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Appendices
Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
6
The medicinal plants selected for the present research work were
Morinda tinctoria and Nerium indicum. Morinda tinctoria belongs to the family
Rubiaceae that grows widely and distributed throughout Southeast Asia. It is
commercially known as Nunaa, indigenous to tropical countries and considered as
an important folklore medicine. In the traditional system of medicine, leaves and
roots of Morinda tinctoria are used as astringent, deobstruent, emmenagogue and
to relieve pain in the gout (Kumaresan and Saravanan, 2009).
Nerium indicum L. is an evergreen shrub belongs to the family
Apocynaceae, and distributed in tropical Asia (Garima and Amla, 2010). Nerium
indicum is commonly known as Indian oleander (Sikarwar et al., 2009) an
important plant used against various disorders in indigenous system of medicine.
The plant originates from the Mediterranean region and is indigenous to
Indo-Pakistan subcontinent (Govind, 2010a). It is a well known ornamental plant
with leathery evergreen leaves and handsome clusters of red, pink or white flowers
(Jawarkar et al., 2012). Leaves are powerful repellent and the decoction of the
leaves has been applied externally in the treatment of scabies and to reduce
swellings. The leaves and the flowers are cardiotonic, diaphoretic, diuretic,
emetic, expectorant and sternutatory (Shah and Chakraborthy, 2010).
So far, no studies were undertaken to explore the antioxidative, anticancer
and antimicrobial activity of Morinda tinctoria and Nerium incdicum leaves
with an in silico approach. This made us to investigate and examine the
“Anticarcinogenic Effect in DLA Transplanted Mice and Antimicrobial
Efficacy of Morinda tinctoria and Nerium indicum and their Characterization
by in silico Studies”
The present study was carried out in five phases with the following objectives:
• To assess the in vitro antioxidative and anticarcinogenic efficacy of
Morinda tinctoria and Nerium indicum leaves.
• To evaluate the in vivo antioxidative and anticarcinogenic potential of
Morinda tinctoria and Nerium indicum leaves in DLA tumour induced Swiss
albino mice.
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
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• To evaluate the antimicrobial activity of Morinda tinctoria and
Nerium indicum leaves against different microorganisms.
• To characterize the phytochemical constituents of Morinda tinctoria and
Nerium indicum leaves.
• To evaluate the anticarcinogenic and antimicrobial effect of active
compounds of Morinda tinctoria and Nerium indicum leaves against cancer
and microbial target proteins using molecular docking software GLIDE.
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
8
RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE
Human history would be incomplete without illustrating the role of plants,
because early man was directly dependent on plants for his needs (Qureshi et al.,
2006 and Rauf et al., 2012). In both developing and developed countries, the
demand for plant-based therapeutics is increasing to a greater extent due to the
growing recognition of their benefits. They are natural products; are neither
sedatives nor tranquilizers; are eco-friendly, producing minimum environmental
hazards; have no adverse side effects; and are reasonably priced (Shariff et al.,
2006).
Cancer is one of the ten leading diseases which cause death and
advancing in rank year by year throughout the world (Sundaram et al., 2012).
Cancer is a group of diseases where cell growth is aggressive, abnormal, invasive
and metastatic many times leading to death (Tongyoo, 2010). Natural products,
especially those from plants, have been a valuable source of new cancer drugs for
many decades. Medicinal plants are the most exclusive source of life saving drugs
for the majority of the world’s population (Thakore et al., 2012).
Oxidative stress is a known mediator of cancer. Medicinal plants are
important source of antioxidants and serve as subcellular messengers of normal
cell function and have a significant protective role against oxidative injury (Upham
and Wagner, 2001). The secondary metabolites from plants have been reported to
be a potent antioxidant and free radical scavengers (Vadnere et al., 2012).
Considering the negative effects of synthetic drugs, people are looking for natural
remedies, which are safe and effective. In this respect, medicinal plants used in
the traditional therapy could be the alternative source for the development of new
therapeutic agents to combat with the resistant organisms. Nowadays, numerous
scientific investigations are going on in isolation of potent phytochemicals as
leading compounds for antimicrobial therapy (Bhattarai and Bhuju, 2011).
2
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
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Molecular docking is a key tool in structural molecular biology and
computer-assisted drug design. The goal of ligand protein docking is to predict
the predominant binding model(s) of a ligand with a protein of known three
dimensional structures (Srivastava et al., 2010).
Information on the Antioxidative, anticancer and antimicrobial role of
Morinda tinctoria and Neriun indicum leaves and its in silico assessment using
glide is still very scanty in literature. The review of literature pertaining to the
present research entitled “Anticarcinogenic Effect in DLA Transplanted Mice
and Antimicrobial Efficacy of Morinda tinctoria and Nerium indicum and their
Characterization by in silico Studies” is appropriately presented under the
following headings:
2.1. Oxidative Stress
2.1.1. Free radicals
2.1.1.1. Reactive oxygen species
2.1.1.2. Reactive nitrogen species
2.1.1.3. Reactive sulphur species
2.1.1.4. Mechanism of formation and damages caused by free radicals
2.1.2. Non-radicals
2.1.2.1. Hydrogen peroxide
2.1.2.2. Nitric oxide
2.1.3. Synthetic free radicals
2.1.3.1. ABTS
2.1.3.2. DPPH
2.1.4. Lipid peroxidation
2.2. Antioxidants
2.2.1. First line defense antioxidants
2.2.1.1. Catalase
2.2.1.2. Glutathione peroxidase
2.2.1.3. Glautathione reductase
2.2.1.4. Superoxide dismutase
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
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2.2.2. Second line defense antioxidants
2.2.2.1. Vitamin A
2.2.2.2. Vitamin E
2.2.2.3. Vitamin C
2.2.2.4. Reduced glutathione
2.3. Anticancer activity of medicinal plants
2.4. Antimicrobial activity of medicinal plants
2.5. Phyochemical constituents of medicinal plants
2.6. In silico drug design and docking for cancer
2.6.1. Histone deacetylase
2.6.2. Tubulin
2.6.3. Aurora kinase A
2.6.4. Protein kinase C
2.7. In silico drug design and docking for microbial infections
2.7.1. Pantothenate Kinase
2.7.2. Deacetoxy C synthase
2.8. Medicinal plants selected for the study
2.8.1. Morinda tinctoria
2.8.2. Nerium indicum
2.1. Oxidative Stress
Oxidative stress is a phenomenon associated with pathogenetic mechanisms
of several diseases including atherosclerosis, neurodegenerative diseases, such
as Alzheimer’s and Parkinson’s disease, cancer, diabetes mellitus, inflammatory
diseases, as well as psychological diseases or aging processes. Oxidative stress
is defined as an imbalance between production of free radicals and reactive
metabolites, so-called oxidants, and their elimination by protective mechanisms,
referred to as antioxidative systems. This imbalance leads to damage of important
biomolecules and organs with potential impact on the whole organism. Oxidative
and antioxidative processes are associated with electron transfer influencing the
redox state of cells and the organism (Durackova, 2010).
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Figure 1
Oxidative stress related diseases
(http://www.google.co.in/imgres)
Uncontrolled generation of ROS can lead to their accumulation causing
oxidative stress in the cells. All forms of life maintain a steady state concentration
of ROS determined by the balance between their rates of production and their
rates of removal by various antioxidants. Thus each cell is characterized by a
particular concentration of reducing species like GSH, NADH, NADPH and FADH
stored in many cellular constituents which determines the redox state of a cell.
By definition redox state is the total reduction potential or the reducing capacity
of all the redox couples such as GSSG/2GSH, NAD+/NADH, Asc•−/AcsH−, NADP+
/ NADPH found in biological fluids, organelles, cells or tissues. Redox state not
only describes the state of a redox pair, but also the redox environment of a cell.
Under normal conditions, the redox state of a biological system is maintained
towards more negative redox potential values. However, with increase in
ROS generation or decrease in antioxidant protection within cells, it is shifted
towards less negative values resulting in the oxidizing environment. This shift from
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
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reducing status to oxidizing status is referred as oxidative stress (Schafer and
Buettner, 2001; Kohen and Nyska, 2002).
Figure 2
Mutual association between oxidants and antioxidants
(Durackova, 2010) 2.1.1. Free radicals
A free radical has one or more unpaired electrons in its outer orbital. Such
unpaired electrons make these species very unstable and therefore quite reactive
with other molecules. They try to pair their electron(s) and generate a more stable
compound (Shekhawat et al., 2010). Reactive oxygen species (ROS) or free
radicals, formed during physiological and pathological conditions in the body, are
extremely reactive and react with proteins, lipids, carbohydrates and nucleic
acids (Yildiz et al., 2011). The various pathways involved in the generation of
free radicals are given in Figure 3.
