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General Introduction and Review of Literature
One never notices what has been done; one can only see what remains to be done.
- Marie Curie
General Introduction
3
General Introduction
Plants have been an important source of medicine for thousands of years. Use
of plants for the treatment of many diseases dated back to prehistory and people of all
continents have this old tradition. Every culture on earth has relied on the vast variety
of natural chemistries’ found in plants for their therapeutic properties (Seyyed et al.,
2010). Today, scientists and the general public recognize their value as a source of
new or complimentary medicinal products. Recently, wide array of research
investigations highlight the potential health beneficial principles from phytal sources.
Interest in medicinal plants has grown enormously from the use of herbal products as
natural cosmetics and for self-medication by the general public to the scientific
investigations of plants for their biological effects in human beings. Beyond this
pharmaceutical approach to plants, there is a wide tendency to utilize herbal product
to supplement the diet, mainly with the intention of improving the quality of life and
preventing the diseases of elderly people (Maffei, 2003). Over the last 20 years there
has been a growing shift in interest towards plants as significant sources of new
pharmaceuticals. Many pharmaceutical companies show interest in plant derived
drugs due to the current widespread belief that ‘Green Medicine’ is safe and more
dependable than the costly synthetic drugs which have adverse side effects.
Despite the remarkable progress in the preparation of synthetic drugs, over
25% of prescribed medicines in industrialized countries are derived directly from
plants (Newman et al., 2000). The World Health Organization (WHO) also considers
phytotherapy in its health programs and suggests basic procedures for validation of
drugs from plant origin (Anushia et al., 2009) and estimates that up to 80% of people
still rely mainly on traditional remedies such as herbs for their medicine (Tripathi and
Tripathi, 2003). Approximately 25-50 % of current pharmaceuticals are derived from
plants.
The use of traditional medicine is widespread in India (Jeyachandran and
Mahesh, 2007). A number of medicinal plants, traditionally used for over 1000 years
named rasayana are present in herbal preparations of Indian traditional health care
systems (Scartezzini and Sproni, 2000). India has been identified as a major
General Introduction
4
resourceful area in the traditional and alternative medicines globally (Premachandra,
2011). The WHO has listed 21,000 plants, which are used for medicinal purposes
around the world. Among these, 2500 species are in India, out of which 150 species
are used commercially on a fairly large scale (Seth and Sharma, 2004). Indigenous
plants are reservoirs of various metabolites and provide unlimited source of important
chemicals that have diverse biological properties (Tomoko et al., 2002; Lico et al.,
2012).
Antimicrobials of plant origin
Plants used for traditional medicine contain a wide range of substances that
can be used to treat chronic as well as infectious diseases. A vast knowledge of how
to use the plants against different illnesses may be expected to have accumulated in
areas where the use of plants is still of great importance (Leelavathi et al., 2010;
Fatima et al., 2011). Plant synthesizes a wide variety of chemical compounds, which
can be sorted by their chemical class, biosynthetic origin and functional groups into
primary and secondary metabolites. Primary metabolites make up the physical
integrity of the plant cell and are involved with the primary metabolite process of
building and maintaining of living cells. Secondary metabolites do not seem to be
vital to the immediate survival of the plant that produces them and are not an essential
part of the process of building and maintaining living cells (Sharanabasappa et al.,
2007). These secondary metabolites of plants serve as self defence mechanism against
predation by many microorganisms, insects and herbivores (Vaghasiya et al., 2011).
A growing body of evidence indicates that secondary plant metabolites play critical
roles in human health and may be nutritionally important (Xiao-Ya et al., 2012). The
most important of these bioactive compounds of plants are alkaloids, flavonoids,
tannins, phenolic compounds, steroids, saponins and glycosides (Duraipandiyan et al.,
2006; Yadav and Agarwala, 2011).
The steadily increasing microbial resistance to existing drugs is a serious
problem in antimicrobial therapy and necessitates continuing research into new classes
of antimicrobials (Essawi and Srour, 2000; Woodford, 2003). One way to prevent
antibiotic resistance of pathogenic species is to use new compounds that are not based
on existing synthetic antimicrobial agents (Shah, 2005; Chanda et al., 2011). Plant and
plant derived agents have long history to clinical relevance as a source of potential
General Introduction
5
chemotherapeutic agents (Cushnie and Lamb, 2005). Many studies have been
undertaken with the aim of determining the antimicrobial and phytochemical
constituents of medicinal plants and using them for the treatment of both topical and
systemic microbial infections as possible alternatives to chemical synthetic drugs to
which many infectious microorganisms have become resistant (Chopra, 2007; Kumar
et al., 2011). The antimicrobial activity of plant extracts is due to different chemical
agents in the extract. These compounds are usually the secondary metabolites, which
function to attract beneficial and repel harmful organisms, serve as phytoprotectants and
respond to environmental changes in plants. In humans, however the compounds have
beneficial effects (Johanna, 2003; Maiyo et al., 2010).
Free radicals, antioxidants and their activities
Life on earth survives only due to the presence of oxygen. Oxygen gives us
energy by oxidation of food which is essential for living. During this process highly
reactive and harmful oxygen species are also generated which can cause damage to living
organisms. The family of free radicals generated from oxygen are called Reactive oxygen
species (ROS) which includes hydroxyl radical, superoxide radical and hydrogen
peroxide (Braca et al., 2002). These ROS have an unpaired electron spinning on the
peripheral layer around the nucleus and cause damage to other molecules by extracting
electrons from them in order to attain stability. Reactive oxygen speices are highly
reactive and potentially damaging transient chemical species that are continuously
produced in the human body, as a consequence of endogenous metabolic processes
involving redox enzymes and bioenergetics electron transfer. They are also produced on
exposure to a plethora of exogenous sources such as radiation, air pollution, tobacco
smoke, organic solvents and pesticides present in our ambient environment (Ali et al.,
2008). Reactive oxygen speices can attack lipids and proteins and destroy membranes.
