CHAPTER 1
INTRODUCTION AND
REVIEW OF LITERATURE
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1.1. INTRODUCTION
India has a unique wealth of biota which includes a large number of
medicinal and aromatic plants. A medicinal plant is a plant which contain substance
that can be used for therapeutic purposes or which are precursors for chemico-
pharmaceutical semi-synthesis. Medicinal plants may therefore be defined as a
group of plants that possess some special properties or virtues that qualify them as
articles of drugs and therapeutic agents, and are used for medicinal purposes. Thus it
is not unreasonable to believe that plant kingdom should yield safe and effective
drugs for most of the human ailments.
Nowadays plants are still important sources of medicines, especially in
developing countries that still use plant-based traditional medicine for their
healthcare. It was estimated in the Bulletin of the World Health Organization
(WHO) that around 80% of the world’s population relied on medicinal plants as
their primary healthcare source.
Plants have formed the basis of sophisticated traditional medicine (TM)
practices that have been used for thousands of years by people in China, India, and
many other countries. Some of the earliest records of the usage of plants as drugs are
found in the Artharvaveda, which is the basis for ayurvedic medicine in India.
Ayurveda has a long tradition in treating various diseases including liver diseases
using herbal medicines. Apart from timely cure the ayurvedic herbs give a
permanent relief from the diseases by removing the metabolic toxins from our body.
Herbal drugs play an important role in healthcare programmes worldwide,
mainly due to the general belief that they are without any side effects, besides being
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cheap and locally available. Ayurveda is a perfect science of life; it works
effectively fighting against various infections and diseases and thereby gaining
quick recovery. Lately there is a resurgence of interest in herbal medicines for
treatment of various ailments including liver disorders.
Liver, the largest organ in the vertebrate body - is the major site of intense
metabolic activities such as drug and xenobiotic metabolism. Liver injury caused by
toxic chemicals and certain drugs has been recognized as a crucial toxicological
problem. In the present world a large number of toxins are introduced daily. So it is
more important than ever to keep the liver healthy and potent. The most important
metabolic function of liver is the detoxification and excretion of toxic chemicals,
drugs and hormones. Liver tissue has the capacity to regenerate, so a moderate cell
injury is not reflected by measurable change in its metabolic function. Due to the
high tolerance of liver, liver disease is seldom detected at the early stage and once
detected treatment faces a poor prognosis in most cases.
Till date, there is no effective medicine for hepatic disorders, such as hepatic
fibrosis and hepatocellular carcinoma. Many plants have been reported for their
antioxidant and hepatoprotective activity and are used in ayurvedic system of
medicine for the treatment of liver disorders. Woodfordia fruticosa is a traditional
medicinal plant and its flowers are used for the preparation of fermented drugs and
for the treatment of various disorders such as dysentery, sprue, rheumatism,
hematuria, hemorrhoids, derangement of liver, disorders of mucous membrane etc.
All parts of the plant possess valuable medicinal properties viz anti inflammatory,
anti tumour, hepatoprotective and free radical scavenging activity but flowers are in
maximum demand. But still there is a paucity of information regarding the potential
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of Woodfordia fruticosa flowers in resisting oxidative stress, hepatic fibrosis and
hepatocellular carcinoma. Hence, this study was undertaken with the following
Objectives.
1. To explore the phytochemical constituents and in vitro antioxidant activity of
Woodfordia fruticosa flowers.
2. To prove the antioxidant effect of this medicinal plant against thioacetamide
induced oxidative stress.
3. To study the effect of W. fruticosa flowers against CCl4 induced hepatic
fibrosis.
4. To explore the anticancer properties of W. fruticosa in combating
hepatocellular carcinoma induced by N-nitrosodiethylamine.
5. To study the chemopreventive effect of the methanolic extract of
W. fruticosa and its sub-fractions on human hepatoma cell line.
1.2. REVIEW OF RELATED LITERATURE
1.2.1. The Liver – Structure and functions
The liver is the largest organ and is the central chemical laboratory in the
body. It is an organ of paramount importance and plays a pivotal role not only in the
metabolism and disposition of exogenous toxins and therapeutic agents responsible
for metabolic derangement, but also in the biochemical regulation of fats,
carbohydrates, amino acids, proteins, blood coagulation and immuno-modulation.
The liver is a major target organ for toxicity of xenobiotics and drugs, because most
of the orally ingested chemicals and drugs first go to liver where they are
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metabolized into toxic intermediates. A large number of xenobiotics are reported to
be potentially hepatotoxic (Ajith et al., 2007).
1.2.1.1. The structure of liver
The liver consists of four sections, or lobes. There are two main lobes - the
right lobe, which is by far the larger, and the left lobe. Two small lobes lie behind
the right lobe.
Each lobe is made up of multisided units called lobules. Most livers have
between 50,000 and 100,000 lobules. Each lobule consists of a central vein
surrounded by tiny liver cells grouped in sheets or bundles. These cells perform the
work of the liver. Cavities known as sinusoids separate the groups of cells within a
lobule. The sinusoids give the liver a spongy texture and enable it to hold large
amounts of blood.
The liver is composed of a number of cell types that function independently.
The most abundant cell is the hepatocyte, comprising approximately 70 percent of
the liver volume and performing the bulk of liver functions. Each hepatocyte is
supplied with nutrient rich portal blood and oxygen rich aortic blood, supporting its
high metabolic and secreatory activity. Another unique feature of the hepatocyte is
its unusually high capacity for proliferation when a portion of the liver is removed or
damaged, which underlies the liver’s regenerative properties.
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Fig. 1.1. Anatomy of liver
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1.2.1.2. Liver Functions
This complex organ performs multiple interrelated functions that are
essential for life, including: (1) uptake, storage, metabolism and release of nutrients
(e.g., amino acids, carbohydrates, lipids, vitamins and minerals); (2) synthesis of bile
salts from cholesterol and their secretion to assist with fat absorption and digestion;
(3) synthesis and secretion of plasma proteins necessary for blood clotting and
transport of molecules through the circulation; (4) detoxification of drugs, hormones
and the end products of metabolism and distribution to the bile or urine for
excretion; and (5) removal of bacteria and dying red blood cells from the circulation.
During the detoxification of xenobiotics, reactive oxygen species (ROS) are
generated which cause oxidative stress (Kohen and Nyska, 2002) and which leads to
the hepatic damage.
1.2.2. Reactive oxygen species and oxidative stress
Oxygen is thought to have been responsible for the expansion of life on
Earth, there are two sides to this molecule: life giving and life taking. Oxygen in the
air we breathe is a relatively nonreactive chemical. However, when oxygen is
exposed to high-energy or electron-transferring chemical reactions, it can be
converted to various highly reactive chemical forms (Fig. 1.2) collectively
designated “reactive oxygen species” (ROS) (Rodriguez and Redman, 2005)
Reactive oxygen species (ROS) known as free radicals, are oxidizing agents
generated as a result of metabolism of oxygen and have at least one unpaired
electron that make them very reactive species. Normally, free radicals attack the
nearest stable molecule, which becomes a free radical itself, beginning a cascade of
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chain reaction. These can very rapidly oxidize biomolecules that they encounter in
their vicinity thus exerting either a positive or a negative influence on normal cell
function (Warren et al., 1987)
Fig. 1.2. Generation of different ROS by energy transfer or sequential univalent reduction of ground-state triplet oxygen
Normal aerobic metabolism is related to optimal levels of ROS because a
balance exists between ROS production and antioxidant activity. Oxidative stress
(OS) is the term applied when oxidants outnumber the antioxidants due to excessive
generation of reactive oxygen species and when antioxidants cannot scavenge these
free radicals (Sharma et al., 1999). Such phenomena cause pathological effects,
damaging cells, tissues and organs (Aitken and Baker 1995).
Reactive oxygen and nitrogen species are physiologically produced during
metabolic processes and especially during electron transport chain reactions (Di
Meo and Venditti, 2001). Another internal source of reactive species is peroxisomes,
small membrane-enclosed organelles containing enzymes important for oxidation
reactions. Furthermore, the enzymes of the Ρ-450 complex generate reactive species
during the detoxification of xenobiotics, such as drugs. There are also external
sources of reactive species related to UV radiation, air pollution, smoking, alcohol
consumption, and exercise (Halliwell and Gutteridge, 2007).
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ROS are potential carcinogens because of their roles in mutagenesis, tumour
promotion, and progression (Dröge, 2003). If not regulated properly, the excess ROS
can damage lipids, protein or DNA, inhibiting normal function (Perry et al., 2000).
Proteins are one of the major targets of reactive species which induce the formation
of carbonyl groups (aldehydes and ketones) in those amino acids that are susceptible
to oxidation, such as histidine, arginine, lysine, and proline. The carbonyl groups are
not metabolized in proteasomes and lysosomes, but are accumulated (Levine, 2002).
Furthermore, the thiol groups (−SH) present in protein molecules are oxidized in
thiol radicals (RS.). Protein oxidation leads to conformational changes which result
in the modification or loss of protein function (Halliwell and Gutteridge, 2007).
Apart from proteins, lipids are vulnerable in reactive species-induced oxidative
damage (Halliwell and Chirico, 1993). Lipid peroxidation increases the permeability
of cellular membranes, resulting in cell death. Reactive species also affect DNA by
causing chain breaks and damaging its repair mechanism (Jenkins, 1988). DNA, and
especially guanine, oxidation results in the production of 8-hydroxy-2-
deoxyguanosine. This by-product, if not repaired, induces DNA mutations that may
cause aging and carcinogenesis (Radak et al., 1999). In addition, excessive
production of reactive species has been implicated in immune system dysfunction
(Schneider and Tiidus, 2007), muscle damage (Nikolaidis et al., 2007a, 2007b), and
fatigue (Betters et al., 2004).
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Fig. 1.3 Organs affected by oxidative stress
1.2.3. Antioxidant defense mechanism
Antioxidants are substances that delay or prevent the oxidation of cellular
oxidizable substrates (Halliwell and Gutteridge, 2007). When the generation of the
active oxygen-free radical exceeds the scavenging ability many degenerative
diseases such as brain dysfunction, cancer, heart diseases, age-related degenerative
conditions, declination of the immune system, gastric ulcer and DNA damage will
arise. Antioxidants can be divided into two categories according to specific
characteristics: - endogenous antioxidants and exogenous antioxidants.
1.2.3.1. Endogenous antioxidants
Endogenous antioxidant systems possess enzymatic and non-enzymatic
antioxidative mechanisms which minimizes the generation of reactive oxygen species.
They include enzymes such as superoxide dismutase (SOD), catalase, glutathione
reductase (GR), and glutathione peroxidase (GPx) and non-enzymatic metabolites
such as glutathione, uric acid, vitamins and polyphenols. Regarding their origin,
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various antioxidants such as glutathione, uric acid, catalase and SOD can be
synthesized in vivo, whereas others namely, polyphenols and β-carotene, are obtained
from food. Based on their physical properties, antioxidants can be divided into water
soluble antioxidants such as uric acid, glutathione and polyphenols or lipid-soluble
antioxidants such as vitamins A, vitamin E and lipoic acid (Veskoukis et al., 2012).
The SOD enzyme catalyzes the dismutation of Ο2. to Η2Ο2. It exists in
mitochondrial form (MnSOD) and in cytoplasmic form (Cu/ZnSOD) that is
primarily found in muscle cells (Das et al., 1997). Catalase is present in almost every
kind of cell, but its concentration is higher in the erythrocytes and liver (Masters et
al., 1986). Its subcellular localization is in peroxisomes, mitochondria and in the
nucleus. It catalyzes the conversion of Η2Ο2, which is produced by SOD to Η2Ο and
Ο2. The antioxidant activity of catalase is of great significance as it prevents the
conversion of Η2Ο2 to the very harmfulOH.. Furthermore, it has been demonstrated
that catalase and SOD activities exhibit a linear correlation with life span in
mammals (Cutler, 1984). GPx, which requires selenium as a cofactor is present in
the cytoplasm and mitochondria and is an alternative route of Η2Ο2 degradation.
Specifically, Η2Ο2 is converted to Η2Ο and Ο2 and oxidizes GSH (reduced form of
glutathione) to GSSG (oxidized form of glutathione).
