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MEDICINAL PLANTS
ANTIOXIDANT PROPERTIES, TRADITIONAL
USES AND CONSERVATION STRATEGIES
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MEDICINAL PLANTS
ANTIOXIDANT PROPERTIES, TRADITIONAL
USES AND CONSERVATION STRATEGIES
New York
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
Chapter 4
117
Chapter 5
143
Chapter 6
165
Chapter 7
Chapter 8
91
183
227
vi
Contents
Chapter 9
Chapter 10
Index
243
259
267
Preface
Nowadays, natural products and in particular medicinal plants, play an important role in
human health and therapeutics. Across the world, several different cultures employ medicinal
plants for the treatment of a wide range of pathological conditions. In this book, the authors
address the antioxidant properties of several medicinal plants, as well as their traditional uses
and conservation strategies. This is, without a doubt, a wonderful opportunity to have a closer
insight into the chemistry, biological properties, conservation and traditional use of medicinal
plants used around the world.
Chapter 1 - Plants have been used for medicinal purposes since the origin of human
civilization and their uses were described by the great civilizations of the ancient Chinese,
Indian and Mediterranean. Nowadays, they continue to be the source of new medicines either
by providing lead molecules or as natural herbal products (teas, tinctures, powders, poultices,
infusions as well as other formulations).
Herbal medicinal products are defined as any medicinal product, exclusively containing
as active ingredients one or more herbal substances or one or more herbal preparations, or one
or more such herbal substances in combination with one or more such herbal preparations.
Compounds produced by plants are divided in two groups: primary and secondary
metabolites. Primary metabolites are compounds that possess fundamental roles in plant
development steps such as phytosterols, acyl lipids, nucleotides, amino acids and organic
acids. Secondary plant metabolites are structurally diverse and many are distributed among a
limited number of plant species. Some of these compounds were found to have a key role in
the protection of plants in several ways. Moreover, there are increasing evidences that modest
long-term intakes of some specific classes of these compounds can favorable reduce and/or
prevent the incidence of cancers and many chronic diseases such as cardiovascular disease,
neurodegenerative disease, type II diabetes and hypertension, as well as the ageing process.
Plant secondary metabolites can be grouped, based on their biosynthetic formation, into
four groups: phenolic compounds, terpenoids, alkaloids and sulphur-containing compounds.
Phenolic compounds are of great interest mainly due to their bioactive functions involved
in human health-related issues.
Oxidative stress and human health, namely in the pathogenesis of various diseases and
disorders are related in different ways. Under stress, the human body will produce more
harmful species, such as reactive oxygen species (ROS) than enzymatic antioxidants and nonenzymatic antioxidants, inducing cell damage. This effect is increased when there are not
enough antioxidants to quench these harmful radicals.
viii
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
serve to be effective against these diseases and their complications. In this perspective,
therapeutic intervention with the ability to reduce oxidative stress can impede or delay the
onset of the metabolic disorder. Thus, agents possessing dual effect such as antidiabetic/anti-obesity and antioxidant activity are greatly in demand. The therapeutic effect of
phytochemicals found in natural products to combat oxidative stress is gaining significance as
they are recognized to be safe with a wide range of biological and pharmacological
activities. Dietary components from plants such as polyphenols (flavonoids), terpenes and
tannins are ubiquitous in nature and can effectively scavenge reactive oxygen and nitrogen
species, thus, modulating the genes associated with metabolism and stress defense. This
chapter discusses the sources of flavonoids, their potential antioxidant properties and the
mechanism through which they exert their pharmacological effects in diabetes and obesity.
Chapter 5 - The prevalence of overweight and obese individuals is increasing at an
alarming rate across the globe. Obesity has become one of the most important avoidable risk
factors for morbidity and mortality. The associated risks with obesity are cancer, diabetes and
heart diseases. According to the World Health Organization, obesity is defined as abnormal or
excessive fat accumulation that may impair health.
In 2008, more than 1.4 billion adults were overweight and more than half a billion were
obese. At least 2.8 million people die each year as a result of being overweight or obese. A
person is considered obese if they possess a body mass index (BMI; a ratio of height to
weight) greater than 30 whereas a healthy BMI should be 18.5 to 24.9. Obesity is the leading
cause of death which can be prevented by diet and lifestyle modifications. Although the exact
link between obesity and its associated risks is not clear, it is known that increased production
of reactive oxygen species (ROS) is associated with cellular damage, including oxidation of
cell membranes and proteins in conjunction with disturbances of cellular redox homeostasis.
Free radicals are known to be involved in a number of human pathologies including
atherosclerosis, cancer and hypertension. Studies have shown that obesity promotes increased
plasma lipid peroxidation. Obesity also increases the mechanical and metabolic loads on the
myocardium, thus increasing myocardial oxygen consumption. Therefore, antioxidants are
capable of reversing these pathways and, in fact, can be helpful in preventing the deleterious
effects caused by reactive oxygen species. However, antioxidants do not reduce obesity per
se. Antioxidants are widely present in the plant kingdom and are known to prevent various
disorders. Flavonoids, especially flavones, flavonols, flavanones, flavanols (catechins),
anthocyanins, isoflavones and chalcones, are considered effective antioxidants associated
with other pharmacological properties such as anti-cancer, anti-diabetic, anti-mutagenic, antithrombotic, anti-inflammatory and anti-HIV activities. Many studies have indicated that
phenolic compounds such as o-coumaric acid, EGCG, esculetin, genistein, procyanidin,
pycnogenol, rutin, and tea catechins, carnitine, CoQ10, choline, inositol and various herbs are
effective in reducing obesity and promoting weight loss. This review will focus on recent
examples of antioxidant nutrients, traditional medicines and foods that have been validated by
scientific evaluation for controlling obesity or promoting weight loss.
Chapter 6 - The use of medicinal plants represents the oldest and most common form of
medication. Among the hundreds of studies published in the last two decades on medicinal
plants research, the quest for new antioxidant drugs has a been pivotal. Some of those plants
with antioxidant activity, as well as their bioactive components, have been in some cases
further analyzed for a hypothetical anti-hemolytic potential. Although oxidative stress is not
the primary etiology of diseases such as hemolytic anemias, it is believed to aggravate them.
Preface
xi
xii
These plants are often administered in many different ways, including decoctions,
macerations, infusions, tinctures or by cooking. Due to their usefulness as medicinal plants
tissue culture has been used as a part of the conservation strategies that have been employed
in preserving and maintaining the islands flora.
ISBN: 978-1-62948-219-4
2014 Nova Science Publishers, Inc.
Chapter 1
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.
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).
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.
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.
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.
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).
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.
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
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
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).
10
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).
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).
11
12
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).
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).
13
14
15
16
(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).
17
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.
18
(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.
19
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.
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,
20
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).
21
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).
22
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).
23
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.
24
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).
25
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.
26
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.
27
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
28
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
29
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).
30
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.
31
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).
32
33
34
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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ISBN: 978-1-62948-219-4
2014 Nova Science Publishers, Inc.
Chapter 2
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
42
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
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.
44
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.
45
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
46
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.
47
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
48
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
Cranberry
Vaccinium
macrocarpon
Fruits
Berries
Rubus coreanus
Rubus idaeus
Rubus fruticosus
Rubus
leucodermis
Grapes
Vitis vinifera
Fruits
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).
Orange
Citrus sinensis
Papaya
Carica papaya
Fruit pulp,
leaves and seeds
Pomegranate
Punica granatum
Fruits
Tangerine
Citrus tangerina
Citrus reticulata
Citrus deliciosa
Broccoli
Brassica oleracea
Cauliflower
Brassica oleracea
Cactus
Opuntia ficusindica
Carrot
Daucus carota
Root
Pepper
Capsicum annuum
Fruit
Spinach
Spinacia oleracea
Leaves
Tomate
Lycopersicum
esculentum
Watercress
Nasturtium
officinale
Fruit
Leaves
49
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).
50
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
51
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).
52
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).
53
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.
54
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.
55
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).
56
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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58
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).
59
60
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).
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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).
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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.
63
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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66
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).
67
68
69
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.
70
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.
71
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).
72
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.
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.
74
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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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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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).
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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.
98
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.
99
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).
100
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.
101
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
102
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
103
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).
104
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).
105
106
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
Salinitya
(NaCl addition)
ns
ns
Low N levelb
(N as NO3-)
ns
+
NH4+ additionb
(constant total N)
_
_
Hypoxiaa
ns
ns
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.
107
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.
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ISBN: 978-1-62948-219-4
2014 Nova Science Publishers, Inc.
Chapter 4
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].
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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.
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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].
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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.
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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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
Citrus foods
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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].
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.
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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].
129
130
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
131
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].
132
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.
133
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].
134
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].
135
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
136
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.
137
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Chapter 5
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,
*
144
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
145
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).
146
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).
147
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).
148
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
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
149
150
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
151
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).
152
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).
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
153
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.
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
154
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
155
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,
156
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
157
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).
158
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).
159
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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ISBN: 978-1-62948-219-4
2014 Nova Science Publishers, Inc.
Chapter 6
Abstract
The use of medicinal plants represents the oldest and most common form of
medication. Among the hundreds of studies published in the last two decades on
medicinal plants research, the quest for new antioxidant drugs has a been pivotal. Some
of those plants with antioxidant activity, as well as their bioactive components, have been
in some cases further analyzed for a hypothetical anti-hemolytic potential. Although
oxidative stress is not the primary etiology of diseases such as hemolytic anemias, it is
believed to aggravate them. Therefore, the use of natural antioxidants, either as additives
or as pharmaceutical supplements, may prevent or at least slow down free radical
reactions that are responsible for provoking damage to essential red blood cell molecules.
In this Chapter, we review the current knowledge regarding the use of medicinal plants as
anti-hemolytic agents. Particular emphasis in the compounds responsible for this activity,
as well as the mechanism of action is given.
Introduction
Since ancient times, humans have been looking for drugs in nature in order to prevent or
heal a wide number of diseases. In earlier stages, medicinal plants were used in an intuitive
and empirical way. Over time, the fundaments for the precise use of certain plants in the
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treatment of specific diseases were being discovered and gradually the empiric use of those
plant species was abandoned and started to be supported by evidences (Halberstein, 2005). In
the last decades, the scientific community has been trying to identify the specific molecules in
herbs that account for their health promotion benefits. This search has been focusing
primarily on plant metabolites (most of them are powerful antioxidants), phytochemicals,
vitamins and fiber content.