They can cause damage to DNA and lead to mutation and chromosomal changes (Valko
et al., 2006). Oxidative damage of proteins, DNA and lipid is associated with chronic
degenerative diseases including diabetes, hypertension, coronary heart disease, cancer and
atherosclerosis (Lee et al., 2000; Ashish et al., 2010).
Oxidative stress depicts the existence of products called free radicals which
are formed under normal physiological conditions but become deleterious when not
being eliminated by the endogenous systems. Mammalian cells possess elaborate
General Introduction
6
defence mechanism for radical detoxification through the action of dietary
antioxidants (α-tocopherol, β-carotene, ascorbic acid, glutathione and uric acid), some
harmones (estrogen and angiotensin) and endogenous enzymes (superoxide
dismutase, catalase and glutathione peroxidise) (Subhasree et al., 2009). In fact,
oxidative stress results from an imbalance between the generation of ROS and
endogenous antioxidant systems (Kaplan et al., 2007). When the mechanism of
antioxidant protection becomes unbalanced by several internal and external factors,
deterioration of physiological functions may occur requiring the system to depend on
exogenous antioxidants from natural sources. Compounds that can scavenge free
radicals have great potential in ameliorating the disease processes (Behera et al.,
2006; Madhulika and Kumar, 2010).
Antioxidants are the compounds which possess the ability to protect the cell
organelles from damage caused by free radicals induced oxidative stress either by
inhibiting the initiation or propagation of oxidative chain reactions (Martino et al.,
2009; Jain et al., 2011). Antioxidants exhibit their antioxidant activity either by
inhibiting lipid peroxidation, by scavenging free radicals and active oxygen species,
preventing the decomposition of hydrogen peroxides into free radicals or by chelating
heavy metal ions (Ghimeray et al., 2009; Ghaffar and El-Elaimy, 2012).
Effective synthetic antioxidants such as Butylated Hydroxytoluene (BHT) and
Butylated Hydroxyanisole (BHA) have been used for industrial processing. But these
are suspected of being responsible for liver damage and carcinogenesis (Barlow,
1990; Miladi and Damak, 2008). Hence, strong restrictions have been placed on their
application and there is a trend to substitute them with naturally occurring
antioxidants (Rahman et al., 2010). The search for antioxidants from natural sources
has received much attention and efforts have been put into identifying compounds that
can act as suitable antioxidants to replace synthetic ones.
Use of medicinal plants as antioxidants
Medicinal plants are an important source of antioxidants (Rice-Evans, 2004).
Ayurveda, Unani and other traditional medical systems provide substantial lead to find
active and therapeutically useful antioxidant compounds from plants (Sati et al., 2010).
General Introduction
7
The role of medicinal plants in disease prevention or control has been attributed to
antioxidant properties of their constituents (Ivanova et al., 2005). The protective
effect of plant products are due to the presence of several components such as
enzymes, proteins, vitamins (Halliwell, 1996), carotenoids (Edge et al., 1997),
flavonoids (Zhang and Wang, 2002) and other phenolic compounds (Angolo et al.,
2004). Besides playing an important role in physiological systems, antioxidants have
been used in the food industry to prolong the shelf life of foods, especially those rich
in polyunsaturated fats (Chinedu et al., 2011). Therefore, researchers have focused on
natural antioxidants and numerous crude extracts and pure natural compounds have
been recognized to have beneficial effects against free radicals in biological systems
as antioxidants (Demiray et al., 2009; Priya et al., 2011).
Diabetes mellitus and its classification
Diabetes mellitus (DM) is a metabolic disorder resulting from a defect in
insulin secretion, insulin action or both. Insulin deficiency in turn leads to chronic
hyperglycemia with disturbances of carbohydrate, fat and protein metabolism. As the
disease progresses tissue or vascular damage ensues leading to severe diabetic
complications such as retinopathy, nephropathy, cardiovascular complications and
microbial complications. Thus, diabetes covers a wide range of heterogenous diseases
(Bastaki, 2005).
The classification and diagnostic criterion of diabetes was first put forward by
the WHO in 1965. The latest recommendations have been published by the American
diabetes association expert committee (Expert committee on the diagonosis and
classification of Diabetes mellitus, 1997) and by the WHO consultation (World
Health Organisation consultation, 1999). The vast majority of diabetic patients are
classified into one of two broad categories: type 1 diabetes (insulin dependent
diabetes mellitus), which is caused by an absolute deficiency of insulin, and type 2
diabetes (non-insulin dependent diabetes mellitus), which is characterized by the
presence of insulin resistance with an inadequate compensatory increase in insulin
secretion. In addition, women who develop diabetes during their pregnancy are
classified as having gestational diabetes. Finally, there are a variety of uncommon and
diverse types of diabetes which are caused by infections, drugs, endocrinopathies,
pancreatic destruction and genetic defects. These unrelated forms of diabetes are
classified separately.
General Introduction
8
Type 2 diabetes, which accounts for 90-95% of cases encompasses individuals
who have insulin resistance and usually have relative (rather than absolute) insulin
deficiency at least initially, and often throughout their life time and do not need
insulin treatment to survive. The risk of developing this form of diabetes increases
with age, obesity and lack of physical activity. It is often associated with strong
genetic predisposition, more so than is the autoimmune form of type 1 diabetes.
