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INTRODUCTION

MEDICINAL PLANT WEALTH OF INDIA

The Indian subcontinent with diversified topography from sea level to the Himalayas

and equally diversified climatic conditions offers excellent environment for diverse

plant species to grow and multiply. India being one of the 12 mega biodiversity

centres has about 45,000 plant species. The diversity is more so in Southern Western

Ghats and Eastern Himalayas. Hooker (1872 - 97) has recorded 14,000 species of

angiosperms in the Indian subcontinent.

A large number of country’s rural population depends on plants of medicinal

importance for treating various diseases (WHO, 2002). Nearly 80 % of world’s

population relies on traditional medicine, particularly plant products for their

primary health care (Kamboj, 2000; Sampson et al., 2000; Natesh, 2000; WHO,

2001; Dubey et al., 2004). About 20,000 plant species are considered to be of

medicinal value (Dev, 1997). Around 500 communities make use of about 800 plant

species as medicinal plants for curing various diseases (Ravikumar and Ved, 2000;

Kamboj, 2000).

Traditional systems such as Ayurveda, Siddha and Unani continue to be taught and

practiced. Indian folk medicine includes several herbal preparations for wound

healing, treating inflammations, skin lesions, leprosy, diarrhea, scabies, venereal

diseases, snakebites and ulcers (Perumalsamy and Gopalakrishnan, 2007).

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Nearly 8000 registered practitioners are there under Indian system of medicine and

about 850 under Homoeopathy. Interest and demand for traditional remedies and plant

based health products are increasing world wide ((Mosihuzzaman and Choudhary,

2008).

About 80 % of the 500 and odd plant species used by contemporary Ayruvedic

industry are procured from the wild (Gupta, 1993). India caters to about 12 % of

global herbal trade and recent years the volume of medicinal plant materials traded

within and outside the country has reached extreme heights (Perumalsamy and

Gopalakrishnan, 2007). This increase in consumption of medicinal plants through

expansion of local, regional and global markets has increased the pressure on the

resource that is largely harvested from depleted wild populations in shrinking wild

habitats (IUCN, 2008).

REVIVAL OF PLANT MEDICINES

Medicinal herbs are moving from fringe to mainstream use with a great number of

people seeking remedies and health approach free from side effects. Recently,

considerable attention has been paid to utilize eco-friendly plant based products for

the prevention and cure of various human diseases. Considering the adverse side

effects of synthetic drugs the western population has switched over to herbal

medicines which are believed to be safe and effective (Pearce, 1993; Dubey et al.,

2004). Medicinal herbs and plant extracts are re-recognized as effective medicines to

be respected, appreciated and they play a major role in modern pharmacy. Although

herbal medicine has existed since the dawn of our civilization, our knowledge of how

plants actually influence human physiology remains largely unexplored. Studies have

pointed out that many drugs that are in use have come from folk-use and use of plants

by indigenous cultures (Anonymous, 1994).

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Although physicians and pharmacists have began to recognize the relative safety and

efficacy of at least some of the herbs better researched on the market, many have

concerns about contradictions, potential adverse reactions, and possible herb-drug

interactions. Solid market statistics are often elusive, but the general trend towards

increased acceptance of herbs by consumers is undeniable. This has been explicitly

reported by Princeton Survey Research Associates that about 49 % (91 million) of the

entire adult Americans have used herbal products during the year 1998-1999. The

survey also showed that about 24 % (44.6 million) have used herbs regularly

(Johnson, 2000).

Interest in the utilization of medicinal and aromatic plants as pharmaceuticals, herbal

remedies, flavourings, perfumes, cosmetics, and other natural products has also

greatly increased in the recent years (Anonymous, 1994; Ayensu, 1996; Salleh et al.,

1997; Kumar et al., 2000). As with many other economic plants that are still being

collected from the wild and exploited by humans unsustainably, threats to genetic

diversity and species survival have also increased in the case of medicinal plants as a

result of habitat destruction, over-exploitation, land use changes and other pressures

(Arora and Engels, 1993). In India alone, less than 10 % of the medicinal plants

traded in the country are cultivated, about 90 % are collected from the wild, very

often in a destructive and unsustainable manner (Natesh, 2000). The number of

organizations conducting research and other activities related to the use of medicinal

and aromatic plants is large and increasing (Ayensu, 1996; Sharma et al., 2002).

Pharmacognosy and documentation of indigenous knowledge (Leaman et al., 1999;

Kshirsagar and Singh, 2001) is advancing to greater level and has surpassed the work

on conservation of medicinal plants resource, particularly at the level of intra-specific

genetic diversity.

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Recently, a regional inventory of medicinal and aromatic plants and polyherbal

formulations dealing with 65 Indian medicinal plants, 10 important Indonesian and 25

medicinal plants of Malaysia, along with important traditional and polyherbal

formulations used in these countries has been brought out by CIMAP and supported

by the Department of Biotechnology, Government of India (Anon,1999). It is only in

the last 40 to 50 years that many of the medicines were produced industrially and sold

in shops and markets with trade names (Sasson, 1996; Natesh, 2000).

TRADITIONAL MEDICINAL PRACTICES

The history of traditional medicine in India can be traced to the remote past. The

earliest mention of the medicinal use of plants was found in the rig Veda, perhaps the

oldest repository of human knowledge (Chopra et al., 1956). The Principal aim of

ancient Hindu medicine was to prolong life. The Chinese believed that for every

illness that there was a corresponding natural remedy.

People living in rural areas of the Asia-Pacific are familiar with the medicinal

properties of local plants. Fresh plant materials collected were used either to obtain

the extract from the whole plant or parts like leaves, roots, flowers or fruits. In case of

woody plants, the bark, roots and other parts are used. Carminatives like ginger, clove

and coriander are also usually added as fresh or dried materials. Information about

medicinal plants were passed by word of mouth to the succeeding generation and

usually maintained as a family secret. Very few records or writings were maintained,

leading to the loss of such precious knowledge, through a number of treatises do exist

in various countries (Rao and Rao, 1998).

Refinement of such practices lead to the well established Asian systems of medicines

including Ayurveda, Siddha of India, Unani system of middle and Far East Asia, Ying

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and Yan principles of Chinese herbal medicines and Jamu of Indonesia (Sharma et al.,

1998; Natesh, 2000). The Foundation for revitalization of local health traditions

(FRLHT) data base lists about 7,361 medicinal plants used in all kinds of traditional

practices in India (Ravikumar and Ved, 2000).

