7

2. REVIEW OF LITERATURE

Ethno pharmacology is a highly diversified approach to drug discovery

involving the observation, description, and experimental investigation of

indigenous drugs and their biological reactions. It is based on botany, chemistry,

biochemistry, pharmacology and many other disciplines that contribute to the

discovery of natural products with biological activity.

Plants are considered to be medicinal if they possess pharmacological

activities of possible therapeutic use. Indian Materia Medica includes about 2000

drugs of natural origin almost all of which are derived from different traditional

systems. These activities are often recognized as a result of millennia of trial and error

but they have to be carefully investigated if new drugs are to be developed for use in

modern treatment.

2.1. MEDICINAL PLANTS SCENARIO IN INDIA:

The rich biodiversity of Indian subcontinent contributes to the wealth of

medicinal plants, which are very much used in traditional medical treatments

(Chopra et al., 1956). India is one of the 12 mega biodiversity centers with over

18,000 plant species. Over 2,500 species are formally recognized as having true

medicinal value. About 7500 plants have been used in local health traditions in rural

and tribal villages of India. Out of these, the medicinal efficacy of 4000 plants is

either little known or unknown to the mainstream population (Pushpangadan, 1996).

8

About 1500 plants with medicinal uses are mentioned in ancient texts such as

Ayurveda, Siddha, Unani and Tibetan medicine (Table: 1). In fact it is the

responsibility of the scientific community to do scientific validation of the medicinal

property of the traditional claims.

TABLE 1: Medicinal plants used in various codified systems of Medicine in India

In recent years, the growing demand for herbal product has led to a quantum

jump in volume of plant materials traded within and across various countries in the

world. Though India has a rich biodiversity, the growing demand is putting a heavy

strain on the existing resources. While the demand for medicinal plants is growing,

some of them are increasingly being threatened in their natural habitat. According to

an all India ethno biological survey carried out by the Ministry of Environment &

S.NO MEDICAL SYSTEM NO.OF PLANTS USED

1 AYURVEDA 1587

2 SIDDHA 1128

3 UNANI 503

4 SOWA-RIGPA 253

5 HOMOEOPATHY 468

6 WESTERN 192

9

Forests, Government of India, there are over 8000 species of plants being used by the

people of India for diseases like breast cancer (12%), liver disease (21%), human

immunodeficiency virus (22%), asthma (24%) and rheumatological disorders (26%).

Some of these plants are commonly used by people as folk medicines for hundreds of

years.

Over 17,500 species of higher plants occur in India, of which approximately

9,000 plants are known to be economically useful. Of these, 7,500 species are used for

healthcare by various ethnical communities in India (Arora, 1997). However various

traditional communities in India are using around 800 plants for curing different

ailments (Kamboj, 2000). India is the world’s largest supplier of raw materials. India

is also known to be exporting a number of medicinal plants. Some of the important

medicinal plants with high trade value are Cuscuta epithymum, Glycyrrhiza glabra,

Lavendula stoechas, Operculina turpethum, Pimpinella anisum, Smilax china, Smilax

ornata and Thymus vulgaris. The annual exports of India’s herbal sector add up to Rs.

807 Crores for the year 2004-05 (Ved and Goraya, 2007). This includes exports worth

Rs. 354.80 Crores related to plant raw drugs, Rs. 161 Crores related to plant extracts

and Rs.291 Crores related to medicants of Ayurvedic, Unani, Siddha and

Homoeopathic systems. It shows that the finished herbal products constitute nearly

36% of the total exports of India’s herbal sector and the balance 64% exports are in

the form of raw materials and extracts.

Three of the widely selling herbal medicines in developed countries, namely

10

Allium sativum, Aloe barbadensis and Panax species are also available in India. India

is the largest grower of Psyllium (Plantago ovata) and Senna (Cassia senna) plants

and one of the largest growers of Castor (Ricinus communis) plant. The plants

Glycyrrhiza glabra, Piper longum, Adhatoda vasica, Withania somnifera, Cyperus

rotundus, Tinospora cordifolia, Berberis aristata, Tribulus terristris, Holarrhena

antidysenterica and Boerhaavia diffusa have been used in 52 to 141 herbal

formulations and triphala (Terminalia chebula, Terminalia bellerica and Emblica

officinalis) alone have been used in 219 formulations (BCIL, 1996).

The turnover of herbal medicines in India as counter products, ethnical and

classical formulations and home remedies of Ayurveda, Unani and Siddha systems of

medicine is about $1 billion with a meager export of about $80 million. Psyllium

seeds and husk, castor oil and opium extract alone account for 60% of the exports.

80% of the exports to developed countries are of crude drugs. However, only a small

proportion of plant species have been thoroughly investigated for their medicinal

properties (Frame et al., 1998) and undoubtedly there are many more novel

biologically active compounds to be discovered.

2.2. Drugs isolated from traditional Medicinal Plants

Natural products derived from medicinal plants play an important role as useful

tools in pharmacological studies. With the technological advancement of

science the isolation, identification and elucidation of chemical principles from

11

natural sources have become much simpler and has contributed significantly to the

development of new drugs from medicinal plants.

There are hundreds of significant drugs and biologically active compounds

developed from the traditional medicinal plants, a few of which are mentioned here;

the antispasmodic agent vasicin isolated from Adhatoda vasica, anticancer agents

such as vincristine, vinblastine and D- Tubocurarine isolated from Catharanthus

roseus (Gurib-Fakim, 2006), antibacterial agents isolated from Diospyros

melanoxylon (Mallavadhani et al, 1998), antimalarial agent isolated from Sida acuta

(Karou et al, 2006), steroid and lancamarone with cardiotonic properties, lantamine

with antipyretic and antispasmodic properties from Lantana camara (Ghisalberti,

2000), antimicrobial agents isolated from Acorus calamus (Chowdhury et al, 1993),

antiviral, antibacterial and anti-inflammatory agents isolated from Urtica dioica

(Harborne and Buxter, 1993), anticancer agents isolated from Andrographis

paniculata, Phyllanthus amarus, Piper longum, Semecarpus anacardium, Withania

somnifera, Moringa oleifera, Aloe vera, Curcuma longa, Allium sativum and

Tinospora cordifolia (Balachandran and Govindarajan, 2005), promising and potent

antimalarial drug artemisinin isolated from Artemesia annua (Dhingra et al, 2000).