Glutathione is considered one of the most important antioxidant metabolites
and is the first line of defense against reactive species. At rest, glutathione is usually
present in the reduced state. GSH is a tripeptide consisting of glutamic acid, cysteine
and glycine. It is the most abundant low-molecular-weight thiol-containing
compound in biological fluids and tissues of mammals. In eukaryotic cells, 90% of
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the intracellular GSH pool resides in the cytoplasm, and the remaining 10% is found
in the mitochondria, endoplasmic reticulum and the nucleus. However, the
biosynthesis of GSH appears to occur exclusively in the cytoplasm (Barycki, 2007).
GSH possess potent antioxidant properties, maintaining the intracellular redox
homeostasis due to the thiol group of cysteine which serves as a substrate of GPX
and contributes to xenobiotic detoxification (Halliwell and Gutteridge, 2007). In
physiological conditions, GSH is in a dynamic equilibrium with GSSG. However, in
the context of oxidative stress GSH works with GPX to efficiently remove
intracellular H2O2. This process protects biomolecules from oxidative modifications,
and GSH is converted to GSSG. Glutathione reductase reduces GSSG to GSH using
NADPH as an electron donor, thus replenishing the GSH pool (Barycki, 2007).
Fig. 1.4. Generation of free radicals exceed the cellular antioxidants leads to cancer
1.2.3.2. Exogenous antioxidants
The cellular antioxidants may not be capable of neutralizing all the free
radicals produced in the body as well as those derived from the environment, so
therefore a need for an external source of antioxidants to neutralize the free radical
load in the body. A large number of antioxidants, both nutritive and nonnutritive,
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occur in foods. Besides β-carotene, vitamin C and vitamin E (which are nutrients),
a number of carotenoids, phenols and flavonoids also occur naturally in foods and
can act as antioxidants. β-carotene is an excellent scavenger of singlet oxygen.
Vitamin C interacts directly with radicals like 2O . and OH. (hydroxyl). Vitamin C
and vitamin E prevent formation of nitrosamine, which is carcinogenic. Vitamin E
also protects selenium against reduction and protects polyunsaturated fatty acids
(PUFA) in the membrane against oxidative damage (Rao, 2003).
1.2.3.3. Natural antioxidants
Natural antioxidants present in fruits, vegetables, cereals and medicinal
plants act as effective free radical scavengers, by donating hydrogen to highly
reactive radicals. They are converted into relatively harmless free radicals, which
may react with other free radicals and inactivate them. Studies reveal that increased
consumption of fruits rich in antioxidant polyphenols lower the risk of degenerative
diseases such as cancer (Patel et al., 2011) and increase the concentration of β-
carotene in the blood. Polyphenolic compounds are plant secondary metabolites
which have at least one aromatic ring in their molecule and usually exist in the form
of glycosides. More than 8,000 different polyphenolic compounds have been
described. They are subdivided into nonflavonoids (e.g., hydrobenzoic acids,
hydroxycinnamic acids, and stilbenes) and flavonoids (e.g., flavonols, flavanals,
isoflavones, and anthocyanins). Flavonoids are composed of more than 4,000
different species that have two aromatic benzene rings linked through three carbons
forming an oxygenated heterocycle.
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Synthetic antioxidants such as propyl gallate, butylated hydroxyanisol
(BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) are
commonly used to control lipid oxidation in foods but are suspected to be
responsible for liver damage and carcinogenesis (Ito et al, 1986; Safer and al-
Nughamish 1999). Recently, interest in finding naturally occurring antioxidants has
increased considerably to replace synthetic antioxidants. All these concerns
regarding the synthetic antioxidants, together with consumers’ preference for natural
food ingredients, have reinforced the current attention toward the development of
alternative natural antioxidants. The use of traditional medicine is widespread, and
plants still present a large source of natural antioxidants. Several medicinal plants
have been screened based on the integrative approaches on drug development from
Ayurveda (Mukherjee and Wahile, 2006). The ability of certain secondary
metabolites present in plants act as scavengers of free radicals, besides their
antioxidant and antimicrobial properties, is raising the possibility of their food and
pharmaceutical applications.
Table 1.1. Selected list of plants with antioxidant activity
SI. No.
Plant Part used Major active compounds
Reference
1. Allium sativum (garlic)
Underground stem
allicin, ajoene, selenium, quercetin
Meriga et al., 2012
2. Avena sativa (oat) endosperm phenolic acids Emmons et al., 1999
3. Cassia sieberiana root polyphenolic compounds
Nartey et al., 2012
4. Cornus capitat adventitious root
ellagic acid derivatives Tanaka et al., 2003
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Table 1.1. (Cont.) Selected list of plants with antioxidant activity
SI. No. Plant Part used Major active compounds Reference
5. Daucus carota (carrot)
root tuber carotenoids Mech-Nowak et al., 2012
6. Fagopyrum esculentum (buckwheat)
endosperm flavonoids Oomah and Mazza, 1996
7. Glycyrrhiza uralensis
licorice root glycyrrhizin Tanaka et al., 2008
8. Malus domestica (apple)
fruit quercetin, catechin, phloridzin, chlorogenic acid
Boyer and Liu, 2004
9. Momordica charantia (bitter melon)
fruit gallic acid, gentisic acid, catechin
Santos et al., 2010; Horaz et al., 2005
10. Moringa oleifera leaf polyphenols, anthocyanin, thiocarbamates
Luqman et al., 2012
11. Oryza sativa (rice)
endosperm quinolone alkaloid Chung and Woo, 2001
12. Piper nigrum (black pepper)
fruit piperine, arbutin, magnoflorine
Singh et al., 2008
13. Psidium guajava (common guava)
fruit carotenoids, polyphenols
Mai et al., 2007
14. Punica granatum (pomegranate)
fruit ellagic acid, ellagitannins, anthocyanins, punicic acid, flavonoids, anthocyanidins, flavones
Karasu et al., 2012
15. Rosmarinus officinalis (rosemary)
- carnosoic acid Kim et al., 2011
16. Rubus idaeus (raspberry)
fruit ellagic acid, phenolic compounds, vitamin C
Liu et al., 2002
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Table 1.1. (Cont.) Selected list of plants with antioxidant activity
SI. No.
Plant Part used Major active compounds
Reference
17. Salvia officinalis (sage)
- carnosol, carnosic acid, rosmanol, apigenin
Walch et al., 2011
18. Solanum lycopersicum (tomato)
fruit lycopene Palozza et al., 2012
19. Solanum tuberosum (potato)
potato peel phenolic compounds
Kanatt et al., 2005
20. Syzygium aromaticum (clove )
Flower bud eugenol, eugenyl acetate
Lee and Shibamoto, 2001
21. Thymus zygis (thyme)
- thymol, carvacrol, terpinene
Youdim et al., 2002
22. Vitis vinifera (grapes)
fruit resveratrol, anthocyanins, catechins
Bunea et al., 2012
23. Zingiber officinale (ginger)
Underground stem (rhizome)
quercetin, catechin, kaempferol
Rahman et al., 2011
1.2.4. Oxidative stress and liver damage
Liver cells possess endogenous antioxidant defense system consisting of
antioxidants such as GSH, GST, ascorbic acid, vitamin E and antioxidant enzymes
such as SOD, Catalase and GPx to protect own cells against oxidative stress, which
causes destruction of cell components and cell death.
The liver is a major target organ for toxicity of xenobiotics and drugs,
because most of the orally ingested chemicals and drugs first go to liver where they
are metabolized into toxic intermediates. A large number of xenobiotics are reported
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to be potentially hepatotoxic (Ajith et al., 2007). It also handles the excretion of
drugs and other xenobiotics from the body thereby providing protection against
foreign substances by detoxifying and eliminating them (Saleem et al., 2010).
Hepatocytes, which make up the majority of the liver structure, are very active in the
metabolism of exogenous chemicals, and this is one of the major reasons why the
liver is a target for toxic substances (Timbrell, 2001). During the detoxification of
xenobiotics, reactive oxygen species (ROS) are generated which cause oxidative
stress (Kohen and Nyska, 2002) which leads to the hepatic damage.
1.2.5. Liver disorders caused by drugs and toxins
Liver disease is one of the major causes of morbidity and mortality in public,
affecting humans of all ages. About 20,000 deaths occur every year due to liver
disorders. Some of the commonly known disorders are viral hepatitis, alcohol liver
disease, non-alcoholic fatty liver disease, autoimmune liver disease, metabolic liver
disease, drug induced liver injury, liver fibrosis, cirrhosis, hepatocellular carcinoma
etc. According to WHO estimates, globally 170 million people are chronically
infected with hepatitis C alone and every year 3–4 millions are newly added into the
list.
Depending on the duration of the disease the liver diseases are classified as
acute or chronic. If the disease does not exceed to months it is considered as acute
liver disorder while diseases of longer duration are classified as chronic. Acute viral
hepatitis and drug reactions account for the majority of cases of acute liver disease.
Hepatitis A, B and E are the commonest causes of viral hepatitis. Hepatitis C is not
usually recognized as an acute infection because it rarely causes jaundice at this
stage.
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Drug/chemical-mediated hepatic injury is the common sign of drug toxicity
(Lee, 2003) and accounts for greater than 50% of acute liver failure cases. Hepatic
damage is the largest obstacle to the development of drugs and is the major reason
for withdrawal of drugs from the market (Cullen and Miller, 2006). Chronic liver
damage is a worldwide common pathology characterized by inflammation and
fibrosis that can lead to chronic hepatitis, cirrhosis and cancer (Tessitore and Bollito,
2006; Kohle et al., 2008). Chronic hepatitis or long term intoxification can severely
injure hepatic cells. Initially, the damaged cells are denatured, but subsequently
transformed to hypertrophic fibrosis and necrosis, and eventually may progress to
hepatoma. Hepatic fibrosis is usually initiated by hepatocyte damage. Biologic
factors such as hepatitis virus, bile duct obstruction, cholesterol overload,
schistosomiasis, etc; or chemical factors such as CCl4 administration, alcohol intake,
etc. were known to contribute to liver fibrosis. In many patients liver damage
become chronic and eventually progresses to more serious liver pathologies such as
fibrosis, cirrhosis or even carciongenesis, causing devastating economic losses and
mortality.
Liver toxicity mainly occurs due to drugs, alcohol, viruses and by chemicals.
The use of drugs like paracetamol and antibiotics cause acute liver damage. Some of
the drugs causing liver damage are listed below:
1.2.5.1. Non-steroidal anti-inflammatory drugs (NSAIDs)
Non-steroidal anti-inflammatory drugs (NSAIDs) are the centre piece of
pharmacotherapy for most rheumatologic disorders, and are used in large numbers
as analgesics and antipyretics, both as prescription drugs and over the counter
purchases. They are the most frequently used medications for the treatment of
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a variety of common chronic and acute inflammatory conditions (Manoukian and
Carson, 1996). Nearly all of the NSAIDs have been implicated in causing liver
injury (Rabinovitz and Van Thiel, 1992). Diclofenac and particularly sulindac, are
reported to be more commonly associated with hepatotoxicity (Bjorkman, 1998).
Several NSAIDs have been withdrawn from clinical use because of associated
hepatotoxicity (Rabkin et al., 1999). The new more selective COX-2 inhibitors (e.g.
celecoxib, rofecoxib, nimesulide) are also associated with hepatotoxicity (Merlani
et al., 2001). Hepatotoxicity from NSAIDs can occur at any time after drug
administration, but like most adverse drug reactions, most commonly occurs within
6–12 weeks of initiation of therapy (Aithal and Day, 1999).