Though herbal medicines used in traditional systems of medicine have been tested

through long historical practice, WHO guidelines (1993) insist on evaluating the

safety and efficacy of herbal medicines is highly necessary and suggests modern

research methodology on pharmacodynamic and general pharmacological studies,

toxicity investigations and clinical trials. Hence, such scientific evaluation will help

us in determining their safety role in health care. To a number of traditional medicinal

formulations importance has not been paid to explore and understand safety and their

genotoxic and mutagenic effects. The damage caused due to the uptake of nature

medicines in the body and consequently in the chromosome has been an efficient and

reliable source of information which was used to measure the genetic toxicity in

organisms. However, medicinal plants, and indeed plants in general, synthesize toxic

substances, which in nature act as a defense against infections, insects and herbivores,

but which often affect the organisms that feed on them. Thus, an assessment of their

cytotoxic and mutagenic potential is necessary to ensure a relatively safe use of

medicinal plants. Therefore, a concern regarding the indiscriminate use of medicinal

plants in folk medicine continues to be extremely necessary (Rosangela et al., 2003;

zeiger, 2002 ).

DIABETES AND HUMAN POPULATION

In the 21st century human beings world wide, have become prone to a major

metabolic disorder – Diabetes. Diabetes mellitus (DM), results from a defect in

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insulin secretion, insulin action, or both (Bastaki, 2005). This is an ancient Greek

word meaning siphon or running through i.e. passing large amount of urine and

mellitus meaning honey–like (referring to the sweetness of the urine due to excess

sugar as identified by ancient physicians (Powers, 1996). In the 21st century more

people appear to be prone to the major metabolic disorder - diabetes. The incidence of

diabetes is increasing in order forever. Sugar is necessary for the human body to

provide energy but, excess sugar in blood leads to diabetes. One form is diabetes

mellitus with excessive presence of sugar (glucose) in the blood.

Diabetes – The World Perspective

Diabetes is one of the major health problems of mankind worldwide and an important

cause of prolonged ill health and early death. It was one of the sixteen leading causes

of global mortality in 1990, accounting for 571,000 deaths (Arunachalam and

Gunasekaran, 2002). The number of people with diabetes is increasing due to

population explosion, ageing, urbanization, and growing prevalence of obesity and

physical inactivity (King and Rewers, 1993). Even if the prevalence of obesity

remains stable until 2030, which seems unlikely, it is anticipated that the number of

people with diabetes will be more than double as a consequence of population aging

and urbanization (WHO, 2001). Unless preventive measures are taken, 366 million

people worldwide will have diabetes by 2030, with the largest increase occurring in

developing countries (Wild et al., 2004)

Types of Diabetes

Diabetes is divided into two types, Type-I and Type-II. Type-I diabetes is referred as

insulin dependent diabetes mellitus (IDDM). Type-I diabetes develops when the

body’s immune system destroys pancreatic beta cells, the only cells in the body that

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make the hormone insulin that regulates blood glucose. This form of diabetes usually

strikes children and young adults, although disease onset can occur at any age. Type-I

diabetes may account for 5 to 10 percent of all diagnosed cases of diabetes. Risk

factors for Type-I diabetes may include autoimmune genetic and environmental

factors (Powers, 1996).

Type-II Diabetes is otherwise called as non–insulin dependent diabetes mellitus.

Type-II diabetes may account for about 90 to 95 percent of all diagnosed cases of

diabetes. Causative reason for this type of diabetes is insufficient secretion of insulin

in the islets of pancreas. As the need for insulin rises the pancreas gradually loses its

ability to produce enough insulin. This is the type of diabetes most prevalent and

occurs over 40 years of age. Due to demand of insulin the stored sugar cannot be

utilized and is represented in the blood and urine as excess sugar. After the onset of

diabetes they may lead to complications like diabetic retinopathy, diabetic

nephropathy and many other disorders (Powers, 1996; Bastaki, 2005).

Treatment and Control of Diabetes

At present there may be no complete cure for diabetes but, may be kept well under

control. People with Type - I diabetes must have insulin delivered by injections or a

pump to keep their level of sugar under control in their blood. People with Type - II

diabetes control their blood glucose by following a careful diet and exercise

programme, losing excess weight and taking oral medication. Some people with

diabetes take medication to control their cholesterol and blood pressure. Diabetes may

cause damage to other parts of body like eyes and lead to vision impairment. Due to

diabetes fat deposition may take place in the arteries and create hindrance in the flow

of blood. Research studies have found that lifestyle changes prevent or delay the onset

of diabetes. Lifestyle interventions including diet control, moderate physical activity

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like walking, and exercises are more vital which can keep diabetes under control.

Diabetic patients are advised to follow a systematic diet plan which includes more of

cereals, pulses, vegetables and moderate amount of fat free milk, meat and fish. Apart

from controlling diabetes through well planned diet, insulin injection has to be taken

according to the advice of the physician. Although large number of chemical drugs

including insulin are already in use for Type - II diabetes, still there are limitations

such as adverse side effects and high rate of secondary failure and no promising

therapy to cure diabetes (Sumana and Suryawanshi, 2001; Xie et al., 2003).

TRADITIONAL SYSTEMS OF TREATING DIABETES

Ayurveda, Siddha and Unani

Ayurveda, Siddha, Unani, and folk medicinal systems are various traditional systems

where several plant species were used in the treatment of different ailments (Rabe and

Staden, 1997). In India Ayurveda, Siddha and Unani systems were the formal and

most organized amongst the traditional systems of medicine.

Ayurveda one of the ancient forms of Indian medicine utilizes the active ingredients

present in plants for treating various diseases and illnesses (Ravikumar and Anuradha,

1999). The western medicine has been considered as the offspring of Ayurveda.

Ayurveda still remains as one of the most ancient and living tradition of medicine

practiced widely in Asian countries including Sri Lanka (Dahanukar and Thatte, 2000;

Chopra and Diophode, 2002). Knowledge of Ayurvedic medicine has unfortunately

been confined to India and not so prevalent in the west. Even in India, this traditional

medical practice is not in use in the urban areas. One of the main reasons for this is

that much of the early and core medical literature on Ayurveda was in Sanskrit, the

ancient language which ceased to be a day-to-day language in this century, except in

extremely small groups of the vast Indian population.

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Since oral hypoglycemic agents cause side effects, there is a growing interest in

herbal remedies for the treatment of diabetes mellitus (Kim et al., 2006). Medicinal

plants play an important role in the management of diabetes mellitus especially in

developing countries where resources are meager. An exhaustive review has been

made on medicinal plants with hypoglycemic effects with the aim of collating all

available data in the public domain and to bring out the importance and the interest

placed on medicinal plants in the drive to demonstrate their antidiabetic effects and to

isolate the bioactive agents (Atta-Ur-Rahman and Zaman, 1989; Ivorra et al., 1989;

Bnouham et al., 2002; Al-Rowais, 2002; Grover et al., 2002; Bnouham et al., 2006;

Garg, and Garg, 2008). About 176 species belonging to 84 families are used by

various medicinal systems across the world as antidiabetic plants. In china at least 250

plants were used to treat diabetes as mentioned in ancient literatures B.C. (Garg, and

Garg, 2008; Garg, 2009).

