanol extracts of Heterotheca inuloides Extract Name Heterotheca inuloides Acetonic extract

Heterotheca inuloides Methanolic extract

Chemical composition (% extract ) Cadalen-15-oic acid, 3,7-dihydroxy-3(4H)-isocadalen-4-one, dicadalenol, 7-hydroxycadalene, 7-hydroxy-4 α H-3,4-dihydro-cadalene, 1α, hydroxy-1(4H)-isocadalen-4-one, 1α-hydroxy-4αh-1,2,3,4-tetrahydrocadalen15oic acid, 7-(3,3-dimethylallyloxy)coumarin, Caryolan-1,9β-diol, Quercetin, Stigmasterol, β-sitosterol. Quercetin (0.19), Quercetin 3-O-glucoside, kaempferol, kaempferol-3-O-glucoside, kaempferol-osophoroside, D-chiro-inositol, Epinasterol, 3-O-β-D-epinasterol glycoside, 7-hydroxy-4 α H-3,4-dihydro-cadalene (0.004), 7-hydroxycadalene (0.002).

Pharmacology propierties Antiinflammatory. COX-1 and COX-2 inhibitory effect. Antioxidant. Inhibition of lipid peroxidation.

References

Inhibition of Tyrosinase activity. Antioxidant. Inhibition of lipid peroxidation. Antiinflamatory.

Haraguchi et al., 1997. Kubo et al., 1994. Delgado et al., 2009 (personal comunication)

Haraguchi et al., 1997. Segura et al., 2000. Delgado et al., 2001.

In the Nineties, four sesquiterpenoids were isolated that exhibited antimicrobial activity against gram positive and two of these also showed anti-fungal activity. These results, as well as investigations of their common use for nosocomial infections, support the use of this plant in skin infections since the use of antibiotics to combat them can induce long-term resistence (Kubo et al., 1994). The sesquiterpenoid 7-hydroxy-3,4-dihydrocadalene isolated from H. inuloides was used against lipid peroxidation in an in vitro model using rat liver microsomes and human erythrocytes in conditions of oxidative stress (Haraguchi et al., 1996). The sesquiterpenoid βcaryophyllene, β-caryophyllene 4,7-oxide 5α-hydroxy-3,4-dihydrocadalene and 7hydroxycadalene also showed cytotoxic activity against several lines of solid tumors (Kubo et al., 1996). Likewise, the 7-hydroxy-3,4-dihydrocadalene and 7-hydroxycadalene in addition to the flavonoids quercetin and kaempferol, were evaluated as antioxidants. These compounds showed potent radical scavenging activity of diphenyl-p-picrylhydrazyl (DPPH●) and radicalanion superoxide in an in vitro model using rat liver microsomes where the best scavengers are flavonoids (Haraguchi et al., 1997).


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Analgesic and anti-inflammatory effect in in vitro and in vivo models was evaluated with different bioactive compounds of Mexican arnica. 7-hydroxy-3,4-dihydrocadalene inhibited in vitro expression of COX-1 and COX-2 catalyzed biosynthesis of prostaglandins, as well as edema of croton oil-induced in an animal model. In addition, it is reported that 1-9 cariolan-βdiol, and quercetin dicadenol also possess anti-inflammatory effect with minimal side effects (Gené et al., 1998; Segura et al., 2000; Delgado et al., 2001).

Oxidative Stress and Mexican Arnica A large number of reports suggest that the use of antioxidant compounds can help to maintain human health. Pharmaceutical, food and cosmetic industries use synthetic or natural antioxidants such as vitamin C, sodium ascorbate (AA1) (Elmore, 2005), thymol, o-cymen (AA2) Drometrizole (AA3) in their preparations (Anderssen, 2006). Therefore, studies are focused on the identification of extracts and naturally occurring metabolites as antioxidant supplements. Most of these natural antioxidants come from fruits, vegetables, spices, grains, and herbs such as ginseng, turmeric, ginkgo, rosemary, green tea, grape, ginger and garlic (Rababah et al., 2004). H. inuloides is a plant widely used in Mexico for its recognized medicinal effects. It is used in traditional medicine for the treatment of thrombophlebitis, acne, bruises and muscle aches and as an anti-inflamatory. The methanolic extract was mainly constituted by flavonoids and terpenes that were found in lower concentrations in the acetonic extract (Delgado et al, 2001). In particular, polyphenols and flavonoids are responsible for the antioxidant activity, predominantly in the methanolic extracts which have high antioxidant capacity (Rice-Evans and Miller, 1996 and 1997; Rice-Evans, 2001). Several constituents of this plant have been previously identified (Table 1). Some of these individual terpenoids and flavonoids are reported to have anti-inflammatory activity and have been found in the acetonic extract, detected by the inhibition of edema in the mouse ear induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) (Delgado, et al., 2001). It has further been reported that H. inuloides sesquiterpenes have potent radical scavenging activity of DPPH●, with no activity against superoxide generated by enzymatic or non-enzymatic pathways (Haraguchi et al., 1997). On the contrary, H. inuloides’s flavonoids are efficient scavengers of the two types of radicals. This characterization is part of the bio-evaluation of its effectiveness as a traditional medicine and identification of potential drug molecules. The polyphenol components of the plant are very important because they have hydroxyl groups that make them able to scavenge radicals and chelate transition metal ions (RiceEvans and Miller, 1997). Moreover, literature data suggests that structural characteristics of flavonoids include: i) a o-diphenol in the B ring, ii) a double bond at position 2 and 3 combined with the 4-oxo function, and iii) the presence of hydroxyl groups at positions 3 and 5. These features are important for their activity as scavengers of FR (Furuno et al., 2002; Wang et al., 2006; Choi et al., 2007). Ascorbic acid is an essential compound in plant tissues, it has been reported to act as an effective scavenger of DPPH● (Soares, et al., 2003), therefore commonly used as reference compound. Haraguchi determined that quercetin glycosides and kaempferol isolated from H.



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inuloides have a DPPH● scavenging activity because they contain a hydroxyl group attached to a methyl group which may contribute to this activity (Haraguchi et al., 1997). Recently, it has been suggested that the flavonoids possessing a catechol-O-group with a double bond at position 2 and 3 on the B ring and bonded to an oxo group at position 4 in the C ring may be responsible for the trapping of DPPH● (Haraguchi et al., 1997). In particular, the ABTS [2-2´-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid)] assay, this has been used for analysis of antioxidant activity of various flavonoids. Pannala reported that flavonoids with 3 hydroxyl in the B ring are better ABTS scavengers, and compounds having one hydroxyl group only take longer or may not react at the same time to reduce the ABTS (Panala et al., 2001). These facts suggest that the B ring and the various substituent hydroxyl groups are important for the antioxidant activity of flavonoids. Superoxide (O2●-) is a ROS, which can cause cell and DNA damage leading to various diseases (Basaga, 1990; Halliwell and Gutteridge, 2006). It has been proposed that inhibition of the xanthine/xanthine oxidase system for various flavonoids may be linked to the hydroxyl groups. In particular this radical is scavenging by acetonic and methanolic extracts of H. inuloides (Cos et al., 1998). Furthermore, it was demonstrated that the extra hydroxyl group in the basic structure of the flavonoids, contribute to the inhibition of the xanthine/xanthine oxidase system and therefore influence the decrease of the IC50 (Coss et al., 1998). Van Hoorn reported the hydroxyl substituents inhibiting decrease in the following order: 5> 7> 4'= 3'. Although Cos demonstrated the importance of the hydroxyl group in the 5 and 7 positions of the A ring and in the 3' and 4´ position of the B ring (Van Hoorn et al., 2002). There are reports that show the scavenging activity of short chain flavonoids, with 5 or more hydroxyl groups in their substituents different structures. For instance, quercetin (flavone), catechin (flavonol) and taxifolin (flavanone) with 5 hydroxyl groups (3, 5, 7, 3', 4') vary in a IC50 range of 3 to 9 mM, or flavonoids with 3 hydroxyl groups (5, 7, 4'), with IC50 of 90 mM. At the same time it continues to support the importance of the hydroxyl group at position 3 on the C ring that interacts with the B ring by binding with the hidroxyl group in 6' and 2'. Furthermore, it was demonstrated in liver microsomes that flavonoids from H. inuloides trap O2●- generated enzymatically or non-enzymatically (Haraguchi et al., 1997). The results reported in our work group showed that quercetin and kaempferol inhibits the production of O2●- generated by xanthine/xanthine- oxidase system (Coballase et al., 2010). These findings suggest that the reduction in superoxide concentration is associated with the presence of these metabolites isolated from H. inuloides. Moreover, the hydroxyl radical (OH●) is associated with various pathologies (Valentão et al., 2002). The OH● is generated by the reaction between the iron/EDTA with H2O2 in the presence of ascorbic acid. We showed that in all cases quercetin and kaempferol had a good radical scavenging capacity (Melidou et al., 2005; Widmer et al., 2010). Its chemical structure is associated to the scavenging of FR, due to the ease in which the hydrogen atom from the aromatic hydroxyl group can be donated to the radical species. The stability of the resultant quinone structure supports an unpaired electron, which stabilizes and relocates the unpaired electron and for its ability to chelate transition metal ions (Cos et al., 1998; Pannala et al., 2001). Quercetin is known to chelate iron and therefore directly inhibit lipoperoxidation (Mathew and Abraham, 1996). Haraguchi demonstrated that quercetine, kaempferol, 7 hidroxy-3,4-dihydrocadalene and 7-hidroxycadalene flavonoids isolated from the dried


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flowers of H. inuloides inhibited lipid peroxidation (Haraguchi et al., 1996 and 1997). These bioactive compounds are found in both extracts. Therefore, this effect can be attributed mainly to the donation of hydrogen and electron transfer capability of hydroxyl groups as substituents (Coballase et al., 2010). Moreover, the hydroxyl radical may come from multiple sources including peroxynitrous acid or peroxide acid decomposition, mediated by a metal. Hydrogen peroxide (H2O2) in vivo is commonly reduced by iron (II), which results in the formation of OH● via Fenton reaction, as described below (Widmer et al., 2010). Fe2++H2O2- Fe3++OH●+OH The iron becomes unable to participate in Fenton reactions and the propagation phase of lipid peroxidation. H2O2 is a weak oxidizing agent that inactivates the enzymes usually by the substantial oxidation of the thiol groups (-SH). Melidou evaluated in Jurkat cells, the ability of flavonoids to protect DNA damage caused by H2O2. Results reported in our work group suggest that flavonoids are able to bind to the iron, mainly by the presence of a hydroxyl group in position 3 and 4 of the C ring. However, the presence of an additional hydroxyl group in position 5 increases protection, this data is consistent with previous reports in the literature (Melidou et al., 2005). HOCl is produced in the body by oxidation of Cl-ions by the enzyme neutrophil myeloperoxidase, and proved to be a powerful oxidant that reacts easily with many important molecules (Arouma et al., 1989). Firuzi reported that the common structure feature of various flavonoids is the presence of a hydroxyl group at 5-position in the A ring and the presence of more than two hydroxyl groups, which confers greater activity against hypochlorous acid (Firuzi et al., 2004). Another observation proposed is the presence of a hydroxyl group in position 3 of the C ring; this probably has a greater effect in the entrapment of HOCl. The blocking of this position in quercetin is associated to a diminishing scavenging activity (RiceEvans et al., 1996; Hirose et al., 2002). Binsack demonstrated that polyphenols react with HOCl and its chlorinated products. Analysis by mass spectrometry indicated that the chlorination takes place at C6 and C8, which increases evidence of its antioxidant capacity (Binsack et al., 2001). Peroxinitrite (ONOO−) is a compound formed by the reaction between the O2●- and nitric oxide (NO●), it can easily cross the biological membrane and interact with target molecules such as DNA, proteins and lipids. In this sense, the efficiency of flavonoids to protect against ONOO− toxicity has been demonstrated in several studies (Heijnen et al., 2001). The ability to react with various flavonoids ONOO− where observed indirectly by measuring the products formed by the nitration of tyrosine. This measurement was done by HPLC and mass spectrometry. The authors conclude that the hydroxyl groups in 3' and 4' position of the B ring, the double bond at carbon 2 and 3 and the carbonyl group in position 4 of the C ring are strongly implicated in the antioxidant activity (Pollard et al., 2006). Singlet oxygen (1O2) is generated in the biological systems by a number of endogenous processes (enzymatic and chemical reactions) and exogenous stimuli (e.g. UV or visible light); their main targets are side chains of proteins (Davies and Truscoot, 2001). There are few reports in which the flavonoids have worked as scavengers, these studies indicate the importance of a catechol group in the B structure and that perhaps the double bond at the C ring may be essential to scavenge the 1O2 (Huvaere et al., 2009). These observations are



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confirmed, mentioning that the number and arrangement of hydroxyl groups, particularly the 3' and 4' of the B ring are essential, in position 4 of the ring C and the oxo group also favors the activity (Huvaere et al., 2009). It has been shown that scavenging of the O2●- by EPR method, supports the fact that both extracts effectively reduced the signal intensity of the adduct DMPO-OOH generated by the xanthine/xanthine oxidase system (Lun-Yi et al., 1999; Ying-Shan et al., 2006). As mentioned above the O2●- can dismute to form H2O2 in the presence of transition metals through the Fenton reaction. Therefore, the protection mechanism of these agents is the elimination of O2●-.

Protective Effects of H. inuloides Recent information regards the H. inuloides antioxidant properties generated in vitro. Our work group focus is to study the effects on oxidative profile of acetone and methanolic extracts of H. inuloides in a hepatotoxicity model where rats are poisoned with carbon tetrachloride (CCl4). Hepatotoxicity induced by CCl4 is developed during the biotransformation of this by cytochrome P-450 forming two FR (Shimizu et al., 2001; Weber et al., 2003). The first radical (CCl3●) forms covalent bonds with lipids and proteins. CCl3● can either interact with O2 to form a second radical (CCl3O●) or lose hydrogen atoms to form chloroform (Clawson, 1989; Szymonik-Lesiuk et al., 2003). These events occur in the membrane and cause liver damage. A first hypothesis of liver damage induced by CCl4 administration was observed by the evaluation of liver injury marker enzymes such as AST and ALT. The transferases activity increases 48 hr after the final treatment. The administration of acetonic or methanolic extracts significantly prevented CCl4-induced elevation of AST and ALT, indicating hepatoprotective activity of the extracts of H. inuloides (Coballase et al., 2010). Lipid peroxidation derived FR from CCl4 (CCl3● and CCl3O2). The administration of this compound is one of the major mechanisms of injury induced in the liver. MDA (malondialdehyde) is a highly reactive aldehyde that appears during peroxidation of fatty acids of biological membranes. In experiments conducted in our laboratory induced toxicity in the group treated with CCl4 caused an increased MDA level compared to the control groups. Treatment with acetonic and methanolic extracts of H. inuloides significantly reversed these changes. The rise in MDA levels in liver may be caused by failure or decreased antioxidant defense mechanisms to prevent the formation of ROS. However, the polyphenols found in both extracts played an important role in cellular protection. CCl4 toxic effect was also confirmed by histological observations showed extensive perivenular macrovesicular steatosis with severe fibrosis and necrosis (Coballase et al., 2010). The pretreatment of rats with acetonic or methanolic extracts clearly protected from CCl4-induced hepatotoxicity. Our results also showed that both extracts slightly affected the normal architecture creating some areas of discontinuity in the cords of hepatocytes. Considering that quercetin is known as a hepatoprotective agent and one of the main components present in the methanolic extract (Table 1), it was included in the experiments. 3nitrotyrosine (3-NT) is thought to be a relatively specific marker of oxidative damage, which is produced by the reaction between O2●- and NO● (Coballase et al., 2010).


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NO● induces damage to proteins, lipids and nucleic acids, which can lead to apoptosis and necrosis. Tyrosine residues of nitrated proteins give rise to the production of 3-NT. The capacity of the methanolic extract of H. inuloides toprevent protein damage has been evaluated by 3-NT immunostaining. The results obtained by our work group clearly indicate that the accumulation of 3-NT in CCl4 treated group was significantly higher than in the groups treated with the methanolic extract and quercetin (Coballase et al., 2010). The results of our in vitro tests confirm the hypothesis that both quercetin and methanolic extracts have scavenging capacity against ONOO− and 1O2 (Coballase et al., 2010). Another commonly used marker of the presence of lipid peroxidation is the 4-hydroxynonenal (4-HNE) (Zarkovic, 2003). Our data is consistent with increased staining of 4-HNE adducts in the group of animals treated with CCl4. In the liver samples from exposed groups to quercetin and methanolic extract, there is no noticeable increase that suggests an improvement of oxidative stress on these components (Coballase et al., 2010). Oxidative stress is generally defined as an excess ROS formation. The effect of antioxidants on oxidative stress is easily measured through certain biomarkers as CAT, SOD, GPx and GR. Several studies have shown that the antioxidant enzymes such as SOD, CAT, GPx and GR represent a protection against oxidative damage to various tissues (Coballase et al., 2013). SOD has a very effective defense, due to the transformation of O2●- to H2O2. CAT is found in all cells and metabolizes H2O2 to oxygen and water. GPx plays an important role in detoxification of xenobiotics in the liver and catalyzes the reduction of H2O2 and hydroperoxides to nontoxic products. GR is a hepatic cytosolic enzyme involved in the detoxification of a range of xenobiotics by conjugation with GSH (Coballase et al., 2013). CCl4 induced a significant decrease in the levels of activity of the antioxidant enzymes CAT, SOD, GPx, GR, probably due to inactivation of proteins by FR. The methanolic extracts and quercetin were able to prevent some of the decreased activity of the antioxidant enzymes, this preventive effect could be reflected in the improved liver histology caused by the extract (Coballase et al., 2011).

