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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


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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

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Copyright 2014 by Nova Science Publishers, Inc.


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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

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Contents
Preface
Chapter 1

Chapter 2

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

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


Joo 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, Noem Crdenas-Rodrguez,
Bernardino Huerta-Gertrudis, Jos Luis Rodrguez-Chvez
and Elvia Coballase-Urrutia

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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

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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.

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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.

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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

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David Alexandre Micael Pereira

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.

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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 worlds 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 - Jamaicas 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.

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xii

David Alexandre Micael Pereira

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 islands flora.

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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, Vtor Spnola and Paula C. Castilho
Centro de Qumica da Madeira, Departamento de Qumica,
Universidade da Madeira, Campus Universitrio 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.

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Sandra C. Gouveia, Vtor Spnola 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 N134 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).

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Phenolic Compounds and Antioxidant Capacity of Medicinal Plants

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.

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Sandra C. Gouveia, Vtor Spnola 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

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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

C6-C1

Phenolic acids

Gallic acid

C6-C2

Acetophenones

Gallacetophenone

C6-C2

Phenylacetic acid

p-Hydroxyphenyl-acetic acid

C6-C3

Hydroxycinnamic
acids

p-Coumaric acid

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.

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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 -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 (Guzmn 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 C6C1
hydroxybenzoates, most notably gallic acid, which is the precursor of hydrolysable tannins,
the C6C3 hydroxycinammates and their conjugated derivatives, and the polyphenolic C6
C2C6 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

p-Hydroxybenzoic acid

OH

OH

Gallic acid

OH

Protocatechuic acid

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 C6C3 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

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 -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 C6C3C6 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).

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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; F3H, 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 liquidliquid 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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(SCCO2), 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 samplesolvent 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 (350365 nm or
250260 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 C (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. -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 (esla 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 (< 1L/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 (Mtt 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
,
,
[MHglycoside]-. 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 -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 4O-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 2position 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), -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) (Magalhes 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.
ETbased 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) (Magalhes 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 (Magalhes 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 (Magalhes 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 samples 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 (Magalhes 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 (Magalhes 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 (Magalhes 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
(Magalhes 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 cyclodextrin as a water solubility enhancer (Huang et al., 2002).

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Sandra C. Gouveia, Vtor Spnola and Paula C. Castilho

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 (Magalhes 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
(Magalhes 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 (Magalhes 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 (Magalhes 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. -Carotene-Linoleic Acid Bleaching Assay
The evaluation of the antioxidant activity by the -Carotene assay is based on the fact
that the free radical linoleic acid attacks the highly unsaturated -Carotene, and the presence
of compounds with antioxidant properties delay the -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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Sandra C. Gouveia, Vtor Spnola and Paula C. Castilho

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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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

Chapter 2

Potential Antioxidant Benefits


of Commonly Used Fruits
and Vegetables around the World
Lourdes Rodrguez-Fragoso1, Ulises Osuna-Martnez2,
Ana Isabel Gonzaga-Morales1 and Jorge Reyes-Esparza1
1

Universidad Autnoma del Estado de Morelos, Facultad de Farmacia,


Cuernavaca, Mxico
2
Universidad Autnoma de Sinaloa,
Facultad de Ciencias Qumico Biolgicas, Cualiacn, Mxico

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: mrodriguezf@uaem.mx.

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L. Rodrguez-Fragoso, Ul. Osuna-Martnez, 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 (DAutreaux 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

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43

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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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-1 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-1 (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: Parkinsons disease, Alzheimers 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 (Bnfi 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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L. Rodrguez-Fragoso, Ul. Osuna-Martnez, Ana Isabel Gonzaga-Morales et al.

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, -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 Womens
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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L. Rodrguez-Fragoso, Ul. Osuna-Martnez, Ana Isabel Gonzaga-Morales et al.

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 Organizations (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
Agricultures 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).
-carotene, lutein, phenolics, flavonoids and anthocyanins
(Vislocky and Fernandez, 2010), carotenoids (Bunea, 2012),
anthocyanins, catechins, resveratrol, phenolic acids, and
procyanidins. (Kammerer et al., 2004).

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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-Garca 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).
-cryptoxanthin esters (Breithaupt and Bramedi, 2001), -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 (Rodrguez-Hernndez 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), - and -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, -carotene,
lycopene, phytoene, phytofluene, quercetin, polyphenols and
kaempferol (Rao and Agarwal, 1999)
Isothiocyanates, flavonoids (quercetin and hydroxycinnamic
acids) (Gill et al, 2007). -carotene, lutein and glucosinolates
(Getahun and Chung, 1999).

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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
(Dcord 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 (Guimares 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-Beltrn 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-Zuiga 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 Gonzlez, 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 (Dcord 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 eect 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 M. 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 eects 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, -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 eects 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 (Morr and Morr, 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 (Assunco 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, -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
(Guimares 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-B), suppresses NF-B activation induced by inflammatory agents, including tumor
nuclear factor alpha (TNF-), 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 worlds 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-Garca 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-B 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, -cryptoxanthin, and -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-B, 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 -cryptoxanthin esters (Breithaupt and Bramedi, 2001). Additionally,
tangerines are a source for -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).
-carotene and -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 -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 -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 20C 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 -cells, allowing them to secrete more insulin, or to a
favorable effect on regenerated -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 -thioglucoside N-hydroxysulfate common structure, containing a -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-Beltrn 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 (Rodrguez-Hernndez 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-Domnguez 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) (Fernndez-Lpez 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 (7.711.2 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 -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 g/g dw). This value is 10 times higher than C. annuum var.
cerasiferum (5.6 g/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 g/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 g/ml and 356.8 g/ml against -amylase
and -glucosidase, respectively, in the lipophilic fraction. This fraction showed phytol,
vitamin E, -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 -glucosidase at 43%
at 200 M (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, -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 -carotene, which might be due to
the combined activities of several phytochemicals, especially lutein and -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-Hernndez et al. showed that arbol and
chipotle varieties presented the highest values of antioxidant activities, followed by morita
and guajillo (Hervert-Hernndez 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-Zuiga 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, -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 -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, -carotene,
and -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 (Polvkovet 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 -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 -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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73

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, pures, 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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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

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
*

rita.maggini@unipi.it.

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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 (Domnguez-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 (Mlgaard 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;
Rodrguez-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; Lonhart 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, Lonhart 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 (Paraikovi 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 (Kovik 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 (Kovik 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
Calendulas 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; Crdenas-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; Klimnkov et al., 2008).
The relative content of each constituent can often enable to differentiate among distinct
cultivars (Klimnkov 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 Bao 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 (-80C) samples, as
desiccation at 70C 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 plants 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 (80C) 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.

