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Chapter 2 Review Of Literature
8
2.0 REVIEW OF LITERATURE
Plants have been an essential part of human society since the
civilization started. A medicinal plant is any plant which, in one or more of its
parts contain substances that can be used for therapeutic purposes or which
are precursors for the synthesis of useful drugs (Perumal et al., 2004;
Oladunmoye, 2007). Recent awareness of therapeutic potential of several
traditionally used plants has opened a new dimension for the study and
research of medicinal plants (Bhandari et al., 2007).
Cancer is one of the most dreaded diseases of the 20th century and
spreading further with continuance of increasing incidence in 21st century
(Balachandran and Govindarajan, 2005). In recent years, a considerable
attention has been placed to identify naturally occurring chemopreventive
substances capable of inhibiting, retarding or reversing the process of
carcinogenesis (Shukla and Kalra, 2007).
Cancer is a hyperproliferative disorder that involves transformation,
dysregulation of apoptosis, proliferation, invasion, angiogenesis and
metastasis (Aggarwal et al., 2006). Chemoprevention by the use of naturally
occurring substances is becoming a promising strategy to prevent cancer
(Chen et al., 2007). Cancer chemopreventive agents are classified as blocking
or suppressing agents if they inhibit initiation or promotion / progression phase
of carcinogenesis, respectively (Moreno et al., 2007).
India is well known for a plethora of medicinal plants. The traditional
Indian medicinal plants act as antiradicals and DNA cleavage protectors.
These plants have also been considered to protect health, longevity,
intelligence, immunosurveillance and body resistance against different
infections and diseases (Manna et al., 2006).
To the possible extent sincere efforts have been made to collect the
relevant literature of the study. After thorough reviewing of all possible sources, it
was observed that very few studies have been conducted earlier on certain
dimensions of the present study in our laboratories. Information on the
Antioxidative, antitumor and immunomodulatory role of C. dactylon and
T. catappa leaves is still very scanty in literature. The review of literature
Chapter 2 Review Of Literature
9
pertaining to the present research entitled “Antioxidative, antitumor and
immunomodulatory efficacy of protein fraction of Cynodon dactylon and
Terminalia catappa leaves on experimentally implanted ELA cells in Swiss
albino mice” is appropriately presented under the following headings:
2.1 Reactive oxygen species
2.1.1 Free radicals
Formation of free radicals
Superoxide anion
Perhydroxyl radical
Hydroxyl radical
DPPH
2.1.2 Non-radicals
Singlet Oxygen
Hydrogen peroxide
Hyphochlorous acid
Nitric oxide
2.2 Lipid peroxidation
2.3 Antioxidants
2.3.1 Enzymic antioxidants
2.3.2 Nonenzymic antioxidants
2.4 Antitumor activity of medicinal plants
2.5 Immunomodulatory activity of medicinal plants
2.6 Medicinal plants selected for the study
2.6.1 Cynodon dactylon
2.6.2 Terminalia catappa
2.1 REACTIVE OXYGEN SPECIES
Reactive Oxygen Species are usually highly reactive and short-lived,
known to cause damage to cellular components including lipid, DNA, protein,
carbohydrate, and other biological molecules. They consequently lead to many
pathological processes such as aging, cancer, cardiovascular diseases, diabetes,
inflammation, neurodegenerative diseases and infertility (Piconi et al., 2003;
Junqueira et al., 2004; Casetta et al., 2005; Singh et al., 2005; Haidara et al.,
2006; Valko et al., 2006; Reddy, 2006; Gupta et al., 2006; Grossman, 2008;
Chapter 2 Review Of Literature
10
Cheung et al., 2008). Fortunately, biological systems can protect themselves
against harmful effects of ROS and free radicals by designing an optimal
nutritional countermeasure and formation of antioxidants (Fang et al., 2002).
The ROS play a major role in tumor promotion by cellular damage. The
biological importance of these radicals arises from the fact that they occur
during the normal metabolic process. The ultimate target of free radicals is the
genome, ie., usually DNA and RNA molecules or it may cause strand breakage
in both nuclear and mitochondrial organelles. The irreversible nature of these
an alteration ultimately leads to malignant and mutagenic states toppling the
whole cellular metabolism (Yu et al., 2007).
Mammalian life depends upon oxygen as the final acceptor of electrons
in mitochondrial electron transport, but the process also generates toxic
metabolites (Phillips et al., 2003). Reactive oxygen species leak from
mitochondria into the cytoplasm where they cause cellular damage by oxidizing
a variety of biologically important molecules, including DNA, proteins, lipids
and carbohydrates (Przekwas et al., 2003). Reactive Oxygen Species produce
cellular injury and necrosis via several mechanisms including peroxidation of
membrane lipids, protein denaturation and DNA damage (Hemnani and
Parihar, 1998; Cuzzocrea et al., 2002). Reactive Oxygen Species mediated
DNA damage has long been thought to play a role in carcinogenesis initiation
and malignant transformation (Valko et al., 2006).
The ROS include both free radicals and non-radicals. Most biologic
molecules are non-radicals containing two electrons per orbital, which is a
stable configuration in a molecule (http://www.cellscience.com). A free radical
is a molecule that can exist independently and contains one or more unpaired
electrons. An unpaired electron means that there is only one electron in an
orbital (shown in red), which is an unstable configuration and makes free
radicals highly reactive (Figure 1). ROS encompasses other reactive species
which are not true radicals but are nevertheless capable of radical formation in the
intra and extra cellular environment. Eg., Hydrogen peroxide, Hypochlorous acid
and singlet oxygen (Chapple and Matthews, 2007).
Chapter 2 Review Of Literature
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FIGURE 1: REACTIVE OXYGEN SPECIES (ROS)
(http://www.cellscience.com)
2.1.1 Free radicals
A free radical can be defined as any atom or molecule possessing one
or more unpaired electrons. A major source of free radicals in biological
systems is molecular oxygen. They are formed when oxygen interacts with
certain molecules. Once formed, these highly reactive radicals can start a
chain reaction hence they have significant biological importance. They are
generally unstable and very reactive. The biologically relevant free radicals
derived from oxygen are the superoxide anion (O2-), the perhydroxyl radical
(protonated superoxide, HO2), the hydroxyl radical (HO.) and free radical nitric
oxide (Cuzzocrea et al., 2001).