Fabricant and Farnsworth (2001) presented 86 important drugs developed from active

compounds and their clinical use, few are listed (Table 2).

12

Table: 2 Drugs derived from plants with their ethno medical correlations and sources

Drug Action or Clinical use Plant source

Acetyldigoxin Cardiotonic Digitalis lanata

Aescin Anti-inflammatory Aescuus hippocastanum

Arecoline Anthelmintic Areca catechu

Aesculetin Anti-dysentery Fraxinus rhynchophylla

Ajamalicine Circulatory disorders Rauvolfia serpentiana

Allyl isothiocyanate Rubefacient Brassica nigra

Andrographolide Bacillary dysentery Andrographis paniculata

Anisodarnine Anticholinergic Anisodus tanguticus

Anisodine Anticholinergic Anisodus tanguticus

Asiaticoside Vulnerary Centella asiatica

Atropine Anticholinergic Atropa belladonna

Berberine Bacillary dysentery Berberis vulgaris

Bergenin Antitussive Ardisia japonica

Bromelain Anti-inflammatory Ananas comosus

Caffeine CNS stimulant Camellia sinensis

(+)-Catechin Haemostatic Potentilla fragaroides

Chymopapain Proteolytic Carica papaya

Cocaine Local anaesthetic Erythroxylum coca

13

Codeine Analgesic; antitussive Papaver somniferum

Colchicine Antitumor agent Colchicum autumnale

Convallotoxin Cardiotonic Convallaria majalis

Curcumin Choleretic Curcuma longa

Cynarin Choleretic Cynara scolymus

Danthron Laxative Cassia Sps.

Deserpidine Antihypertensive Rauvolfia canesceas

Deslanoside Cardiotonic Digitalis lanata

Digitalin Cardiotonic Digitalis purpurea

Digitoxin Cardiotonic Digitalis purpurea

Digoxin Cardiotonic Digitalis lanata

Emetine Amoebicide; emetic Cephaelis ipecacuanha

Ephedrine Sympathetomimetic Ephedra sinica

Etoposide Antitumour agent Podophyllum peltatom

Gitalin Cardiotonic Digitalis purpurea

Glaucaroubin Amoebicide Simarouba glauca

Glycyrrhizin Sweetener Glycyrrhiza glabra

Gossypol Male contraceptive Gossypium Sps.

14

2.3. Traditional uses of different species of Cassia

Cassia species have been of medical interest due to their good therapeutic

value in folk medicine. Cassia genus consists of more than 600 species throughout the

world. Some important species are Cassia fistula, Cassia grandis, Cassia hirsutica,

Cassia sieberiena, Cassia alata, Cassia tora, Cassia occidentalis, C. auriculata, C.

nigricans.

The anti-inflammatory and hepatoprotective activity of Cassia occidentalis, C.

fistula and C. sophera are also reported1 (Jafri et al., 1999; Burkill H.M, 1997). C.

sophora, C. italica, C. pumila are reported to have CNS depressant, anxiolytic and

hypnotic activity (Jain, 1996). The seeds of Cassia tora have good binding and

suspending property and are used as a substitute for coffee. Cassia mimosoides is

reported to have antiobesity activity (Caceres, 1991). Cassia tora, C. auriculata, C.

fistula, C. alata were reported to have anti-diabetic and antioxidant activity (Juvekar,

2006). The seeds of Cassia tora have good binding and suspending property and also

used as a substitute for coffee.

Among the species, Cassia auriculata L. (Leguminaceae) plant known for

curing diabetes, rheumatism, fever, leprosy, skin diseases, conjunctivitis, constipation

etc. has been chosen for detailed investigation of some pharmacological parameters

and toxicity studies. In the present study an attempt has been made to screen the

leaves of Cassia auriculata and to asses various pharmacological activity and toxicity

15

of the isolated active molecules.

On the basis of the use of Cassia auriculata by the traditional healers all over

the world, research has been carried out in various pharmacological studies of this

plant such as toxicity, anti-diabetic, anti-inflammatory and antipyretic activities.

2.4. Cassia auriculata L.

Figure 1: Cassia auriculata L.

Cassia auriculata L. belonging to Leguminosae family is native of India and

Sri Lanka. It is commonly known as tangedu in Telugu and tanner’s cassia & avaram

senna in English in India. It is fast growing branched evergreen shrub with reddish

brown branches and vivid yellow flowers (Figure 1). It mainly occurs in the dry

regions of India and Sri Lanka. It is common along the sea coast and the dry zone in

Sri Lanka. The leaves are alternate, stipulate, paripinnately compound, slender,

16

pubescent, 2-2.5cms long and 1-1.3cms broad. Its flowers are irregular, bisexual,

bright yellow and large (nearly 5cms across), the pedicels glabrous and 2.5cms long

the fruit is a short legume, 7.5-11cms long, 1.5cms broad, flat, thin, pale brown. 12-20

seeds per fruit are carried each in its separate cavity. This plant is said to contain

cardiac glycoside (sennapicrin) and sap, leaves and bark yield anthraquinones, while

the latter contains tannins.

Cassia auriculata has been widely used in traditional medicine. Its leaves,

flowers, roots and pods are employed in herbal medicine in and around the world. The

leaves have anthelmintic, anti-diabetic, laxative properties (Malhotra and Mishra,

1982). The root is used in decoctions against fevers, diseases of urinary system,

constipation, rheumatism, skin diseases, leprosy, conjunctivitis and diabetes (Joshi,

2000). The dried flowers are used as a substitute for tea in case of diabetes patients

and its leaves are also used for anti-diabetic, antioxidant (Juvekar and Halade, 2006;

Sawhney et al., 1978). Seeds are used in ophthalmia and dysentery (Kirtikar and

Basu, 1980; Vaidya 1998).

