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Medicinal Plants as Antioxidant Agents: Understanding Their Mechanism of Action and Therapeutic
Efficacy, 2012: 97-145 ISBN: 978-81-308-0509-2 Editor: Anna Capasso
6. Medicinal plants as antioxidant agents:
Understanding their mechanism of action and
therapeutic efficacy
1
Daniel Seifu1, Freshet Assefa2 and Solomon M. Abay3
Addis Ababa University, School of Medicine, Department of Biochemistry, Addis Ababa, Ethiopia
2
Hawassa University, College of Health Sciences, Department of Biomedical Sciences
Ethiopia; 3Addis Ababa University, School of Medicine, Department of
Pharmacology, Addis Ababa, Ethiopia
Abstract. The present review focuses on the antioxidant activities
of the various medicinal plants, their bio-availability, mechanism
of action and therapeutic efficacy. Antioxidant of medicinal plant
origin may exert their effects on biological systems by different
mechanisms. Efforts have been made to explore the structure and
functional groups that involve removing the oxidants that most
often occurs in the biological systems. Some of the mechanisms
have been dealt deeply in this review. Many oxidants have been
implicated in a number of disease, removal or minimization of
oxidants exposure and at the same time increasing the antioxidant
ability of the biological system may reduce the damage.
Medicinal plants produce significant amounts of antioxidants
such as flavonoids, phenolics and polyphenolics compounds to
prevent the body from oxidative stress that could be caused by
reactive oxygen and nitrogen species. Therefore, this review
assessed the role of important antioxidants to combat these reactive
species.
Correspondence/Reprint request: Dr. Daniel Seifu, Addis Ababa University, School of Medicine, Department of
Biochemistry; Addis Ababa, Ethiopia. E-mail: [email protected]
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1. Introduction to medicinal plants
The use of plants as medicines goes back to early man. Certainly the
great civilizations of the ancient Chinese, Indians, and North Africans
provided written evidence of man's ingenuity in utilizing plants for the
treatment of a wide variety of diseases. In ancient Greece, for example,
scholars classified plants and gave descriptions of them thus aiding the
identification process. Theophrastus has been described by some as the father
of botany but little, if anything, has been recorded on his distant relative J.B.
Theophrastus who extolled the virtues of medicinal plants and forecast the
possibility of discovering flavonoids. As Europe entered the dark ages much
of this information would have been lost had it not been for the monasteries
that acted as centers for the production of medicinal plants which were used
to heal the suffering of mankind.
It was not until the 19th century that man began to isolate the active
principles of medicinal plants and one particular landmark was the discovery
of quinine from Cinchona bark by the French scientists Caventou and
Pelletier. Much less is known about the isolation of quinine by J.B. Caventou
and J.B. Pelletier. Such discoveries led to an interest in plants from the New
World and expeditions scoured the almost impenetrable jungles and forests in
the quest for new medicines [1].
Like other Plants, medicinal plants synthesize a vast range of organic
compounds that are traditionally classified as primary and secondary
metabolites although the precise boundaries between the two groups can in
some instances be somewhat blurred. Primary metabolites are compounds
that have essential roles associated with photosynthesis, respiration, and growth
and development. These include phytosterols, acyl lipids, nucleotides, amino
acids and organic acids. Other phytochemicals, many of which accumulate in
surprisingly high concentrations in some species, are referred to as secondary
metabolites. These are structurally diverse and many are distributed among a
very limited number of species within the plant kingdom. Although ignored for
long, their function in plants is now attracting attention as some appear to have
a key role in protecting plants from herbivores and microbial infection, as
attractants for pollinators and seed-dispersing animals, as allelopathic agents,
ultra violet (UV) protectants and signal molecules in the formation of nitrogenfixing root nodules in legumes. Secondary metabolites are also of interest
because of their use as dyes, fibers, glues, oils, waxes, flavoring agents, drugs
and perfumes, and they are viewed as potential sources of new natural drugs,
antibiotics, insecticides and herbicides.
In recent years the role of some secondary metabolites as protective
dietary constituents has become an increasingly important area of human
Medicinal plants as antioxidant agents
99
nutrition research. Unlike the traditional vitamins they are not essential for
short-term well-being, but there is increasing evidence that modest long-term
intakes can have favorable impacts on the incidence of cancers and many
chronic diseases, including cardiovascular disease and Type II diabetes [2].
Medicinal plants antioxidant activity is mainly due to the presence of
secondary metabolites [3]. Based on their biosynthetic origins, plant
secondary metabolites can be divided into three major groups: flavonoids and
allied phenolic and polyphenolic compounds, terpenoids and nitrogencontaining alkaloids and sulphur-containing compounds [2].
1.1. Phenolic compounds (flavonoids and phenolic acids)
Phenolic compounds possess one or more aromatic rings and one or more
hydroxyl groups. They are the products of secondary metabolism in plants,
providing essential functions in the reproduction and the growth of the plants;
acting as defense mechanisms against pathogens, parasites, and predators, as
well as contributing to the color of plants. In addition to their roles in plants,
phenolic compounds in diet provide health benefits. They can be grouped in
to two major categories:
Cranberry, apple, red grape, strawberry, pineapple, banana, peach,
lemon, orange, pea grape fruit, broccoli, spinach, yellow onion, red pepper,
carrot, cabbage, potato, lettuce, celery, cucumber and others, shown on figure
1.1, are some of the best food sources of phenolic compounds [4].
Figure 1.1. Best dietary sources of phenolic compounds.
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1.1.1. Phenolic acids
The simplest Phenolic compounds commonly found in plants. Generally,
they can be classified in two broad categories based on their chemical nature:
Benzoic acid derivatives and cinnamic acid derivatives as elucidated in figure
1.2.
1.1.2. Flavonoids
Flavonoids are a group of polyphenolic compounds, which are widely
distributed throughout the plant kingdom. Many have low toxicity in
mammals. Flavonoids exhibit several biological effects such as antiinflammatory, anti-hepatotoxic and anti-ulcer actions. They also inhibit
enzymes such as aldose reductase and xanthine oxidase. They are potent
antioxidants and have free radical scavenging abilities. Many have antiviral
actions and some of them provide protection against cardiovascular mortality.
They have been shown to inhibit the growth of various cancer cell lines in
vitro, and reduce tumour development in experimental animals.
Flavonoids occur as aglycones, glycosides and methylated derivatives.
The flavonoid aglycone consists of a benzene ring (A) condensed with a six
memberring (C), which in the 2-position carries a phenyl ring (B) as a
substituent (Figure 1.3). Six-member ring condensed with the benzene ring is
either a flavonols and flavonones or its dihydroderivative (flavanols and
flavanones). The position of the benzenoid substituent divides the flavonoid
Phenolic acids
Cinnamic acid derivatives
Benzoic acid derivatives
R
R
COOH
COOH OH
OH
R1
R1
R=R1=H; P-Hydroxybenzoic Acid
R=R1=H; Coumaric Acid
R=OCH3, R1=H; vanillic Acid
R=OH, R1=H; Caffeic Acid
R=R1=OH; Gallic Acid
R=OCH3, R1=H; Ferulic Acid
R=R1=OCH3; Syringic Acid
R=R 1=OCH3; Sinapic Acid
Figure 1.2. Two major classes of Phenolic acids with examples.
Medicinal plants as antioxidant agents
101
Figure 1.3. Six major classes of flavonoids with examples.
class into flavonoids (2-position) and isoflavonoids (3-position). Flavonols
differ from flavonones by hydroxyl group the 3-position and C2-C3 double
bonds. Flavonoids are often hydroxylated in position methyl ethers and acetyl
esters of the alcohol group are known to occur in nature. When glycosides are
formed, the glycosidic linkage is normally located in positions 3 or 7 and the
carbohydrate can be L-rhamnose, D-glucose,gluco-rhamnose, galactose or
arabinose [5].
1.2. Terpenoids
The term terpene refers to a hydrocarbon molecule, while terpenoid
refers to a terpene that has been modified, for example by the addition of
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oxygen [6]. Terpenes or isoprenoids, are one of the most diverse classes of
secondary metabolites which play variety functional roles in plants as
hormones (gibberellins, abscisic acid), photosynthetic pigments (phytol,
carotenoids), electron carriers (ubiquinone, plastoquinone), mediators of
polysaccharide assembly (polyprenyl phosphates), and structural components
of membranes (phytosterols). More than 55,000 different terpenoids have
been isolated, and this number has almost doubled each decade, many of
which are of plant origin. Terpenoids are essential for plant growth,
development, and general metabolism. Terpenoids are found in almost all
plant species [7, 8, 9].
In plants, terpenoid biosynthesis occurs by two different pathways to
synthesize the main building block Inositol pyrophosphate (IPP), (a) the
Mevalonic acid pathway or HMG-CoA reductase pathway that occurs in
cytosol and produces IPP for sesquiterpenoids, (b) methylerythritol
phosphate/1-deoxy-D-xylulose (MEP/DOX) pathway forms IPP in the
chloroplast for mono and diterpenoids [10].
Generally based on the number of building blocks, i.e., the isoprenoid
units, terpenoids are classified into several classes, such as Hemeterpene,
monoterpenes (e.g., carvone, geraniol, d-limonene, and perillyl alcohol),
diterpenes (e.g. retinol and trans-retinoic acid), triterpenes [e.g., Betulinic
Acid (BA), lupeol, oleanic acid, and Ursolic Acid (UA)], and tetraterpenes
(e.g., α-carotene, β-carotene, lutein, and lycopene) [11].Different terpenoids
molecules have antioxidant, antiviral, antibacterial, antimalarial,
antiinflammatory, inhibition of cholesterol synthesis, antiallergenic,
antihyperglycemic, immunomodulatory and anticancer activities [12, 13].
1.3. Alkaloids
Alkaloids are a diverse group of low molecular weight, nitrogencontaining compounds mostly derived from amino acids. Alkaloids are
thought to play a defensive role in the plant against herbivores and pathogens.