Countless studies have been performed and published upon the role of specific plant
metabolites in disease prevention or treatment, mainly in diseases originated by reactive
oxygen species (ROS). Coronary heart diseases, ulcers, cancers and neurodegenerative
diseases (e.g. 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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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
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.
Botanical name
Part used
State
RBC
Activity*
Concentration
Ref
Agrimony
Agrimonia eupatoria L
Leaf
Aqueous extract
Human
+++
0.005%
Aleppo oak
ND
Human
++
<0.25%
(Sangkitikomol, 2012)
Avocado
Persea Americana L
Leaf
Aqueous extract
Human
++
0.1%
Silk bananas
Musa sapientum
Leaf
Methanol extract
Human
++
0.1%
Banyan tree
Ficus bengalensis L.
Aerial root
Acetone extract
Cow
++
0.05%
Methanol extract
Cow
++
0.05%
Aqueous extract
Quails
++
0.175%
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
Human
++
<0.25%
(Sangkitikomol, 2012)
Chamberbitter
Phyllanthus urinaria L
Leaf
Human
++
<0.25%
(Sangkitikomol, 2012)
Human
++
<0.25%
(Sangkitikomol, 2012)
++
0.02%
(Rajeshwari et al.,
2012)
Clove
Buds
Human
Coriander
Coriandrum sativum
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%
++
<0.25%
(Sangkitikomol, 2012)
++
0.0384%
++
0.01%
(Sabuncuolu &
hretolu, 2012)
++
0,0025%
Eucalyptus
Eucalyptus globules L
Leaf
Eugenia
Leaf
Human
Gamak
Aerial parts
Ethanol extract
Rat
Genarium
Geranium tuberosum L
Flower
Methanol extract
Ginkgo
Ginkgo biloba L
Leaf
Aqueous extract
Human
Human
*Activity classification: (+) - < 40% hemolysis inhibition; (++) 40% < hemolysis inhibition < 80%; (+++) < 80% hemolysis inhibition.
Common name
Botanical name
Part used
State
Hexane Extract
Goat weed
Scoparia dulcis L
Plant
Chloroform extract
Methanol extract
RBC
Rat
Rat
Rat
Human
Golden root
Rhodiola rsea L.
Root
Aqueous extract
Green tea
Camellia sinensis L.
Leaf
Aqueous extract
Heath
Calluna vulgaris L
Hyssopus angustifolius
Leaf
Flower
Stem
Leaf
Ficus racemosa L
Stem bark
Indian lotus
Flower
Aqueous extract
Methanol extract
Methanol extract
Methanol extract
Acetone extract
Methanol extract
Aqueous/methanol extract (80:20)
Peppermint
Mentha piperita L
Aerial parts
Ethanol extract
Mexican marigold
Tagetes erecta L
Leaf
Seed
Aqueous extract
Seed
Aqueous extract
Hyssop
Milk thistle
Silybum marinum L
Myrtle
Neem
Myrtus communis L
Azadirachta indica A Juss
Leaf
Leaf
Aqueous extract
Aqueous/methanol extract (80:20)
Nilgiri barberry
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.
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
Leaf
Rose
Sage
Savory
Screwpine
Sweet-amber
Thyme
Walnut-tree
Yarrow
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.
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)
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
176
possess an excellent anti-hemolytic capacity. Costa et al. (2009) also reported the antioxidant
capacity of green tea extracts upon AAPH radicals, by inhibiting hemolysis in more than 50%
at a concentration of 25 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.
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
178
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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ISBN: 978-1-62948-219-4
2014 Nova Science Publishers, Inc.
Chapter 7
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.
184
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
185
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.
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
186
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.
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,
187
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.
188
189
Local names
Plant part
Application
References
Horoz ibii
LF
Sterility
Infusion
Kizvan, nervend
LX, FR
(immature)
GM
FR
Chewing
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
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
Kerme
Rizyane
Rizyane
RS
SD
AE
wound healing
Abdominal pain in children
Stomach ache
Hellis
Soy
LF
AE
Wound healing
Diabetes
Serzer
AE
Wound healing
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
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
Application
Fresh ( on wounds)
References
Kaval, 2011
Karibel
RT
Diabetes
Decoction (int.)
Guhktk
RT,
AE
Wound healing,
erythematic,
Poultice (on
wounds), ointment
(poultice mixed with
honey)
Kaval, 2011
BD,
FR, LF
BD
Pounded (ext.),
crushed (ext.)
Pounded (ext.)
FL, LF,
AE
FL,
LF,
WP
ASCLEPIDACEAE
Vinetoxicum canescens (Wild.) Decne.
subsp. canescens
Vinetoxicum tmoleum Boiss.
ASTERACEAE
Achillea biebersteinii Afan.
Ar iei, bovijan
Civan peremi,
herezan
Civan peremi,
bovijan
Papatya
Papatya
Sar papatya
Sar papatya
Nuserk, top telli,
kriz, lolek
WP,
AE
AE
AE
FL
FL
LF,
FL,
RT
Lolek
LF
Sunstroke
Top telli,
LF,
RT
AE
Swelling of stomach,
abscesses,
Dyspnoea and diabetes
Zilasur
Bevjana kuvi
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
Kelembek
ermnik
aladir
LF
LF
WP
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
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.)
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
Sevka a
Guyazerk
BB
AE
Diabetes
Kidney stones, cholesterol, diabetes
Included in meals,
coffee substitute, ,
decoction (int.),
direct application
(ext.)
Eaten fresh
Decoction (int.)
Gllga zer,
herdemtaze
ngz, peniruk
AE, SH, FL
Kidney stones
AE
Haemorrhoids
Andz
RT
Chest pain
Decoction, infusion
(int.)
Ointment ( mixed
with Vaseline/cream)
Infusion (int.)
Family/Species name
Lactuca saligna L.
Lactuca serriola L.
Senecio vernalis
Scorzonera latifolia
(Fisch. & C.A. Mey.) DC.
Local names
Tehlika geva
Kaju
Gurutik, kanok,
nermend
Plant part
BD
AE
BR+FL, AE
LX, RT,
WP
Scorzonera tomentosa L.
Arvent
LX
Wound healing
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
Fresh on skin
Kaval, 2011
Jaundince in animals
Orexigenic
Epilepsy
Eaten fresh,
decoction (int.)
Int. (by licking)
Eaten fresh
Decoction (int.)
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)
Havacva otu,
hovacov
Mjmejok
RT
Wound healing
FL
Echium italicum L.
RT
Wound healing
Bostan otu
BR+FL
Cancer
Kaval, 2011
Table 1. (Continued)
Family/Species name
Nonea pulla (L.) DC.
Local names
Gzrik, mjmj
Plant part
LF,
RT
Application
Eaten fresh, poultice
(ext.)
References
Kaval, 2011; Altundag and
Ozturk, 2011
Keselmehmut, ulik
Decoction, powdered
(ext.)
Ointment (with
butter,), pounded
(ext.)
Moistened, with
sugar (ext.)
Decoction (int.)
Infusion (int.)
Powder (ext.),
infusion (int.)
BRASSICACEAE
Alyssum pateri Nyr.
subsp. pateri
Asymnea rigidum Grosh.
Nujdan
AE,
WP
LX
Brassica oleracea L.
Lahana
LF
Abscesses
Munzur otu
Nujdar
AE
AE
LF
Diabetes
Kidney stones
Wound healing, vulnerary
Mayasl otu
LF
Haemorrhoids
Boiled (ext.)
RT
Sterility
Boiled (int.)
Stomach disorders,
constipation,hypertension
Decoction, fresh,
powder (int.)
Dikenli ard
FR
Rheumatism
Topalak
RT
Diuretic
Infusion (int.)
Puk, buki
LX
Antiseptic
Gevrik
LX
Directly on skin
(ext.)
As above
LX
Constipation
Eaten fresh
CARYOPHYLACEAE
Telephium imperati L. subsp. orientale
(Boiss) Nyman
CRASSULACEA
Umblicus erectus DC
CUCURBITACEAE
Bryonia multiflora
Boiss. & Heldr.
CUPRESSACEAE
Juniperus oxycedrus L.
CYPERACEAE
Cyperus rotundus
DIPSACACEAE
Scabiosa sulfurea Boiss. et Huet
Cephalaria sparsipilosa
Matthews
EUPHORBIACEAE
Euphorbia sp.
Tabata et al.,1994
Tuzlac and Doan, 2010
Tuzlac and Doan, 2010
Tabata et al., 1994; zgke
and zelik, 2004; Altundag
and Ozturk, 2011
Family/Species name
Euphorbia denticulata Lam.
Local names
Hekletis
Plant part
LX
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
Fresh (ext.)
Kaval, 2011
LX
ilomali
LX
Constipation
Directly on skin
(ext.)
Eaten fresh (with
sugar)
Kaval, 2011
Urinary problems,
diuretic
Hypertension, kidney pain
Decoction, Infusion
(int.)
Decoction
RT
Decoction (int.)
Hespst
Sebelk nefel
WH
AE
Astringent
Stomach disorders
Poultice (ext.)
Decoction
Pltan
SH
Hypoglycaemic
Pounded, decoction
(int.)
Tolk
RT
Decoction
Kaval, 2011
Ahu
LF
Parasites
Infusion (int.)
Binbir delikotu,
kantaron
WP,
FL
Decoction (int.),
infusion (int.)
Pizak
RT
Kidney stones
Decoction
Kaval, 2011
Table 1. (Continued)
Family/Species name
JUNGLADACEAE
Juglans regia L.
Local names
Plant part
Application
References
Gz, ceviz
SD,
LF,
FR,
BA, CO
LABIATEA
Mentha longifolia (L.)
Hud. subsp. longifolia
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.
Decoction (int.)
Origanum acutidens
(Hand-mazz) letswaart
Mentha spicata L.
Zemul
WP,
FL
BR+FL
Decoction (int.)
WP, AE, LF
Mercankk
Catr, ku zemulu
Cantir, cantirik
ST
WP, LF,
BR+FL
WP
Decoction [(int./ext.
(bath)]
Eaten fresh
Decoction (int.),
infusion (int.)