The nitrogen derived free radicals are nitric oxide (NO), peroxy nitrite anion
(ONOO), Nitrogen dioxide (NO2) and Dinitrogen trioxide (N2O3). The thiol derived
free radicals include sulphite (SO32-), disulfide S oxide (DSSO), sulfenic acid
(RSOH) and sulfenyl (RS.) radicals (Panchawat et al., 2010 and Mathew et al.,
2011). When an overload of free radicals cannot gradually be destroyed,
their accumulation in the body generates a phenomenon called oxidative stress.
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
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Figure 3
Production of free radicals
(Kunwar and Priyadarsini, 2011)
This process plays a major role in the development of chronic and
degenerative illness such as cancer, autoimmune disorders, aging, cataract,
rheumatoid arthritis, cardiovascular and neurodegenerative diseases (Pham-Huy
et al., 2008).
2.1.1.1. Reactive oxygen species (ROS)
Reactive oxygen species is a collective term that describes the chemical
species that are formed upon incomplete reduction of oxygen. ROS are thought to
mediate the toxicity of oxygen because of their greater chemical reactivity with
regard to oxygen (Autreaux and Toledano, 2007). The sequential reduction of
oxygen through the addition of electrons lead to the formation of a number of
ROS including: superoxide, hydrogen peroxide, hydroxyl radical, hydroxyl
ion, peroxyl and alkoxyl radicals. Most reactive oxygen species are generated as
by-products during mitochondrial electron transport. In addition, ROS are formed
as necessary intermediates of metal catalyzed oxidation reactions (Dolai et al.,
2012 and http://www.biotek.com/resources/articles/reactive-oxygen-species.html).
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Figure 4
Generation of reactive oxygen species
(http://www.rndsystems.com/cb_detail_objectname_SP96_FreeRadicalsOxidativeStree.aspx)
The ROS may be very damaging, since they can attack lipids in cell
membranes, proteins or enzymes in tissues, carbohydrates and DNA, to induce
oxidations, which may causes cancer, atherosclerosis, aging, immunosuppression,
inflammation, ischemic heart disease, diabetes, hair loss and neurodegenerative
disorders such as Alzheimer’s disease and Parkinson’s disease (Zadak et al.,
2009 and Naskar et al., 2010). Living cells possess a protective system of
antioxidants which prevents excessive formation and enables the inactivation of
ROS. The antioxidants protect from the potentially damaging oxidative stress,
which is a result of an imbalance between the formation of ROS and the
antioxidant defense of the body (Kratchanova et al., 2010).
The exogenous sources of ROS include electromagnetic radiation,
cosmic radiation, UV-light, ozone, cigarette smoke and low wavelength
electromagnetic radiations; and endogenous sources are mitochondrial
electron transport chain and β-oxidation of fat (Panchawat et al., 2010). ROS
can cause tissue damage by reacting with lipids in cellular membranes,
nucleotides in DNA (Ahsan et al., 2003), sulphydryl groups in protein and cross-
linking/fragmentation of ribonucleoproteins (Knight, 1995; Waris and Alam, 1998).
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
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The role of reactive oxygen species (ROS) in carcinogenesis has been
extensively investigated in several cellular and animal models, and various
connections have been established (Gibellini et al., 2010). In cancer cells, ROS
increase the rate of mutagenicity, which leads to DNA damage and chromosomal
instability, and thereby accelerates cancer progression (Radisky et al., 2005). On
the one hand, appropriate levels of ROS play an important role in the modulation
of several physiologic responses in signalling network regulating cell function. On
the other hand, excessive intracellular levels of ROS, as well as a defective
antioxidant system, can give rise to pathological conditions, and even encourage
the progression of these conditions (Rhee, 2006).
Hydroxyl radicals
Among ROS, hydroxyl radical (OH.) is the most reactive oxidant. Hydroxyl
radical is a highly reactive radical formed in biological systems and capable of
damaging almost every molecule found in living cells. This radical has the capacity
to induce carcinogenesis, mutagenesis and rapidly initiates lipid peroxidation
(Manian et al., 2008). In the Haber-Weiss reaction hydroxyl radicals are generated
in the presence of hydrogen peroxide and iron. The first step involves reduction of
ferric into ferrous ion and the second step is the Fenton reaction. The generation
of hydroxyl radicals catalyzed by ferric ions without any additional redox agent, this
can be considered as a special case of the Fenton reaction. Here, one electron
from the hydroxyl group of water is transferred to the ferric ion with the formation of
a divalent iron and a hydroxyl radical (Lipinski, 2011).
Fe3+ +•O2
- Fe2+ +O2.
Fe2+ +H2O2 Fe3+ +OH− +OH•
Fe3+ +HO− Fe2+ +OH•
Superoxide anions
Superoxide anion radical is known as an initial radical and plays an
important role in the formation of other reactive oxygen species, such as hydrogen
peroxide, or singlet oxygen in living systems (Stief, 2003). It plays an important
role in oxidative stress and related to the pathogenesis of various important
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diseases (Kumar and Kumar, 2009). Superoxide is biologically important since it
can be decomposed to form stronger oxidative species such as singlet oxygen and
hydroxyl radicals (Pari and Amudha, 2011). Superoxide dismutase catalyzes the
dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen
(Sharma et al., 2011).
The most commonly occurring cellular free radical is superoxide radical,
which is produced when an oxygen molecule gains one electron from another
substance (Sherki et al., 2002). The superoxide anion radical is dismutated to
hydrogen peroxide (H2O2). H2O2 is converted to the hydroxyl radical in the
presence of a transition metal such as iron; this is identified as the Fenton reaction
(Reiter, 2000).
(Kyaw et al., 2004)
2.1.1.2. Reactive nitrogen species
Nitrogen-derived free radicals are called reactive nitrogen species (RNS) and
their utmost representative precursors are nitric oxide (NO) and peroxynitrite
(ONOO−). NO is well known to be a product of the catalytic action of the nitric
oxide synthase (NOS) enzyme family on L-arginine (Espey et al., 2002; Li and
Poulos, 2005). However, evidence suggests that it can also be formed by
reduction of nitrite, which can arise in the body by ingestion or from bacterial
metabolism (Lundberg and Weitzberg, 2005). Low levels of both ROS and RNS
are continuously produced in mammalian cells and play important physiological
roles (Gutteridge and Halliwell, 2000). These include processes as diverse as
gene expression (Allen and Tresini, 2000), cell proliferation and survival (Kamata
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
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and Hirata, 1999), pathogen clearance by the immune system, and blood vessel
permeability. Reactive nitrogen species (RNS) such as nitrogen dioxide (•NO2),
peroxynitrite (ONOO–) and nitrosoperoxycarbonate (ONOOCO2–) are among the
most damaging species present in biological systems due to their ability to cause
modification of key biomolecular systems through oxidation, nitrosylation, and
nitration (Nash et al., 2012).
Figure 5
Generation of reactive nitrogen species
(Eleuteri et al., 2009)
2.1.1.3. Reactive sulfur species (RSS)
Sulfur is an essential and quantitatively important element for living
organisms. Sulfur is a constituent of many organic molecules, for example amino
acids such as cysteine and methionine and the small tripeptide glutathione, but
sulfur is also essential in the form of Fe–S clusters for the activity of many
enzymes, particularly those involved in redox reactions. Sulfur chemistry is
therefore important. In particular, sulfur in the form of thiol groups is central to
manifold aspects of metabolism. Because thiol groups are oxidized and reduced
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easily and reversibly, the redox control of cellular metabolism has become an
increasing focus of research. In the same way that oxygen and nitrogen have
reactive species (ROS and RNS), sulfur too can form reactive molecular species
(RSS), for example when a –SH group is oxidized. Indeed, several redox reactions
occur via RSS intermediates. Furthermore, RSS can also be used as redox-active
pharmacological tools to study cell metabolism (Gruhlke and Slusarenk, 2012).
Figure 6
Generation of reactive sulfur species
(Gruhlke and Slusarenk, 2012)
Free radicals are formed from molecules via the breakage of a chemical
bond such that each fragment keeps one electron, by cleavage of a radical to give
another radical and also via redox reactions (Bahorun et al., 2006). Oxidation is
one of the most important free radical producing processes in food, chemicals and
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Anticarcinogenic effect in DLA transplanted mice and antimicrobial efficacy of Morinda tinctoria and Nerium indicum and their characterization by in silico studies
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even in the living system. Free radicals play an important role in food and chemical
material degradation, and they also contribute to more than one hundred disorders
in humans (Gorghiu et al., 2004; Ye and Song, 2008).