There are two main clinical patterns of hepatotoxicity due to NSAIDs
(Rabinovitz and Van Thiel, 1992; Aithal and Day, 1999). The first is an acute
hepatitis with jaundice, fever, nausea, greatly elevated transaminases and sometimes
eosinophilia. The alternative pattern is with serological and histological (periportal
inflammation with plasma and lymphocyte infiltration and fibrosis extending into
the lobule) features of chronic active hepatitis. Some of the NSAIDs which cause
liver damage are listed below.
a. Diclofenac
Diclofenac sodium has antipyretic, analgesic and anti-inflammatory effects
but significant incidence of hepatotoxicity. In many cases clinical and biochemical
features of diclofenac hepatotoxicity suggest the involvement of reactive or toxic
metabolites. These products presumably were formed via the hepatic cytochrome
P450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that
are trapped by GSH (glutathione) conjugation. It is therefore possible that reactive
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benzoquinone imines may be formed and contribute to diclofenac mediated hepatic
injury (Tang et al., 1999).
b. Sulindac
Liver injury from sulindac appears within a few days to six weeks after
therapy is initiated. Fever, rash, eosinophilia, and edema are frequently found in
association with evidence of liver injury.
c. Nimesulide
It is an antiinflamatory drug and is almost exclusively metabolized and
cleared by the liver (Chatterjee and Sil, 2007). The drug can cause several types of
liver damage, ranging from mild abnormal function such as increase in serum amino
transferase activity to severe organ injuries such as hepatocellular necrosis or
intrahepatic cholestasis (Lucena et al., 2001).
d. Bromfenac
This acetic acid derivative was introduced in 1997 as non-narcotic for short
term pain relief, but was removed from the market in 1998 owing to several
instances of fulminant hepatic failure (FHF) leading to death or transplant that
occurred after prolonged administration (Goldkind and Laine, 2006).
e. Indomethacin
Indomethacin has produced hepatocellular necrosis, sometimes accompanied
by microvesicular steatosis and striking cholestasis (Fenech et al., 1967)
f. Ibuprofen
Ibuprofen was withdrawn from use in the 1960s because of fatal
hepatocellular injury.
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1.2.5.2. Acetaminophen
Acetaminophen, a widely used analgesic and antipyretic drug commonly
used for pain and fever relief (Whitcomb, 1994). It is commonly considered as a
“safe drug” when take within the suggested therapeutic dose. But in higher doses it
cause hepatotoxiciy in humans and experimental models. As the ingested dose of
acetaminophen is increased, hepatic glutathione stores become progressively depleted.
Consequently glutathione available for scavenging oxygen radicals are brought down,
resulting in an increase in reactive oxygen. This will be thus associated with
concurrent increase in lipid peroxidation and other hydroperoxides. When the
formation of N-acetyl-p-benzoquinoneimine (NAPQI) is of sufficient magnitude,
glutathione stores will fall below a critical level that is no longer adequate to sustain
detoxification of NAPQI. At this point, the disruption of cellular structure and
function occurs due to the covalent binding of NAPQI to cellular macromolecules
such as proteins and lipids thereby leading to hepatic necrosis (Dahlin et al., 1984).
1.2.5.3. Alcohol
Alcohol consumption causes accumulation of reactive oxygen species, which
in turn causes lipid peroxidation of cellular membranes, proteins and DNA oxidation
resulting in hepatocyte injury (Zhou et al., 2002). Alcohol treatment of rats is known
to cause the translocation of fat from the peripheral adipose tissue to liver, kidney
and brain for accumulation (Nadro et al., 2006).
1.2.5.4. Antibiotics
Tetracycline, Erythromycin, Nitrofurantoin, Ampicillin, Sulphonamides and
Lincomycin
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1.2.5.5. Anti-tubercular drugs
Para amino salicylate, Isoniazid, Rifampicin, Pyrazinamide, Ethionamide and
Ethanobutol
1.2.5.6. Anaesthetics
Halothane and chloroform
1.2.5.7. Anti-convulsants and anti-depressants
Phenobarbitone, Trimethadione, Tricyclic anti-depressants (eg. Amitrypyline),
Chloridiazepoxide, Monamine oxide inhibitors (eg. Iproniazid) and Phenothiazine.
Poisons affecting the liver are of three main classes, namely physical, biological
and chemical.
(i) Physical toxins
Hyperthermia, Burns and Irradiation
(ii)Biological toxins
Aflatoxin, Senecio alkaloid, and Amanita mushrooms.
(iii) Chemical toxins
Carbon tetrachloride, Tetrachloroethane, Chlorophenithone (DDT), Benzene
derivatives, Trinitrotoluene, Tannic acid, Phosphorus, Iron, Beryllium and Arsenic
1.2.6. Hepatic xenobiotic metabolism
On exposure to xenobiotics, the liver of vertebrates manages to eliminate
such foreign compounds as early as possible. This is accomplished by making use of
the normally existing biochemical mechanisms in the tissue. Certain enzymes and
other endogenous biomolecules which are actually meant for the metabolism of
endogenous substrates may be utilized for this purpose. Biotransformation of a
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xenobiotic compound following its exposure can alter its distribution and action
leading to its detoxification and excretion or enhance its toxicity due to the
activation of the compound (Athar et al., 1997).
Biotransformation of xenobiotics usually occurs in two phases.
Phase I: The main drug metabolizing system reside in the microsomal fraction of
the liver cell (smooth endoplasmic reticulum). The enzymes concerned are mixed
function monooxygenase, cytochrome c-reductase, and cytochrome P-450 (Mitchell
et al., 1973). NADPH in the cytosol is a cofactor. The drug is rendered more polar
by hydroxylation or oxidation. Alternative phase I drug metabolizing reactions
include the conversion of alcohol to aldehyde by alcohol dehydrogenase found
mainly in the cytosolic fraction.
Phase II: These biotransformations involve conjugation of the drug or drug
metabolite with a small endogenous molecule. The enzymes concerned are usually
not confined to the liver, but are present there in high concentration. An active
transport system is located at the biliary pole of the hepatocytes and this system
regulates the transport of drug molecules in and out of the hepatocytes.
1.2.7. Hepatotoxins and their effect
A number of pharmacological and chemical agents act as hepatotoxins and
produce a variety of liver ailments. They include industrial toxins, the heat-stable
toxic bicyclic octapeptides of certain species (Amanita and Galerina), chemicals and
various pharmacological agents. In general there are two major types of chemical
hepatotoxins, namely direct and indirect hepatotoxins. The most common direct
hepatotoxins are carbon tetrachloride, thioacetamide, paracetamol, galactosamine,
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
25
fulvine, phalloidin, ethyl alcohol, aflatoxins etc. Some examples of indirect
hepatotoxins are methyl testosterone, chlorpropamide, tetracycline, halothane,
phenytoin, methyldopa, amanita phalloides, acetaminophen, sulphonamides,
allopurinol, rifampicin etc. Thioacetamide, carbon tetrachloride and
N-nitrosodiethylamine were the hepatotoxins used in this study.
1.2.7.1. Thioacetamide
Thioacetamide is a compound endowed with liver damaging and
carcinogenic activity. Shortly after its administration thioacetamide is metabolized
to acetamide and thioacetamide-5-oxide. The latter binds to tissue macromolecules
responsible for the change in cell permeability, increased intracellular concentration
of Ca++, increase in nuclear volume and enlargement of nucleoli and inhibits
mitochondrial activity eventually leading to hepatic necrosis (Bautista et al 2010).
Thioacetamide on prolonged exposures causes cirrhosis (Bruck et al., 2001) by
inhibiting the respiratory metabolism of the liver through uncontrolled entry of Ca++
into hepatocytes. The final result is the inhibition of oxidative phosphorylation.
1.2.7.2. Carbon tetrachloride (CCl4)
Carbon tetrachloride (CCl4) has been one of the most intensively studied
hepatotoxicants to date and provides a relevant model for other halogenated
hydrocarbons that are used widely (Dahm et al., 1996; Weber et al., 2003). It
consistently produces liver injury in many species, including non-human primates
and man (Kumar et al., 1972). Acute poisoning with CCl4 becomes manifested as a
multisystem disorder, involving the liver, kidneys, brain, lungs, adrenal glands, and
the myocardium (Gitlin, 1996). CCl4 is a potent hepatotoxin and a single exposure
can rapidly lead to severe centrizonal necrosis and steatosis (Recknagel et al., 1973).
Chapter 1Chapter 1Chapter 1Chapter 1
26
Mechanism of CCl4 toxicity
Carbon tetracholoride is lipophilic and because of this property CC14 is
absorbed from the skin and gastrointestinal tract as well as the lungs, although the
rate of absorption by the separate routes is different. Nowadays, CCl4 is used as a
classical hepatotoxicant for experimental liver functions.
CC14 is first metabolised by cytochrome P-450 in the liver endoplasmic
reticulum to the highly reactive 3CCl . radical (Recknagal, 1967). This free radical
may react again with oxygen to form trichloromethylperoxy radical 3(CCl OO). . The
free radicals thus formed can attack lipids on the membrane of endoplasmic
reticulum more readily than the 3CCl . free radical. The trichloromethylperoxy free
radical leads to elicit lipid peroxidation, the distruption of Ca2+ homeostasis and
finally results in cell death (Recknagal, 1983). As a consequence of this necrotic
behavior, leakage of large quantities of enzymes into the blood stream is often
associated with CCl4 toxicity.
1.2.7.3. N-nitrosodiethylamine (NDEA)
N-nitrosodiethylamine (NDEA) is a potent carcinogenic dialkylnitrosoamine
present in tobacco smoke, cheddar cheese, cured and fried meals and in a number of
alcoholic beverages. It is a hepatocarcinogen producing reproducible HCC after
repeated administration and is the most important environmental carcinogen among
N-niroso compounds (Singh et al., 2009). Administration of NDEA to animals
causes cancer in liver and at low incidence in other organs also. The formation of
reactive oxygen species (ROS) during the metabolism of NDEA may be one of the
key factors in the etiology of cancer (Bansal et al., 2005).
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
27
The mechanism of action is due to metabolism of NDEA to alkylating agents
and reactive oxygen species and further interaction with DNA molecule, forming
various DNA adducts that can lead to mutations (Jeena et al.,1999). The O4 – ethyl
deoxythymidine adduct (O4- Etdt) accumulates in hepatocyte DNA following NDEA
administration which is thought to be important in tumor initiation (Sivalokanathan
et al., 2006).
1.2.8. Types of hepatic diseases
Mainly there are four types of hepatic diseases:
1. Hepatitis (an inflammatory liver disease)
2. Hepatosis (non inflammatory disease)
3. Cirrhosis (a degenerative disease)
4. Hepatocellular carcinoma
1.2.9. Management of Hepatic diseases
Drugs that stimulates liver function offers protection to the liver from
damage or helps regeneration of hepatic cells. As it is the function of
hepatoprotective agents to interfere with these pathological processes by blocking
their evolution and helping recovery, the development of new antihepatotoxic drugs
is the need of the hour.
Since increase in the use of synthetic drugs in therapy leads to many side
effects and undesirable hazards, there is a worldwide trend to go back to natural
resources (mainly traditional plants) which is both culturally acceptable and
economically viable. Thousands of plants are used in this world to prevent or cure
Chapter 1Chapter 1Chapter 1Chapter 1
28
diseases, but the biochemical basis of protective action which is necessary for the
rational development of safe and potent drugs, is lacking in most of the cases.
Ayurveda, an indigenous system of medicine in India, has a long tradition of
treating liver disorders with plant drugs. This ancient system of medicine makes use
of active principles present in plants for treating diseases. Medicinal herbs provide
protection against hepatotoxins in various ways: by enhancing the functioning of the
hepatic glutathione antioxidant system, inhibiting cytochrome P450, promoting
glucuronidation, stimulating hepatic regeneration, activating functions of reticulo-
endothelial systems, inhibiting biosynthesis of cytochrome P450 (Rao and Mishra,
1998) preventing lipid peroxidation, stabilizing hepatocellular membrane, enhancing
protein biosynthesis (Lin et al., 1997); accelerating the regeneration of parenchymal
cells and thus protecting against membrane fragility, decreasing the leakage of
marker enzymes into the circulation, interfering with the microsomal activation of
CC14 and/or accelerating detoxification (Bishayee et al., 1995); counteracting the
hepatic lysosomal enzymes (Saxena et al., 1993).
1.2.9.1. Drugs for liver diseases
Conventional and synthetic drugs used in the treatment of liver diseases are
sometimes inadequate and can have serious adverse effects. Steroids, vaccines, and
antiviral drugs, have been used as therapies for liver pathologies, have potential
adverse side-effects, especially if administered chronically or sub-chronically.