Therapeutic Relevance The use of medicinal plants has been one of the major therapeutic tools to treat human diseases. This knowledge has been systematized with increasing experimental approaches that allows us to elucidate the mechanisms of action by which these plants exert their therapeutic capacity. It has been shown that the scavenging ability of these agents makes them powerful antioxidant therapeutic agents for diseases that afflict all of our body systems, which can become chronic and degenerative diseases, while not excluding those non-pathological physiological processes such as aging processes. In particular, the evidence shows that Mexican arnica (H. inuloides) has a potent antioxidant capacity that allows us to understand its use in more than one tratment for diseases. It is precisely this ability that gives us the possibility of using it as a therapeutic agent in other diseases in which it has not yet been used.



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Conclusion The results reported in our work and the ones described in literature show that methanolic and acetonic extracts isolated from H. inuloides are efficient FR scavengers. Scavenging properties of H. inuloides described in this chapter, may explain some of the diseases caused by ROS. The antioxidant activity against some ROS resides mainly in the methanolic extract, particularly in its metabolites spinasterol and quercetin. These metabolites have higher FR scavenging properties and protect the liver and brain against oxidative damage induced by CCl4. It is suggested that methanolic and acetonic extracts of H. inuloides could confer protection against acute hepatotoxicity induced not only by CCl4, but also by other environmental or biological agents capable of inducing FR. The results described in this chapter support the biomedical properties attributed to this plant and warrant further research into its potential use as a preventive agent in human populations.

Acknowledgments We thank Biotechnology Engineer Arantxa Romero-Toledo and Professor Aristides III Sampieri-Hernández for their technical assistance.

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Chapter 9

Meconopsis: Traditional Uses, Chemistry and Pharmacology

1

Haifeng Wu1, Xiaopo Zhang1, Yan Zhou4, Xiaofeng Zhang2, Yao Li3, Jingyi Zhang3, Lisheng Ding4, Junshan Yang1 and Xudong Xu1,*

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China 2 Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China 3 College of Oceanography, Hainan University, Haikou, China 4 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China

Abstract As the second-largest genus in the family Papaveraceae, Meconopsis comprises about 57 species among which 32 species are distributed in Qinghai-Tibet Plateau. The plants of Meconopsis have been prescribed as popular Tibetan medicine for the treatment of tuberculosis and hepatitis. The chemical constituents have been examined and the isolation of alkaloids, flavonoids and essential oils has been reported. Pharmacological activities include hepatoprotection and analgesic effects. The phytochemical and pharmacological studies on medicinal plants of Meconopsis genus have been reviewed in this chapter.

Keywords: Papaveraceae, Meconopsis, Alkaloids, Pharmacological activity

Introduction Meconopsis Vig., the second largest genus in the family Papaveraceae, comprises about 57 monocarpic or polycarpic species mainly distributed discontinuously in both East Asia and *



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Haifeng Wu, Xiaopo Zhang, Yan Zhou et al.

West Europe. Except M. cambria indigenous to England, Wales, Ireland, and the fringes of Western Europe, most species are commonly found in the region of the Himalayas and China. About three fifths of these species is distributed in Qinghai–Tibet plateau area including seven endemic species (An et al., 2009; Egan, 2011; Li et al., 2012; Ohba et al., 2009; Prain, 1906; Sulaiman and Babu, 1996; Wu and Zhuang, 1980; Yoshida et al., 2012; Zhuang, 1981). The plants of the genus Meconopsis (Himalayan poppy), annual or perennial poppy-like alpine herbs with attractive flowers, are growing in a variety of habitats ranging from temperate forests and pastures below the tree line at around 2500 m, to alpine meadows, screes and nival zones up to 5500 m (Luo et al., 1984). Some plants have been individually used in Tibetan Sowa-Rigpa Medicine (commonly known as 'Amchi') and the Bhutanese traditional medicine as a chief ingredient of polyherbal formulations for the treatment of hepatitis, tuberculosis, headaches and fractures, etc (Wang et al., 2003; Northwest Plateau Institute of Biology, 1991). The medicinal application of Meconopsis was recorded in the traditional Tibetan pharmacopeia, e.g. Yue Wang Yao Zhen, Si Bu Yi Dian, and Jing Zhu Ben Cao. M. quintuplinervia, M. integrifolia, M. punicea, M. horridula, and M. racemosa were most commonly used Tibetan herbs. The genus Meconopsis has been extensively investigated for the presence of alkaloids in its many species. Besides, chemical investigation on the genus indicated the presence of flavonoids, essential oils, steroids, triterpenoids and other constituents. Pharmacological research demonstrated that extract of some Meconopsis exhibited significant antioxidant, hepatoprotection and analgesic effects. Herein, the research reported over the past decades on the isolation and pharmacology is summarized in this chapter.

Phytochemistry Previous phytochemical investigation on the genus Meconopis resulted in the isolation of alkaloids, flavonoids, essential oils, and other constituents, etc.

1. Alkaloids Alkaloids, nitrogen-containing heterocycles, have attracted the interest of scientists in the chemical fields for a long time. Within the class of alkaloids, the isoquinoline alkaloids are of particular interest, owing to their tremendous potential chemical variations of the basic precursor molecules and on the other hand comprise some of the most important drugs for therapy and euphoria (e.g. morphine and its chemical derivatives, papaverine, berberine, dimeric bisbenzylisoquinolines) (Zenk, 1994). Isoquinoline alkaloids with aromatic tetracyclic backbone are characteristic constituents of the genus Meconopsis. Until now, about 58 alkaloids belonging to ten different skeletal types (i.e. rhoeadines, protoberberines, pavines and isopavines, aporphines, proaporphines, protopines, benzophenanthridines, indoles, benzylphenethylamines, and others), have been obtained from plants of nineteen species, showing antimicrobial, anti-inflammatory and analgesic activity. Most of these alkaloids are tertiary, the rest being quarternary followed by secondary alkaloids. The first reported isolations of alkaloids in Meconopsis date back to the first half of the 20th century. A



245

number of alkaloids from different species were isolated by Slavík and coworkers (Slavík, 1960, 1965; Slavík and Slavíková, 1976, 1977, 1996). Subsequently, Hemingway et al. reported the isolation of several alkaloids from M. cambrica in Europe (Hemingway and Phillipson, 1975; Hemingway et al., 1981). Besides, Allais et al. obtained a new alkaloid from M. villosa (Allais, 1983). Most recently, Tatsis et al. isolated a series of nudicaulins, a group of alkaloids with a unique pentacyclic skeleton composed of an indole ring and a polyphenolic moiety from M. cambrica (Tatsis et al., 2013). Chinese researchers started research into alkaloids in Meconopsis in 1980s. A series of alkaloids were discovered from eight species (Gao et al., 1997; Li, 2007; Liu and Wang, 1986; Shang et al., 2002a, 2003a, 2003b; Shang, 2002b; Wang et al., 1991; Wang and Chen1995; Wang et al., 1994; Wang and Ding, 1996; Wu et al., 2007, 2009, 2011; Xie et al., 2001). Zhou et al. used response surface methodology (a statistic approach) for optimization of UHPLC–QTOF conditions to analyze the main alkaloids of Meconopsis species (Zhou et al., 2009). Recently, two new proaporphine alkaloids, (+)-Des-N-methylcryprochine (38) (Li, 2007) and 8, 9dihydroprooxocryptochine (39) (Wu et al., 2009), were obtained by Li et al. and our group, respectively. 1.1. Rhoeadines Rhoeadine alkaloids that have only been found among the family Papaveraceae and are biosynthetic derivatives of protopines, are cyclic acetals or hemiacetals having the (R) configuration at C-2 and are dextrorotatory, bearing oxygen substituent at C-7, C-8, C-12, and C-13. However, no rhoeadine alkaloids have been found with an oxygen substituent at C-9. The acid-catalyzed rearrangement and dehydration of rhoeadine alkaloids can result in a red irniniurn salt. Six rhoeadine alkaloids are reported, including rhoeadine (1), isorhoeadine (2), papaverrubine A (3), papaverrubine C (4), papaverrubine D (5), and papaverrubine E (6) (Slavík and Slavíková, 1976, 1977, 1996) (Figure 1). O O

6

9

H

B NCH3 1 H 10

O C

14

H3CO

R1

O

4

A

O

H

D O

NCH3 H O

H3CO

O

1

HO

H

O

O

2

H3CO

NH H

H

O

H3CO

O

O

R2

H

NH H O

H3CO

O

4

O

3 R1 + R2 = OCH2O 5 R1 = OCH3, R2 = OH

NH H

H3CO

O

O

O

6

Figure 1. Structures of rhoeadine alkaloids (1–6).


246


1.2. Protoberberines Protoberberines present themselves as tetrahydroprotoberberines and protoberberine salts. Twelve protoberberines, coptisine (7), cheilanthifoline (8), corysamine (9), alborine (10), berberine (11), mequinine (12), palmatine (13) (–)-mecambridine (14), (–)-Omethylmecambridine (15), (–)-N-methylmecambridinium (16), karachine (17), and valachine (18) were isolated (Slavík and Slavíková, 1977, 1996; Hemingway and Phillipson, 1975; Hemingway et al., 1981; Gertig, 1996; Liu and Wang, 1986; Wang et al., 1991; Shang et al., 2003b; Wu et al., 2011) (Fig. 2). Mecambridine showed antihistamine, anti-inflammation effects, and inhibition of human drug metabolizing cytochrome P450 enzymes (Hemingway and Phillipson, 1975; Salminen et al., 2011). R3 N+

R2 R1

7 9 10 11 12 13

R4 R5

R9

R6

R8

R1 = R4 = R7 = R8 = R9 = H, R2+R3 = R5+R6 = OCH2O R1= R4 = R7 = R8 = H, R2+R3 = R5+R6 = OCH2O, R9 = CH3 R1 = R6 = R7 = OCH3, R2+R3 = OCH2O, R4 = R9 = H, R8 = CH2OH R1 = R4 = R7 = R8 = R9 = H, R2 + R3= OCH2O, R5+ R6 =OCH3 R1 = R2 = R4 = R6 = R7 = OCH3, R3 = OH, R5 = R9 = H, R8 = CH2OH R1 = R4 = R7 = R8 = R9 = H, R2=R3 = R5 = R6 = OCH3

R7

R4 R3 N

R2 R1

H

R5 R8

8 R1 = R4 = R7 = R8 = H, R2 = OH, R3 = OCH3, R5 + R6 = OCH2O 14 R1 = R6= R7 = OCH3, R2 = R3 = OCH2O, R4 = R5 = H, R8 = CH2OH 15 R1 = R4 = R6= R7 = OCH3, R2 = R3 = OCH2O, R5 = H, R8 = CH2OH

R6 R7

O O

O N+

O

O O

O N

O N

OCH3 OCH3

CH3

OH OCH3 16

OCH3 OCH3

17

OCH3 OCH3 18

Figure 2. Structures of protoberberine alkaloids (7–18).

1.3. Pavines and Isopavines Pavines and isopavine alkaloids are probably derived biosynthetically from the tetrahydrobenzylisoquinoline, frequently found in four plant families, namely the Papaveraceae, Berberidaceae, Lauraceae, and Ranunculaceae. Ten alkaloids, (–)-reframoline (19), (–)-amurensinine (20), N-oxide amurensinine A (21), N-oxide amurensinine B (22), amurensinine methohydroxide (23), (–)-flavinantine (24), O-methylflavinantine (25), (–)amurine (26), meconoquintupline (27), and O-methylpallidine N-Oxide (28) (Hemingway et al., 1981; Slavík and Slavíková, 1976, 1996; Wang et al., 1991; Wang and Chen, 1995; Wang et al, 1994, 1996; Wu et al., 2009) were obtained (Fig. 3).


Meconopsis: Traditional Uses, Chemistry and Pharmacology R1 NCH3

R2

R3

O

R4

O

R1

19 R1 = OH, R2 = OCH3, R3+R4 = OCH2O 20 R1+R2= OCH2O, R3 + R4 = OCH3 NCH3

NCH3 H O

R1 R2

OCH3

O

OCH3

+ R2

N

OCH3

21 R1 = O, R2 = CH3 22 R1 = CH3, R2 = O 23 R1 = R2= CH3

O

O

OCH3

N O

H3CO

O OCH3

H3CO

27

24 R1 = OCH3, R2 = OH 25 R1 = R2 = OCH3 26 R1 = R2 = OCH2O

247

28

Figure 3. Structures of pavine and isopavine alkaloids (19–28).

1.4. Aporphines Aporphines, derived from benzyltetrahydroisoquinolines, are a diverse family of isoquinoline alkaloids with more than 300 members, sharing a characteristic tetracyclic motif with different levels of oxidation on both aromatic rings. A range of interesting biological activities has been documented, including serotonergic, antiplatelet, anticancer, antimalarial and vasorelaxing activity. Five alkaloids, (+)-magnoflorine (29), (+)-corytuberine (30), (+)mecambroline (31), roemeroline (32), and (+)-roemerine (33), were reported (Gertig, 1996; Hemingway and Phillipson, 1975; Slavík et al., 1965; Slavík and Slavíková, 1976, 1977, 1996) (Fig. 4). H3CO HO HO H3CO

O

H3CO N+ CH3 H CH3

N CH3 H

HO HO

R1

H3CO 30

29

N CH3 H

O

R2

31 R1 = OH, R2 = H 32 R1 = H, R2 = OH 33 R1 = R2 = H

Figure 4. Structures of aporphine alkaloids (29–33).

1.5. Proaporphines Proaporphine alkaloids are probably intermediates in the conversion of benzylisoquinoline into aporphines. The dienone group in ring D was produced through oxidation of benzylisoquinoline. Six proaporphine alkaloids, (–)-mecambrine (34), (–)-Nmethylcrotonosine (35), (–)-pronuciferine (36), and glaziovine (37), (+)-Des-N-


248


methylcryprochine (38) and 8, 9-dihydroprooxocryptochine (39), were obtained (Li, 2007; Hemingway and Phillipson, 1975; Slavík, 1960, 1965; Wu et al., 2009) (Fig. 5). R1

H3CO NCH3

R2

H3CO NCH3

R

H

H3CO NH

H3CO

H

H

N

HO

O O

O

34 R1+R2 = OCH2O 35 R1 = OH, R2 = OCH3

HO

HO

36 R = OCH3 37 R= OH

39

38

Figure 5. Structures of proaporphine alkaloids (34–39).

1.6. Protopines The difference between protopines and proberberine is the cleavage of C–N into three rings system in protopine alkaloids. Three protopine alkaloids, protopine (40), cryptopine (41), and allocryptopine (42), were reported (Gao et al, 1997; Hemingway and Phillipson, 1975; Hemingway et al., 1981; Shang et al., 2003b; Wu et al., 2007, 2011) (Fig. 6). R1 N

R2

CH3

O

R3

R5

R4

40 R1+R2 = R3+R4 = OCH2O, R5 = H 41 R1 = R2 = OCH3, R3+R4= OCH2O, R5= H 42 R1+R2 = OCH2O, R3= R4 = OCH3, R5 = H Figure 6. Structures of protopine alkaloids (40–42).

1.7. Benzophenanthridines Benzophenanthridine could be derived from proberberine by the cleavage of C-6–N bond and formation of C-6–C-13 bond, consisting of four fused ring including aromatic rings A and D. Five alkaloids, sanguinarine (43), norsanguinarine (44), chelerthrine (45), dihydrosanguinarine (46), and 6-acetonyl-5,6-dihydrosanguinarine (47) were obtained (Hemingway et al., 1975, 1981; Shang et al., 2003b) (Fig. 7). O

O

O

O

O

O

O

+

O

NCH3

O 43

O

N O

H3CO H3CO

44

N CH3

O O

N O

45

Figure 7. Structures of benzophenanthridine alkaloids (43–47).


R

CH3

46 R = H 47 R = Acetonyl


249

1.8. Indoles A indole alkaloid, 6-methoxy- 2-methyl-1,2,3,4-tetrahydro-β-carboline (48), was isolated (Slavík, 1960, 1965). Most recently, a series of nudicaulins (49–54) were isolated from M. cambrica (Tatsis et al., 2013) (Fig. 8). H3CO N

N H

HO HO

O

H

OR2 HO HO OH HO HO

HO HO

OH

O

OH

OR1

48

OR1

N

OH

O HMG

O

O O O

COOH OH Mal

OH 49 R1=R2= H 51 R1=Mal, R2= H 53 R1=HMG, R2= H

OH

O

H

OR2

COOH

O O

O

HO HO OH HO HO

N

O O

O O

O

OH

OH 50 R1=R2= H 52 R1=Mal, R2= H 54 R1=HMG, R2= H

Figure 8. Structures of indole alkaloids (48–54).

1.9. Benzylphenethylamines A benzylphenethylamine, lycorine (55), was isolated from M. paniculata and M. simplisifolia (Hemingway and Phillipson, 1975; Slavík and Slavíková, 1996) (Fig. 9).

OH HO O O

H H

N

55 Figure 9. Structures of benzylphenethylamine alkaloid (55).

1.10. Others One new alkaloid, himalayamine (56) was isolated from M. villosa in the northern India (Allais et al., 1983). Oleracein E (57) (Wu et al., 2007, 2011) and anhydroberberillic acid (58) (Wu et al., 2011) was obtained from M. integrifolia and M. punicea, respectively (Fig. 10).


250

O O

Haifeng Wu, Xiaopo Zhang, Yan Zhou et al. OH H CH3 N O

56

HO N

HO

O

O O

O

H3CO

O

N O

57

O HO

OCH3

O

58

Figure 10. Structures of benzylphenethylamine alkaloid (56–58).