However, the genetics of this form of diabetes is complex and not clearly defined.
Prevalence of diabetes mellitus
The world prevalence of diabetes among adults (aged 20-79 years) was
estimated to be 6.4%, affecting 285 million adults, in 2010, and will increase to 7.7%
and 439 million adults by 2030 (Shaw et al., 2010). Globally, diabetes prevalence is
similar in men and women but it is slightly higher in men <60 years of age and in
women at older ages (International Diabetes Federation, 2006). According to Shaw
et al. (2010), India had 50.8 million diabetic subjects in the year 2010 and this number
would increase to 87 million by the year 2030.
Table R1 : Top 10 countries for number of people aged 20-79 years with diabetes
in 2010 and 2030
Ranking
2010 2030
Country
No. of adults
with diabetes
(millions)
Country
No. of adults
with diabetes
(millions)
1 India 50.8 India 87.0
2 China 43.2 China 62.6
3 U.S.A 26.8 U.S.A. 36.0
4 Russian Federation 9.6 Pakistan 13.8
5 Brazil 7.6 Brazil 12.7
6 Germany 7.5 Indonesia 12.0
7 Pakistan 7.1 Mexico 11.9
8 Japan 7.1 Bangladesh 10.4
9 Indonesia 7.0 Russian Federation 10.3
10 Mexico 6.8 Egypt 8.6
Source: Shaw et al. (2010)
General Introduction
9
Diagnostic criteria for diabetes mellitus
There are three ways to diagnose diabetes mellitus and each, in the absence of
unequivocal hyperglycemia, on a subsequent day, by anyone of the three methods
given below (Expert committee on the diagnosis and classification of diabetes
mellitus, 1997). The use of glycated hemoglobin for the diagnosis of diabetes mellitus
is not recommended at this time.
1. Symptoms of diabetes plus causal plasma glucose concentration ≥ 200 mg/dl
(11.1 mmol/l). Causal is defined as any time of the day without regard to time
since last meal. The classic symptoms of diabetes mellitus include polyuria,
polydipsia and unexplained weight loss.
2. Fasting plasma glucose (FPG) ≥ 126 mg/dl (7.0 mmol/l). Fasting is defined as
no caloric intake for at least 8 h.
3. Post load glucose (2 h) ≥ 200 mg/dl (11.1 mmol/l) during an oral glucose
tolerance test (OGTT). The test should be performed as described by WHO,
using a glucose load containing the equivalent of 75 g anhydrous glucose
dissolved in water. This test is not recommended for routine clinical use.
Complications of diabetes mellitus
Diabetes mellitus is a chronic condition that can lead to complications over a
period of time (Fowler, 2008). These complications include:
Nephropathy (disease of the kidney), which can lead to kidney failure and the
need for dialysis
Retinopathy (disease of the eye), which can lead to blindness
Neuropathy (disease of the nerves), which can lead to, among other things,
ulceration of the foot requiring amputation
Coronary heart disease, which can lead to heart attack
Cerebrovascular disease, which can lead to stroke
Microbial complications
Patients with diabetes are prone to infection with certain microorganisms.
Diabetes is a common predisposing factor for urinary tract infections caused by
Escherichia coli, Proteus sp., Candida sp. and other Gram-negative bacilli. Foot
infections are the most common soft tissue infections in diabetic patients caused by
General Introduction
10
Gram-positive Staphylococcus aureus, Streptococcus and Enterococcus, Gram-negative
E. coli, Enterobacter, Klebsiella, Pseudomonas and Proteus and anaerobes such as
Bacteriods, Clostridium and Peptostreptococcus (Kompoti, 2004). Ring worm or tinea
is a common fungal infection seen in diabetic patients. The fungi, Trichophyton,
Epidermophyton and Microsporum are commonly isolated from infected lesions (John,
2004). Patients with diabetes are prone to oral cavity infections caused by species of
Candida and Aspergillus (Joshi et al., 1999).
Many of these complications show no symptoms at early stage and most can
be prevented or minimized with a combination of regular medical care, regulated diet,
exercise and blood sugar monitoring.
Experimental diabetic animal model
Diabetes can be induced by pharmacological, surgical or genetic
manipulations in several animal species. Most experiments in diabetes are carried out
on rodents, although some studies are still performed on large animals. Streptozotocin
(STZ) and alloxan are by far the most frequently used diabetogenic drugs and this
model has been useful for the study of multiple aspects of the disease. The cytotoxic
action of these diabetogenic agents is mediated by ROS. But both drugs differ in their
mechanism of action (Lei et al., 2005). Streptozotocin enters the pancreatic β cell via
a glucose transporter-GLUT2 and causes alkylation of DNA. Furthermore, STZ
induces activation of poly adenosine diphosphate ribosylation and nitric oxide release.
As a result of STZ action, pancreatic β cells are destroyed by necrosis (Mythili et al.,
2004). Pancreatic β cells have been proposed to be selectively sensitive to STZ
because the enzyme N-acetylglucosamine transferase is expressed at higher levels in
the β cell than in any other cell (Hanover et al., 1999).
Treatment of diabetes mellitus
The main goals of the treatment of hyperglycemia are: 1) Alleviating
symptoms, 2) Minimizing acute complications, 3) Increasing the sense of well being
and quality of life and 4) Eliminating or minimizing chronic complications. Current
therapeutic agents available for type 2 diabetes include sulfonyl ureas and related
compounds, biguanides, α-glucosidase inhibitors and thiazolidinediones. A rational
approach would be to begin with the agents particularly suited to the stage and nature
of the disease, progressing, if necessary, to combination therapy. The treatment of
type 1 diabetes includes the use of exogenous insulin.