Current medical treatments for these liver diseases are often ineffective, and
therefore efforts are being made to seek new effective medications (Seeff et al.,
2001). Developing pharmacologically effective agents from natural products has
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
29
become a new trend by virtue of their little toxicity or few side effects. There are
few plant derived drugs in the market which are used for the liver disorders.
(a) Silymarin
Silymarin, derived from the seeds of Silybum marianum L. (Family:
Asteraceae or Compositae), commonly known as milk thistle, has been used for
centuries as a natural remedy for liver and biliary tract diseases (Saller et al., 2001).
Milk thistle protects and regenerates the liver in most liver diseases such as
cirrhosis, jaundice, and hepatitis (Flora et al., 1998). Silymarin offers good
protection in various models of experimental liver disease. It has antioxidative,
antilipid peroxidative (Pascual et al., 1993), antifibrotic (Mourelle et al., 1989),
membrane stabilizing, immunomodulatory and liver regenerating mechanisms
(Pradhan and Girish, 2006).
Limitations
Silymarin is insoluble in water and typically administered as a sugar coated
tablet (Thakur, 2002) or as an encapsulated standardized extract. The absorption by
oral route is as low as 2-3 percent of the silybin recovered from rat bile in 24 h.
About 20- 40 percent of the administered dose of silymarin is excreted in bile as
sulphates and glucuronide conjugates in human beings (Saller et al., 2001).
Side Effects
Silymarin has low level of toxicity. Although, silymarin has a good safety
record, there are few reports of gastrointestinal disturbances and allergic skin rashes
(Negi et al., 2008).
Chapter 1Chapter 1Chapter 1Chapter 1
30
(b) Liv – 52
Liv-52 was introduced in 1954 as a specially formulated Ayurvedic herbal
remedy for the treatment of viral hepatitis and has been widely prescribed for
infective hepatitis since then (Mukerjee and Dasgupta, 1970). Experimentally, Liv-
52 prevented injurious effects of carbon tetrachloride and other toxic substances on
the liver.
Liv.52 is available as tablets and syrup containing the following herbs:
Capparis spinosa; Cichorium intybus; Solanum nigrum; Terminalia arjuna; Cassia
occidentalis, Achillea millefolium; Tamarix galica and Phyllanthus amarus. These
herbs are processed and formulated according to the principles of Ayurveda, which
are aimed at enhancing efficacy and avoiding toxicity (Charak and vimanasthan,
1981).
1.2.9.2. Herbal medicine
India is a rich source of medicinal plants and a number of plant derived
extracts are used against diseases in various systems of medicine such as Ayurveda,
Unani and Siddha. Use of herbal medicines can be traced back as far as 2100 B.C. in
ancient China (Xia dynasty) and India (Vedic period). The first written reports date
back to 600 B.C. with the Charaka Samhita of India and the early notes of the
Eastern Zhou dynasty of China that became systematized around 400 B.C. The
recipes, once formulated, were usually expanded rather than abandoned during
subsequent centuries.
The use of medicinal plants in curing diseases is as old as man. The World
Health organization (WHO) has long recognized and drawn the attention of many
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
31
countries to the ever increasing interest of the public in the use of medicinal plants
and their products in the treatment of various ailments. It is estimated that 70-80% of
people worldwide rely on traditional herbal medicine to meet their primary health
care needs (Uprety et al., 2012). The use of herbal medicines presents unique clinical
and pharmacological challenges that are not encountered with conventional single-
compound medicines. These medicines are usually complex mixtures of many
bioactive compounds and conventional “indications and uses” criteria devised for
single compound entities may not be applicable to this system in a significant number
of ways. Compared to single-agent pharmaceuticals, phytomedicines may differ in the
different mechanisms of action of bioactive constituents, in their dose-response
relationships, and in the synergistic/combinatorial effects of the many bioactive
compounds found in herbal extracts” (Yong et al., 2004).
India has been identified as one of the top twelve mega bio-diversity center
of the world. This is because India has a vast area with wide variation in climate,
soil, altitude and latitude (Tiwari, 2008). India is rich in all the three levels of
biodiversity, namely species diversity, genetic diversity and habitat diversity. In
India thousands of species are known to have medicinal value and the use of
different parts of several medicinal plants to cure specific ailments has been in
vogue since ancient times (Parekh et al., 2005). India with its biggest repository of
medicinal plants in the world may maintain an important position in the production
of raw materials either directly for crude drugs or as the bioactive compounds in the
formulation of pharmaceuticals and cosmetics etc (Tiwari, 2008). Extraction of
bioactive compounds from medicinal plants permits the demonstration of their
Chapter 1Chapter 1Chapter 1Chapter 1
32
physiological activity. It also facilitates pharmacology studies leading to synthesis of
a more potent drug with reduced toxicity.
Plant derived natural products such as flavonoids, terpenes and alkaloids
(Shukla et al., 2010) have received considerable attention due to their diverse
pharmacological properties including inflammatory, antipyretic and analgesic
activities. Consumption of natural products reduce the risk of developing
pathological conditions, including cancer, nervous system disorders, hepatic
damage, cardiovascular, genetic, and inflammatory diseases (Newman and Cragg,
2007; Jurenka, 2009). Plants contain numerous bioactive molecules that can improve
the body’s resistance to cellular stress and prevent the cytotoxicity of various agents.
Many of the active ingredients for health care products are directly or indirectly
derived from plants (Newman et al., 2000). However, many high value plant-derived
natural products remain undiscovered or unexplored for their pharmacological
activity (Raskin et al., 2002).
1.2.10. Hepatoprotective Study
The Ayurveda has a long tradition of treating liver diseases using herbal
medicines, and the control of liver diseases has become a major goal of modern
medicine. As it is the function of hepatoprotective agents to interfere with these
pathological processes by blocking their evolution and helping recovery, the
development of new antihepatotoxic drugs is the need of the hour. Traditional
medicine all over the world is nowadays being re-evaluated by extensive research on
different plant species with reference to their therapeutic principles. The 21st century
has seen a paradigm shift towards therapeutic evaluation of herbal products in liver
disease models by carefully synergizing the strength of the traditional systems of
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
33
medicine with that of the modern concept of evidence based medicinal evaluation,
standardization and randomized placebo controlled clinical trials to support clinical
efficacy (Thyagarajan et al., 2002).
A large number of compounds are capable of causing liver injury. To
evaluate the hepatoprotective potential of medicinal plants in animal models some
chemical compounds like acetaminophen, carbon tetrachloride, galactosamine and
thioacetamide are used as a toxicants to induce liver damage (Subramoniam and
Pushpangadan, 1999).
Hepatotoxins are of two major groups namely direct and indirect hepatotoxins.
Direct hepatotoxic agents damage the membrane of hepatocytes directly resulting
interference in cell metabolism. The indirect hepatotoxins cause hepatic injury as a
result of selective interference with metabolic pathways or selective binding to or
alteration of a specific component. The degree of injury varies from alteration of
only one metabolic function without structural change. The liver injury may
progress to chronic liver disease, fulminant hepatic failure, cirrhosis or malignancy
in due course.
Table 1.2. List of hepatoprotective medicinal plants
Sl.No Plant (family) Part used Solvent used
Hepatotoxicant used References
1. Acalypha racemosa Wall. (Euphorbiaceae)
Leaves Methanol CCl4 Iniaghe et al., 2008
2. Actinidia deliciosa Chev. Actinidiaceae)
Roots Ethanol CCl4 Bai et al., 2007
3. Adhatoda vasica Nees (Acanthaceae)
Leaves Water Galactosamine Bhattacharyya et al., 2005
Chapter 1Chapter 1Chapter 1Chapter 1
34
Table 1.2. (Cont.) List of hepatoprotective medicinal plants
Sl.No Plant (family) Part used Solvent used
Hepatotoxicant used References
4. Aloe barbadensis
Mill. (Liliaceae)
Aerial parts Water CCl4 Chandan et al., 2007
5. Andrographis lineate Fam. (Acanthaceae)
Leaves Methanol, Water
CCl4 Sangameswaran
et al., 2008
6. Anoectochilus formosanus Hayata. (Orchidaceae)
Whole plant Water CCl4 Fang et al.,
2008
7. Apium graveolens Linn. (Apiaceae) Croton oblongifolius Roxb. (Euphorbiaceae)
Seeds Petroleum ether, Acetone, Methanol
CCl4 Ahmed et al.,
2002
8. Artemisia absinthium L. (Asteraceae)
Aerial part Water CCl4 Amat et al., 2010
9. Azadirachta indica Juss. (Meliaceae)
Leaves Fresh juice
Acetaminophen Yanpallewar
et al., 2002
10. Bauhinia variegate L. (Leguminosae)
Stem bark Alcohol CCl4 Bodakhe and
Ram, 2007
11. Berberis tinctoria Lisch. (Berberidaceae)
Leaves Methanol Acetaminophen Murugesh et al., 2005
12. Boerhaavia diffusa Linn. (Nynctaginaceae)
Leaves Ethanol Acetaminophen Olaleye et al.,
2010
13. Boswellia serrata Roxb. (Burseraceae)
Oleo gum resin
n- Hexane CCl4,
Thioacetamide Jyothi et al., 2006
14. Camellia sinensis
Linn. (Theaceae)
Leaves Water Sodium oxalate Oyejide and
Olushola, 2005
15. Cassia tora L.
(Caesalpiniaceae)
Ononitol
Monohydrate from Leaves
- CCl4 Dhanasekaran
et al., 2009
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
35
Table 1.2. (Cont.) List of hepatoprotective medicinal plants
Sl.No Plant (family) Part used Solvent used
Hepatotoxicant used References
16. Cleome viscosa Linn.(Capparidaceae)
Leaves Ethanol Thioacetamide Gupta and Dixit, 2009
17. Cistus laurifolius
L. (Cistaceae)
Isolation of flavonoid from Leaves
Ethanol Acetaminophen Kupeli et al.,
2006
18. Citrus limon L.
Burm. (Rutaceae)
Fruits 70%
Ethanol
CCl4 Bhavsar et al.,
2007
19. Curculigo orchioides Gaertn. (Amaryllidaceae)
Rhizome methanol CCl4 Venukumar and Latha, 2002
20. Curcuma longa Linn. (Zingiberaceae)
Rhizome Ethanol,
Water
Diclofenac Hamza, 2007
21. Cuscuta chinensis Lam. (Convolvulaceae)
Seeds Ethanol,
Water
Acetaminophen Yen et al.,
2007
22. Cytisus scoparius L. (Leguminosae)
Aerial part Ethanol: Water (7:3)
CCl4 Raja et al., 2007
23. Decalepis
hamiltonii Wight.
(Asclepiadaceae)
Root Water CCl4 Srivastava and
Shivanandappa,
2010
24. Diospyros
malabarica Kostel.
(Ebenaceae)
Bark Methanol CCl4 Mondal et al.,
2005
25. Enicostemma
axillare Raynal.
(Gentianaceae)
Swertiamarin
from
Whole plant
Ethyl
acetate
D-galactosamine Jaishree and
Badami, 2010
26. Epaltes divaricata
L. (Compositae)
Whole plant Water CCl4 Hewawasam
et al., 2004
Chapter 1Chapter 1Chapter 1Chapter 1
36
Table 1.2. (Cont.) List of hepatoprotective medicinal plants
Sl.No Plant (family) Part used Solvent used
Hepatotoxicant used References
27. Euphorbia
fusiformis D.Don.
(Euphorbiaceae)
Tubers Ethanol Rifampicin Anusuya et al., 2010
28. Ginkgo biloba Linn. (Ginkgoaceae)
Leaves Water CCl4 Shenoy et al., 2001
29. Glycyrrhiza glabra
L. (Leguminosae)
Glycyrrhizin
from Root
Methanol CCl4 Lee et al.,
2007
30. Halenia elliptica
(Gentianaceae)
Whole plant 70%
Methanol
CCl4 Huang et al.,
2010a
31. Hibiscus sabdariffa
L. (Malvaceae)
Flowers
Water Azathioprine Amin and
Hamza, 2005
32 Rosmarinus
officinalis L.
(Lamiaceae)
Leaves
Water Azathioprine Amin and
Hamza, 2005
33 Salvia officinalis
L. (Lamiaceae)
Leaves Water Azathioprine Amin and
Hamza, 2005
34. Hybrophila
auriculata Heine.