2.5. Toxicity

The active principles of medicinal plants may sometimes have deleterious

toxic effect on the physiological functioning of organisms. Hence there is a need for

scientific study of acute and chronic toxicity. Seventy five medicinal plants

of the traditional Ayurvedic pharmacopeia of Sri Lanka have been screened

chemically for alkaloids and pyrrolizidine alkaloids. Of these, Crotolaria juncea L.

17

was found to contain pyrrolizidine alkaloids with biological effects consistent with

pyrrolizidine alkaloid toxicity. Mattei et al. (1998) reported the toxic effects and

behavioral level of Paullinia cupana in rats and mice and showed that, percentage

mortality was equivalent in control and treated groups. Similarly Hellion-lbarrola et al.

(1999) studied the acute toxicity and general pharmacological activities of the crude

hydroalcoholic rhizome extract of Kyllinga brevifolia. The LD50 was found to be 575

mg/kg.

Muko and Ohiri (2000) studied the toxicity by oral administration of aqueous

extracts of Emilia sonchifolia in rats arid concluded that the acute toxicity showed

LD50 of 860 mg/kg and 780 mg/kg for methanol and aqueous extracts respectively.

Rezaeipoor et al. (2000) reported the effect of Isatis cappadocica on humoral immune

response in mice. In particular, the dose of 0.25 g/kg suppressed the primary immune

response, while the dose of 0.5 g/kg stimulated the secondary immune response.

Monteiro (2001) evaluated the toxicity levels of Vernonia condensate (Asteraceae) at

a dose of 5000 mg/kg. The results suggested that V. condensate aqueous extracts

contained low acute toxicity and possed neither teratogenic nor mutagenic risks.

Marcelo et al. (2002) investigated the acute toxicity of

Stryphnodendron adstringen, after oral administration to mice, and its effect on certain

biochemical parameters were assessed in plasma of rats after 30 days of

administration. The results showed that the extract administered in a prolonged period

18

produced toxic effects in the experimental animals. In another study, the total

alcoholic, total aqueous, and methanolic extracts isolated from the leaves of A.

marmelos were studied for their toxic effects. There were no remarkable changes

noticed in the histopathological studies after 50 mg/kg body weight of the

extracts of A. marmelos when administered intraperitoneally for 14 days

successively (Veerappan et al., 2007).

Toxicity studies were carried out for six plants used in the traditional Arab system

of medicine, namely: Aloe vera, Ammi majus, Areca catechu, Citrullus colocynthis,

Cuminum cyminum and Zizyphus spina-christi. In the acute toxicity test, C. colocynthis

showed a dose dependent toxic effect. On chronic treatment, per cent lethality was

found to be significant in A. catechu and C. colocynthis treated groups where

haematological changes were also observed (Shah, 2003).

The cytotoxicity of aqueous extracts (AE) and hydroalcoholic extracts (HAE) of

Phyllanthus amarus on Caco-2 cells were evaluated using neutral red uptake and

MTT test. A single oral dose of the extracts at 5 g/kg b.wt did not produce mortality

or any significant change in treated animals over a 14 day observation period (Lawson

et al., 2008). Various concentrations of aqueous extract of Phyllanthus niruri

were investigated in diabetic Wistar strain rats by Nwanjo (2007). Alterations were

observed in glucose level and some hepatospecific markers in a dose dependent

manner.

19

2.6. Anti-diabetic activity

Diabetes Mellitus (DM) describes a metabolic disorder of multiple etiologies

characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and

protein metabolism resulting from defects in insulin secretion, insulin action, or both

(Andrade-Cetto and Heinrich, 2005). It is caused by inherited and/or acquired

deficiency in the production of insulin by the pancreas, or by ineffectiveness of the

insulin produced and such a deficiency results in increased concentrations of glucose

in the blood, which in turn damage the body system, in particular the blood vessels and

nerves (Mukherjee et al., 2006).

2.6.1. Indigenous Treatment for Diabetes mellitus

The use of plants as therapeutic tools, especially those used to relieve

chronic pathologies, have had a remarkable role in the popular medicine of

different countries (Lee et al., 1999). Plant derivatives with purported hypoglycemic

properties have been used in folk medicine and traditional healing systems around

the world (Yeh et al., 2003) and they represent a vast source of potentially useful

dietary supplements for improving blood glucose control and preventing long-term

complications in type 2 diabetes mellitus (Gallagher et al., 2003). Since time

immemorial, various plants and plant derived compounds have been used in the

treatment of diabetes to control the blood sugar of the patients. In the period of

1907-1988, anti-diabetic activity was reported for about 343 plants at global level

and those are mostly used in the indigenous system of medicine as well as

20

scientifically proven plants (Rahman and Zaman, 1989). Before the introduction of

insulin in 1922 the treatment of diabetes mellitus relied heavily on dietary

measures which included the use of traditional plant therapies (Gray and Flatt,

1999).

Before two decades, more than 400 traditional plants were recorded for the

treatment of diabetes mellitus with only a small number of scientific and medical

evaluations to assess their efficacy (Bailey and Day, 1989). In traditional medicine,

diabetes mellitus is treated with diet, physical exercise and medicinal plants, even

though, more than 1200 plants are used around the world in the empirical

control of diabetes mellitus and approximately 30% of the traditionally used

anti-diabetic plants were pharmacologically and chemically investigated (Alarcon-

Aguilar et al., 1998). On the other hand, potential hypoglycaemic agents have also

been detected for more than 100 plants used in anti-diabetic therapy.

Hypoglycemic action from some treatments has been confirmed in animal

models and non-insulin dependent diabetic patients and now a day a number of

hypoglycemic compounds have been identified.