References
Abeles, FB; Morgan, PW; Saltveit, ME, Jr. Ethylene in plant biology. San Diego, CA, USA:
Academic Press; 1992.
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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

Chapter 4

Flavonoids as Antioxidant Therapy


for Metabolic Disorders
B. S. Lakshmi1, K. N. Sangeetha2 and K. Shilpa1
1

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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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 [krovnkov
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 [krovnkov et al., 2012]. These medicinal
plants are a reservoir of secondary metabolites, which act either individually, additively or in
synergy with several dierent 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 -tocopherol (vitamin E) based on in vitro antioxidant assays because of their strong
capacity to donate electrons or hydrogen atoms [Hernandez 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 plantpathogen 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 Hernndez 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--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 [Ami 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 flavonoids six-membered ring substituted with -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) [Hernandez 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) [Tomas-Barberan 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
B (NF-B)-mediated cytokine production. Nevertheless, hyperglycemic condition activates
production of ROS thereby stimulating the stress related signaling mechanisms such as NFB, 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-), 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 [Fernndez 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- [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. -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- -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-()-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--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--L-fucopyranoside and apigenin6-C-(2-O- -L-rhamnopyranosyl)- -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--Larabinopyranosyl--D-glucopyranoside along with the known flavonoid glycosides such as

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kaempferol 3-O--D-glucopyranoside exerted promising inhibition of porcine lens aldose


reductase activity [Kim et al., 2004]. Likewise, engeletin, astilbin (dihydroflavonol
glycosides), isorhamnetin 3-O--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, 3,4dihydroxyflavone, 3,4,7-trihydroxyflavone, luteolin, luteolin 7-O- -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 -glucosidases hydrolyze oligosaccharides,
trisaccharides and disaccharides to glucose and other monosaccharides in the small intestine.
Hence, as a therapeutic measure for diabetes, -glucosidase inhibitors are in demand to
reduce the impact of carbohydrates on blood sugar. Certain flavonoids and its derivatives
have been reported to exhibit -glucosidase inhibitory activity. Hydroxy flavonoids such as 6hydroxyapigenin (scutellarein), 6-hydroxyapigenin-7-O--D-glucopyranoside,6-hydroxyluteolin-7-O- -D-glucopyranoside, feruloylglucosides such as 6-hydroxyapigenin-7-O-(6-Oferuloyl)--D-glucopyranoside,6-hydroxyluteolin-7-O-(6-O-feruloyl)--D-glucopyranoside
isolated from Origanum majorana and flavonoid 6-hydroxyluteolin and 5,6,7trihydroxyflavone (baicalein) from Scutellaria baicalensis showed rat intestinal -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 -glucosidase, -glucosidase, and -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- -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- -D-glucopyranoside (isoquercetin) from the leaves of Eucommia
ulmoides were found to be acting as glycation inhibitors [Kim et al., 2004]. Luteolin 6-C-(6O-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, -tocopherol and carotenoids from
oxidation, regeneration of vitamin E from oxidized -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 -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 -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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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

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,
*

Corresponding author: vgulati@swin.edu.au.

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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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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

Cl

Orlistat

Lipase
inhibitor

Fecal
urgency,
oily stools,
abdominal
pain, flatus

H3C

(H2C)10
CH3

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)- 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)
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/EBP (CCAAT-enhancer-binding protein)
mediated induction of downstream genes, notably PPAR, prevents entry of the preadipocytes
into the growth-arrested phase and interacts with activators of PPAR 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 PPAR and C/EBP 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 = 28kgm2) participated. The trial
was randomized into three groups, receiving either one tablet of multivitamin and mineral
supplement (MMS), or Ca 162mg 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-,
activate PPAR- and PPAR-, 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, -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

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

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).

HO

OH

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 (6.2550 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 mat) 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-
and downregulated the expression of TNF-. 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).

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 bodymass index, waisthip 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 SpragueDawley 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 -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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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

Chapter 6

Application of Antioxidant Plants


as Anti-Hemolytic Agents
1

Joo 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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Joo C. Fernandes and David M. Pereira

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. Parkinsons and Alzheimers), 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) (OBrien 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 (OBrien 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 (Mar 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 (Mar 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

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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 Gio 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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Joo C. Fernandes and David M. Pereira

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 g/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 g/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 L of golden root aqueous extract
(41.6 mg/mL) was sufficient to reduce hemolysis in 50%; 200 L 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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169

hemolysis; both plants showed anti-hemolytic activity, however cumin possessed higher
capacity over 60% inhibition at 100 M 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--D-glucoside, phenolic compounds from ginger (Zingiber officinale Roscoe) and rhubarb
(Rheum rhabarbarum L.), respectively. At 50 M, 3,3,5-trihydroxy-4-methoxystilbene 3-D-glucoside showed higher protection (65%) than trolox (40%) in AAPH-induced hemolysis
in RBC. Differently, 6-gingerol only exerted some effect when at 100 M. 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 -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>ECGEC>GA.

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Figure 1. ROS generation in RBC and its defenses.

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

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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%

(Gio 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%

(Gio et al., 2010)

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 m

(Atrooz, 2013)

Black tea

Camellia sinensis L.

Leaf

Caraway

Carum carvi L.

Seed

Methanol extract

Human

++

100 m

(Atrooz, 2013)

Ceylon ironwood

Mesua frrea L

Flower

Aqueous/methanol extract (80:20)

Human

++

<0.25%

(Sangkitikomol, 2012)

Chamberbitter

Phyllanthus urinaria L

Leaf

Aqueous/methanol extract (80:20)

Human

++

<0.25%

(Sangkitikomol, 2012)

Aqueous/methanol extract (80:20)

Human

++

<0.25%

(Sangkitikomol, 2012)

++

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 m

(Atrooz, 2013)

Methanol extract

Human

++

100 m

(Atrooz, 2013)

Aqueous extract

Human

++

0.05%

(Gio et al., 2010)

++

<0.25%

(Sangkitikomol, 2012)

++

0.0384%

(Nabavi et al., 2012)

++

0.01%

(Sabuncuolu &
hretolu, 2012)

++

0,0025%

(He et al., 2008)

Eucalyptus

Eucalyptus globules L

Leaf

Eugenia

Syzygium gratum Wight

Leaf

Aqueous/methanol extract (80:20)

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.

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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 rsea 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

Aqueous/methanol extract (80:20)

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

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

100l of
41.6mg/ml
200l of
41.6mg/ml
0.054%
0.0025%
20m
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

++
+++

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

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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)
(Gio 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)
(Gio 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%

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

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

Application of Antioxidant Plants as Anti-Hemolytic Agents

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 20M, 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

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Joo C. Fernandes and David M. Pereira

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 g/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 g/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 didnt seem to have any effect. He et al. (2008) reported a dual
effect of G. biloba leaf extract on the amyloid peptide (A25-35)-induced hemolysis. In the
presence of G. biloba leaf extract, the hemolytic rate was inhibited by 70% at a dose of
approximately 25 g/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 M) 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.