Plant-derived alkaloids currently in clinical use include analgesics, antineoplastic agent, gout suppressant, muscle relaxants, antiviral, cytotoxic,
antinociceptive, anticholinergic, antiinflammatory and DNA-binding
activities and some of them have also been used in the treatment of
Alzheimer‟s disease, myasthenia gravis and myopathy [14,15].
Alkaloids can be classified into families, on the basis of structural
similarities and the amino acids that are used for their biosynthesis. Some
alkaloids are also produced using building blocks derived from other
secondary metabolic pathways, such as terpenoids, polyketides and peptides
[16]. Some of the important classes of alkaloid are shown below: Terpenoid
Medicinal plants as antioxidant agents
103
Indole Alkaloids (TIAs) comprise a family of greater than 3000 compounds
that includes the antineoplastic agent‟s vinblastine and camptothecin, the
antimalarial drug quinine, and the rat poison strychnine. Some TIAs have
been proposed to play a role in the defense of plants against pests and
pathogens. TIAs consist of an indole moiety provided by tryptamine and a
terpenoid component derived from the iridoid glucoside secologanin [17].
The benzylisoquinoline alkaloids are a very large and diverse class of
alkaloids with > 2500 defined structure. This family contains such varied
physiologically active members as emetine (an antiamoebic), colchicines (a
microtubule disrupter and gout suppressant), berberine (an antimicrobial
against eye and intestinal infections), morphine (a narcotic analgesic),
codeine (a narcotic analgesic and antitussive), and sanguinarine (an
antimicrobial used in oral hygiene) [18].
Tropane alkaloids (TPAs) occur mainly in the Solanaceae. There
principal characterstics pyrollic ring derived from the ornithine and arginine
aminoacids by a chemical reaction catalysed by ornithine decaboxylase and
Arginine decarboxylase respectively. This class of alkaloid includes the
anticholinergic drugs atropine, hyoscyamine, and scopolamine, and the
narcotic tropical anesthetic cocaine. Although nicotine is not a member of the
tropane class, the N-methyl-∆1-pyrrolinium cation involved in TPA
biosynthesis is also an intermediate in the nicotine pathway [17, 19].
Purine alkaloids such as caffeine, theobromine, and theacrine are widely
distributed in the plant kingdom. Caffeine, a nonselective adenosine A1 and
A2A receptor antagonist, is the most widely used psychoactive substance in
the world. Evidence demonstrates that caffeine and selective adenosine A 2A
antagonists interact with the neuronal systems involved in drug
reinforcement, locomotors sensitization, and therapeutic effect in Parkinson's
disease (PD) [20].
2. Metabolism of phenolic compounds
2.1. Intakes
Flavonoids intake seem to vary greatly between countries; the lowest
intakes (2.6 mg/d) are in Finland and the highest intakes (68.2 mg/d) are in
Japan. Quercetin is the most important contributor to the estimated intake of
flavonoids, mainly from the consumption of apples and onions. A major
problem in cohort studies of flavonoids intake is that only a limited number
of flavonoids can be measured in biological samples, and more importantly,
only a relatively small number of fruit and vegetables are used to make an
accurate estimation [21].
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2.2. Intestinal absorption
Much about the intestinal mechanisms of the gastrointestinal absorption
of polyphenols remains unknown. Most polyphenols are probably too
hydrophilic to penetrate the gut wall by passive diffusion, but the membrane
carriers that could be involved in polyphenol absorption have not been
identified. The unique active transport mechanism that has been described is
a Na+-dependent saturable transport mechanism involved in cinnamic and
ferulic acid absorption in the rat jejunum [22].
In foods, all flavonoids except flavanols are found in glycosylated forms,
and glycosylation influences absorption. The fate of glycosides in the
stomach is not clear. Experiments using surgically treated rats in which
absorption was restricted to the stomach showed that absorption at the gastric
level is possible for some flavonoids, such as quercetin and daidzein, but not
for their glycosides. Most of the glycosides probably resist acid hydrolysis in
the stomach and thus arrive intact in the duodenum. Only aglycones and
some glucosides can be absorbed in the small intestine, whereas polyphenols
linked to a rhamnose moiety must reach the colon and be hydrolyzed by
rhamnosidases of the microflora before absorption. The same probably
applies to polyphenols linked to arabinose or xylose, although question has
not been specifically studied. Because absorption occurs less readily in the
colon than in the small intestine because of a smaller exchange area and a
lower density of transport systems, as a general rule, glycosides with
rhamnose are absorbed less rapidly and less efficiently than are aglycones
and glucosides. This has been clearly shown in humans for quercetin
glycosides: maximum absorption occurs 0.5–0.7 hour after ingestion of
quercetin 4'-glucoside and 6–9 hours after ingestion of the samequantity of
rutin (quercetin-3-rutinoside). The bioavailability of rutin is only 15–20% of
that of quercetin 4'-glucoside, its structure shown in figure 2.1. Similarly,
absorption of quercetin is more rapid and efficient after ingestion of onions,
which are rich in glucosides, than after ingestion of apples containing both
OH
O
OH
glucose
O
OH O
OH
Figure 2.1. Structure of quercitin 4'- glucoside.
Medicinal plants as antioxidant agents
105
glucosides and various other glycosides. In the case of quercetin glucosides,
absorption occurs in the small intestine, and the efficiency of absorption is
higher than that for the aglycone itself. The underlying mechanism by which
glucosylation facilitates quercetin absorption has been partly elucidated.
Glucosides could be transported into enterocytes by the sodium-dependent
glucose transporter (SGLT1). They could then be hydrolyzed inside the cells
by a cytosolic-glucosidase [23].
Another pathway involves the lactase phloridzine hydrolase, a
glucosidase of the brush border membrane of the small intestine that
catalyzes extracellular hydrolysis of some glucosides, which is followed by
diffusion of the aglycone across the brush border. Both enzymes are probably
involved, but their relative contribution for the various glucosides remains to
be clarified. Quercetin 3-glucoside, which is not a substrate for cytosolicglucosidase, is certainly absorbed after hydrolysis by lactase phloridzine
hydrolase, at least in rats, whereas hydrolysis of quercetin 4'-glucoside seems
to involve both pathways. In humans, whatever the mechanism of
deglucosylation, the kinetics of plasma concentrations is similar after ingestion
of quercetin 3-glucoside or quercetin 4'-glucoside. Isoflavone glycosides
present in soya products can also be deglycosylated by glucosidases from the
human small intestine. However, the effect of glucosylation on absorption is
less clear for isoflavones than for quercetin [24].
2.3. Metabolism
2.3.1. Flavonoids conjugation
A. Glucuronidation of flavonoids in the intestine
Although flavonoids aglycons were supposed to be rapidly absorbed after
oral ingestion, their plasma concentrations are found to be very low whereas
the phase II metabolites such as glucuronides, sulfates, and methylated
conjugates seem to be predominant in blood circulation. Therefore, liver and
intestine are thought to be responsible for the extensive first-pass metabolism
of flavonoids, and glucuronidation mediated by various UDP
glucuronosyltransferases (UGTs) is suggested to be one of the most
important metabolic pathways of flavonoids in both liver and intestine. Quite
a number of studies in human have demonstrated the contribution of UGTs to
the first-pass glucuronidation of flavonoids. For instance, after intake of
kaempferol in human, 3-O-glucuronide conjugate of kaempferol was found to
be the predominant form in plasma. Epicatechin glucuronide was detected as
the main metabolite in human plasma after ingestion of flavonoid
procyanidins and flavan-3-ols enriched cocoa milk drinks. The conjugate
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metabolites, namely epicatechin-3'-O-glucuronide, 4′-O-methyl- epicatechin3'-O -glucuronide, and 4'-O-methyl-epicatechin- 5 or 7-O-glucuronide, were
identified in human after intake of epicatechin. It was also found that
quercetin-3-O-glucuronide
together
with
quercetin-3'-Osulfateandisorhamnetin- 3-O-glucuronide was dominant in human plasma
after oral administration of quercetin [25].
B. Glucuronidation of flavonoids in the liver
Comparing with the investigation of intestinal first-pass metabolism of
flavonoids, studies on the hepatic first-pass glucuronidation of flavonoids
were limited. By comparing the concentrations of Quercetin after its
intravenous and intra portal administration to rats, the hepatic extraction ratio
was determined to be about 52.6%, and extensive hepatic glucuronidation of
Quercetin was suggested due to the finding of glucuronides as the major
metabolites of quercetin. Although limited studies were designed to
specifically evaluate the contribution of glucuronidation of flavonoid in the
liver, its importance should be aware of due to high content of UGTs present
in the liver [26].
C. Enzymes mediating glucuronidation of flavonoids
Glucuronidation is a process of metabolism catalyzed by UDPglucuronosyltransferases (UGTs). To date, more than 20 UGT isoforms have
been identified from the endoplasmic reticulum of different tissues
responding for catalyzing the biotransformation of hydrophobic substrates to
hydrophilic glucuronides. Liver was found to contain most of UGT isoforms
and UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15 are thought to be the
most important for the drug glucuronidation in the liver. Studies on the
extrahepatic distribution discovered that intestine also contains a large
number of UGTs. For example, UGT 1A1, 1A3, 1A4, 1A6, 2B15, and 2B4
were also revealed in the intestine, whereas UGT 1A7, 1A8, and 1A10 were
found only expressed in the intestine but not in the liver. By using
recombinant human UGTs, specific isoforms of UGT have been identified for
the glucuronidation of various flavonoids. UGT 1A3 was reported to mediate
the glucuronidation of flavonoids including naringenin, apigenin, galangin,
fisetin, 7-hydroxyflavone, genestein and quercetin. Kaempferol and quercetin
were demonstrated to be the substrate of UGT 1A9. UGT 1A9, 1A1, and
2B15 were reported to catalyze the glucuronidation of galangin.UGT 1A1,
1A8, and 1A9 were involved in the glucuronidation of luteolin and quercetin.
Moreover, UGT 1A7 displayed differential activities toward flavonoids such
Medicinal plants as antioxidant agents
107
as chrysin, apigenin, galangin, fisetin kaempferol, morin, quercetin, etc.