Yeh et al., (2003) reviewed and analyzed, a total of 108 trails examining 36

herbs (single or in combination) and 9 vitamin/ mineral supplements, involving 4,565

patients with diabetes or impaired glucose tolerance. In their study there were 58

controlled clinical trials involving individuals with diabetes or impaired glucose

tolerance and most studies involved patients with type 2 diabetes. Of these 58 trials,

21

the direction of the evidence for improved glucose control was positive in 76% and

very few adverse effects were also reported by them. There have been many studies

on hypoglycaemic plants and a great variety of compounds have been isolated (Table

3)(alkaloids, glycosides, terpenes, flavonoids, etc.), but the main fact is the further

development of such leads into clinically useful medicines and especially

phytomedicines or adequate nutritional supplements, which would be of direct

benefits to patients (Andrade-Cetto and Heinrich, 2005).

Table 3: Plants with significant Anti-diabetic compounds and their mode of action

Plant name Class of

Compound

Anti-diabetic

Compound

Mode of action

Abelmoscus moschatus Flavonoid Myricetin

(3,5,7,3’,4’,5’-

hexahydroxyflavone)

Anti-diabetic effect (Liu

et al., 2005)

Alpinia galanga

Polysaccharides

Protein-bound

polysaccharide

Increase serum insulin

levels, reduce blood

glucose levels and

improve glucose tolerance

(Quanhong et al., 2005).

Andrographis paniculata Diterpenoid Andrographolide Hypoglycemic activity

(Yu et al., 2003).

Citrus aurantifolia Polymethoxylated

flavones

Insulin resistance (IR)

model of hamsters

Increases the insulin-

sensitizing effects (Li et

al., 2006)

Curcuma longa

Ferulic acid 4-hydroxy-3-

methoxycinnamic acid

Hypoglycemic effect

(Ohnishi et al., 2004)

Stimulate insulin

22

secretion (Nomura et al.,

2003).

Gymnema sylvestre Triterpenoid Gymnemic acid IV Hypoglycemic activity

(Sugihara et al., 2000).

Momordica charantia

Sterol Charantin Insulin-like activity and

stimulate insulin release

(Ng et al., 1986)

Punica granatum Tanins Gallic acid

Enhances cardiac PPAR-γ

mRNA expression and

cardiac glucose

transporter (GLUT)-4.

(Huang et al., 2005)

Tinospora cordifolia

Alkaloid Berberine

Hypoglycemic activity

and Alpha -glucosidase

inhibitor and decrease

glucose transport (Singh

et al., 2003).

Tribulus terrestris

Imidazoline

compounds

Harmane, norharmane

and pinoline

Increase insulin secretion

(Nadkarni, 1976; Cooper

et al., 2003).

In the Chinese traditional medical treatment of diabetes, compound recipes are

often used more than simple recipes (prescription with one medicine) due to

consideration of integrated effects of different medicines. There are hundreds of

prescriptions to aim directly at different symptoms of diabetes, and about 100 of

natural medicines and preparations are used in these prescriptions or folk simple

recipes and diets for diabetes care in China, most of which come from plants (Li et

al., 2004). More than 100 medicinal plants are mentioned in the Indian system of

23

medicines including folk medicines for the management of diabetes, which are

effective either separately or in combinations (Kar et al, 2003). In India, the

plants such as Eugenia jambolana, Coccinia indica, Gymnema sylvestre, Momordica

charantia and Tigonella foenum-graecum are widely used for the treatment of diabetes

in traditional as well as modern medicine. At the present time,(Table 4) the plants like

Gymnema, fenugreek, bitter melon, ginseng, nopal, Aloe, bilberry, milk thistle,

garlic and ginkgo are the most active plants and have many active constituents with

anti-diabetic properties (McWhorter, 2001).

Table: 4 Plants with significant anti-diabetic activity in experimental animals

Botanical Name Parts Anti-diabetic mechanism References used

Aloe vera Leaf gel Hypoglycemic effects Roman-Ramos et al.,,

1995.

Annona squamosa Leaf Hypoglycaemic effect Gupta et al., 2005.

Azadirachta indica Leaf Hepatoprotective Chattopadhyay, 2005.

Eugenia jambolana Seed Anti-hyperglycaemic effect Grover et al., 2000.

Gymnema sylvestre Leaf Anti peroxidative,

hypoglycaemic and cortisol

lowering activities

Gholap and Kar, 2003.

Tinospora cordifolia Root Hypoglycaemic and

hypolipidaemic effects

Stanely and Menon,

2003.

Trigonella foenum

graecum

Seeds Anti-hyperglycemic and

hypoglycemic effects

Hannan et al, 2003.

24

Panax ginseng Whole

plant

Hypoglycemic and

hypolipidemic effects

Jang et al., 2001.

Rhus barb Rhizome Anti-diabetic Lee et al, 2008.

Experimental diabetes has the advantage that it allows the analysis of the

biochemical, hormonal and morphological events that take place not only during the

induction of a diabetic state but also after it has become established and during its

evolution to a severe insulin deficiency or even death (Silva et al., 2002).

Streptozotocin has been extensively studied and has yielded the vast majority of

information relevant to human diabetes due to its wide range of safety compared to

other chemical agents capable of inducing diabetes. The effective dose (ED50) is 4-5

times lower than the lethal dose (LD50).

Table: 5 Mechanism of action of some Anti-diabetic principles isolated from

medicinal plants

Name of the

Plant

Compound Experimental

Model

Molecular level

functional properties in

the treatment of

diabetes & References

Citrus aurantifolia Polymethoxylated

flavone

Insulin resistance (

IR) model of hamster

Increases the insulin-

sensitizing effects might

occur as a consequence

of adipocytokine

regulation and increases

protein expression of

25

PPAR-α and PPar-γ in

the liver(Li et al., 2006).

Ervatamia

microphylla

Conophylline

vinca alkaloid

AR42J-progenitor

cells, Neonatal

Streptozotocin –

treated rats.

Effectively inducing

differentiation of

pancreatic ductal cells

to β–cells (Kojima &

Umezawa, 2006.)