Free radicals are very unstable and react quickly with other compounds,
trying to capture the needed electron for gaining stability. Generally, free radicals
attack the nearest stable molecule, and “steal” its electron. When the “attacked”
molecule loses its electron, it becomes a free radical itself, and begins a chain
reaction. Once the process is started, it can cascade, finally resulting in the
disruption of a living cell. Some free radicals arise normally during metabolism.
Sometimes the body’s immune system’s cells purposefully create them to
neutralize viruses and bacteria. However, environmental factors such as pollution,
radiation, cigarette smoke and herbicides can also spawn free radicals
(http://www.sstwo-mall.com/index.php/free-radicals/).
2.1.1.4. Mechanism of formation and damages caused by free radicals
Free radicals can be formed by three ways.
• By homolytic cleavage of covalent bond of normal molecule, with each
fragment retaining one of paired electrons.
X: Y X* + Y*
• By the loss of single electron from normal molecule.
X: Y X+ + Y-
• By addition of single electron to normal molecule.
X + e- X (Kumar, 2011)
� Oxidative damages to lipids
All of the most important classes of biomolecules may be attacked by free
radicals but lipids are probably the most sensitive. Cell membranes are rich
sources of polyunsaturated fatty acids (PUFAs), which are readily attacked by
oxidising radicals. The oxidative destruction of PUFAs, known as lipid
peroxidation, is particularly damaging because it proceeds as a self-perpetuating
chain-reaction (Palmieri and Sblendorio, 2006).
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� Oxidative damages to proteins
Oxidative attack on proteins results in site-specific amino acid modification,
fragmentation of the peptide chain, aggregation of cross linked reaction products,
altered electrical charges and increased susceptibility to proteolysis (Farr and
Kogoma, 1991).
� Oxidative damage to DNA
Activated oxygen and agents that generate oxygen free radicals, such as
ionizing radiations, induce numerous lesions in DNA that causes deletion,
mutations and other lethal genetic effects. Characterization of this damage to DNA
has indicated that both sugar and base moieties are susceptible to oxidation,
causing base degradation, single strand breakage and cross links to proteins
(Imlay and Linn, 1988).
2.1.2. Non radicals
Non radicals containing two electrons per orbital, which is a stable
configuration in a molecule, include hydrogen peroxide and nitric oxide.
2.1.2.1. Hydrogen peroxide
Hydrogen peroxide is the two electron reduction product of O2. It is
potentially reactive oxygen, but not a free radical (Halliwell et al., 2000). By
comparison with superoxide and certainly by comparison with the hydroxyl radical,
H2O2 is relatively “safe” in the absence of transition metals, it is stable and
unreactive, even at concentrations much higher than a biological system would
ever generate (John, 2007). Hydrogen peroxide is a weak oxidizing agent that
inactivates a few enzymes directly, usually by oxidation of essential thiol (-SH)
groups. It can cross cell membranes rapidly; once inside the cell, it can probably
react with Fe2+ and possibly Cu2+ ions to form hydroxyl radicals and this may be
the origin of many of its toxic effects (Miller et al., 1993). Although hydrogen
peroxide itself is not very reactive, it may convert into more reactive species such
as singlet oxygen and hydroxyl radicals (Bhattacharjee et al., 2011).
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2.1.2.2. Nitric oxide
Nitric oxide (NO) has emerged as one of the most intriguing molecules in
vertebrate biology in recent years (Reeves et al., 2008). NO is lipophilic and highly
diffusible solute forms within the cell and its actions are concentration dependent
(Kim et al., 2001). In addition to reactive oxygen species, nitric oxide is also
implicated in inflammation and other pathological conditions (Nabavi et al., 2008a).
NO has been associated with a variety of physiological processes in the human
body since it was identified as a novel signal molecule. It transmits signals from
vascular endothelial cells to vascular smooth muscle cells and causes vascular
dilation. It also plays an important role in physiological functions in respiratory,
immune, neuromuscular and other systems (Ebrahimzadeh et al., 2010).
2.1.3. Synthetic free radicals
2.1.3.1. 2, 2′-Azinobis (3-Ethylbenzothizoline-6-Sulfonicacid) (ABTS+·)
Bleaching of a preformed solution of the blue-green radical cation 2, 2′-
azinobis (3-ethylbenzothizoline-6-sulfonic acid) (ABTS+·) has been extensively
used to evaluate the antioxidant capacity of complex mixtures and individual
compounds (Henriquez et al., 2002). ABTS-, the oxidant is generated by persulfate
oxidation of 2, 2′-azinobis (3-ethylbenzothizoline-6-sulfonic acid) - (ABTS2-).During
this reaction, the blue ABTS radical cation is converted back to its colorless neutral
form. The reaction may be monitored spectrophotometrically. This assay is often
referred to as the trolox equivalent antioxidant capacity (TEAC) assay (Barclay et
al., 1985 and Baskar et al., 2008). The ABTS radical cation is reactive towards
most antioxidants including phenolics, thiols and Vitamin C (Richard and Jace,
2009).
(Zulueta et al., 2009)
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2.1.3.2. Diphenylpicrylhydrazyl radical (DPPH)
DPPH (1, 1-diphenyl-2-picryl-hydrazyl) is a relatively stable nitrogen centered
free radical that easily accepts an electron by reacting with suitable reducing
agents (Narayanaswamy et al., 2011). It is an excellent tool for determining the
antioxidant activity of hydrogen donating oxidants and of chain breaking
antioxidants (Karthika et al., 2012). DPPH radical is a commonly used substrate
for fast evaluation of antioxidant activity because of its stability in the radical form
and simplicity of the assay (Bozin et al., 2008). This assay is known to give
reliable information concerning the antioxidant ability of the tested compounds
(Huang et al., 2005). The principle behind this assay is the colour change
of DPPH solution from purple to yellow as the radical is quenched by the
antioxidant (Karagozler et al., 2008).
Figure 7
Diphenylpicrylhydrazyl Radical (DPPH)
a) Diphenylpicrylhydrazyl b) Diphenylpicrylhydrazine (non radical) (free radical)
(Molyneux, 2004)
When a solution of DPPH is mixed with substance that can donate a
hydrogen atom, that give rise to the reduced form (Figure 7 b) with the loss of
violet colour (although there may be a residual pale yellow colour from the picryl
group still present). Representing the DPPH radical by Z• and the donor molecule
by AH, the primary reaction is
Z• + AH ZH + A•
Where, ZH is the reduced form and A• is free radical produced in this first
step (Molyneux, 2004).
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2.1.4. Lipid peroxidation
Lipid peroxidation has gained more importance today because of its
involvement in pathogenesis of many diseases like atherosclerosis, cancer,
hepatitis, diabetes mellitus, myocardial infarction and also ageing. Free radicals or
Reactive Oxygen Species (ROS) are produced in vivo from various biochemical
reactions and also from the respiratory chain as a result of occasional leakage.
These free radicals are the main agents in lipid peroxidation (Donfack et al., 2011).
Lipid peroxidation is initiated by the attack on a fatty acid or fatty acyl side chain of
any chemical species. Especially the group of polyunsaturated fatty acids (PUFAs)
is highly susceptible to reactions with free radicals. Peroxidation of fatty acids in
lipids may lead to a radical chain reaction (Rajeshwari and Andallu, 2011).
(Novo and Parola, 2008)
Enhanced production of oxygen free radicals is responsible for peroxidation
of membrane lipids and the degree of peroxides damage of cell was controlled by
the potency of peroxidase enzyme system (Sairam and Tyagi, 2004).
Malondialdehyde (MDA) is a peroxidative decomposition product of polyenoic fatty
acids, and increase in tissue levels indicates an expansive lipid peroxidation (Cavit
et al., 2010).
Unsaturated fatty acids such as those present in cellular membranes are a
common target for free radicals. Reactions typically occur as a chain reaction
where a free radical will capture a hydrogen moiety from an unsaturated carbon to
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form water. This leaves an unpaired electron on the fatty acid and that is
then capable of capturing oxygen, forming a peroxy radical. Lipid peroxides
are unstable and decompose to form a complex series of compounds,
which include reactive carbonyl compounds such as malondialdehyde (MDA)
(http://www.biotek.com/resources /articles/ reactive-oxygen-species.html).