Antidiabetic effect of Swertia densifolia (shilajit) and Ficus bengalensis (Banyan)

have been found to be associated with their weight promoting, anabolic and

pancreatrophic effects. In spite of the long tradition of use very few plants have been

tested for their efficacy by present day technology. Diabecon (D-400) formulation is

based on the ancient ayurvedic references corroborated through modern research and

clinical trials (Dubey et al., 1983). This consists of a mixture of powders from 10

different antidiabetic plants viz., Gymnema sylvestre, Vinca rosea and Curcuma longa

- 80 mg each; Azadirachta indica, Pterocarpus marsupium, Momordica charantia,

Syzygium cumini, Acacia arabica and Tinospora cordifolia - 40 mg each; Zingiber

officinale- 20 mg (Ganguly, 1995).

The Siddha system of medicine is predominantly followed in the state of Tamil Nadu

and is also popular in Andhra Pradesh, Karnataka and Kerala. The Siddha system is

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also practiced in Sri Lanka, Malaysia and Singapore. The name Siddha oushadha

(siddha medicine) relates to the earlier esoteric medicinal postulates concerning

longevity as conceived and practiced by 18 Siddhars, lived in Tamil Nadu. It is for

this reason the entire Siddha medical literature is in Tamil. The Siddha system has

about 800 texts of which 180 are in print. The Siddha system of medicine is rooted in

the Dravidian culture, of the pre-Vedic period (Kurup, 1983). Siddha system

incorporates minerals and toxic metals like mercury, sulphur, arsenic, and vegetable

poisons. Independent of the Siddha system, India has developed its own tradition of

alchemy and iatrochemistry, called Rasashashtra or Rasa vaidya that employed

minerals, metals and some plants (Bhat and Rao, 1993).

To treat diabetes mellitus Siddha system has recorded a formulation of plant drugs

which includes Avaarai (Cassia auriculata), Kondrai (Cassia fistula), naval

(Syzygium cumini) Ekanayakam (Salacia reticulata), korai kizhangu (Cyperus

rotundus), marutham (Terminalia arjuna), kizhanelli (Phyllanthus amarus). This

formulation seems to be highly effective in controlling diabetes if taken for about four

months without any break and with strict diet control (Nadkarni, 2005).

‘Solaimalai Sarkaraikolli neerizhu churnam’ - a siddha preparation for treating

diabetes consist about 48 herbal mix, marketed by Solaimalai Indian herbal drugs,

Paramakudi, Tamil Nadu. Powder of 3 g has been prescribed before meals (twice a

day) to remedy diabetes. Though the mixture has herbal powders and largely sold in

markets, its efficacy and safety has not been scientifically validated. Similarly there

are several formulations based on sidhha literature prepared in a secrete way to treat

diabetes and marketed. Siddha system does not advocate the use of Gymnema

sylvestre for treating diabetes. However, the other plants such as Terminalia chebula

and Syzygium cumini have been widely used in Siddha preparations to cure diabetes.

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The Unani Tibb system of medicine is traced to the system of Greek medicine

developed during the Arab civilization and is also called the Greco-Arab system.

Though the European historians call it Arab medicine, now it is prevalent in India,

Pakistan and Bangladesh. Detailed general information on various aspects of the

Unani system of medicine has been reviewed by Baquai, (1977), Said (1983) and

Bhat and Rao, (1993). A very useful glossary of plants used in Unani medicine has

been compiled by Fathima (1994), which includes sources of therapeutic information.

Emblica officinalis has been used as herbal remedy to treat diabetic retinopathy based

on Unani. The tannoids extracted from this plant inhibit aldose reductase, which is

known to be involved in the secondary complications of diabetes such as cataract

(Suryanarayana et al., 2004). Various plants have now been shown to affect

glycaemia. Fenugreek leaves have also shown to affect hyperglycaemia,

hypoinsulinaemia and glycosylated haemoglobin levels in diabetic rats. Key

metabolic enzymes were affected and the effect appeared to be similar to that of the

drug glibenclamide (Devi et al., 2003). The anti-hyperglycaemic effects of three

extracts of the bitter gourd Momordica charantia have been successfully documented

(Virdi et al., 2003). The aqueous extract of the unripe fruit reduces fasting blood

glucose by 48 %, which is equivalent to the figures obtained with glibenclamide.

Anti-diabetic effect of Costus pictus leaves in normal and streptozotocin-induced

diabetic rats has been reported and the leaf extracts do not have any toxicity effect

(Jayasri et al., 2008).

Plant products investigated for anti-diabetic effect have been exhaustively reviewed

and the inhibitory effect of some of the common plant extracts against hyperglycemic

response of anterior pituitary extract in glucose fed rats has been compared (Bnouham

et al., 2006; Garg, 2009). Many ethnobotanical (Atta-Ur-Rahman and Zaman, 1989;

Al-Rowais, 2002; Lin, 1992; Mahabir and Gulliford, 1997) and traditional medicinal

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plants (Day, 1998; Bailey and Day, 1989; Gupta, 1993) reports on antidiabetic

medicinal plants of the local population have been performed in different parts of the

world. Many studies have confirmed the benefits of medicinal plants with

hypoglycemic effects in the management of diabetes mellitus. The effects of these

plants may delay the development of diabetic complications and correct the metabolic

abnormalities. Moreover, during the past few years some of the new bioactive drugs

isolated from hypoglycemic plants showed antidiabetic activity with more efficacy

than oral hypoglycemic agents used in clinical therapy (Bnouham et al., 2006). It has

been reported that some of the plant extracts restrict the rise of blood sugar caused by

the pituitary hormones responsible for inhibiting peripheral utilization of glucose as

well as causing glycogenolysis in maturity onset diabetes (Garg, 2009).

Leaf extract of Gymnema sylvestre with its constituent gymnemic acid inhibits the

stress responses mediated through the adrenohypophyseal axis 74 as well as the

hyperglycaemic response to adrenaline, corticotropin and somatotropin (Gupta and

Variyar, 1964). This might help in unrestricted utilization of glucose by the peripheral

tissues. Tinospora cordifolia has been found to inhibit hepatic glucose release caused

by adrenaline, (Gupta et al., 1967) while Coccinia indica, Casearia esculenta and

Pterocarpus marsupium besides reducing blood sugar have also been reported to

block glucose absorption from the gut. A flavanoid-epicatechin isolated from P.

marsupium has been reported to promote regeneration of beta cells of islets of

Langerhans in pancreas and on clinical trials beneficial effects have been reported in

maturity onset diabetes (Shanmugasundaram et al., 1990b). Tinospora cordifolia and

Casearia esculenta have been reported that they promote insulin induced glucose

uptake by the tissues and improve glucose tolerance, (Gupta et al., 1967) while M.

charantia also promotes peripheral utilization of glucose. The fresh juice of M.

charantia has been reported to potentiate tolbutamide actions and a crystalline

fraction isolated from fruits of this plant possesses hypoglycemic effect similar to

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insulin in juvenile diabetes. Many herbal products including several metals and

minerals have been described for the care of diabetes mellitus (Chattopadhyay, 1998a;

Hakkim et al., 2007; Garg, 2009).