UGT 1A10 mainly found in gastrointestinal tract catalyzed the
glucuronidation of a number of flavonoids, including apigenin, chrysin,
luteilin morin, daidzine genistein naringenin. Recent study on baicalein was
found that it is the substrate of various UGT isozymes including UGT 1A1,
1A3, 1A8, 1A7, 1A9, and 2B15 [27].
D. Tissue uptake of flavonoids
When single doses of radiolabeled polyphenols (quercetin,
epigallocatechin gallate, quercetin 4'-glucoside, resveratrol) are given to rats
or mice killed 1–6 hour later, radioactivity is mainly recovered in blood and
in tissues of the digestive system, such as the stomach, intestine, and liver.
However, polyphenols have been detected by high performance liquid
chromatography (HPLC) analysis in a wide range of tissues in mice and rats,
including brain, endothelial cells, heart, kidney, spleen, pancreas, prostate,
uterus, ovary, mammary gland, testes, bladder, bone, and skin. The
concentrations obtained in these tissues ranged from 30 to 3000 ng aglycone
Equivalents/g tissue depending on the dose administered and the tissue
considered. It is still difficult to say whether some polyphenols accumulate in
specific target organs. A few studies seem to indicate that some cells may
readily incorporate polyphenols by specific mechanisms. The endothelium is
likely to be one of the primary sites of flavonoid action. Energy-dependent
transport system is active in aortic endothelial cells for the uptake of morin.
This system may also transport other hydroxylated phenolic compounds.
Microautoradiography of mice tissues after administration of radiolabeled
epigallocatechin gallate or resveratrol indicated that radioactivity is unequally
incorporated into the cells of organs. Regional selectivity has also been
observed in the prostate and the brain [24].
E. Elimination of flavonoids
Metabolites of polyphenols may follow two pathways of excretion, i.e.,
via the biliary or the urinary route. Large, extensively conjugated metabolites
are more likely to be eliminated in the bile, whereas small conjugates such as
monosulfates are preferentially excreted in urine. In laboratory animals, the
relative magnitude of urinary and biliary excretion varies from one
polyphenols to another. Biliary excretion seems to be a major pathway for the
elimination of genistein, epigallocatechin gallate, and eriodictyol. Biliary
excretion of polyphenols in humans may differ greatly from that in rats
because of the existence of the gall bladder in humans; however, this has
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never been examined. Intestinal bacteria possess-glucuronidases that are able
to release free aglycones from conjugated metabolites secreted in bile.
Aglycones can be reabsorbed, which results in enterohepatic cycling [24].
The general metabolism process, oral ingestion, absorption, Glucuronidation,
tissue uptake and elimination of flavonoids are elucidated in figure 2.2.
F. Toxicity of flavonoids
There is much controversy regarding the purported toxic or even
mutagenic properties of quercetin. At a conference of the Federation of
American Societies for Experimental Biology in 1984 on mutagenic food
flavonoids, carcinogenicity was reported in just 1 of 17 feeding studies
conducted in laboratory animals. Furthermore, at early time researchers also
reported that high doses of quercetin over several years might result in the
formation of tumors in mice. However, in other long-term studies, no
carcinogenicity was found. In contrast with the potential mutagenic effects of
flavonoids in earlier studies, several more recent reports indicate that
flavonoids, including quercetin, seem to be antimutagenic in vivo. A large
clinical study, in which 9959 men and women were followed for 24 years,
showed an inverse relation between the intake of flavonoids (for example
quercetin) and lung cancer. One possible explanation for these conflicting
data is that flavonoids are toxic to cancer cells or to immortalized cells, but
are not toxic or are less toxic to normal cells. If this is true, flavonoids might
play a role in the prevention of cancer [21].
Figure 2.2. Possible routs of dietary flavonoids after oral injection [28].
Medicinal plants as antioxidant agents
109
3. Free radicals and oxidative stress
3.1. Free radicals
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are
the terms collectively describing free radicals and other non-radical reactive
derivatives also called oxidants. Radicals are less stable than non-radical
species, although their reactivity is generally stronger. A molecule with one
or more unpaired electron in its outer shell is called a free radical. They
include hydroxyl (OH•), superoxide (O2•ˉ), nitric oxide (NO•), nitrogen
dioxide (NO2•), peroxyl (ROO•) and lipid peroxyl (LOO •). Also, hydrogen
peroxide (H2O2), ozone (O3), singlet oxygen (1O2), hypochlorous acid
(HOCl), nitrous acid (HNO2), peroxynitrite (ONOOˉ), dinitrogen trioxide
(N2O3), lipid peroxide (LOOH), are not free radicals and generally called
oxidants, but can easily lead to free radical reactions in living organisms [29].
Biological free radicals are thus highly unstable, which seek stability through
electron pairing with biological macromolecules such as proteins, lipids and
DNA in healthy human cells, cause protein and DNA damage along with
lipid peroxidation. These changes contribute to cancer, atherosclerosis,
cardiovascular diseases, ageing, and inflammatory diseases [30].
Reactive oxygen species (ROS) mostly originate from three sources: the
mitochondrial electron transport chain, NADPH oxidase and myeloperoxidase
of neutrophils and xanthine oxidase of endothelial cells (figure 3.1). However,
Figure 3.1. Describes the generation of free radicals and some mechanism of removal
of ROS [34].
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Primary sources of ROS are the mitochondrial respiratory chain and xanthine
oxidase. There is also a delayed and amplified generation of ROS due to the
inflammatory response initiated by cytokines released from the damaged cells
[31].
To deal with reactive species (RS), the body is equipped with an
effective defense system, which includes: enzymes such as superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GP), and
glutathione reductase (GR); high molecular weight antioxidants such as
albumin, ceruloplasmin, and ferritin; and an array of low-molecular-weight
antioxidants such as ascorbic acid, α-tocopherol, β-carotene, glutathione
(GSH), and uric acid [32].
In health, balance between production of ROS/RNS and antioxidant
defenses lies slightly in favour of ROS/RNS production. Oxidative stress
occurs when there is an imbalance between free radical reactions and the
scavenging capacity of antioxidative defense mechanism of the organism
(figure 3.1).
Oxidative stress is a severe disruption of balance in favour of ROS/RNS.
In principle, oxidative stress can result from increased production of
ROS/RNS, excessive activation of phagocytic cells in chronic inflammatory
diseases, diminished antioxidants. For example, mutations affecting
antioxidant defense systems and depletions of dietary antioxidants and
micronutrients [33].
3.2. Antioxidant activities of phenolic compounds
Antioxidants may intervene at different levels in the oxidative process
(for example, by scavenging for free radicals and lipid peroxyl radicals ,
Cellular
defenses
ROS
or/and
RNS
Figure 3.2. Oxidative stress: occurs when there is an imbalance between the
production of ROS and /or RNS at one side and cellular defenses in the other.
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Medicinal plants as antioxidant agents
removing oxidatively damaged bio-molecules, and having other types of
action). Antioxidants can be grouped in to two: synthetic and natural
antioxidants [35]. Many synthetic antioxidants such as butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate
(PG) have been used to retard the oxidation process [36].
In recent years, there has been a growing interest in the search for natural
antioxidants, especially secondary metabolites, for three principal reasons:
A. Numerous clinical and epidemiological studies have demonstrated that
consumption of fruits and vegetables is associated with reduced risks of
developing chronic diseases such as cancer, cardio-vascular disorders
and diabetes;
B. Safety consideration regarding the potential harmful effects of the
chronic consumption of synthetic antioxidants in foods and beverages;
and
C. The public‟s perception that natural and dietary antioxidant are safer than
synthetic analogues. The result has been an increased interest in spices,
aromatic and medicinal plants as sources of natural antioxidants [37].
3.2.1. Phenolic acids
The antioxidant activity of Phenolic acids is related to the acid moiety
and the number and the relative positions of hydroxyl groups on the aromatic
ring structure. Hydroxycinnanic acids are more effective antioxidant than
hydroxybenzoic acids due to the increased possibilities for delocalization of
phenoxyl radical (figure 3.3). Benzoic and cinnamic acid, neither of which
possesses free hydroxyl groups, have no free radical scavenging activities.
Di- and tri-hydroxylation increase the activity over a single hydroxyl group
with the position of the hydroxyl groups being the most important factor.
HO
HO
COOH
OH
COOH
HO
OH
HO
(a)
(b)
Figure 3.3. (a) polyhydroxybenzoic acid and (b) polyhdroxycinnamic acid.
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Hydroxylation in the 2- and 4- positions or in the 3-, 4-, 5-positions confer
the greatest antioxidant activity. As substituents increase the electron density
on the hydroxyl group cause a decrease in the dissociation energy of the O-H
bond. Therefore electron-donating substituents will increase the antioxidant
activity, as in case of vanillic acid relative to the p-hydroxybenzoic acid.
Hydroxycinnamic acid esters, such as caffeoyltaric acid,
p-coumaroyltartaric acid and chorogenic acid, exhibit greater antioxidant
activity than the parent hydroxycinnamic acids, possibly due to increased
possibilities for electron delocalization [38].
3.2.2. Flavonoids
Flavonoids have long been acknowledged for their unique antioxidant
properties. They may prevent production of oxidants (e.g. by inhibition of
xanthine oxidase and chelation of transition metals), inhibit oxidants from
attacking cellular targets (e.g. by electron donation and scavenging
activities), block propagation of oxidative reactions (by chain-breaking
antioxidant activity), and reinforce cellular antioxidant capacity (through
sparing effects on other antioxidants and inducing expression of endogenous
antioxidants). Flavonoids also possess anti- inflammatory and anti-platelet
aggregation effects through inhibiting relevant enzymes and signaling
pathways, resulting ultimately in lower oxidant production. Finally,
flavonoids used as vasodilator and have a lot medicinal values [30].
3.2.2.1. Free radical scavenging activity of flavonoids
Flavonoid antioxidants function as scavengers of free radicals by rapid
donation of a hydrogen atom to radicals. In general, the radical-scavenging
activity of flavonoids depends on the molecular structure and the substitution
pattern of hydroxyl groups, that is, on the availability of phenolic hydrogens
and on the possibility of stabilization of the resulting phenoxyl radicals via
hydrogen bonding or by expanded electron delocalization (figure 3.4).