Eucommia

ulmoides

Flavanol

glycoside and

quercitine 3-O-α-

1-arabino

pyranosyl-(1-2)-β-

D-gluco

pyranoside

Bovine serum

albumin (10 mg/dl)

was incubated with

250 mMD-fructose

in the presence or

absence of test

material for 5 days

Inhibits the advanced

advanced glycation in in

vitro condition, and

decrease the secondary

complications (Kim et

al., 2004)

Hyssopus

officinalis

(7S,8S)-

Syringoylglycerol-

9-O-(60-O-

cinnamoyl)-b-d-

gluco pyranoside

Rat small intestine Α-Glucosidase inhibitor

activity(Matsuura et al.,

2004)

Pterocarpus

marsupium

7-O-α-1-rham

nopyranosyl oxy-

4-menthoxy-5-

hydroxy

isoflavone

L6 myotube &

adipocytic cell-line

3T3LI

Activation of PPARγ

through PPARγ agonists

are known to increase

the glucose uptake

through induction of

GLUT-4 mRNA

expression

(Anandharajan et al.,

2005)

26

Punica granatum Galic acid (3,4,5-

trihydroxybenzoi

c acid)

Zucker diabetic fatty

rats, a genetic animal

model for type 2

diabetes

Enhanced cardiac

PPAR-γ mRNA

expression and restored

the down regulated

cardiac glucose

transporter(GLUT-4)

mRNA (Desrosis et al.,

2004).

Vitis vinifera Procyanidins

(flavonoids)

Streptozotocin-

induced diabetic rats,

L6E9 myotubes and

3T3-L1 adipocytes

Stimulate the glucose

uptake in adipocytes and

myotubes acts through

glucose transporter,

GLUT-4, in the plama

membrane (Pinent et al.,

2004).

2.6.2. Structure of Streptozotocin

Streptozotocin (STZ; N-nitro derivative of glucosamine) is a naturally

occurring, broad-spectrum antibiotic and cyto-toxic chemical that is particularly toxic

to the pancreatic, insulin producing β-cells The diabetogenicity of streptozotocin has

been correlated with a rapid reduction in pancreatic islet pyridine nucleotide

concentration and subsequent beta cell necrosis (Hayashi et al., 2006).The chemical

name is 2-deoxy-2-(3-methyl-3-nitrosourido)-O-glucopyranose. Molecular formula is

C8H15N3O7 (Fig.2). The structure is composed of a nitrosourea moiety with a methyl

group attached at one end and a glucose molecule at the other end (Weiss, 1982).

27

Fig. 2. Structure of Streptozotocin

Diabetes is induced by single intraperitonial injection of freshly prepared

streptozotocin (55 mg kg-1

bw) in 0.1M citrate buffer (PH-4.5) in a volume of 1 ml

Kg-1

rats (Siddique et al., 1987). In case of mice the dose 175-200 mg kg-1

and Dog

15 mg kg-1

for 3 days). Streptozotocin also induces diabetes in Hamster, monkey and

Guinea pig (Chattopadhyay et al., 1997).

2.6.3. Mechanism of action

STZ is diabetogenic because it selectively destroys the insulin-producing beta

cells by inducing necrosis. It is postulated that the selective beta-cell toxicity of STZ

is related to the glucose moiety in its chemical structure, which enables STZ to enter

the cell via the low affinity glucose transporter GLUT2 in the plasma membrane

(Elsner, 2000). It is generally accepted that the cytotoxicity produced by STZ depends

on DNA alkylation and subsequent activation of poly ADP-ribose synthetase causes

CH2 OH

OH OH

O H

OH

NH

C=0

H

H

N-NO

CH3

H

28

rapid and lethal depletion of NAD in pancreatic β-islets, thereby causing cell death

(Fig.3).

Several lines of evidences indicate that free radicals, highly reactive carbonium

radicals originating from the decay of STZ molecules might increase the production

of oxygen free radicals including hydroxyl radicals and nitric oxide, may play an

essential role in the mechanism of β-cell damage and diabetogenic effect of STZ

(Halliwell & Gutteridge, 1990).

2.6.4. Treatment for diabetes mellitus

Diabetes is a multifactorial disease leading to several complications, and

therefore demands a multiple therapeutic approach. Currently available drug regimes

for management of diabetes mellitus have certain drawbacks and therefore, there is a

need for safer and more effective anti-diabetic drugs. Many oral hypoglycaemic

agents, such as biguanides and sulfonylurea are available along with insulin for the

Fig. 3: Mechanism of action of Streptozotocin.

29

treatment of diabetes mellitus, but these synthetic agents can produce serious side

effects, and in addition, they are not suitable for use during pregnancy. Even though

several therapies are in use for treatment, there are certain limitations due to high cost

and side effects such as development of hypoglycemia, weight gain, gastrointestinal

disturbances, liver toxicity etc (Dey et al., 2002). Based on recent advances and

involvement of oxidative stress in complicating diabetes mellitus, efforts are on to

find suitable anti-diabetic and antioxidant therapy.

2.6.5. Herbal Support for Diabetes

In the late 1980s, Chinese doctors became alarmed by a huge increase in the

incidence of diabetes in China. Observing that this previously rare condition appeared

to be linked with newly adopted Western eating habits, they initially relied on

Western diabetes drugs to treat what was perceived to be a primarily Western

disorder. Eventually medical experts turned away from this approach after noting that,

in addition to serious side effects, modern pharmaceuticals also failed to address the

underlying cause of the problem. A central tenet of Chinese healing is to treat both

acute symptoms and the underlying cause of an illness. Based on this principle,

leading Chinese researchers turned their attention to traditional herbal remedies used

in China for thousands of years (Richards 2007).