(Acanthaceae)
Root Water CCl4 Shanmugasund
aram and
Venkataraman,
2006
35. Indigofera pinnatifida Cass. (Asteraceae)
Leaves and roots
Chloroform
Methanol
Paracetamol Kumar et al., 2008
36. Justicia simplex D.
Don. (Acanthaceae)
Whole plant
(Isolated
lignans)
Petroleum
ether
CCl4 Jasemine et al., 2007
37. Kyllinga nemoralis
L. (Cyperaceae)
Rhizome Petroleum ether, Ethanol
CCl4 Somasundaram
et al., 2010
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
37
Table 1.2. (Cont.) List of hepatoprotective medicinal plants
Sl.No Plant (family) Part used Solvent used
Hepatotoxicant used References
38. Ligustrum
robustum Roxb.
(Oleaceae)
Leaves Water CCl4 Lau et al.,
2002
39. Lygodium
flexuosum (L.) Sw.
(Lygodiaceae)
Whole plant Hexane CCl4 Wills and
Asha, 2006a
40. Moringa oleifera
Lam. (Moringeaceae)
Seed 70%
Ethanol
CCl4 Hamza, 2010
41. Nelumbo nucifera
Gaertn.
(Nelumbonaceae)
Leaves 60%
Ethanol
CCl4 Huang et al.,
2010b
42. Operculina
turpethum L.
(Convolvulaceae)
Root Ethanol Acetaminophen Suresh kumar
et al., 2006
43. Phyllanthus niruri
L.(Euphorbiaceae)
Leaves,
Stem
PO4 buffer
Nimesulide Chatterjee and
Sil, 2007
44. Phyllanthus
urinaria L.
(Euphorbiaceae)
Whole plant 80%
Ethanol
Acetaminophen Hau et al.,
2009
45. Physalis peruviana
L. (Solanaceae)
Whole plant Water Acetaminophen Chang et al.,
2008
46. Polyalthia longifolia
var. pendula.
(Annonaceae)
Leaves Methanol Diclofenac Tanna et al.,
2009
47. Punica granatum
Linn. (Punicaceae)
Water CCl4 Celik et al.,
2009
Chapter 1Chapter 1Chapter 1Chapter 1
38
Table 1.2. (Cont.) List of hepatoprotective medicinal plants
Sl.No Plant (Family) Part used Solvent used
Hepatotoxicant used References
48. Rhoicissus tridentata
Wild. (Vitaceae)
Root Water CCl4 Opoku et al.,
2007
49. Sida acuta Burm. f.
(Malvaceae)
Root Methanol Acetaminophen Sreedevi et al., 2009
50. Syzygium cumini
L. (Myrtaceae)
Leaves Water CCl4 Moresco et al., 2007
51. Terminalia arjuna
Bedd.
(Combretaceae)
Bark Water CCl4 Manna et al.,
2006
52. Terminalia belerica Roxb. (Combretaceae)
Fruit Ethanol CCl4 Jodan et al. 2007
53. Terminalia
catappa L.
(Combretaceae)
Leaves Ethanol CCl4 Gao et al.,
2004
54. Vernonia
amygdalina Delile.
(Astereaceae)
Leaves 90%
Methanol
CCl4 Adesanoye
and Farombi,
2010
55. Vitis vinifera L.
(Vitaceae)
Leaves 80%
Ethanol
CCl4 Orhan et al.,
2007
56. Zanthoxylum
armatum DC.
(Rutaceae)
Bark 70%
Ethanol
CCl4 Ranawat et al., 2010
57. Zingiber officinale
Roscoe.
(Zingiberaceae)
Rhizome 50%
Ethanol
Acetaminophen Ajith et al.,
2007
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
39
1.2.11. Hepatic Fibrosis and Cirrhosis
Hepatic fibrosis is a precursor of cirrhosis, developing in response to chronic
hepatocellular injury show general features of a wound repair process characterized by
specific cellular reactions. Hepatic fibrosis is usually initiated by hepatocyte damage.
Biologic factors such as hepatitis virus, bile duct obstruction, cholesterol overload,
schistosomiasis, etc; or chemical factors such as CCl4 administration, alcohol intake,
etc. were known to contribute to liver fibrosis. Hepatic fibrosis is major features of a
wide range of chronic liver injuries including metabolic, viral, cholestatic and
genetic disease. The failure of bile salt excretion in cholestasis leads to retention of
hydrophobic bile salts within the hepatocytes and causes apoptosis and/or necrosis
(Miyoshi et al., 1999).
Hepatic fibrosis is characterized by the excessive deposition of extracellular
matrix (ECM) proteins including collagen, fibronectin, laminin and proteoglycans
(Wills and Asha, 2006b). Activated hepatic stellate cells, portal fibroblasts and
myofibroblasts of bone marrow origin have been identified as major collagen-
producing cells in the injured liver. Excess depositions of ECM proteins disrupt the
normal functioning of the liver, ultimately leading to patho-physiological damage to
the organ, which has high mortality rate (Wills and Asha, 2007). Reactive oxygen
free radicals have been known to produce tissue injury through covalent binding and
lipid peroxidation and have been shown to augment fibrosis as seen from increased
collagen synthesis (Geesin et al., 1990). As these processes continue, accompanying
fibrosis interfere with blood flow through the liver resulting in severe
pathophysiological consequences such as portal hypertension, hepatic insufficiency,
jaundice and ascites.
Chapter 1Chapter 1Chapter 1Chapter 1
40
Fig. 1.5. Schematic representation of the development of hepatic fibrosis
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
41
1.2.11.1. Pathogenesis of hepatic fibrosis
Hepatic fibrosis is the result of the wound-healing response of the liver to
repeated injury.
Fig. 1.6. Changes in hepatic architecture associated with advanced hepatic fibrosis
After an acute liver injury (e.g., viral hepatitis), parenchymal cells regenerate
and replace the necrotic or apoptotic cells. This process is associated with an
inflammatory response and a limited deposition of ECM. If the hepatic injury
persists, then eventually the liver regeneration fails, and hepatocytes are substituted
with abundant ECM, including fibrillar collagen. In chronic liver injury,
inflammatory lymphocytes infiltrate the hepatic parenchyma. Some hepatocytes
undergo apoptosis, and Kupffer cells activate, releasing fibrogenic mediators. HSCs
proliferate and undergo a dramatic phenotypical activation, secreting large amounts
of extracellular matrix proteins. The distribution of this fibrous material depends on
Chapter 1Chapter 1Chapter 1Chapter 1
42
the origin of the liver injury. In chronic viral hepatitis and chronic cholestatic
disorders, the fibrotic tissue is initially located around portal tracts, while in alcohol-
induced liver disease; it locates in pericentral and perisinusoidal areas (Pinzani,
1999). Sinusoidal endothelial cells lose their fenestrations, and the tonic contraction
of HSCs causes increased resistance to blood flow in the hepatic sinusoid. As
fibrotic liver diseases advance, disease progression from collagen bands to bridging
fibrosis to frank cirrhosis occurs.
Liver fibrosis is associated with major alterations in both the quantity and
composition of ECM. In advanced stages, the liver contains approximately 6 times
more ECM than normal, including collagens (I, III, and IV), fibronectin, undulin,
elastin, laminin, hyaluronan, and proteoglycans. HSCs are the main ECM-producing
cells in the injured liver (Gabele, 2003). In the normal liver, HSCs reside in the
space of Disse and are the major storage sites of vitamin A. Following chronic
injury, HSCs activate or transdifferentiate into myofibroblast-like cells. Activated
HSCs migrate and accumulate at the sites of tissue repair, secreting large amounts of
ECM and regulating ECM degradation. PDGF, mainly produced by kupffer cells, is
the predominant mitogen for activated HSCs. Collagen synthesis in HSCs is
regulated at the transcriptional and post-transcriptional levels (Lindquist, 2000).
Although fibrosis and cirrhosis are of high incidence worldwide, therapeutic
management of these diseases still remains insufficient. These therapeutic concepts
focus mainly on symptoms rather than on blocking central fibrogenic mechanisms.
Progress in the understanding of the pathological mechanisms may open new
strategies with which to interfere, at early steps, in the development of these diseases
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
43
(Gebhardt, 2002; Tsukada et al., 2006). Detection of the expression of liver cytokines
is useful in exploring the probable mechanisms of anti-fibrotic drugs.
Fig.1.7. Cellular mechanism of hepatic fibrosis
The CCl4-treated rat is frequently used as an experimental model to study
hepatic fibrosis (Oh et al., 2003; Inao et al., 2004). Reversibility of advanced liver
fibrosis in patients has been recently documented, which has stimulated to develop
antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the
accumulation of fibrogenic cells and/or preventing the deposition of extracellular
matrix proteins in the liver (Bataller and Brenner, 2005).
Several herbal drugs have been investigated for their antifibrotic effects on
chemically induced hepatic fibrosis in rats. Some of them are listed in the Table 1.4.
Traditional plant drugs have been found to be effective in preventing fibrogenesis
and other chronic liver injury which develops a more hopeful future in controlling
liver fibrosis, cirrhosis and hepatocarcinogenesis (Lee et al., 2003; Yao et al., 2005).
Chapter 1Chapter 1Chapter 1Chapter 1
44
Table 1.3. List of medicinal plants with antifibrotic activity
Sl. No Plant Part used/compound
References
1. Artemisia capillaries Entire plant Wang et al., 2012
2. Artemisia iwayomogi Entire plant Wang et al., 2012
3. Curcuma longa Curcumin Bruck et al., 2007
4. Ginkgo biloba - Luo et al., 2004
5. Lygodium flexuosam entire plant Wills and Asha, 2006b
6. Regimen (combination of Salvia miltiorrhiza, Ligusticum chuanxiong, Glycyrrhiza glabra
Entire plant Lin et al., 2008
7. Rheum palmantum L Emodin Hu et al., 2009
8. Rheum officinale Rhein Guo et al., 2002
9. Rhus verniciflua Butein Lee et al., 2003
10. Salvia miltiorrhiza Bge. Salvianolic acid B
Hu et al., 2009; Hsu et al., 2005
11. Scutellaria baicalensis Georgi
Baicalin Hu et al., 2009
12. Tinospora crispa stem Kadir et al., 2011
1.2.12. Cancer
Cancer is a class of disease or disorder characterized by uncontrolled
division of cells and has the ability to invade other tissues, either by direct growth
into adjacent tissue through invasion or by implantation into distant sites by
metastasis. If the spread is not controlled, it can result in death (American Cancer
Society, 2011). Occasionaly, dividing and differentiating cells deviate from their
normal genetic program and give rise to tissues called tumours or neoplasm. This
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
45
process by which a cell loses its ability to remain constrained in its growth
properties is called transformation. If the transformed cells stay together in a single
mass, the tumour is said to be benign and the cells of a tumour can invade and
disrupt surrounding tissues, the tumour is said to be malignant and is identified as a
cancer. Cells from malignant tumours can break off and move through the blood
and lymphatic system, forming new tumours at other locations in the body.
Malignancy can result in death due to damage to critical organs, starvation,
secondary infection, metabolic problems or haemorrhage (Karp, 1996). Metastasis is
defined as the stage in which cancer cells are transported through the blood or
lymphatic system. It is the sum of all processes which transform normal healthy
alive cells into abnormal damaged denatured cells. Genetic alteration is the
fundamental underlying process that allows a normal cell to evolve into cancerous
one. Critical events in the evolution of the neoplastic disease include the loss of
proliferative control, the failure to undergo programmed cell death (apoptosis), the
onset of neoangiogenesis, tissue remodeling, invasion of tumour cells into
surrounding tissue and finally metastatic distribution of tumour cells to distant
organs (Herzig and Christofori, 2002).
1.2.12.1. Etiology of cancer
The causes of cancer have been determined to be the result of genetic
predisposition, environmental exposure, infection by suitable agent or a combination
of these. The agents which cause cancer are called carcinogens. Carcinogenesis in
multicellular organism can result from anyone or a combination of genetic (DNA
damage or gene mutation), chemical (drugs, tobacco, alcohol, diet etc.), physical
(radiation- X rays, UV rays) and/or biological (infections due to DNA and RNA
Chapter 1Chapter 1Chapter 1Chapter 1
46
viruses) insults to cells. These obviously are implicated as causal agents of
mammalian cancers (Sakarkar and Deshmukh, 2011).