Formation of free radicals
Living cells are exposed to oxidants originating from a large variety of
exogenous or endogenous sources. Exogenous sources - air pollutants,
ozone, radiation, chemicals, toxins, pathogenic microorganisms. Endogenous
sources-due to leaks in electron transport chain in mitochondria during
oxidation of food stuffs and inflammatory cells produce free radicals by a
process of respiratory burst (Figure 2).
Chapter 2 Review Of Literature
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FIGURE 2: FORMATION OF FREE RADICALS
(http://www.smokersrx.com)
Free radicals cause tissue damage by a variety of different mechanisms
which include DNA damage, protein damage, lipid peroxidation, enzyme oxidation
and stimulation of proinflammatory cytokines release (Pauwels et al., 2007).
The role of free radicals has been implicated in the causation of several
pathophysiological disorders such as liver cirrhosis, atherosclerosis, cancer,
aging, rheumatoid arthritis, diabetes and neuro degeneration (Finkel and
Chapter 2 Review Of Literature
13
Holbrook, 2000; Droge, 2002) and the compounds that scavenge free radicals
have great potential in ameliorating these disease processes. The human body
has inherent mechanisms to reduced free radical induced injury by glutathione,
arginine, citrulline, taurine, creatine, selenium, zinc, vitamin E, vitamin C, vitamin A
and tea polyphenols and endogenous enzymes such as superoxide dismutase.
Sometimes these protective mechanisms are found not to be sufficient when
compared to the insult produced to the body; hence the search for exogenous
antioxidants is continued (Shirwaikar et al., 2004).
Reactive Oxygen Species are emerging as critical signaling molecules
(Pouyssegur et al., 2006). Redox balance, the ratio between oxidizing and
reducing species within the cell, plays a significant role in the regulation of signaling
pathways, including kinase and phosphatase activity and gene expression through
modulation of transcription factor function (Biswas et al., 2006).
Free radicals do their damage with a sequence of changes resulting
from an injury (burn or thermal shock) and ultimately oxidative stress from the
depletion of antioxidant defense mechanisms. Oxidative stress can damage
many biological molecules. Proteins and DNA appear to be some significant
targets of cellular injury (Gradelet et al., 1998; Glei et al., 2000; Collins, 2001).
Condition of oxidative stress arises either from the overproduction of free
radicals of oxygen or from the deficiency of antioxidant defenses or repair
mechanisms and results in reversible or irreversible tissue injury (Athar, 2002;
Dey and Cederbaum, 2006). It was also showed that oxidative stress from
chronic inflammation favours cancer development in many organs
(Valko et al., 2007). Evidences have accumulated to suggest that ROS play an
important role in tumor initiation by enhancing or facilitating the metabolic
activation and / or initiating effect of carcinogens. It also occurs due to an
imbalance in prooxidant and antioxidant levels (Zablocka and Janusz, 2008). The
formation of excessive amounts of ROS including superoxide anions is toxic to the
cell. Hence, metabolizing and scavenging systems to remove them are
functionally critical and tightly controlled in the cells (Mullineaux and Creissen,
1996).
Chapter 2 Review Of Literature
14
Superoxide anion (O2-)
The superoxide free radical anion is formed when oxygen is reduced by
the transfer of a single electron to its outer shell. The major source of
superoxide in vivo is the electron leakage that results from the electron transfer
chain of the mitochondria. Superoxide anion plays an important role in the
formation of more reactive species such as hydrogen peroxide, hydroxyl
radical, and singlet oxygen, which induce oxidative damage in lipids, proteins
and DNA (Wu and Cederbaum, 2004).
A major portion of the biological consumption of molecular oxygen
occurs during reduction to water via oxidative phosphorylation in mitochondria.
However a small portion of the total oxygen consumed is reduced in a specific
pathway yielding superoxide and hydroxyl free radical, all of which can be
potentially damaging to respiring cells (Vuillaume, 1987).
O2-
(+H+) e-
O2 (+H+) e- O2- (+H+) e- O2
- (+H+) e- OH (+H+) e- H2O
hv or other energy
Perhydroxyl radical (OOH.)
Perhydroxyl radical, the conjugate acid of O2- play an important role in
oxidant damage. Protonation of O2- gives rise to HOO' radical which can
initiate peroxidation. It is also known as hydroperoxyl radical. Perhydroxyl
radical [HOO', the conjugate acid of superoxide (O2-)], initiates fatty acid
peroxidation (a model for biological lipid peroxidation) by two parallel
pathways: fatty acid hydroperoxide (LOOH)-independent and LOOH-
dependent. The superoxide anion and the perhydroxyl radical are in
equilibrium in aqueous solution (http://en.wikipedia.org/wiki/Hydroperoxyl).
Hydroxyl radical (OH.)
The hydroxyl radical is an extremely reactive oxidizing radical that will react
to most biomolecules at diffusion controlled rates, which means that reactions will
occur immediately with biomolecules. The hydroxyl radical is important in
radiobiological damage and is more reactive towards cellular constituents than
Chapter 2 Review Of Literature
15
superoxide radicals. When superoxide and hydrogen peroxide react together they
produce hydroxyl radicals (Coyle and Puttfarcken, 1993).
_22
_2 OHOHOHO _
DPPH
A stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical has long been
used as a convenient method for the antioxidant assay of biological materials
such as cysteine, glutathione, ascorbic acid, tocopherol and polyhydroxy
aromatic compounds (Suja et al., 2004; Nishizawa et al., 2005). DPPH is a
commercially available free radical which is soluble in ethanol
(Summa et al., 2006). This method is based on the reduction of alcoholic
DPPH solution at 517nm in the presence of hydrogen donating antioxidant
(AH) due to the formation of non-radical form DPPH-H by the reaction:
AHDPPHAHDPPH _ (Koleva et al., 2002)
The sensitivity of the method is determined by the strong absorption of
DPPH. This method is rapid, sample analysis takes only 15 minutes and little
manpower, no expensive reagents or sophisticated instruments are required
(Koleva et al., 2002). The oils and aqueous extracts of leaves and flowers of
Bidens pilosa were subjected to screening for their possible antioxidant activities
by using DPPH and β-carotene bleaching methods (Deba et al., 2008).