2. Flavonoids Flavonoids are a diverse group of polyphenolic compounds widely distributed in the plant kingdom, possessing a wide range of bioactive capacities which includes antioxidant and antibacterial properties, as well as protective effects on cardiovascular systems. As antioxidants, flavonoids can scavenge free radicals, increase the levels of antioxidant enzymes, and thus protect body systems against damage from free oxygen species (Sandhar et al., 2011). Yue et al. suggested that flavonoids could be considered as the main characteristics for the quality control of Tibetan medicine M. quintuplinervia (Yue et al., 2010). Till now, twenty-eight flavonoids divided into flavonols, flavones, and anthocyanidin, as well as flavonoid glycosides, have been isolated from thirteen species of Meconopsis (Fig. 11), including isorhamnetin (59), herbacetin (60), quercetin (61), quercetin 3-O--Dglucopyranoside (62), quercetin 3-O--D-galacopyranoside (63), kaempferol-3-gentiobioside (64) and kaempferol 3-xylosylgentiobioside (65), kaempferol 3-O-(6-O--D-glucopyranosyl)-D-galactopyranoside (66), quercetin 3-O-[-D-galactopyranosyl-(16)]-Dglucopyranoside (67), isorhamnetin 3-O-[-D-galactopyranosyl-(16)]--D glucopyranoside (68), kaempferol 3-O-[-D-glucopyranosyl-(12)]--D- glucopyranoside] (69), isorhamnetin 3-O-[-D-glucopyranosyl-(16)]--D-galactoside (70), kaempferol 3-Oβ-D-glucopyranoside (71), tricin 7-O--D-glucopyranoside (72), eriodictyol (73), dihydroquercetin (74), chrysoeriol (75), luteolin (76), apigenin (77), tricin (78), huazhongilexone (79), hydnocarpin (80), quercetin 3-O-[2′′′-O-acetyl--D-glucopyranosyl(16)--D-glucopyranoside] (81), quercetin 3-O-[2′′′,6′′′-O-diacetyl--D-glucopyranosyl(16)--D-glucopyranoside] (82), isorhamnetin 3-O-[2′′′-O-acetyl--D-glucopyranosyl(16)--D-glucopyranoside] (83), quercetin3-O-[2′′′-O-acetyl-α-L-glucopyranosyl -(16)-D-glucopyranoside] (84), cyanidin 3-O-(6-O-malonyl-β-sambubioside) -7-O--Dglucopyranoside (85) and 3-O-(2-O--D-xylopyranosyl)--D-glucopyranosyl-7-O--Dglucopyranosyl-cyanidin (86) (Fu et al., 2010; Liu and Wang, 1986; Wang et al., 1991; Wu et al., 2011; Shang et al., 2002a, 2006b; Shang, 2002b; Takeda et al., 1996; Tanaka et al., 2001; Yoshida et al., 2006).


Meconopsis: Traditional Uses, Chemistry and Pharmacology R1

R5O

R2 O

OH O 59 60 61 62 63 64 65 66 67 68 69 70 71 72

HO

R4

O

OH O

R1 = R3 = R4= R5 =H, R2 = OCH3 R1 = OH, R2 = R3 =R4= R5 = H R1 = R3 =R4= R5 = H, R2 = OH R1 = R4= R5 =H, R2 = OH, R3 = glc R1 = R4= R5 =H, R2 = OH, R3 = gal R1 = R2 =R4= R5 = H, R3 = -glc-glc R1 = R2 =R4= R5 = H, R3 = -glc-glc-xyl R1 = R2 =R4= R5 = H, R3 = -glc-gal R1 = R4= R5 =H, R2 = OH, R3 = -glc-gal R1 =R4= R5 = H, R2 = OCH3, R3= -glc-gal 2 = -glc glc R1 = R2 =R4= R5 = H, R 3 R1 = R4= R5 =H, R2 = OCH3, R3 = -gal-glc R1 = R2 = R4= R5 =H, R3 = glc R1 = R2 = H, R3 = glc, R4=OCH3, R5 = glc

R2 R1

O OH O O

R4R5 R3 HO 81 82 83 84

O O HOHO OR2

HO

OH

O

R2

R3 OH O

73 R1 = R3= H, R2 =OH 74 R1 = R2 = OH, R3= H 79 R1 = R2 = H, R3= OH

75 76 77 78 O

HO

O

R1 = OCH3, R2 = H R1 = OH, R2 = H R1 = R2 = H R1 = R2 = OCH3

CH2OH OCH3

O

OH

OH O 80

OH HO

R1

OH OH

OR3

251

OR1 O OH

R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OH R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OH R1 = CH3, R2 = Ac, R3 = OH, R4 = H, R5 = CH2OH R1 = R3 = H, R2 = Ac, R4 = OH, R5 = H

OH O HO HO

OH

O

O+

OH

OH OH RO

O O

HO O

HO HO

O

OH OH

85 R = malonyl 86 R = OH

Figure 11. Structures of flavonoids (59–86).

3. Essential Oils Essential oils have been popularly used in pharmaceutical, sanitary, cosmetic, and agricultural and food industries for bactericidal, virucidal, fungicidal, antiparasitic and insecticidal properties. Usually obtained by steam distillation from aromatic plants, they contain a variety of volatile constituents such as terpenoids, phenol-derived aromatic components and aliphatic components. Analysis on essential oils of several species of Meconopsis has been conducted using GC–MS (Chen and Zhang, 1989; Gao et al., 2013; Guan et al., 2007; Wu et al., 2006; Yuan et al., 2003). Most of chemical constituents of essential oils are aliphatic components. For example, n-hexadecanoic acid (27.653%) and 6,10,14-trimethyl-2-pentadecanone (16.330%) were the main components in M. oliverana essential oils that showed strong antioxidant activity (Gao et al., 2013).


252


4. Steroids Three steroids, β-sitosterol (87), stigmasterol (88), and daucosterol (89), were reported (Guo et al., 2003; Pan, 1998; Shang et al., 2006b; Wu et al., 2011; Zhang et al., 1997) (Fig. 12). R2

R3

R1O

HO

88 R1 = R2 = R3 = H 89 R1 = glc, R2 = R3 = H

87 Figure 12. Structures of steroids (87–89).

5. Triterpenoids Four triterpenoids, 21α-hydroxy ursolic acid (90), β-amyrin (91), ursolic acid (92), and 3hydroxyolean-12(13)-en-24-oic acid (93) were reported (Fu et al., 2010; Guo et al., 2003; Pan, 1998; Shang et al., 2006b; Zhang et al., 1997) (Fig. 13). R1

R2

R4 HO

R3 90 91 92 93

R1 = CH3, R2 = H, R3 = OH, R4 = COOH R1 = R3 = H, R2 = R4 = CH3 R1 = CH3, R2 = R3 = H, R4 = COOH R1 = R3 = H, R2 = COOH, R4 = CH3

Figure 13. Structures of triterpenoids (90–93).

6. Miscellaneous Constituents In addition, cinnamic acid (94), caffeic acid (95), p-hydroxycinnamic acid (96), protocatechuic acid (97), coumaric acid (98), 2-(3,4-dihydroxyphenyl)-ethyl-O-β-Dglucopyranoside (99), p-hydroxybenzoyl- β-D-glucopyranoside (100), 4-O-β-Dglucopyranosyl-(Z)-p-coumaric acid (101), 5, 7-dihydroxy-4H-4-chromenone (102), 2hydroxyacetylfuran (103), 5, 5′-bis(oxymethyl)- furanaldehyde (104), and 2, 3-hydroxypropyl myristate (105), uracil (106), and 2-methoxyphenyl-β-D-glucopyranoside (107) are also



253

obtained (Fu et al., 2010; Guo et al., 2003; Pan, 1998; Shang et al., 2002a, 2006b; Shang, 2002b; Zhang et al., 1997; Wu et al., 2011) (Fig. 14) . R1

COOH

COOH

R2

97 R = OH 98 R = OCH3

O

CHO

O

100

HO

HO

O

Oglc O

OH O

101

102

104

103

O

OH HO

Oglc

Oglc 99

OH

H3CO H3CO

COCH2OH

HO

HO

R

94 R1 = R2 = H 95 R1 = R2 = OH 96 R1 = H, R2 = OH

O

HO

NH

O O

N H 105

O

HOH2C HO HO

106

H3CO O

O

OH 107

Figure 14. Miscellaneous compounds (94–107).

Biological Activity Although some Meconopsis species are in clinically prescribed in form of complex preparations for the treatment of hepatitis, enteritis and diarrhea by Tibetan medical practitioner, pharmacological activities of Meconopsis were rarely investigated. Some Meconopsis herbs reportedly exhibited significant antioxidant, anti-inflammatory, analgesic, antidiarrheal and hepatoprotective effects (Ding and Li, 2007; Guo et al., 2003; Wang et al., 2011, 2013; Wangchuk et al., 2011, 2013; Zhou et al., 2013). Antioxidants extracted from plants could play an important role in liver protection (Akanitapichat et al., 2010). Recently, two reports focused on antioxidant properties of Meconopsis species. He et al. evaluated the antioxidant potential of ethanolic extract of M. quintuplinervia using various established systems (He et al., 2012). The extract showed strong in vitro antioxidant ability. In the in vivo study, CCl4-induced oxidative stress caused significant decreases in the superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels and a significant increase in the malondialdehyde (MDA) level, most of which were significantly reversed (except for SOD in the liver) by treatment with the extract and standard Vitamin E. In another study, Zhou et al. investigated the hepatoprotective and antioxidant effects of ethanolic extract of M. integrifolia (MIE) in vitro and in vivo (Zhou et al., 2013). MIE exhibited strong antioxidant ability in vitro. In the rats with CCl4-induced liver injury, the groups treated with MIE and silymarin showed significantly lower levels of glutamate pyruvate transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin


254


(TB). MIE demonstrated good antioxidant activities in both the liver and kidney of the rats in vivo. Wangchuk and colleagues found that M. simplicifolia used as Bhutanese showed strong antiplasmodial activity with IC50 values of 0.40 μg/ml against TM4/8.2 strain (a wild type chloroquine and antifolate sensitive strain) and 6.39 μg/ml against K1CB1 (multidrug resistant strain) (Wangchuk et al., 2011). In another study, crude extracts of M. simplicifolia exhibited good inhibition of TNF-α production in LPS-activated THP-1 monocytic cells (Wangchuk et al., 2013).

Conservation Strategies Populations of many medicinal plant species, particularly threatened endemic taxa, have sharply declined in the last few decades as a result of habitat destruction, deforestation and overexploitation. Meconopsis is an endangered genus of ornamental and medicinal value, most species of which inhabit open alpine slopes (Dar et al., 2010). The increasing use has exerted much more pressure on the wild plant population through persistent collection practices. It is imperative to cultivate Meconopsis; however, it is difficult for cultivation of Meconopsis at lower altitudes owing to its intolerance to hot summers. By comparison of the photosynthetic capacity of M. integrifolia and M. horridula as well as their photosynthetic responses to light and temperature in the nursery at an altitude of 3260 m, Zhang et al. found that at lower altitudes, introduction and cultivation of M. horridula could be easier due to its wider physiological adaptation (Zhang and Hu, 2008). In another study, it was deduced that the poor photosynthetic performance at high temperature be what limits M. horridula cultivation at low altitude (Zhang, 2010).

Conclusion The scientific validation and the quality assurance of the ethno-medicinal plants requires an integrated multidisciplinary approaches including the ethno-medical assessment, botanical identification, phytochemical investigation, pharmacological evaluation, and the verification of ethnopharmacological uses of both the ingredient and the multi-herbs formulations. The indepth phytochemical and pharmacological investigations are required toward quest to unearth the scientific basis for medicinal use of Meconopsis. So far, more than 107 compounds have been reported from nineteen species of Meconopsis genus. Discovery of new species are ongoing. It is highly likely that further phytochemical investigations on the other species will result in much more isolations of bioactive constituents. The biological activities of alkaloids in Meconopsis should be extensively investigated. In addition, plants of the genus Meconopsis produce the structurally diverse and complex alkaloids, which should play a vital role for the plant itself from the evolutionary perspective. Biological/ecological role of these alkaloids in the life cycle of the plant should also be paid much more attentions.



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Acknowledgments Financial support from the National Natural Science Foundation of China (No. 81102770) is gratefully acknowledged.

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Chapter 10

A Case Study of Indigenous Medicinal Plants: Antioxidant Properties, Traditional Uses and Conservation Strategies Henry Lowe and Joseph Bryant Bio-Tech R&D Institute, Jamaica

Abstract Jamaica’s flora has a rich source of medicinal plants, with over 2900 species of identified flowering plants; 1788 plants have been identified to contain two or more bioactive compounds, of these 51 possessing antioxidant properties. Herbal medicine has been the source for many pharmaceutical and nutraceutical products based on wellresearched and developed ethno-medicinal practices worldwide. It provides an alternative method for the management of various diseases, such as cancer, diabetes, hypertension among others, which are all alleviated by antioxidant compounds. Antioxidant activity is often times found in those plants that are edible and as such is able to alleviate oxidative stress when eaten. The Petiveria alliacea (guinea hen weed), the green coffee beans decoction (Coffee arabica), infusion, Hibiscus sabdariffa (Jamaican sorrel), the Eupatorium odoratum (jack in the bush) and Momordica charantia (cerasee) are among the common plants used on the island to promote oxidative relief. These plants are often administered in many different ways, including decoctions, macerations, infusions, tinctures or by cooking. Due to their usefulness as medicinal plants tissue culture has been used as a part of the conservation strategies that have been employed in preserving and maintaining the island’s flora.


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Henry Lowe and Joseph Bryant

Indigenous Plants with Antioxidant Activity The antioxidant effect of plants provides a wide array of medicinal uses that aids in the combating of various diseases. Antioxidant compounds are those inhibitory molecules that are able to bind to free radicals and thus prevent physiological damage which also inhibits interference of biochemical reactions within the body. The elimination of free radicals from the body is imperative in maintaining health from the cellular level which will thus be reflected throughout the body. Plants within the Jamaican flora with such properties have been in use for decades from generation to generation. The incorporation of science has now been able to validate the uses of these plants in treating these ailments. Approximately 51 plants have been known to contain antioxidant properties, some of which are common and more frequently studied. These include garlic (Allium sativa), guinea hen weed (Petiveria alliacea), green coffee (Coffea arabica) beans, Aloe vera, chocho (Sechium edule), cinnamon (Cinnamomun verum), watermelon (citrillus lanatus), Spiny pigweed (Amaranthus spinosus), cashew (Anarcardium occidentale), soursop (Annona muricata), Spanish needle (Bidens pilosa), pignut (Hyptis suaveolens), Jack-in-thebush (Euphorium odoratum), and Jamaican sorrel (Hibiscus sabdariffa) as shown in Figure 1 below along with the known antioxidant portion of the plant.

Antioxidant Compounds in Medicinal Plants The active compounds that contribute to the antioxidant effect are usually due to the polyphenols, flavonoids, terpenes, tocopherols, ascorbic acid and polyunsaturated fatty acids present. These phytochemical compounds have been shown to reduce oxidative stress and thus retards the progression of chronic diseases such cancer, diabetes, hypertension. According to Richard et al. (2008), in vitro studies showed that the polyunsaturated fatty acids (PUFAs) had significant antioxidant activity as the PUFAs were able to stimulate the production of reactive oxygen/nitrogen species (ROS/RNS) from free radical within human aortic endothelial cells, thus initiating their removal from the body. As a result, this would again prevent further deterioration of cells, as inflammation is reduced and subsequently the prevention of cardiovascular diseases such as atherosclerosis.

Plant Compounds As Antioxidants Polyphenols are natural phenolic compounds that possess antioxidant properties and are common among many plants, especially those in the diet. There are over 4000 known polyphenols that have been extracted from various plants. Fruits are usually rich in polyphenols and therefore are usually included in fruit drinks or as dietary supplement. Polyphenol extracted from green coffee beans also show significant antioxidant activity. Other phytochemicals with antioxidant activity are the terpenes. Terpenes have been shown to protect the cells from oxidative stress using different mechanisms by scavenging for free radicals or by increasing the enzymatic and non-enzymatic antioxidant status within the cell (Gonzalez-Burgos et al., 2012). Azadirachta indica (neem plant) has been known to have


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many medicinal advantages which may be attributed to the active compounds azadirachtin and nimbin. Its antioxidant activity is one such reason for its effectiveness against many diseases. The antioxidant property of the seed, leaves and bark were found to contain high quantities of polyphenols and flavonoids when tested using the 1, 1-diphenyl-2-picrylhyazyl (DPPH) method (Ghimeray, et al., 2009). The results showed that the bark had a higher quantity of total polyphenols present (23.85 – 237.00 µg/mg), while the leaves contain between 66.63 – 629.04 µg/mg (tannic acid equivalents). The total flavonoids content was also assessed and expressed in equivalents to quercetine, which showed that the leaves contained more flavonoids contributing to the overall antioxidant property of the plant (Ghimeray, et al., 2009). Flavonoids are another type secondary metabolite in plants with over 8000 already identified (Pietta, 2000). They aid in pigmentation and act as a chemical messenger throughout the plant and have been known to reduce free radicals present and therefore have contributed to the antioxidant activity of many plants (Hernandez et al., 2009; Pannala et al., 2001; Pietta, 2000; Jovanovic et al., 1994). One such plant is the Aloe vera which is known for its numerous medicinal properties. It is an antidiabetic agent in that the oral administration of extract was able to significantly reduce the blood glucose and lipid levels in diabetic and hyperlipidaemia patients (Vogler and Ernst, 1999). These effects may also be as a result of the high flavonoid and polysaccharide content, which has been shown to contribute to significantly increase antioxidant activity the plant exhibits and the growth stage also played an important role in the level of antioxidant activity, as a three (3) year old aloe vera plant had more antioxidant activity when compared with the two and four years old plants (Hu et al., 2003). Terpenoids such as monoterpenes and diterpenes contribute the unique flavours found in plants, especially herbs. They are able to remove free radicals from the body and have been prospective treatment for many ailments (Grassmann, 2005).