Previous structure-activity relationship (SAR) studies of flavonoids have
pointed to the importance of the number and location of the phenolic OH
groups present for the antiradical efficacy. The structural requirement
considered to be essential for effective radical scavenging by flavonoids is
the presence of a 3‟, 4‟-dihydroxy, i.e., a O-dihydroxy group (catechol
structure) in the B ring, possessing electron donating properties and being a
radical target.
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Medicinal plants as antioxidant agents
Prevention
of redox
reaction
Metal
chelation
Direct scavenging
of ROS
Prevention
of ROS
formation
Inhibition of
xanthine oxidase
Flavonoids
Inhibition of
NADPH oxidase
Protection
of lipophilic
antioxidants
Induction of
antioxidant
enzymes
Figure 3.4. Mechanism of antioxidant effect of flavonoids.
Also, the 3-OH moiety of the C ring is also beneficial for the antioxidant
activity of flavonoids.The C2-C3 double bond conjugated with a 4-keto group,
which is responsible for electron delocalization from the B ring, enhances
further the radical-scavenging capacity, and saturation of the 2, 3-double bond
is believed to cause a loss of activity potential. Also, the presence of both 3-OH
and 5-OH groups in combination with a 4-carbonyl function and C2-C3 double
bond increases the radical scavenging activity. In the absence of the
O-dihydroxy structure in the B ring, hydroxyl substituent in a catechol structure
on the A-ring was able to compensate and become a larger determinant of
flavonoid antiradical activity. Figure 3.5, summarizes the structural criteria that
modulate the free radical scavenging activity of flavonoids [39].
In summary, these structural features contribute to the increase of the
phenoxyl radical stability, that is, the radical scavenging activity of the parent
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Daniel Seifu et al.
flavonoid. DPPH• is a free radical compound and it has been widely used to
test the free radical scavenging ability of flavonoids. The scavenging of
DPPH• by flavonoid (free radical scavenger) can be represented as depicted
in figure 3.6 and 3.7 [40].
3.2.2.2. Chelation of transition metals by flavonoids
The antioxidant activity of flavonoids can be explained through their
chelating action (figure 3.8). They bind with transition metal particularly iron
and copper and thus inhibit of transition metal-catalysed free radical
formation. The two most likely points of attachment of transition metal ions
to the flavonoids are the o-diphenolic groups at the 3', 4',-dihydroxy positions
OH
3'
2'
OH
9
7
A
6
5
HO
B
5'
1
8
OH
OH
4'
O
1'
2
C
10
6'
4
OH
3
O
Figure 3.5. Structure of representative flavonoid.
R
R
OH
O
OH
O
OH
HO
+
x
OH
O
O
+
O
HO
Favonol free radical
Oxidized flavonol
XH
O
Reduced free radical
Figure 3.6. Structure of flavonols and their oxidized form.
Figure 3.7. In vitro free radical scavenging activity of flavonoids.
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Medicinal plants as antioxidant agents
OH
OH
HO
O
OH
H
O
O
Mn+
or
OH
H
Mn+
O
OH
HO
O
O
HO
OH
OH
H
O
Mn+
OH
O
OH
O
OH
OH
or
HO
O
O
OH
H
O
Mn+
Figure 3.8. Chelation of transition metals by flavonoids (M n+ = transition metal ions
like Fe2+ and Cu+ ions).
in the B ring and the ketol structures, 4-keto, 3-hydroxy or 4-keto, 5-hydroxy
in the C ring. Chelated in this way, transition metals would be unavailable to
interact with other compounds and initiate biologically damaging reactions.
Flavonoids inhibit lipid peroxidation, oxidation of linoleic acid and Fe +2
catalyzed oxidation of glutamine synthase, through free radical scavenging
and removal of metal ions from catalytic sites via chelation [41]. Due to their
reducing powerthese phytochemicals act as both antioxidant and pro-oxidant
depending upon the exposed environment. They act as prooxidant in the
absence of free radicals. The classical antioxidants, α-tocopherol and vitamin
C, are also reported to behave in a similar fashion. Catechins, abundant in
green tea, also possess the antioxidative and pro-oxidative characteristics of
Cu+2 induced low density lipoprotein (LDL) oxidation [42].
3.2.2.3. Inhibition of xanthine oxidase activity by flavonoids
The xanthine oxidase pathway has been implicated as an important route
in the oxidative injury to tissues, especially after ischemia-reperfusion. Both
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Daniel Seifu et al.
xanthine dehydrogenase and xanthine oxidase are involved in the metabolism
of xanthine to uric acid in the last two steps of purin metabolism. Xanthine
dehydrogenase is the form of the enzyme present under physiologic
conditions, but its configuration is changed to xanthine oxidase during
ischemic conditions. Xanthine oxidase is a source of oxygen free radicals. In
the reperfusion phase (i.e. reoxygenation), xanthine oxidase reacts with
molecular oxygen, thereby releasing superoxide free radicals (figure 3.9 and
3.10) [21].
Flavonoids such as luteolin, silibinin, and quercetin inhibit xanthine
oxidase, and suggested that these compounds, or derivatives of them, may be
useful leads in the development of clinically useful inhibitors of XO.
Quercetin and luteolin have stronger inhibition effect relative to silibinin.
It seems likely that luteolin, silibinin, and quercetin position in the active site
of XO with the dihydroxybenzene functionality of their benzopyran moiety
directed toward the molybdenum cofactor, their benzopyran intercalated
between Phe914 and Phe1009, their C1 carbonyl groups directed toward
Arg880 [43].
O
O
NAD+
N
NADH + H
+
N
NH
NH
O
xanthine
dehydrogenase
N
NAD+
N
NH
N
H
O
NADH + H+
N
H
N
H
xanthine dehydrogenase
N
H
O
Xanthine
Hypoxanthine
N
H
O
Uric Acid
Figure 3.9. Metabolism of hypoxanthine xanthine to uric acid under normal
physiological condition.
quercentin
quercentin
O
N
N
H
O
O2
O2 H2O2
NH
O
O2
N
O2
Hypoxanthine
N
NH
O
xanthine oxidase
N
H2O2
NH
N
H
Xanthine
N
H
xanthine oxidase
O
N
H
N
H
O
Uric Acid
Figure 3.10. Reperfusion phase of ischemic condition and xanthine oxidase inhibition
by quercentin.
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Medicinal plants as antioxidant agents
3.2.2.4. Inhibition of NADPH oxidases activity by flavonoids
Initially, NADPH oxidases were thought to be enzymes present only in
phagocytes of the innate immune response, where they are responsible for
generating large amounts of O2•− to kill invading pathogens (“oxidative
burst”). Upon activation, O2 is reduced to O2•− by the transfer of one electron
from the reducing equivalent NADH or NADPH (figure 3.11). However,
these enzymes that exist only to produce free radicals appeared unexpectedly
in nonphagocytic cells, and it became evident that NADPH oxidases exist in
various cell types [44]. Recently, a novel flavonoid derivative S17834
[6,8- diallyl 5, 7-dihydroxy 2-(2-allyl 3-hydroxy 4-methoxyphenyl) 1-H
benzo(b)pyran-4-one] has been reported to directly inhibit vascular NADPH
oxidase in vitro [45]. Although not yet investigated for this mechanism in
ischemia– reperfusion, flavonoids have shown ability to suppress enzyme
activity and/or expression of NADPH oxidases [30].
NADPH
O2
NADPH
oxidase
O2
NADP
Plasma membrane
Figure 3.11. Generation of superoxide by NADPH oxidases.
3.2.2.5. Reinforcement of cellular antioxidants by flavonoids
Human studies have shown depletion of non-enzymatic antioxidants such
as glutathione, ascorbic acid, and vitamin E following myocardial ischemia–
reperfusion. Hydrophilic antioxidants, such as ascorbate and glutathione,
have shown to work at the front line of defense against oxidative stress,
protecting lipophilic antioxidants such as ubiquinol and vitamin E from
oxidation. Ascorbic acid also helps to regenerate vitamin E from its oxidized
form, and is in turn recycled by glutathione, although vitamin C is also
needed for the recovery of glutathione from its oxidized form. In such a
network, flavonoids are proposed to act as intermediate antioxidants,
protecting lipophilic antioxidants and being protected by hydrophilic
antioxidants [30].
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3.2.2.6. Induction of phase two enzymes activities of flavonoids
The antioxidant effect of flavonoids and other phytochemicals may be
exerted indirectly through induction of phase two enzymes. Phase two
enzymes are proteins whose expression is coordinately regulated by an
antioxidant response element (ARE) located in the promoter region of the
corresponding genes. Since phase two enzymes are committed to
neutralization and detoxification of xenobiotics and electrophiles, inducers of
such genes may deliver protection against oxidative stress. One of the phase
two enzymes, heme oxygenase-1, has been recognized as an important
mediator of the delayed phase of ischemia preconditioning, and its over
expression has led to reduced ventricular remodeling and hypertrophy and
better myocardial recovery and contractile function.
Over the last decade, a large number of investigations have indicated the
ability of flavonoids to induce phase two enzymes in animals and human cell
cultures. This ability of epigallocatechin gallate has recently been reviewed.
However, whether flavonoids can induce phase two enzymes in heart and
thereby provide advance protection against ischemia–reperfusion injury is not
yet investigated [46].
3.3. Antioxidant activity of terpenoids: Carotinoids
Carotenoids are natural pigments synthesized by plants and
microorganisms, but not by animals. Carotenoids are classified as follows: 1)
Carotenoid hydrocarbons are known as carotenes and contain specific end
groups. Lycopenes have two acyclic end groups. β-Carotene has two
cyclohexene type end groups. 2) Oxygenated carotenoids are known as
xanthophyls. Examples of these compounds are a zeaxanthin and lutein
(hydroxy), b) spirilloxanthin (methoxy), c) echinenone (oxo), and d)
antheraxanthin (epoxy) [47].