The DPPH radical scavenging activity of the ethanol and aqueous extracts
of aerial parts of Varthemia iphionoides Boiss (Al- Dabbas et al., 2006), ethanol
extract of aerial parts of herb Poeonia emodi (Khan et al., 2005), aqueous extract
of the herb Thymus fallax Fisch and Mey (Ozgen et al., 2006), D. hamiltonii roots
and Choerospondias axillaries fruits (Wang et al., 2008) were reported.
2.1.2 Non-radicals
Non-radicals containing two electrons per orbital, which is a stable
configuration in a molecule, include singlet oxygen, hydrogen peroxide,
hypochlorous acid and nitric oxide.
Singlet oxygen (O2.)
Singlet oxygen is a nonradical reactive oxygen species often associated
with oxygen free radicals that has strong oxidizing activity. It is an electronically
excited and mutagenic form of oxygen. It is generated by input of energy,
Chapter 2 Review Of Literature
16
example radiation, but can also be generated enzymatically by the action of
peroxidases or lipoxygenases or by the reaction of hydrogen peroxide with
hypochlorite or peroxynitrite, thermo-decomposition of dioxetanes or during the
respiratory burst of phagocytes. They are also generated in biological systems in
a number of pigment reactions including chlorophylls, retinal and flavins when
they are illuminated in the presence of oxygen (Prakash et al., 1998).
Hydrogen peroxide (H2O2)
Hydrogen peroxide is not a free radical but falls in the category of reactive
oxygen species. It is an oxidizing agent that is not particularly reactive but its
main significance lies in that it is the main source of hydroxyl radicals in the
presence of transition metal ions. Hydrogen peroxide can be generated from the
two electron reduction of oxygen. In biological systems hydrogen peroxide is
generated by the production of superoxide: two superoxide molecules can react
together to form hydrogen peroxide and oxygen (Winston and Di Giulio, 1991).
222_
2 OOH2H2O
Hypochlorous acid (HOCl.)
Hypochlorous acid is a chemically reactive oxidant. It is an important
component of the inflammatory response and may contribute to carcinogenesis.
Stimulation of phagocytosis triggers a membrane-associated NADPH oxidase,
which reduces molecular oxygen to superoxide. The latter dismutates to
hydrogen peroxide. Phagocytic cell myeloperoxidase catalyzes the formation of
HOCl. from hydrogen peroxide and chloride ions. It induces oxidative injury
during phagocytosis (Lakshmi et al., 2000).
Nitric oxide (NO)
Saha et al. (2008), stated that NO is an important chemical mediator
generated by endothelial cells, macrophages, neurons and involved in the
regulation of various physiological processes. Overproduction of NO can mediate
toxic effects such as DNA fragmentation, cell damage and neuronal cell death.
NO does not interact with the bioorganic macromolecules such as the DNA or
proteins directly. During infections and inflammations, formation of NO is elevated
and may bring about some undesired deleterious effects like tumor growth. The
peroxynitrite produced during the reaction of NO with O2- is probably responsible
for genetic damage (Roberfroid and Calderon, 2008).
Chapter 2 Review Of Literature
17
2.2 LIPID PEROXIDATION
Lipid peroxidation has been defined as “Oxidative deterioration of
polyunsaturated fatty acids”, i.e. more than one carbon-carbon double bonds. It
lowers the nutritional quality of food (Gulcin et al., 2005). Initiation of peroxidation
in a membrane or polyunsaturated fatty acid is due to the attack of any species
that can “pull off” a hydrogen atom from one of the -CH2- groups in the carbon
chain. Many observations support the notion that lipid peroxidation plays an
important role in carcinogenesis (Gulcin, 2006).
Lipid peroxidation is usually initiated by the interaction of a ROS or other
free radical with polyunsaturated fatty acids and exacerbated by the presence
of divalent metal ions. During lipid peroxidation, polyunsaturated fatty acids
are oxidized to produce lipid peroxyl radicals that in turn lead to further
oxidation of polyunsaturated fatty acids in a perpetuating chain reaction as
follows (Novo and Parola, 2008):
RHLRadicalLH radicalperoxyllipidLOOOL 2
npropagatiolipidtoleadingLOOHLLOOLH nterminatiochaintantioxidanLOOHtantioxidanLOO
Lipid peroxides are potentially toxic and possess the capacity to damage
most cells (Halliwell, 1994) by inactivating membrane enzymes and receptors,
depolymerising polysaccharide and cross-linking and fragmenting protein. As a
result, the structure and fluidity of the membrane are damaged and normal cell
function is lost (Ng et al., 2005). Membranes and lipids are particularly
susceptible to the oxidant process and to the peroxidative reaction induced by free
radicals (Rahman, 2003). Great emphasis has recently been placed on the
significant contribution of lipid peroxidation to the development of cancer (Dreher
and Junod, 1996) and atherosclerosis. Lipid peroxidation may be prevented at
the initiation stage by free radical scavengers, while the chain propagation
reaction can be intercepted by peroxy-radical scavengers such as phenolic
antioxidants (Magnani et al., 2000; Sun et al., 2000; Dangles et al., 2000).
Chapter 2 Review Of Literature
18
Lipid peroxidation in biological systems has been considered as one of the
major mechanisms of cell injury in aerobic organisms subjected to oxidation
stress. Lipid peroxidation products such as malondialdehyde (MDA) and
4-hydroxy-2-nonenal (HNE), are closely related to carcinogenesis as they are
potent mutagens and they have been suggested as modulators of signal
pathways related to proliferation and apoptosis, two processes implicated in
cancer development (Olalye and Rocha, 2007).