2.2. Antioxidants
Antioxidants are substances that may protect human body cells from the
damages caused by unstable free radicals and they are highly reactive chemicals
that play a part in some forms of cancer. Antioxidants interact with and stabilize
free radicals and may prevent some of the damages created by free radicals.
Propagation and initiation of free radicals chain reaction can be delayed or
minimized by the donation of hydrogen from the antioxidants (Hamid et al., 2010).
Considerable laboratory evidence from chemical, cell culture, and animal studies
indicates that antioxidants may slow or possibly prevent the development of
cancer (http://www.cancer.gov/cancertopics/factsheet/prevention/antioxidants).
Antioxidants are grouped as endogenous or exogenous. The endogenous
group includes metallo enzymes superoxide dismutase (zinc, manganese, and
copper), glutathione peroxidase (selenium) and catalase, and proteins like
albumin, transferrin, ceruloplasmin, metallothionein and haptoglobin. The most
important exogenous antioxidants are dietary phytochemicals (such as
polyphenols, quinones, flavonoids, catechins, coumarins, terpenoids) and the
smaller molecules like ascorbic acid (Vitamin C), alpha-tocopherol, beta-carotene,
Vitamin-E and their supplements (Bizimenyera et al., 2007).
The extracts from number of medicinal plants which are known to have
some biologically active principles are used in ayurvedic preparations and these
extracts are prepared in bulk for commercial purpose (Sathisha et al., 2011).
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 GPx
and GST regulate inner mitochondrial membrane permeability by detoxifying ROS
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produced during electron transport and confer protection against lipid peroxidative
damage (Andreyev et al., 2005).
Antioxidants can be classified into three main types: first line defense
antioxidants, second line defense antioxidants and third line defense antioxidants.
Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT),
Glutathione reductase (GR) and some minerals like Se, Mn, Cu and Zn comes
under first line defense antioxidants (Figure 8). Reduced glutathione (GSH),
Vitamin C, Vitamin E, uric acid, carotenoids, albumin, bilurubin, Vitamin A and
flavonoids come under second line defense antioxidants (Gupta and Sharma,
2006). Lipase, proteases, DNA repair enzymes, transferases and methionine
sulphoxide reductase come under third line defense antioxidants (Irshad and
Chaudhuri, 2002).
2.2.1. First line defense antioxidants
Antioxidant enzymes are able to catalytically remove free radicals and other
reactive species (Isai et al., 2009). Free radical scavenging enzymes such as
catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD)
are the first line of cellular defense against oxidative injury (Burlakova et al., 2010).
Figure 8
Antioxidant enzyme system
(Pandey and Rizvi, 2010)
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2.2.1.1. Catalase (CAT)
Catalase is an antioxidant enzyme widely distributed in all animal tissues.
The enzyme is known to protect the system from highly reactive hydroxyl radicals
through hydrogen peroxide decomposition. Depletion of this enzyme may enhance
the cellular damage caused by assimilation of superoxide and hydrogen peroxide
(Oyedemi and Afolayan, 2011). Among the antioxidant enzymes, CATs are
ubiquitous heme enzymes that are found in aerobic organisms, ranging from
bacteria to higher plants and animals (Lee et al., 2003). CAT has one of the
highest turnover numbers of all enzymes; one molecule of CAT can convert
millions of molecules of hydrogen peroxide to water and oxygen per second
(David, 2004). Therefore reduction in the activity of CAT may result in a number of
deleterious effects due to the assimilation of superoxide radical and hydrogen
peroxide (Palanivel et al., 2008).
2H2O2 CAT O2 + 2H2O (Lenzen, 2008)
2.2.1.2. Glutathione peroxidise (GPx)
Glutathione peroxidases (GPxs) are members of the family of antioxidant
enzymes that scavenge hydrogen peroxide in the presence of reduced glutathione,
and seven isoforms having different substrate specificities and tissue distribution
have been identified (Takebe et al., 2002 and Drevet, 2006). GPx is a selenium-
dependent enzyme that contains a selenium atom incorporated within the
selenocysteine residue (Kryukov et al., 2003). GPx is one of the most important
enzymes in human. The enzyme pays an important role in peroxide detoxification.
GPx utilize the reducing equivalents of glutathione to reduce hydrogen peroxide
and it may be the main mechanism for protection against the deleterious effects of
hydroperoxides (Manjusha et al., 2011).
H2O2 + 2GSH GPx GSSG + 2H2O (Finaud et al., 2006)
2.2.1.3. Glutathione reductase (GR)
Glutathione reductase is a flavine nucleotide dependent enzyme and its
predominant subcellular distribution is in the cytosol and mitochondria (Gururaj
et al., 2004). GR is a key enzyme of the antioxidative system that protects cells
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against free radicals. GR inhibition disturbs cellular prooxidant antioxidant balance
and may contribute to the genesis of many diseases (Tandogan and Ulusu, 2006).
It maintains the cellular thiol redox state by catalyzing the reduction of glutathione
disulfide (GSSG) to glutathione (GSH) with NADPH as the reducing cofactor (Qiao
et al., 2007). All GR family members share a similar three dimensional structure in
their FAD binding domain as well as atleast one conserved sequence motif (Dym
and Eisenberg, 2001).
GSSG + NADPH + H+ GR 2GSH + NADP+ (Casao et al., 2010)
2.2.1.4. Superoxide dismutase (SOD)
Superoxide dismutase is a group of metallo-enzymes that play a critical
role in the first line of defense against oxidative stress caused by free radicals in
many organisms. SOD protects cells and cell components against reactive oxygen
species (ROS) by catalysing the conversion of oxygen radicals to hydrogen
peroxide and molecular oxygen, and thus provides a protective role against
oxidative stress (Lester et al., 2009). Three types of SODs have been
characterized according to their metal content; copper-zinc SOD is located in
the cytosol, the manganese SOD is primarily a mitochondrial enzyme, and
extracellular SOD is usually found on the outside of the plasma membrane (Suzy
and Serpil, 2002). Mutations in the cytoplasmic or mitochondrial form of SODs
result in ageing, neurodegenerative diseases and carcinogenesis (Bonatto, 2007).
O2•- +O2
•-+ 2H+ SOD H2O2 +O2 (Lenzen, 2008) 2.2.2. Second line defense antioxidants
Reduced glutathione (GSH), Vitamin C, Vitamin E, uric acid, carotenoids,
albumin, bilurubin, Vitamin A and flavonoids come under second line defense
antioxidants (Gupta and Sharma, 2006).
2.2.2.1. Vitamin A
Vitamin A is a generic term for a group of compounds with similar biologic
activity such as retinol, retinal, and retinoic acid. The term ‘retinoids,’ on its turn,
comprises both these natural forms of Vitamin A and many synthetic analogues to
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retinol. Retinol absorbed may be released directly in extra-hepatic tissues or
captured by the liver or returned to the blood flow to supply the organism’s needs
(Debier and Larondelle, 2005). Vitamin A is a fat-soluble vitamin present in many
lipid substances. β-carotene, present in cell membranes, is converted into vitamin
A when the body needs it (Finaud et al., 2006). Vitamin A also acts a neutralizing
agent for the free radicals (Selvi et al., 2007).It acts as a powerful, free radical
scavenger (Singlet oxygen) and chain breaking antioxidant (Raghavan and
Kumari, 2006).
2.2.2.2. Vitamin E Vitamin E, a component of the total peroxyl radical-trapping antioxidant
system reacts directly with peroxyl and superoxide radicals and singlet oxygen and
protects membranes from lipid peroxidation. The deficiency of Vitamin E is
concurrent with increased peroxides and aldehydes in many tissues (Maritim et al.,
2003). The most well-known function of Vitamin E is that of a chain-breaking
antioxidant that prevents the cyclic propagation of lipid peroxidation (Mustacich
et al., 2007).
Figure 9
Mechanism of action of Vitamin E
(http://www.cnsforum.com/imagebank/item/MOA_VITE/default.aspx)
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Vitamin E also plays a role in neurological functions (Muller, 2010) and
inhibition of platelet aggregation (Brigelius and Davies, 2007; Atkinson et al.,
2008). Antioxidants, such as Vitamin E, prevent cell damage by binding to the free
radical and neutralising its unpaired electron. For example, when Vitamin E
binds to OO· or O2· they form an intermediate structure that is converted to
alpha tocopherylquinone as seen in the Figure 9 (http://www.cnsforum.com/
imagebank /item/MOA_VITE/default.aspx).