Carotenoids exert many important functions, among which are the
outstanding antioxidant effects in lipid phases by free radical scavenging or
singlet oxygen quenching. With regard to antioxidants activity in biological
systems, carotenoids appear to be involved protection against both singlet and
triple oxygen (as radical chain- breaking antioxidants). The best documented
antioxidant action of carotenoids is their ability to quench singlet oxygen
(which is known to be capable of damaging lipids, DNA and of being
mutagenic). This results in an excited carotenoid, which has the ability to
dissipate newly acquired energy through a series of rotational and vibrational
interactions with the solvent, thus regenerating the original unexcited
carotenoid, which can be reused for further cycles of singlet oxygen
Medicinal plants as antioxidant agents
119
quenching: 1O2 + Q O2 + 3Q where 1O2 represents singlet oxygen, Q denotes
quencher molecules, O2 and 3Q denotes triple oxygen and quencher
respectively. The quenching activity of a carotenoid mainly depends on the
number of conjugated double bonds of the molecule and is influenced to a
lesser extent by carotenoid end groups (cyclic or acyclic) or the nature of
substituents in carotenoids containing cyclic end groups. Lycopene (eleven
conjugated and two non conjugated double bonds) is among the most
efficient singlet oxygen quenchers of the natural carotenoids. The prevention
of lipid peroxidation by carotenoids has been suggested to be mainly via
singlet oxygen quenching [48, 49].
β -Carotene is also scavenger of peroxyl radicals, especially at low
oxygen tension. This activity may be also exhibited by others carotenoids.
The interactions of carotenoids with peroxyl radicals may precede via an
unstable β-carotene radical adduct. Carotenoid adduct radicals have been
shown to be highly resonance stabilized and are predicted to be relatively
unreactive. They may further undergo decay to generate non radical products
and may terminate radical reactions by binding to the attacking free radicals.
Carotenoids act as antioxidants by reacting more rapidly with peroxyl
radicals than do unsaturated acyl chains. In this process, carotenoids are
destroyed [50].
3.4. Antioxidant activities of alkaloids: Berberine
The antioxidant activity of berberine has been widely demonstrated.
First, it was reported that berberine (fig.3.12) can scavenge reactive oxygen
species (ROS) and reactive nitrogen species (RNS) in similar fashion with
flavonoids. For instance, among the RNS, peroxynitrites (ONOO−) generated
through the reaction between nitric oxide (NO·) and superoxide anion radical.
Secondly, berberine can inhibit lipid peroxidation and show protective effects
against low-density lipoprotein (LDL) oxidation. Thirdly, it has inhibitory
effects on lipoxygenase and xanthine oxidase, two important ROS-derived
Figure 3.12. Molecular structure of berberine [53].
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Daniel Seifu et al.
sources. Berberine also significantly increased superoxide dismutase activity
and decreased superoxide anion and malondialdehyde (MDA) formation In
addition, it was found that berberine can also bind catalyzing metal ions
(transition metals like iron and cupper ions), which can reduce the
concentration of metal ions in lipid peroxidation [51,52].
4. Other medicinal efficacy of secondary metabolites of
medicinal plants
4.1. Flavonoids
4.1.1. Flavonoids anti-inflammatory activity
Flavonoids have shown the capacity to inhibit enzymes involved in
eicosanoid pathways, including phospholipase A2, cyclooxygenasesand
lipoxygenases, thereby limiting production of inflammatorymediators such as
prostaglandins and leukotrienes. Flavonoids can also inhibit production of
pro-inflammatorycytokines, such as tumor necrosis factor -α (TNF α)-,
interleukin (IL)-1β, IL-6, and interferon-γ, as well as chemotactic agents
(figure 18) [30].
Cyclooxygenase and lipoxygenase play an important role as
inflammatory mediators. They are involved in the release of arachidonic acid,
which is a starting point for a general inflammatory response. Neutrophils
containing lipoxygenase create chemotactic compounds from arachidonic
acid. They also provoke the release of cytokines. Selected phenolic
compounds were shown to inhibit both the cyclooxygenase and
5-lipoxygenase pathways. This inhibition reduces the release of arachidonic
acid. The exact mechanism by which flavonoids inhibit these enzymes is not
clear. Quercetin, in particular, inhibits both cyclooxygenase and lipoxygenase
activities, thus diminishing the formation of these inflammatory metabolites.
Flavonoids also inhibit both cytosolic and membrane tyrosine kinase.
Integral membrane proteins, such as tyrosine 3-monooxygenase kinase, are
involved in a variety of functions, such as enzyme catalysis, transport across
membranes, and transduction of signals that function as receptors of
hormones and growth factors, and energy transfer in ATP synthesis.Inhibition
of these proteins results in inhibition of uncontrolled cell growth and
proliferation. Tyrosine kinase substrates seem to play key roles in the signal
transduction pathway that regulates cell proliferation. Another antiinflammatory property of flavonoids is their suggested ability to inhibit
neutrophil degranulation. This is a direct way to diminish the release of
arachidonic acid by neutrophils and other immune cells [21].
Medicinal plants as antioxidant agents
121
4.1.2. Antiplatelet aggregation activities of flavonoids
Thromboxane A2 (TxA2) is a powerful unstable inducer of platelet
activation and aggregation produced by sequential arachidonic acid
metabolism by cyclooxygenase and thromboxane synthase, upon activation
of platelets with agonists such as adenosine diphosphate, thrombin or
collagen. Once generated, TxA2 acts in an autocrine and paracrine manner,
increasing activation and recruitment of the surrounding platelets to the site
of vascular damage.
TxA2 effects on platelets, and on other target cells, are mediated via
interaction with specific seven- transmembrane G-protein-coupled receptors
(GPCR). The TxA2 receptor (TP) is encoded by a single gene that is
alternatively spliced in the carboxyl terminus resulting in two variants, TPa
(343 residues) and TPb (407 residues), that share the first 328 amino acids.
Several studies have shown that flavonoids impair agonistinduced TxA2
formation through the inhibition of arachidonic acid liberation and
metabolism by cyclooxygenase and TxA2 synthase activities[55]. Moreover,
studies using TxA2 analogues indicate that certain flavonoids, apigenin and
genistein, may behave as TP antagonists (figure 4.1) [56].
Figure 4.1.Structural features explaining the TP antagonistic activity of
flavonoids: (A) flavonoid active core believed to interact with the
thromboxane A2receptor; (B) specular relationship between certain elements
within the structure of thromboxane A2 (the heterocyclic ring conjugated
with a double bondand the adjacent hydroxyl group) and apigenin (γ-pyrone
side of the A and C conjugated rings) [57].
Figure 4.1. The proposed action of flavonoids. Flavonoids „F‟ “=” and “ ”denote
enzyme inhibition and down regulation of expression, respectively [54].
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Daniel Seifu et al.
4.1.3. Flavonoids against cardiovascular disease
The potential of photochemical constituents of plant material for the
maintenance of health and protection from coronary heart disease is also
raising interest among scientists and food manufactures as consumers move
towards functional foods with specific health effects. Cardiovascular diseases
(CVD) is the name for the group of disorders of the heart and blood vessels
and include hypertension (high blood pressure), coronary heart disease (heart
attack), cerebrovascular disease (stroke), heart failure, peripheral vascular
disease [21].
4.1.4. Flavonoids activities against hypertension
Flavanoids in plants available as flavones (containing the flavonoid
apigenin found in chamomile); flavanones (hesperidin - citrus fruits; silybinmilk thistle flavonols (tea: quercetin, kaempferol and rutin grapefruit;
rutinbuckwheat; ginkgo flavonglycosides - ginkgo), play a major role in
curing the cardiovascular diseases. Flavonoids block the angiotensinconverting enzyme (ACE) that raises blood pressure (figure 4.2). Flavonoids
also protect the vascular system and strengthen the tiny capillaries that carry
oxygen and essential nutrients to all cells [21].
Liver
Kidney
Angiotensinogen
Renin
Angiotensin I
Lung
ACE
F
Sympathetic
nervous system
activity
Vasoconstriction
Angiotensin II
Hypertension
Aldosterone
secretion
Figure 4.2. Flavonoids inhibit Angiotensin Converting Enzyme that raises blood
pressure.
4.1.5. Flavonoids activities against coronary heart disease and
vascular disorder
Increased low density lipoprotein (LDL) and especially oxidized LDL
are recognized as risk factors in coronary artery disease (CAD). Certain
Medicinal plants as antioxidant agents
123
flavonoids were potent inhibitors of the modification of LDL. Flavonoids
also inhibited the cell-free oxidation of LDL mediated by CuSO4. The
flavonoids appeared to act by protecting LDL against oxidation caused by the
macrophages, as they inhibited the generation of lipid hydroperoxides and
protected α-tocopherol, a major lipophilic antioxidant carried in lipoproteins,
from being consumed by oxidation in the LDL. Thus the flavonoids protected
α-tocopherol (and possibly other endogenousantioxidants) in LDL from
oxidation, maintained their levels for longer periods of time, and delayed the
onset of lipid peroxidation. While the mechanisms by which flavonoids
inhibit LDL oxidation are not certain, the following possibilities have been
advanced. First, they may reduce the generation or release of free radicals in
the macrophages or may protect the α-tocopherol in LDL from oxidation by
being oxidized by free radicals themselves. Second, flavonoids could
regenerate active α-tocopherol by donating a hydrogen atom to the
α-tocopheryl radical; the latter is formed when it transfers its own hydroxyl
hydrogen atom to a lipid peroxyl radical to terminate the chain reaction of
lipid peroxidation. Third, flavonoids may sequester metal ions, such as iron
and copper, thereby diminishing the engendered free radicals in the medium.
Preliminary evidence indicated that the isoflavone genistein inhibits Cumediated LDL oxidation in a time- and concentration-dependent fashion [25].
4.1.6. Antitumor activities of flavonoids
Cancer has emerged as a major public health problem in developing
countries, matching the industrialized nations. A healthy lifestyle and diet can
help in preventing cancer. Chronic inflammation is associated with a high
cancer risk. At the molecular level, free radicals and aldehydes, produced
during chronic inflammation, can induce deleterious gene mutation and
posttranslational modifications of key cancer-related proteins. Chronic
inflammation is also associated with immune suppression, which is a risk
factor for cancer.