2.3 ANTIOXIDANTS
The body is usually under a dynamic equilibrium between free radical
generation and quenching. The physiological defense systems to counteract
free radicals encompass endogenous antioxidant enzyme systems such as
catalase, glutathione reductase and superoxide dismutase as well as
glutathione, urate and coenzyme Q or exogenous factors such as β-carotene,
vitamin C, vitamin E and selenium. All these molecules have an antioxidant
effect due to their ability to transform ROS into stable and harmless
compounds or by scavenging both ROS and reactive nitrogen species (RNS)
with a redox based mechanism (Valko et al., 2006).
Under normal physiological conditions, the highly toxic ROS are quenched by
the mitochondrial antioxidant defense systems. In particular, mitochondrial catalase,
manganese superoxide dismutase, as well as glutathione in conjunction with
glutathione peroxidase and glutathione S-transferase regulate inner mitochondrial
membrane permeability by detoxifying ROS produced during electron transport and
confer protection against lipid peroxidative damage (Andreyev et al., 2005).
The plants are susceptible to damage caused by active oxygen and thus
develop numerous antioxidant defense systems resulting in the formation of
numerous potent antioxidants. Many aromatic and spice plants contain compounds
that possess confirmed strong antioxidative components. The essential oils derived
from aromatic plants not only serve as fragrance and flavor agents but also as
dietary antioxidant expected to prevent several diseases caused by free radicals
(Mishra et al., 2007).
Plant extracts with antioxidant activity are traditionally used to
strengthen the natural immune defenses. Many studies have focused on the
antioxidant effects of flavonoids resulting in their identification as potential
Chapter 2 Review Of Literature
19
antioxidants and anticancer agents (Lee et al., 2004). Antioxidant properties of
water and ethanol extract of Day lily flowers were evaluated in terms of total
antioxidant activity, reducing capacity and metal chelating activity and the
ethanolic extract showed strong antioxidant activity which is further evaluated
by feeding mice for 60 days which significantly increased superoxide
dismutase activity and decreased lipid peroxidation in both blood and liver of
mice (Que et al., 2007).
The ethanolic extract of the plant Cytisus scoparius L., showed potent
free radical scavenging and antioxidant activity which might be helpful in
preventing or slowing the progress of various oxidative stress related diseases
(Sundararajan et al., 2006). The methanolic extract of the plants Cassia
spectabilis and Cassia fatula were identified as potentially novel sources of the
radical scavenging compounds (Nehru et al., 2008).
Silymarin
Silymarin, a standardized extract obtained from seeds of Silybum
marianum (Asteraceae or Compositae) is widely used in treatment of liver
diseases of varying origins and cancer (El-Samaligy et al., 2006; Dixit et al.,
2007). It consists of a mixture of three bioflavonoids found in the fruit, seeds and
leaves of this plant namely Silybin, Silydianin and Silychristine (Khan et al.,
2006). Seeds of S. marianum have been used to treat liver and gall bladder
disorders, including hepatitis, cirrhosis and jaundice and to protect the liver
against poisoning from chemicals, environmental toxins, snake bites, insect
stings, mushroom poisoning and alcohols (Ball and Kowdley, 2005; Kren and
Walterova, 2005). It also protects liver cells directly by stabilizing the membrane
permeability through inhibiting lipid peroxidation and preventing liver glutathione
depletion (Skottova et al., 2003).
Silymarin is an important bioactive principle having anticancer,
antiinflammatory, antioxidant and immunomodulatory effects (Okawa et al., 2001;
Lebedev et al., 2001; Tyagi et al., 2002; Kohno et al., 2002; Johnson et al., 2003;
Katiyar, 2005). It is also useful to treat alcoholic DNA damage, in addition to
alcoholic liver injury (Saravanan and Pugalendi, 2005). Silymarin promoted UV-
irradiated A375-S2 cell survival partly. It also modulated the distribution of the cell
cycle to allow more time for the damaged cells to repair. This result may broaden
Chapter 2 Review Of Literature
20
silymarin's potential therapeutic use for many diseases in the future (Li et al., 2007).
The dietary silymarin exerts a chemopreventive effect on 4-Nitroquinoline 1-oxide-
induced rat tongue carcinogenesis, when fed during the promotion phase. This
cancer protective effect of silymarin might relate to the control of carcinogen-
induced hyper-cell proliferation and/or alteration of the amino acid metabolic
pathway (Yanaida et al., 2002).
Recent studies suggested that silymarin and its polyphenolic fraction could
have beneficial effects on some risk factors of atherosclerosis. The results have
shown that silymarin has hypolipidemic effect (Sobolova et al., 2006) and
preventive effect on low density lipoprotein peroxidation in vitro. It also has
protective effects against stress-induced gastric ulcers and induces recovery of
pancreatic function after alloxan damage in rats (Soto et al., 2004).
It has been introduced fairly recently that silymarin can be used as a
hepatoprotectant for acute viral hepatitis, poisoning by Amanita phalloides,
ethanol, paracetamol and carbon tetrachloride (Fraschini et al., 2002). The
silymarin, a single herbal drug formulation which is mostly used in liver disease
amounts to about 180 million dollars in Germany alone (Thakur et al., 2007).
The protection to immune system provided by silymarin appears to rest
on four properties (Figure 3):
activity against lipid peroxidation as a result of free radical
scavenging and the ability to increase the cellular content of GSH
ability to regulate membrane permeability and to increase membrane
stability in the presence of xenobiotic damage
capacity to regulate nuclear expression by means of a steroid-like
effect and
inhibition of the transformation of stellate hepatocytes into
myofibroblasts, which are responsible for the deposition of collagen
fibres leading to cirrhosis.
Chapter 2 Review Of Literature
21
FIGURE 3: MECHANISM OF ACTION OF SILYMARIN
(Valenzuela and Garrido, 1994)
Silymarin and silibinin inhibit the absorption of toxins such as phalloidin or
amanitin, preventing them from binding to the cell surface and inhibiting
membrane transport systems. Furthermore, silymarin and silibinin, by interacting
with the lipid component of cell membranes, can influence their chemical and
physical properties. Studies in erythrocytes, mast cells, leucocytes, macrophages
and hepatocytes have shown that silymarin renders cell membranes more
resistant to lesions (Mourelle et al., 1989).