2.2.2.3. Vitamin C
Vitamin C (ascorbic acid) is a six-carbon lactone that is synthesized from
glucose in the liver. Vitamin C is an electron donor and therefore it is the reducing
agent. All known physiological and biochemical actions of Vitamin C are due to its
action as an electron donor. Ascorbic acid donates two electrons from a double
bond between the second and third carbons of the 6-carbon molecule. Vitamin C is
called an antioxidant because, by donating its electrons, it prevents other
compounds from being oxidized (Padayatty et al., 2003).
Vitamin C has also been used as a dietary supplement intended to prevent
oxidative stress–mediated chronic diseases such as cancer and cardiovascular
diseases (Khaw et al., 2001) and neurodegenerative disorders (Engelhart et al.,
2002). Although it has generally been acknowledged that Vitamin C protects cells
from oxidative DNA damage, thereby block the initiation of carcinogenesis.
Moreover, the chemopreventive mechanism of Vitamin C may be linked to the
inhibition of other processes particularly tumour promotion rather than to that of
tumour initiation (Neeraj et al., 2010).
2.2.2.4. Reduced glutathione (GSH)
Reduced glutathione (GSH) a sulphydryl containing tripeptide (gamma
glutamyl cystenyl glycine) is an important endogenous antioxidant in human which
plays a central role in the defense against oxidative damage and toxins. It serves
as a co-factor for GPx and glutathione S- transferase and can react directly with
hydrogen peroxide, super oxide anion, hydroxyl and alkoxyl radicals by its free
sulphydryl groups (Chaudhari et al., 2008). When present extracellularly, GSH is
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able to react directly with cytotoxic aldehydes produced during lipid peroxidation
(Eskiocak et al., 2005).
Figure 10
Non enzymic antioxidant system
(http:// www.biochem.arizona.edu/classes/bioc460/summer/.../pentose.ppt)
2.3. Anticancer activity of medicinal plants
Cancer is the second leading cause of death in the Western world
(Madhusudan and Middleton, 2005). Cancer is one of the life threatening diseases
with more than 200 different types. A tumour, or mass of cells, formed the
abnormal cells may remain within the tissue in which it originated (a condition
called in situ cancer), or it may begin to invade nearby tissues (a condition called
invasive cancer). An invasive tumour is said to be malignant, and cells shed into
the blood or lymph from a malignant tumour are likely to establish new tumours
(metastases) throughout the body. Tumours threaten an individual's life when their
growth disrupts the tissues and organs needed for survival (Jena et al., 2012).
Due to lack of effective drugs, expensive cost of chemotherapeutic agents
and side effects of anticancer drugs, cancer can be a cause of death. Therefore
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efforts are still being made for the search of effective naturally occurring
anticarcinogen that would prevent, slow or reverse cancer development. Plants
have a special place in the treatment of cancer (Deepa et al., 2011). Plant
materials have been used for the treatment of malignant diseases for centuries.
Recent phytochemical examination of plants which have a suitable history of use
in folklore for the treatment of cancer had induced and often resulted in the
isolation of principles with antitumour activity (Dhanamani et al., 2011).
Ancient herbal medicines may have some advantages over single purified
chemicals (Vickers, 2002). Often the different components in a herb have
synergistic activities or buffer toxic effects. Mixtures of herbs are even more
complex and so might have more therapeutic or preventive activity than single
products alone. In fact, several studies have demonstrated that extracts from
several herbal medicines or mixtures had an anticancer potential in vitro or in vivo
(Bonham et al., 2002; Hu et al., 2002; Lee et al., 2002 and El-Shemy et al., 2007).
Higher plants have been one of the largest sources of new compounds with
pharmacological activity. For example, the species Catharanthus roseus (L.)
G. Don (Apocynaceae) produces several alkaloids, two of which, vincristine and
vinblastine have anticancer activity (Santos and Elisabetsky, 1999).
Antitumour activity of Mylabris cichorii extracts against murine Ascites
Dalton’s Lymphoma was studied by Prasad et al. (2010). Terminalia arjuna,
Dillenia indica and Oroxylum indicum were screened for anticancer efficacy
against Dalton’s lymphoma (Brahma et al., 2011). Antiproliferative and antioxidant
activity of Aegle marmelos (Linn.) leaves in Dalton's Lymphoma Ascites
transplanted mice was reported by Chockalingam et al. (2012). The methanol
extract of stem bark of Dillenia pentagons appears to be more active against
Dalton’s Lymphoma (Rosangkenia and Prasad, 2004). The Saponins from the
plant of china, Clematis manshrica has obvious antitumour effects against
various transplanted tumour on mice (Zhao et al., 2005). The antineoplastic activity
of methanolic extracts of five medicinal plants that are native to Iran including
Galium mite, Ferula Angulata, Stachys obtuscrena, Grsium bracteosum, and
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Echinophora Cinerea was investigated and proved to have antitumour activity
(Amirghofran et al., 2006). The anti-neoplastic activity of guduchi (Tinospora
cordifolia) on Ehrlich ascites carcinoma was proved by Jagetia and Rao (2006).
Methanol extract of Ledum groelandicum Retzius (Labrador tea) leaf twig extract
showed anticancer activity (Dufour et al., 2007).The methanolic extracts of
Dendrosicyos Socotrana, Withania aduensis, Withania riebeckii, Dracena
Cinnabari and Buxus hildebrandlii exhibited the highest toxicity on all tumour cell
lines (Mothana et al., 2007).
The methanol extract of Bauhinia racemosa stem bark exhibited antitumour
effect in EAC bearing mice (Gupta et al., 2004a). An alcoholic extract of
Biorhythms sensitivum showed antitumour activity by inhibiting the solid tumour
development in mice induced with Dalton’s lymphoma ascites (DLA) cells and
increase the life span of mice bearing Ehrlich ascites carcinoma (EAC) tumours
(Guruvayoorappan and Kuttan, 2007). The Careya arborea bark significantly
reduced the solid tumour volume induced by DLA cells (Natesan et al., 2007).
2.4. Antimicrobial activity of medicinal plants
In the worldwide as well as in the developing countries, the most human
died due to infectious bacterial diseases (Nathan, 2004). The bacterial organisms
including Gram positive and Gram negative like different species of Bacillus,
Staphylococcus, Salmonella and Pseudomonas are the main source to cause
severe infections in humans. Bacterial diseases include any type of illness caused
by bacteria. Bacteria are a type of microorganism, which are tiny forms of life that
can only be seen with a microscope. Millions of bacteria normally live on the skin,
in the intestines, and on the genitalia. The vast majority of bacteria do not cause
disease, and many bacteria are actually helpful and even necessary for good
health. These bacteria are sometimes referred to as “good bacteria” or “healthy
bacteria’’. Common pathogenic bacteria and the types of bacterial diseases they
cause include: Escherichia coli and Salmonella that cause food poisoning,
Staphylococcus aureus causes a variety of infections in the body, including boils,
cellulitis, abscesses, wound infections, toxic shock syndrome, pneumonia, and
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food poisoning (http://www.localhealth.com/article/bacterial-diseases) and Shigella
causes diarrhea and fever (Uyigue and Anukam, 2011).
Proteus vulgaris is a rod-shaped, Gram negative bacterium that inhabits the
intestinal tracts of humans and animals. It can be found in soil, water and fecal
matter. It is grouped with the enterobacteriaceae and is an opportunistic pathogen
of humans. It is known to cause urinary tract infections and wound infections
(http://en.wikipedia.org/wiki/Proteus_vulgaris). It may also cause respiratory
infections that persist even after antibiotic treatment (http://thejediknight4.
iwarp.com/ Proteus%20vulgaris.pdf). Pseudomonas aeruginosa is a Gram-
negative, rod-shaped, asporogenous, and monoflagellated bacterium that has an
incredible nutritional versatility. P. aeruginosa a very ubiquitous microorganism, for
it has been found in environments such as soil, water, humans, animals, plants,
sewage, and hospitals (http://microbewiki.kenyon.edu/index.php/Pseudomonas_
aeruginosa). It is a frequent cause of nosocomial infections such as pneumonia,
urinary tract infections, and bacteremia. Pseudomonal infections are complicated
and can be life threatening (http://emedicine.medscape.com/article/226748-
overview).