(a). Genetic factors
The majority of recognized carcinogens cause genetic mutations. Changes in
gene expression in somatic cells, mostly due to mutation, are thought to be the basis
for malignant transformation; there may be one or more, rare, dominantly inherited
susceptibilities to every type of cancer. The contribution made by these highly
penetrant, dominant susceptibilities to the total incidence of cancer has been
estimated at 2–5% of fatal cancers. Genetic variation in susceptibility to cancer may
also arise because of genetic polymorphism affecting the absorption, transport,
metabolic activation, or detoxification of environmental carcinogens.
(b). Tobacco
Tobacco smoking is the largest single avoidable cause of premature death
and the most important known carcinogen. Based on proportions of cancers of lung,
larynx, oral cavity and pharynx, oesophagus, pancreas, kidney, and bladder due to
smoking, 15% (1.1 million new cases per year) of all cancer cases worldwide are
attributed to smoking (25% of cases worldwide in men, 4% in women).
(c). Alcohol
Free radicals generated as a result of the metabolism of alcohol are shown to
be responsible for augmentation of hepatic lipid peroxidation and ethanol mediated
liver carcinogenesis. The main effect of alcohol is a joint effect with tobacco
smoking in cancers of the oral cavity, pharynx, larynx, and oesophagus.
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
47
(d). Diet
High intake of vegetables and fruit show a consistent inverse relationship
with cancer of the larynx, lung, oesophagus, and stomach, and there is weaker
evidence that this is the case also for cancer of the mouth and pharynx, pancreas, and
colon. Low levels of consumption of fruit and vegetables, high levels of meat
consumption, obesity, and lack of regular physical activity tend to be aspects of a
lifestyle more typical of developed than of developing countries. Nowadays majority
of our vegetables, fruits and rice are contaminated with pesticides and toxic
chemicals. All these orally ingested chemicals first go to liver because liver is the
major target organ for toxicity of xenobiotics. A large number of xenobiotics are
reported to be potentially hepatotoxic, this increases the risk of liver cancer.
(e). Drugs
Intake of acetaminophen like drugs and certain chemicals may also lead to
hepatocellular carcinoma. Long term use of analgesics and antipyretics cause hepatic
injury and on prolonged conditions it leads to cancer.
(f). Infections
Viruses may be the cause of at least 15% of all human cancers. Human
papillomavirus (HPV) of any type accounts for 82% of cervical cancers in
developed countries and 91% in developing countries. The human papilloma viruses
occur in 70 different types. The strongest evidence for carcinogenicity is for
HPV types 16 and 18. 81% of cases of liver cancer are attributable to chronic
infection with hepatitis B or hepatitis C.
Chapter 1Chapter 1Chapter 1Chapter 1
48
Strong evidence supports a causal relationship between chronic infection
with the bacterium Helicobacter pylori and the development of gastric
adenocarcinoma, and there is some evidence for gastric lymphoma. 60% of cases of
gastric cancer in developed countries, and 53% in developing countries, may be
attributable to Helicobacter pylori.
(f). Environmental factors
The incidence of many types of cancer varies greatly between geographical
areas. There are changes of rates following migration between areas of contrasting
incidence, changes in incidence over time, and variation within populations
according to socio-economic status. Thus environmental factors appear to have a
major role in the aetiology of most types of cancer, accounting for over 80% of
human cancer.
(g). Solar exposure
The 1996 Harvard Report on Cancer Prevention concluded that over 90% of
malignant melanoma is attributable to solar radiation. Malignant melanoma
accounted for just over 1% of the world cancer burden in 1985. Uncertainties
remain, even though it is widely assumed that exposure to solar radiation also
accounts for the great majority of cases of basal cell and squamous cell carcinoma.
(h). Other exposures
Other exposures account for 5% or less of the cancer burden. Occupational
exposures have been linked with lung, bladder, and haematopoietic malignancies.
Breast cancer has consistently been associated with early age at menarche, late age
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
49
at first birth, and late age at menopause with relative risks of the order of 2.0 or less.
Parity is associated inversely with endometrial and ovarian cancer.
Although most types of cancer are more common in urban than in rural
areas, few causal links with environmental pollutants have been firmly established.
It has been estimated that 1% of lung cancer deaths in the US are attributable to air
pollution. While exposure to ionizing radiation at doses of 500–2000 mSv is known
to be carcinogenic, exposures of this magnitude are unusual—about 1% of the
deaths of the Japanese atomic bomb survivors could be attributed to radiation. The
average per capita dose from all sources of ionizing radiation is about 3.4 mSv per
year, of which about 88% is from natural sources and the remainder primarily from
medical exposures. Extrapolation from data on people exposed to doses of 500 mSv
or more suggests that 1–3% of all cancers might be attributable to radiation arising
largely from natural sources. No clear association with exposure to extremely low
frequency magnetic fields has been established.
Some pharmaceutical agents (e.g. immunosuppressive agents, anti-neoplastic
drugs, and hormonal preparations) are human carcinogens.
1.2.12.2. Stages of Carcinogenesis
The process of carcinogenesis may be divided into at least three stages:
initiation, promotion and progression. Cancer development is now commonly
recognized as a microevolutionary process that requires the cumulative
accumulation of multiple events. These events may occur in a single cell clone and
can be explained by a simplified three stage model. These stages include initiation of
DNA mutation in a somatic cell is known as initiation, stimulation of initiated cell
Chapter 1Chapter 1Chapter 1Chapter 1
50
and its clonal expression referred to as promotion and conversion of benign tumor
into malignant termed as progression (Athar, 2002). The initiation phase appears to
be irreversible and relatively easily induced by DNA damaging agents, mutagenesis
has been implied underlying mechanism responsible for this step. The promotion
phase of carcinogenesis, operationally is an interruptible process (and reversible up
to certain point). This implies that the initiated cell can be stimulated to proliferate
but will not terminally differentiate. The promotion process can be implied to be an
epigenetic process. Mitogenesis, rather than mutagenesis, best describe the
promotion process.
Fig. 1.8. Stages of carcinogenesis and the occurrences involved in each one
1.2.12.3 Cancer prevention and treatment
Cancers due to use of tobacco, alcohol, exogenous hormones and exposure of
environmental carcinogen can be effectively prevented through education and social
policies that discourage unhealthy practices. Certain cancers that are related to
infectious agents such hepatitis B (HBV), human immunodeficiency virus (HIV),
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
51
human papilloma virus (HPV) and Helicobacter pylori could be prevented through
known interventions such as vaccines, antibiotics, improved sanitation and
education. Through regular screening and examination many cancers can be
diagnosed and easily cured at the early stages of their development.
Several methods exist for treatment of cancer in modern medicine, which
include chemotherapy, radiotherapy, surgery, hormonal therapy and immunotherapy.
Selective killing or removal of the cancer cells without affecting the rest of the body
is the goal of cancer treatment.
(a). Chemotherapy
Chemotherapy remains as the treatment choice for most advanced cancers or
as an adjunct to other treatment modalities like surgery and radiotherapy, but severe
toxic effects towards normal tissues also limit its use (Baxevanis et al., 2009).
It involves the use of cytotoxic agents which are transported by the bloodstream to
different parts of the body to destroy cancer cells. The first uses of chemotherapy to
control cancer were reported in the 1940s, and in the decades since, treatment of
patients with broadly toxic chemicals have represented a mainstay of medical
oncology, in spite of the frequent severe side effects associated with such treatments.
Many chemotherapeutic agents used to treat malignant diseases damage
lymphocytes and consequently suppress cell-mediated immunity (Bagnyukova et al.,
2010).
(b). Radiotherapy
Radiotherapy is effectively used to reduce the initial tumour load. It involves
the use of ionizing radiation to kill cancer cells and shrink tumours and also
Chapter 1Chapter 1Chapter 1Chapter 1
52
developing as a clinically essential part of cancer therapy for the majority of solid
malignant neoplasm including brain tumours. It has been proved to be a fundamental
tool available in the battlefield against cancer, offering a clear survival benefit in
most cases. However, numerous studies have associated tumour irradiation with
enhanced aggressive phenotype of the remaining cancer cells. A cell population
manages to survive after the exposure, because it either receives sub lethal doses or
it successfully utilizes the repair mechanisms. The biology of irradiated cells is
altered leading to up-regulation of genes that favor cell survival, invasion and
angiogenesis (Kargiotis et al., 2010).
(c). Surgery
Surgery is the oldest treatment of cancer. Surgical interventions can be used
for diagnosis, treatment of precancerous lesions or for removal of normal organs
which are at an elevated risk of developing cancers. As long as the growth of the
tumour remains localized, it can usually be treated and cured by surgical removal of
the tumour and surrounding tissue, but the tendency of cancers to invade adjacent
tissue or to spread to distant sites by metastasis makes complete surgical excision of
the cancer usually impossible (Ozgediz et al., 2008).
(d). Immunotherapy
Historically, the first successful immunotherapy to treat cancer involved the
use of toxins from Streptococcus erysipelatis and Bacillus prodigious by William
Coley in the 1890's (Coley, 1991). Toll-like receptor agonists have been shown to
boost immune responses toward tumours. Also, a wide array of cell based immune
therapies utilizing T cells, NK cells and DC cells have been established.
Furthermore, a rapidly expanding repertoire of monoclonal antibodies is being
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
53
developed to treat tumours, and many of the available antibodies have demonstrated
impressive clinical responses (Borghaei et al., 2009). More recently, the
development of vaccines to tumour-causing hepatitis B virus and papilloma virus are
contributing significantly to prevent cancer in a large portion of the human
population (Blumberg, 1997; Rogers et al., 2008). In addition to vaccines interferon
and interleukin 2 are also used in immunotherapy.
(e). Hormonal therapy
Hormonal therapy is also used for certain type of cancers. Hormones
commonly used in the cancer therapy are steroids, anti-estrogens, anti-androgens,
LH-RH analogues and anti-aromatase agents.
(f). Complementary and Alternative Medicine
Recently, complementary and alternative medicine (CAM) is becoming a
popular treatment for various cancers. Among the CAMs, herbal medicine is one of
the methods used in cancer therapy (Cassileth, 1999). CAM has been defined as a
group of diverse medical and healthcare systems, practices and products that are not
presently considered to be part of conventional medicine. In the last three decades,
the use of CAM has increased in popularity in both the worldwide general
population and in patients with cancer (Yildirim, 2010). The goals of CAM are to
increase the efficacy of conventional cancer treatment programs, reduce symptoms,
and improve quality of life for patients with cancer (Levine, 2010).
(g). Natural products
The natural products have afforded a rich source of compounds that have
found many applications in the fields of medicine, pharmacy and biology and are
Chapter 1Chapter 1Chapter 1Chapter 1
54
being used to treat a wide variety of clinical conditions, with relatively little
knowledge of their modes of action. Within the sphere of cancer, a number of
important new commercialized drugs have been obtained from natural sources, by
structural modification of natural compounds, or by the synthesis of new compounds
and by designing as natural compound as model (Gordaliza, 2007). Currently,
numerous scientific studies support herbal medicine as a potent anticancer drug.
However, herbal remedies are yet to be integrated into main stream medicine mainly
due to lack of experimental and clinical studies on their safety, efficacy, quality
control and pharmacological mechanisms. Careful in vitro and in vivo studies will be
essential and necessary to evaluate their efficacy and safety before clinical trials can
be contemplated (Buchanan et al., 2005; Kwon et al., 2009).
Table 1.4. Selected lists of plants with anticancer activity
SI. No Plant Major active compound Reference
1. Aglaia foveolata Silvestrol Kim et al., 2007
2. Allium sativum (Garlic)
Diallyl sulfide, Diallyl disulfide, Diallyl trisulfide
Choi and Park, 2012
3. Aloe barbadenis
(Aloe vera) Aloe-emodin, Emodin
Chiu et al., 2009
4. Ananas comosus (Pine apple)
Bromelain Hale et al., 2010
5. Berberis amurensis (Berberis)
Berbamine Wei et al., 2009
6. Boswellia serrata Boswellic acid Agrawal et al., 2011
7. Brassica oleracea (Cabbage)
Indole-3-carbinol Wu et al., 2010
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
55
Table 1.4. (Cont.) Selected lists of plants with anticancer activity
SI. No Plant Major active compound Reference
8. Catharanthus roseus (Vinca)
Vinblastine, Vincristine , Alstonine, Ajmalicine.