Decoction (int.)
WP
Wound healing
Prunella vulgaris L.
Phlomis armeniaca Wild.
Sosn Belgsesing
alba, alba
Abdominal pain
Antipyretic, colds, asthma, bronchitis
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.)
Origanum majorana L.
Origanum vulgare L. subsp. gracile (C.
Koch) letswaart
Origanum vulgare L.
subsp. viride
Origanum vulgare L. subsp. vulgare
Family/Species name
Salvia nemorosa L.
Local names
Gemda
Plant part
WP
Application
Pounded (ext.)
Salvia sclarea L.
Da ay
BR+FL, LF
Salvia verticillata L.
Hart, ada ay
LF
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
Gurnik
FR
Cardiac diseases
Sirmuk
Eaten fresh
Da sarmsa
RT,
LF
BB
Crushed (int.)
Corin
Yara otu
LF
RT
Anaemia
Wound healing
Gulik
RT, LF (juice)
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.
Decoction (int.),
eaten fresh,
pounded (ext.)
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
Table 1. (Continued)
Family/Species name
MALVACEAE
Alcea apterocarpa (Fenzl) Boiss.
Local names
Plant part
Application
Huri
Kidney stone
Govik
RT,
SH,
WP
RT,
WP
LF
Infusion (int.),
decoction (ext.)
Poultice (ext.)
Amervans
Hatmi
FL, LF
RT
Expectorant, cold
Kidney stone, abscesses
Alcea flavovirens
(Boiss. & Buhse.) Iljin
Alcea kurdica
RT, WP, LF
Infusion (int.)
Decoction (int.),
residue (ext.)
Decoction (int.)
Hero
Decoction (int.)
Hatmi
Hero
RT,
LF
LF
LF,
RT
WP
LF,
AE,
WP
Infusion (int.)
Decoction (int.)
Infusion (int.)
Poultice (ext.),
decoction [(int.-ext.
(bath)], infusion (int.)
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
Gulor, glhor
AE
Diabetes
Infusion (int.),
decoction (int.)
Sinir otu
LF
Wound healing
Fresh (ext.)
Family/Species name
Plantago lanceolata L.
Local names
Plant part
Giyamambel,
LF
giyabironug, sinirotu,
belhevzar, mambel,
ilan dili, zimanugmari
Belhevzar, belgbirim, LF
belghavz, belghevizar,
boa yapra, amin
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
Infusion (int.)
RT,
SD,
SH,
LF, ST
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.)
Troka kera
LF
Poultice (ext.)
Kaval, 2011
Prpar, pimpirim,
semiz otu,
parpar, pirpirim
AE
Decoction (int.),
crushed (ext.),
infusion (int.)
Cngk
rekotu
LF
SD
Rheumatism
Diabetes, ulcer
Poultice (ext.)
Decoction (int.)
Kaval, 2011
zgke and zelik, 2004
Muhabbet iei
RT
Stomach pains
Eaten fresh
Gyabirin
Helhelok
LF
FR
Astringent
Prostate
Poultice (ext.)
Eaten fresh
Kaval, 2011
Kaval, 2011
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
Application
Eaten fresh,
decoction, poultice
(ext.), fresh on skin
(ext.)
Poultice (fresh),
powder (dry), fresh
on skin (ext.)
Table 1. (Continued)
Family/Species name
Cerasus mahalep L.
Crataegus monogyna Jacq.
Pyrus syriaca Boiss. var. syriaca
Rosa canina L.
Local names
Mahlep
Al
ekok
ilank,
ilan,
kuburnu,
domuzturpu
ilank
Plant part
FR
FR
FR
FR,
RT,
LF
Application
Infusion (int.)
Eaten fresh
Infusion (int.)
Decoction (int.),
stewed (int.), infusion
(int.)
FR
Decoction
Ddrk,
drne,
mmirk, ttrk
SD,
FR
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
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)
Masicerk
BR+FL
Masicark
AE
Rheumatism
Sprge out
LF
Inflammed wounds
RUBIACEAE
Galium consanguineum Boiss.
Galium verum L. subsp. glabrescens
Ehrend
SCROPHULARIACEAE
Verbascum cheiranthifolium
Boiss. var. cheiranthifolium
Verbascum speciosum Schrad.
Scrophularia libanotica
Boiss, var. urartuensis R. Mill.
References
Cakilcioglu et al., 2011
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
Family/Species name
SOLONACEAE
Hyoscyamus niger L.
Local names
Plant part
Application
References
Hrdal, hargel,
harundol, ban out,
batbat
Dadoan
SD
Inhaled, directly on
tooth (ext.)
RT+SD
Intoxication
Eaten fresh
Ttn
LF
Wound healing
Powder (ext.)
Tevri
BR,
BK
Fresh, decoction
powder (ext.)
Kaval, 2011
Derdoan
FR
Diarrhoea
Eaten fresh
Geznk, geznek,
yn, dezink,
geznk, srgan otu,
gezik, jrtken,
inar, drk
SD,
AE,
WP,
LF
Urtica urens L.
LF,
WP
VIOLACEAE
Viola odorata L.
Bnevok
WH
VALERIANACEAE
Valeriana officinalis L.
Valeriana alliarifolia Adams
ZYGOPHYLLACEAE
Tribulus terrestris L.
Kediotu
AE
RT
Kidney stone
Sedative, antispasmodic
Decoction (int.)
Tea (int.)
Decoction (int.)
Peganum harmala
zerlik
AE,
FR
RT,
SD
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
AE: aerial part; BB: bulb; BA: bark; BR: branch; BD: bud; BK: bark; CO: cortex; GM: gum; FL: Flower; FR: fruit; LF: leaf; LX: latex; RT: root; RS: root sap;
SD: seed; SH: shoot, SL: stylus; ST: stem; WH: whole plant. Ext: external, Int: internal.
Table 2. Selected ailments and their traditional remedies used in Eastern Anatolia
Health condition
Wounds, injuries
Stomach disorders
Abdominal pain
Carminative /flatulence
Constipation
Haemorrhoids
Diabetes
Rheumatism
Diuretic
Kidney pain
Kidney stones
Gallbladder disorders
Urinary inflammation
Cardiac disorders
Eye problems
Common cold
Traditional remedy
Nicotiana tabacum, Scrophularia libanotica, Rumex tuberosus, Portulaca oleracea, Plantago major, Plantago atrata, Plantago lanceolata, Malva
neglecta, Asphodeline tenuior, Salvia nemorosa, Origanum vulgare, Juglans regia, Euphorbia macrocarpa, Lepidium latifolum, Asymnea rigidum,
Alyssum pateri, Echium italicum, Alkanna orientalis, Alkanna megacarpa, Taraxacum montanum, Tanacetum balsamita, Scorzonera tomentosa,
Cichorium intybus, Centaurea karduchorum, Aristolochia bottae, Pistacia atlantica, Ferula orientalis, Ferula haussknechtii, Heracleum antasiaticum,
Johrenia dichotoma, Prangos pabularia
Pistacia khinjuk, Eryngium billardieri, Ferula orientalis, Grammosciadium platycarpum, Achillea biebersteinii, Achillea millefolium, Achillea
vermicularis, Anthemis nobilis, Arctium minus (Hill.) Bernh. subsp. pubens, Arctium tomentosum, Artemisia spicigera, Centaurea virgata, Anchusa
azurea Mill. var. azurea, Alyssum pateri 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
Health condition
Malaria
Hypocholesterolemia
Hypertension
Burns
Antiseptic
Toothache
Tonsilitis
Parasites
Venome poisoning
Traditional remedy
Centaurea solstitialis L. subsp. solstitialis
Anethum graveolens, Helichrysum armenium D.C. subsp. armenium, Cichorium intybus
Equisetum fluviatile
Rhus coriaria, Alkanna megacarpa, Asymnea rigidum, Galium verum
Rhus coriaria L., Scabiosa sulfurea, Cephalaria sparsipilosa
Daphne mucronata, Hyoscyamus niger, Salix alba, Salix aegyptiaca
Rubus caesius
Pelargonium quercetorum,Globularia trichosantha, Allium macrochaetum
Centaurea iberica, Nonea pulla
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Table 2 presents the list of selected ailments and medicinal plants used in their treatment.
This table shows that frequently multiple herbs have been identified to treat the same health
disorder. For example, 32 herbs (17.6% of the total number) have been identified for the
treatment of wounds and 33 herbs (18.1%) for the treatment of stomach disorders. The
percentages of herbs used to treat these two common health conditions listed in this chapter
are higher than those cited by Kaval: 10.1% for wound healing and 12.8% for stomach
disorders (Kaval, 2011).
Diabetes and cancer are two prevailing health conditions of the contemporary society. In
Eastern Anatolia a high number of plant sources have also been identified for their treatment.
Twenty five herbs (13.7%) are in use to alleviate the symptoms of diabetes. The percentage of
herbs to treat diabetes presented in this listing is double that reported earlier (7.3%) by Kaval
(2011).
A relatively high number of plants (12 plants; 6.6%) are applied in cancer treatment.
Eryngium bornmuelleri is used specifically to cure stomach cancer, while Plantago
lanceolata and Plantago maritime are used to treat the cancer of uterus. Galium verum L.
subsp. glabrescens Ehrend is used for the treatment of all types of cancers. These plants
represent extremely valuable resources for future research aimed at identification of active
constituents and possibly - development of new anti-diabetic or anti-cancer agents.
Table 2 presents also 15 medicinal plants (8.2%) utilized for the treatment of kidney
disorders, and additional 13 plants (7.1%) identified to cure specifically kidney stones. In the
last group six plants belong to the Malvaceae family. Alternative uses of a few members of
the same plant family to cure one ailment may indicate a presence of the same active
components.
Relatively high number of plants (14; 7.7%) are used for the treatments of rheumatism;
the percentage of plants utilized to cure this condition is in agreement with earlier report by
Kaval (2011).
The application of herbal remedies from Eastern Anatolia listed in this chapter addresses
common illnesses (e.g. abdominal pain or common cold) as well as specific conditions, such
as gall bladder stones, tonsillitis, and stomach cancer. Two common chronic conditions
related to the modern lifestyle are also treated; hypertension with Equisetum fluviatile, and
hypocholesterolemia with Anethum graveolens, Helichrysum armenium D.C. subsp.