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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 l and 60 l/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 M; barbaloin IC50 = 31.7M) (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 M) (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 (200g/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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Joo C. Fernandes and David M. Pereira

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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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

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

CSIRO Animal, Food and Health Sciences, North Ryde, NSW, Australia
YzncYl 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 worlds 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: Izabela.Konczak@csiro.au; rzekarega@hotmail.com.

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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 (Aaolu 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 (Baar, 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 1990s the research teams of E. Yesilada and E. Sezik of the Gazi University,
Ankara, Turkey, in collaboration with M. Tabatas 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, Erz ncan, Ari, Kars, Idir 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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Izabela Konczak, Abdullah Dalar and Konrad A. Konczak-Islam

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;
zgke and zelik, 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 (Cokun and
Genler zkan, 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 aseyh) 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. Barnabys 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 1990s and data
published by other authors have revealed that a large number of medicinal plants are found in
the states of Agri, Ardahan, Bingl, Bitlis, Elazig, Erzincan, Erzurum, Hakkari, Igdir, Kars,
Malatya, Mu, 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,
Erz ncan, Ari, Kars and Idir 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 (zdnan-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 (Mkemre, 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; zgke and zelik, 2004; Tabata et al.,
1994; Tuzlac and Doan, 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 summers 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.

Complimentary Contributor Copy

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 ibii

LF

Sterility

Infusion

zgke and zelik, 2004

Kizvan, nervend

LX, FR
(immature)
GM
FR

Keratoma, wound healing, mouth


diseases
Stomach disorders
Common colds, diuretic, urinary
inflammations
Burns, antiseptic, diarrhoea

Chewing

Tuzlac and Doan, 2010

Pistacia khinjuk Stocks


Pistacia terebinthus L.

Gezan,
Menegi, edene

Eaten fresh
Decoction (int.)

Kaval, 2011
zgke and zelik, 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.

Sbt

AE

Hypercholesterolemia

Anthriscus slyvestris (L.) Hoffm.


Diplotaenia cachrydifolia Boiss.
Eryngium billardieri Delar.

Gmgmi
Siyabu
Ts, gelenk

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

Eryngium bornmuelleri
Ferula orientalis L.

Tusi
Heliz, air,
Kngor,

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.

Kerme
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

Mkemre, 2013
Kaval, 2011;
Tuzlac and Doan, 2010,
Mkemre, 2013
Kaval, 2011
Kaval, 2011
Kaval, 2011

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

Complimentary Contributor Copy

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.)

zgke and zelik, 2004;


Altundag and Ozturk, 2011

Guhktk

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.)

zgke and zelik, 2004;


Tuzlac and Doan, 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.

Ar iei, bovijan

Achillea millefolium L. subsp.


millefolium

Civan peremi,
herezan

Achillea vermicularis Trin

Civan peremi,
bovijan
Papatya
Papatya
Sar papatya
Sar papatya
Nuserk, top telli,
kriz, lolek

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

Lolek

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 (Krnicke) Arenes
Artemisia absinthium L.

Zilasur

Bevjana kuvi

Tuzlac and Doan, 2010;


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

Complimentary Contributor Copy

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 gz,
Gya brinok
Tahlisk

FL
WP
AE

Centaurea iberica Trev. ex Spreng.


Centaurea pterocaula Trautv.
Centaurea virgata Lam.

Kelembek
ermnik
aladir

LF
LF
WP

Centaurea solstitialis L. subsp.


solstitialis
Cichorium intybus L.

Belhok, zerik

AE

Kani,
talik,
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
zgke and zelik, 2004
Mkemre, 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
Tuzlac and Doan, 2010;
Altundag and Ozturk, 2011
Kaval, 2011; zgke and
zelik, 2004; Tabata et al.,
1994; Tetik et al., 2013;
Altundag and Ozturk, 2011;
Mkemre 2013
Tuzlac and Doan, 2010
Kaval, 2011

SH,
SD,
LX,
RT, ST

Vitiligo, cold, catarrh, aphrodisiac,


diarrhoea, diabetes, orexigenic

Sevka a
Guyazerk

BB
AE

Diabetes
Kidney stones, cholesterol, diabetes

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

zgke and zelik, 2004;


Tabata et al., 1994; Tetik et
al., 2013; Cakilcioglu et al.,
2011; Altundag and Ozturk,
2011
Kaval, 2011
Mkemre, 2013

Gllga zer,
herdemtaze
ngz, peniruk

AE, SH, FL

Kidney stones

AE

Haemorrhoids

Kaval, 2011; zgke and


zelik, 2004
Kaval, 2011

Andz

RT

Chest pain

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

Complimentary Contributor Copy

zgke and zelik, 2004

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

Local names
Tehlika 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.

Bevjan

AE

Diabetes

Gyakek

LF

Wound healing

Bevjan

AE

Ptot

Zirin

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

References
Kaval, 2011
Tetik et al., 2013
Tuzlac and Doan, 2010
Kaval, 2011; zgke and
zelik, 2004; Mkemre,
2013
Ointment (with butter Tuzlac and Doan, 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
Galatrpenk
Krkba otu

FR,
RT
RT
ST
RT

Havaciva

RT (bark)

Wound healing, burns

Alkanna orientalis (L.)


Boiss. var. orientalis
Anchusa azurea Mill. var. azurea

Havacva otu,
hovacov
Mjmejok

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

Complimentary Contributor Copy

Kaval, 2011

zgke and zelik, 2004


Altundag and Ozturk, 2011
zgke and zelik, 2004

zgke and zelik, 2004;


Tabata et al., 1994
Mkemre, 2013
zgke and zelik, 2004;
Tabata et al., 1994
Tuzlac and Doan, 2010

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

Local names
Gzrik, mjmj

Plant part
LF,
RT

Therapeutic effect/ailments treated


Venom,
antiseptic

Application
Eaten fresh, poultice
(ext.)

References
Kaval, 2011; Altundag and
Ozturk, 2011

Keselmehmut, ulik

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 Nyr.
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

Mayasl otu

LF

Haemorrhoids

Boiled (ext.)

zgke and zelik, 2004

RT

Sterility

Boiled (int.)

zgke and zelik, 2004

Abdulselam, daraling, RT,


juri ruvi, trye ruvi
FR

Stomach disorders,
constipation,hypertension

Decoction, fresh,
powder (int.)

Kaval, 2011, Mkemre,


2013

Dikenli ard

FR

Rheumatism

Decoction (ext. bath)

zgke and zelik, 2004

Topalak

RT

Diuretic

Infusion (int.)

zgke and zelik, 2004

Puk, buki

LX

Antiseptic

Tabata et al., 1994

Gevrik

LX

Antiseptic for wounds and cuts

Directly on skin
(ext.)
As above

Keringan, hoil, huil

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.