Van Der Heijden et al., 2004
9. Citrullus lanatus (Watermelon)
Lycopene Ilic et al., 2011
10. Citrus reticulate (Mandarin orange)
Tangeretin, Nobiletin, Hesperetin, hesperidin, Naringenin, Naringin
Meiyanto et al., 2012
11. Curcuma longa (Turmeric)
Curcumin, Tumerone Manikandan et al., 2012
12. Glycine max (Soyabean)
Genistein Li et al., 2012
13. Malus domestica (Apple)
Ursolic acid Gayathri et al., 2009
14. Rheum rhabarbarum (Rhubarb)
Emodin Huang et al., 2009
15. Saussurea lappa Confertin, myricetin, diaminobutryic acid
Thara and Zuhara, 2012
16. Solanum lycopersicum (Tomato)
Lycopene Tang et al., 2009
17. Solanum pseudocapsicum
O-methylsolanocapsine Dongre et al., 2007
18. Taxus brevifolia (Pacific Yew)
Paclitaxel Kingston., 2007
19. Typhonium flagelliforme (Rodent tuber)
Pheophorbide-a, Pheophorbide-a', Pyropheophorbide-a, Methyl pyropheophorbide-a
Lai et al., 2010
Chapter 1Chapter 1Chapter 1Chapter 1
56
Table 1.4. (Cont.) Selected lists of plants with anticancer activity
SI. No Plant Major active compound Reference
20. Vicia faba (Fava bean)
Diadzein, Genistein Kaufman et al., 1997
21. Vitis vinifera (Grapes)
Resveratrol, Piceatannol Kita et al.,2012
22. Wikstroemia indica (Indian stringbush)
Daphnoretin Lu et al., 2011
23. Zingiber officinale (Ginger)
Curcumin, gingerenone A, gingerols, 6-shogaol, 10-gingerol, enone-diarylheptanoid
Peng et al.,2012
1.2.13. Hepatocellular Carcinoma
Hepatocellular carcinoma (HCC) is the fifth most common ubiquitous
deadliest cancer worldwide with poor diagnosis and accounts for approximately
about 500,000 to 1,000,000 new cases per year and accounting for more than
600,000 deaths each year (American Cancer Society, 2012). Generally, HCC is more
frequent in men than in women and the incidence increases with age (Levrero,
2006). Liver is often abused by environmental and biological toxins, poor eating
habits, consumption of alcohol, prescription and over-the-counter drug use, and
viruses which can damage and weaken the liver. These factors eventually lead to
hepatitis, cirrhosis, alcoholic liver disease and hepatocellular carcinoma (Gitlin, 1996).
1.2.13.1. Risk factors of hepatocellular carcinoma
There are multiple etiological agents that are associated with the
development of HCC, the most frequent being chronic hepatitis B virus (HBV),
hepatitis C virus (HCV) infections, long-term exposure to the mycotoxin and
aflatoxin B1.
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
57
Fig.1.9. Risk factors that leads to HCC
(a). Hepatitis B Virus (HBV)
HBV is a DNA virus and is recognized as a major etiological factor in the
development of diseases such as fatty liver (steatosis), cirrhosis, hepatocellular
adenoma and HCC. The risk of HCC in chronic HBV carriers is more than 100
times greater than in uninfected individuals (Beasley et al., 1981; Ito et al., 2010).
(b). Hepatitis C Virus (HCV)
HCV is an enveloped positive stranded RNA virus belongs to the genus
Hepacivirus. It is a completely cytoplasmic-replicating virus that induces oncogenic
transformation (Tellinghuisen and Rice, 2002). Chronic HCV infection mostly leads
to hepatic cirrhosis before developing HCC (Donato et al., 1997). Generally, the
prevalence of HCV-infection is accepted to be a major morbidity factor in hepatic
carcinogenesis. However, it also now established that many viral proteins are
implicated in malignant transformation and HCC development. Of these proteins,
Chapter 1Chapter 1Chapter 1Chapter 1
58
core proteins, NS3 an dNS4, were shown to have transformation potential in tissue
culture (Sakamuro et al., 1995; Gale et al., 1999; Park et al., 2000). These viral
proteins, in addition to the viral RNA, interact with many host-cell factors, while
still regulating the viral life cycle. They modulate host-cell activities such as cell
signaling, transcription, transformation, apoptosis, membrane rearrangement,
vesicular trafficking and protein translation. This ultimately misleads the host
transcription factors, disturbing cell mitosis and protein synthesis, leading to
carcinogenesis (Levrero, 2006).
Fig. 1.10. Progression of HCV infection to HCC
(c). Diabetes mellitus
Many studies around the world have found a significant relationship between
diabetes and the development of HCC (Lagiou et al., 2000). Between 10 and 20% of
cirrhosis patients have overt diabetes and a higher percentage present impaired
glucose tolerance.
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
59
(d). Exposure to environmental pollutants
An environmental pollutant such as aflatoxins is a type of mycotoxin, toxic
chemicals made by some types of fungi. Aflatoxin is produced by Aspergillus fungi
when the fungus grows on improperly stored food products. Aflatoxins are capable
of causing DNA mutations, including the tumor suppressor, TP53 (p53). Aflatoxins
may be found in peanuts, tree nuts, corn, wheat and other grains, and oil seeds.
Other known chemical carcinogens are chlorination byproducts in drinking water.
Uncontrolled water chlorination converts many organic traces in water into
dangerous intermediates, such as di-and tri-chloroacetic acids, which are
experimentally known to induce HCC. Many other chemical contaminants, such as
solvents, food additives, drugs and hormones are also thought to contribute to HCC
(Abdel-Hamid, 2009).
(e). Alcoholism
Alcohol is the second most common risk factor for HCC after infection with
hepatits virus, steatohepatitis (fatty liver) and cirrhosis (Donato et al., 2002). In
developed countries, alcohol drinking seems to be the most common source for
HCC. Alcohol either directly initiates HCC after its oxidation into acetaldehyde,
which is genotoxic, or indirectly through the development of cirrhosis (London
et al., 1996).
Chapter 1Chapter 1Chapter 1Chapter 1
60
(f). Obesity
Obesity showed a 5 fold increase in cancer mortality in people with great
body mass index in contrast to those who had a normal body mass index. Liver
cancer is frequently found in patients with metabolic disarrangements.
(g). Hereditary hemochromatosis
Hereditary hemochromatosis is an autosomal recessive condition
characterized by excessive iron deposition in hepatocytes due to an increased
intestinal absorption. Among hemochromatotic patients, 6% of men and 1.5% of
women are at absolute risk of liver cancer (Elmberg et al., 2003).
1.2.13.2. Developmental stages of hepatocellular carcinoma
Fig. 1.11. Histopathological progression and molecular features of HCC
After hepatic injury incurred by any one of several factors (hepatitis B virus
(HBV), hepatitis C virus (HCV), alcohol and aflatoxin B1), there is necrosis
followed by hepatocyte proliferation. Continuous cycles of this destructive–
regenerative process foster a chronic liver disease condition that culminates in liver
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
61
cirrhosis. Chronic hepatitis leads to cirrhosis within 15-40 years. Mostly, HCC
develops among 70%-90% of cirrhotic patients, while only 10% of HCC patients
have a non-cirrhotic liver, or even have inflammatory lesions. Cirrhosis is
characterized by abnormal liver nodule formation surrounded by collagen deposition
and scarring of the liver. Subsequently, hyperplastic nodules are observed, followed
by dysplastic nodules and ultimately hepatocellular carcinoma (HCC), which can be
further classified into well differentiated, moderately differentiated and poorly
differentiated tumours - the last of which represents the most malignant form of
primary HCC. (Levrero, 2006; Farazi and DePinho, 2006).
1.2.13.3. Experimental model – NDEA induced hepatocellular carcinoma
N-nitrosodiethylamine (NDEA) is a potent carcinogenic dialkylnitrosoamine
frequently used to induce liver cancer in animal models. NDEA belongs to the group
of N-nitrosamines, causing a wide range of tumors in all animal species and
suspected to be health hazards to man (Loeppky, 1999; Pandi perumal et al., 2006).
It is found in a wide variety of foods such as cheese, soybeans, smoked, salted and
dried fish, cured meat and alcoholic beverages and producing reproducible
hepatocellular carcinoma after repeated administration (Singh et al., 2009).
NDEA becomes metabolically active in the liver by the action of cytochrome
P450 enzymes to produce reactive electrophiles, which increase oxidative stress
level leading to cytotoxicity, mutagenecity and carcinogenicity (Archer, 1989).
It has been shown that the mechanism of action is due to metabolism of NDEA to
alkylating agents and reactive oxygen species and further interaction with DNA
molecule, forming various DNA adducts that can lead to mutations (Jeena et al,
1999). ROS are continuously generated in vivo as a result of NDEA administration
Chapter 1Chapter 1Chapter 1Chapter 1
62
causing oxidative stress that seriously damaged the biological systems by injuring
tissues, altering biochemical compounds, causing chromosomal instability, eroding
cell membranes and mutation, which are involved in all steps of carcinogenesis, i.e.
initiation, promotion and progression (Karbownik et al., 2001).
The conventional therapy of hepatocarcinoma including chemotherapy,
radiation, surgical resection and ablation gives little hope for restoration of health
because of poor diagnosis and serious side effects. Liver transplantation is
considered to be the most effective treatment for patients with hepatocarcinoma.
However, low availability of organs limits the offer of this option to all candidates,
and the high risk of tumor recurrence after transplantation further com-promises its
efficiency.
Numerous components of plants, collectively termed “phytochemicals” have
been reported to possess substantial chemopreventive properties. For many years
cancer chemotherapy has been dominated by potent drugs that either interrupt the
synthesis of DNA or destroy its structure once it has formed. Unfortunately, their
toxicity is not limited to cancer cells and normal cells are also harmed. So efforts to
develop less toxic drugs that affect only malignant cells and mechanism based
approach are necessary in cancer therapy (Sivalokanathan et al., 2006). Several
herbal drugs have been investigated for their chemopreventive potential against
hepatocellular carcinoma.
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
63
Table 1.5. Chemopreventive effects of certain plants against hepatocellular carcinoma
SI. No Plant /Compound Experimental model Reference
1. Abrus precatorius Hep G2 cell lines and NDEA induced hepatocellular carcinoma in rats
Kartik et al., 2010
2. Acacia nilotica (polyphenolics)
NDEA induced hepatocellular carcinoma in rats
Singh et al., 2009
3. Achyranthes aspera NDEA and CCl4 induced hepatocellular carcinoma in rats
Kartik et al., 2010b
4. Alocasia macrorrhiza Hepatocellular carcinoma cell lines SMMC-7721 and Murine hepatoma H22 cell lines
Fang et al., 2012
5. Callilepis laureola Human hepatoma Hep G2 cells
Popat et al., 2002
6. Cuphea hyssopifolia (Cuphiin D1, cuphiin D2, woodfordin C, oenothein B)
Hepatoma Hep 3B cells Wang et al., 1999
7. Emblica officinalis NDEA induced hepatocellular carcinoma in rats
Jeena et al., 1999
8. Feronia limmonia Human liver hepatoma cells Hep G2
Jain et al., 2011
9. Genistein DEN induced hepatocellular carcinoma in rats
Chodon et al., 2007
10. Glycyrrhizin NDEA induced hepatocellular carcinoma in rats
Shiota et al., 1999
11. Gynandropsis gynandra Aflatoxin B1 induced hepatocellular carcinoma in rats
Sivanesan and Begum, 2007
Chapter 1Chapter 1Chapter 1Chapter 1
64
Table 1.5. (Cont.) Chemopreventive effect of certain plants against hepatocellular carcinoma
SI. No Plant /Compound Experimental model Reference
12. Lygodium flexuosum
NDEA induced hepatocellular carcinoma in rats
Wills et al., 2006
13. Morin NDEA induced hepatocellular carcinoma in rats
Sivaramakrishnan et al., 2008
14. Phyllanthus amarus NDEA induced hepatocellular carcinoma in rats
Rajeshkumar and Kuttan, 2000
15. Picrorrhiza kurroa NDEA induced hepatocellular carcinoma in rats
Jeena et al., 1999
16. Scutia myrtina NDEA induced hepatocellular carcinoma in rats
Ramanathan et al., 2011
17. Silymarin NDEA induced hepatocellular carcinoma in rats
Ramakrishnan et al., 2008; Ramakrishnan et al., 2009
18. Strychnos nux-vomica (Brucine, Strychnine, Brucine N-oxide, isostrychnine)
Human hepatoma HepG2 cell lines
Deng et al., 2006
19. Terminalia arjuna NDEA induced hepatocellular carcinoma in rats
Sivalokanathan et al., 2006
1.2.14. Selection of the plant for present study
When selecting a plant for pharmacological activities, four basic methods are
usually followed (Suffness and Douros, 1979):
Introduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of LiteratureIntroduction and Review of Literature
65
a. Random choice of plant species
b. Choice based on ethnomedical use
c. Follow up of existing literature on the use of the species
d. Chemotaxonomic approaches
Based on the ethnopharmacological relevance and reported activities
Woodfordia fruticosa Kurz flowers were selected for the study.