Armenium, and Cichorium intybus. Natural medicines to treat both conditions are in high
demand. Traditional medicinal plants presented in this chapter may provide valuable leads for
identification of physiologically active natural compounds for pharmaceutical uses.
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206
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
207
208
209
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
210
[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.
211
212
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.
Results
Compounds identified
References
Phenolics
Phenolics
Phenolics
Dalar, unpublished
result
Phenolics
Phenolics
Phenolics
Phenolics
Phenolics
Phenolics
Phenolics
Dalar, unpublished
result
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
Phenolics
Aqueous methanol
Phenolics
Lyophilized ethanol
Phenolics
References
Dalar and Konczak,
2012
Dalar and Konczak,
2012
Dalar and Konczak,
2012
Dalar and Konczak,
2013
Ethanol
Acetone
Pure water
Lyophilized ethanol
Lyophilized ethanol
Aqueous methanol
Phenolics
Aqueous methanol
Phenolics
Aqueous methanol
Phenolics
Dalar, unpublished
result
Aqueous methanol
Phenolic compounds
Aqueous methanol
Flavonoid glucosides,
chlorogenic acid
Flavonod glucosides
Dalar, unpublished
results
Dalar, unpublished
results
Phenolic compounds
Antioxidant test
Verbascum cheiranthifolium
plant parts (stem, leaf, flower)
Malva neglecta tea
Extraction
Aqueous methanol
Lyophilized ethanol
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
Ethanol
Acetone
Pure water
Lyophilized ethanol
Lyophilized ethanol
Lyophilized methanol
Heracleum persicum
Prangos ferulacea
Chaerophyllum macropodum
Rheum ribes stem
Lyophilized methanol
Lyophilized methanol
Lyophilized methanol
Lyophilized chloroform
Lyophilized methanol
Lyophilized chloroform
Lyophilized methanol
Lyophilized methanol
Lyophilized methanol
Compounds identified
Phenolic compounds
Phenolic compounds
References
Dalar and Konczak,
2012
Dalar and Konczak,
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
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
Phenolic compounds
Phenolic compounds
Phenolic compounds
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
Compounds identified
References
Phenolic compounds
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
Compounds identified
Rutin, flavonoid glucosides,
phenolic acids
References
Dalar et al., 2013
Phenolic compounds
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
Phenolic compounds
IC50: 1.680.17
Flavonoid glucosides
IC50: 6.040.05
Dalar, unpublished
data
Dalar, unpublished
data
Dalar, unpublished
data
Phenolic compounds
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
Table 4. (Continued)
Enzyme inhibitory assay
Cichorium intybus whole plant
Extraction
Lyophilized
ethanol
Compounds identified
Chicoric acid, caftaric acid,
chlorogenic acid, flavonoid
glucosides
References
Dalar, unpublished
data
Phenolic compounds
34.02.0
21.02.0
35.02.0
27.02.0
21.02.0
23.04.0
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.
220
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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.
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.
228
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).
229
people living in rural and urban communities. This is considered evidence of their
effectiveness (Almaguer, 2001; Martnez, 1984 and 1989) .
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.
230
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.
231
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).
232
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).
233
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
234
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
235
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-.
236
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.
237
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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ISBN: 978-1-62948-219-4
2014 Nova Science Publishers, Inc.
Chapter 9
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.
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
*
244
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
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
246
1.2. Protoberberines
Protoberberines present themselves as tetrahydroprotoberberines and protoberberine salts.
Twelve protoberberines, coptisine (7), cheilanthifoline (8), corysamine (9), alborine (10),
berberine (11), mequinine (12), palmatine (13) ()-mecambridine (14), ()-Omethylmecambridine (15), ()-N-methylmecambridinium (16), karachine (17), and valachine
(18) were isolated (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
R7
R4
R3
N
R2
R1
R5
R8
R6
R7
O
O
O
N+
O
O
O
N
O
N
OCH3
OCH3
CH3
OH OCH3
16
OCH3
OCH3
17
OCH3
OCH3
18
R2
R3
R4
R1
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
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
1.5. Proaporphines
Proaporphine alkaloids are probably intermediates in the conversion of
benzylisoquinoline into aporphines. The dienone group in ring D was produced through
oxidation of benzylisoquinoline. Six proaporphine alkaloids, ()-mecambrine (34), ()-Nmethylcrotonosine (35), ()-pronuciferine (36), and glaziovine (37), (+)-Des-N-
248
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
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
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
CH3
46 R = H
47 R = Acetonyl
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
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).
250
O
O
56
HO
N
HO
O
O
H3CO
N
O
57
O
HO
OCH3
58
2. Flavonoids
Flavonoids are a diverse group of polyphenolic compounds widely distributed in the plant
kingdom, possessing a wide range of bioactive capacities which includes antioxidant and
antibacterial properties, as well as protective effects on cardiovascular systems. As
antioxidants, flavonoids can scavenge free radicals, increase the levels of antioxidant
enzymes, and thus protect body systems against damage from free oxygen species (Sandhar et
al., 2011). Yue et al. suggested that flavonoids could be considered as the main characteristics
for the quality control of Tibetan medicine M. quintuplinervia (Yue et al., 2010). Till now,
twenty-eight flavonoids divided into flavonols, flavones, and anthocyanidin, as well as
flavonoid glycosides, have been isolated from thirteen species of Meconopsis (Fig. 11),
including isorhamnetin (59), herbacetin (60), quercetin (61), quercetin 3-O--Dglucopyranoside (62), quercetin 3-O--D-galacopyranoside (63), kaempferol-3-gentiobioside
(64) and kaempferol 3-xylosylgentiobioside (65), kaempferol 3-O-(6-O--D-glucopyranosyl)-D-galactopyranoside
(66),
quercetin
3-O-[-D-galactopyranosyl-(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).
R5O
R2
O
OH O
59
60
61
62
63
64
65
66
67
68
69
70
71
72
HO
R4
OH O
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
OH
O
HO
HO
OH
O+
OH
OH
OH
RO
O
O
HO
O
HO HO
OH
OH
85 R = malonyl
86 R = OH
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).
252
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
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
253
obtained (Fu et al., 2010; Guo et al., 2003; Pan, 1998; Shang et al., 2002a, 2006b; Shang,
2002b; Zhang et al., 1997; Wu et al., 2011) (Fig. 14) .
R1
COOH
COOH
R2
97 R = OH
98 R = OCH3
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
Biological Activity
Although some Meconopsis species are in clinically prescribed in form of complex
preparations for the treatment of hepatitis, enteritis and diarrhea by Tibetan medical
practitioner, pharmacological activities of Meconopsis were rarely investigated. Some
Meconopsis herbs reportedly exhibited significant antioxidant, anti-inflammatory, analgesic,
antidiarrheal and hepatoprotective effects (Ding and Li, 2007; Guo et al., 2003; Wang et al.,
2011, 2013; Wangchuk et al., 2011, 2013; Zhou et al., 2013). Antioxidants extracted from
plants could play an important role in liver protection (Akanitapichat et al., 2010).
Recently, two reports focused on antioxidant properties of Meconopsis species. He et al.
evaluated the antioxidant potential of ethanolic extract of M. quintuplinervia using various
established systems (He et al., 2012). The extract showed strong in vitro antioxidant ability.
In the in vivo study, CCl4-induced oxidative stress caused significant decreases in the
superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels and a significant
increase in the malondialdehyde (MDA) level, most of which were significantly reversed
(except for SOD in the liver) by treatment with the extract and standard Vitamin E. In another
study, Zhou et al. investigated the hepatoprotective and antioxidant effects of ethanolic
extract of M. integrifolia (MIE) in vitro and in vivo (Zhou et al., 2013). MIE exhibited strong
antioxidant ability in vitro. In the rats with CCl4-induced liver injury, the groups treated with
MIE and silymarin showed significantly lower levels of glutamate pyruvate transaminase
(ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin
254
(TB). MIE demonstrated good antioxidant activities in both the liver and kidney of the rats in
vivo.
Wangchuk and colleagues found that M. simplicifolia used as Bhutanese showed strong
antiplasmodial activity with IC50 values of 0.40 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.
255
Acknowledgments
Financial support from the National Natural Science Foundation of China (No.
81102770) is gratefully acknowledged.
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ISBN: 978-1-62948-219-4
2014 Nova Science Publishers, Inc.
Chapter 10
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.
260
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).
262
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
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
263
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
264
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.