Plant Extractions of Antioxidant Compounds Antioxidant compounds are found in the polar extractions that are usually done using methanol, ethanol, acetone or water. The type of polar solvent used is dependent on the type of plant, the region of the plant that the isolation will be done, that is the root, leaves, bark, fruit or inflourescent. The crude ethanol extract of the leaves and the water extract of both the leaves and seeds of S. edule showed significant hydrogen donating properties when reacted with DDPH stable radicals. The antioxidant activity of S. edule is due to the compound alpha rho. No. 11, a bio-catalyser that can also extracted from the fermentation of Carica papaya Linn. and Penisetum pupureum. (Ordonez et al., 2006). Petiveria alliacea (guineahen weed) is a potential anticancer drug as research has shown the anti-tumour effect on various cancer cell lines (Uruena, 2008; Williams et al.,). It also acts as an immune system booster. Other commonly used plants with antioxidant properties include the Jamaican sorrel (Hibiscus sabdariffa) which has been shown to reduce the free radical content; and has subsequently being added to juices which increase their antioxidant properties. Hibiscus sabdariffa (roselle/sorrel) exhibits significant antioxidative properties as the crude methanol extract of the calyx had a better effect in linoleic acid model system. This


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was as a result of the formation of conjugated diene compounds and the TBARS (thiobarbituric acid) when the extract was administered. This was compared with two known antioxidant compounds (α-tocopherol and butylated hydroxyl-anisole, BHA) which had an increased antioxidant property. This level of antioxidant activity was also significantly more than grapes when tested similarly (Tee et. al, 2002). Eupatorium odoratum (jack in the bush) is a member of the Asteraceae family and have been used for various ailments. The leaves in particular are usually harvested for their antiinflammatory,antidiabetic, antihypertensive, analgesic among other properties such as antifungal,antibacterial, antiinflammatory and antiandrogenic effects when the aqueous extract of the leaves were tested (Sofowora, 1993). It was investigated and thus authenticated its use as an antioxidant. The crude extracts of the leaves obtained from ethanol and water were assessed using various in vitro radical induced assays (DPPH, nitric oxide and hydroxyl) and showed that there was significant antioxidant activity (Chakraborty et al., 2010). The antioxidant activity of E. odoratum was said to be attributed to the phenolic content with in the leaves (Luximon-Ramma et al., 2002). The crude methanol extract of the bark of Swietenia mahagoni (West Indian mahogany) was able to significantly lower the thiobarbituric acid reactive substances (TBARS) while increasing the glutathione and catalase levels in STZ-induced diabetic rats(Prasa d Panda, et al., 2010) and therefore confirms the use of this plant as an antioxidant. Table 1 below lists other plants with antioxidant properties that are commonly used in ethnomedicinal therapy for various ailments. Table 1. Some indigenous plants with antioxidant properties obtained from polar extractions Plant

Common Names

A. cavaliers A. sativum A. spinosus A. occidentalis C cajan C. papaya

Maiden fern Garlic Spiny pigweed Cashew Gungo papaw

C. roseous

periwinkle

C. lanatus C. nucifera

watermelon coconut

E. odoratum

Bitter bush, jackin-the-bush Horseradish tree(moringa) pignut

M. oleifera H. suaveolens S. mahagoni H. sabdariffa P. alliacea

West Indian Mahogany Sorrel, roselle Guineahen weed

Class of Antioxidant Compound Flavonoids phenolics phenolics Flavonoid Vitamin C, mallic and citric acids, glucose, vanillic acid, p-hydroxybenzoic Phenolics, alkaloids

Cultivation for antioxidant activity Entire fern Bulb leaves shoot leaves Pulp and seeds

ethanol methanol methanol methanol Ethanol n-butanol ethyl acetate

Phenolics Ascorbic acid and other vitamins phenolics

Aerial water

Phosphate buffer Methanol NA

leaves

ethanol

Flavonoid, phenolics and ascorbic acid Alkaloids, flavanol, terpenes

leaves

methanol

root

methanol

bark

methanol

calyx Leaves and roots

methanol methanol

flavonoids Thiosulfate derivative

leaves

Crude Extract



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Traditional Uses and Preparations These would include examples of endemic plants with antioxidant properties commonly used to treat various ailments, including cancer. These plants are traditional prepared in various ways, these include: In decoctions the plants are either dried or placed as fresh samples in warm water. Many plants show antioxidant properties when administered via decoction. Research done showed that cerasee significantly reduced oxidative stress when compared to the control. Research done by Richard et al found that teas were able to significantly promote the release of insulin when epididymal fat cells were assessed. The antioxidant activity of the teas was mainly due to the polyphenol content which reduces oxidative damage (Anderson, et. al, 2002). Eupatorium odoratum also showed marked antioxidant activity in lab tests using a variety of methods. This plant is traditionally prepared as an infusion of the leaves and therefore, the antioxidant effect would be similar to that done experimentally with the ethanol and water as shown by Chakraborty et al. in 2010. ). The cerasee plant has long been used for various ailments via infusion. The crude extract of the fruit was prepared via an aqueous extraction and was subsequently tested on STZ-induced diabetic rats, which salso confirmed its antioxidant activity coupled with an increased antihyperglycaemic effect (Sitasawad et. al., 2000). An infusion of aqueous B. pilosa which showed the health benefits of infusion as the extract which showed significant antioxidant activity due to the flavonoids (Abajo et al., 2004). Tinctures, macerations and cooking are not commonly used to extract and obtain an antioxidant effect from medicinal plants. However, the compounds are still able to elicit some effect if not destroyed, for example by heat during the cooking of fruits and vegetables.

Conservation Strategies The use of plants as medicine has significantly impacted the pharmaceutical society as they form the basis for many medicines used in modern medicine and traditional treatment. For example, Biotech R&D Institute Ltd, Jamaica has capsulated a number of plants with medicinal properties, Aloe vera and P. alliacea are two such plants that also contain antioxidant activity. As such, it is imperative to implement various strategic methods that will always provide the necessary compounds with antioxidant properties that will reduce the free radicals within the body that often times interrupt the normal functioning of the organs which eventually leads to physiological diseases. Plants with bioactivity are screened for their active components, and thus further isolations can be done. Based on HPLC and other chromatographic analysis, these plants can then be culture and modified to increase the percentage yield of the active compounds. The conservation of medicinal plants is imperative to the development of products and improved scientific research and as such protection includes the implementation of various types of gene banks whether they are in situ, ex situ or in vitro. In situ conservation carried out in Jamaica and other regions of the Caribbean involves the protection these folkloric medicinal and agricultural plants within high conservation areas such as their natural habitats (Prendergast et al., 1993). This type of conservation is carried out by monitoring the forest cover and reducing deforestation for wood, farming, charcoal


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etc… and by promoting the growth and sustainability of our Jamaican forests which houses many of our endemic species. Fencing is also a common method used in other areas to prevent the continuous grazing and thus the loss of some of our valued ethnomedicinal plants. The Azadirachta indica (neem tree), Eucalyptus globulus (eucalyptus), Psidium guajava (guava), Eugenia spp., Anarcardium occidentale (cashew) and E. odoratum are among some of the trees that can be found in our island’s forests containing antioxidant properties (Dudonne et al., 2009; sultana et al., 2007; Vasquez et al., 2009). The Ministry of Agriculture via the Forestry Department ensures the conservation of these plants and constantly replants any species that has become endangered after continuous research. These medicinal plants may have been lost due to natural disasters such as hurricane, soil erosion, fire and as such research is conducted in order to monitor the flora and its numerous natural products they contain. The protection of the medicinal plant species can also be done by placing them in a new location with additional benefits which will enhance their survival is referred to as ex situ conservation (Heywoods et al., 2003). This can be done colony relocation, where plants with medicinal properties are replanted in mined out lands as a method of reforestation and as such increasing the quantity of these plants found in different parts of the island with improved technological. The neem and Cassia plants are commonly planted. The smaller medicinal plants are often replanted in botanical gardens or in horticultural greenhouses. These prevent species from becoming extinct as they are constantly being regenerated and provides for continuous research can be done. Some of medicinal plants are stored in seed banks, germplasm banks and other in vitro methods. Tissue culture technique is often employed as one of the main measures in preserving these plants. Tissue culture creates the avenue for species regeneration especially with the tropical climatic conditions. Medicinal plants can be harvested in small quantities and the regeneration of plantlets is done using various parts of the plant depending on the species and the media being used. For example, Aloe vera has numerous medicinal properties and is often used for diabetes, hypertension, and as an anticancer due to its antioxidant properties (Hu et al., 2003). The active compounds towards these diseases are found in the succulent leaves of the plant. These leaves are often regenerated by tissue culture for research, re-planting or for modification and extraction of phytochemicals. Medicinal plant research also creates the avenue for conservation as knowledge of the usefulness of these plants are then published and thus increase knowledge of the uses of these plants as well as creates the avenue for additional study. Various parts of the plant contain activity and thus, these parts of the plants are also stored for further regeneration of plantlets. This prevents extinction of species, as a result, a seed bank helps to alleviate this. Periodical testing of the stored seed are done to ensure viability and thus sustainability of medicinal plants. The conservation and sustainment of the rich medicinal source from nature can also be further creates medicines to alleviate various diseases. Subsequent implementation of products to the market by nutraceutical companies e.g. Biotech R&D Institute can then be done to preserve the use of the plant products.



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References Abajo, C., Boffill, Del Campo, J., Mendez, M. A., Gonzalez, Y., Mitians, M. and Vinardell, M.P. (2004). In vitro study of the antioxidant and immunodulatory activity of aqueous infusion of Bidens pilosa. Journal of Ethnopharmacology, 93 (2-3): 319 – 323. Ahmad, J., Khan, I., Ahmad, A. and Imam, K. (2012). In vitro Antioxidant and Antimicrobial Activity of Methanolic Root Extracts of Hyptis suaveolens. Research Journal for Recent Sciences, 2: 41 – 46. Amatya, S & Tuladhar, SM – In Vitro Antioxidant Activity of Extracts from Eupatorium Odoratum – www.scialert.net (14/07/2010) Anderson, R.A. and Polansky, M.M. (2002). Tea Enhances Insulin Activity. Journal of Agriculture and Food Chemistry, 50 (24): 7182 – 7186. DOI: 10.1021/jf020514c Asprey, GF & Thornton, P - Medicinal Plants of Jamaica Parts 1-4 - West Indian Journal of Medicine vol. 2-4 (1953-1955) Arora, R., Gill, N. S., Kaur, S. and Jain, A. D. (2011). "Phytopharmacological evaluation of ethanolic extract of the seeds of Abrus precatorius linn". Journal of Pharmacology and Toxicology 6 (6): 580–588. Chakraborty, A. K., Roy, H. and Bastia, S. (2010). Evaluation of Antioxidant Activity of the Leaves of Eupatorium odoratum Linn. International Journal of Pharmacy and Pharmaceutical Sciences, 2 (4):77 – 79. Dudonne, S., Vitrac, X., Coutiere, P., Woillez, M. and Merillon, J.M. (2009). Comparative Study of Antioxidant Properties and Total Phenolic Content of 30 Plant Extracts of Industrial Interest Using DPPH, ABTS, FRAP, SOD and ORAC Assays. J. Agric.Food Chem, 57 (5): 1768 – 1774 Ghimeray, A.K., Jin, C., Ghimirez, B.K. and Ha Cho, D. (2009). Antioxidant Activity and Quantitative Estimation of azadirachtin and nimbin in Azadirachta indica A. Juss. Grown in Foothills of Nepal. African Journal of Biotechnology, 8(13): 3084 – 3091. Gonzalez-Burgos, E. and Gomez-Serranillos, M.P. (2012). Terpene compounds in nature: A Review of their potential Antioxidant Activity. Curr Med Chem, 19 (31): 5319 – 5341.Inya-agha, SI et al – Phytpochemical and Antibacterial Studies on the Essential Oil of Eupatorium Odoratum – Pharmaceutical Biology – (1987) Vol 25(1) pp 49-52. Grassmann, J. (2005). Terpenoids as Plant Antioxidants. Vitamins & Hormones, 72: 505 – 535.Hernandez, I., Alegre, L. and Breusegem, F. V. and Munne-Bosch, S. (2009). How relevant are flavonoids as antioxidants in plants? Trends in Plant Science, 14 (3): 125 – 132. Heywood, V. H. and Iriondo, J.M. (2003) Plant Conservation: old problems, new perspectives. Biological Conservation, 113 (3): 321 – 335. Hu, Y, Xu, J. and Hu, O. (2003). Evaluation of Antioxidant Potential of Aloe vera (Aloe barbadensis Miller) Extracts. Agric. Food Chem.,51 (26): 7788–7791 Jovanovic, S.V., Steenken, S., Tosic, N., Marjanovic, B. and Simic, M. (1994). Flavonoids as Antioxidants. J. Am. Chem. Soc., 116 : 4846 – 4851. Luximon-Ramma, A., Bahorun, T., Soobrattee, M.A. and Aruoma, O.I. (2002). Antioxidant Activities of Phenolic, Proanthocyanidin and Flavonoid Components in Extracts of Cassia fistula. J Agric Food Chem, 50 (18): 5042 – 5047.


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Mitchell & Ahmad - A Review of Medicinal Plant Research at the University of the West Indies, Jamaica 1948-2001 - WI Med J (2006);55(4):243 Ordonez, A.A.L., Gomez, J. D., Vattuone, M.A. and Isla, M. I. (2006). Antioxidant Activities of Sechium edule (Jacq.) Swartz extracts. Food Chemistry, 97 (3): 452 – 458. Pannala, A. S., Chan, T.S., O’Brien, P. and Rice-Evans, C.A. (2001). Flavonoid B-Ring Chemistry and Antioxidant Activity: Fast Reaction Kinetics. Biochemical and Biophysical Research Communications, 282 (5): 1161 – 1168. Phan, TT et al - An aqueous extract of the leaves of Eupatorium Odoratum (Eupolin) inhibits hydrated collagen lattice contraction by normal human dermal fibroblasts – J. Altern Complement Med. (1996) Fall; 2 (3): pp 335-343 Pietta, P. (2000). Flavonoids as antioxidants. J. Nat. Prod., 63 (7): 1035 – 1042. Prasa d Panda, S., Haldar, P.K., Bera, S. Adhikary, S and Kandar, C.C. (2010). Antidiabetic and Antioxidant activity of Swietenia mahagoni in streptozotocin-induced diabetic rats. Pharmaceutical Biology, 48 (9): 974 – 979. Prendergast, J.R., Quinn, R.M., Lawton, J.H., Eversham, B.C. and Gibbons, D.W. (1993). Rare species, the coincidence of diversity hotspots and conservation strategies. Nature, 365: 335 – 337. Richard, D., Kefi, K., Barbe, U, Bausero, P. and Visioli, F. (2008). Polyunsaturated fatty acids as Antioxidants. Pharmacol. Res. 57 (6): 451 – 455. Sitasawad, S.L., Shewade and Y., Bhonde. (2000). Role of Bittergourd Fruit Juice in STZ induced Diabetic State in vivo and in vitro. Journal of Ethnopharmacology, 73 (1-2): 71 – 79. Sofowora, L. A. (1993). Medicinal plants and traditional medicine in Africa. Harborne: Spectrum Books Limited., Ibaban. Sultana, B., Anwar, F. and Przybylski, R. (2007). Antioxidant activity of phenolic components present in barks of Azadirachta indica, Terminalia arjuna, Acacia nilotica and Eugenia jambolana lam. Trees. Food Chemistry, 104 (3): 1106 – 1114. Tee, P., Yusof, S. and Mohamed, S. (2002). Antioxidative Properties of Roselle (Hibiscus sabdariffa L.) in Linoleic Acid Model System. Nutrition and Food Science, 32 (1): 17 – 20. Vazquez, G., Gonzalez-Alvarez, J., Santos, J., Freire, M.S. and Antorrena, G. (2009). Evaluation of potential applications for chestnut (Castanea sativa) shell and eucalyptus (Eucalyptus globulus) bark extracts. Industrial Crops and Products, 29 (2-3): 364 – 370. Vogler, B.K. and Ernst, E. (1999). Aloe vera: A systematic Review of its Clinical Effectiveness. Br. J. Gen. Pract., 49 (447): 823 – 828. Winston, C. J. (1999). Health-promoting properties of common herbs. Am. J. Clin. Nutr., 70 (3): 491 – 499. Zheng, W. and Wang, S.H. (2001). Antioxidant Activity and Phenolic Compounds in Selected Herbs. J. Agric. Food Chem., 49 (11): 5165 – 5170.