Furthermore, the well documented scavenging activity of silymarin and
silibinin can explain the protection afforded by these substances against
Chapter 2 Review Of Literature
22
hepatotoxic agents. Silymarin and silibinin may exert their action by acting as free
radical scavengers and interrupting the lipid peroxidation processes involved in
the hepatic injury produced by toxic agents.
Types of antioxidants
Antioxidants are chemical substances that donate an electron to the free
radical and convert it to harmless molecule. Antioxidants are molecules, which
can safely interact with free radicals and terminate the chain reaction before
vital molecules are damaged. Plants have evolved different phytochemicals
and enzymes as antioxidant defense to maintain growth and metabolism.
Antioxidants are produced in leaves and protect the plants from damage by
quenching free radicals (Pandhair and Sekhon, 2006). As a defensive
strategy, cells are capable of inducing antioxidant enzymes such as superoxide
dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), to
remove harmful ROS. Although there are several nonenzymic systems in the
body that scavenge free radicals, the principle micronutrient antioxidants are
vitamin E, vitamin C and beta-carotene (Huang et al., 2005).
Antioxidants of plants experimentally proved to have effective protective
agents against oxidative stress (Rekha et al., 2001). The extracts of Withania
somnifera increase the action of antioxidants CAT, SOD and GPx (Kaur et al., 2004).
Aglaia roxburghiana increase the level of antioxidants by scavenging oxide free
radicals (Chakrabarty et al., 2004). Calotropis species has reported to have very
high antioxidant activity (Mueen et al., 2003). Andrographis paniculata activate
antioxidant enzymes thereby protecting the tissues from free radicals. Leaf
extracts of Aloe vera, Allium sativum, Azadirachta indica, Emblica officinals and
Tinospora cordifolia are some other plants which have been reported to have
antioxidant activity (Govindarajan et al., 2005).
2.3.1 Enzymic antioxidants
The first line of defense is the preventive antioxidants, which suppress
formation of free radical (enzymes such as Catalase, Superoxide dismutase
and Glutathione peroxidase). The first line of defense against and hydrogen
peroxide mediated injury (Figure 4) are antioxidant enzymes like CAT, SOD
and GPx (Bukan et al., 2003).
Chapter 2 Review Of Literature
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FIGURE 4: ACTION OF ENZYMIC ANTIOXIDANTS
(http://www.redlabs.be)
Catalase (CAT)
Catalase is an enzymatic antioxidant widely distributed in all animal tissues
and the highest activity is found in the red cells and in liver and localized mainly in
peroxisome (Prakash et al., 2009). CAT decomposes H2O2 and protects the
tissue from highly reactive hydroxyl radicals (Darlington and Stone, 2001; Edwin
and Jarald, 2005). Therefore, the reduction in the activity of this enzyme may
result in a number of deleterious effects due to the accumulation of superoxide
radicals and hydrogen radicals (Szymonik et al., 2003). Overexpression of
catalase targeted to mitochondria showed extension of murine life span (Schriner
et al., 2005). Catalase contains heme as prosthetic group and can act as
peroxidase when the concentration of H2O2 is low and the concentration of electron
donors is high. They are tetramers each subunit possessing a protoporphyrin IX as
prosthetic group with one Fe (III). It is involved in β-oxidation of fatty acids,
glyoxylate cycle and purine metabolism (Munne-Bosch and Falk, 2004).
Chapter 2 Review Of Literature
24
Catalase has one of the highest turnover rates for all enzymes i.e., one molecule
of CAT can convert 6 million molecules of H2O2 to water and oxygen each minute.
Catalase has a double function, because it catalyses the following reactions.
1) Decomposition of H2O2 to give
2222 OO2HO2H
2) Oxidation of H donors, for example, methanol, ethanol, formic acid,
phenol with the consumption of 1 mole of peroxide.
AROHOHAHROOH 22
Superoxide dismutase (SOD)
Superoxide dismutase is one of the important intracellular antioxidant enzymes;
present in all aerobic cells (Arteel, 2003) has an antitoxic effect against superoxide
anion and forms hydrogen peroxide and molecular oxygen (Loki and Rajamohan, 2003;
Kerksick and Willoughby, 2005; Jyothi et al., 2008; Garg et al., 2008).
22222 OOH2HOO
It is considered to be stress protein, which is synthesized in response to
oxidative stress (Oberley and Oberely, 2006). Mutations in the cytoplasmic or
mitochondrial form of SODs result in aging, neurodegenerative diseases and
carcinogenesis (Bonatto, 2007).
Glutathione peroxidase (Gpx)
The Gpx competes with CAT for H2O2 as a substrate and is the major
source of protection against low levels of oxidative stress. It converts reduced
glutathione into oxidized glutathione and also removes lipid peroxides and
H2O2 along with catalase (Kashyap et al., 2005) leading to the maintenance of
membrane integrity and increased tolerance to oxidative stress caused by
various stress conditions (Yoshimura et al., 2004).
O2HGSSG2GSHOH 222
NADP2GSHHNADPHGSSG
This system forms an excellent protection against lipid peroxidation by
scavenging the lipid peroxides (Figure 5).
OHROHGSSG2GSHROOH 2
Chapter 2 Review Of Literature
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FIGURE 5: ACTION OF GLUTATHIONE PEROXIDASE
(http://www.google.co.in/images)
It also scavenges H2O2, which is responsible for the initiation of lipid
peroxidation (Silva and Jerald, 2005). H2O2 formed by the enzymatic processes
of SOD and peroxidase is reduced to H2O by the reaction of glutathione
peroxidase (GPx) with GSH, which is oxidized to GSSG (Dringen, 2000).