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
268
Index
analgesic, xi, 66, 126, 156, 227, 230, 238, 243, 244,
253, 262
anemia, 167, 180, 209
angiogenesis, 45, 54
angiotensin converting enzyme, 76
angiotensin II, 128
anhydrase, 132
anorexia, 206
ANS, 13
anthocyanin, 9, 53, 60, 257
antibiotic, 10
anticancer, x, 143, 158, 204, 261, 264
anticancer activity, 58, 67, 75
anticancer drug, 97
antiinflammatory drugs, 209
antioxidative activity, 90
antioxidative potential, 87
antispasmodic, 126
antitumor, 58, 69
apoptosis, viii, 33, 38, 41, 54, 56, 60, 64, 87, 90, 145,
146, 151, 152, 153, 159, 164, 236
appetite, 131, 147, 151, 154, 158, 159, 161, 163, 230
apples, 51, 52, 53, 79, 81, 121, 123
arginine, 151, 162
argon, 21
Armenia, 115, 186
aromatic compounds, 155
aromatic rings, 122, 247, 248
arres(s)t, 56, 60, 78
arteries, 45, 56
artery, 76
arthritis, 119, 220
ascites, 221
ascorbic acid, viii, 26, 29, 32, 41, 42, 44, 49, 57, 58,
59, 62, 63, 65, 66, 69, 71, 78, 85, 169, 228, 229,
233, 260, 262
aseptic, 97
Asia, xi, 64, 183, 187, 206, 220, 222
aspartate, 152, 156, 253
assessment, 3, 95, 237, 238, 254
assimilation, 102
asthma, 188, 192, 193, 196, 198, 200, 205, 206
astringent, 191
atherogenesis, 137
atherosclerosis, x, 50, 55, 56, 59, 62, 78, 129, 138,
143, 152, 159, 228, 229, 260
atherosclerotic plaque, 57, 131
atmosphere, 16
atmospheric pressure, 20, 21, 35
ATP, 102, 128, 130
atrophy, 70
authorities, 3
autism, 45
automation, 16
autooxidation, 50, 55
awareness, 118
azadirachtin, 261, 265
B
bacteria, 136, 205
bacterial fermentation, 219
bacterial infection, 167, 237
bacteriophage, 179
ban, 201
banks, 263, 264
base, 9, 135
beef, 87, 162
beer, 121
Beijing, 243
beneficial effect, viii, ix, 41, 48, 56, 59, 63, 69, 70,
91, 92, 136, 149, 158, 176, 177, 210
benefits, viii, 3, 14, 33, 34, 42, 43, 48, 50, 54, 57, 59,
63, 71, 73, 74, 82, 132, 146, 149, 166, 205, 228,
239, 263, 264
benign, 16
benzene, 93, 104, 123
benzo(a)pyrene, 75
beta-carotene, 87
beverages, viii, xi, 2, 15, 26, 74, 121, 150, 205, 227
bile, 65
bilirubin, 132, 166, 228, 229, 253
bioactive secondary metabolite, ix, 92, 100
bioavailability, 14, 37, 54, 71, 81, 131
biochemical processes, 118
biochemistry, 39, 108, 127, 137, 139, 180
biodiversity, 186
bioflavonoids, 136
biological activities, 37, 64, 104, 126, 132, 136, 247,
254, 257
biological activity, 54, 83, 87
biological samples, 22, 27
biological systems, 234, 240
biologically active compounds, 57, 111
biomarkers, 55, 79, 146, 236
biomass, 95, 96, 97, 98, 99, 100, 102, 104, 105, 106,
107, 108, 109, 115
biomolecules, 28, 44, 135
biosynthesis, 3, 11, 12, 13, 36, 37, 61, 65, 73, 79,
102, 105, 112, 114, 122, 125, 142, 167, 232
biosynthetic pathways, 96
biotechnology, 109, 220
biotic, 92, 121
birds, 92
Black Sea region, 184
black tea, 189, 212, 240
Index
bladder stones, 204, 207, 209
bleeding, 196, 197
blindness, 45
blood, xi, 50, 52, 55, 59, 62, 63, 70, 76, 77, 80, 83,
84, 87, 127, 134, 148, 151, 155, 156, 157, 163,
165, 175, 177, 178, 261
blood pressure, 76, 87, 148, 151, 156, 163
BMI, x, 143, 144, 147, 151, 154, 157, 158
body composition, 87, 158, 160
body fat, 129, 144, 147, 154, 155, 157, 158
body weight, 70, 129, 150, 151, 152, 154, 155, 156,
157, 158, 159, 163
bonding, 168, 169
bonds, 22, 23, 42, 136
bone, 57, 78, 166
bone resorption, 57
brain, xi, 44, 45, 61, 74, 84, 227, 237
Brazil, 2, 93, 149, 160
breakdown, 64, 71
breast cancer, 57, 58, 64, 71, 90
Britain, 82
bronchitis, 190, 195, 196, 198, 200, 205, 207, 209
bursa, 194, 202
butadiene, 23
butylated hydroxyanisole (BHA), viii, 2, 26
butylated hydroxytoluene (BHT), viii, 2, 26
buyers, 97
by-products, 26, 43, 57, 58, 118, 164
C
Ca2+, 128
cabbage, 84
caffeine, 154, 161
calcium, 47, 48, 53, 57, 144, 163
calibration, 29, 31
caloric intake, 144, 146, 156
calorie, 86, 151
calyx, 261, 262
cancer, viii, x, xi, 14, 33, 39, 42, 45, 48, 51, 53, 54,
55, 56, 58, 62, 64, 66, 67, 69, 71, 72, 74, 75, 79,
80, 81, 82, 83, 87, 90, 93, 119, 120, 143, 163,
181, 189, 190, 197, 204, 205, 206, 209, 211, 219,
221, 222, 228, 229, 230, 239, 259, 260, 261, 263
cancer cells, 51, 56, 64, 87
cancer progression, 56
candidates, 65, 133, 136
candidiasis, 198, 207, 209
capillary, 18, 21, 34, 35
capsule, 156
carbohydrate, 68, 129, 133, 141, 144, 154, 155, 158
carbohydrate metabolism, 133
carbohydrates, 42, 92, 100, 134, 144, 154, 219
269
270
Index
childhood, 150
children, 144, 150, 190, 195
Chile, 108
China, 2, 82, 243, 244, 255, 256, 258
Chinese medicine, 176, 212
Chinese women, 90, 151, 162
chiral center, 7
chlorination, 234, 237
chloroform, 16, 209, 215, 225, 235
chlorophyll, 115
cholesterol, 53, 56, 57, 60, 61, 62, 63, 65, 67, 69, 75,
79, 88, 131, 147, 154, 155, 156, 158, 192, 207,
210, 222
choline, x, 144
chromatographic technique, 16, 37
chromatography, 16, 17, 18, 35, 36, 38
chromium, 153
chronic diseases, vii, 1, 4, 48, 51, 55, 67, 69, 85, 90,
135, 228, 260
chronic illness, 71
cigarette smoke, 166
circulation, 56, 166, 177
civilization, vii, 1, 227
classes, vii, 1, 4, 6, 7, 12, 57, 62, 99, 121, 122, 177,
209, 257
classification, 140, 172, 173, 174, 238
cleavage, 22, 23, 24, 36, 222, 248
cleavages, 24
climate(s), 100, 113, 189, 230
clinical trials, 51, 59, 61, 64, 152, 154
clustering, 129
CO2, 16, 23, 25
cobalt, 139
coenzyme, 47, 49, 57, 60, 105, 129
coffee, xi, 25, 37, 38, 121, 192, 206, 259, 260
cognitive function, 44
colic, 206
collaboration, 184
collagen, 56, 266
colon, 34, 56, 58, 61, 72, 78, 86, 136, 219, 222
colon cancer, 34, 58, 78, 222
color, 11, 28, 29, 32, 54, 60, 68, 71, 77, 121, 125,
258
commercial, ix, 25, 91, 94, 95, 97, 98, 107
communities, 229
community, 26, 34, 159, 166
comparative analysis, 115
compatibility, 16
compilation, 39
complications, viii, x, 41, 45, 56, 117, 119, 127, 128,
129, 133, 138, 140, 144, 150, 155, 159
composition, ix, 20, 36, 67, 69, 71, 78, 79, 80, 89,
91, 96, 100, 102, 103, 106, 108, 110, 113, 157,
205, 208, 209, 210, 219, 231, 258
condensation, 12, 123
configuration, 6, 8, 38, 155, 245
conjugated dienes, 131
conjugation, 12, 19, 70, 236
conservation, vii, xii, 94, 224, 259, 263, 264, 266
constipation, 144, 189, 194, 199
constituents, xi, 44, 45, 48, 54, 68, 72, 79, 80, 86, 90,
103, 113, 120, 134, 136, 140, 158, 167, 176, 188,
204, 221, 231, 232, 238, 239, 243, 244, 251, 254,
255, 256, 257, 258
construction, 29
consumers, viii, ix, 41, 74, 91, 94, 95
consumption, 2, 14, 16, 19, 42, 48, 50, 52, 53, 54,
56, 58, 59, 61, 63, 64, 70, 72, 74, 76, 79, 80, 83,
85, 86, 87, 90, 94, 121, 131, 135, 149, 150, 154,
160, 162, 176, 189, 209
contaminant, 108
contamination, 21, 95, 96
control group, 70, 150, 235
controlled trials, 79, 149
cooking, xi, 69, 78, 82, 259, 263
coordination, 44
copper, 53, 93, 135
corona discharge, 21
coronary artery disease, 54, 82, 150
coronary heart disease, 34, 56, 59, 146, 205, 210
correlation, 27, 32, 53, 101
correlations, 59, 209
cortex, 60, 188, 201
cortisol, 147
cosmetic(s), 94, 103, 120, 209, 232, 238, 251
cost, 16, 19, 21, 94, 95, 97, 107, 108, 111, 184
cough, 192, 195, 196, 198, 200, 201, 205
coumarins, 6, 120, 207
counterirritant, 68
covalent bond, 135, 235
covalent bonding, 135
Croatia, 37, 181
crop, 101, 102, 110, 111
crop production, 111
crops, 58, 94, 97, 98, 100, 103, 109, 111, 113, 115
CRP, 146
CT, 110
Cuba, 58, 87
cues, 122
cultivars, 66, 68, 71, 76, 82, 83, 89, 90, 102, 103,
104, 111
cultivation, ix, 82, 88, 91, 95, 96, 97, 98, 99, 100,
105, 106, 107, 109, 111, 115, 220, 254
Index
culture, xi, 37, 61, 85, 94, 96, 97, 98, 99, 100, 101,
102, 103, 105, 106, 108, 109, 111, 112, 224, 259,