Index A ABA, 110 Abraham, 233, 239 abstraction, 29 access, 2, 15, 21, 168 accessions, 89, 109, 110 accounting, 33 acetaminophen, 82 acetic acid, 5 acetogenins, 49, 62 acetone, 15, 31, 231, 235, 261 acetonitrile, 15, 19 acidic, 9, 30 acidity, 59, 74 acne, 232 active compound, 3, 94, 113, 260, 261, 263, 264 active oxygen, 84 active site, 155 acupuncture, 3 acute lymphob adaptation, ix, 26, 91, 92, 100, 102, 109, 254, 258 additives, viii, xi, 2, 26, 120, 165, 205 adenine, 128 adenocarcinoma, 69 adenosine, 128, 130, 134, 150, 153 adenosine triphosphate, 128, 153 adhesion, 33, 86 adipocyte, 129, 145, 146, 147, 149, 153, 160, 163, 164 adiponectin, 129, 151, 152, 154 adipose, 45, 59, 129, 131, 137, 144, 145, 149, 150, 151, 155, 157, 158, 159, 163 adipose tissue, 45, 59, 129, 131, 137, 144, 145, 149, 150, 151, 155, 157, 158, 159, 163 adiposity, 131, 146, 147, 149, 151, 162 ADP, 130, 135 adrenaline, 89, 148

adsorption, 15, 19, 39 adults, x, 45, 48, 80, 85, 87, 143, 144, 149, 153, 157, 163, 178 advanced glycation end product (AGE), ix, 117 adverse effects, 2, 153, 158, 228 aflatoxin, 66, 77 Africa, 65, 187, 266 age, 45, 46, 52, 58, 60, 69, 131, 135, 144, 187, 219 age-related diseases, 46 aggression, 121 aging process, 44, 80, 86, 228, 236 agriculture, 15, 96, 209 AIDS, 206 alanine, 152, 156 alanine aminotransferase, 152 albumin, 131, 132, 139 alcohols, 9, 44, 177 aldehydes, 135 aldose reductase pathway, ix, 117 alfalfa, 38 alkaloids, vii, xi, 1, 4, 49, 59, 99, 101, 102, 154, 156, 228, 243, 244, 245, 246, 247, 248, 249, 254, 255, 256, 257, 258, 262 alkylation, 6 allergic rhinitis, 209, 221 aloe, 176, 261 alpha-tocopherol, 60, 75, 78, 83 ALT, 235, 253 alternative medicine, 159 alters, 75, 80, 108 amines, viii, 2, 26, 32 amino, vii, 1, 4, 17, 39, 44, 64, 65, 151, 167 amino acid(s), vii, 1, 4, 44, 64, 65, 151, 167 ammonia, 12, 102, 105, 111, 151 ammonium, 101, 109, 114 amylase, 68, 88, 155, 163, 216, 219 amyotrophic lateral sclerosis, 45


268

Index

analgesic, xi, 66, 126, 156, 227, 230, 238, 243, 244, 253, 262 anemia, 167, 180, 209 angiogenesis, 45, 54 angiotensin converting enzyme, 76 angiotensin II, 128 anhydrase, 132 anorexia, 206 ANS, 13 anthocyanin, 9, 53, 60, 257 antibiotic, 10 anticancer, x, 143, 158, 204, 261, 264 anticancer activity, 58, 67, 75 anticancer drug, 97 antiinflammatory drugs, 209 antioxidative activity, 90 antioxidative potential, 87 antispasmodic, 126 antitumor, 58, 69 apoptosis, viii, 33, 38, 41, 54, 56, 60, 64, 87, 90, 145, 146, 151, 152, 153, 159, 164, 236 appetite, 131, 147, 151, 154, 158, 159, 161, 163, 230 apples, 51, 52, 53, 79, 81, 121, 123 arginine, 151, 162 argon, 21 Armenia, 115, 186 aromatic compounds, 155 aromatic rings, 122, 247, 248 arres(s)t, 56, 60, 78 arteries, 45, 56 artery, 76 arthritis, 119, 220 ascites, 221 ascorbic acid, viii, 26, 29, 32, 41, 42, 44, 49, 57, 58, 59, 62, 63, 65, 66, 69, 71, 78, 85, 169, 228, 229, 233, 260, 262 aseptic, 97 Asia, xi, 64, 183, 187, 206, 220, 222 aspartate, 152, 156, 253 assessment, 3, 95, 237, 238, 254 assimilation, 102 asthma, 188, 192, 193, 196, 198, 200, 205, 206 astringent, 191 atherogenesis, 137 atherosclerosis, x, 50, 55, 56, 59, 62, 78, 129, 138, 143, 152, 159, 228, 229, 260 atherosclerotic plaque, 57, 131 atmosphere, 16 atmospheric pressure, 20, 21, 35 ATP, 102, 128, 130 atrophy, 70 authorities, 3 autism, 45

automation, 16 autooxidation, 50, 55 awareness, 118 azadirachtin, 261, 265

B bacteria, 136, 205 bacterial fermentation, 219 bacterial infection, 167, 237 bacteriophage, 179 ban, 201 banks, 263, 264 base, 9, 135 beef, 87, 162 beer, 121 Beijing, 243 beneficial effect, viii, ix, 41, 48, 56, 59, 63, 69, 70, 91, 92, 136, 149, 158, 176, 177, 210 benefits, viii, 3, 14, 33, 34, 42, 43, 48, 50, 54, 57, 59, 63, 71, 73, 74, 82, 132, 146, 149, 166, 205, 228, 239, 263, 264 benign, 16 benzene, 93, 104, 123 benzo(a)pyrene, 75 beta-carotene, 87 beverages, viii, xi, 2, 15, 26, 74, 121, 150, 205, 227 bile, 65 bilirubin, 132, 166, 228, 229, 253 bioactive secondary metabolite, ix, 92, 100 bioavailability, 14, 37, 54, 71, 81, 131 biochemical processes, 118 biochemistry, 39, 108, 127, 137, 139, 180 biodiversity, 186 bioflavonoids, 136 biological activities, 37, 64, 104, 126, 132, 136, 247, 254, 257 biological activity, 54, 83, 87 biological samples, 22, 27 biological systems, 234, 240 biologically active compounds, 57, 111 biomarkers, 55, 79, 146, 236 biomass, 95, 96, 97, 98, 99, 100, 102, 104, 105, 106, 107, 108, 109, 115 biomolecules, 28, 44, 135 biosynthesis, 3, 11, 12, 13, 36, 37, 61, 65, 73, 79, 102, 105, 112, 114, 122, 125, 142, 167, 232 biosynthetic pathways, 96 biotechnology, 109, 220 biotic, 92, 121 birds, 92 Black Sea region, 184 black tea, 189, 212, 240


Index bladder stones, 204, 207, 209 bleeding, 196, 197 blindness, 45 blood, xi, 50, 52, 55, 59, 62, 63, 70, 76, 77, 80, 83, 84, 87, 127, 134, 148, 151, 155, 156, 157, 163, 165, 175, 177, 178, 261 blood pressure, 76, 87, 148, 151, 156, 163 BMI, x, 143, 144, 147, 151, 154, 157, 158 body composition, 87, 158, 160 body fat, 129, 144, 147, 154, 155, 157, 158 body weight, 70, 129, 150, 151, 152, 154, 155, 156, 157, 158, 159, 163 bonding, 168, 169 bonds, 22, 23, 42, 136 bone, 57, 78, 166 bone resorption, 57 brain, xi, 44, 45, 61, 74, 84, 227, 237 Brazil, 2, 93, 149, 160 breakdown, 64, 71 breast cancer, 57, 58, 64, 71, 90 Britain, 82 bronchitis, 190, 195, 196, 198, 200, 205, 207, 209 bursa, 194, 202 butadiene, 23 butylated hydroxyanisole (BHA), viii, 2, 26 butylated hydroxytoluene (BHT), viii, 2, 26 buyers, 97 by-products, 26, 43, 57, 58, 118, 164

C Ca2+, 128 cabbage, 84 caffeine, 154, 161 calcium, 47, 48, 53, 57, 144, 163 calibration, 29, 31 caloric intake, 144, 146, 156 calorie, 86, 151 calyx, 261, 262 cancer, viii, x, xi, 14, 33, 39, 42, 45, 48, 51, 53, 54, 55, 56, 58, 62, 64, 66, 67, 69, 71, 72, 74, 75, 79, 80, 81, 82, 83, 87, 90, 93, 119, 120, 143, 163, 181, 189, 190, 197, 204, 205, 206, 209, 211, 219, 221, 222, 228, 229, 230, 239, 259, 260, 261, 263 cancer cells, 51, 56, 64, 87 cancer progression, 56 candidates, 65, 133, 136 candidiasis, 198, 207, 209 capillary, 18, 21, 34, 35 capsule, 156 carbohydrate, 68, 129, 133, 141, 144, 154, 155, 158 carbohydrate metabolism, 133 carbohydrates, 42, 92, 100, 134, 144, 154, 219

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carbon, 4, 6, 7, 15, 54, 79, 92, 159, 168, 234, 235, 238, 240 carbon atoms, 4 carbon dioxide, 15, 159 carbon tetrachloride, 79, 235, 238, 240 carbon-centered radicals, 168 carboxyl, 25 carboxylic groups, 55 carcinogen, 33, 181 carcinogenesis, 56 carcinoma, 56, 69, 221 cardiotonic, 206 cardiovascular disease, vii, viii, 1, 4, 14, 42, 45, 55, 62, 69, 71, 83, 86, 88, 121, 137, 181, 260 cardiovascular risk, 59, 67, 70, 77, 79 cardiovascular system, 67, 250 Caribbean, 263 carotene, 47, 48, 49, 55, 59, 61, 62, 67, 69, 70, 72, 77, 132, 135, 210 carotenoids, viii, 26, 29, 41, 42, 44, 48, 49, 51, 57, 58, 59, 62, 66, 67, 68, 69, 70, 71, 72, 78, 83, 88, 89, 132, 135, 146, 166, 209, 210, 219, 221, 228, 229 cascades, viii, 41 case study, ix, 91, 109 cataract, 45, 78, 127 Catharanthus roseus, 102, 110, 118 cation, 8, 30, 212 cattle, 87 C-C, 23, 24 cell culture, 88, 96, 111, 114, 115, 224, 239 cell cycle, 33, 56, 60, 64, 78, 83 cell death, 43, 58, 79, 142, 145, 238 cell division, 33, 145, 151 cell line(s), 58, 61, 67, 86, 261 cell membranes, x, 118, 143, 150, 176 cell size, 157 cellulose, 17 Central Europe, 111 central nervous system, 147 cervix, 56 chain propagation, 132 challenges, 97, 109, 160, 223 chemical, ix, xi, 4, 8, 14, 15, 17, 20, 27, 28, 30, 32, 35, 37, 51, 55, 62, 64, 67, 68, 71, 76, 87, 89, 95, 108, 113, 117, 119, 167, 228, 229, 233, 234, 240, 243, 244, 251, 255, 256, 257, 261 chemical reactions, 28, 71, 234 chemical reactivity, 27 chemical structures, 14 chemicals, 211 chemoprevention, 33, 39, 81, 84, 90, 152 chemotherapeutic agent, 58


270

Index

childhood, 150 children, 144, 150, 190, 195 Chile, 108 China, 2, 82, 243, 244, 255, 256, 258 Chinese medicine, 176, 212 Chinese women, 90, 151, 162 chiral center, 7 chlorination, 234, 237 chloroform, 16, 209, 215, 225, 235 chlorophyll, 115 cholesterol, 53, 56, 57, 60, 61, 62, 63, 65, 67, 69, 75, 79, 88, 131, 147, 154, 155, 156, 158, 192, 207, 210, 222 choline, x, 144 chromatographic technique, 16, 37 chromatography, 16, 17, 18, 35, 36, 38 chromium, 153 chronic diseases, vii, 1, 4, 48, 51, 55, 67, 69, 85, 90, 135, 228, 260 chronic illness, 71 cigarette smoke, 166 circulation, 56, 166, 177 civilization, vii, 1, 227 classes, vii, 1, 4, 6, 7, 12, 57, 62, 99, 121, 122, 177, 209, 257 classification, 140, 172, 173, 174, 238 cleavage, 22, 23, 24, 36, 222, 248 cleavages, 24 climate(s), 100, 113, 189, 230 clinical trials, 51, 59, 61, 64, 152, 154 clustering, 129 CO2, 16, 23, 25 cobalt, 139 coenzyme, 47, 49, 57, 60, 105, 129 coffee, xi, 25, 37, 38, 121, 192, 206, 259, 260 cognitive function, 44 colic, 206 collaboration, 184 collagen, 56, 266 colon, 34, 56, 58, 61, 72, 78, 86, 136, 219, 222 colon cancer, 34, 58, 78, 222 color, 11, 28, 29, 32, 54, 60, 68, 71, 77, 121, 125, 258 commercial, ix, 25, 91, 94, 95, 97, 98, 107 communities, 229 community, 26, 34, 159, 166 comparative analysis, 115 compatibility, 16 compilation, 39 complications, viii, x, 41, 45, 56, 117, 119, 127, 128, 129, 133, 138, 140, 144, 150, 155, 159

composition, ix, 20, 36, 67, 69, 71, 78, 79, 80, 89, 91, 96, 100, 102, 103, 106, 108, 110, 113, 157, 205, 208, 209, 210, 219, 231, 258 condensation, 12, 123 configuration, 6, 8, 38, 155, 245 conjugated dienes, 131 conjugation, 12, 19, 70, 236 conservation, vii, xii, 94, 224, 259, 263, 264, 266 constipation, 144, 189, 194, 199 constituents, xi, 44, 45, 48, 54, 68, 72, 79, 80, 86, 90, 103, 113, 120, 134, 136, 140, 158, 167, 176, 188, 204, 221, 231, 232, 238, 239, 243, 244, 251, 254, 255, 256, 257, 258 construction, 29 consumers, viii, ix, 41, 74, 91, 94, 95 consumption, 2, 14, 16, 19, 42, 48, 50, 52, 53, 54, 56, 58, 59, 61, 63, 64, 70, 72, 74, 76, 79, 80, 83, 85, 86, 87, 90, 94, 121, 131, 135, 149, 150, 154, 160, 162, 176, 189, 209 contaminant, 108 contamination, 21, 95, 96 control group, 70, 150, 235 controlled trials, 79, 149 cooking, xi, 69, 78, 82, 259, 263 coordination, 44 copper, 53, 93, 135 corona discharge, 21 coronary artery disease, 54, 82, 150 coronary heart disease, 34, 56, 59, 146, 205, 210 correlation, 27, 32, 53, 101 correlations, 59, 209 cortex, 60, 188, 201 cortisol, 147 cosmetic(s), 94, 103, 120, 209, 232, 238, 251 cost, 16, 19, 21, 94, 95, 97, 107, 108, 111, 184 cough, 192, 195, 196, 198, 200, 201, 205 coumarins, 6, 120, 207 counterirritant, 68 covalent bond, 135, 235 covalent bonding, 135 Croatia, 37, 181 crop, 101, 102, 110, 111 crop production, 111 crops, 58, 94, 97, 98, 100, 103, 109, 111, 113, 115 CRP, 146 CT, 110 Cuba, 58, 87 cues, 122 cultivars, 66, 68, 71, 76, 82, 83, 89, 90, 102, 103, 104, 111 cultivation, ix, 82, 88, 91, 95, 96, 97, 98, 99, 100, 105, 106, 107, 109, 111, 115, 220, 254


Index culture, xi, 37, 61, 85, 94, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 108, 109, 111, 112, 224, 259, 263, 264 curcumin, 33 cure, xi, 73, 183, 184, 189, 204, 205, 219 CV, 142 CVD, 45, 55, 56, 57 CWA, 139 cycles, 121 cyclophosphamide, 63, 85 Cydonia oblonga, 168, 174, 178 cysteine, 44, 45, 49, 65 cystic fibrosis, 152 cytochrome, 43, 235, 246, 256 cytokines, 33, 45, 70, 146, 159 cytoskeleton, 176 cytotoxicity, viii, 2, 26, 38, 101

D damages, 75 danger, 94 database, 81 decay, 31, 34 decomposition, 19, 28, 31, 234 defence, 93, 150, 151, 180 defense mechanisms, 118, 211, 228, 229, 235 deficiency, 62, 100, 102, 105, 109, 129, 150, 166, 178 deficit, 102, 113 deforestation, 254, 263 deformability, 167, 168, 176, 177, 180 degradation, 47, 61, 73, 107, 131, 176, 238 dehydration, 245 deoxyribonucleic acid, 118 Department of Agriculture, 48, 91 deposition, 57, 159 deposits, 159 depression, 45 deprivation, 121 depth, 210, 254 derivatives, 8, 9, 10, 17, 33, 38, 39, 49, 62, 64, 65, 70, 93, 100, 102, 112, 114, 118, 120, 122, 133, 134, 135, 140, 141, 207, 209, 223, 244, 245 desiccation, 104, 107 desorption, 20 destruction, 15, 42, 94, 254 detection, viii, 2, 17, 18, 19, 20, 30, 35, 36, 37, 39, 79, 89, 141 detoxification, 44, 63, 81, 166, 236 developed countries, ix, 3, 91, 94, 95, 144 developing countries, 94, 95, 108, 111, 120, 136, 144