Shigella flexneri is a human intestinal pathogen, causing dysentery by
invading the epithelium of the colon and is responsible, worldwide, for an
estimated 165 million episodes of shigellosis and 1.5 million deaths per year. The
bacterium is commonly found in water polluted with human faeces. It is transmitted
in contaminated food or water and through contact between people. Upon
infection, humans develop severe abdominal cramps, fever, and frequent
passage of bloody stools. Shigellosis is not only a significant cause of infant
mortality in developing nations but maintains endemic levels of infection worldwide
(http://www.ebi.ac.uk/2can/genomes/bacteria/Shigella_flexneri.html). Klebsiella
pneumoniae is among the most common Gram-negative bacteria. It is a common
hospital-acquired pathogen, causing urinary tract infections, nosocomial
pneumonia, and intra abdominal infections. K. pneumoniae is also a potential
community-acquired pathogen (http://www.phagetherapycenter.com/pii/Patient
Servlet? command= static_klebsiella).
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These organisms have the ability to survive in harsh condition due to their
multiple environmental habitats (Ahameethunisa and Hoper, 2010). Herbal
treatment would promise a greater viable solution for effective treatment of
diseases caused by bacteria (Khan et al., 2007; Rahman and Hossain, 2010).
Mohamed et al. (2010) reported antimicrobial activity of methanol extracts of
Andrographis paniculata (leaves), Eugenia jambolana (kernel), Cassia auriculata
(flowers), Murraya koenigii (leaves), Salvadora persica (stem) and Ipomoea
batatas (leaves) against two Gram-positive bacteria (Staphylococcus aureus and
Staphylococcus epidermitis) and three Gram-negative bacteria (Escherichia coli,
Klebsiella pneumoniae and Pseudomonas aeruginosa). Antimicrobial activities
have been studied with the methanolic plant extracts of Abultilon indicum,
Adenocalymma alliaceum, Carica papaya, Crotolaria laburnifilia, Croton
bonplandianum, Derris scandens, Eichornia crassipes, Iopomea hispida, Moringa
heterohylla and Peltophorum pterocarpum (Vadlapudi, 2010).
2.5. Phytochemical constituents of medicinal plants
The medicinal value of the plants lies in some chemical substances that
produce a definite physiological action on the human body (Edeoga et al., 2005).
Phytochemicals, the natural bioactive compounds of plants are divided into two
groups, which are primary and secondary constituents according to their functions
in plant metabolism. Primary constituents comprise common sugars, amino acids,
proteins and chlorophyll while secondary constituents consist of alkaloids,
terpenoids, phenolic compounds, tannins and so on (Krishnaiah et al., 2007).
These phytoconstituents work with nutrients and fibres to form an integrated part
of defense system against various diseases and stress conditions (Koche et al.,
2010).
Phytochemicals have complementary and overlapping mechanisms of
action, including gene expression in cell proliferation, cell differentiation,
oncogenes and tumour suppressor genes, induction of cell-cycle arrest and
apoptosis, modulation of enzyme activities in detoxification, oxidation and
reduction, stimulation of the immune system and regulation of hormone
metabolism. They also have antimicrobial effects (Sun et al., 2002).
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All plants produce an amazing diversity of secondary metabolites. One of the
most important groups of these metabolites is phenolic compounds. Phenolics are
characterized by at least one aromatic ring (C6) bearing one or more hydroxyl
groups. They are mainly synthetized from cinnamic acid, which is formed from
phenylalanine by the action of L-phenylalanine ammonia-lyase (EC 4.3.1.5), the
branch point enzyme between primary (shikimate pathway) and secondary
(phenylpropanoid) metabolism (Dixon and Paiva, 1995). The conception of
antioxidant action of phenolic compounds is not novel (Bors et al., 1990).
Antioxidant action of phenolic compounds is due to their high tendency to chelate
metals. Phenolics possess hydroxyl and carboxyl groups, able to bind particularly
iron and copper (Jun et al., 2003).
Phenolics are also a kind of natural product and antioxidant substance
capable of scavenging free superoxide radicals, anti-aging and reducing the risk of
cancer (Ghasemzadeh and Ghasemzadeh, 2011). The antioxidant properties of
phenolic and flavonoid compounds are mediated by scavenging radical species
such as ROS/ RNS; suppressing ROS/RNS formation by inhibiting some enzymes
or chelating trace metals involved in free radical production and up regulating or
protecting antioxidant defense (Cotelle, 2001). The reduction activity of phenolic
and flavonoid compounds depends on the number of free hydroxyl groups in the
molecular structure (Rice-Evans et al., 1996). Isolated polyphenols from different
plants have been considered in a number of cancer cell lines at different stages of
cancer growth. For example, the isolated polyphenols from strawberry including
kaempferol, quercetin, anthocyanins, coumaric acid and ellagic acid, were shown
to inhibit the growth of human breast (MCF-7), oral (KB, CAL-27), colon (HT-29,
HCT-116), and prostate (LNCaP, DU-145) tumour cell lines (Damianaki et al.,
2000 and Zhang et al., 2008).
Silymarin is the bioactive extract from Silybum marianum L. seeds
(Asteraceae) and contains 65-85% flavonolignans like silychristin, isosilychristin,
silydianin, silybin A and B, isosilybin A and B, and also 20- 35% fatty acids,
flavonoids, and other polyphenolics. The major source of silymarin is fruits and
seeds from this plant, but traces of these compounds can occur in all plants
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(Ramasamy and Agarwal, 2008). Silymarin has been used medicinally to treat liver
disorders, including acute and chronic viral hepatitis, toxin/drug-induced hepatitis,
and cirrhosis and alcoholic liver diseases. It has been reported to be effective in
certain cancers. Its mechanism of action includes inhibition of hepatotoxin binding
to receptor sites on the hepatocyte membrane; reduction of glutathione oxidation
to enhance its level in the liver and intestine; antioxidant activity; and stimulation of
ribosomal RNA polymerase and subsequent protein synthesis, leading to
enhanced hepatocyte regeneration (Dixit et al., 2007).
Silymarin’s hepatoprotective effects are accomplished via several
mechanisms. These include: antioxidation (Mirguez et al., 1994), Inhibition of lipid
peroxidation (Bosisio et al., 1992), enhanced liver detoxification via inhibition of
phase detoxification (Halim et al., 1997 and Baer-Dubowska et al., 1998),
protection from glutathione depletion (Campos et al., 1989), anticarcinogenesis by
inhibition of cyclin dependent kinases and arrest of cancer cell growth. Silymarin
also found to have immunomodulatory effects on the diseased liver (Deak et al.,
1990 and Lang et al., 1990).
Figure 11
Anticancer mechanism of silymarin
(Ramasamy and Agarwal, 2008)
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Numbers of studies have established the cancer chemopreventive role of
silymarin in both in vivo and in vitro models. Silymarin modulates imbalance
between cell survival and apoptosis through interference with the expressions
of cell cycle regulators and proteins involved in apoptosis. In addition, silymarin
also showed anti-inflammatory as well as antimetastatic activity (Figure 11)
(Ramasamy and Agarwal, 2008).
Hiremath and Urmila (2012) explored that methanol and aqueous extract of
Amaranthus caudatus leaves contain carbohydrates, proteins, amino acids,
saponins, glycosides, phenoilcs and tannins; chloroform and acetone extracts
contains glycosides and saponins and petroleum ether extract of leaves contain
saponins. Kondongala et al. (2012) found that phytochemical investigation of
methanolic extract of stem and bark of Bauhinia purpurea showed the presence
of carbohydrates, glycosides, saponins, sterols and triterpinoids. Sheela et al.
(2012) reported the presence of alkaloids, flavonoids, saponins, tannins,
carotinoids and phytates in Sanseiveria roxburghiana leaf extract.
Venkateshwaralu et al. (2012) reported that leaves of methanolic extract of
Ximenia americana Linn. showed the presence of alkaloids, steroids, sugars,
saponins, tannins and terpenoids.
2.6. In silico drug design and docking for cancer In silico methods can help in identifying drug targets via bioinformatics
tools. They can also be used to analyze the target structures for possible binding/
active sites, generate candidate molecules, check for their drug likeness, dock
these molecules with the target, rank them according to their binding affinities,
further optimize the molecules to improve binding characteristics. The use of
computers and computational methods permeates all aspects of drug discovery
today and forms the core of structure-based drug design. The use of
complementary experimental and informatics techniques increases the chance of
success in many stages of the discovery process, from the identification of novel
targets and elucidation of their functions to the discovery and development of lead
compounds with desired properties. Computational tools offer the advantage of
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delivering new drug candidates more quickly at a lower cost. Major roles of
computation in drug discovery are; (1) Virtual screening and de novo design, (2) in
silico Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME/T)
prediction and (3) Advanced methods for determining protein-ligand binding
(http://www.scfbio-iitd.res.in/tutorial/drugdiscovery.htm).