GSSG is then reduced back to GSH; a step catalyzed by glutathione reductase
(GSSG - R) with NADPH, and is then reused as a GPx substrate. This ensures a
steady supply of the reductive substrate (NADPH) to glutathione Peroxidase
(Agarwal and Prabakaran, 2005).
2.3.2 Nonenzymic antioxidants
The antioxidant enzymes are complemented by small molecule
antioxidants. The small molecule antioxidants are present extra and
intracellularly and include Vitamin A, E and Glutathione.
Chapter 2 Review Of Literature
26
Vitamin A
Vitamin A is a fat soluble vitamin is essential for the normal functioning of
the visual system, epithelial cell integrity and growth, immunity and reproduction
(Maciel et al., 2007). It also plays an important role in respiratory diseases.
Vitamin A maintains the integrity of the epithelial tissue and retinoids, either
topically or orally administered, were able to induce complete remission in a high
proportion of patients with basal cell or advanced squamous cell carcinoma. It is
also involved in the regulation of lipid peroxidation in plasma (Zobali et al., 2002).
Retinoic acid is an active metabolite of vitamin A (Ren et al., 2007) and serves as
a hormone like nutrient in cellular differentiation and proliferation in various tissues
including the small intestine (Ogura et al., 2005).
Liver contain the highest concentrations of vitamin A, followed by epidermis
and serum. Vitamin A is an essential micronutrient to the normal brain function.
However, there is an increasing concern regarding the use of Vitamin A high
doses even therapeutically (Oliveira and Moreira, 2007). This enzyme catalyses
the decomposition of H2O2 to water and oxygen and protects the cell oxidative
damage by H2O2 and OH (Schunemann et al., 2002).
Vitamin E
Vitamin E (á-tocopherol) is a major lipid-soluble antioxidant found in
cells. Vitamin E is the most effective chain-breaking antioxidant within the cell
membranes and lipoproteins. Its main antioxidative function is protection
against lipid peroxidation by scavenging peroxyl radical intermediates in the
chain reaction (Ganapathi and Jagetia, 1995; Prior and Cao, 2000). Vitamin E
protects the cells against the effect of dangerous free radicals and potentially
damaging product of our body metabolism (Pryor, 2000; Dwivedi et al., 2005).
Many studies have suggested that high intake of Vitamin E may slow down the
development and progression of atherosclerosis. Some clinical trials also
reported beneficial effects of Vitamin E supplementation in the secondary
prevention of cardiovascular events (Meydani, 2004). It prevents the attack of
ROS of the membrane PUFA (Kamal and Appelqvist, 1996). It protects the
membranes, lipids and lipoprotein (Van Bakl et al., 2000).
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Reduced Glutathione
Reduced glutathione (GSH) found in most tissues, and is present in
millimeter concentrations in some tissues (Townsend and Tew, 2003) cells and
sub cellular compartments, scavenges H2O2, reacts non-enzymatically with singlet
oxygen, superoxide radical and hydroxyl radical (Gomez et al., 2004). It is the
most abundant non protein thiol (Guven and Gulmez, 2004). Halliwell and
Gutteridge (1984) identified that GSH is important in redox regulation of
transcription factors and enzymes for signal transduction. Polyphenols mediated
regulation of GSH alters the cellular processes. GSH is probably the most
important antioxidant present in cells. It is an essential cofactor for GST, which
helps to remove reactive molecules from the cells. Moreover, GSH can interact
directly with certain hydroxyl radical to detoxify them. Mahakunakorn et al. (2004)
stated that GSH functions as a catalyst in disulfide exchange reactions. During
oxidative stress, -SH groups become oxidized to form disulfide links known as
GSSG (Sinha et al., 2007). Gupta et al. (2008) found that glutathione is one of
the most abundant tripeptide L-glutamyl-L-cysteinyl glycine, a non-enzymatic
biological antioxidant present in the liver. Its functions are concerned with the
removal of free radical species and maintenance of thiol proteins and as a
substrate for GPx and GST.
2.4 ANTITUMOR ACTIVITY OF MEDICINAL PLANTS
Isolation and identification of some potent antitumor compounds from plants
has encouraged scientists to screen different parts of plant species against cancer
cell lines (Emami et al., 2005). Increasing DNA repair (folic acid); changing
immunological response (carotenoids, vitamins C and E, selenium and zinc);
inhibition of cyclooxygenase (resveratrol); restriction of caloric intake and
absorption; decreasing time for transit of intestinal bulk, avoiding carcinogen
formation and absorption (fibers); inhibition of angiogenesis and abrogation of tumor
cells proliferation (by suppressing telomerase or induction of apoptosis) also
constitute important antitumor properties of functional foods (Kelloff et al., 2000;
Ferrari and Torres, 2003).
Numerous drugs and compounds have been reported to have antitumor
effects on different organ cancer such as lung, liver, breast and ovarian
(Llovet et al., 2003). Qin et al. (2006) reported that vaccination with
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pSLC-3P-Fc (DNA vaccine) by gene gun inoculation induced a strong
antitumor response in a mouse tumor model, which significantly inhibited tumor
growth and prolonged the survival time of the tumor-bearing mice.
Derivatisation of diospyrin, a bisnaphthoquinonoid isolated from Diospyros
montana Roxb., led to the modification of its inhibitory activity towards a murine
tumor model, Ehrlich ascites carcinoma (ELA) and two human cancer cell
lines: (A375) malignant skin melanoma and (Hep2) epidermoid laryngeal
carcinoma (Sarma et al., 2007).
The effects of the anticancer drug irinotecan combined with ethanolic
extract of propolis, a water-soluble derivative of propolis, quercetin and
naringin on the growth of Ehrlich ascites tumor and the life span of tumor-
bearing Swiss albino mice may be beneficial in maximizing antitumor activity
and minimizing post-chemotherapeutic reactions to the cytostatic drug
(Benkovic et al., 2007). Similar antitumor properties of Zanthoxylum rhoifolium
Lam leaves was investigated in vitro and in vivo using the Ehrlich ascites tumor
model (Silva et al., 2007). The extract of Tinospora cordifolia (Guduchi)
against Ehrlich ascites tumor (ELA) in mice resulted in growth inhibition and
induction of apoptosis in a dose-dependent manner (Thippeswamy and
Salimath, 2007). The aqueous extract from the roots of Glycyrrhiza glabra
inhibits the in vivo and in vitro proliferation of Ehrlich ascites tumor cells and
may be used as a potential supplemental source for cancer therapy
(Sheela et al., 2006).