Allopathy

Allopathy is also called ‘modern’, ‘western’, or ‘scientific’ medicine. Allopathy is

now both biomedicine and clinical medicine. Developments of the past 50 years or so

in anatomy, biochemistry, physiology, pharmacology, physics, biology, electronics

and engineering have made allopathy radically different not only from the other

systems of medicine but also from its own earlier versions. Descriptions of several

disease conditions like diabetes in the earliest works on Allopathy treatments are also

found in the Vedic hymns of the predecessors of Ayurveda (Canary, 1983).

Allopathy, with very impressive advancements in its numerous areas of specialization,

has performed wonders and achieved a phenomenal popularity throughout the world,

much to the detriment of the indigenous/traditional systems of medicine. However, it

has become increasingly inaccessible to the majority of the world population due to

its dependence on expensive instrumentation and very high costs of drugs and

services. Allopathy is firmly rooted in the products of synthetic chemistry, as its drug

arsenal. Nevertheless, in the rich world 25 per cent of all medical drugs are still plant

based and in the poorer world this is closer to 75 per cent (Principe, 1991). Allopathy

will continue to depend on plants for its drugs and this dependence is more likely to

increase rather than decrease. Thus Allopathy continues to be an area of interest to

those who work on medicinal plants.

A number of allopathic chemical drugs are being used to treat diabetes and are

available in the market. Various oral antidiabetic drugs have been used in the control

of hyperglycemia. The drugs administered are basically classified into Sulphonylureas

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and nonsulphonylureas (ICMR, 2005). Second generation Sulphonylureas drugs are

currently used in the treatment of diabetes. A few Sulphonylureas drugs used are

glipizide, glibenclamide, glipizide XL, gliclazide MR and gliclazide. The duration of

these drugs ranges between 6 and 24 h. The other groups of drugs are the non-

sulphonylurease drugs including repaglinide, metformin, phenformin, acarbose and

other drugs are currently in use (ICMR, 2005) to treat diabetes.

SIDE EFFECT OF DRUGS

One of the persistent challenges faced by professionals and regulators is on obtaining

information regarding adverse effects potentially associated with herbal medicines

and then, sharing this information in a meaningful way with the public. A much

repeated and significant charge against modern medicine is that, it is fraught with

serious side effects and the defense of traditional medicine is that, it has no side

effects. A side effect of a medicine is the one other than the therapeutic effect desired

in a particular context. Thus, because of less or no side effects with plant based

medicines there is a great deal of revival in usage and drug discovery (Mosihuzzaman

and Choudhary, 2008).

Most of the chemical drugs have been reported to cause side effects by causing cell,

tissue, organ and whole body to react in abnormal condition. A single chemical

compound would have one or several effects on the body and was aimed at using the

most appropriate effect in a given context (disease state). Side effects are also caused

by the administration of an improper drug or by a wrong dosage. All drugs have

certain contraindications, when they should not be used.

Recently the herb Ephedra sinica has received substantial scrutiny regarding its

adverse effect profile. The adverse effect of this plant has been explicitly documented

through legitimate clinical trials (Kingston and Blumenthal, 2003). Ginkgo products

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were found to contain colchicine (26 µg per tablet) which was observed in blood

samples of pregnant women (Petty et al., 2001).

Since the action of all drugs, whether modern or traditional, is rooted in chemical

interactions between the drug and the body, all drugs, from whatever the system of

medicine, have side effects, small or big. One needs to understand the complete

picture of action of drug in each case to avoid, or at least minimize the side effects

and derive the desired therapeutic benefits.

Ayurvedic drugs were not given much emphasis to evaluate the adverse side reactions

and genotoxicity effect. The efficacy of some herbal products is beyond doubt.

Mutagenic and carcinogenic effects have been reported in some of the herbal

preparations (Dubey et al., 2004; Shah, 1997). In general if a drug is said to combat

severe disease, it must be reasonably powerful. Such drugs will naturally have some

dangerous side effects and after effects. Certain Siddha medicines, particularly more

effective ones consist of metals, minerals and other poisonous ingredients. There is a

chance of these ingredients turning more dangerous than the disease (Pillai, 1979).

Some plants have been used for hundreds of years together as in India and China to

treat diabetes and still they are considered as safe and efficient. Though, it is highly

necessary that more research is needed to understand in most cases, the active

principles and mode of action against diabetes (Vogel, 1991; Patwardhan et al., 2004;

Garg, 2009).

Indiscriminate use of crude extracts and over doses of nature medicines have lead to

several severe damages and unknown consequences including irreparable mutations

(Bhisey, 2000). Thousands of tonnes of dried plant materials are sent every year to the

developed countries for this purpose. International export trade in medicinal plants

has been dominated by China which exported 121 900 tonnes a year and India which

exported 32 600 tonnes a year (Rajasekharan and Ganeshan, 2002). More number of

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researchers and institutions need to be seriously involved in medicinal plants research

and development, not only for the intellectual challenges involved but also the huge

possible profits obtainable over a period of time (Latif, 1997; Osman, 1996; Rates,

2000).

PLANTS SELECTED FOR EXPERIMENTATION IN THE PRESENT STUDY

A large number of plants have found application to treat diabetes. List of antidiabetic

plants as mentioned earlier show that more than 23 of plants used by traditional health

practitioners in the treatment of diabetes. The following three plants viz, Gymnema

sylvestre, Syzygium cumini and Terminalia chebula are used by different systems for

which not much information is available on the safety and efficacy of these plants and

hence the present study was envisaged.

Gymnema sylvestre (Retz) Sm.

Gymnema sylvestre is a large woody climber, belonging to the family Asclepiadaceae.

The climber is extensive, much-branched and some times twining shrubs. Leaves 3-6

x 2-3 cm, simple opposite, ovate or elliptic-oblong, apiculate, rounded at base, sub-

coriaceous. Flowers minute, greenish-yellow, spirally arranged in lateral pedunculate

or nearly sessile cymes. Corolla lobes imbricate. Flowering: August- November;

Fruiting: April – June. Fruits a pair of follicles, up to 8 x 0.7 cm, terete, lanceolate,

straight or slightly curved, glabrous. Seeds ovate-oblong, glabrous, winged brown

(Gamble, 1928; Mabberley, 2005).

This plant grows in the wild as a climber. It is found in the plains in scrub jungles and

in thickets, distributed in the tropical forests of central and southern India, Western

Ghats, Konkan, Deccan peninsula, Assam, extending to parts of northern and western

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India and some parts of Africa. This has been occasionally cultivated as a resourceful

medicinal plant.

Sushruta describes Gymnema sylvestre as a destroyer of madhumeha (glycosuria) and

other urinary disorders (MacFarlance et al., 1997). The plant is stomachic, emetic,

expectorant, stimulant, laxative, diuretic, refrigerant, astringent and tonic. It is also

said to be useful in cough, biliousness and sore eyes. The leaves when chewed have

the remarkable property of paralyzing the sense of taste for sweet and bitter

substances for few hours (Gamble, 1928; Chopra et al., 1956; Warren et al., 1969).