Bioinformatics is seen as an emerging field with the potential to significantly
prove how drugs are found, brought to the clinical trials and eventually released to
the marketplace. Computer Aided Drug Design (CADD) is a specialized discipline
that uses computational methods to simulate drug – receptor interactions. One of
those methods is called docking. The site of drug action, which is ultimately
responsible for the pharmaceutical effect, is a receptor. Docking allows the
scientist to virtually screen a database of compounds and predict the strongest
binders based on various scoring functions (Virupakshaiah et al., 2007).
Docking explores the ways in which two molecules, such as drugs and an
enzyme receptor fit together and dock to each other well. The molecules binding to
a receptor, inhibit its function, and thus act as drug. Complexes were identified via
docking and their relative stabilities were evaluated using molecular dynamics and
their binding affinities, using free energy simulations (Babu et al., 2008).
2.6.1. Histone deacetylase (HDAC)
For the last four decades, a number of potential approaches have been
proposed for the treatment of cancer. One of the recent targets is histone
deacetylase (Saha et al., 2010). It has been widely recognized in recent years that
HDACs are promising targets for therapeutic interventions intended to reverse
aberrant epigenetic states associated with cancer (Pandolfi, 2001; Baylin and
Ohm, 2006). HDAC is an enzyme that removes an acetyl group from histones,
which allows them to bind DNA and inhibit gene transcription (Elaut et al., 2007
and Santini et al., 2007). Acetylation and deacetylation of chromatin histone
protein by HDAC alters chromatin structure and dynamically affects transcriptional
regulation (Liu et al., 2006a).
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Histone deacetylase inhibitors (HDACIs) are emerging as a new class of
anticancer agents. HDACIs have shown activity against diverse cancer types and
notable effects on tumour cell proliferation, programmed cell death, differentiation
and angiogenesis in vitro and in vivo. Currently, there are more than a dozen of
phase I and II clinical trials involving the use of HDACIs in patients with
haematological and solid malignancies (Marks et al., 2004).
Figure 12
Role of Histone deacetylase inhibitors
(http://englishclass.jp/reading/topic/Histone_deacetylase)
In preclinical studies several classes of HDACIs have been found to
have potent anticancer activities, with remarkable tumour specificity, and some
have demonstrated promising therapeutic potential in early-phase clinical trials
for haematological malignancies such as cutaneous T-cell lymphoma,
myelodysplastic syndromes and diffuse B-cell lymphoma (Lindemann et al., 2004;
Jabbour and Giles, 2005; Marks and Jiang, 2005).
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2.6.2. Tubulin
Microtubules the key components of the cytoskeleton are long, filamentous,
tube-shaped protein polymers that are essential in all eukaryotic cells. They are
crucial in the development and maintenance of cell shape, in the transport of
vesicles, mitochondria and other components throughout cells, in cell signalling,
and in cell division and mitosis. Their importance in mitosis and cell division makes
microtubules an important target for anticancer drugs (Jordan and Wilson, 2004).
Figure 13
Role of Tubulin in the inhibition of mitosis
(http://www.photobiology.info/Christensen.html).
Microtubule inhibitors disrupt microtubule dynamics of tubulin
polymerization and depolymerization, which results in the inhibition of
chromosome segregation in mitosis and consequently the inhibition of cell division
(Mulligan et al., 2006). In normal mitosis, the chromosomes are pulled by
microtubules, formed from tubulin, towards the two centrioles, marking the location
where the nuclei of the two daughter cells will be formed. After that, the cell
membrane is pinched off, and the chromosomes decondense in the newly formed
cells. If the chromosomes are not separated by the "ropes" formed from tubulin,
the normal process of mitosis will not be completed, and cell death may result. An
inhibition of the tubulin function leads to an arrest of the cells in mitosis, and no
further cell division as long as the chromosomes are not transported to the two
poles. A large number of cells have been shown to die while they are arrested in
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this phase of cell division. A mitotic arrest is the mechanism behind the
function of mitotic inhibitors commonly used in cancer therapy (Figure 13)
(http://www.photobiology. info/ Christensen.html).
2.6.3. Aurora kinase A
Aurora-A kinase (officially known as Serine/threonine-protein kinase 6) is an
important regulator of cell division and acts in several aspects of spindle formation
and function (Barr and Gergely, 2007). Aurora family kinases play roles in several
mitotic processes, including the G2/M transition, mitotic spindle organization,
chromosome segregation and cytokinesis (Andrews et al., 2003; Crane et al.,
2003; Katayama et al., 2003 and Meraldi et al., 2004). Aurora A is found in the
cytoplasm and at centrosomes during interphase; during mitosis, it also localizes
to microtubules near the spindle poles. Aurora A interacts with several different
proteins that are required for proper centrosome maturation and spindle function
(Gadea and Ruderman, 2005).
Figure 14
Mechanism of action of Aurora kinase
(http://cancergrace.org/cancer-treatments/tag/targeted-therapy/)
The levels of Aurora A are abnormally high in many tumour types, and
altered abundance is thought to be of relevance for oncogenic transformation
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(Gautschi et al., 2008; Vader and Lens, 2008). Its abundance in cell cycle
regulated, with a peak in G2 and M phases, followed by regulated proteolysis at
the end of mitosis (Giubettini et al., 2010). Indeed, Aurora-A has recently been
proposed as a potential target in anticancer therapy; inhibitors of its activity have
been synthesised, some of which are currently being tested in clinical trials
(Castro et al., 2008 and Karthigeyan et al., 2010).
2.6.4. Protein kinase C
Protein kinase C (PKC) was originally discovered by Yasutomi Nishizuka in
1977 as a histone protein kinase activated by calcium and diacylglycerol (DAG),
phospholipids and/or phorbol esters (Takai et al., 1977).It is known that the PKC
family consists of serine/threonine-specific protein kinases that differ in their
structure, cofactor requirement and substrate specificity (Takai et al., 1979).Due to
biochemical properties and sequence homologies, PKCs are divided into three
subfamilies: firstly, classical or conventional, secondly, novel and finally, atypical
(Nishizuka, 1992; Newton, 1995; Schenk and Snaar-Jagalska, 1999).
Figure 15
Mechanism of action of kinase inhibitors
(Kondapalli et al., 2005)
Pre-clinical and clinical data has suggested that protein kinase C (PKC)
may represent an attractive target for cancer therapy (Marengo et al., 2011). PKC
regulates tumour promotion and cell growth by inducing activation of
transcriptional factors, such as activator protein-1 (AP-1) and nuclear factor- kappa
B (NF- kappa B), and by increasing the expression of key enzymes, such as
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ornithine decarboxylase, inducible nitric oxide synthase, and cyclooxygenase-2
(Amstad et al., 1992; Fischer et al., 1993; Stauble et al., 1994 and Klein et al.,
2000). PKC has unique structural aspects that render it susceptible to activation by
oxidant tumour promoters, such as H2O2, periodate, and tobacco related tumour
promoters (Gopalakrishna and Anderson, 1989; Gopalakrishna and Anderson,
1991; Gopalakrishna et al., 1994).
2.7. In silico drug design and docking for microbial infections
2.7.1. Pantothenate Kinase
Coenzyme A (CoA) is a key component of cellular metabolism and is
essential for bacterial viability since disruption of the CoA biosynthetic pathway is
lethal. CoA synthesis begins with the phosphorylation of pantothenate (Vitamin B5)
by pantothenate kinase (Dunster et al., 2002). Pantothenate kinase, which triggers
the first step in the production of coenzyme A (CoA), a molecule that is
indispensable to all forms of life. CoA plays a pivotal role in the cells' ability to
extract energy from fatty acids and carbohydrates; bacteria need CoA to make
their cell walls. The job of pantothenate kinase is to grab a molecule of pantothenic
acid (Vitamin B5) and another molecule that contains a chemical group called
"phosphate." The enzyme then removes the phosphate group from that molecule
and sticks it onto pantothenic acid. In humans, certain mutations in this enzyme
block its ability to put the phosphate group onto pantothenic acid. That diminishes
the production of CoA and causes the pantothenate kinase associated
neurodegenerative disease (Hong et al., 2006).
The pantothenate kinase is a key rate-determining enzyme of this pathway
and become a prime target for its inhibition. By doing so, we are not only inhibiting
this CoA biosynthetic pathway, but also inhibiting the microbial growth. It should
therefore be possible to develop selective small molecule inhibitors for the
pantothenate kinases that are expressed by the pathogenic microorganisms of
interest (Leonardi et al., 2005a).