263, 264
curcumin, 33
cure, xi, 73, 183, 184, 189, 204, 205, 219
CV, 142
CVD, 45, 55, 56, 57
CWA, 139
cycles, 121
cyclophosphamide, 63, 85
Cydonia oblonga, 168, 174, 178
cysteine, 44, 45, 49, 65
cystic fibrosis, 152
cytochrome, 43, 235, 246, 256
cytokines, 33, 45, 70, 146, 159
cytoskeleton, 176
cytotoxicity, viii, 2, 26, 38, 101
D
damages, 75
danger, 94
database, 81
decay, 31, 34
decomposition, 19, 28, 31, 234
defence, 93, 150, 151, 180
defense mechanisms, 118, 211, 228, 229, 235
deficiency, 62, 100, 102, 105, 109, 129, 150, 166,
178
deficit, 102, 113
deforestation, 254, 263
deformability, 167, 168, 176, 177, 180
degradation, 47, 61, 73, 107, 131, 176, 238
dehydration, 245
deoxyribonucleic acid, 118
Department of Agriculture, 48, 91
deposition, 57, 159
deposits, 159
depression, 45
deprivation, 121
depth, 210, 254
derivatives, 8, 9, 10, 17, 33, 38, 39, 49, 62, 64, 65,
70, 93, 100, 102, 112, 114, 118, 120, 122, 133,
134, 135, 140, 141, 207, 209, 223, 244, 245
desiccation, 104, 107
desorption, 20
destruction, 15, 42, 94, 254
detection, viii, 2, 17, 18, 19, 20, 30, 35, 36, 37, 39,
79, 89, 141
detoxification, 44, 63, 81, 166, 236
developed countries, ix, 3, 91, 94, 95, 144
developing countries, 94, 95, 108, 111, 120, 136, 144
271
diabetes, vii, ix, x, xi, 1, 4, 14, 34, 48, 56, 82, 86, 93,
117, 119, 120, 121, 127, 128, 132, 133, 134, 136,
138, 139, 140, 142, 143, 150, 151, 152, 156, 159,
161, 176, 189, 190, 191, 192, 199, 201, 204, 206,
210, 211, 219, 220, 222, 228, 229, 259, 260, 264
diabetic nephropathy, 163
diabetic patients, 61, 86, 87, 142, 149, 150
diacylglycerol, 70
diarrhea, 219, 253
diet, x, 6, 14, 26, 42, 44, 46, 48, 50, 52, 53, 66, 70,
71, 72, 74, 77, 78, 80, 82, 129, 143, 144, 146,
147, 149, 150, 151, 152, 153, 154, 155, 156, 157,
158, 160, 162, 163, 164, 228, 229, 260
dietary antioxidants, viii, 41, 131, 223, 240
dietary fat, 155
dietary fiber, 63
dietary habits, 74, 144
dietary intake, 56, 71
dietary supplementation, 56
diffusion, 176
digestion, 58, 87, 199, 206
dimethylformamide, 15
dipole moments, 16
discontinuity, 235
discrimination, 25
disease-reduction benefits., viii, 42
disorder, 204
dispersion, 34, 92
disposition, 80
dissociation, 21, 35
distillation, 251
distribution, 19, 27, 90, 109, 125, 147, 159, 208, 258
diuretic, 190, 192, 195, 198, 199, 201, 206
diversity, 9, 18, 76, 119, 121, 136, 184, 266
DNA, 33, 45, 50, 51, 52, 55, 56, 59, 61, 66, 67, 72,
75, 80, 83, 89, 93, 118, 126, 132, 140, 160, 211,
228, 229, 233, 234, 238, 239, 240
DNA damage, 50, 51, 52, 55, 56, 59, 61, 67, 72, 75,
80, 89, 140, 160, 233, 234, 239, 240
DNA repair, 89, 132
DNA strand breaks, 52, 118
docosahexaenoic acid, 152, 175
doctors, 94
DOI, 265
donors, 128, 167
double bonds, 19, 71
down-regulation, 58, 158
drainage, 112
drought, 93, 114, 121, 205
drug discovery, 119, 136
drug interaction, 2
drugs, x, 2, 3, 36, 81, 93, 94, 114, 118, 119, 120,
138, 139, 148, 149, 159, 163, 165, 244
272
Index
E
East Asia, 243
ecology, 38, 255
eczema, 188, 190, 196, 197, 209
edema, 56, 232
editors, 109, 110, 111, 114, 115, 180
Egypt, 206
eicosapentaenoic acid, 152
electric field, 17, 21
electrical conductivity, 97, 101
electron(s), 17, 23, 28, 29, 32, 42, 43, 53, 70, 93,
121, 128, 131, 208, 211, 212, 233, 234
electrophoresis, 17, 18, 21, 34
electrophoretic separation, 18
emission, 20, 112
endangered, 254, 256, 264
endocrine, 129, 137, 147, 186, 238
endocrine disorders, 238
endothelial cells, 43, 78, 177, 260
endothelial dysfunction, 131, 146
endothelium, 129, 131
endurance, 154
energy, 16, 43, 46, 76, 87, 102, 108, 118, 130, 144,
145, 147, 148, 149, 151, 158, 160, 162, 163, 166
energy density, 46
energy expenditure, 147, 151, 162
England, 244
enteritis, 253
entrapment, 102, 234
environment, ix, 16, 27, 43, 91, 92, 97, 109, 110,
116, 118, 131, 176, 229
environmental conditions, viii, 42, 93, 96, 98, 104
environmental impact, 101, 106
environmental influences, 147
environmental issues, 94, 112
environmental stress, 205
EPA, 152
epidemic, 136
epidemiologic, 81, 139, 150
epidemiologic studies, 139
epidemiology, 79
epilepsy, 189, 192, 196, 206, 228
epithelial cells, 54, 90
EPR, 235
equilibrium, 18, 132
erythrocyte membranes, 177
erythrocytes, 50, 51, 52, 58, 66, 75, 167, 179, 180,
181, 231
erythroid cells, 166
ESI, 18, 20, 21, 22, 25, 36, 38
ester, 9, 70
ester bonds, 9
ethanol, 15, 29, 66, 75, 78, 81, 209, 213, 214, 215,
216, 217, 218, 225, 261, 262, 263
ethers, 142
ethnic groups, 187
ethyl acetate, 15, 156, 262
ethylene, 23, 102
etiology, x, 165, 166
EU, 2, 3, 13
eucalyptus, 167, 264, 266
eukaryotic, 43
euphoria, 244
Europe, 2, 3, 64, 82, 95, 184, 187, 206, 229, 244, 245
European Union, 94, 115
evaporation, 21
evidence, xi, 14, 42, 44, 45, 47, 54, 69, 73, 74, 81,
84, 88, 107, 122, 126, 128, 144, 147, 159, 187,
227, 228, 229, 234, 236
evolution, 135, 258
excitation, 20, 31
excretion, 57, 149, 222
exercise, 44, 147, 151
expectorant, 191, 199
experimental condition, 29, 58
experimental design, 258
expertise, 3
exposure, 15, 31, 52, 167, 211
extinction, 94, 264
extracellular matrix, 43
extracellular signal regulated kinases, 134
extraction, viii, ix, 2, 15, 16, 34, 37, 38, 57, 58, 75,
91, 95, 97, 104, 107, 112, 157, 221, 263, 264
F
factories, 114
FAD, 128
families, 6, 103, 185, 187, 189, 246
FAS, 157
fasting, 150, 154, 155, 156
fat, x, 72, 77, 129, 130, 141, 143, 144, 145, 147, 149,
150, 151, 152, 153, 154, 155, 157, 158, 159, 160,
161, 162, 163, 164, 263
fat reduction, 151
fatty acids, ix, 45, 75, 117, 129, 131, 144, 146, 151,
152, 209, 210, 219, 235, 260, 266
fauna, 187
FDA, 3
Index
feelings, 158, 162
female rat, 74
fermentation, 60, 87, 261
ferritin, 166
fertilization, 96, 113
fertilizers, 96, 105
fever(s), 67, 68, 144
fiber, 46, 51, 166
fiber content, 51, 166
fibrinogen, 131
fibroblasts, 266
fibrosis, 235, 257
field crops, ix, 91, 95, 96
filtration, 157
Finland, 113
flammability, 16
flatulence, 154, 202, 206, 219
flavonol, 6, 13, 14, 24, 34, 39, 54, 76, 133, 135, 168,
233, 256
flavo(u)r, 68, 121, 156, 205
flight, 21
flooding, 115
floods, 128
flora, xi, xii, 81, 184, 186, 187, 259, 260, 264
flour, 188, 200
flowers, 48, 76, 103, 109, 121, 142, 184, 189, 205,
206, 211, 212, 230, 234, 239, 244, 257
fluctuations, 96
fluid, 15, 157
fluid extract, 157
fluorescence, 18, 19, 20, 31, 115
folic acid, 48, 53, 58, 69
folklore, 81
food, viii, 3, 15, 27, 34, 37, 39, 41, 42, 47, 48, 53,
55, 57, 58, 60, 64, 65, 67, 68, 73, 74, 81, 82, 84,
87, 88, 94, 95, 103, 120, 121, 124, 140, 147, 148,
150, 154, 156, 162, 187, 189, 207, 209, 211, 220,
222, 232, 240, 251
Food and Drug Administration, 3
food intake, 148, 154
food products, 42, 55, 88
force, 18
formation, vii, ix, 1, 4, 15, 16, 18, 23, 24, 26, 57, 61,
63, 69, 77, 92, 100, 102, 117, 120, 122, 128, 131,
135, 139, 140, 141, 142, 168, 181, 211, 234, 235,
236, 248, 258, 262
formula, 21
fossils, 187
fractures, 244
fragility, 176
fragments, 21, 23, 24
France, 156
273
free radicals, ix, 14, 27, 28, 29, 32, 34, 43, 50, 51,
57, 60, 61, 62, 63, 69, 80, 117, 118, 120, 121,
122, 127, 131, 132, 135, 136, 137, 166, 169, 176,
205, 211, 228, 237, 240, 250, 260, 261, 263
fructose, 150
fruits, viii, 6, 8, 9, 14, 36, 38, 42, 44, 48, 50, 51, 53,
54, 57, 58, 61, 62, 63, 65, 66, 68, 69, 73, 74, 75,
77, 79, 80, 81, 82, 85, 87, 88, 90, 121, 122, 124,
125, 136, 157, 158, 161, 175, 184, 189, 228, 232,
239, 263
functional food, 85
fungal infection, 188, 190, 191, 195, 196, 197
fungi, 205
furan, 62
G
gamma rays, 33
gas diffusion, 102
gastric ulcer, 65
gastritis, 230
gastrointestinal tract, 149