2.7. ANTI-INFLAMMATORY ACTIVITY

Inflammation is the body’s way of dealing with infections and tissue damage,

but there is a fine balance between the beneficial effects of inflammation cascades and

30

their potential for long-term tissue destruction (Simmons, 2006). Studies have been

continuing on anti-inflammatory drugs to treat inflammatory diseases in various

countries. Inflammation can be defined as a generalized, on specific, beneficial

response of tissues to injury. The inflammatory process involves a complex interplay

between cells of the blood, the blood vessels and the cells of the involved tissue. The

process can be seen as a coordinated response of a large number of cells to an initial

stimulus. Early changes in inflammatory tissues are involved in the release of various

biologically active materials from polymorph nuclear leukocytes and lysosomal

enzymes.

The vascular effects are primarily mediated by kinins, prostaglandins and vaso-

active amines (e.g. histamine, released by mast cells), which cause increased vascular

permeability leading to plasma exudation .This complex reaction of inflammation is a

consequence of the materials that release into the extra cellular environment. Such

materials include histotoxic agents such as proteases and oxygen metabolites (Henson

and Murphy, 1989).

The inflammatory reaction is characterized by blush, heat, tumor, pain and lost

function (Dassoler et al., 2004). There are many causes for the inflammations, but the

mechanisms are common to all. In spite of the discovery of several newer agents, the

search for better anti-inflammatory drugs continues because they have many known

side effects and none of them is suitable for prolonged use (Ramprasath et al, 2004).

31

Plant drugs with anti-inflammatory, antioxidant activity can bring relief to

conditions like hemorrhoids, varicose veins and other conditions that involve a better

flow of blood. The anti-inflammatory activities are often attributed to the presence of

saponins while the antioxidant activity attributed to the presence of flavonoids and

other molecules having antioxidant activities (Gurib-Fakim, 2006).

Over the past 20 years there has been a significant increase in knowledge about

immunology, both in terms of molecular targets and molecular mechanisms. The

processes of inflammation fall into four major groups: changes in blood flow caused

by changes in smooth muscle cell function causing vasodilatation, alterations in

vascular permeability by cytoskeletal contraction in endothelial cells, migration of

phagocytic leukocytes to the site of inflammation and phagocytosis (Denko, 1992;

Evans and Whicher, 1992).

Inflammatory disorders can be studied in two different models

(1) Acute inflammatory model

(2) Chronic inflammatory model.

Acute models are designed to test drugs that modulate blood flow (erythema),

changes in vascular permeability, leukocyte migration and chemotaxis, phagocytosis

and other phagocytic cells, measurement of local pain, antipyretic activity, local

analgesic action and rat paw edema. Chronic models are designed to find drugs that

may modulate the disease process and these include sponge and pellet implants and

granuloma pouches which deposit granulation tissue (Lewis, 1989). Different

32

approaches used to analyze the anti-inflammatory potential of plants and plant derived

compounds in the past years.

Desmarchelier et al. (2000) reported that the oral administration of methanol

extract of the leaves of Pothomorphe peltata at a dose of 20 mg/kg exhibited

significant anti-inflammatory activity. The leaves of Bouchea fluminensis contain

iridoid and steroid glycosides that are present in the form of crude triterpene mixture

having anti-inflammatory property and the purified fraction of the plant was found to

contain ursolic, oleanolic and micromeric acids (Costa et al., 2003).

The leaves of Gochnatia polymorpha contain sesquiterpenes and lactone

derivatives and the ethanolic fractions at a dose level of 200 mg/ kg showed

significant inhibition on rat paw edema induced by carregeenan (Moreira et al., 2000).

The leaf extract of Hyptis pectinata administered orally at a dose of 600 mg/kg

exhibited a significant antidermatogenic activity (Bispo et al., 2001). The

intraperitoneal administration of the extract at the dose of 300 mg/kg inhibited the rat

paw edema by 33.8% and the results showed that the aqueous extract of H. pectinata

acted on both the cyclooxygenase and lipoxygenase pathways.

Franzotti et al. (2000) studied the aqueous extract of leaves of Sida cordifolia

to assess the anti-inflammatory properties using the Carrageenan and arachidonic acid

induced rat paw edema model. Oral administration at a dose level of 400 mg/kg of the

extracts howed 28.31% reduction in edema,whereas the dose level of 200 mg/kg was

33

ineffective in reducing edema and the dose of 800 mg/kg inhibited 7.55% in the

model of Carrageenan induced edema.

Bani et al. (2000) studied that the hydrosoluble fraction of Euphorbia royleana

latex was active in suppressing the paw edema in both acute and chronic models of

inflammation and this was further supported by the poor action of the extract in cotton

pellet granuloma test in rats which was found to be highly sensitive to steroidal type

of drugs. Anti-inflammatory activity of the leaves of Dalbergia was investigated by

Hajare et al. (2001) and Carrageenan, kaolin and nystatin were used to induce paw

edema. The 90% ethanolic extract of the plant when administered orally at doses of

100, 300 and 1000 mg/kg showed significant inhibition of inflammation.

Leaves of Cassia fistula were tested for anti-inflammatory effects as compared

with phenylbutazone using carrageen, histamine and dextran induced paw edema in

rats and potent anti-inflammatory activity against all phlogistic agents was noted

(Bhakta et al., 2000). The anti-inflammatory activity of heat treated Cassia alata leaf

extract and kaempferol 3-O-gentiobioside isolated from C. alata as an abundant

flavonoid glycoside were studied by evaluating by their activities with sun dried C.

alata leaf extract. The heat treated C. alata leaf extract exhibited stronger inhibitory

effects than the effects of the sun dried leaf extract (Moriyama et al., 2003).

Devi (2004) evaluated the anti-inflammatory activity of the aqueous extract of

Spilanthes acmella in Carrageenan induced rat paw edema. The aqueous extract in

34

doses of 100, 200 and 400 mg/kg showed 52.6, 54.4 and 56.1% inhibition of paw

edema. Karina et al. (2007) investigated the effect of Brazilian polyherbal formulation

for inflammatory conditions. In their investigation, the formulation at a dose of 26

ml/kg inhibited capsaicin induced ear edema.