2.7.2. Deacetoxy C synthase
Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from
Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the
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ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation
of the latter to deacetylcephalosporin C (Wu et al., 2011). Deacetoxycephalosporin
C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring
expansion of the penicillin nucleus into the nucleus of cephalosporins. The
reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a
reactive iron-oxygen intermediate from a ferrous iron in the active site (Oster et al.,
2004).
2.8. Medicinal plants selected for the study
2.8.1. Morinda tinctoria
The genus Morinda, belonging to the family Rubiaceae, grows wild and is
distributed throughout Southeast Asia, commercially known as Nunaa, is
indigenous to tropical countries and is considered an important traditional folk
medicine (Sivaraman and Muralidharan, 2011). Morinda tinctoria also commonly
known as Aal or Indian Mulberry is a species of flowering plant. Its common name
also refers to Morinda citrifolia (http://en.wikipedia.org/wiki/Morinda_tinctoria). It is
an evergreen shrub or small tree growing to 5-10 m tall. The Leaves are glabrous
of slightly pubescent or only hairy in the axils underneath, from broad-ovate to
elliptic-lanceolate, acuminated at the apex, generally about 4-5 in. long by 1 1/4 - 2
1/4 broad, but sometimes larger; petioles 1/2-1 in. long; stipules membranous
broad, entire or bifid, variable in size. The fruit is a green of each flower-head
united in a compound succulent berry (syncarpium) forming a pulpy mass, about 1
in. diameter., including a number of hard, 1-seeded pyrenes, orbicular, flattened,
usually 2-4 from each flower (Nisha et al., 2011). All parts of Morinda tinctoria have
medicinal properties (Shanthi et al., 2012).
In India Morinda is widely grown under natural conditions in Andaman and
Nicobar Islands. It is seen throughout the coastal region along fences and road
sides due to its wider adaptability to hardy environment. In the main land of India it
is found along the coastal areas of Kerala, Karnataka, Tamil Nadu and many other
places. Survey of Morinda in south India indicated that 12 different species or
varieties of Morinda are distributed throughout Tamil Nadu and Kerala. However,
the species Morinda tinctoria is present abundantly in most parts of Tamil Nadu
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and in some parts of Kerala. An unidentified Morinda species with large and
leathery leaves was reported in the Dhandakaranya forest area of Malkanagiri
district in Orissa (Singh et al., 2007).
There is a greater demand for fruit extract of Morinda species in treatment
for different kinds of illness such as arthritis, cancer, gastric ulcer and other heart
diseases (Narayanasamy et al., 2006). Leaves are useful as tonic, febrifuge,
deostruent and emmenagogue. It is also used for curing dyspepsia, diarrhoea,
ulceration, stomatitis, digestion, wound and fever. The leaf juice is useful as a local
application. Root is used to cure inflammation and boils. Unripe fruit is used to
cure rheumatism. Ash of the fruit prevents dysentery, vomiting, diarrhoea and
cholera (Kanchanapoom, 2001).The major components have been identified in the
Nunaa plant which includes octoanic acid, potassium, Vitamin C, terpenoids,
scopoletin, flavonesglycosides, lineoleicacid, anthraquinones, morindone, rubiadin
and alizarin (Moorthy and Reddy, 1970; Singh and Tiwari, 1976; Duduku et al.,
2007).
Many species of Morinda are available in India, of which Morinda tinctoria
predominantly grows as a weed tree in vacant agricultural land and especially on
uncultivated lands and along the boundaries of the cultivated fields. Ancient
writings reveal that Morinda has long been cultivated in different parts of Tamil
Nadu state in India. Although the south Indian ancestors realized the therapeutic
value of M. tinctoria and used it in the traditional Indian medicinal systems like
siddha, lack of proper documentation resulted in loss of that knowledge (Jeyabalan
and Palayan, 2009).
Fruit extract of Morinda tinctoria was found to accelerate wound healing in
rats (Mathivanan et al., 2006). The fruit extracts of Morinda tinctoria showed
effective antidiabetic activity in alloxan induced experimental rats (Mathivanan and
Surendiran, 2006). Ethanol extract of Morinda tinctoria Roxb leaves found to
possess antiulcer activity in rats (Vadivu et al., 2008). Jeyabalan and Palayan
(2009) have reported the analgesic and anti-inflammatory activity of leaves of
Morinda tinctoria Roxb. Anticonvulsant activity of Morinda tinctoria has been
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reported by Kumaresan and Saravanan (2009). Fruit extract of Morinda tinctoria
showed antihyperglycemic and antidiabetic effects in streptozotocin (STZ)-induced
diabetic rats (Muralidharan and Sivaraman, 2009). Antidiabetic and antioxidant
activity of Morinda tinctoria roxb fruit extract in streptozotocin induced diabetic rats
were also reported (Pattabiraman and Muthukumaran, 2011). Cytoprotective effect
of Morinda tinctoria Roxb. against surgical and chemical factor induced gastric and
duodenal ulcers in rats were studied by Sivaraman and Muralidharan (2011).
Pharmacognostical studies on Morinda tinctoria (Roxb) was reported by Shanthi
et al. (2012).
2.8.2. Nerium indicum
Nerium indicum (family: Apocynaceae) has been traditionally attributed
with several medicinal properties (Banerjee et al., 2011). It is commonly known as
Indian oleander (http://www.flower-knowledge.com/2011/10/nerium-indicum-
flower.html), soland, lorier bol, rosebay, and rose laurel and kaner (Ansford and
Morris, 1981). Nerium indicum is an erect, smooth shrub, 1.5 to 3 meters height
with a cream colored sticky resinous juice. Leaves are in whorls of 3 or 4, linear-
lanceolate, 10-15cm long, with numerous horizontal nerves. Flowers are showy,
sweet-scented, single or double, 4-5 cm in diameter, white, pink or red, borne in
terminal inflorescence (cymes). Fruit is cylindrical, paired, with deep linear
striations, 1.5-2.0 cm long. Seeds are numerous and compressed, with a tuft of
fine, shining, white, silky hairs (Vijayvergia and Kumar, 2007).
The flowers of this plant are hermaphrodite (Pendse and Dutt, 1934). All
parts of the plant are reputed therapeutic agents and have been used in folklore in
a variety of disease (Simon, 1942). Decoction of leaves has been applied
externally in the treatment of scabies and to reduce swellings (Wasif et al., 2008).
The leaves and flowers are cardiotonic, diaphoretic, diuretic (promotes excretion),
emetic, expectorant, sternutatory as well as for treatment of malaria and
abortifacient (Govind, 2010a).
Antiinflammatory and antinociceptive activity was reported by Erdemoglu
et al. (2003). Ahmed et al. (2006) reported the analgesic activity of Nerium indicum
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Mill. Primary Metabolites of Nerium indicum Mill was quantified by Vijayvergia and
Kumar (2007). Protective potential of Nerium indicum extract on lipid profile, body
growth rate, and renal function in streptozotocin induced diabetic rats were
reported (Yassin and Mwafy, 2007). It also possess to have antiulcer (Govind and
Saurabh, 2010), neuroprotective (Man-shan et al., 2007), molluscicidal (Zhang et
al., 2009), piscicidal (Sudhanshu and Ajay, 2009) and antiviral (Rajbhandari et al.,
2001) activities. Methanolic flowers extract of Nerium indicum was evaluated for its
hepatoprotective effect in rats (Govind, 2010b). Antihyperlipidemic activity of
Nerium indicum leaves extracts in hyperlipidimic rats has been studied by Sikarwar
and Patil (2011).
The molluscicidal activity of Nerium indicum bark against Lymnaea
acuminata snails was studied (Singh and Singh, 1998). Polysaccharides from the
flowers of Nerium indicum showed neuroprotective effects (Yu et al., 2004).
Insecticidal potentialities of Nerium indicum leaves extracts against Epilachna
28-punctata (F.) was reported by Ranjana and Anil (2005). N-butanol extracts
and water extracts of Nerium indicum also shown to possess molluscicidal
activity (Wang et al., 2006). New polysaccharide from Nerium indicum
protected neurons via stress kinase signaling pathway (Yu et al., 2007). Nerium
indicum leaf extract showed antidiabetic activity in alloxan induced diabetic rats
(Sikarwar et al., 2009).Cardiac glycosides from fresh leaves of Nerium indicum
were also evaluated for its molluscicidal activity against Pomacea canaliculata
(Dai et al., 2011).