General Introduction
11
Use of medicinal plants for the treatment of diabetes mellitus
Management of diabetes without any side effects is still a challenge in the
medical field, as presently available drugs for diabetes have one or more adverse
effects (Bohannon, 2002). Since the existing drugs for the treatment of DM do not
satisfy our need completely, the search for new drugs continues. In recent years,
herbal remedies for the unsolved medical problems have been gaining importance in
the research field. Apart from the currently available therapeutic options, many herbal
medicines have been recommended for the treatment of diabetes. Herbal drugs are
prescribed widely because of their effectiveness, less side effects and relatively low
cost (Venkatesh et al., 2003; Devaki et al., 2011).
Plants have always been an exemplary source of drugs and many of the
currently available drugs have been derived directly or indirectly from them. The
ethnobotanical information reports about 800 plants that may possess antidiabetic
potential (Alarcon-Aguilara et al., 1998). Several such herbs have shown antidiabetic
activity when assessed using presently available experimental techniques (Jafri et al.,
2000). India has a rich history of using various potent herbs and herbal preparations
for treating diabetes. In India, indigenous remedies have been used in the treatment of
diabetes since the time of Charaka and Sushrutha (Grover et al., 2001). There have
been several reviews on the hypoglycemic medicinal plants more particularly use of
Indian botanicals for hypoglycemic activity (Saxena and Vikram, 2004; Sudha et al.,
2011). Many Indian plants have been investigated for their beneficial use in different
types of diabetes and are reported in numerous scientific journals (Joy and Kuttan,
1999; Nagarajan et al., 2005; Verma et al., 2010). To date, over 400 traditional plant
treatments for diabetes have been reported in India (Modak et al., 2007). The use of
medicinal plants in modern medicine suffers from the fact that though hundreds of
plants are used in the world to prevent or to cure diseases, scientific evidence in terms
of modern medicine is lacking in most cases. However, today it is necessary to
provide scientific proof to justify the use of plant as well as its active principles.
Review of Literature
12
Review of Literature
Plants contain a wide range of bioactive compounds such as phenolics,
carbohydrates, flavonoids, terpenoids, carotenoids, saponins, alkaloids, flavours and
fragnances. Plant extracts with these bioactive molecules are widely used in the food,
pharmaceutical and cosmetic industries. Extraction techniques have been widely
investigated to obtain such valuable natural compounds from plants for
commercialization (Wang and Weller, 2006). There is an increasing interest both in
the industry and in scientific research for herbs because of their functional properties,
which exceed many currently available synthetic compounds.
Extensive review of literature has indicated an accumulation of voluminous
literature on functional properties in various spices, vegetables, fruits and herbs. The
present review encompasses an up to-date published literature from various sources
on different aspects of the selected three plants viz., Andrographis paniculata Nees.,
Tinospora cordifolia Miers. and Trigonella foenum-graecum L. However, relevant
earlier works from other plant sources have been copiously cited for critical
evaluation and meaningful conclusion of our results.
Our study aimed at identifying the antimicrobial, antioxidant and antidiabetic
activities of the selected three plants (A. paniculata, T. cordifolia and T. foenum-
graecum) in different solvent systems and to determine the solvent that best extracts
the compounds responsible for these activities. The results from the present study
would aid in further studies on isolation and characterization of these compounds.
Review of Literature
13
Andrographis paniculata Nees.
Andrographis paniculata (Burm. f.) Wall. ex. Nees. is an erect annual herb
which is extremely bitter in taste in all parts of the plant body. The plant is known in
north-eastern India as Maha-tita, which literally means "king of bitters". As an
Ayurvedic herb it is known as Kalmegh or Kalamegha, which means "dark cloud". It
is also known as Bhui-neem, which means "neem of the ground", since the plant,
though being a small annual herb, has a similar strong bitter taste as that of the large
neem tree (Azadirachta indica). The genus Andrographis consists of 28 species
essentially distributed in tropical Asia. Only a few species are medicinal, of which
A. paniculata is the most popular (Mitra and Bal, 1950).
Classification (Pullaiah, 1998)
The taxonomical hierarchy of A. paniculata is as follows:
Kingdom : Plantae
Division : Angiosperma
Class : Dicotyledonae
Order : Personales
Family : Acanthaceae
Genus : Andrographis
Species : Andrographis paniculata Nees.
Vernacular names of Andrographis paniculata
Vernacular names of A. paniculata in different parts of India are given in
Table R2.
Table R2 : Vernacular names of Andrographis paniculata
State Language Vernacular name
Karnataka Kannada Nelaberu
Tamilnadu Tamil Nilavembu
Andhra Pradesh Telugu Nelavemaa
Kerala Malayalam Kiriyattu
Uttar Pradesh Hindi Kirayat
Maharastra Marathi Oli-Kiryata
West Bengal Bengali Kalmegh
Gujarath Gujarathi Kariyatu
Review of Literature
14
Botanical description
Andrographis paniculata is an annual, branched, erect herb of 30-110 cm
height (Fig. R1). The stem is dark green, 2-6 mm in diameter, quadrangular with
longitudinal furrows and wings at angles of the younger parts, slightly enlarged at the
nodes. Leaves are lanceolate measuring up to 8 cm long by 2.5 cm wide, acute,
glabrous, slightly undulated, pale beneath with base tapering. Flowers are small,
solitary and borne in spreading racemes. The fruit is a capsule and contain numerous
yellow-brown seeds (Gamble, 1935; Gogte, 2000).