On account of its property of abolishing the taste of sugar it has been named as

‘gurmar’ meaning ‘sugar destroyer’ and it is believed therefore that it might neutralize

the excess of sugar present in the body as in Diabetes mellitus (Warren et al., 1969).

Even though there is no scientific evidence for the antidiabetic effect of G. sylvestre

the plant has found its continuous usage in the modern age. It has been reported to

increase urine output and reduce hyperglycemia in both animal and human (Bhakuni

et al., 1971).

Phytochemical Components of Gymnema sylvestre

G. sylvestre constituents include two resins (one soluble in alcohol), gymnemic acids,

tartaric acid, gurmarin, calcium oxalate, glucose, saponins, stigmasterol, quercitol,

and the amino acid derivatives betaine, choline and trimethylamine. Many compounds

of G. sylvestre including gymnemic acid are derivatives of triterpene glycosides. It

has long been known that chewing G. sylvestre leaves is a folkloric remedy for

diabetes mellitus in India, and causes temporary loss of the sweet taste (Liu et al.,

1992; Warren et al., 1969). The antisweet principle was precipitated by acidification

with mineral acid was named as ‘gymnemic acid’ (Liu et al., 1992). Antisweet

property of gymnemic acid can be destroyed by boiling with dilute hydrochloric acid.

The effect of antisweetness of gymnemic acid varies from species to species even in

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mammals (Glaser et al., 1984; Hellekant, 1976; Hellekant and Gopal, 1976). A new

substance gurmarin was isolated from leaves of G. sylvestre. This is a polypeptide

which consists of 35 amino acid residues including three intramolecular disulfide

bonds. Suppression of sweet taste responses in the rat was found to be due to the hot

water extract of the leaves of G. sylvestre (Imato et al., 1991). The inhibitory effect of

gurmarin was highly specific to the sweet taste response to sucrose, glucose, glycine

and saccharin so that the response to the salty taste of NaCl, the sour taste of HCl and

the bitter taste of quinine were hardly affected. The inhibitory effect of gurmarin was

reversible, but complete recovery was studied in rats and the suppressed response

required approximately 2 hours for recovery (Ninomiya and Imoto, 1995).

Gurmarin suppresses the sweet taste response in rats. It is interesting to know that

gurmarin showed either a weak or no effect on the sweet taste sensation in human

beings. Besides gymnemic acid, certain other materials that reduce sweet taste

recognition are known. They include ziziphin from Ziziphus jujube (Meisalman et al.,

1976), another compound hodulcin from Hovenia dulcis and escin from Aesculus

hippocastanum (Miyasaka and Imoto, 1988).

Antidiabetic Activity

G. sylvestre has been widely used in many traditional and folk medicines except for

Siddha to treat diabetes mellitus although its use in Ayurvedha is known from

antiquity. Several studies have been conducted and reported on the antidiabetic

properties and efficacy of G. sylvestre (Baskaran et al., 1990; Shanmugasundaram et

al., 1990a; Murakami, 1996; Chattopadhyaya, 1998). The choice of drug was mostly

crude extract of leaves (Rastogi and Dhawan, 1982) which contains about 70 % of

gymnemic acid. So far no study was performed with isolated pure gymnemic acid.

For such studies only animal models were used to assess the cytotoxic and genotoxic

properties. The mode of treatment had been mostly intra peritoneal and rarely oral

21

feeding (Gupta and Seth, 1962). Gymnema was singly or combination with other

antidiabetic plants such as Momordica charantia, coccinia indica and Pterocarpus

marsupium were tested (Gupta, 1963). Gymnema leaves whether extracted or infused

in to a tea suppress glucose absorption and reduce the sensation of sweetness in foods-

effects that may deliver important health benefits for individual who want to reduce

blood sugar level or body weight. Modern dietary supplements containing G. sylvestre

are typically intended for control of sugar cravings, and weight loss, particularly in

patients with diabetes (Shanmugasundaram, 1990).

Pharmacological, phytochemical and pharmacognosical and clinical trials on G.

sylvestre have been reviewed recently (Gurav et al., 2006). Pharmacological studies

have revealed that the water extracts of the leaves of G. sylvestre was effective to treat

diabetes mellitus (Potwale et al., 2008). Similarly water extract of G. sylvestre leaves

inhibited the absorption of glucose in the small intestine and suppressed the increase

of blood glucose value after administration of sucrose in rat (Miyoshi et al., 1987).

Animals treated with ethanol extract of G. sylvestre at a dosage of 100 mg/kg body

weight resulted in insignificant reduction of blood sugar in normal rats, and

significant reduction in anterior pituitary treated hyperglycaemic rats. Effect of the

drug was comparable to that of tolbutamide (50 mg/kg) in the hyperglycaemic rats.

The drug had influenced the disturbed carbohydrate metabolism in hyperglycaemic

animals by limiting the carbohydrate turnover and thus inhibiting the vicious cycle of

hyperglycaemia (Gupta and Seth, 1962). Further studies on albino rats established the

antidiabetic activity of G. sylvestre, which was compared with other conventional

indigenous oral antidiabetic drugs like Coccinia indica, Pterocarpus marsupium,

Momordica charantia. In such a study animals were subjected to subcutaneous

injection with 100 mg/kg dose of the anterior pituitary extract and fed with alcoholic

extract of G. sylvestre and Coccinia indica (100 mg/kg each), aqueous infusion of P.

marsupium (20 ml/kg), extract of M. charantia (5 ml/kg) and tolbutamide (50 mg/kg)

22

orally. Inhibition of the hyperglycaemic response of the anterior pituitary extract at 6,

12 and 24 hours was most marked in tolbutamide and G. sylvestre. The inhibitory

effect was highly significant in G. sylvestre when compared with P. marsupium and

M. charantia (Gupta, 1963).

The importance of G. sylvestre therapy in alloxan induced diabetic rabbits was

investigated by feeding crude leaf powder at a dosage of 250 mg/kg body weight once

a day. This therapy not only produced blood glucose homeostasis but also increased

the activities of enzymes affording the utilization of glucose by insulin dependent

pathways; it controlled phosphorylase levels, gluconeogenic enzymes and sorbitol

dehydrogenase. The uptake and incorporation of glucose into the glycogen and

proteins had increased in the liver, kidney and muscle in diabetic animals when

compared to control diabetic animals (Shanmugasundaram et al., 1983). Prakash et al.

(1986) have reported that the rats fed with G. sylvestre leaf powder during the diet for

10 days prior and 15 days after the treatment of beryllium nitrate significantly

protected the animals from the full fall of blood glucose.