1.2.14.1. Plant profile
Botanical name : Woodfordia fruticosa (Linn.) Kurz
: Woodfordia floribunda (Salib.)
Family : Lythraceae
Vernacular names : Eng. : Fire-flame bush, Shiranjitea, Woodfordia
Hin. : Davi, Tavi
Kan : Bela, Tamrapurpi
Mal : Tatri, Tatirippu
San : Dhataki, Madaniyahetu
Tam : Dhattari, Jargi, Velakkai
Tel : Dhataki, Jargi, Serinji
Distribution : Throughout India, but more abundant in north India, ascending up to
an altitude of about 1600 m, and also in a majority of the countries of South East and
Far East Asia like Malaysia, Indonesia, Sri Lanka, China, Japan and Pakistan as well
as Tropical Africa (Kirtikar and Basu, 1935).
Chapter 1Chapter 1Chapter 1Chapter 1
66
The plant: A much branched beautiful deciduous shrub attaining a height of 3-7m
with much long arching branches and bark of the plant, characteristically cinnamon-
brown coloured and smooth, peels off in fibres and the young shoots are terete, often
clothed with fine white pubescence. Leaves simple, opposite or sub- opposite, entire,
ovate - lanceolate, acute, subcoriaceous with black granular dots on the under
surface. Flowers numerious, brilliant, red in dense, axillary, paniculate - cymose
clusters. Fruits ellipsoid, irregularly dehiscent capsule about 1cm long.
Parts used : Flowers
Properties : The flowers are astringent, acrid, refrigerant, stimulant, depurative,
styptic, uterine sedative, anthelmintic, constipating, antibacterial, vulnerary,
corrective of urinary pigments, alexeteric and febrifuge. They are used in vitiated
condition of kapha and pitta, leprosy, skin disease, burning sensation, haemorrhages,
menorrhagia, leucorrhoea, haemoptysis, erysipelas, diarrhea, dysentery, foul ulcers,
diabetes, bilious fever, hepatopathy and verminosis. They are an important
ingredient of Aristam and Asavam as they aid in fermentation; they are also highly
valued as a stimulant in pregnancy.
1.2.14.2. Reported activities of Woodfordia fruticosa
In India, Woodfordia fruticosa Kurz is a much used medicinal plant in
Ayurvedic and Unani systems of medicines (Chopra et al., 1956; Watt, 1972;
Dymock et al., 1995). Although all parts of this plant possess valuable medicinal
properties, there is a heavy demand for the flowers, both in domestic and
international markets specialized in the preparation of herbal medicines
(Oudhia, 2003).
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67
The flower is pungent, acrid, cooling, toxic, sedative and anthelmintic, and is
useful in thirst, dysentery, leprosy, erysipelas, blood diseases, leucorrhoea,
menorrhagia and toothache. It is considered as ‘Kapha’ (mucilage type body
secretion) and ‘Pitta’ (energy-dependent metabolic activity) suppressant in the
Ayurvedic concepts of medicine. Many marketed drugs comprise flowers, fruits,
leaves and buds mixed with pedicels and thinner twigs of the plant (Chopra et al.,
1956).
The flowers are used in the preparation of Ayurvedic fermented drugs called
“Aristhas” (hot extraction followed by month-long slow fermentation) and “Asavas”
(cold percolation followed by month-long slow fermentation) (Atal et al., 1982).
Aristhas are believed to be general health tonics in nature, having overall health
stimulating properties via ameliorating and/or delaying one or other systemic
disorders. Of the 18 aristhas mentioned in the Indian Ministry of Health & Family
Welfare’s monograph (CCRIMH, 1978), 17 have been found to contain Woodfordia
fruticosa. Tribal people in the Chhattisgarh district of central India uses fresh
flowers to stop bleeding in emergency cuts, but they prefer to employ dried flower
powder to heal wounds more efficiently. It is also one of the ingredients of a
preparation used to increase fertility in women (Burkill, 1966; Dey, 1984).
Flowers used for the ayurvedic preperation “Kutajarista” for Sprue,
dysentery, diarrhoea (Shenoy and Yoganarasimhan, 2008), “Lukol” for the
leucorrhoea DUB (dysfunctional uterine bleeding) and symptoms of pelvic
inflammatory disease. Oil based flower extract has always been recommended for
open wounds (Tewari et al., 2001; Das et al., 2007). The dried flowers powder
sprinkled over ulcers and wounds to diminish discharge and promote granulation
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(Khorya and Katrak, 1984). They are also used as tonic in disorders of mucous
membranes, hemorrhoids and in derangement of the liver (Chopra et al., 1956; The
Wealth of India, 1988; Mhaskar et al., 2000). An Ayurvedic medicine called
“Balarishta”, a drug of ‘Asava’ and ‘Aristha’ group, contains Woodfordia fruticosa
flowers as one of the major constituents and is used for burning sensation in stomach
(Agnimandya), weakness (Daurbalya) and rheumatic diseases (Vataja roga)
(Anonymous, 1978). A popular crude drug (Sidowaya or Sidawayah) of Indonesia
and Malaysia mainly contains dried flowers of Woodfordia fruticosa (Burkill, 1966).
It has been used as an astringent to treat dysentery and sprue and also for the
treatment of bowel complaints, rheumatism, dysuria and hematuria in many
Southeast Asian countries.
Table 1.6. Reported activities of Woodfordia fruticosa Kurz with part and solvent used
SI.No Activity Part used Solvent used Reference
1. Antibacterial Flowers Petroleum ether, Chloroform, Methanol Ethanol, Water
Kumaraswamy et al., 2008
2. Antibacterial Flowers Methanol Parekh and Chanda, 2007
3. Antibacterial Leaves Ethanol Bajracharya et al., 2008
4. Antibacterial Leaves - Chougale et al., 2009
5. Antibacterial Kutajarista- An ayurvedic
preparation
- Shenoy and Yoganarasimhan, 2009
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69
Table 1.6. (Cont.) Reported activities of Woodfordia fruticosa Kurz with part and solvent used
SI.No Activity Part used Solvent used Reference
6. Antidiarrhoeal Kutajarista- An ayurvedic
preparation
- Shenoy and
Yoganarasimhan,
2008
7. Antifertility Flowers Alcohol Khushalani et al.,
2006
8. Antimicrobial Flowers Hexane, Chloroform,
Acetone, Methanol,
Water
Dabur et al., 2007
9. Antimicrobial Leaves Essential oil and Hexane, Methanol, Acetone
Kaur and Kaur,
2010
10. Antioxidant Flowers Petroleum ether, Chloroform, Methanol, Water
Kumaraswamy and Satish, 2008
11. Antioxidant Flowers (gallic acid) Petroleum ether, Chloroform, Methanol, Water
Lok Ranjam Bhatt, 2005
12. Antitumor Flowers(WoodfordinA, B and C dimeric hydrolysable tannins)
- Yoshida et al., 1989
14. Antitumor Isolated compound - Kuramochi-Motegi et al., 1992
15. Antiulcer Roots Ethanol Mihira et al., 2011
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Table 1.6 (Cont.) Reported activities of Woodfordia fruticosa Kurz with part and solvent used
SI.No Activity Part used Solvent used Reference
16. Hepatoprotective
Flowers 50 % Alcohol Brindha and
Geetha, 2009
17. Hepatoprotective
Flowers Water Chandan et al.,
2008
18. Hepatoprotective
Flowers Methanol Baravalia et al., 2011
19. Immunomodulatory
Fermented product from flowers
- Kroes et al., 1993
20. Immunostimulatory
Flowers Ethanol Shah and Juvekar,
2010
1.2.14.3. Chemical constituents of Woodfordia fruticosa
The extracts of Woodfordia fruticosa flowers showed the presence of
carbohydrates, gums, flavonoids, sterols and phenolic compounds/tannins
(Khushalani et al., 2006). A series of publications have appeared on the structural
characterization of the secondary metabolites of the plant. The compounds identified
are predominantly phenolics, particularly hydrolysable tannins and flavonoids. The
following chemical constituents are found in different part of the Woodfordia
fruticosa.
Octacosanol and β-sitosterol (Chauhan et al., 1979a), steroid sapogenin
hecogenin and meso-inositol from the flowers (Chauhan et al., 1979b), lupeol,
betulin, betulinic acid, oleanolic acid and ursolic acid from the leaves (Dan and Dan,
1984) .
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71
The phenolic constituents include gallic acid in leaves and stems (Kalidhar
et al., 1981; Kadota et al., 1990), ellagic acid in leaves and flowers (Nair et al.,
1976), bergenin (a C-glycoside of gallic acid) and the norbergenin in stems
(Kalidhar et al., 1981), chrysophanol-8-O-β-D-glucopyranoside in flowers
(Chauhan et al., 1979a), and the naphthaquinone pigment lawsone in leaves (Saoji
et al., 1972).
The flavonoid constituents: six quercetin glycosides; 3-rhamnoside from
flowers (Chauhan et al., 1979b), 3-β-L-arabinoside (polystachoside) from flowers
and leaves (Nair et al., 1976), and 3-O-α-L-arabinopyranoside, 3-O-β-D-
xylopyranoside, 3-O- (6″-galloyl)-β-D-glucopyranoside from leaves (Kadota et
al., 1990). Three myricetin glycosides; 3-O-β-D-galactoside in flowers and leaves
(Nair et al., 1976), and 3-O-α- L-arabinopyranoside, 3-O-(6″-galloyl)-β-D-
galactopyranoside in leaves (Kadota et al., 1990), as also naringenin 7-glucoside and
kaempferol 3-O-glucoside in flowers (Chauhan et al., 1979b).
A large number of new and known hydrolysable tannins have been isolated
from the flowers. The known tannins reported are: 1,2,3,6-tetra-O-galloyl-β-D-
glucose, 1,2,4,6-tetra-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-
glucose, tellimagrandin, gemin D, heterophyllin A and oenothein B (Yoshida et al.,
1989, Yoshida et al., 1990), woodfordins A- C (Yoshida et al., 1989, Yoshida et al.,
1990), woodfordin D, oenothein A (Yoshida et al., 1991), as also isoschimawalin A
and woodfordins E-I (Yoshida et al., 1992; Kuramochi-Motegi et al., 1992)
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The traditional system of medicine in India recommends the hepatoprotective
potential of this plant. Yet there is paucity of information regarding the liver
protective efficacy of Woodfordia fruticosa. Hence this study was undertaken to fill
the lacuna in this regard.
So the thesis embodies the study regarding the antioxidative, antifibrotic and
anticancer activities of Woodfordia fruticosa in experimental animals. The study
pertaining to the isolation and identification of the active phytochemical constituents
responsible for these effects too forms a portion of this thesis.
Fig. 1.12. Woodfordia fruticosa Habit
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73
Fig. 1.13. Woodfordia fruticosa flowers
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