Complimentary Contributor Copy

Tabata et al.,1994
Tuzlac and Doan, 2010
Tuzlac and Doan, 2010
Tabata et al., 1994; zgke
and zelik, 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.

Stleen

LX

Huil

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

Stleen, ylan otu

LX

Eczema, fungal infection

Tuzlac and Doan, 2010

ilomali

LX

Constipation

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

zgke and zelik, 2004

Kaval, 2011

Giyagezk, at kuyruu AE,


WP
Getgedok
AE

Urinary problems,
diuretic
Hypertension, kidney pain

Decoction, Infusion
(int.)
Decoction

Kaval, 2011; zgke and


zelik, 2004
Kaval, 2011

Mekik kk, meyan,


sus, meyan kk

RT

Diabetes, heartache, cough, bronchitis,


stomach ache, diarrhoea, digestive

Decoction (int.)

Hespst
Sebelk nefel

WH
AE

Astringent
Stomach disorders

Poultice (ext.)
Decoction

Kaval, 2011; zgke and


zelik, 2004; Sezik et al.,
1997; Tetik et al., 2013
Kaval, 2011
Kaval, 2011

Pltan

SH

Hypoglycaemic

Pounded, decoction
(int.)

zgke and zelik, 2004

Tolk

RT

Intestinal worm in children

Decoction

Kaval, 2011

Ahu

LF

Parasites

Infusion (int.)

zgke and zelik, 2004

Binbir delikotu,
kantaron

WP,
FL

Stomach ache, sedative

Decoction (int.),
infusion (int.)

zgke and zelik, 2004;


Cakilcioglu et al., 2011

Pizak

RT

Kidney stones

Decoction

Kaval, 2011

Complimentary Contributor Copy

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

Local names

Plant part

Therapeutic effect/ailments treated

Application

References

Gz, 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; zgke and


zelik, 2004; Tabata et al.,
1994; Tuzlac and Doan,
2010

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

Png, tyl nane,


punk, yarpuz, pnk

AE,
LF,
WP

Decoction [int./ext.
Tabata et al., 1994; Kaval,
(bath)], infusion (int.) 2011; zgke and zelik,
2004; Tuzlac and Doan,
2010; Sezik et al., 1997;
Cakilcioglu et al., 2011

Mentha pulegium L.

Pjan, havan, 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; zgke


and zelik, 2004;
Tuzlac and Doan, 2010

Pnk, pune, nane

WP, AE, LF

Rheumatism, antispasmodic, colds, flu

Mercankk
Catr, ku 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
zgke and zelik, 2004;
zgke and zelik, 2004;
Tuzlac and Doan, 2010
Tabata et al., 1994

Canter, kekik otu

WP

Wound healing

Tabata et al., 1994

Prunella vulgaris L.
Phlomis armeniaca Wild.

Sosn Belgsesing
alba, alba

Abdominal pain
Antipyretic, colds, asthma, bronchitis

Phlomis pungens Wild.


Salvia candidissima
Vahl. subsp. candidissima
Salvia multicaulis Vahl.

alba
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;
Mkemre, 2013
Altundag and Ozturk, 2011
Tuzlac and Doan, 2010

Adaay

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

Complimentary Contributor Copy

Family/Species name
Salvia nemorosa L.

Local names
Gemda

Plant part
WP

Therapeutic effect/ailments treated


Bleeding, wounds

Application
Pounded (ext.)

Salvia sclarea L.

Da ay

BR+FL, LF

Respiratory system diseases, cold

Salvia verticillata L.

Hart, ada ay

LF

Catarrh, common colds, nausea

Infusion (int.), with


lemon juice)
Decoction (int.)

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

Anh, da kekii
BR+FL
Keselmehmut, derman AE,
WP
Keselmehmut,
LF,
beyaz ot,
AE,
merve, neman,
ST
bovijana in
Zahter
WP
atra 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
Tuzlac and Doan, 2010
(int.)
Infusion (int.)
Tuzlac and Doan, 2010

Sirmuk

Digestive, urinary inflammations

Eaten fresh

Tetik et al., 2013

Da sarmsa

RT,
LF
BB

Hypotensive , cancer, ringworm

Crushed (int.)

Tuzlac and Doan, 2010

Corin
Yara otu

LF
RT

Anaemia
Wound healing

Altundag and Ozturk, 2011


Tuzlac and Doan, 2010

Gulik

RT, LF (juice)

Diabetes, fungal infection, eczema

Gl falc

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. Tuzlac var.
tenuiflora
Eremurus spectabilis Bieb.
Merendera trigyna
(Steven ex Adam) Stapf.

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

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References
Sezik et al., 1997; Altundag
and Ozturk, 2011
Tuzlac and Doan, 2010
Tabata et al., 1994; zgke
and zelik, 2004; Tuzlac
and Doan, 2010
Tuzlac and Doan, 2010
Kaval, 2011; zgke and
zelik, 2004
Tabata et al., 1994; Kaval,
2011; zgke and zelik,
2004; Sezik et al., 1997
Mkemre, 2013
zgke and zelik, 2004
Kaval, 2011

Tuzlac and Doan, 2010


zgke and zelik, 2004;

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

Local names

Plant part

Therapeutic effect/ailments treated

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.

Hra iei, 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
Mkemre, 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.)

zgke and zelik, 2004


Kaval, 2011

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

zgke and zelik, 2004


Tabata et al., 1994; Kaval,
2011; zgke and zelik,
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,
Tolgakvi,
ebegmeci,
hiru

Injury, asthma

References

zgke and zelik, 2004;


Altundag and Ozturk, 2011
Tuzlac and Doan, 2010;
Altundag and Ozturk, 2011
Tuzlac and Doan, 2010
zgke and zelik, 2004

Gulor, glhor

AE

Diabetes

Infusion (int.),
decoction (int.)

Tuzlac and Doan, 2010

Sinir otu

LF

Wound healing

Fresh (ext.)

zgke and zelik, 2004

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Family/Species name
Plantago lanceolata L.

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

Therapeutic effect/ailments treated


Stoma disorders, diabetes, haemorrhoids,
wound healing, abscesses, recovery of
incisions, cancerous uterus

Ylan dili

LF

Cancer of uterus

Decoction (ext.)

References
Kaval, 2011;
zgke and zelik, 2004;
Tabata et al., 1994;
Mkemre, 2013
Tabata et al., 1994; Kaval,
2011; zgke and zelik,
2004; Tuzlac and Doan,
2010; Sezik et al., 1997
zgke and zelik, 2004

Msr

SL

Antilithic, diuretic, haemorrhoids

Infusion (int.)

zgke and zelik, 2004

Revas, ribes, gn,


ukun, 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.

Tirok
Kuzu kula

ST (juice), AE
LF

Chewing fresh
Pounded (ext.)