Fig. R1 : Andrographis paniculata plant
Origin and distribution
Native populations of A. paniculata are spread throughout south India and Sri
Lanka which perhaps represent the centre of origin and diversity of the species. It is
distributed in tropical Asian countries, often in isolated patches. It can be found in a
variety of habitats, such as plains, hillsides and coastlines. The species also occurs in
Hong Kong, Thailand, Brunei, Singapore, and other parts of Asia where it may or may
not be native. The herb is an introduced species in northern parts of India, Java,
Malaysia, Indonesia, West Indies and America (Kirtikar and Basu, 1984; Gogte, 2000).
Uses of Andrographis paniculata in traditional system of medicine
Andrographis paniculata has traditionally been used over centuries in Asia as
a folklore medicine for a variety of ailments or as herbal supplements for health
promotion. Indian pharmacopoeia narrates that it is a predominant constituent of
Review of Literature
15
at least 26 Ayurvedic formulations. According to Ayurveda, plant is bitter, acrid,
cooling, laxative, vulnerary, antipyretic, antiperiodic, antiinflammatory, expectorant,
depurative, soporific, antihelmintic, digestive and useful in hyperdispsia, burning
sensation, wounds, ulcers, chronic fever, malarial and intermittent fevers,
inflammations, cough, bronchitis, skin diseases, leprosy, colic, flatulence, diarrhoea,
dysentery, diabetes and hemorrhoids. In Unani system, green leaves are used as a
tonic and alternative in syphilitic cachexia and foul syphilitic ulcers (Kirtikar and
Basu, 1984; Council of Scientific and Industrial Research, 1985; Kapoor, 1990). In
traditional Chinese medicine, it is used to treat fever and to dispel toxins from the
body. In Scandinavian countries, it is commonly used to prevent and treat common
colds (Mishra et al., 2007).
Current status
A number of diterpenoids and diterpenoid glycosides of similar carbon
skeleton have been isolated from A. paniculata and the bitterest compounds among
them are andrographolide, neoandrographolide and deoxyandrograpolide. Other such
phytochemicals amassed by the plant are 14-deoxyandrographolide, 14-deoxy-11, 12-
didehydroandrographolide, andrographiside, deoxyandrographiside, andrographan,
andrographon and stigmasterol (Siripong et al., 1992). The leaves of A. paniculata
contain most medicinally active phytochemical in the plant, while the seeds contain
the lowest (Sharma et al., 1992). Extensive review of literature on A. paniculata has
revealed a wide range of pharmacological activities. Diterpenoids and flavonoids are
the main chemical constituents responsible for most of the biological activities of this
plant (Tang and Eisenbrand, 1992). Some of the functional activities accounted for
A. paniculata are as follows:
Anti-human immunodeficiency virus (HIV) activity
Chang and Yeung (1988) have shown the inhibition ability of aqueous extract of
the leaves against HIV-1 infection and replication in the lymphoid cell line MOLT-4.
Immunostimulatory activity
Intragastric administration of ethanol extract of aerial parts (25 mg/kg b.w.) or
purified andrographolides (1 mg/kg b.w.) in mice stimulated antibody production and
delayed hypersensitivity response to sheep red blood cells. The extract was more
Review of Literature
16
effective than either andrographolide or neoandrographolide alone, suggesting that
other constituents may be involved in the immunostimulant response (Puri et al.,
1993).
Antipyretic activity
Intragastric administration of ethanol extract of aerial parts (500 mg/kg b.w.)
to rats’ decreased yeast induced pyrexia. The extract was reported to be as effective as
200 mg/kg b.w. asprin (Vedavathy and Rao, 1991).
Antidiarrhoel activity
Ethanol, chloroform or butanol extract of the aerial parts inhibited E. coli
enterotoxin induced secretory response in rabbit and guinea ileal loop assay (Gupta
et al., 1990; Gupta et al., 1993).
Antiinflammatory activity
Intragastric administration of purified andrographolides to rats inhibited
inflammatory responses by rat neutrophils (Shen et al., 2002).
Antimalarial activity
A 50% ethanol extract of the aerial parts inhibited the growth of Plasmodium
berghei both in in vitro (100 mg/ml) and in mice (1g/kg b.w.) (Mishra et al., 1992).
Antimicrobial activity
Methanol extract of A. paniculata has shown antibacterial activity against
bacteria causing skin diseases (Sule et al., 2010). Extracts of aerial parts have shown
the antifungal activity against field fungi (Wanchaitanawong et al., 2005).
Antivenom activity
Intraperitoneal injection of ethanol extract of the aerial parts (25 g/kg b.w.) to
mice poisoned with cobra venom markedly delayed the occurrence of respiratory
failure and death (Chang and But, 1987).
Review of Literature
17
Antihepatotoxic activity
The methanol extract of aerial parts and their constituent andrographolides
have shown antihepatotoxic activity both in in vitro and in vivo models (Kapil, 1993).