The inhibitory effects of G. sylvestre and purified gymnemic acid on Gastric

Inhibitory Peptide (GIP) release was studied in rats. The GIP gets released into the

portal vein in response to duodenal infusion of D-glucose in presence of leaf extract

of G. sylvestre at a dosage of 0.5 ml/kg. The inhibition of GIP release by gymnemic

acid was attributed to the interaction with the glucose receptor for GIP release which

was similar in specificity to the active glucose transport system. The results suggested

that a glucose receptor interacts with the leaf extracts of G. sylvestre and purified

gymnemic acid (Fushiki et al., 1992).

Gymnemic acid is referred to as GS. Two water soluble extracts GS3 and GS4 obtained

from the leaves of G. sylvestre, were tested in streptozotocin treated rats for their

23

effects on blood glucose homeostasis and pancreatic endocrine tissue. In diabetic rats,

fasting blood glucose levels returned to normal after 60 days of GS3 and after 20 days

of GS4 oral feeding. Administration of 10 g /ml of gurmarin significantly depressed 40

- 50 % of taste response ability to sugar and saccharin in rats. In diabetic rat pancreas,

both therapies led to a rise in serum insulin to levels close to normal testing levels.

Both GS3 and GS4 doubled the islet number and beta cell number in the pancreas of

diabetic rats. This herbal therapy appeared to bring about blood glucose homeostasis

through increased serum insulin levels provided by repair of the endocrine pancreas

(Shanmugasundaram et al., 1990a).

Clinical Trials

As G. sylvestre has been known to control diabetes in traditional systems of medicine,

little scientific validation has been done to ascertain the fact by conducting clinical

trials with diabetic patients (Baskaran et al., 1990). All such trials were performed

using either crude extracts or whole leaf powder among the clinical trials. The results

of such trials do support the antidiabetic properties of G. sylvestre either by increased

hypoglycemic activity or enhanced insulin levels to certain extent. Khare et al. (1983)

reported the hypoglycemic activity of G. sylvestre in ten normal and six diabetic

patients. They were subjected to glucose tolerance test and their blood samples were

collected at 30 minutes intervals up to 2 hrs. Aqueous decoction of the leaves was

administered at a dosage of 2 g thrice a day for a period of 10 days. Administration of

the extract brought about a significant reduction in the fasting blood sugar levels in

normal and diabetic patients, which suggested a definite hypoglycemic activity.

Similarly a clinical trial conducted on 27 patients with insulin-dependent diabetes

mellitus (IDDM) with the administration of GS4 - a water soluble extract of the leaves

of G. sylvestre at a dosage of 400 mg/day for each patient. The level of insulin got

reduced together with fasting blood glucose and glycosylated haemoglobin (HBA1c)

24

and glycosylated plasma protein levels, while serum lipids returned to near normal

level with GS4 therapy. Treatment with GS4 appeared to enhance endogenous insulin,

possibly by regeneration or revitalization of the residual beta cells in insulin-

dependent diabetes mellitus (Shanmugasundaram et al., 1990 b).

Further clinical studies were conducted to test the effectiveness of GS4 therapy an

extract from leaves of G. sylvestre in controlling hyperglycemia in 22 patients with

Type –II diabetes (NIDDM -Non-insulin dependent Diabetes mellitus) on

conventional oral anti-hyperglycemic agents. GS4 (400 mg/day) was administered for

18 - 20 months as a supplement to the conventional oral drugs. During GS4

supplementation, the patients continued to show a significant reduction in blood

glucose level. Five of the 22 diabetic patients were able to discontinue their

conventional drug and maintain their blood glucose homeostasis with GS4 alone.

These evidences suggest the beta cells regeneration and repair in type-II diabetic

patients on GS4 supplementation. This is also supported by the appearance of raised

level of insulin in the serum of patients after GS4 supplementation (Baskaran et al.,

1990).

In another study hypoglycemic effect of G. sylvestre was studied in 16 normal non

diabetic persons and 43 mildly diabetic patients. All the subjects were administered

with leaf powder 10 g per day in divided doses for a period of 7 days. The results

indicate that G. sylvestre leaf powder has a hypoglycemic effect comparable to

tolbutamide. Serum triacylglycerol, free fatty acids and cholesterol levels in normal

subjects were unaffected where as in diabetic patients it was significantly decreased.

Ascorbic acid and iron levels were elevated significantly in both groups, where as

excretion of creatine decreased in diabetic patients, this remained unaffected in

normal healthy volunteers (Balasubramaniam et al., 1992). Absence of antidiabetic

25

and hypolipidemic effect of G. sylvestre in non-diabetic and alloxan-diabetic rats has

also been reported (Galletto et al., 2004).

Syzygium cumini (L.) Skeels

Syzygium cumini (= Eugenia jambolana Lam) is a big tree belonging to Myrtaceae

family, native to southern Asia. This is a smooth tree 4 to 15 m in height. The leaves

are leathery, oblong-ovate to elliptic or obovate-elliptic, and 6 to 12 cm long. The tip

of the leaf is broad and shortly pointed. The panicles are borne mostly from the

branchlets below the leaves, often being axillary or terminal, and are 4 to 6 cm long.

The flowers are numerous, scented, pink or nearly white, without stalks, and borne in

crowded fascicles on the ends of the branchlets. The calyx is funnel-shaped, about 4

mm long, and toothed. The petals cohere and fall all together as a small disk. The

stamens are numerous and about as long as the calyx. The fruit is oval to elliptic, 1.5

to 3.5 cm long, dark-purple or nearly black, luscious, fleshy, and edible; it contains a

single large seed.

The bark of the tree is used against dysentery, hemorrhage and leucorrhea. It is also

used to treat non-insulin dependent Type-II diabetes, because it lowers the blood

glucose level to normal (Moreira, 1985). It is used to treat diarrhea and infections

from the upper-respiratory-tract since it has an antimicrobial property. The seeds and

fruits of this plant have been frequently used to treat diabetes. It has been shown that

the bark, fruits, seeds or leaves of this plant collected from diverse regions of the

world are administered in different pharmaceutical preparations eg., tinctures and

aqueous extracts. Also, infusions (simple aqueous extracts prepared with hot water

but without boiling) and decoctions (boiled infusions) of S. cumini have been used in

popular medicine for the treatment of diabetes mellitus (Elisabetsky, 1987; Pepato et

al., 2001).

26

Phytochemical Components of Syzygium cumini

The seeds do not contain an alkaloid nor an enzyme, but abundance of starch and

tannin. The seeds and bark contain tannins, resins (gambol), terpenes (�-pigeon, �-

pigeon, limonene), acids (gallic, palmitic, stearic, oleic), steroids (phytosterol),

saponinic glycosides (antimelin) and flavanols (Albuquerque, 1989). The interesting

constituent of the resin, however, is a new phenolic substance possessing the

empirical formula C16

H8O

9, which has been designated as ‘Jambulol’. This substance

is a light-brown powder, which is insoluble, or nearly so, in the usual organic

solvents, but separates on crystallization. Another substance acetyljambulol,

C16

H3O

9(CO.CH

3)5, forms pale brown platelets. Pentabenoyl jambulol,

C16

H3O

9(CO.C

6H

5)

5, was obtained in small colorless plates (Anon, 1986).