2.5 IMMUNOMODULATORY ACTIVITY OF MEDICINAL PLANTS
An immunomodulator is a substance which has an effect on the immune
system. There are two types of effects-immunostimulation and
immunosuppression. Immunostimulants primarily have the stimulant effect and
immunosuppressants primarily have the suppressant effect. Immunomodulation is
a process that can alter the immune system of an organism by interfering with its
functions (Shivaprasad et al., 2006). The immune system may be the last line of
defense against cancer development. According to the most recent point of view
about cancer immunology, the key issue is whether recognition of tumor antigens
by the immune system leads to activation (i.e. surveillance) or tolerance
(Pardoll, 2003).
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The natural resistance of the body against infection can be enhanced by
the use of herbal drugs. Several herbal preparations that can enhance the body’s
immune status are extensively being used in the indigenous system of medicines.
There is an upsurge in the clinical usage of indigenous drugs as they are free from
serious side effects. The term immunomodulatory means regulation of the
immune system by suppression and stimulation of the cells and organs of the
immune system (Bafna and Mishra, 2005). Immunostimulation in a drug-induced
immunosuppression model and immunosuppression in an experimental hyper-
reactivity model by the same preparation can be said to be true
immunomodulation (Patwardhan et al., 1990). Certain agents have been shown
to possess activity to normalize or modulate pathophysiological processes and
are hence called immunomodulatory agents. A number of plant products are
being investigated for immune response modifying activity. Modification of
immune functions by pharmacological agents is emerging as a major area of
therapeutics (Upadhyay, 1997).
Immunomodulators are used as an adjuvant in conditions of
immunodeficiency in cancer and to a limited extent in acquired immunodeficiency
syndrome (Malfitano et al., 2006). Immunomodulatory agents of plant and animal
origin enhance the immune response of an organism against a pathogen by
activating the immune system. However these agents should be subjected to
systematic studies to substantiate the therapeutic claims made with regard to their
clinical utility (Fulzele et al., 2003). The immune system is known to be involved in
the aetiology as well as pathophysiologic mechanism of many diseases.
Immunology is thus probably one of the most rapidly developing areas of biomedical
research and has great promises with regard to prevention and treatment of a wide
range of disorders (Ziauddin et al., 1996). Immunomodulators of herbal origin appear
to be a better alternative to overcome the above problem. Herbal drugs are known to
possess immunomodulatory properties and generally act by stimulating both specific
and non-specific immunity (Patwardhan et al., 1991).
Many plants used in traditional medicine have immunomodulating
activities. Some of these stimulate both humoral and cell-mediated immunity,
while others activate only the cellular components of the immune system. Some
of these plants also suppress both humoral and cell-mediated immunity. The
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30
great majority of chemicals identified as cytotoxic to cancer cells are generally
also toxic to normal cells (Kim et al., 1996). Nevertheless, the potentiation of host
defense mechanisms has been recognized as a possible means of inhibiting
tumor growth without harming the host (Ameho et al., 1997). Therefore,
searching for immunomodulatory materials from natural herbs and characterizing
the immune enhancement effects may have great potential in cancer treatment,
based on a combination of time honored traditional usage and ongoing scientific
research (Rivera et al., 2003).
Acidic polysaccharides isolated from Tanacetum vulgare L. suggesting the
modulation of immune system (Xie et al., 2007). The administration of crude
extracts of Nyctanthes arbortristis to Swiss albino rats showed significant increase
in RBC and WBC counts. The ethanolic extract of Nyctanthes arbortristis
screened in rats for humoral and cell mediated immunity showed enhanced
circulating antibody titer and delayed type hypersensitivity reactions (Kannan
et al., 2007). Immunostimulatory activities of Chlorophytum borvilionum was
evident when ethanolic extract of roots were administered to the wistar strain
albino rats to asses their immunomodulatory activity. The studies showed that
there is improved survival against Candidia albicans and showed increase in
delayed type hypersensitivity response, percentage neutrophil adhesion and in
vivo phagocytosis (Thakur et al., 2007).
The methanolic extract of Haussknechtia elymatica showed inhibitory
effects on both humoral and cell mediated immune response in a dose dependent
manner (Amirghofran et al., 2007). The aqueous extract of Actinidia
macrosperma on the growth of ascite tumor showed it exerts in vivo
immunomodulatory activities on S180-bearing mice in a dose dependent manner.
The assessment of immunomodulatory activity against tumor was carried out by
testing the humoral, cellular and non-specific immune response to the antigenic
challenge by sheep RBC, lymphocyte proliferation and natural killer cell
cytotoxicity and by macrophages function test showed significant increase in the
immune functions (Lu et al., 2007).
The immunomodulatory activity determined by administration of
Kalpaamrutha which is composed of Semecarpus anacardium nut milk, Phyllanthus
emblica and honey into Wistar albino rats and the humoral, cell mediated and non-
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31
specific immunity assessed by haemagglutination titer, delayed type
hypersensitivity and phagocytic index respectively showed immunostimulatory role
(Sujatha and Sachdanadam, 2004; Arulkumaran et al., 2007). Butanol and ethyl
acetate soluble fractions of methanolic extracts of Lagenaria sinceraria fruits
significantly inhibited sheep RBC induced delayed type hypersensitivity response.
Both these fractions significantly increased haemagglutination antibody titer in a
dose dependent fashion. Both the fractions significantly increased total WBC,
neutrophil and lymphocyte counts, while insignificant change was observed in
monocyte, eosinophil and basophil counts (Gangwal et al., 2008).