gene expression, 33, 44, 72, 75, 90, 93, 118, 134,
151, 210
gene regulation, 69
genes, x, 117, 122, 150
genetic background, 90
genetic defect, 33
genetic factors, 147
genetic marker, 147
genotype, 72, 104, 107
genus, xi, 8, 62, 94, 103, 230, 243, 244, 254, 256,
257
geometry, 21
Georgia, 186
Germany, 2, 52
germination, 92, 98
ginger, 120, 158, 160, 169, 232, 240
ginseng, 232
glasses, 150
glaucoma, 127
glucagon, 149
gluconeogenesis, 127, 157
glucose, ix, 11, 44, 45, 61, 63, 64, 86, 87, 117, 127,
128, 129, 133, 134, 137, 140, 141, 144, 150, 151,
152, 154, 155, 157, 158, 163, 166, 178, 261, 262
glucose tolerance, 133
glucose-induced insulin secretion, 133
glucosidases, 134
glucoside, 10, 51, 133, 169, 205, 207, 209, 221, 231
glucosinolates, 49, 64, 65, 72, 79, 89
GLUT4, 134
glutamate, 44, 253
274
Index
glutathione, viii, 26, 41, 43, 44, 50, 51, 56, 61, 82,
84, 129, 131, 132, 149, 150, 160, 169, 176, 178,
179, 180, 220, 228, 229, 240, 253, 262
glycerol, 150
glycine, 44
glycogen, 127, 134
glycolysis, 127, 128, 178
glycoproteins, 155
glycoside, 5, 22, 24, 64, 133, 177, 231
glycosylation, 5, 6, 7, 8, 34, 35, 122
gout, 206, 209
grass, 38
grazing, 264
Greeks, 187
greenhouse, 97, 98, 99, 100, 101, 103, 104, 106, 107,
108, 109, 111, 112, 113
greenhouses, 97, 107, 109, 264
growth, viii, 4, 11, 36, 41, 51, 54, 58, 60, 61, 65, 79,
86, 87, 92, 94, 95, 97, 98, 99, 100, 101, 102, 104,
105, 106, 108, 109, 110, 111, 112, 113, 114, 115,
118, 147, 150, 159, 205, 261, 264
growth factor, viii, 41
growth hormone, 147
growth rate, 99, 108
guidance, 3
H
H. sabdariffa, 262
habitat, 254
habitats, 103, 244
harmful effects, 205
harmonization, 3
harvesting, ix, 91, 94, 96, 98, 100, 104, 230
HCC, 33
head and neck cancer, 71, 79
headache, 144, 192, 193, 196, 205
healing, viii, 41, 59, 73, 82, 190, 191, 192, 193, 194,
196, 197, 198, 199, 201
health, viii, x, 3, 14, 33, 34, 37, 41, 42, 44, 46, 47,
48, 54, 55, 56, 58, 60, 62, 63, 71, 73, 74, 77, 81,
82, 95, 121, 132, 142, 143, 146, 159, 162, 166,
176, 184, 185, 204, 205, 210, 211, 219, 222, 224,
227, 228, 260, 263
health care, 3, 184, 224
health care system, 224
health condition, 166, 204, 219
health effects, 142
health problems, 42, 185
health promotion, 166
hearing loss, 45
heart attack, 56
heart disease, x, 48, 61, 79, 143, 166
Index
hydrophobicity, 18
hydroponics, ix, 91, 96, 97, 98, 99, 101, 102, 104,
107, 111, 114, 115
hydroquinone, viii, 2, 26
hydroxyacids, 10
hydroxyl, 4, 5, 6, 12, 23, 24, 26, 55, 70, 92, 93, 104,
118, 122, 123, 132, 166, 169, 178, 232, 233, 234,
235, 237, 239, 240, 262
hydroxyl groups, 5, 6, 23, 70, 93, 104, 122, 232, 233,
234, 235, 239
hypercholesterolemia, 62, 82, 150, 189, 192, 206,
207, 219
hyperglycemia, 129, 138, 141, 155, 219, 222
hyperinsulinemia, 142
hyperlipemia, 138
hyperlipidemia, 53, 57, 158
hypertension, vii, x, xi, 1, 4, 45, 48, 56, 57, 129, 143,
144, 148, 150, 159, 189, 192, 194, 204, 206, 211,
219, 220, 222, 259, 260, 264
hyperthyroidism, 79
hypertrophy, 45, 146
hypotensive, 63
hypothalamus, 147, 149
hypothesis, 88, 92, 100, 235, 236
hypothyroidism, 79
hypoxia, 44, 97, 102, 104, 105, 106, 109, 110, 111
hypoxia-inducible factor, 44
I
ideal, 119, 144, 148, 159
identification, 16, 19, 20, 21, 22, 23, 25, 34, 35, 36,
37, 55, 89, 90, 94, 119, 204, 219, 232, 254
identity, 184, 204
ileum, 207
images, 175
imbalances, 92
immune function, 166, 210
immune regulation, 33
immune response, 44
immune system, 34, 186, 189, 228, 261
immunomodulation, 60
immunomodulatory, 207, 220
immunostimulant, 126, 200
impotence, 206
improvements, 65, 149, 156
in vivo, 26, 28, 38, 50, 51, 53, 54, 56, 58, 60, 61, 64,
71, 72, 76, 81, 83, 89, 141, 155, 167, 175, 177,
228, 229, 232, 234, 239, 240, 253, 255, 258, 266
incidence, vii, 1, 4, 14, 56, 62, 74, 95
income, 144
India, 117, 156, 181, 223, 249, 255
Indians, 154
275
individuality, 4
individuals, x, 72, 86, 143, 151, 154, 158
induction, 33, 64, 70, 72, 101, 150, 153, 157, 177
industrial processing, 88
industries, 15, 184, 232, 251
industry, 57, 59, 95, 96, 103, 120, 209
infection, 4, 109, 118, 121, 199
infertility, 198, 207, 209
inflammasome, 90
inflammation, viii, 14, 33, 42, 45, 51, 54, 59, 67, 83,
89, 120, 129, 130, 144, 191, 202, 228, 230, 246,
260
inflammatory cells, 159
inflammatory disease, 93
inflammatory mediators, 131
inflammatory responses, viii, 41
infrastructure, 97
ingestion, 79, 167, 175
ingredients, vii, 1, 3, 48, 73, 85, 178, 189, 255
inhibition, 32, 50, 51, 53, 54, 60, 64, 65, 68, 71, 72,
133, 134, 135, 136, 155, 157, 161, 167, 169, 172,
173, 174, 175, 177, 205, 209, 219, 220, 232, 233,
238, 240, 246, 254
inhibitor, 51, 71, 129, 139, 148, 157, 163, 219
initiation, 14, 145, 169
injuries, 56, 202, 205
injury, 79, 87, 109, 137, 138, 150, 177, 178, 205,
229, 235, 240, 253
inner ear, 76
inositol, x, 144, 231
insects, 92
insomnia, 206
insulin, 63, 68, 90, 127, 133, 134, 136, 142, 144,
147, 148, 150, 151, 152, 154, 159, 263
insulin resistance, 144, 150, 159
insulin sensitivity, 150, 151
insulin signaling, 136
integrity, 176
interface, 18
interference, 30, 260
intervention, x, 52, 55, 67, 72, 76, 83, 86, 117, 149,
150, 151
intestine, 14
intima, 76
intoxication, 240
investment, 19, 97, 98
ion channels, 43, 150
ionization, 17, 18, 20, 21, 27, 34, 35, 36, 89, 222
ionizing radiation, 166
ions, 17, 21, 22, 23, 24, 25, 101, 105, 126, 135, 166,
234, 258
Iowa, 47, 84
Iran, 110, 186, 222
276
Index
Iraq, 186
Ireland, 244
iron, 52, 60, 81, 140, 209, 233, 234, 239
irradiation, 33
irrigation, 96, 101, 106
ischemia, 150, 152, 228
ischemia reperfusion injury, 152, 228
Islam, v
isoflavone, 13, 135
isoflavonoids, 4, 125
isolation, xi, 3, 16, 122, 243, 244, 245, 261
isomers, 8, 10, 22, 24, 25, 35, 71, 153, 175, 181, 210
isoprene, 206
issues, vii, ix, 1, 4, 91, 97, 223
Italy, 91, 104
J
Jamaica, xi, 259, 263, 265, 266
Japan, 60, 184, 256
jaundice, 206
jejunum, 70, 207
joint pain, 201
Jordan, 126, 139
K
kaempferol, 6, 20, 24, 48, 49, 53, 54, 65, 66, 68, 70,
123, 133, 134, 135, 138, 169, 176, 209, 230, 231,
232, 233, 250
ketones, 114, 155
kidney, 64, 136, 142, 154, 189, 192, 194, 195, 197,
198, 200, 201, 204, 206, 207, 209, 230, 254
kidney stones, 198, 200, 204
kinase activity, 150
kinetics, 28, 78, 239
Korea, 81, 158
L
lactoferrin, 166
L-arginine, 128, 132
LC-MS, 35
LDL, 50, 51, 52, 53, 55, 61, 63, 65, 67, 76, 79, 86,
131, 132, 135, 137, 147, 155, 157, 158
leaching, 101, 106, 113
lead, vii, 1, 15, 29, 43, 54, 62, 118, 135, 144, 149,
159, 167, 236
lead molecules, vii, 1
leakage, 131
lean body mass, 147, 151
legislation, 94
legume, 181
lens, 134
leprosy, 206
leptin, 129, 146, 147, 149, 151, 154, 157
lesions, 52, 83, 93
leukocytes, 83
life cycle, 163, 254
lifestyle changes, 144
light, xi, 16, 31, 58, 71, 93, 100, 108, 110, 115, 126,
166, 183, 187, 205, 234, 254
light conditions, 100
lignans, 4, 120, 121, 177
lignin, 9, 11, 36, 93
linoleic acid, 32, 69, 261
lipases, 132
lipid metabolism, 14, 157, 158
lipid oxidation, 50, 51, 56, 60, 66, 71, 76, 89, 149,
167
lipid peroxidation, x, 50, 51, 53, 54, 56, 59, 62, 66,
67, 69, 71, 76, 84, 85, 86, 93, 101, 118, 132, 135,
136, 137, 143, 146, 167, 169, 175, 176, 177, 180,
231, 234, 236, 239
lipid peroxides, 132, 135
lipids, vii, 1, 4, 15, 42, 45, 51, 52, 61, 66, 93, 118,
128, 131, 132, 156, 166, 168, 177, 211, 228, 229,
234, 235, 236
lipolysis, 146, 147, 149, 151, 153, 155, 158, 159
lipoproteins, 76, 118, 131
liquid chromatography, 19, 34, 35, 36, 39, 77, 79, 89,
258
liquid phase, 18
lithium, 32
liver, xi, 39, 50, 52, 56, 60, 63, 64, 66, 67, 72, 75, 79,
82, 83, 136, 146, 148, 152, 155, 157, 206, 207,