Table R3 : Consolidated report of important functional properties of Andrographis
paniculata
Sl. No. Functional Properties References
1 Antiinflammatory activity Amroyan et al. (1999)
2 Antidiarrhoel activity Yin and Guo (1993)
3 Antiviral activity Wiart et al. (2005)
4 Hepatoprotective activity Trivedi and Rawal (2005)
5 Anticancer activity Zhou et al. (2006)
6 Immune stimulatory activity Iruretagoyena et al. (2005)
7 Antisnake bite activity Samy et al. (2008)
8 Antibacterial activity Prajjal et al. (2003)
9 Antimalarial activity Rahman et al. (1999); Dua et al. (2004)
10 Antifungal activity Mathur et al. (2011)
11 Antithrombotic activity Zhao and Fang (1991)
12 Antihyperglycemic activity Zhang and Tan (2000b)
13 Cardioprotective activity Guo et al. (1994); Ojha et al. (2009)
Due to various functional properties of A. paniculata, the plant was selected
for the present study. The earlier studies on antimicrobial, antioxidant and antidiabetic
activities of this plant have relied on the extracts of the whole plant. None of the
earlier studies have shown the antidermatophytic activity of A. paniculata leaf
extracts. Moreover, there are no studies which show the antimicrobial, antioxidant and
antidiabetic activities of A. paniculata leaves both in vitro and in vivo in a single
solvent system. For the reasons elaborated above, we were keen to study the activity
of its leaves.
Review of Literature
18
Tinospora cordifolia Miers.
Tinospora cordifolia (Thunb.) Miers commonly known as Amrita (Guduchi)
is a widely used plant in folk and Ayurvedic systems of medicine. In Hindi, the plant
is commonly known as ‘Giloya’ which is a Hindu mythological term that refers to the
heavenly elixir which has saved celestial beings from old age and kept them eternally
young. ‘Guduchi’, the Sanskrit name means one which protects the entire body. The
term ‘Amrita’ is attributed to its ability to impart youthfulness, vitality and longevity
(Bhandari, 2006).
Classification (Pullaiah, 1998)
The taxonomical hierarchy of T. cordifolia is as follows:
Kingdom : Plantae
Division : Angiosperma
Class : Dicotyledonae
Order : Ranunculales
Family : Menispermaceae
Genus : Tinospora
Species : Tinospora cordifolia Miers.
Vernacular names of Tinospora cordifolia
Vernacular names of T. cordifolia in different parts of India are given in Table R4.
Table R4 : Vernacular names of Tinospora cordifolia
State Language Vernacular name
Karnataka Kannada Amrita Balli
Tamilnadu Tamil Shindilakodi
Andhra Pradesh Telugu Tippaatigo
Kerala Malayalam Ambrithu
Uttar Pradesh Hindi Giloya, Amrita
Maharastra Marathi Gulavel
West Bengal Bengali Gulancha
Gujarath Gujarathi Galo
Review of Literature
19
Botanical description
Tinospora cordifolia is a large, deciduous, extensively spreading and climbing
shrub with several elongated twining branches. Stems are fleshy with thin, greyish or
creamy white coloured bark and with long, filiform and fleshy aerial roots from the
branches. Leaves are simple, alternate, ovate or ovate chordate which is 10-20 cm
long and 8-15 cm broad. The flowers are unisexual, small on separate plants and
greenish yellow. In axillary and terminal racemes or racemose panicles, the male
flowers are clustered and female flowers are usually solitary. The drupes are ovoid,
glossy, succulent, red, pea sized and occur in winter (Khosa and Prasad, 1971;
Chopra, 1994).
Fig. R2 : Tinospora cordifolia plant
Origin and distribution
Tinospora cordifolia is indigenous to the tropical areas of India, Myanmar and
Sri Lanka ascending to an altitude of 1200 m. It is a fairly common plant of deciduous
and dry forests, growing over hedges and small trees (Kirti et al., 2004; Bhandari,
2006).
Uses of Tinospora cordifolia in traditional system of medicine
Guduchi is mentioned in various texts of Ayurvedic systems of medicine, viz.,
Charaka (Charaka, 1961), Sushruta (Sushruta, 1992) and other treaties like Bhava
Prakash (Mishra, 1969) and Dhanvantari Nighantu (Aiyer and Kolammal, 1963). In
Ayurveda, its roots, leaves and stem are claimed to be useful in treating leprosy,
Review of Literature
20
jaundice, diabetes, dysentery, chronic diarrhoea, skin infections and bleeding piles
(Singh et al., 2003; Chunekar and Pandey, 2006). It is reported to be a potent tonic for
promoting longevity and works as an antiperiodic, astringent and diuretic. In
Ayurvedic literature, it is used as a constituent of several compound formulations
such as churna, taila and sattva (Sharma et al., 2001). In Unani system, it is used as a
febrifuge and general tonic (Thakur et al., 1989). Tinospora cordifolia finds a special
mention in tribal or folk medicine in different parts of the country in the treatment of
fever, jaundice, chronic diarrhoea, general debility, asthma and skin disease (Singh
and Maheshwari, 1983; Singh et al., 2003).
Current status
A number of chemical constituents belonging to different groups, viz.,
terpenoids like tinosporide (Khuda et al., 1964), tinosporaside (Hanuman et al.,
1986), furanolactone diterpene (Khan et al., 1989), alkaloids like tinosporine (Khuda
et al., 1964), berbrine (Pachey and Schneidir, 1981) and steroids like β sitosterol
(Khaleque et al., 1970) have been isolated from T. cordifolia stem and roots. Some of
the functional properties attributed to T. cordifolia are as follows:
Anticancer activity
Administration of intraperitoneal injection T. cordifolia stem methanol extract
to BALB/c mice (200 mg/kg b.w.) for five days increased the total WBC count
significantly. It reduced solid tumor growth and synergistically acted with
cyclophosphamide in reducing the animal tumors (Mathew and Kuttan, 1999).