Immunomodulatory function of Tinospora cordifolia has been reported
on increased total WBC count, using Balb/C mice and enhancement in
antibody titre, macrophage activation (Kuttan, 2000). The antibody titre,
spleenocytes and peripheral immune cells showed a significant result of
Nigella sativa immunomodulatory effect against thyroid antigen
(Newaz et al., 2005). The aqueous extract of tea, Camellia sinensis possesses
the immunomodulatory effect (Zvetkova et al., 2001).
2.6 MEDICINAL PLANTS SELECTED FOR THE STUDY
2.6.1 Cynodon dactylon
Cynodon dactylon (Poaceae), a hardy perennial grass, forming thick
mats by means of stolons and rhizomes (Gibbs Russell et al., 1991), eight to
40 culms (stems), rarely to 90 cm high; leaves hairy or glabrous, three to seven
spikes (rarely two), usually 3-6 cm long and in one whorl, or in robust forms up
to ten spikes and is one of the most commonly occurring weeds in India.
Although a problem for farmers, dhoub grass is a valuable herbal medicinal
plant and used as first aid for minor injuries (Oudhia, 1999a and Oudhia,
1999b). Bermuda grass is a perennial weed growing largely from root stocks
and stolons (Oudhia, 2001; Oudhia, 2002).
The aqueous fluid extract of the rhizome is used as anti-inflammatory,
diuretic, anti-emetic, purifying agent and also in dysentery (Ahmed et al., 1994;
Singh et al., 2008). As medicine Cynodon dactylon holds a reputed position in all
systems of medicine in India. According to Ayurveda, India’s traditional
pharmacopoeia, Cynodon dactylon plant is pungent, bitter, fragrant, heating,
appetizer, vulnary, antihelminthic and antipyretic. It destroys foulness of breath,
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32
useful in leucoderma, bronchitis, piles, asthma, tumors and enlargement of the
spleen (Mahesh and Brahatheeswarn, 2007). According to Unani system of
medicine, Cynodon dactylon is bitterish, vulnerary, expectorant and useful in
vomiting, diarrhoea, burning sensation, blood disorders, stomatitis, biliousness
and hiccup (Oudhia, 2003).
The ethanol extract of aerial parts of Cynodon dactylon showed marked
CNS depressant activities compared to other extracts of it in preliminary
pharmacological screening. The plant Cynodon dactylon has been reported for
antiatherosclerotic, antioxidant, Helicobacter pylori activities and traditionally, for
management of neurodegenerative diseases (Surendra et al., 2008).
Singh et al. (2007) revealed that the Cynodon dactylon aqueous extract has
remarkable effects on blood glucose level and marked improvement on
hyperlipidemia due to diabetes. Its specific effect on HDL has additional
advantage in checking coronary risks. Virus-affected discolored leaves of
Cynodon are used for the treatment of liver complaints. In Homoeopathic
systems of medicine, it is used to treat all types of bleeding and skin troubles
(Oudhia et al., 1998). Farmers traditionally apply crushed leaves to minor wounds
as a styptic to stop bleeding similar to Tridax procumbens, Achyranthes aspera
and Blumea iacera
(Oudhia and Pal, 2000).
2.6.2 Terminalia catappa
Terminalia catappa is a large deciduous tree, originally from India, growing
up to 9.0 feet tall with horizontal whorls of branches offering clusters of foot long,
obovate leaves. Its bark is brownish black and its fresh leaves are green in colors
which turn pink-red to red-yellow before falling. The leaves turn brown before
decaying. This changing of leaf’s color is caused by the present of pigments like
violaxanthin, lutein and zeaxanthin. The leaves are quickly replaced by new
growth so the tree is bare for only a short period of time (Gilman and Watson,
2007). It alternates fragrant white tomentose flower where its female and male
flowers are on the same tree (Lopez-Hernandez et al., 2001; Chen and Nicholas,
2007). These flowers are not very showy. The tree has large nutty edible fruits
that taste very much like commercially grown almonds and the color of the fruit is
green, yellow or reddish.
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The best Terminalia catappa Linn habitat is at an area that receives full
sun, moist, well drained soil, has salt and drought tolerance. The more specific
habitats are at sandy seashores, beaches with humid climate, village and grassy
village commonly. It can shadow large area underneath it till many people
consider T. catappa as a desirable shade tree. This tree is quite popular in
medical world whether in modern or traditional one. The most important and
useful part is definitely its leaves. In Taiwan, the fallen leaves are used as an
herbal drug in the treatment of liver related diseases.
The extract of T. catappa leaves inhibit Lewis lung carcinoma cells that
contribute to lung cancer (Chu et al., 2007). It means T. catappa is an anticancer
agent. In another study conducted by Ahmed et al. (2005) showed that the
extracts of T. catappa produced a significant antidiabetic activity. The various
extract of leaves is also reported to be anticarcinogenic and antioxidant. The
antioxidant in T. catappa gives anticlastogenic effect - a process which causes
breaks in chromosomes. While in traditional treatment, a tea from the leaves is
used against diarrhea and dysentery. The extract of T. catappa showed
antimicrobial activities against Escherichia coli, Staphylococcus aureus,
Pseudomonas aeruginosa, Candida albicans, Trichophyton mentagrophytes,
Pityrospon ovale and Microspoum gypseum (Suganda et al., 2004).
In view of its chemical composition, antioxidative potential of the plant has
been evaluated and proved by many workers (Wang et al., 2000; Ko et al., 2002).
The plant is very well known for its therapeutic values since long and has proved
by many researchers to be useful as antiinflammatory (Fan et al., 2004),
anticancer (Kandil et al., 1999), antihepatotoxic (Lin et al., 2001), antigenotoxic
(Chen et al., 2000), anticlastogenic (Liu et al., 1996), for the treatment of skin
aging, irritation, hyperpigmentation, allergy (Renimel et al., 1998) and bronchial
asthma in children (Prazeres, 1995). It also exhibits antimicrobial (Pawar and Pal,
2002; Naz et al., 2007; Nair and Chanda, 2008), insecticidal and molluscicidal
activities (Jayasinghe et al., 2000).