222, 227, 228, 229, 231, 233, 235, 236, 237, 240,
253, 257
liver cancer, 39
liver cells, 66
liver cirrhosis, 228
liver damage, 82, 207, 235
liver disease, 152, 229
localization, 43, 44
low fat diet, 146
low risk, 19
low temperatures, 62, 102
low-density lipoprotein, 50, 52, 55, 71, 78, 88, 131,
156, 162, 179
lung cancer, 33, 61
Luo, 109, 244, 256
lutein, 48, 49, 55, 61, 69, 72, 87, 210
lycopene, 49, 59, 67, 71, 72, 84, 85, 86, 135, 210
lymphatic system, 72
lymphocytes, 52, 83, 89
Index
lymphoid, 56, 186
lysis, 167, 169, 176, 181
lysozyme, 33, 39
M
machinery, 110, 126, 131
macromolecules, 43, 101
macrophages, 45, 61, 76, 79, 86, 131
macular degeneration, 59
magnesium, 57, 61
magnitude, 43, 71
Maillard reaction, 135
majority, 17, 28, 104, 187, 188, 204, 219
MALDI, 21
management, xi, 96, 114, 149, 152, 155, 161, 162,
163, 220, 222, 259
manganese, 77
manipulation, ix, 91, 107
manufacturing, 55, 74, 115
marketplace, 2
mass, x, 17, 18, 19, 20, 21, 22, 25, 34, 35, 36, 37, 89,
104, 143, 144, 147, 150, 151, 156, 158, 159, 222,
231, 234, 258
mass spectrometry, 17, 19, 20, 21, 22, 25, 34, 35, 36,
37, 89, 222, 234, 258
materials, 2, 48, 184
matrix, 15, 18, 20, 21, 34, 53, 54, 82, 230
matrix metalloproteinase, 54, 82
matrixes, 16
matter, 178
MB, 58, 137, 141
measurement(s), 31, 155, 234
media, 30, 76, 96, 97, 99, 116, 264
medical, 42, 48, 94, 178, 184, 187, 189, 207, 211,
229, 253, 254
medical history, 229
medication, x, 165
medicine, viii, xi, 2, 3, 41, 59, 60, 65, 68, 73, 74, 99,
118, 119, 120, 176, 179, 183, 184, 185, 187, 189,
207, 209, 221, 222, 223, 224, 225, 227, 228, 229,
230, 232, 243, 244, 250, 256, 258, 259, 263, 266
Mediterranean, vii, 1, 65, 83, 86, 184, 206, 224
Mediterranean climate, 86
MEK, 134
mellitus, 120, 140, 142, 151, 152, 179
membrane permeability, 93, 101
membranes, 50, 55, 125, 168, 169, 177, 235
memory, 83
mental illness, 3
mesangial cells, 137
mesophyll, 126
messengers, ix, 76, 86, 117
277
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
O
obesity, ix, x, 34, 53, 56, 59, 77, 90, 117, 121, 129,
130, 131, 136, 137, 138, 139, 140, 142, 143, 144,
145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
155, 157, 158, 159, 160, 161, 162, 163, 164, 210,
219
OH, 8, 9, 10, 20, 24, 28, 118, 127, 150, 166, 168,
233, 234
oil, 56, 61, 67, 75, 78, 87, 88, 95, 99, 102, 108, 112,
113, 114, 206, 232, 255
oil production, 108, 112
oleic acid, 69, 210
oligomers, 55
olive oil, 38, 179, 180
Index
omega-3, 144, 146, 152
opportunities, ix, 92, 97, 109, 162
optimization, 95, 96, 245
organ, 104, 111, 130
organelle(s), 46
organic compounds, 64
organism, 50, 92
organs, xi, 45, 102, 103, 127, 208, 227, 263
ornamental plants, 103, 114
osmotic stress, 101
osteoarthritis, 87
osteoporosis, 78, 121
ovaries, 230
overlap, 148
overproduction, 128
overweight, x, 143, 144, 149, 154, 155, 156, 157,
158, 160, 161, 162, 163
overweight adults, 160
ox, 49, 70
oxidation, viii, ix, x, 2, 26, 27, 28, 29, 32, 33, 39, 47,
50, 51, 52, 53, 54, 55, 60, 61, 67, 69, 71, 76, 85,
117, 122, 129, 131, 135, 137, 141, 143, 151, 157,
158, 167, 168, 169, 175, 176, 177, 234, 238, 239,
247
oxidation products, 239
oxidative agents, 177
oxidative damage, viii, ix, 41, 44, 50, 52, 53, 55, 57,
58, 63, 66, 69, 75, 86, 90, 93, 101, 117, 126, 131,
135, 139, 166, 167, 176, 177, 180, 235, 236, 237,
239, 263
oxygen, viii, x, 15, 26, 31, 32, 41, 43, 44, 51, 55, 66,
70, 76, 77, 83, 86, 102, 105, 109, 110, 112, 117,
118, 120, 122, 126, 128, 131, 132, 138, 140, 142,
143, 167, 179, 180, 181, 205, 208, 212, 234, 236,
245, 250
oxygen consumption, x, 143
ozone, 16, 126
P
Pacific, 115, 180
pain, 148, 189, 190, 192, 194, 195, 196, 197, 198,
201, 202, 204, 205, 206, 207, 209, 230
Pakistan, 81, 113
palpitations, 196, 205
pancreas, 56, 60, 148
parallel, 149
parents, 59
participants, 52, 149, 151, 152, 153, 154, 155, 176
partition, 18
pastures, 244
pathogenesis, vii, 2, 26, 42, 45
pathogens, 11, 92, 95, 121, 205
279
280
Index
Q
quality assurance, 112, 254
quality control, 95, 250
quality of life, 144
quality standards, 95
quantification, viii, 2, 17, 19, 31, 36
quartile, 149
quercetin, 20, 23, 34, 38, 48, 49, 51, 52, 53, 54, 57,
63, 65, 66, 68, 70, 72, 90, 123, 133, 135, 142,
145, 146, 152, 169, 176, 177, 205, 207, 209, 222,
230, 231, 232, 233, 234, 235, 236, 237, 250
questionnaire, 39
quinone, 29, 233
R
radiation, 4, 17, 121, 139, 205, 211, 220
radical formation, 129, 167
radical reactions, xi, 165, 167
radicals, vii, ix, x, 2, 26, 28, 31, 34, 43, 51, 66, 68,
79, 84, 117, 118, 127, 131, 132, 143, 166, 168,
169, 175, 178, 179, 205, 211, 212, 232, 238, 260,
261
rape, 90
rash, 230
Index
RE, 208
reaction mechanism, 29, 31
reaction medium, 31
reaction time, 30
reactions, 11, 23, 27, 28, 43, 118, 129, 135, 140, 234,
260
reactive oxygen, vii, ix, x, 2, 14, 26, 42, 50, 86, 92,
100, 102, 117, 118, 138, 141, 143, 166, 205, 211,
212, 228, 229, 239, 240, 260
reactivity, 43, 45
reagents, 140
receptors, 6, 128, 131, 150
recognition, 17
recovery, 19, 199
red blood cells, 135, 166, 176, 178, 179, 181
red wine, 8, 47, 56, 61, 90, 121, 122, 124, 152
reducing sugars, 57
redundancy, 148
regeneration, 75, 135, 264
regions of the world, 209
relevance, 38
relief, xi, 259
renal cell carcinoma, 45
repair, 26, 132, 167
reproduction, 4, 11, 92
requirements, 95, 96, 115, 149
researchers, 33, 73, 94, 177, 245
reserves, 160
residues, 4, 25, 45, 64, 75, 236
resistance, 93, 150, 158, 160, 176, 205
resolution, 17, 21
resources, 184, 204, 220, 224
respiration, 4, 92, 102
response, viii, ix, 4, 26, 41, 55, 65, 72, 91, 92, 101,
102, 104, 111, 112, 115, 120, 121, 122, 131, 149,
152, 245, 258
restoration, 66
restrictions, 156
resveratrol, 10, 33, 48, 55, 56, 71, 75, 85, 146, 152,
240
reticulum, 125
retinol, 78, 150
retinopathy, 45, 127, 176, 179
revenue, 2
riboflavin, 33
rings, 4, 22, 23, 121, 122, 248
ringworm, 197
risk(s), viii, x, 33, 34, 42, 47, 48, 50, 51, 55, 56, 57,
59, 61, 66, 67, 71, 72, 74, 79, 81, 82, 83, 87, 90,
94, 139, 143, 144, 146, 149, 150, 161, 181, 210
risk factors, x, 48, 61, 82, 83, 143, 146, 150, 161
ROOH, 28
room temperature, 188
281
root(s), 48, 78, 83, 97, 98, 99, 100, 101, 102, 103,
104, 105, 108, 109, 110, 111, 112, 115, 121, 134,
139, 140, 168, 172, 173, 184, 188, 201, 206, 207,
208, 210, 213, 214, 215, 216, 221, 222, 223, 255,
261, 262
root growth, 97, 102, 105
root system, 98, 102
routes, 11, 93
Royal Society, 110
S
safety, 3, 33, 42, 50, 60, 73, 94, 149, 163, 184, 237,
238
salinity, 101, 104, 106, 111, 113, 114, 115
saliva, 137
salt tolerance, 101
salts, 8, 55, 108, 246
saponin, 155
saturated fat, 48, 129, 210
saturated fatty acids, 129, 210
scavengers, viii, 2, 26, 44, 50, 56, 70, 120, 121, 231,
232, 233, 234, 237, 238, 239
schizophrenia, 45
science, 139, 260
scope, 138
secondary metabolism, ix, 91, 92, 96, 100, 102
secrete, 63
secretion, 89, 90, 127, 147
security, 3
sedative, 195
seed, 39, 56, 58, 63, 74, 78, 79, 83, 84, 88, 89, 92,
121, 152, 163, 178, 188, 201, 215, 216, 240, 261,
264
seedlings, 110
selectivity, 18, 149
selenium, 48, 53, 149, 160
sensation, 206
sensing, 44, 115
sensitivity, 18, 33, 102
serum, 57, 61, 65, 68, 70, 76, 84, 86, 87, 150, 151,
152, 154, 156, 158, 163, 207, 222
sesquiterpenoid, 207, 231, 239
severe stress, 122
sex, 147
sex steroid, 147
shape, 145, 167, 176
shelf life, 62
shoot, 99, 101, 102, 104, 105, 188, 201, 262
shoots, 102, 105
showing, 149, 152, 244
sickle cell, 166, 178
sickle cell anemia, 166, 178
282
Index
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
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