Antidiabetic and genotoxic properties of Syzygium cumini

Achrekar et al. (1991) have reported the hypoglycemic activity of the extract of

Syzigium cumini seeds and fruits produced the same effect after 24 hrs. The oral

administration of the extract resulted in the enhancement of insulinemia in

normoglycaemic and diabetic rats. The incubation of isolated pancreatic islet cells of

normal and diabetic animals with this plant extracts resulted in increased insulin

secretion. This extract inhibited insulinase activity from liver and kidney. Conversely

oral administration of 2.5 and 5.0 g/kg body weight of the aqueous extract of the

seeds of S. cumini for six weeks in alloxan-diabetic rats resulted in a significant

reduction in blood glucose concentration and an increase in total haemoglobin, but in

the case of 7.5 g/kg body weight, the effect was not significant. It also resulted in

decreased free radical formation in tissues (Prince et al., 1998). The isolated

compound mycaminose and crude extracts of S. cumini seeds in ethyl acetate [EA]

27

and methanol [ME] were reported for the anti-diabetic activity against streptozotocin

(STZ) induced diabetic rats. The compound ‘mycaminose’, crude extracts and

methanol extracts were shown to cause significant reduction in blood glucose level

and the anti-diabetic effects of the seed extracts were authenticated (Kumar et al.,

2008; Rajasekar et al., 2009). In human phytotherapy about 1.9 g of seed powder or

30 seeds per day was recommended as daily dose. LD-50 values were determined as 4

g/kg bw for oral administration and 0.4g/kg bw for intra peritoneal administration in

mice (EMEA, 1999). Genotoxic assessments were reported by Vicentini et al. (2001)

where very mild dose of 0.07mg/ml and 0.7mg/ml were injected intraperitoneally. At

these concentrations S. cumini neither induced chromosomal damage nor altered the

cell division cycle in both plant and animal models.

Terminalia chebula (Gaertner) Retz.

Terminalia chebula belongs to the family Combretaceae. It is a flowering evergreen

commonly known as the black myrobalan. It is also known as Haritaki (Sanskrit and

Bengali), Harad (Hindi), Karkchettu (Telugu), Kadukkai (Tamil), and Harada

(Marathi and Gujarati). It is native to Indian subcontinent and the adjacent areas such

as Pakistan, Nepal and the South-West of China and Sri Lanka where it is called

Aralu. The flowers are monoecious, dull white to yellow, with a strong unpleasant

odour, borne in terminal spikes or short panicles. The fruits are glabrous, ellipsoid to

ovoid drupes, yellow to orange brown in colour. The fruits give a valuable tannin

material and a yellow dye. T. chebula is found throughout the deciduous forests of the

Indian subcontinent, on dry slopes up to 900 m elevation (Gamble, 1928;

Chattopadhyay and Bhattacharyya, 2007).

Phytochemical Components

28

The presence of large amount of tannins in the fruit and bark is a characteristic feature

of this genus (Evans, 1996). The chief constituents of tannin are chebulic acid,

chebulagic acid, corilagin and gallic acid and it is of pyrogallol (hydrolyzable) type

(Bruneton, 1995; Chevallier, 1996). Fructose, amino acids, succinic acid,

betasitosterol, resin and purgative principle of anthroquinone and sennoside are also

present (Creencia et al., 1996). The tannin content of T. chebula varies with

geographical gap (Jayaramkumar, 2006).

Antidiabetic and genotoxic properties of Terminalia chebula

The seeds of this tree are used in the treatment of diabetes. The fruits were reported to

have dose dependent reduction in blood glucose of streptozotocin induced diabetic

rats both in short term and long term study and also had retinoprotective activity (Rao

and Srinavas, 2006; Periasamy et al., 2006). Methanolic extract of T. chebula, T.

belerica and Emblica officinalis in combination named ‘Triphala’ (equal proportion of

above three plant extracts) are being used extensively in Indian system of medicine.

They are known to inhibit lipid peroxide formation and to scavenge hydroxyl and

superoxide radicals in vitro. Oral administration of the plant extracts reduced the

blood sugar level in normal and in alloxan induced diabetic rats significantly within 4

hrs (Sabu and Kuttan, 2002). Arseculeratne et al. (1985) have stated that feeding trials

in rats with T. chebula produced hepatic lesions that included central vein

abnormalities and marked renal lesions. Cytotoxicity and genotoxicity studies based

on Ames test were reported by Mehmood and Mohammad (1998) and Arora et al.

(2005). In both the studies no cytotoxic and genotoxic effects of T. chebula was

reported.

TOXICOLOGY AND GENOTOXICITY ASSESMENTS

29

Since time immemorial, toxins in general have been recognized by the human beings,

and used both beneficially and destructively. Like morphine and other pain relievers,

toxins have often been used in medicine to combat various diseases or disorders.

From the ancient Vedic time, toxins in various herbs and plants have been most

thoroughly studied by Indian herbal physicians (known as the Vaidyas) and their

acquired knowledge (Ayurveda) is still a yardstick for the (western as well as oriental)

medical system; the basic difference between the two lies in the accidental use of

toxic chemical compounds (inorganic toxins) instead of the organic toxins in herb and

plants. The entire foundation of the homeopathic medicinal system is based on the

impact of toxins on human body as well as mind, and is a classical example, how a

form of a toxic element can be used to nullify some other toxicity in our body.

Although there are some controversies over the doctrine of homeopathic medicine

system, there is no doubt about the neutralizing capacities of toxins of different kinds.

Toxins are characterized by their toxicity, in some form or the other, in relation to

their reactions on living organisms including man. In a traditional sense, toxicity

relates to the poisonous effects which are associated with specific substances such as

potassium cyanide. Rather, the more complex types that often progress slowly and

invisibly, and in that process invite other activities, such as genotoxicity and

carcinogenicity, have a far greater need for statistical appraisal (Sen, 2001).

Genotoxicity assay is a useful technique in ascertaining the genetic damage directly or

indirectly caused to organisms by any chemical and physical agent. There is a

growing concern about the genotoxic effects induced by a number of chemical

substances such as herbicides, pesticides, synthetic medicines, herbal medicines,

cosmetic products, food and dietary supplements as we use in every day life. The

assessment helps to identify the interaction of physical and chemical agents with the

genetic material of living organisms. Chemical substances which induce cytotoxicity,

genotoxicity and chromosomal damage are designated as genotoxins (Grant, 1978;

30

ADA, 1997; Albertini et al., 2000). Genotoxic monitoring finds its way in assessing

the limitation in usage of chemical substances. Genotoxic responses focus on

cytological end points like chromosomal aberrations (CA), micronucleus (MN) and

sister chromatid exchange (SCE) (Bolognesi, 2003). Genotoxicity assessments have

been carried out by methods which include apoptosis (Kerr et al., 1972),

micronucleus assay (Schmid, 1975) and higher plant genetic system (Mazrooei and

Kabarity, 1984), animal models (Sing et al., 2003) and Ames test using microbes (Lah

et al., 2005), DNA fragmentation (Gavrieli et al., 1992), and the advanced technique -

comet assay (Singh et al., 1988).