271

diabetes, vii, ix, x, xi, 1, 4, 14, 34, 48, 56, 82, 86, 93, 117, 119, 120, 121, 127, 128, 132, 133, 134, 136, 138, 139, 140, 142, 143, 150, 151, 152, 156, 159, 161, 176, 189, 190, 191, 192, 199, 201, 204, 206, 210, 211, 219, 220, 222, 228, 229, 259, 260, 264 diabetic nephropathy, 163 diabetic patients, 61, 86, 87, 142, 149, 150 diacylglycerol, 70 diarrhea, 219, 253 diet, x, 6, 14, 26, 42, 44, 46, 48, 50, 52, 53, 66, 70, 71, 72, 74, 77, 78, 80, 82, 129, 143, 144, 146, 147, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 160, 162, 163, 164, 228, 229, 260 dietary antioxidants, viii, 41, 131, 223, 240 dietary fat, 155 dietary fiber, 63 dietary habits, 74, 144 dietary intake, 56, 71 dietary supplementation, 56 diffusion, 176 digestion, 58, 87, 199, 206 dimethylformamide, 15 dipole moments, 16 discontinuity, 235 discrimination, 25 disease-reduction benefits., viii, 42 disorder, 204 dispersion, 34, 92 disposition, 80 dissociation, 21, 35 distillation, 251 distribution, 19, 27, 90, 109, 125, 147, 159, 208, 258 diuretic, 190, 192, 195, 198, 199, 201, 206 diversity, 9, 18, 76, 119, 121, 136, 184, 266 DNA, 33, 45, 50, 51, 52, 55, 56, 59, 61, 66, 67, 72, 75, 80, 83, 89, 93, 118, 126, 132, 140, 160, 211, 228, 229, 233, 234, 238, 239, 240 DNA damage, 50, 51, 52, 55, 56, 59, 61, 67, 72, 75, 80, 89, 140, 160, 233, 234, 239, 240 DNA repair, 89, 132 DNA strand breaks, 52, 118 docosahexaenoic acid, 152, 175 doctors, 94 DOI, 265 donors, 128, 167 double bonds, 19, 71 down-regulation, 58, 158 drainage, 112 drought, 93, 114, 121, 205 drug discovery, 119, 136 drug interaction, 2 drugs, x, 2, 3, 36, 81, 93, 94, 114, 118, 119, 120, 138, 139, 148, 149, 159, 163, 165, 244


272

Index

dry matter, 69, 105 drying, 16, 68, 78, 82, 157, 188 dyslipidemia, 55, 150 dyspepsia, 68, 206

E East Asia, 243 ecology, 38, 255 eczema, 188, 190, 196, 197, 209 edema, 56, 232 editors, 109, 110, 111, 114, 115, 180 Egypt, 206 eicosapentaenoic acid, 152 electric field, 17, 21 electrical conductivity, 97, 101 electron(s), 17, 23, 28, 29, 32, 42, 43, 53, 70, 93, 121, 128, 131, 208, 211, 212, 233, 234 electrophoresis, 17, 18, 21, 34 electrophoretic separation, 18 emission, 20, 112 endangered, 254, 256, 264 endocrine, 129, 137, 147, 186, 238 endocrine disorders, 238 endothelial cells, 43, 78, 177, 260 endothelial dysfunction, 131, 146 endothelium, 129, 131 endurance, 154 energy, 16, 43, 46, 76, 87, 102, 108, 118, 130, 144, 145, 147, 148, 149, 151, 158, 160, 162, 163, 166 energy density, 46 energy expenditure, 147, 151, 162 England, 244 enteritis, 253 entrapment, 102, 234 environment, ix, 16, 27, 43, 91, 92, 97, 109, 110, 116, 118, 131, 176, 229 environmental conditions, viii, 42, 93, 96, 98, 104 environmental impact, 101, 106 environmental influences, 147 environmental issues, 94, 112 environmental stress, 205 EPA, 152 epidemic, 136 epidemiologic, 81, 139, 150 epidemiologic studies, 139 epidemiology, 79 epilepsy, 189, 192, 196, 206, 228 epithelial cells, 54, 90 EPR, 235 equilibrium, 18, 132 erythrocyte membranes, 177

erythrocytes, 50, 51, 52, 58, 66, 75, 167, 179, 180, 181, 231 erythroid cells, 166 ESI, 18, 20, 21, 22, 25, 36, 38 ester, 9, 70 ester bonds, 9 ethanol, 15, 29, 66, 75, 78, 81, 209, 213, 214, 215, 216, 217, 218, 225, 261, 262, 263 ethers, 142 ethnic groups, 187 ethyl acetate, 15, 156, 262 ethylene, 23, 102 etiology, x, 165, 166 EU, 2, 3, 13 eucalyptus, 167, 264, 266 eukaryotic, 43 euphoria, 244 Europe, 2, 3, 64, 82, 95, 184, 187, 206, 229, 244, 245 European Union, 94, 115 evaporation, 21 evidence, xi, 14, 42, 44, 45, 47, 54, 69, 73, 74, 81, 84, 88, 107, 122, 126, 128, 144, 147, 159, 187, 227, 228, 229, 234, 236 evolution, 135, 258 excitation, 20, 31 excretion, 57, 149, 222 exercise, 44, 147, 151 expectorant, 191, 199 experimental condition, 29, 58 experimental design, 258 expertise, 3 exposure, 15, 31, 52, 167, 211 extinction, 94, 264 extracellular matrix, 43 extracellular signal regulated kinases, 134 extraction, viii, ix, 2, 15, 16, 34, 37, 38, 57, 58, 75, 91, 95, 97, 104, 107, 112, 157, 221, 263, 264

F factories, 114 FAD, 128 families, 6, 103, 185, 187, 189, 246 FAS, 157 fasting, 150, 154, 155, 156 fat, x, 72, 77, 129, 130, 141, 143, 144, 145, 147, 149, 150, 151, 152, 153, 154, 155, 157, 158, 159, 160, 161, 162, 163, 164, 263 fat reduction, 151 fatty acids, ix, 45, 75, 117, 129, 131, 144, 146, 151, 152, 209, 210, 219, 235, 260, 266 fauna, 187 FDA, 3


Index feelings, 158, 162 female rat, 74 fermentation, 60, 87, 261 ferritin, 166 fertilization, 96, 113 fertilizers, 96, 105 fever(s), 67, 68, 144 fiber, 46, 51, 166 fiber content, 51, 166 fibrinogen, 131 fibroblasts, 266 fibrosis, 235, 257 field crops, ix, 91, 95, 96 filtration, 157 Finland, 113 flammability, 16 flatulence, 154, 202, 206, 219 flavonol, 6, 13, 14, 24, 34, 39, 54, 76, 133, 135, 168, 233, 256 flavo(u)r, 68, 121, 156, 205 flight, 21 flooding, 115 floods, 128 flora, xi, xii, 81, 184, 186, 187, 259, 260, 264 flour, 188, 200 flowers, 48, 76, 103, 109, 121, 142, 184, 189, 205, 206, 211, 212, 230, 234, 239, 244, 257 fluctuations, 96 fluid, 15, 157 fluid extract, 157 fluorescence, 18, 19, 20, 31, 115 folic acid, 48, 53, 58, 69 folklore, 81 food, viii, 3, 15, 27, 34, 37, 39, 41, 42, 47, 48, 53, 55, 57, 58, 60, 64, 65, 67, 68, 73, 74, 81, 82, 84, 87, 88, 94, 95, 103, 120, 121, 124, 140, 147, 148, 150, 154, 156, 162, 187, 189, 207, 209, 211, 220, 222, 232, 240, 251 Food and Drug Administration, 3 food intake, 148, 154 food products, 42, 55, 88 force, 18 formation, vii, ix, 1, 4, 15, 16, 18, 23, 24, 26, 57, 61, 63, 69, 77, 92, 100, 102, 117, 120, 122, 128, 131, 135, 139, 140, 141, 142, 168, 181, 211, 234, 235, 236, 248, 258, 262 formula, 21 fossils, 187 fractures, 244 fragility, 176 fragments, 21, 23, 24 France, 156

273

free radicals, ix, 14, 27, 28, 29, 32, 34, 43, 50, 51, 57, 60, 61, 62, 63, 69, 80, 117, 118, 120, 121, 122, 127, 131, 132, 135, 136, 137, 166, 169, 176, 205, 211, 228, 237, 240, 250, 260, 261, 263 fructose, 150 fruits, viii, 6, 8, 9, 14, 36, 38, 42, 44, 48, 50, 51, 53, 54, 57, 58, 61, 62, 63, 65, 66, 68, 69, 73, 74, 75, 77, 79, 80, 81, 82, 85, 87, 88, 90, 121, 122, 124, 125, 136, 157, 158, 161, 175, 184, 189, 228, 232, 239, 263 functional food, 85 fungal infection, 188, 190, 191, 195, 196, 197 fungi, 205 furan, 62

G gamma rays, 33 gas diffusion, 102 gastric ulcer, 65 gastritis, 230 gastrointestinal tract, 149 gene expression, 33, 44, 72, 75, 90, 93, 118, 134, 151, 210 gene regulation, 69 genes, x, 117, 122, 150 genetic background, 90 genetic defect, 33 genetic factors, 147 genetic marker, 147 genotype, 72, 104, 107 genus, xi, 8, 62, 94, 103, 230, 243, 244, 254, 256, 257 geometry, 21 Georgia, 186 Germany, 2, 52 germination, 92, 98 ginger, 120, 158, 160, 169, 232, 240 ginseng, 232 glasses, 150 glaucoma, 127 glucagon, 149 gluconeogenesis, 127, 157 glucose, ix, 11, 44, 45, 61, 63, 64, 86, 87, 117, 127, 128, 129, 133, 134, 137, 140, 141, 144, 150, 151, 152, 154, 155, 157, 158, 163, 166, 178, 261, 262 glucose tolerance, 133 glucose-induced insulin secretion, 133 glucosidases, 134 glucoside, 10, 51, 133, 169, 205, 207, 209, 221, 231 glucosinolates, 49, 64, 65, 72, 79, 89 GLUT4, 134 glutamate, 44, 253


274

Index

glutathione, viii, 26, 41, 43, 44, 50, 51, 56, 61, 82, 84, 129, 131, 132, 149, 150, 160, 169, 176, 178, 179, 180, 220, 228, 229, 240, 253, 262 glycerol, 150 glycine, 44 glycogen, 127, 134 glycolysis, 127, 128, 178 glycoproteins, 155 glycoside, 5, 22, 24, 64, 133, 177, 231 glycosylation, 5, 6, 7, 8, 34, 35, 122 gout, 206, 209 grass, 38 grazing, 264 Greeks, 187 greenhouse, 97, 98, 99, 100, 101, 103, 104, 106, 107, 108, 109, 111, 112, 113 greenhouses, 97, 107, 109, 264 growth, viii, 4, 11, 36, 41, 51, 54, 58, 60, 61, 65, 79, 86, 87, 92, 94, 95, 97, 98, 99, 100, 101, 102, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 115, 118, 147, 150, 159, 205, 261, 264 growth factor, viii, 41 growth hormone, 147 growth rate, 99, 108 guidance, 3

H H. sabdariffa, 262 habitat, 254 habitats, 103, 244 harmful effects, 205 harmonization, 3 harvesting, ix, 91, 94, 96, 98, 100, 104, 230 HCC, 33 head and neck cancer, 71, 79 headache, 144, 192, 193, 196, 205 healing, viii, 41, 59, 73, 82, 190, 191, 192, 193, 194, 196, 197, 198, 199, 201 health, viii, x, 3, 14, 33, 34, 37, 41, 42, 44, 46, 47, 48, 54, 55, 56, 58, 60, 62, 63, 71, 73, 74, 77, 81, 82, 95, 121, 132, 142, 143, 146, 159, 162, 166, 176, 184, 185, 204, 205, 210, 211, 219, 222, 224, 227, 228, 260, 263 health care, 3, 184, 224 health care system, 224 health condition, 166, 204, 219 health effects, 142 health problems, 42, 185 health promotion, 166 hearing loss, 45 heart attack, 56 heart disease, x, 48, 61, 79, 143, 166

heart rate, 148 heartburn, 230 heavy metals, 94, 114, 205 height, x, 143, 144, 205, 206 helium, 21 heme, 128 hemoglobin, 166, 167, 176, 177 hemoglobinopathies, 166 hemolytic anemia, x, 165, 166, 179 hemorrhoids, 188, 189, 205, 206 hepatic injury, 255 hepatitis, xi, 243, 244, 253 hepatocytes, 235 hepatotoxicity, 235, 237, 238 herbal medicine, 2, 33, 94, 103, 184, 186, 206, 209, 221 herbal teas, xi, 183, 189, 205, 211, 212, 219, 220, 221 hereditary spherocytosis, 166 heredity, 147 hexane, 16, 177 high density lipoprotein, 158 high fat, 129, 152, 154, 155, 156, 157, 158, 160 Highlands, 212 hippocampus, 60 histamine, 210 histology, 236 history, 50, 148, 187, 209, 227 HIV, x, 126, 144 homeostasis, x, 44, 78, 127, 129, 143 homocysteine, 87, 151 homolytic, 24 hormone(s), 44, 67, 102, 131, 147148 horticultural crops, 78 host, 58 hotspots, 266 House, 137, 256 HPV, 113 human body, vii, 2, 26, 161 human health, vii, viii, ix, 1, 2, 4, 14, 26, 41, 62, 81, 87, 89, 91, 92, 101, 108, 118, 120, 126, 141, 162, 223, 232 human subjects, 56, 176 hybrid, 59, 86 hydrazine, 29 hydrogen, viii, 26, 28, 29, 31, 41, 42, 43, 44, 50, 55, 70, 72, 75, 80, 84, 92, 121, 125, 140, 166, 167, 169, 205, 208, 212, 233, 234, 237, 239, 240, 261 hydrogen atoms, 121, 235 hydrogen peroxide, viii, 26, 41, 42, 43, 44, 50, 55, 72, 75, 80, 84, 92, 125, 140, 166, 237, 239, 240 hydrolysis, 14, 25, 64, 65, 136, 177 hydroperoxides, viii, 41, 44, 69, 131, 236


Index hydrophobicity, 18 hydroponics, ix, 91, 96, 97, 98, 99, 101, 102, 104, 107, 111, 114, 115 hydroquinone, viii, 2, 26 hydroxyacids, 10 hydroxyl, 4, 5, 6, 12, 23, 24, 26, 55, 70, 92, 93, 104, 118, 122, 123, 132, 166, 169, 178, 232, 233, 234, 235, 237, 239, 240, 262 hydroxyl groups, 5, 6, 23, 70, 93, 104, 122, 232, 233, 234, 235, 239 hypercholesterolemia, 62, 82, 150, 189, 192, 206, 207, 219 hyperglycemia, 129, 138, 141, 155, 219, 222 hyperinsulinemia, 142 hyperlipemia, 138 hyperlipidemia, 53, 57, 158 hypertension, vii, x, xi, 1, 4, 45, 48, 56, 57, 129, 143, 144, 148, 150, 159, 189, 192, 194, 204, 206, 211, 219, 220, 222, 259, 260, 264 hyperthyroidism, 79 hypertrophy, 45, 146 hypotensive, 63 hypothalamus, 147, 149 hypothesis, 88, 92, 100, 235, 236 hypothyroidism, 79 hypoxia, 44, 97, 102, 104, 105, 106, 109, 110, 111 hypoxia-inducible factor, 44

I ideal, 119, 144, 148, 159 identification, 16, 19, 20, 21, 22, 23, 25, 34, 35, 36, 37, 55, 89, 90, 94, 119, 204, 219, 232, 254 identity, 184, 204 ileum, 207 images, 175 imbalances, 92 immune function, 166, 210 immune regulation, 33 immune response, 44 immune system, 34, 186, 189, 228, 261 immunomodulation, 60 immunomodulatory, 207, 220 immunostimulant, 126, 200 impotence, 206 improvements, 65, 149, 156 in vivo, 26, 28, 38, 50, 51, 53, 54, 56, 58, 60, 61, 64, 71, 72, 76, 81, 83, 89, 141, 155, 167, 175, 177, 228, 229, 232, 234, 239, 240, 253, 255, 258, 266 incidence, vii, 1, 4, 14, 56, 62, 74, 95 income, 144 India, 117, 156, 181, 223, 249, 255 Indians, 154

275

individuality, 4 individuals, x, 72, 86, 143, 151, 154, 158 induction, 33, 64, 70, 72, 101, 150, 153, 157, 177 industrial processing, 88 industries, 15, 184, 232, 251 industry, 57, 59, 95, 96, 103, 120, 209 infection, 4, 109, 118, 121, 199 infertility, 198, 207, 209 inflammasome, 90 inflammation, viii, 14, 33, 42, 45, 51, 54, 59, 67, 83, 89, 120, 129, 130, 144, 191, 202, 228, 230, 246, 260 inflammatory cells, 159 inflammatory disease, 93 inflammatory mediators, 131 inflammatory responses, viii, 41 infrastructure, 97 ingestion, 79, 167, 175 ingredients, vii, 1, 3, 48, 73, 85, 178, 189, 255 inhibition, 32, 50, 51, 53, 54, 60, 64, 65, 68, 71, 72, 133, 134, 135, 136, 155, 157, 161, 167, 169, 172, 173, 174, 175, 177, 205, 209, 219, 220, 232, 233, 238, 240, 246, 254 inhibitor, 51, 71, 129, 139, 148, 157, 163, 219 initiation, 14, 145, 169 injuries, 56, 202, 205 injury, 79, 87, 109, 137, 138, 150, 177, 178, 205, 229, 235, 240, 253 inner ear, 76 inositol, x, 144, 231 insects, 92 insomnia, 206 insulin, 63, 68, 90, 127, 133, 134, 136, 142, 144, 147, 148, 150, 151, 152, 154, 159, 263 insulin resistance, 144, 150, 159 insulin sensitivity, 150, 151 insulin signaling, 136 integrity, 176 interface, 18 interference, 30, 260 intervention, x, 52, 55, 67, 72, 76, 83, 86, 117, 149, 150, 151 intestine, 14 intima, 76 intoxication, 240 investment, 19, 97, 98 ion channels, 43, 150 ionization, 17, 18, 20, 21, 27, 34, 35, 36, 89, 222 ionizing radiation, 166 ions, 17, 21, 22, 23, 24, 25, 101, 105, 126, 135, 166, 234, 258 Iowa, 47, 84 Iran, 110, 186, 222