Rumex tuberosus L. subsp. horizontalis


(Koch.) Rech.
PORTULACACEAE
Portulaca oleracea

Troka kera

LF

Diabetes, stomach ache


Haemorrhoids, anti-inflammatory,
antiphlogistic, antirheumatic
wound healing

Kaval, 2011; zgke and


zelik, 2004; Tabata et al.,
1994; Tuzlac and Doan,
2010;
Mkemre, 2013
Tuzlac and Doan, 2010
zgke and zelik, 2004

Poultice (ext.)

Kaval, 2011

Prpar, pimpirim,
semiz otu,
parpar, pirpirim

AE

Urethra infection, inflamed wounds,


cardiac disorders, diabetes, constipation

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

Tuzlac and Doan, 2010;


Tetik et al., 2013

Cngk
rekotu

LF
SD

Rheumatism
Diabetes, ulcer

Poultice (ext.)
Decoction (int.)

Kaval, 2011
zgke and zelik, 2004

Muhabbet iei

RT

Stomach pains

Eaten fresh

zgke and zelik, 2004

Gyabirin
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.)

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

Local names
Mahlep
Al
ekok
ilank,
ilan,
kuburnu,
domuzturpu
ilank

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

Ddrk,
drne,
mmirk, ttrk

SD,
FR

Tonsillitis, sore throat, strengthen of


eyes, kidney stones

Eaten fresh, dry


powder in water
(gargle),

Tabata et al., 1994; Kaval,


2011

Babelisk
Yourt otu, yanikotu

AE
FL

Haemorrhoids
All cancers, burns

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

Kaval, 2011
Tabata et al., 1994; zgke
and zelik, 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, st

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

Tuzlac and Doan, 2010;


Mkemre, 2013
Kaval, 2011

Sprge 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 (Crp.) . 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.

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References
Cakilcioglu et al., 2011
zgke and zelik, 2004
Tuzlac and Doan, 2010
Tabata et al., 1994; Kaval,
2011; zgke and zelik,
2004; Tuzlac and Doan,
2010; Sezik et al., 1997
Kaval, 2011

zgke and zelik, 2004

Family/Species name
SOLONACEAE
Hyoscyamus niger L.

Local names

Plant part

Therapeutic effect/ailments treated

Application

References

Hrdal, hargel,
harundol, ban out,
batbat
Dadoan

SD

Toothache, tooth cavity

Inhaled, directly on
tooth (ext.)

RT+SD

Intoxication

Eaten fresh

Ttn

LF

Wound healing

Powder (ext.)

Tabata et al., 1994; zgke


and zelik, 2004; Sezik et
al., 1997
Tabata et al., 1994; zgke
and zelik, 2004;
Tabata et al., 1994

Tevri

BR,
BK

Toothache, rheumatism, cough

Fresh, decoction
powder (ext.)

Kaval, 2011

Derdoan

FR

Diarrhoea

Eaten fresh

Tuzlac and Doan, 2010

Geznk, geznek,
yn, dezink,
geznk, srgan otu,
gezik, jrtken,
inar, drk

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.

Isrgan otu, inar

LF,
WP

Diarrhoea, diuretic, rheumatism

VIOLACEAE
Viola odorata L.

Bnevok

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.)

Tuzlac and Doan, 2010


zgke and zelik, 2004;

Kidney stone, diarrhoea

Decoction (int.)

Peganum harmala

zerlik

AE,
FR
RT,
SD

Haemorrhoids, prostatitis, urinary


incontinence, stomach ache

Decoction (int.),
roasted (int.)

Kaval, 2011;
Cakilcioglu et al., 2011
Tabata et al., 1994; zgke
and zelik, 2004, Sezik et
al., 1997

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

Seddan, ptrak

Kaval, 2011; zgke and


zelik, 2004; Tabata et al.,
1994; Tuzlac and Doan,
2010; Sezik et al., 1997;
Cakilcioglu et al., 2011;
Mkemre, 2013
Tea (int.), fresh (ext.) Tabata et al., 1994; zgke
and zelik, 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.

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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 Nyr. 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

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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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204

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

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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Health Attributes, Antioxidant Properties and Phytochemical Composition

205

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 Anatolias 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 107.2134.2 mg GAE (gallic acid equivalent)/g DW (dry weight) and flavonoid content is
42.477.97 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 M/plate) to 91.1% (S. typhimurium TA1537: 0.2 M/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), -caryophyllene (2.6%), camphor (1.6%), -muurolene (1.1%), and
piperitenone (1.0%). The remaining 33 compounds (-pinene, -pinene, -myrcene, 3octanol, -terpinene, p-cymene, limonene, (Z)--ocimene, (E)--ocimene, -terpinene, cissabinene hydrate, linalool, pinocamphone, terpinen-4-ol, -terpineol, dihydro carvone, bornyl
acetate, sabinyl acetate, thymol acetate, -bourbonene, nepetalactone, -copaene, caryophyllene, bicyclogermacrene, -bisabolene, -cadinene, d-cadinene, spathulenol,
caryophyllene oxide, salvial-4(14)-en-1-one, -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--d-glucoside), delphinidin 3-O-(6-O-malonyl--d-glucoside)-5-O--dglucoside, delphinidin 3-O--d-glucoside-5-O-(6-O-malonyl--d-glucoside) and delphinidin
3,5-di-O--d-glucoside (Nrbk 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 -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 tolk or ebegmeci)
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 3.40.3 (root) to 17.40.3 (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 0.970.02 (root) to 7.210.28 (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<stem
<fruit<flower<leaf. 4-Hydroxycinnamic acids made the second largest group of phenolic
compounds and contributed approximately 10 to 18% of the total phenolics. In the different
parts of M. neglecta plant their level ranged from 0.480.04 (root) to 2.560.07 (leaf) mg
CAE (caffeic acid equivalent)/g DW, which represented 14.3% (root) and 14.7% (leaf) of the
total phenolics, respectively. With regards to the distribution of 4-hydroxy-cinnamic acids in
various plant organs the following order was observed: rootstemfruit flowerleaf. This
indicates that the distribution pattern of 4-hydroxycinnamic acids was identical to that of total
phenolics and total flavonoids (Dalar et al., 2012).
Antioxidant capacity of M. neglecta has been investigated using two reagent-based assys:
oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP).
These two assays are based on two different mechanisms of action: ORAC is based on a
hydrogen electron transfer (HAT), and FRAP on a single electron transfer (SET) mechanism.
It can be expected that plants sources that exhibit potent antioxidant capacities in both assays,
involving oxygen radical scavenging and reduction of oxidizing agents, may offer more
efficient protection from oxidative stress. Alcohol-based (80% methanol and 1% HCl (v/v) in
water) extracts of various plant parts exhibited comparable antioxidant capacity to that of
traditional Chinese medicinal plants. M. neglecta leaf extract had the highest total reducing
capacity (FRAP assay) of 190.36.7 mol Fe+2/g DW and was followed by flower (109.81.3
mol Fe+2/g DW), fruit (56.43.6 mol Fe+2/g DW), stem (51.51.3 mol Fe+2/g DW) and
root (39.21.2 mol Fe+2/g DW; Dalar et al., 2012). The FRAP values of M. neglecta were
comparable with those of the following Chinese medicinal plants: Portulaca oleracea
(common purslane) (54.120.19 mol Fe+2/g DW), Prunella vulgaris L. (common selfheal)
(56.080.56 mol Fe+2/g DW) and Taraxacum mongolicum Hand (dandelion) (54.912.45
mol Fe+2/g DW). Flowers of M. neglecta exhibited a similar total reducing capacity to
Coptis ohinensis Franch (112.223.28 mol Fe+2/g DW), and the leaf had a similar reducing
capacity as Viola yedoensis Mak. (199.101.39 mol Fe+2/g DW) (Dalar et al., 2012).
The oxygen radical scavenging capacities (ORAC assay) of the root [425.36.7 mol TE
(trolox equivalent)/g DW], stem (506.20.8 mol TE/g DW) and fruit (448.11.6 mol TE/g
DW) of M. neglecta alcoholic extracts were similar to those of celery (419.516 mol TE/g
DW) and parsley (529.384.8 mol TE/g DW). The flower (795.214.6 mol TE/g DW) and
leaf (898.914.9 mol TE/g DW) of M. neglecta had similar ORAC values to those of basil
(7024 mol TE/g DW) and thyme (8252.8 mol TE/g DW) (Dalar et al., 2012).
A strong positive correlation between the levels of total phenolics, as well as various
groups of phenolic compounds, and antioxidant capacities of the M. neglecta extracts
indicates that phenolic compounds are the sources of antioxidant capacity (Dalar et al., 2012).
The highest correlations were obtained for hydroxycinnamic acids and antioxidant capacities,