Antidiabetic and antihyperglycemic activity
Alcoholic extracts of the stem at a concentration of 50, 100 and 200 mg/kg
b.w. caused a reduction in the fasting blood sugar in the alloxan induced diabetic
rabbits (Wadood et al., 1992). Daily oral feeding of T. cordifolia extracts for 40 days
in streptozotocin diabetic mice caused a reduction in blood glucose concentration and
also prevented polyurea (Grover et al., 2002).
Antiinflammatory activity
The stem extract of T. cordifolia showed antiinflammatory activity on
carrageenin induced hind paw edema in rats (Sharma and Singh, 1980).
Review of Literature
21
Antioxidant activity
Alcoholic extract of T. cordifolia roots administered at a dose of 100 mg/kg
b.w. orally to diabetic rats for six weeks normalized the antioxidant status of liver and
kidney (Prince et al., 2004a).
Hypolipidaemic activity
Administration of aqueous extract of roots of T. cordifolia at a concentration of
5 g/kg b.w. for six weeks resulted in a significant reduction in serum and tissue cholesterol
phospholipids and free fatty acids in alloxan diabetic rats (Stanely et al., 1999).
Immunomodulatory activity
The water and ethanol stem extracts of T. cordifolia inhibited immunosuppression
produced by cyclophosphamide (Manjreker et al., 2000).
Hepatoprotective activity
The hepatoprotective activity of T. cordifolia extract has been demonstrated in CCl4
induced liver damage in rats. The extract was found to normalize the liver function as
assessed by morphological, biochemical and functional tests (Bishayi et al., 2002).
Table R5 : Consolidated report of important functional properties of Tinospora
cordifolia
Sl. No. Functional Properties References
1 Antistress activity Patil et al. (1997)
2 Antiinflammatory activity Wesley et al. (2008)
3 Antiallergic activity Badar et al. (2005)
4 Antioxidant activity Singh et al. (2006)
5 Antineoplastic activity Jagetia and Rao (2006)
6 Antiinfective activity Jeyachandran et al. (2003)
7 Antipyretic activity Vedavathy and Rao (1991)
8 Hepatoprotective activity Adhvaryu et al. (2008)
9 Antihyperglycemic activity Grover et al. (2002a)
10 Immunomodulatory activity Ranjith et al. (2008)
11 Diuretic activity Nagaraja et al. (2007)
12 Cardioprotective activity Nagaraja et al. (2008)
13 Antiulcer activity Bafna and Balaraman (2005)
14 Antifertility activity Gupta and Sharma (2003)
15 Osteoprotective activity Kapur et al. (2008)
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22
Tinospora cordifolia was selected for our study due to its various functional
properties as listed above and also due to its wide usage in Ayurvedic formulations.
In vivo and in vitro research investigations on T. cordifolia have shown the use of its
stem and roots and not the leaves. Not many studies have been carried out to show the
antimicrobial, antioxidant and antidiabetic activities of its leaves. Antifungal activity
studies against dermatophytes have not been proved till now even though its activity
against the field fungi have been carried out. Moreover, in this study we are trying to
select a suitable solvent extract of leaves which has antimicrobial, antioxidant and
antidiabetic activity. All these facts have prompted us to select only the leaves of
T. cordifolia for our study.
Review of Literature
23
Trigonella foenum-graecum L.
Trigonella or fenugreek or Greek Hayes is cultivated as a leafy vegetable,
condiment and as a medicinal plant. The seeds are used as spices worldwide, whereas
the leaves are used as green leafy vegetables in the diet. Fenugreek has been in use for
over 2500 years. India is the major producer of fenugreek and its main consumer for
culinary and medicinal uses (Srinivasan, 2006).
Classification (Pullaiah, 1998)
The taxonomical hierarchy of T. foenum-graecum is as follows:
Kingdom : Plantae
Division : Angiosperma
Class : Dicotyledonae
Order : Fabales
Family : Fabaceae
Genus : Trigonella
Species : Trigonella foenum-graecum L.
Vernacular names of Trigonella foenum-graecum
Vernacular names of T. foenum-graecum in different parts of India are given
in Table R6.
Table R6 : Vernacular names of Trigonella foenum-graecum
State Language Vernacular name
Karnataka Kannada Menthya
Tamilnadu Tamil Venthiyam
Andhra Pradesh Telugu Mentulu
Kerala Malayalam Uluva
Uttar Pradesh Hindi Methi
Maharastra Marathi Methi
West Bengal Bengali Methi
Gujarath Gujarathi Methi
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24
Botanical description
Trigonella foenum-graecum is an annual and aromatic herb up to 60 cm tall.
Stem is solitary or basally branched and green to purple. Leaves are petiolate,
alternate and trifoliolate. Flowers are axillary, subsessile and whitish to pale yellow in
colour. Its pods are curved with narrow ends. These pods contain 10-12 seeds which
are lozenge-shaped with brown or yellowish in colour (Kirtikar and Basu, 1991; The
Ayurvedic Pharmacopoeia of India, 2000).
Fig. R3 : Trigonella foenum-graecum plant
Origin and distribution
Fenugreek is a native of South Eastern Europe and West Asia, now cultivated
in India, Argentina, Egypt and Mediterranean countries. India is the largest producer
of fenugreek in the world where Rajasthan, Gujarat, Uttaranchal, Uttar Pradesh,
Madhya Pradesh, Maharashtra, Haryana and Punjab are the major fenugreek
producing states (Rajagopalan, 2001).