276

Index

Iraq, 186 Ireland, 244 iron, 52, 60, 81, 140, 209, 233, 234, 239 irradiation, 33 irrigation, 96, 101, 106 ischemia, 150, 152, 228 ischemia reperfusion injury, 152, 228 Islam, v isoflavone, 13, 135 isoflavonoids, 4, 125 isolation, xi, 3, 16, 122, 243, 244, 245, 261 isomers, 8, 10, 22, 24, 25, 35, 71, 153, 175, 181, 210 isoprene, 206 issues, vii, ix, 1, 4, 91, 97, 223 Italy, 91, 104

J Jamaica, xi, 259, 263, 265, 266 Japan, 60, 184, 256 jaundice, 206 jejunum, 70, 207 joint pain, 201 Jordan, 126, 139

K kaempferol, 6, 20, 24, 48, 49, 53, 54, 65, 66, 68, 70, 123, 133, 134, 135, 138, 169, 176, 209, 230, 231, 232, 233, 250 ketones, 114, 155 kidney, 64, 136, 142, 154, 189, 192, 194, 195, 197, 198, 200, 201, 204, 206, 207, 209, 230, 254 kidney stones, 198, 200, 204 kinase activity, 150 kinetics, 28, 78, 239 Korea, 81, 158

L lactoferrin, 166 L-arginine, 128, 132 LC-MS, 35 LDL, 50, 51, 52, 53, 55, 61, 63, 65, 67, 76, 79, 86, 131, 132, 135, 137, 147, 155, 157, 158 leaching, 101, 106, 113 lead, vii, 1, 15, 29, 43, 54, 62, 118, 135, 144, 149, 159, 167, 236 lead molecules, vii, 1 leakage, 131 lean body mass, 147, 151 legislation, 94

legume, 181 lens, 134 leprosy, 206 leptin, 129, 146, 147, 149, 151, 154, 157 lesions, 52, 83, 93 leukocytes, 83 life cycle, 163, 254 lifestyle changes, 144 light, xi, 16, 31, 58, 71, 93, 100, 108, 110, 115, 126, 166, 183, 187, 205, 234, 254 light conditions, 100 lignans, 4, 120, 121, 177 lignin, 9, 11, 36, 93 linoleic acid, 32, 69, 261 lipases, 132 lipid metabolism, 14, 157, 158 lipid oxidation, 50, 51, 56, 60, 66, 71, 76, 89, 149, 167 lipid peroxidation, x, 50, 51, 53, 54, 56, 59, 62, 66, 67, 69, 71, 76, 84, 85, 86, 93, 101, 118, 132, 135, 136, 137, 143, 146, 167, 169, 175, 176, 177, 180, 231, 234, 236, 239 lipid peroxides, 132, 135 lipids, vii, 1, 4, 15, 42, 45, 51, 52, 61, 66, 93, 118, 128, 131, 132, 156, 166, 168, 177, 211, 228, 229, 234, 235, 236 lipolysis, 146, 147, 149, 151, 153, 155, 158, 159 lipoproteins, 76, 118, 131 liquid chromatography, 19, 34, 35, 36, 39, 77, 79, 89, 258 liquid phase, 18 lithium, 32 liver, xi, 39, 50, 52, 56, 60, 63, 64, 66, 67, 72, 75, 79, 82, 83, 136, 146, 148, 152, 155, 157, 206, 207, 222, 227, 228, 229, 231, 233, 235, 236, 237, 240, 253, 257 liver cancer, 39 liver cells, 66 liver cirrhosis, 228 liver damage, 82, 207, 235 liver disease, 152, 229 localization, 43, 44 low fat diet, 146 low risk, 19 low temperatures, 62, 102 low-density lipoprotein, 50, 52, 55, 71, 78, 88, 131, 156, 162, 179 lung cancer, 33, 61 Luo, 109, 244, 256 lutein, 48, 49, 55, 61, 69, 72, 87, 210 lycopene, 49, 59, 67, 71, 72, 84, 85, 86, 135, 210 lymphatic system, 72 lymphocytes, 52, 83, 89


Index lymphoid, 56, 186 lysis, 167, 169, 176, 181 lysozyme, 33, 39

M machinery, 110, 126, 131 macromolecules, 43, 101 macrophages, 45, 61, 76, 79, 86, 131 macular degeneration, 59 magnesium, 57, 61 magnitude, 43, 71 Maillard reaction, 135 majority, 17, 28, 104, 187, 188, 204, 219 MALDI, 21 management, xi, 96, 114, 149, 152, 155, 161, 162, 163, 220, 222, 259 manganese, 77 manipulation, ix, 91, 107 manufacturing, 55, 74, 115 marketplace, 2 mass, x, 17, 18, 19, 20, 21, 22, 25, 34, 35, 36, 37, 89, 104, 143, 144, 147, 150, 151, 156, 158, 159, 222, 231, 234, 258 mass spectrometry, 17, 19, 20, 21, 22, 25, 34, 35, 36, 37, 89, 222, 234, 258 materials, 2, 48, 184 matrix, 15, 18, 20, 21, 34, 53, 54, 82, 230 matrix metalloproteinase, 54, 82 matrixes, 16 matter, 178 MB, 58, 137, 141 measurement(s), 31, 155, 234 media, 30, 76, 96, 97, 99, 116, 264 medical, 42, 48, 94, 178, 184, 187, 189, 207, 211, 229, 253, 254 medical history, 229 medication, x, 165 medicine, viii, xi, 2, 3, 41, 59, 60, 65, 68, 73, 74, 99, 118, 119, 120, 176, 179, 183, 184, 185, 187, 189, 207, 209, 221, 222, 223, 224, 225, 227, 228, 229, 230, 232, 243, 244, 250, 256, 258, 259, 263, 266 Mediterranean, vii, 1, 65, 83, 86, 184, 206, 224 Mediterranean climate, 86 MEK, 134 mellitus, 120, 140, 142, 151, 152, 179 membrane permeability, 93, 101 membranes, 50, 55, 125, 168, 169, 177, 235 memory, 83 mental illness, 3 mesangial cells, 137 mesophyll, 126 messengers, ix, 76, 86, 117

277

meta-analysis, 79, 149, 154 Metabolic, v, ix, 117, 132, 136, 147, 161, 163 metabolic changes, 144 metabolic disorder(s), x, 59, 117, 119, 120, 127, 136, 144 metabolic dysfunction, 144 metabolic pathways, 102 metabolic syndrome, 50, 53, 54, 76, 138, 144, 145, 150, 151, 155, 159, 160, 161, 178, 219, 221 metabolism, ix, 27, 33, 35, 46, 58, 65, 79, 93, 96, 100, 102, 109, 110, 117, 128, 133, 136, 138, 142, 147, 149, 155, 166, 180, 181, 210, 211 metabolites, vii, ix, 1, 4, 14, 39, 54, 64, 76, 91, 92, 93, 95, 96, 98, 99, 100, 101, 102, 104, 107, 108, 110, 111, 113, 114, 116, 120, 121, 122, 166, 180, 204, 206, 210, 231, 232, 233, 237, 238, 239 metabolized, 11, 136 metabolizing, 43, 64, 246, 256 metal ion(s), 14, 93 metals, 120, 166, 211 metastasis, 33, 45 meth, 94 methanol, 15, 16, 19, 29, 63, 67, 172, 173, 174, 180, 208, 211, 213, 214, 215, 216, 221, 223, 231, 261, 262 methodology, 17, 112, 245 methyl group(s), 5, 233 methylation, 5, 6, 8, 11, 17 Mexico, 65, 228, 229, 230, 232 mice, 63, 66, 76, 84, 90, 136, 139, 149, 154, 155, 156, 158, 159, 162, 163, 164, 176, 181, 221 microcirculation, 177 microemulsion, 36 micronutrients, ix, 46, 117 microorganisms, 136 microscopy, 175 microsomes, 231, 233 microwaves, 16 Middle East, 60 migration, viii, 33, 41, 45 mitochondria, 43, 90, 118, 128, 130 mitochondrial DNA, 131 mitogen, 128, 134 mixing, 18 MMP, 50, 54, 60 MMP-2, 50, 54 MMP-9, 60 MMS, 151 model system, 55, 107, 180, 261 models, xi, 27, 39, 56, 64, 136, 146, 151, 152, 181, 227, 232 modifications, x, 106, 121, 143, 150, 167 molasses, 83


278

Index

molecular biology, 11 molecular mass, 18 molecular oxygen, 132 molecular structure, 21, 122 molecular weight, 4, 21, 57, 63, 122, 167 molecules, vii, ix, xi, 1, 8, 18, 20, 21, 24, 26, 27, 29, 35, 43, 44, 45, 47, 50, 54, 55, 64, 78, 80, 91, 92, 93, 94, 96, 97, 100, 103, 107, 117, 118, 119, 132, 135, 146, 165, 166, 167, 211, 228, 229, 232, 234, 244, 260 molybdenum, 32 MOM, 112 monomers, 8 monounsaturated fatty acids, 49, 210 Montana, 77, 174 Moon, 57, 81, 87, 90, 140 morbidity, x, 143 morphine, 102, 118, 244 morphology, 11, 103 mortality, x, 47, 48, 84, 143 mortality rate, 84 mortality risk, 47 motif, 247 mountain ranges, 186 MR, 108, 114, 115, 140 mutagen, 104 mutation(s), 45, 118, 211 myoblasts, 145 myocardium, x, 143

N Na+, 101 NaCl, 101, 104, 106, 111 NAD, 51, 64, 128, 129, 133, 135 NADH, 128, 135 natural compound, viii, 2, 3, 26, 75, 146, 204, 219 natural disaster(s), 264 natural habitats, 263 natural herbal products, vii, 1 nausea, 197 necrosis, 150, 235, 236 negative effects, 14 Nepal, 255, 265 nephropathy, 127 nervous system, 147, 186 Netherlands, 39, 114 neurodegenerative diseases, 53, 66, 71, 86, 119, 121, 166, 229 neurodegenerative disorders, 45, 74 neuroendocrine system, 147 neurological disease, 45, 60 neuronal apoptosis, 44

neuronal cells, 50, 60, 80, 84 neurons, 129 neuropathy, 127 neurotoxicity, 45, 55 neutral, 18, 25 New South Wales, 183 niacin, 153 nicotinamide, 128 nitric oxide, ix, 43, 47, 61, 78, 117, 118, 128, 166, 178, 234, 262 nitric oxide synthase, ix, 43, 78, 117, 128, 166 nitrogen, x, 21, 26, 64, 92, 108, 109, 110, 111, 112, 113, 114, 117, 118, 142, 209, 211, 228, 244, 260 NMR, 19, 21, 22, 37, 81 non-enzymatic antioxidants, vii, 2, 26, 53, 132 non-polar, 15, 16, 17 non-smokers, 65 norepinephrine, 155 normal aging, 229 North Africa, 118 North America, 94, 99, 206 Northeast Asia, 77 Nrf2, 44, 81 nuclear magnetic resonance, 19, 231 nuclei, 126, 141 nucleic acid, 42, 118, 150, 236 nucleotides, vii, 1, 4 nucleus, 25, 126 Nuevo León, 230 nutraceutical, xi, 163, 259, 264 nutrient, ix, 11, 54, 91, 96, 97, 98, 100, 101, 102, 104, 105, 106, 107, 108, 109, 112, 114, 116, 121, 132 nutrient imbalance, 97 nutrients, x, 57, 62, 65, 102, 144, 149 nutrition, 48, 90, 93, 96, 100, 104, 105, 109, 110, 111, 112, 129, 140, 163, 210, 238

O obesity, ix, x, 34, 53, 56, 59, 77, 90, 117, 121, 129, 130, 131, 136, 137, 138, 139, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 157, 158, 159, 160, 161, 162, 163, 164, 210, 219 OH, 8, 9, 10, 20, 24, 28, 118, 127, 150, 166, 168, 233, 234 oil, 56, 61, 67, 75, 78, 87, 88, 95, 99, 102, 108, 112, 113, 114, 206, 232, 255 oil production, 108, 112 oleic acid, 69, 210 oligomers, 55 olive oil, 38, 179, 180


Index omega-3, 144, 146, 152 opportunities, ix, 92, 97, 109, 162 optimization, 95, 96, 245 organ, 104, 111, 130 organelle(s), 46 organic compounds, 64 organism, 50, 92 organs, xi, 45, 102, 103, 127, 208, 227, 263 ornamental plants, 103, 114 osmotic stress, 101 osteoarthritis, 87 osteoporosis, 78, 121 ovaries, 230 overlap, 148 overproduction, 128 overweight, x, 143, 144, 149, 154, 155, 156, 157, 158, 160, 161, 162, 163 overweight adults, 160 ox, 49, 70 oxidation, viii, ix, x, 2, 26, 27, 28, 29, 32, 33, 39, 47, 50, 51, 52, 53, 54, 55, 60, 61, 67, 69, 71, 76, 85, 117, 122, 129, 131, 135, 137, 141, 143, 151, 157, 158, 167, 168, 169, 175, 176, 177, 234, 238, 239, 247 oxidation products, 239 oxidative agents, 177 oxidative damage, viii, ix, 41, 44, 50, 52, 53, 55, 57, 58, 63, 66, 69, 75, 86, 90, 93, 101, 117, 126, 131, 135, 139, 166, 167, 176, 177, 180, 235, 236, 237, 239, 263 oxygen, viii, x, 15, 26, 31, 32, 41, 43, 44, 51, 55, 66, 70, 76, 77, 83, 86, 102, 105, 109, 110, 112, 117, 118, 120, 122, 126, 128, 131, 132, 138, 140, 142, 143, 167, 179, 180, 181, 205, 208, 212, 234, 236, 245, 250 oxygen consumption, x, 143 ozone, 16, 126

P Pacific, 115, 180 pain, 148, 189, 190, 192, 194, 195, 196, 197, 198, 201, 202, 204, 205, 206, 207, 209, 230 Pakistan, 81, 113 palpitations, 196, 205 pancreas, 56, 60, 148 parallel, 149 parents, 59 participants, 52, 149, 151, 152, 153, 154, 155, 176 partition, 18 pastures, 244 pathogenesis, vii, 2, 26, 42, 45 pathogens, 11, 92, 95, 121, 205

279

pathophysiological, 45, 137, 159, 241 pathways, x, 11, 12, 13, 50, 54, 74, 92, 93, 122, 127, 128, 129, 141, 143, 148, 151, 232 pemphigus, 74 peptic ulcer, 198, 207, 209 peptide, 148, 149, 176 peripheral blood, 52, 72, 81 peripheral nervous system, 228 peroxidation, 67, 131, 132, 135, 168, 177, 235, 237 peroxide, 43, 44, 81, 131, 166, 234 peroxynitrite, 55, 166, 177, 178, 239, 240 personality, 147 personality characteristics, 147 pesticide, 98 pH, 19, 28, 31, 53, 97, 102, 111 pharmaceutical, ix, xi, 3, 48, 58, 91, 92, 93, 94, 95, 96, 97, 98, 100, 101, 103, 112, 165, 204, 219, 251, 259, 263 pharmaceuticals, 34, 184 pharmacokinetics, 136 pharmacological treatment, 219 pharmacology, 137, 163, 244, 256 pharyngitis, 206 phenol, 4, 28, 63, 65, 68, 69, 93, 121, 251 phenylalanine, 9, 11, 12, 64, 102, 104, 105, 121 pheochromocytoma, 50, 59 phosphate, 127, 128, 150, 166, 178 phosphatidylcholine, 50, 55 phosphoenolpyruvate, 134, 157 phosphorus, 101, 108, 115 phosphorylation, ix, 45, 117, 128, 129, 131, 134, 135, 150 photobleaching, 31 photooxidation, 126 photosynthesis, 4, 92, 100, 115, 258 photosynthetic performance, 254 phycoerythrin, 31, 32 physical activity, 144, 147 physical properties, 87 Physiological, 110, 204 physiology, 11, 108, 120 phytomedicine, 3 phytosterols, vii, 1, 70, 155 phytotherapy, 3, 220 PI3K, 134 pigmentation, 126, 261 pilot study, 162, 209, 220 placebo, 3, 55, 149, 151, 152, 153, 154, 155, 156, 157, 161, 162 plant growth, ix, 76, 91, 92, 96, 99, 100, 101, 102, 105, 106, 113 plaque, 138 plasma levels, 151