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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
(Martnez-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, carotene and -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 -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 plants 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 905.05.3 (stem) to 2262.68.5
(flower) mol 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)
(233064 mol TE/g DW) and Rosa damascena (Damascus rose) (238262 mol TE/g DW)
(Dudonn 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;
368.45.0 and 369.119.4 mol 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)
(2409 mol Fe+2/g DW) and Cananga odorata (cananga tree) (3702 mol Fe+2/g DW)
(Dudonn 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 27.90.4 mg GAE/g DW (M. neglecta) to 80.41.8 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 (84.98.03 mg GAE/g DW). V. cheiranthifolium had a similar level of
total phenolics to that of black tea Cameron Highlands trade (60.65.4 mg GAE/g DW); P.
armeniaca and S. limbata had higher or comparable phenolics levels to that of oregano
(58.63.7 mg GAE/g DW), while A. elichrysifolium and M. neglecta had similar phenolics
levels to rosemary (28.10.8 mg GAE/g DW) (Dalar and Konczak, 2013).
In another study ahin 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 (oruh 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.

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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

425.36.7 898.914.9 mol Trolox


eq./gDW

Phenolics

Dalar et al., 2012

920.83.0 1625.015.5 mol Trolox


eq./gDW

Phenolics

Dalar et al., 2012

823.910.6 1307.717.4 mol Trolox


eq./gDW

Phenolics

Dalar, unpublished
result

406.63.1 1489.017.0 mol Trolox


eq./gDW
326.97.2 674.713.7 mol Trolox
eq./gDW
905.05.3 22628.5 mol Trolox
eq./gDW
1638.4218.3 mol Trolox eq./gDW

Phenolics

Dalar and Konczak,


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

Phenolics
Phenolics
Phenolics

3343.9215.2 mol Trolox eq./gDW


3602.952.1 mol Trolox eq./gDW
2978.974.6 mol Trolox eq./gDW
2239.5241.5 mol Trolox eq./gDW
4265.9132.9 mol Trolox eq./gDW
39.21.2 190.36.7 mol Fe2+/gDW

Phenolics

Dalar et al., 2012

130.411.5 369.119.4 mol Fe2+/gDW

Phenolics

Dalar et al., 2012

90.19.5 251.69.7 mol Fe2+/gDW

Phenolics

Dalar, unpublished
result

Complimentary Contributor Copy

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
55.25.9 250.32.2 mol Fe2+/gDW

Compounds identified
Phenolics

Aqueous methanol

42.11.6 67.71.4 mol Fe2+/gDW

Phenolics

Aqueous methanol

223.83.6 368.45.0 mol Fe2+/gDW

Phenolics

Lyophilized ethanol

390.813.5 mol Fe2+/gDW

Phenolics

1130.848.2 mol Fe2+/gDW


930.418.8 mol Fe2+/gDW
853.08.9 mol Fe2+/gDW
402.87.9 mol Fe2+/gDW
1123.552.4 mol Fe2+/gDW
435.418.5 909.137.5 mol 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

305.913.7 mol Trolox eq./gDW

Aqueous methanol

3.40.3 17.40.3 mg Gallic acid


Eq./gDW

Phenolics

Dalar et al., 2012

Aqueous methanol

12.21.7 35.32.8 mg Gallic acid


Eq./gDW

Phenolics

Dalar et al., 2012

Aqueous methanol

9.30.7 22.61.0 mg Gallic acid


Eq./gDW

Phenolics

Dalar, unpublished
result

Aqueous methanol

3.60.3 25.80.2 mg Gallic acid


Eq./gDW
4.40.1 7.40.1 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

813.639.9 mol 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

Complimentary Contributor Copy

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
20.20.6 33.10.4 mg Gallic acid
Eq./gDW
27.90.4 mg Gallic acid Eq./gDW
80.41.8 mg Gallic acid Eq./gDW
56.00.9 mg Gallic acid Eq./gDW
60.23.5 mg Gallic acid Eq./gDW
31.20.2 mg Gallic acid Eq./gDW
67.03.3 mg Gallic acid Eq./gDW
44.41.7 91.94.4 mg Gallic acid
Eq./gDW

Erygium bornmuelleri leaf

Ethanol
Acetone
Pure water
Lyophilized ethanol

Centaurea karduchorum leaf

Lyophilized ethanol

26.91.1 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: 64.44.8 mg Gallic acid


Eq./gDW
Seed: 105.18.7 mg Gallic acid Eq./gDW
65.16.4 mg Gallic acid Eq./gDW
34.07.0 mg Gallic acid Eq./gDW
59.62.8 mg Gallic acid Eq./gDW
22.681.10 mg Gallic acid Eq./gDW