Ethanolic extracts of the roots of Cissampelos pareira at the doses of 200, 400

mg/kg exhibited significant anti-inflammatory activity (Amresh, 2007). The methanol

extract of the leaves of Jatropha curcas exhibited significant anti-inflammatory

activity in acute Carrageenan induced rat paw edema and cotton pellet induced

granular tissue formation after oral treatment for 7 days in albino rats (Mujumdar and

Misar, 2004).

The methanol extract of Ionidium suffruticosam was evaluated on Carrageenan,

histamine and serotonin-induced rat hind paw edema acute models (Boominathana et

al., 2004). The extract at the doses of 200 and 400 mg/kg has been found to possess

significant anti-inflammatory activity on the tested experimental models.

The anti-inflammatory activity of the leaves of Cleome gynandra was assessed

by paw volume measurement and the extract showed significant effect on inhibiting

the paw edema (Narendrakannan and Subramanian,2007). Anti-inflammatory activity

of the ethanolic extract of the leaves of Morus indica was studied in wistar rats using

the Carrageenan induced pleurisy and cotton pellet induced granuloma model

(Balasubramanian and Ramalingam, 2005). The extract (100 mg/kg) inhibited

35

Carrageenan induced rat paw edema and it also showed an inhibitory effect on

leukocyte migration and a reduction on the pleural exudates as well as reduction on

the granuloma weight in the cotton pellet granuloma method.

Table 6: Plants with potential anti-inflammatory principles

NAME OF THE

PLANT

ACTIVE

COMPOUND

ANTI-INFLAMMATORY

MECHANISM

REFERENCES

Allium sativum Allicin and its

precursor alliin

S-allyl cysteine

Scavenge hydroxyl

radicals inhibit NFB

activation in T cells

Rabinkov et al.

(1998);

Geng et al (1997)

Artocarpus

heterophyllus

Cycloheterophyllin

artonins

Inhibit lipid peroxidation

and directly scavenge

stable free radicals

Ko et al. (1998)

Ginkgo biloba Flavonoids,

terpenoids,

ginkgolide B

Scavenge superoxide

anions, hydroxyl radicals,

and peroxy radicals

Inhibit the activity of key

enzymes involved in the

inflammatory process

Kobuchi et al.

(1992)

Magnolia obovata Magnolol Inhibits lipid peroxidation

Prevents

ischemicreperfusion

injury

Hong et al.(1996)

Panax ginseng Ginseng Inhibits decomposition of

fatty acids, which are

highly cytotoxic, due to

iron-mediated lipid

peroxidation

Zhang et al.

(1996)

36

Suba et al. (2005) studied the anti-inflammatory efficacy of the methanol

extract of the aerial parts of Barleria lupulina in acute and sub-acute inflammatory

models of albino rats. In all the tested dose levels, methanol extract exhibited

significant inhibition of Carrageenan and serotonin induced paw edema volumes. The

analgesic and anti-inflammatory activities of a new alkaloid (5′-Hydroxymethyl-1′-(1,

2, 3, 9-tetrahydro-pyrrole [2, 1-b] quinazolin-1-yl)-heptan-1-one) isolated from Sida

cordifolia was produced significant (p<0.01) analgesic activity in animal models

(Sutradhar et al., 2007).

Emmanuel et al. (2006) studied the anti-inflammatory effect of solasodine,

sobatum and methanol extract of Solanum trilobatum and the results showed

significant anti-inflammatory activity. Ratheesh and Helen (2007) carried out anti-

inflammatory tests in Wistar male rats using aqueous, ethanolic and methanolic

extracts of Ruta graveolens. These extracts were administered in the concentrations of

20 and 50 mg/kg b.wt before Carrageenan injection. Methanolic extracts of R.

graveolens with a concentration of 20 mg/kg b.w. and ethanolic extract with a

concentration of 50 mg/kg b.wt showed maximum (90.9%) inhibition on Carrageenan

induced rat paw edema. Biren et al. (2007) studied the ethanolic extract of the leaves

of Colocasia esculenta in Carrageenan induced rat paw edema and the extract

produced significant (p<0.05) anti-inflammatory activity (100 mg/kg, p.o.) induced by

Carrageenan.

37

Yam et al. (2008) reported that the methanol: water (50:50 vol/vol) extracts of

the leaves of Orthosiphon stamineus significantly reduced the hind paw edema in rats

at 3 and 5 hours after Carrageenan administration at the doses of 500 and 1,000

mg/kg. The aqueous extract of the stems of Gynandropsis pentaphylla showed

significant dose dependent anti inflammatory activity at the doses of 100 mg/kg i.p

(Mule et al., 2008).

Lee et al. (2008) studied the effect of ethanol extract of Asparagus

cochinchinensis on skin inflammation in mice. The extract significantly inhibited the

ear edema of mouse at the doses of 200 mg/kg. Oral administration of the ethanolic

extract (200 and 400 mg/kg.) and its fractions (200 mg/kg each) of the aerial parts of

Cleome rutidosperma showed significant analgesic activity in acetic acid induced

writhing and tail immersion tests, anti-inflammatory effect against Carrageenan

induced inflammation (Bose et al., 2007). Anti-inflammatory activity of ethanol

extract of Dalbergia lanceolaria exhibited significant systemic anti-inflammatory

activity in Carrageenan induced rat paw edema by inhibition of histamine and

prostaglandin phases of acute inflammation (Kale et al., 2007).

Methanol extract of Phyllanthus amarus on different phases of inflammation

were examined using different phlogistic agents induced paw edema, Carrageenan

induced air-pouch inflammation and cotton pellet granuloma in rats. Methanol extract

of Phyllanthus amarus significantly inhibited Carrageenan, bradykinin, serotonin and

prostaglandin E1-induced paw edema (Mahat and Patil, 2007). Anti-inflammatory

38

effect of the extracts and purified lignans obtained from the plant, Phyllanthus amarus

inhibited the Carrageenan induced paw edema (Kassuya et al., 2005).