Flavonoids of medicinal plants are proposed as an antitumor agent in
various researches. There mechanism of actions could be: by inhibition of
protein Kinase activities, antiprolifration activities, induction of apoptosis,
and inhibition of metastasis, migration and angiogenesis of the tumor cell
(figure 4.3) [58].
4.1.6.1. Inhibition of protein kinase activity
Protein kinase C (PKC), the ubiquitous, Ca 2+ and phospholipiddependent, multifunctional serine- and threonine- phosphorylating enzyme,
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Daniel Seifu et al.
Figure 4.3. Antitumor activities of Flavonoids.
plays a role in a gamut of cellular activities, including tumor promotion,
mitogenesis, secretary processes, inflammatory cell function and T
lymphocyte function. Certain dietary flavonoids turned out to be potent
inhibitors of PKC in vitro. Out of different flavonoids examined, quercetin
was the most efficient inhibitor of PKC by competitively blocking the ATP
binding site on the catalytic unit of PKC. Flavonoids, impairing the activities
of other ATP-utilizing enzymes, cause inhibition by competitively binding to
the ATP binding site [59, 60].
4.1.6.2. Induction of apoptosis
Cell death in multicellular organisms occurs by two distinct mechanisms,
apoptosis and necrosis. Apoptosis, also called programmed cell death, plays a
cardinal role in embryonic development, metamophorphosis, hormonedependent atrophy, as well as in the maintenance of tissue homeostasis.
Apoptosis is the result of complex signal transduction pathways, bringing
about gene mediated cell death. Being a process regulated by specific gene
activity, apoptosis is sensitive to mutations.
Apoptosis is one of the important pathways through which anticancer
agents inhibit the growth of tumor cells. Resistance of tumor cells to
cytostatic agents is a major problem in the treatment of advanced cancers.
Understanding the signaling pathways that control cytostatic agent induced
apoptosis in tumor cells is critical to ultimately improving anticancer therapy.
At present, only a few potential anticancer agents such as the flavonoids seem
Medicinal plants as antioxidant agents
125
to cause apoptosis. Quercetin flavonoids induced apoptosis, characterized by
typical morphological changes, in certain tumor cell lines. Quercetin also
inhibited the synthesis of heat shock protein (HSP) 70 in these cell lines.
There was an association between this effect and the induction of Quercetin
induced apoptosis. Furthermore, quercetin and luteolin induced apoptosis in a
wide range of tumor cells such as A431, MiaPaCa-2, Hep G2 and MCF 7.
The citrus flavone, tangeretin (5,6,7,8,4'- pentamethoxyflavone), induced
apoptosis in HL-60 cells, at concentrations greater than 2.7 µM, the flavone
had little effect on the mitogen stimulated blastogenic response of human
peripheral blood mononuclear cells [61].
4.1.6.3. Inhibition of metastasis, migration and angiogenesis
The spread of cancer through metastasis represents one of the gravest
dangers of the disease. In human cancers of the breast, liver, colon, lung and
ovary, the production of certain matrix metalloproteinases (MMPs) correlates
with cancer invasion/metastasis. At least 20 genes encoded different MMPs,
which one can categorize into four subclasses based on structural
organization and substrate specificity: collagenases, gelatinases, stromelysins
and membrane-type MMPs. MMPs belong to a rapid growing family of zincdependent endopeptidases that are capable of degrading a variety of
extracellular matrix (ECMs). Collectively, MMPs degrade most components
of the extracellular matrix. Tumor cells probably need more than one MMP,
as well as more general degradative enzymes to cross the tissue barriers they
encounter. There are multiple levels in the regulation of the activities of
MMPs, including the expression and secretion of MMPs, and the activation
processes of MMPs. Endogenous inhibitors such as 2-macroglobulin, and the
tissue inhibitors of metalloproteinases (TIMP) cause inhibition of MMPs in
vivo, once activated. Four structure-related tissue inhibitors, TIMP-1 to
TIMP-4, regulate MMP activity. The secretion of MMPs is necessary for
tumor invasion, as indicated by the observations that treatment with
antibodies or inhibitors against MMPs abolished the invasive behavior of
certain tumor cells. Therefore, one would expect to limit the metastatic
potential of cancer cells by the suppression of the secretion and of the action
of the activated MMPs in cancers. Certain studies report that flavonoids
likeluteolin and quercetin influence the level of MMPs [62].
4.1.6.4. Antiproliferative activities
Dysregulated proliferation appears to be a hallmark of susceptibility to
neoplasia. Cancer prevention is generally associated with inhibition, reversion
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or retardation of cellular hyperproliferation. The molecular mechanism of
antiproliferation may involve the inhibition of the prooxidant process that
causes tumor promotion. It is generally believed that the formation of growth
promoting oxidants (reactive oxygen species, ROS) is a major „„catalyst‟‟ of
the tumor promotion and progression stages, which follow the initiation stage
(carcinogen metabolic activation to mutagens). The prooxidant enzymes
induced or activated by various tumor promoters, for example, phorbolesters,
include the arachidonate metabolizing enzymes, cyclooxygenases (COX), and
lipoxygenases (LOX). In addition, inhibition of polyamine biosynthesis could
be a contributing mechanism to theantiproliferative activities of flavonoids.
Ornithine decarboxylase is a rate-limiting enzyme inpolyamine biosynthesis,
which has been correlated with the rate of DNA synthesis and cell proliferation
in several tissues. Several experiments show that flavonoids can inhibit
ornithine decarboxylase induced by tumor promoters, and thus cause a
subsequent decrease in polyamine and inhibition of DNA/protein synthesis.
Furthermore, flavonoids are also effective at inhibiting signal transduction
enzymes, forexample, protein tyrosine kinase (PTK), protein kinase C (PKC),
and phosphoinositide3-kinases (PIP3), which are involved in the regulation of
cell proliferation [63].
4.1.7. Flavonoids protection against myocardial ischemia–
reperfusion injury
Besides antioxidant effects, flavonoids possess other properties that
alleviate ischemia–reperfusion injury; for instance they help to better reestablish blood flow in post-ischemic hearts. A variety of flavonoids and
polyphenols have shown the capacity to dilate blood vessels. Their
mechanism of action is various and may be exerted in endotheliumdependent and/or -independent manners.
Some polyphenols, such as quercetin and resveratrol, can induce
vasorelaxation by both mechanisms, although in the absence of endothelium
much higher concentrations of polyphenols are probably required. The
endothelium-dependent relaxation effect of polyphenols is mediated by nitric
oxide. Nitric oxide (NO˙) is an important signaling molecule with
vasodilatory, anti-inflammatory, and anti-platelet activities. The upregulatory effect of polyphenols on NO˙ levels occurs through either
activation of endothelium nitric oxide synthase (eNOS) or by removing O2˙−
and thereby inhibiting consumption of NO˙. Other than increasing eNOS
activity, flavonoids may additionally induce eNOS expression. It has been
reported that in ischemic-reperfused hearts a part of beneficial effect of
epigallocatechin gallate is mediated through induction of eNOS.
Medicinal plants as antioxidant agents
127
Intraperitoneal injection 1 mg/kg resveratrol onehour before coronary ligation
in rats induced expression of eNOS and nNOS (neuronal NOS) while
blocking expression of inducible nitrogen oxide synthase (iNOS) which
contrary to eNOS produces excessive amounts of NO˙ associated with
formation of peroxynitrite and oxidative stress [64,65].
As eNOS is a calcium-dependent enzyme, elevation of intracellular Ca2+
has been suggested as the mechanism of the endothelium dependent NO˙mediated vasorelaxation by polyphenols (figure 4.4). Polyphenols likely
increase intracellular Ca2+ by stimulating both Ca2+ entry from extracellular
milieu and Ca2+ release from intracellular Ca2+ stores. Surprisingly, the rise of
Ca2+ by polyphenols occurs as a result of increased production of O2˙ –
asapplication of superoxide dismutase plus catalase attenuated the
Ca2+elevation. These results suggest that the effect of polyphenols on NO˙
levels can occur both through stimulating O2˙ − production inside endothelial
cells (stimulating eNOS activity), and through scavenging O2˙− in the
interstitial fluid (preserving NO˙).
Figure 4.4. Effect of flavonoids on endothelium-dependent vasorelaxation (31).
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NO˙ is generally produced by eNOS attached to the endothelium plasma
membrane and delivered to smooth muscle cells where it manifests its
biological functions. In smooth muscle cells, NOU activates guanylate
cyclase which synthesizes cyclic GMP (cGMP), an important mediator of
vasodilation. cGMP acts by activating protein kinase G which affects a
number of target proteins including those involved in Ca 2+ channels,
decreasing cytosolic Ca2+ through activating endoplasmic reticulum Ca2+
uptake and inhibiting extracellular Ca2+ entry . The eventual low intracellular
Ca2+ in smooth muscle cells mitigates cellular contractility and yields
relaxation. In contrast to the aforementioned polyphenol-induced
vasorelaxation, inhibition of NO˙-cGMP-mediated vasorelaxation has also
been observed with some flavonoids [66].
The mechanism of endothelium-independent relaxation by polyphenols is
yet uncertain, but signaling pathways downstream of cGMP might be
activated in smooth muscle cells independently of NO˙. Among downstream
mechanisms are inhibition of protein kinase C and phosphodiesterases
(a family of enzymes responsible for the breakdown of the vasorelaxants
cyclic AMP (cAMP) and cGMP), inhibition of Ca2+ influx from extracellular
and intracellular resources and activation of voltage-gated K+ channels [67].
The blockage of extracellular Ca2+ influx and endoplasmic reticulum
2+
Ca release by polyphenols is appealing as it could be one of the possible
mechanisms of polyphenol protection of hearts from Ca 2+ overload in states
of ischemia–reperfusion. Flavonoids may also promote vasorelaxation by
stimulating production of prostacyclins by endothelial cells. It is found that 3
weeks oral administration of grape seed proanthocyanidins increased
production of prostacyclins in ischemic and ischemic-reperfused hearts.