280

Index

plasma proteins, 69, 82 plasminogen, 129 platelet aggregation, 56, 71, 209, 221 platelets, 166 platform, 98 playing, 60 PM, 109 poison, 197 polar, 16, 19, 57, 58, 261, 262 polarity, 8, 21 pollen, 187 pollination, 92, 121 pollinators, 4, 11 pollutants, 95, 126, 166 pollution, 211 polymerization, 54 polymers, 9, 21, 120 polymorphism(s), 82, 160 polyphenols, viii, x, 9, 30, 31, 33, 34, 39, 41, 42, 44, 46, 47, 48, 49, 51, 52, 53, 56, 57, 58, 60, 61, 67, 69, 72, 77, 78, 117, 120, 121, 136, 144, 146, 158, 166, 168, 169, 175, 177, 179, 180, 211, 223, 232, 234, 235, 260, 261 polysaccharide(s), 9, 14, 155, 261 polystyrene, 97 polyunsaturated fat, 132, 175, 260 polyunsaturated fatty acids, 132, 175, 260 pools, 43 population, 3, 39, 78, 120, 175, 184, 186, 211, 254 Portugal, 1, 33, 34, 165 positive correlation, 66, 208 potassium, 30, 58, 61, 101, 108 potassium persulfate, 30 precipitation, 31 pregnancy, 147 preparation, xi, 52, 60, 65, 74, 75, 81, 103, 183, 184, 186, 188, 189, 206, 209, 212 preservative, 120 prevention, viii, 33, 42, 48, 54, 56, 58, 60, 69, 73, 75, 76, 79, 80, 83, 85, 88, 93, 146, 149, 154, 158, 166, 179, 209, 222, 228, 239, 260 primary function, 189 principles, 92, 94, 95, 96, 118, 140 probability, 28 probe, 28, 31, 32, 122 production costs, 97 profitability, 97, 107 pro-inflammatory, 146 proliferation, viii, 33, 38, 41, 43, 45, 54, 57, 63, 90, 129, 131, 146, 147 propagation, 234 prophylactic, 54, 60, 85 propyl gallate (Pg), viii, 2, 26

prostaglandins, 152, 232 prostate cancer, 61, 88 prostate specific antigen, 61 prostatitis, 201 protection, vii, 1, 4, 39, 48, 50, 55, 58, 64, 71, 75, 84, 85, 121, 122, 133, 135, 146, 167, 169, 175, 177, 179, 205, 208, 212, 228, 229, 234, 235, 236, 237, 253, 263, 264 protection of plants, vii, 1, 4, 121 protective mechanisms, 92 protective role, 168, 180 protein folding, 131 protein kinase C(PKC), ix, 45, 117 protein oxidation, 167 protein synthesis, 11 proteins, x, 14, 15, 33, 42, 44, 45, 51, 66, 93, 118, 128, 129, 131, 132, 143, 150, 166, 167, 211, 228, 229, 234, 235, 236, 238 proteolytic enzyme, 59 pruning, 138 Pseudomonas aeruginosa, 209 public health, 94 pulp, 48, 49, 58, 63, 78, 79, 88, 157, 160, 176 purification, 16, 39 purity, 97

Q quality assurance, 112, 254 quality control, 95, 250 quality of life, 144 quality standards, 95 quantification, viii, 2, 17, 19, 31, 36 quartile, 149 quercetin, 20, 23, 34, 38, 48, 49, 51, 52, 53, 54, 57, 63, 65, 66, 68, 70, 72, 90, 123, 133, 135, 142, 145, 146, 152, 169, 176, 177, 205, 207, 209, 222, 230, 231, 232, 233, 234, 235, 236, 237, 250 questionnaire, 39 quinone, 29, 233

R radiation, 4, 17, 121, 139, 205, 211, 220 radical formation, 129, 167 radical reactions, xi, 165, 167 radicals, vii, ix, x, 2, 26, 28, 31, 34, 43, 51, 66, 68, 79, 84, 117, 118, 127, 131, 132, 143, 166, 168, 169, 175, 178, 179, 205, 211, 212, 232, 238, 260, 261 rape, 90 rash, 230


Index RE, 208 reaction mechanism, 29, 31 reaction medium, 31 reaction time, 30 reactions, 11, 23, 27, 28, 43, 118, 129, 135, 140, 234, 260 reactive oxygen, vii, ix, x, 2, 14, 26, 42, 50, 86, 92, 100, 102, 117, 118, 138, 141, 143, 166, 205, 211, 212, 228, 229, 239, 240, 260 reactivity, 43, 45 reagents, 140 receptors, 6, 128, 131, 150 recognition, 17 recovery, 19, 199 red blood cells, 135, 166, 176, 178, 179, 181 red wine, 8, 47, 56, 61, 90, 121, 122, 124, 152 reducing sugars, 57 redundancy, 148 regeneration, 75, 135, 264 regions of the world, 209 relevance, 38 relief, xi, 259 renal cell carcinoma, 45 repair, 26, 132, 167 reproduction, 4, 11, 92 requirements, 95, 96, 115, 149 researchers, 33, 73, 94, 177, 245 reserves, 160 residues, 4, 25, 45, 64, 75, 236 resistance, 93, 150, 158, 160, 176, 205 resolution, 17, 21 resources, 184, 204, 220, 224 respiration, 4, 92, 102 response, viii, ix, 4, 26, 41, 55, 65, 72, 91, 92, 101, 102, 104, 111, 112, 115, 120, 121, 122, 131, 149, 152, 245, 258 restoration, 66 restrictions, 156 resveratrol, 10, 33, 48, 55, 56, 71, 75, 85, 146, 152, 240 reticulum, 125 retinol, 78, 150 retinopathy, 45, 127, 176, 179 revenue, 2 riboflavin, 33 rings, 4, 22, 23, 121, 122, 248 ringworm, 197 risk(s), viii, x, 33, 34, 42, 47, 48, 50, 51, 55, 56, 57, 59, 61, 66, 67, 71, 72, 74, 79, 81, 82, 83, 87, 90, 94, 139, 143, 144, 146, 149, 150, 161, 181, 210 risk factors, x, 48, 61, 82, 83, 143, 146, 150, 161 ROOH, 28 room temperature, 188

281

root(s), 48, 78, 83, 97, 98, 99, 100, 101, 102, 103, 104, 105, 108, 109, 110, 111, 112, 115, 121, 134, 139, 140, 168, 172, 173, 184, 188, 201, 206, 207, 208, 210, 213, 214, 215, 216, 221, 222, 223, 255, 261, 262 root growth, 97, 102, 105 root system, 98, 102 routes, 11, 93 Royal Society, 110

S safety, 3, 33, 42, 50, 60, 73, 94, 149, 163, 184, 237, 238 salinity, 101, 104, 106, 111, 113, 114, 115 saliva, 137 salt tolerance, 101 salts, 8, 55, 108, 246 saponin, 155 saturated fat, 48, 129, 210 saturated fatty acids, 129, 210 scavengers, viii, 2, 26, 44, 50, 56, 70, 120, 121, 231, 232, 233, 234, 237, 238, 239 schizophrenia, 45 science, 139, 260 scope, 138 secondary metabolism, ix, 91, 92, 96, 100, 102 secrete, 63 secretion, 89, 90, 127, 147 security, 3 sedative, 195 seed, 39, 56, 58, 63, 74, 78, 79, 83, 84, 88, 89, 92, 121, 152, 163, 178, 188, 201, 215, 216, 240, 261, 264 seedlings, 110 selectivity, 18, 149 selenium, 48, 53, 149, 160 sensation, 206 sensing, 44, 115 sensitivity, 18, 33, 102 serum, 57, 61, 65, 68, 70, 76, 84, 86, 87, 150, 151, 152, 154, 156, 158, 163, 207, 222 sesquiterpenoid, 207, 231, 239 severe stress, 122 sex, 147 sex steroid, 147 shape, 145, 167, 176 shelf life, 62 shoot, 99, 101, 102, 104, 105, 188, 201, 262 shoots, 102, 105 showing, 149, 152, 244 sickle cell, 166, 178 sickle cell anemia, 166, 178


282

Index

side chain, 234 side effects, 3, 94, 119, 148, 184, 219, 232 signal transduction, 44, 116, 134 signaling pathway, ix, 33, 43, 117, 129, 222 signalling, 78, 93, 149, 151 signals, 144 silica, 17 sinusitis, 190, 193 skeletal muscle, 44, 148 skeleton, 5, 13, 121, 245 skin, 37, 55, 56, 59, 60, 61, 71, 83, 147, 188, 193, 194, 195, 198, 199, 200, 206, 230, 231 skin cancer, 60 skin diseases, 206 small intestine, 14, 134, 148 smoking, 65, 188, 190, 211 smooth muscle, 45 smooth muscle cells, 45 snacking, 156, 161 society, 204, 263 sodium, 18, 32, 101, 115, 232, 237 sodium dodecyl sulfate (SDS), 18 soil erosion, 264 soil particles, 95 soleus, 134 solid phase, 15 solid tumors, 231 solubility, 5, 15, 31, 47, 102, 125 solution, ix, 17, 19, 21, 30, 31, 32, 68, 91, 96, 97, 98, 100, 101, 102, 104, 105, 106, 107, 114, 152, 239 solvents, 15, 16, 17, 18, 19, 29, 37 South Africa, 154 soybeans, 7 specialty crop, 97, 109 specifications, 94 spectrophotometry, 28 spectroscopy, 21, 231 spleen, 50, 55 splenitis, 206 splenomegaly, 206 Sprague-Dawley rats, 155 sprouting, 86 squamous cell, 54, 88 squamous cell carcinoma, 54, 88 SS, 13, 110, 112, 142 stability, 21, 44, 83, 121, 138, 233 stabilization, 177 stable radicals, 66, 261 stamens, 230 standardization, 3, 34, 96 starvation, 100 state(s), 3, 29, 30, 44, 45, 46, 48, 61, 77, 94, 128, 129, 130, 139, 147, 166, 187, 230

stem cells, 145 stenosis, 76 steroids, 6, 155, 244, 252 sterols, 49, 85, 231 stigma, 156 stimulant, 68 stimulation, viii, 33, 41, 96, 129, 132 stoma, 190, 191 stomach, 56, 64, 68, 189, 190, 191, 192, 194, 195, 196, 197, 198, 199, 200, 201, 204, 205, 206, 207, 209, 230 storage, 62, 63, 70, 76, 77, 79, 80, 81, 85, 86, 89, 107, 129, 130, 145 stress, vii, ix, x, 2, 4, 26, 45, 50, 53, 59, 60, 61, 73, 76, 81, 91, 92, 93, 96, 101, 102, 104, 105, 108, 110, 113, 117, 118, 119, 120, 121, 122, 128, 129, 130, 131, 136, 137, 138, 139, 140, 142, 144, 166, 167, 176, 177, 180, 181, 204, 211, 221, 229, 236 stress factors, 96 stress response, 76, 138 stroke, 120, 152, 191 structural changes, 118 structural characteristics, 232 structure, 4, 5, 10, 12, 22, 24, 25, 27, 29, 64, 70, 101, 121, 122, 123, 150, 167, 177, 206, 222, 233, 234, 240 subgroups, 9 substitution, 7, 8, 25, 122, 125 substitutions, 6, 122 substrate(s), viii, 2, 26, 38, 96, 98, 128, 134 sucrose, 15 sulfate, 5, 32 sulfur, 32, 64, 151, 158, 162 sulfur dioxide, 32 sulphur, vii, 1, 4, 126 Sun, 88, 139, 162, 179, 256, 258 supplementation, 47, 53, 56, 65, 72, 80, 83, 86, 89, 139, 150, 151, 156, 158, 160, 162, 163, 178 suppression, 158, 209 surface area, 52, 108 surfactants, 18 surplus, 136 survival, 33, 44, 92, 166, 180, 264 susceptibility, 109 sustainability, 264 Sustainable Development, 113 swelling, 56, 191 Switzerland, 90 symptomatic treatment, 206 symptoms, 70, 155, 204 synaptic plasticity, 83 syndrome, 147, 150, 159 synergistic effect, 48, 71


Index synthesis, ix, 11, 44, 61, 65, 91, 92, 95, 101, 104, 107, 122, 127, 134, 150, 158 systolic blood pressure, 61, 76

T Taiwan, 142 tannins, x, 8, 9, 11, 12, 17, 38, 48, 49, 53, 60, 93, 99, 101, 117, 120, 122, 156, 205 target, 17, 28, 32, 101, 148, 167, 234 taxa, 184, 186, 187, 223, 254 teams, 184 technical assistance, 237 techniques, 15, 16, 20, 22, 37, 94, 95, 96, 258 technologies, 113 technology, 88, 97, 107, 108, 111, 113 temperature, 16, 17, 31, 35, 62, 93, 98, 100, 113, 254 terpenes, x, 117, 176, 206, 219, 232, 260, 262 tert-butyl hydroquinone (TBHQ), viii, 2, 26 testing, 149, 211, 212, 264 textiles, 209 texture, 68 TGF, 257 Thailand, 62, 88 thalassemia, 166 therapeutic agents, 46, 48, 115, 236 therapeutic approaches, 136 therapeutic effects, viii, 42 therapeutic targets, 88 therapeutic use, 3 therapeutics, vii, 142 therapy, 57, 75, 76, 80, 119, 146, 151, 164, 178, 219, 228, 238, 244, 262 thermal energy, 71 thrombophlebitis, 232 thyroid, 44, 56, 147 Tibet, xi, 243, 244, 258 time periods, 70 tissue, xi, 45, 47, 56, 61, 65, 72, 92, 94, 96, 101, 108, 111, 127, 129, 130, 131, 146, 148, 150, 155, 159, 204, 229, 237, 259, 264 TLR, 77 TNF, 58, 129, 131, 150, 152, 155, 159, 254, 257 TNF-α, 58, 129, 131, 152, 155, 254, 257 tobacco, 65 tobacco smoke, 65 tocopherols, viii, 41, 42, 120, 260 Tonga, 178 tonic, 156, 206 tonsillitis, 204 tooth, 201 total cholesterol, 53, 63, 152, 156, 157, 158, 162 toxic effect, 42, 101, 235

283

toxic substances, 92 toxicity, viii, 2, 14, 16, 26, 86, 93, 101, 108, 111, 115, 121, 141, 179, 234, 235, 238 TPA, 232 trace elements, 132 trade, 212 training, 151, 158, 160 traits, 103, 109 transaminases, 156 transcription, 44, 85, 128, 158, 209, 223 transcription factors, 158 transducer, 128 transduction, 115, 151 transferrin, 166 transformation, 9, 15, 132, 236 transition metal, 126, 132, 135, 166, 179, 232, 235 transition metal ions, 132, 135, 232, 233 translocation, 155 transplantation, 82 transport, 43, 86, 108, 125, 128 transportation, 107 treatment, vii, viii, xi, 33, 42, 48, 56, 57, 59, 63, 79, 87, 93, 104, 115, 118, 120, 132, 136, 142, 145, 149, 152, 153, 154, 156, 157, 158, 166, 177, 184, 188, 189, 204, 206, 209, 219, 221, 232, 235, 243, 244, 253, 261, 263 trial, 56, 61, 70, 83, 120, 149, 150, 151, 152, 153, 155, 156, 160, 161, 163, 187, 205 tricarboxylic acid, 127 tricarboxylic acid cycle, 127 triggers, 131 triglycerides, 62, 130, 145, 151, 152, 154, 156, 158 tropical rain forests, 120 tryptophan, 64 tuberculosis, xi, 243, 244 tumor, 33, 44, 45, 50, 54, 56, 58, 59, 69, 90, 129 tumor cells, 50, 54, 56, 59 tumor development, 54 tumor growth, 45 tumor necrosis factor, 129 tumorigenesis, 33, 56, 57 tumors, 56, 61 Turkey, 52, 67, 183, 184, 185, 186, 207, 209, 212, 220, 221, 222, 223, 224, 225 type 2 diabetes, 90, 179 tyrosine, 43, 104, 134, 150, 234 Tyrosine, 236

U UK, 35 ulcer, 199 ultrasound, 15


284

Index

underlying mechanisms, 33 United States, 2, 64, 79 urban, 229 uric acid, 44, 129, 132, 166, 228, 229 urinary tract, 54, 70, 86 urinary tract infection, 54, 86 urine, 53, 162 USA, 2, 3, 108, 110, 178, 180 uterus, 188, 199, 204 UV, 4, 11, 17, 18, 19, 20, 22, 26, 28, 29, 64, 92, 121, 126, 141, 205, 231, 234 UV irradiation, 26 UV light, 17 UV radiation, 4, 11, 92, 126

V valence, 42 validation, 3, 254 valve, 21 vanadium, 133, 138 variables, 65, 98, 147 variations, viii, 15, 42, 95, 98, 110, 244 varieties, 37, 39, 42, 58, 66, 69, 75, 78, 79, 80, 81, 82, 103, 113, 122, 157, 230, 255 vasculature, 142 vasodilator, 126, 166 vegetables, viii, 6, 8, 14, 36, 38, 42, 44, 46, 47, 48, 50, 51, 64, 65, 66, 68, 73, 74, 75, 77, 80, 81, 82, 87, 97, 98, 101, 107, 114, 121, 177, 181, 207, 228, 232, 239, 263 viral pathogens, 10 viscera, 159 visceral adiposity, 150 vitamin A, 62, 67, 209 vitamin B6, 47 vitamin C, 26, 44, 47, 49, 57, 58, 59, 60, 62, 64, 66, 70, 72, 77, 78, 79, 86, 99, 101, 121, 132, 169, 209, 228, 229, 232 Vitamin C, 49, 51, 132, 166, 262 vitamin D, 47, 149, 150, 163 vitamin E, 26, 44, 49, 58, 60, 62, 66, 68, 70, 121, 132, 135, 139, 169, 228, 229, 240 vitamins, 43, 48, 49, 53, 55, 61, 65, 66, 67, 69, 122, 146, 166, 207, 240, 262 volatility, 17 vomiting, 206

W Wales, 244 walking, 147 Washington, 110 waste, 15, 26, 57 waste water, 15 wavelengths, 19, 30 wealth, 48 weight control, 147 weight gain, 147, 154, 157 weight loss, x, 87, 144, 146, 148, 149, 151, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163 weight management, 163 weight reduction, 153, 156 West Indies, 266 Western Europe, 2, 244 WHO, 3, 48, 90, 120, 144, 163 wild type, 254 wood, 263 workers, 25, 33, 149, 168, 169, 188, 205 World Health Organization, x, 48, 90, 94, 143, 144, 223 worldwide, xi, 45, 74, 94, 103, 184, 259 wound healing, 190, 192, 193, 194, 195, 198, 199, 204, 206 wound infection, 209, 225

X xanthones, 49, 57

Y yeast, 60, 97, 205 yellow fever, 68 yield, 59, 65, 96, 97, 99, 107, 108, 112, 113, 115, 168, 263

Z Zimbabwe, 77 zinc, 150


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