Uses of Trigonella foenum-graecum in traditional system of medicine
A wide range of uses are described in Ayurveda for methi. The seeds and the
leaves are with a bitter taste and used medicinally as antipyretic, antihelminthic, to
increase the appetite, as astringent to the bowels, to cure leprosy, vomiting, bronchitis
and piles, to remove bad taste from the mouth and useful in treating heart diseases and
diabetes (The Ayurvedic Pharmacopoeia of India, 2000). In Unani system of
medicine, the leaves and the seeds are used as diuretic, emmenagogue, useful in
Review of Literature
25
dropsy, chronic cough, enlargement of the liver and the spleen, external and internal
swellings and burns and to prevent the hair fall (Prajapati et al., 2004). In traditional
Chinese medicine, it is also used for kidney problems and conditions affecting the
male reproductive tract (Toppo et al., 2009).
Current status
The use of fenugreek seeds and leaves are diverse. Fenugreek seeds are a good
source of proteins, fats, minerals and dietary fibers. Fresh and dried leaves provide a
good amount of various minerals and vitamins and they are especially rich in choline
(Srinivasan, 2005). The notable chemical constituents of T. foenum-graecum are
proteins rich in lysine and tryptophan, fibers, flavonoids, saponins, polyphenols,
alkaloid trigonelline and phytic acid (Billaud and Adrain, 2001). Some of the
functional properties attributed to T. foenum-graecum are as follows:
Antihyperglycemic activity
Ethanol extract of T. foenum-graecum seeds at a concentration of 1 g/kg b.w.
showed significant activity against diabetic state induced by alloxan in rats (Mowla
et al., 2009).
Hypocholesterolemic activity
Dietary fenugreek countered the elevation of lipids and lipogenic enzymes in
liver and kidneys (Yadav et al., 2004).
Antioxidant activity
Enhanced lipid peroxidation associated with the depletion of antioxidants in
liver, kidney and pancreas in diabetic rats were normalized on supplementation of
fenugreek seed powder in the diet for 30 days at a dosage of 2 g/kg b.w. (Anuradha
and Ravikumar, 2001).
Gastroprotective activity
An aqueous extract and a gel fraction of fenugreek seeds were shown to
protect the gastric mucosa more efficiently than omeprazole in rats from HCl-ethanol
induced gastric ulcers (Pandian et al., 2002).
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26
Immunomodulatory activity
Aqueous extract (50, 100 and 250 mg/kg b.w.) of fenugreek seeds were
evaluated in male Swiss albino mice for 10 days. Fenugreek showed stimulatory
effect on immune functions as indicated by cellularity of lymphoid organs, delayed
type of hypersensitivity response, phagocytosis and lymph proliferation (Bin-Hafeez
et al., 2003).
Influence on digestion
Dietary intake of fenugreek significantly increased pancreatic lipase activity
by 43% in experimental rats. There was also an increase in chymotrypsin activity in
animals fed with fenugreek (Platel and Srinivasan, 2000).
Table R7 : Consolidated report of important functional properties of Trigonella
foenum-graecum
Sl. No. Functional Properties References
1 Antihyperglycemic activity Vats et al. (2002); Kumar et al. (2005)
2 Hypocholesterolemic activity Annida et al. (2004)
3 Antioxidant activity Genet et al. (2002)
4 Gastroprotective activity Thirunavukkarasu et al. (2003)
5 Immunomodulatory activity Hamden et al. (2010)
6 Influence on digestion Rao et al. (2003)
7 Antihyperthyroidic activity Tahiliani and Kar (2003)
8 Antibacterial activity Sharma and Patel (2009)
9 Wound healing activity Alam et al. (2011)
10 Estrogenic activity Sreeja et al. (2010)
11 Chemoprotective activity Amin et al. (2005)
A systematic review of literature has revealed various functional activities of
T. foenum-graecum. Though, both seeds and leaves of Trigonella are used extensively
in Ayurveda and also in other traditional system of medicines for treating various
ailments, most of the studies (in vivo and in vitro) carried out till now are with that of
the seeds and not the leaves. Studies showing the antimicrobial, antioxidant and
antidiabetic activities of its leaves in different solvent systems have not been carried
out. As the leaves of Trigonella are extensively used as a green vegetable in India, our
study has extreme relevance regarding its activities.
Scope and Objectives of the Present Investigation
Don’t be afraid to take a big step when one is indicated. You can’t cross a chasm in two small jumps
- David Lloyd George
Scope and Objectives
27
Scope and Objectives of the present investigation
Andrographis paniculata, Tinospora cordifolia and Trigonella foenum-graecum
are the plants profusely used in Ayurveda and other traditional system of medicines to
cure both infectious and degenerative diseases. Lack of earlier reports on the use of
leaves of these plants showing antimicrobial, antioxidant and antidiabetic activities in
different solvent systems offered sufficient scope to undertake this research work.
Selection of a single suitable solvent which shows all these activities is of commercial
importance and is critical for the development of products of health benefits. In vitro
study is the initial stage of any study and in vivo study involving experimental animals
is the next stage by which one can be sure of proving the activity of the plant extracts.
This natural scientific inquisitiveness was ardently attended by undertaking the in vivo
investigation of the plant extracts in animal models.
The present investigation was carried out with the leaves of these plants to
investigate the antimicrobial, antioxidant and antidiabetic activities with the following
objectives:
To evaluate the in vitro antimicrobial activities of the extracts
To evaluate the in vitro antioxidant activities of the extracts
To study the in vivo antidiabetic activities of the selected extract on
rats
The results of scientific pursuit carried out in this regard have been presented
in three chapters for the convenience of reading:
1. In vitro antimicrobial activity studies
2. In vitro antioxidant activity studies
3. In vivo antidiabetic activity studies
Reprints of publications are furnished after references.