Proanthocyanidins can also cause vasodilation through suppressing the
rennin–angiotensin system by acting as angiotensin receptor antagonist as
well as inhibiting angiotensin converting enzyme. Furthermore, vasodilatory
effects of flavonoids may partly be exerted by scavenging peroxynitrite and
therefore preserving tetrahydrobiopterin from oxidation. Alternatively,
resveratrol has shown to elevate tetrahydrobiopterin levels by increasing
activity of the rate limiting enzyme in tetrahydrobiopterin synthesis [31].
4.1.8. Inhibition of metalloproteinases by flavonoids
Matrix metalloproteinases (MMP) are a family of proteases that play a
major role in protein degradation and tissue remodeling. Elevation of plasma
levels of MMP has been documented after ischemia–reperfusion-related
morbidities such as myocardial infarction, restenosis, and heart failure. Since
increased activity of MMP is associated with ventricular dilation and cardiac
Medicinal plants as antioxidant agents
129
remodeling, inhibitors of MMP may play as effective strategies to prevent
chronic consequences of the injury [68].
Polyphenolic compounds in red wine and green tea have shown ability to
inhibit activation of metalloproteinase-2. In green tea, the inhibitory effect
seemed to correlate with the gallic acid moiety of the catechins as the
inhibitory activity of epigallocatechin gallate and epicatechin gallate was
more than that of epigallocatechin while catechin and epicatechin showed the
least effect. Epigallocatechin gallate dose-dependently decreased activation
of metalloproteinase- 2 in human umbilical endothelial cells. Similarly,
quercetin dose-dependently decreased expression of metalloproteinase-9 in
human aortic smooth muscle cells. The flavonoids inhibition of
metalloproteinases has also been demonstrated in ischemic-reperfused hearts.
The inhibition of metalloproteinases by phenolic compounds has been
speculated to occur transcriptionally through suppression of DNA binding
activity of NF-κB and AP-1. Moreover, quercetin has shown to stimulate
expression of metalloproteinase-1 tissue inhibitor in human vascular
endothelial cells treated with oxidized LDL. It has been suggested that high
doses of polyphenols inhibit activation of metalloproteinases and prevent
angiogenesis, while low doses of polyphenols show angiogenic effects
without altering activity of metalloproteinases [21].
4.2. Terpenoids
4.2.1. Anti-inflammatory effect of terpenoids
4.2.1.1. Inflammation
Inflammation is caused by a variety of stimuli including physical
damage, ultra violet irradiation, microbial invasion, and immune reactions.
The classical key features of inflammation are redness, warmth, swelling, and
pain. Inflammation cascades can lead to the development of diseases such as
chronic asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel
disease, and psoriasis [69].
Transcription factors, such as nuclear factor kappa-B (NF-kB), activator
protein-1 (AP-1), or signal transducer and activator of transcription (STAT),
are involved in the induced expression of a variety of proteins, and especially
cytokines controlling the inflammatory response. NF-kB is present in the
cytosol of many cell types, usually as a heterodimer composed of p50 and
p65 subunits, held in the inactive state by IkB inhibitory subunit. During
physical damage, microbial invasion, stress and immune reaction, IkB
inducibily phosphorylated, and subsequent ubiquitinylated. These posttranslational modifications tag the molecule for the subsequent proteolytical
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Daniel Seifu et al.
degradation by the ubiquitin-26 S proteasome pathway. This induced
degradation of IkB proteins unmasks the nuclear localization sequences of
the DNA-binding subunits of the NF-kB dimer and allows NF-kB
(heterodimer p50/p65) to enter the nucleus, to bind to its DNA sequence, and
to induce transcription. The target genes whose transcription is mainly
regulated by NF-kB include many cytokines, cell adhesion molecules, such
as intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion
molecule 1, as well as acute-phase proteins and immunoreceptors (described
in figure 4.5). AP-1 refers to a family of protein dimers, usually composed of
c-jun/c-fos subunits, which after stress-induced phosphorylation enter the
nucleus and due to binding to consensus sequences enhance the expression of
the appropriate genes. Activation of NF-kB and/or AP-1 is often induced by
the proinflammatory cytokines, such as IL-1 or TNF-α. On the other hand,
STAT3 belongs to the family of transcription factors activated by such
cytokines as IL-6 and is involved in the up-regulation of acute phase protein
synthesis in liver cells [70, 71].
Figure 4.5. Schematic overview of NF-kB signaling pathways [16].
4.2.1.2. Terpenoids as modulators of NF-kB signaling pathways
The molecular cascade of signaling events involved in NF-kB activation,
provides several steps for specific inhibition of NF-kB activity. Inhibition of
NF-kB activation can occur by different mechanisms including (a) inhibiting
the activation of IKK complex, (b) targeting the proteasomal degradation of
Medicinal plants as antioxidant agents
131
or (c) interfering the translocation of NF-kB to the nucleus, or the binding of
NF-kB to DNA. Several agents including natural products such as
sesquiterpene lactones helenalin, 11α, 13-dihydrohelenalin, and chamissonolid,
sesterterpenes (cyclolinteinone) and ent-kaurane diterpenes (oridonin and
ponicidin) (figure 4.6) have been shown to inhibit either nuclear translocation
of p65 or binding of NF-kB to DNA. However, the molecular target through
which these natural compounds exert their effect not clearly understood.
Nevertheless, the most effective and selective approach for the inhibition of
NF-kB activation is provided by inhibitors of the IKK activity by
sesquiterpene lactones such as parthenolide, kaurane diterpenes such as
kamebakaurin; and labdane diterpenes such as hispanolone derivatives are
examples of the specifically targeting of IKK kinase activity [72,73].
Consequently, downstream events, such as release of cytokines IL-1b, IL-6 or
TNF-α production and lymphocyte proliferation are also inhibited [74].
Figure 4.6. Structures of the investigated sesquiterpene lactones [73].
4.2.2. Terpenoids as anticancer agents
Many studies have shown that several of the dietary monoterpenes are
effective in the prevention and treatment of cancer. Among these, Perillyl
alcohol
(POH)
(p-metha,
1,
7-diene-6-ol
or
4-isopropenylCyclohexenecarbinol) found in the essential oils of several plants (lavendin,
mints, cherries, etc.) and synthesized by the mevalonate pathway. It has well
established chemopreventive activity in rodent mammary, skin, liver, lung,
colon, and fore stomach cancers and also chemotherapeutic activity in
pancreatic, mammary, and prostatic animal tumor models, leading to
regression of existing malignant tumors. Several mechanisms have been
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Daniel Seifu et al.
proposed to mediate the antitumor effects of POH. It has been shown that
POH affects the expression of several regulators of cell cycle and apoptosis.
There is also evidence that POH inhibits the post-translational isoprenylation
of the Ras small GTPase superfamily of proteins, which are known to play a
key role in many signal transduction pathways, including those that stimulate
tumor-associated angiogenesis [75].
Limonene ((1-methyl-4-(1-methylethenyl) cyclohexane), monoterpene,
has well established chemopreventive activity against many cancer types.
Limonene (figure 4.7) has been shown to inhibit the development of
spontaneous neoplasms in mice; dietary limonene also reduces the incidence
of spontaneous lymphomas in p53 mice. Furthermore, when administered
either in pure form or as orange peel oil (95% d-limonene), limonene inhibits
the development of chemically induced rodent mammary, skin, liver, lung
and fore stomach cancers [76].
D-limonene induces phase I and phase II carcinogen-metabolizing
enzymes (cytochrome p450), which metabolize carcinogens to less toxic
forms and prevent the interaction of chemical carcinogens with DNA. It also
inhibits tumor cell proliferation, acceleration of the rate of tumor cell death
and/or induction of tumor cell differentiation. Furthermore, as Preclinical
data indicates d-limonene and its metabolites modulate Ras prenylation via
farnesyl transferase inhibition. Many prenylated proteins regulate cell growth
and/or transformation. Impairment of prenylation of one or more of these
proteins might account for the antitumor activity of d-limonene. It was found
that d-limonene attenuates gastric cancer through increasing apoptosis, while
decreasing DNA synthesis and ornithine decarboxylase activity of cancer
cells. D-limonene inhibits hepatocarcinogenesis via inhibition of cell
proliferation, enhancement of apoptosis, and blockage of oncogene
expression [77, 78].
Terpenes, such as farnesol a sesquiterpene and geraniol a monoterpene,
have also been shown to have chemotherapeutic activities towards cancer
cells. Although cell culture studies utilizing diverse cancer cell lines
demonstrated the pronounced effects of farnesol (FOH) (figure 4.8 A) and
Figure 4.7. Structure of Limonene.
Medicinal plants as antioxidant agents
133
geraniol (GOH) (Figure 4.8 B) on the inhibition of cell proliferation, there is
scarce information on the in vivo cancer chemopreventive potential of these
acyclic isoprenoids. In rats FOH inhibited chemically induced colon and
pancreatic carcinogenesis and GOH inihibited mammary carcinogenesis. FOH
and GOH cell proliferation inhibition is based on their ability to
posttranscriptionally inhibit 3-hydroxy-3- methylglutaryl coenzyme A (HMGCoA) reductase activities, thus reducing synthesis of cholesterol and
intermediaries of the mevalonate pathway, such as farnesyl and geranylgeranyl
pyrophosphates. These mevalonate derivatives are important for protein
farnesylation and geranylgeranylation of certain proto-oncogenes. Differently
from normal cells, preneoplastic and neoplastic liver lesions present a loss in
the transcriptional down regulation mechanism of HMG-CoA reductase by
sterols and of cholesterogenesis. However, in these lesions this enzyme retains
sensitivity to isoprenoid-mediated inhibition. Thus isoprenoids could represent
potential chemopreventive agents against hepatocarcinogenesis. It was
proposed that FOH could exert anticarcinogenic actions through farnesoid X
activated receptor (FXR) - mediated gene expression. This nuclear receptor was
initially shown to be activated by FOH but not by GOH. Subsequently bile
acids were identified as the endogenous ligands for FXR, which plays a central
role in bile acids and cholesterol metabolism. Activators of FXR such as FOH
are potent cell proliferation inhibitors and apoptosis inducers [13, 79].