Rheum ribes stem

Lyophilized methanol

35.711.23 mg Gallic acid Eq./gDW

Rheum ribes root

Lyophilized chloroform

48.661.23 mg Gallic acid Eq./gDW

Rheum ribes root

Lyophilized methanol

25.911.09 mg Gallic acid Eq./gDW

Mentha longifolia L. subsp. longifolia

Lyophilized methanol

45 mg Gallic acid Eq./gDW

Origanum vulgare subsp. vulgare

Lyophilized methanol

220 mg Gallic acid Eq./gDW

63.51.4 mg Gallic acid Eq./gDW

Compounds identified
Phenolic compounds
Phenolic compounds

References
Dalar and Konczak,
2012
Dalar and Konczak,
2013

Rutin, flavonoid glucosides,


phenolic acids

Dalar et al., 2013

Flavonoid glucosides,
chlorogenic acid
Flavonod glucosides

Dalar, unpublished
results
Dalar, unpublished
results
oruh 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

Complimentary Contributor Copy

oruh et al., 2007b


oruh et al., 2007b
oruh et al., 2007b
ztrk et al., 2007
ztrk et al., 2007
ztrk et al., 2007
ztrk et al., 2007
Gulluce et al., 2007
ahin 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
87.070.54 % at 100 g concentration
Rheum ribes root
Lyophilized methanol
High radical scavenging activities
60.600.86 % at 100 g concentration
Antioxidant test
Extraction
Results
Rheum ribes root
Lyophilized methanol
High radical scavenging activities
50.870.30 % at 100 g concentration
Mentha longifolia L. subsp. longifolia
Lyophilized methanol
High radical scavenging activities
IC50: 57.4 g/ml
Origanum vulgare subsp. vulgare
Lyophilized methanol
High radical scavenging activities
IC50: 9.9 g/ml

Compounds identified

References

Phenolic compounds

oruh et al., 2007

Phenolic compounds

oruh et al., 2007

Phenolic compounds

oruh et al., 2007

Phenolic compounds

oruh 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

ztrk et al., 2007


ztrk et al., 2007
References
ztrk et al., 2007
Gulluce et al., 2007
ahin 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

Compounds identified

References

Inhibition (%) at concentration 4 mg/ml


23.60.5
32.30.4
28.31.0
13.50.4
22.40.9
67.81.1

Phenolic compounds

Dalar and Konczak,


2013

Complimentary Contributor Copy

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*: 8.30.1 mg/ml
IC50: 9.60.2 mg/ml
IC50:19.40.4 mg/ml

Compounds identified
Rutin, flavonoid glucosides,
phenolic acids

References
Dalar et al., 2013

Phenolic compounds

Dalar and Konczak,


2013

IC50: 13.830.17
IC50: 1.430.04
IC50: 3.280.23
IC50: 2.540.02
IC50: 12.530.28
IC50: 2.030.10
IC50: 8.50.1 mg/ml
IC50: 10.40.3 mg/ml
IC50:19.10.6 mg/ml
IC50: 10.020.05

Rutin, flavonoid glucosides,


phenolic acids

Dalar et al., 2013

Phenolic compounds

IC50: 1.680.17

Flavonoid glucosides

IC50: 6.040.05

Chicoric acid, caftaric acid,


chlorogenic acid, flavonoid
glucosides

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

Phenolic compounds

Dalar and Konczak,


2013

Rutin, flavonoid glucosides,


phenolic acids

Dalar et al., 2013

Phenolic compounds

Dalar, unpublished
data
Dalar, unpublished
data

IC50: 10.210.08
IC50: 4.530.08
IC50: 6.720.09
IC50: 8.110.04
IC50: 8.560.10
IC50: 4.760.07
IC50: 5.90.1 mg/ml
IC50: 5.00.1 mg/ml
IC50: 6.50.2 mg/ml
IC50: 12.960.38
IC50: 6.210.06

Flavonoid glucosides

Complimentary Contributor Copy

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: 3.970.20

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

References
Dalar, unpublished
data

Inhibition (%) at concentration 0.6 mg/ml

Phenolic compounds

Dalar and Konczak,


2013

34.02.0
21.02.0
35.02.0
27.02.0
21.02.0
23.04.0

*IC50 half maximum inhibitory concentration.

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Health Attributes, Antioxidant Properties and Phytochemical Composition

219

The same extracts commonly consumed in the Van province as herbal teas successfully
suppressed the activities of key enzymes involved in metabolic syndrome: -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 -amylase (Table 4).
Natural -amylase and -glucosidase inhibitors from plant sources offer an attractive strategy
to control postprandial hyperglycemia. These inhibitors in particular, which have a lower
activity against -amylase and a stronger activity against -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 amylase and pronounced inhibitory activities against -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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220

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

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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

Chapter 8

Hetherotheca inuloides
(Mexican Arnica) a Potent Antioxidant
Effect as Neuro and Hepato-Protective
Liliana Carmona-Aparicio, Noem Crdenas-Rodrguez,
Bernardino Huerta-Gertrudis, Jos Luis Rodrguez-Chvez
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:elcoballase@yahoo.com.mx.

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L. Carmona-Aparicio, N. Crdenas-Rodrguez, 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 (Szab 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;
Crdenas-Rodrguez, 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 (Mats and Snchez-Jimnez 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; Martnez, 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 (Mats and Snchez-Jimnez, 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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L. Carmona-Aparicio, N. Crdenas-Rodrguez, B. Huerta-Gertrudis et al.

These plants are included in the genus Mentzelia, Thitonia, Trixis, Verbesina, Helenium, and
Heterotheca (Villaseor Ros and Espinosa Garca, 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
tllyetl (Martnez, 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, Michoacn (Zitacuaro,
Tacuaro, Coalcoman Vazquez), Hidalgo (Gangueo), Morelia (Ocampo), Oaxaca, Puebla,
Nuevo Len, Zacatecas, San Luis Potos, Veracruz, Colima, Tlaxcala, Puebla, Distrito
Federal, Guanajuato, Morelos, Quertaro, Nayarit and Estado de Mxico (Argueta et al.,
1994; Villaseor Ros and Espinosa Garca, 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 (Villaseor Ros and Espinosa
Garca, 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 (Villaseor Ros and Espinosa Garca, 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 H-3,4-dihydro-cadalene,
1, hydroxy-1(4H)-isocadalen-4-one,
1-hydroxy-4h-1,2,3,4-tetrahydrocadalen15oic acid,
7-(3,3-dimethylallyloxy)coumarin,
Caryolan-1,9-diol,
Quercetin,
Stigmasterol,
-sitosterol.
Quercetin (0.19),
Quercetin 3-O-glucoside,
kaempferol,
kaempferol-3-O-glucoside,
kaempferol-osophoroside,
D-chiro-inositol,
Epinasterol,
3-O--D-epinasterol glycoside,
7-hydroxy-4 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 caryophyllene, -caryophyllene 4,7-oxide 5-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-diol, and quercetin dicadenol also possess anti-inflammatory effect with minimal side effects
(Gen 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. inuloidess 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 (Valento
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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L. Carmona-Aparicio, N. Crdenas-Rodrguez, B. Huerta-Gertrudis et al.