2.8. ANTIPYRETIC ACTIVITY

In the traditional system of medicine, many medicinal plants have been

reported to possess the potentiality to cure fever. In the Chinese medicine system the

antipyretic plants are of five types (Dong et al., 1998).

a) the herbs that clear intense internal heat

b) the herbs that clear intense heat in the blood systems

c) the herbs that reduce dry internal dampness or heat in the viscera or bowels

d) the herbs that reduce internal noxious heat or toxins

e) the herbs that clear interior heat of the deficient type

The ethanolic extracts of Ailanthus excelsa, Toddalia asiatica and Araucaria

bidwilli showed moderate to significant degree of antipyretic activity in an

experimental rat model of 20% yeast suspension induced hyperthermia (Suresh et al.,

1995). Methanolic extract of the rhizome of Nelumbo nucifera produced a significant

dose dependent lowering of pyretic effect in pyretic rats (Mukherjee et al., 1996).

Rhynchosia cana showed significant antipyretic activity in rats (Vimala et al., 1997).

Alcoholic extract of the roots of Clerodendron serratum showed significant

antipyretic activity and result was found to be almost equal to paracetamol

(Narayanan et al., 1999). Panax ginseng showed hyperthermic effect and attenuated

39

the hypothermic response of reserpine and 5-HTP induced hyperthermia in animals

(Mitra et al., 1996). Significant antipyretic activity was observed in hexane,

chloroform and water soluble extracts of Artemisia absinthium, Viola odorata, Melia

azadirachta and Fumaria parviflora and comparable in potency aspirin. The Pyresis

was induced by subcutaneous yeast injections and the antipyretic activity was more

prominent in hexane soluble portions (Khattak et al., 1985). Some important

medicinal plants with antipyretic activity are listed in Table 7.

Table 7: Some important medicinal plants with Anti-pyretic activity

Botanical Name Parts used References

Adansonia digitata Fruit pulp Ramadan et al. (1994)

Ageratum conyzoides Essential oil Abena et al. (1996)

Astragalus siculus Whole plant Bisignano et al. (1994)

Buddleia cordata Leaves Martinez-Vazquez et al. (1996)

Euphorbia hirta Whole plant Lanhers et al. (1991)

Lawsonia inermis Leaves Ali et al. (1995)

Maytenus boaria Leaves Backhouse et al. (1994)

Michelia champaca Leaves Vimala et al. (1997)

40

Oral administration of Taxus wallichiana produced significant antipyretic

activity in yeast induced pyrexia model at a dose of 200 mg/kg dose (Nisar et al.,

2008). Amiruddin et al. (2008) investigated the leaves of Dicranopteris linearis for its

antinociceptive, anti-inflammatory and antipyretic properties in experimental animals.

The antipyretic activity was measured using brewer’s yeast-induced pyrexia. The

aqueous extract of Dicranopteris linearis was also found to have significant

antipyretic activity.

Salawu et al. (2008) evaluated the methanolic extract of Crossopteryx

febrifuga for analgesic, anti-inflammatory, antipyretic and antiplasmodial activities in

rodents. The extract significantly diminished antipyretic activity in mice and rats in a

dose-related manner. In another investigation it was observed that oral administration

of the ethanolic extract (200 and 400 mg/kg, p.o) and its fractions (200 mg/kg each)

of the aerial parts of Cleome rutidosperma produced significant antipyretic activity

against yeast induced pyrexia (Bose et al., 2007).

Mucuna pruriens Seeds Jauk et al. (1993)

Ocimum sanctum Leaves Godhwani et al. (1987)

Premna herbacea Root Narayanan et al. (2000)

Solanum ligustrinum Leaves Delporte et al. (1998)

Teclea nobilis Whole plant Mascolo et al. (1998)

41

Garcinia hanburyi was assessed for its antipyretic activity by Panthong et al.

(2007) using experimental animal model and they observed that G. hanburyi

possessed significant antipyretic effect when tested in yeast induced hyperthermic

rats. Oral administration of the aqueous extract of the stem of Urtica macrorrhiza

(200–400 mg/kg) was dose dependently suppressed the yeast induced fever in rats

(Yongna et al., 2005).

The antipyretic activity of methanol extract of Cleome viscosa was investigated

by Devi et al. (2003) for its activity on normal body temperature and yeast induced

pyrexia in albino rats. The extract at doses of 200, 300 and 400 mg/kg b.wt showed

significant reduction in normal body temperature and yeast provoked elevated

temperature in a dose dependent manner. Chattopadhyaya et al. (2002) were studied

the antipyretic potential of the methanol extract of Mallotus peltatus on normal body

temperature and yeast induced pyrexia in Wister albino rats. The leaf extract at oral

doses of 100, 200 and 300 mg/ kg showed significant reduction in normal body

temperature and yeast provoked elevated temperature in a dose dependent manner.

Archana et al. (2005) evaluated the antipyretic and analgesic activities of

ethanolic extract of the seed kernel of Caesalpinia bonducella in adult albino rats at a

dose of 100 and 300 mg/kg administered orally. They observed that antipyretic

activity against Brewer's yeast induced pyrexia in rats. The ethanol (100 and

200 mg/kg) and water extracts (200 mg/kg) of Capparis zeylanica leaves showed dose

dependent analgesic and antipyretic activities (Ghule et al., 2007).

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The chloroform, methanolic and ether extracts of the leaves of Vernonia

cinerea at the doses of 100, 200 and 400 mg/kg intraperitoneally significantly

suppressed the brewer’s yeast induced pyrexia in rats (P<0.05) compared to the

control (Iwalewa et al., 2003). Ahmadiani et al. (2001) examined the Trigonella

foenum-graecum leaves extract for its antipyretic activity. In their study, hyperthermia

was induced by intraperitoneal injection of 20% (w/v) aqueous suspension of brewer's

yeast. Leaf extract of T. foenum-graecum significantly reduced the hyperthermia

induced by brewer's yeast in 1 and 2 hrs after their administration.