Moreover, monoterpenes such as carveol, uroterpenol, and sobrerol have
shown activity against mammary carcinomas. Carvone has been analyzed as an
agent reducing pulmonary adenoma and fore-stomach tumor formation. Several
plant triterpenes exhibited in Vitro antitumor activity. Betulinic acid has been
shown to induce apoptosis of several human tumor cells; including melanoma
and glioma, and ursolic acid and oleanolic acid reduced leukemia cell growth
and inhibited the proliferation of several transplantable tumors in animals [13].
Plant derived diterpenoids are the most effective anticancer agents
approved by the Food and drug administration (FDA). Taxol, registered as a
Figure 4.8. Chemical structure of fernesol (A) a sesquiterpene and geraniol (B)
a monoterpene.
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Daniel Seifu et al.
trademark Taxol® by Bristol-Myers Squibb, and known generically as
paclitaxel, is a secondary compound with a very complex chemical structure
and one of the most effective anti-cancer drugs ever developed ( Figure 4.9).
Taxol extracted and identified in 1971 from the inner bark of Taxus
brevifolia (the pacific yew tree), is a potent antimitotic agent with excellent
activity against breast and ovarian cancers. Taxol exhibits a unique mode of
action. It acts as microtubulin stabilizing agent while the other anticancer
agents destabilize this process. Actually, tubulin polymerizes to microtubulin
and again microtubulin converts into tubulin. In a normal case, this process is
in equilibrium later on, fixed-size 24-nm microtubulin bundles are formed
and the cell multiplication process takes place, whereas taxol makes stabler
bundles of microtubulins of size 22 nm (Figure 4.10). Due to this, a defective
polymerization process occurs and thus, these cells have unnatural „bundles‟
of microtubules and no mitotic spindle. The cancerous cells lack a check
point to detect the absenceof a spindle and attempt to continue the cell cycle,
which leads to cell death. Because of this reason, taxol is sometimes also
referred as a „spindle poison‟ [80, 81].
4.2.3. Antiparasitic and antibacterial activity of terpenoids
Increased resistance in many pathogens towards currently used
medicines, creating a serious threat to the treatment of infectious diseases.
Figure 4.9. Brief structural and activities relationship of Taxol [78].
Medicinal plants as antioxidant agents
135
Figure 4.10. Mechanism of action of taxol on the cell microtubular system. MT=
microtubular [80].
Drug resistance is one of the most serious global threats to the treatment of
infectious diseases. In addition to resulting in significant increases in costs
and toxicity of newer drugs, antibiotic resistance is eroding our therapeutic
armamentarium. Resistant strains of bacteria are continuing to increase, both
in number and in variety, but not significantly different newer antibiotics are
yet available. Treatment of infections caused by these resistant bacteria has
become very difficult. Since they are resistant to many antibiotics,
therapeutic options have become limited. Therefore, alternative methods of
treatment are sought after. For over several years medicinal plants have
served as the models for many clinically proven drugs, and are now being
reassessed as antimicrobial agents [82].
As a result of monoterpenes lipophilic character, they preferentially
partition from an aqueous phase into membrane structures. This results in
membrane expansion, increased membrane fluidity and permeability,
disturbance of membrane-embedded proteins, inhibition of respiration, and
alteration of ion transport processes in gram-negative bacteria, evidencing
that monoterpene uptake is also determined by the permeability of the outer
envelope of the target microorganism. However, specific mechanisms involved
in the antimicrobial action of monoterpenes remain poorly characterized [83].
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4.3. Alkaloids
4.3.1. Antitumor activities of alkaloids
The antitumor properties of the alkaloids extracted from Amaryllidaceae
are well known. The first described compound with a cytostatic effect was
lycorine (figure 4.11), which has been shown to exhibit antitumor activities ,
e.g. it can suppress leukaemia cell growth, reduce cell survival by arresting
the cell cycle and inducing the apoptosis of tumor cells and it can also induce
apoptosis and cell cycle arrest in a pre-B lymphoid cell line (KM3). Other
types of Amaryllidaceae alkaloids likewise exhibit cytostatic effects, e.g.
those of tazettine or crinine type. However, the most promising candidates
are the narciclasin type pancratistatin and narciclasine [80]. Berberine is the
major constituent of Coptis Chinese, a yellow benzylisoquinoline alkaloid, in
vitro, induces apoptosis in both HL-60 and WEHI-3 cell lines in association
with caspase-3 activity and MMP depolarization. In both berberine-treated
cell lines, berberine increased accumulation of Bax and Bad, but decreased
the expression of Bcl-2 and led to the depolarization of mitochondrial
membrane potential (MMP), thus further enhancing the release of
cytochrome c and increasing the activation of caspase-3, finally leading to
apoptosis (figure 4.12). This suggests the potential application of berberine in
the treatment of leukemia cells [85].
Cryptolepine, which is the main indoloquinoline alkaloid found in the
roots of the climbing shrub Cryptolepis sanguinolenta (Periplocaceae), has
cytotoxic properties in several cancer cell lines ( for example B16 melanoma
cells) , and the mechanisms involved may include intercalation into DNA and
inhibit its synthesis. In addition, it stabilizes topoisomerase II-DNA covalent
complexes and stimulates the cutting of DNA at a subset of preexisting
topoisomerase II cleavage sites. The mode of cryptolepine intercalation into
DNA is peculiar in that it occurs at non alternating CG rich sequences
[86, 87].
Figure 4.11. Structure of lycorine [84].
Medicinal plants as antioxidant agents
137
Figure 4.12. A proposed model for the berberine mechanism of apoptosis in leukemia
cells [85].
4.3.2. Gout suppressant activity of alkaloids
Acute gouty arthritis (GA) is characterized by intense inflammation
induced by monosodium urate (MSU) crystal deposition in articular joints
and periarticular tissues. Interactions of MSU crystals with synovial lining
cells, macrophages and other synovial cells attract a massive neutrophils
influx into the joints via interleukin 8 (IL-8) chemotaxis, which drives
episodes of acute gouty inflammation. Ingestion of MSU crystals by
phagocytes after CD14 and Toll-like receptor (TLR) 4 engagements also
elicits production of proinflammatory mediators that propagate and sustain
intense acute GA [88].
Leukocyte chemotaxis in general has been shown to be receptor
mediated, and the activated receptor in stimulated cells is believed to deliver
a signal to the motility elements of the cells (such as microfilaments and
microtubules), resulting in their orientation and subsequent directional
migration towards the chemoattractant. The evidence for a functional role of
microtubules in leukocyte chemotaxis has been somewhat controversial.
However, work from several laboratories has suggested that microtubules
play an important role in leukocyte chemotaxis. It was shown that exposure
of leukocytes to attractants induces microtubule assembly before locomotion.
This assembly was blocked by colchicine, which also prevented their proper
orientation toward chemoattractants (figure. 4.13) [89].
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Daniel Seifu et al.
Colchicine and vinca alkaloids each have a specific binding site on the
tubulin dimer. Both compounds cause metaphase arrest, and either can be
used to treat acute gouty arthritis. Structural studies show unfolding of a
small region in the carboxy terminal of beta tubulin induced by colchicines.
Such unfolding is thought to prevent the necessary contacts for microtubule
polymerization through GTP hydrolysis. Colchicine and other tubulin
disruptive molecules inhibit a characteristic and specific protein
phosphorylation pattern that occurs in neutrophils exposed to monosodium
urate [90].
Colchicine may also alter the distribution of adhesion molecules on the
surface of both neutrophils and endothelial cells, leading to a significant
inhibition of interaction between white blood cells (WBC) and endothelial
cells interfering with their transmigration [91].
Cholchicine
Interaction with
tubulins
Suppresses
microtubules
dynamics
Altering
distribution of
Systems
Inhibition of
neutrophils
adhesion molecules
Immediate antiinflammatory
Profile Effect
Suppress
Gout
Figure 4.13. A model of the mechanism of action of colchicines [92].
Medicinal plants as antioxidant agents
139
4.3.3. Antimalarial activity of alkaloids
Malaria, a tropical blood-borne protozoan disease caused by parasites of
the genus Plasmodium, is one of the most important infectious diseases in the
World. Nowadays, new antimalarial drugs have become an urgent need
because of the declining efficiency of classical medication, and the rapid
extension of chloroquine resistant strains of Plasmodium falciparum. Drug
resistance is responsible for the spread of malaria to new areas, the recurrence
of malaria in areas where the disease had been eradicated and plays an
important role in the occurrence and severity of epidemics in some parts of
the World [93, 94].
Cryptolepine, which has cytotoxic effect for some cancer cell lines, also
has antimalarial activity for drug resistant strains of plasmodium falciparum.
Investigations into its molecular mechanisms of action have revealed that the
antiplasmodial mode of action may be different from the cytotoxic mode of
action. Cryptolepine shares with chloroquine, and related quinoline
antimalarial drugs, the property of binding to heme and prevents its
conversion to hemazoin. The drug-heme complex is considered to be toxic to
the parasite [95].
4.3.4. Alkaloids for Alzheimer’s disease treatment
Alzheimer‟s disease (AD) is the most frequent form of dementia
characterized by memory loss and abnormal mental and physical behavioral
changes. It is a progressive disorder leading β-amyloid plaque formation
neurofibrillary tangle formation, oxidative and inflammatory processes and
deficiency in the neurotransmitter called acetylcholine in the brain [96].
A consistent neuropathological occurrence associated with memory loss
is a cholinergic deficit, which has been correlated with the severity of AD.
Therefore attempts to restore cholinergic function have been a rational target
for drugs used to treat the symptoms of AD. Approaches to enhance
cholinergic function in AD have included stimulation of cholinergic receptors
(e.g. the stimulation of nicotinic receptors by nicotine), or by prolonging the
availability of acetylcholine (ACh) released into the neuronal synaptic cleft.
This may occur by inhibiting ACh hydrolysis by acetylcholinesterase
(AChE), through the use of AChE inhibitors [97]. Molecules including
physostigmine, galanthamine, and huperzine A are the alkaloid-type of
compounds isolated from the plants which possess remarkable
anticholinesterase (inhibition of acetylcholinesterase responsible for
hydrolysis of acetylcholine) effects to treat AD [83, 98].
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