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-Hernndez for their technical assistance.

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Mol Aspects Med. 24(4-5), 281-291.

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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

Chapter 9

Meconopsis: Traditional Uses,


Chemistry and Pharmacology

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
*

Corresponding author: xdxu@implad.ac.cn.

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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 QinghaiTibet 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

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245

number of alkaloids from different species were isolated by Slavk and coworkers (Slavk,
1960, 1965; Slavk and Slavkov, 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 UHPLCQTOF 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)
(Slavk and Slavkov, 1976, 1977, 1996) (Figure 1).
O
O

B NCH3
1
H 10

O C

14

H3CO

R1

D
O

NCH3
H
O

H3CO

HO

H3CO

NH
H

H3CO

R2

NH
H
O

H3CO

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

NH
H

H3CO

Figure 1. Structures of rhoeadine alkaloids (16).

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Haifeng Wu, Xiaopo Zhang, Yan Zhou et al.

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 (Slavk and Slavkov, 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

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
N

O
N

OCH3
OCH3

CH3

OH OCH3
16

OCH3
OCH3

17

OCH3
OCH3
18

Figure 2. Structures of protoberberine alkaloids (718).

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; Slavk and Slavkov, 1976, 1996; Wang et al., 1991; Wang and Chen, 1995; Wang
et al, 1994, 1996; Wu et al., 2009) were obtained (Fig. 3).

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R1
NCH3

R2

R3

R4

R1

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


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

NCH3
H
O

R1
R2

OCH3

OCH3

+ R2

OCH3

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

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 (1928).

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; Slavk et al., 1965; Slavk and Slavkov, 1976, 1977,
1996) (Fig. 4).
H3CO
HO
HO
H3CO

H3CO
N+ CH3
H CH3

N
CH3
H

HO
HO

R1

H3CO
30

29

N
CH3
H

R2

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

Figure 4. Structures of aporphine alkaloids (2933).

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-

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Haifeng Wu, Xiaopo Zhang, Yan Zhou et al.

methylcryprochine (38) and 8, 9-dihydroprooxocryptochine (39), were obtained (Li, 2007;


Hemingway and Phillipson, 1975; Slavk, 1960, 1965; Wu et al., 2009) (Fig. 5).
R1

H3CO
NCH3

R2

H3CO
NCH3

H3CO
NH

H3CO

HO

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 (3439).

1.6. Protopines
The difference between protopines and proberberine is the cleavage of CN 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

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 (4042).

1.7. Benzophenanthridines
Benzophenanthridine could be derived from proberberine by the cleavage of C-6N bond
and formation of C-6C-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

NCH3

O
43

N
O

H3CO
H3CO

44

N CH3

O
O

N
O

45

Figure 7. Structures of benzophenanthridine alkaloids (4347).

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CH3

46 R = H
47 R = Acetonyl

Meconopsis: Traditional Uses, Chemistry and Pharmacology

249

1.8. Indoles
A indole alkaloid, 6-methoxy- 2-methyl-1,2,3,4-tetrahydro--carboline (48), was isolated
(Slavk, 1960, 1965). Most recently, a series of nudicaulins (4954) were isolated from M.
cambrica (Tatsis et al., 2013) (Fig. 8).
H3CO
N

N
H

HO
HO

OR2
HO
HO
OH
HO
HO

HO
HO

OH

OH

OR1

48

OR1

OH

O
HMG

O
O O

COOH
OH
Mal

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

OH

OR2

COOH

O
O

HO
HO
OH
HO
HO

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 (4854).

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

OH
HO
O
O

H
H

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).

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O
O

Haifeng Wu, Xiaopo Zhang, Yan Zhou et al.


OH
H CH3
N
O

56

HO
N

HO

O
O

H3CO

N
O

57

O
HO

OCH3

58

Figure 10. Structures of benzylphenethylamine alkaloid (5658).

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-(16)]-Dglucopyranoside
(67),
isorhamnetin
3-O-[-D-galactopyranosyl-(16)]--D
glucopyranoside (68), kaempferol 3-O-[-D-glucopyranosyl-(12)]--D- glucopyranoside]
(69), isorhamnetin 3-O-[-D-glucopyranosyl-(16)]--D-galactoside (70), kaempferol 3-O-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(16)--D-glucopyranoside] (81), quercetin 3-O-[2,6-O-diacetyl--D-glucopyranosyl(16)--D-glucopyranoside] (82), isorhamnetin 3-O-[2-O-acetyl--D-glucopyranosyl(16)--D-glucopyranoside] (83), quercetin3-O-[2-O-acetyl--L-glucopyranosyl -(16)-D-glucopyranoside] (84), cyanidin 3-O-(6-O-malonyl--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).

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R1

R5O

R2
O

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

HO

R4

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

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

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

CH2OH
OCH3

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+

OH

OH
OH
RO

O
O

HO
O

HO HO

OH
OH

85 R = malonyl
86 R = OH

Figure 11. Structures of flavonoids (5986).

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 GCMS (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).

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Haifeng Wu, Xiaopo Zhang, Yan Zhou et al.

4. Steroids
Three steroids, -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 (8789).

5. Triterpenoids
Four triterpenoids, 21-hydroxy ursolic acid (90), -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 (9093).

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--Dglucopyranoside (99), p-hydroxybenzoyl- -D-glucopyranoside (100), 4-O--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--D-glucopyranoside (107) are also

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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

CHO

100

HO

HO

Oglc
O

OH O

101

102

104

103

OH
HO

Oglc

Oglc
99

OH

H3CO
H3CO

COCH2OH

HO

HO

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

HO

NH

O
O

N
H
105

HOH2C
HO
HO

106

H3CO
O

OH
107

Figure 14. Miscellaneous compounds (94107).

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

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Haifeng Wu, Xiaopo Zhang, Yan Zhou et al.

(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 g/ml against TM4/8.2 strain (a wild type
chloroquine and antifolate sensitive strain) and 6.39 g/ml against K1CB1 (multidrug
resistant strain) (Wangchuk et al., 2011). In another study, crude extracts of M. simplicifolia
exhibited good inhibition of TNF- 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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255

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

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In: Medicinal Plants


Editor: David Alexandre Micael Pereira

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

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
Jamaicas 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 islands 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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261

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 g/mg), while the leaves contain between 66.63
629.04 g/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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Henry Lowe and Joseph Bryant

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 (-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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263

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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264

Henry Lowe and Joseph Bryant

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 islands 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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265

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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

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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

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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

269

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

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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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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 Len, 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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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

Complimentary Contributor Copy

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-, 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

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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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