Moreover, such toxicity studies at the molecular level may sometimes provide clues

for cancer etiology, and modern molecular biology has indeed come up with a

challenging task to incorporate genetic toxicology in deeper understanding of

progression of genotoxicity in living organisms (MacGregor, 2003).

Genotoxicity Assay Using Plant Models

Genotoxicity occurs commonly in human beings due to the continuous usage of

clastogenic substances either from nature or synthetically manufactured. Assays for

genotoxicity are used as predictors or biomarkers of disease, especially cancer (Hulka

and Margolin, 1992; Sen et al., 1995). For the evaluation of genotoxicity, higher

plants have been used as test organisms. Allium cepa is considered as one of the best

models to detect the chromosomal aberrations induced by different agents (Levan,

1938). Apart from Allium cepa, mutagenic activity of various chemicals including

medicines (Mazrooei and Kabarity, 1984), herbicides (Yoshida et al.,1983), pesticides

(Bolognesi, 2003) and herbal drugs (Sreeranjini and Thoppil, 2001) have been

analyzed in different plant systems such as Vicia faba, Arabidopsis thaliana and

Hordeum vulgare. Plant extracts have been found to cause some chromosomal

31

aberrations because of the presence of strong chemical substances which inhibit

mitosis (Sreeranjini and Thoppil, 2001).

The chromosome abnormalities produced by copper fungicides in somatic cells in

plant models have been documented (Tara Mohan, 1979; Sahu et al., 1981). Topsin a

fungicide has been used to control the fungal infections in crop plants. Even at lowest

concentration (100 µg/ml) topsin has induced severe antimitotic effect. This also

interferes with the spindle fibers and produces C– metaphase (Somashekar et al.,

1984). Chromotoxic activity and mitodepressiveness in crop plants have been widely

reported. Turbutryn is an herbicide used to eradicate a number of dicotyledonous

weeds. This has induced a wide range of chromosomal abnormalities in the root tips

of Vicia faba (Badr, 1986).

Herbicides have also been reported to cause chromosomal abnormalities by producing

stickiness, spindle poisoning and alterations in the number of chromosomes (Liang et

al., 1967). The herbicide carbamate is known to cause disorganized chromosomes in

root tip cells of Vicia faba. Yoshida et al. (1983) have reported that increase in the

duration of treatment using carbamate inhibits the growth of root tip cells and

autoradiographic studies revealed suppression of RNA synthesis.

Majority of insecticides have a direct or an indirect effect on plant systems.

Metasystox -R is an organophosphate contact insecticide, which controls sap feeding

insects and mites. The potential cytogenetic activity of this insecticide and pollen

fertility was evaluated in Allium cepa where in a number of somatic cytogenetic

abnormalities have been reported (Pandita, 1986).

Zinc is an essential element for the growth of plants. The toxicity of zinc in Nigella

sativa and Triticum aestivum was studied in concentrations higher than 25 mg/l.

32

Reduction in mitotic index in T. aestivum was more evident than in N. sativa. A

variety of mitotic anomalies including C–metaphase, lag chromosome and multipolar

anaphase were frequently observed (EL-Ghamery et al., 2003).

Administration of synthetic drugs has become a serious problem in human society. A

number of analgesics are presently being used on medical dependents. Parahypon is a

strong analgesic which is largely made up of paracetomol and caffeine. In Allium cepa

dividing and non dividing cells and their nucleic acid synthesis were inhibited due to

parahypon treatment (Mazrooei and Kabarity, 1984).

Plant substances have also been found to induce abnormalities in chromosomes

resulting in cytotoxic or genotoxic effects in plant models. Spilanthes ciliata is a

common weed from which an oil spilanthol is derived and used to treat toothache.

Severe mitotic inhibition and several other aberrations were produced by this plant

extract in Allium cepa (Sreeranjini and Thoppil, 2001). Cytotoxic effect of leaf extract

of Ipomoea carnea on root tips of Trigonella foenum-graceum has been reported. The

leaf extract of this plant produced considerable abnormalities in the root meristem of

T. foenum-graceum and mitotic frequency was also inhibited (Anis et al., 1999).

Ophioglossum oil extracted from species Ophioglossum costatum has been found to

influence the mitotic cell division in Allium cepa and Vicia faba. A complete arrest in

the cell division has been observed indicating, a concentration that promotes

inhibition of growth in roots (Kandhelwal, 1986). The water extract of Ammi majus

seeds is found effective in the treatment of leucodermia and certain other skin

diseases. Extract of Ammi seeds caused delay in completion of mitotic cycle and

prominent abnormalities in the chromosomes (Shehab et al., 1984).

Genotoxicity Assay Using Animal Models

33

Wide ranges of genotoxic substances are present in the environment and influence

human health, thus many eukaryotic systems were developed for evaluation of DNA

damage. The response of plant cells used in bioassays should be as close as possible

to that of animal cells, and especially to human beings (Juchimiuk et al., 2006). For

cytogenetic tests, the commonly used cells are derived from Chinese hamster ovaries,

human lymphocytes and bone marrow cells of rats and mice.

Fungicides constitute an important group of environmental contaminants and

genotoxicants. Cytogenetic effect of a fungicide ediphenphos has been studied on the

bone marrow cells of mice. Dose, route and duration of exposure influence the

percent of aberrations (Bhunya and Behera, 1984). The genotoxic effect of pesticides

used in Italy has been documented in the lymphocytes obtained from 5 healthy donors

were nonsynchronous centeromeric separation was reported. Severe disturbance to the

spindle fibre was the reason for the centeromeric separation (Dolara et al., 1994). The

pesticide benomyl was also embryotoxic and teratogenic (Hogenboom et al., 1991;

Cummings et al., 1992). In human beings agricultural pesticides have increase the

frequency of sister chromatid exchange (Carbonell et al., 1990).

Genotoxic effect of lomefloxacin - an antidiabetic drug has been studied in germ cells

of Swiss albino mice. The capacity to reduce mitotic index and also increase

frequency of chromosomal aberrations was reported and the agent was projected as a

weak clastogen in the bone marrow cells as it did not have any genotoxic effect in

germ cells (Singh et al., 2003).

The clastogenic effect of Rosmarinus officinalis extract was studied in Wistar rats. No

significant activity was reported confirming that the extract was non cytotoxic (Gaiani

et al., 2006). 2-Hydroxy -1, 4-napthoquinone (HNQ) - a hair dye extracted from

34

Lawsonia inermis at about 20 g of leaf paste preparation or about 200-300 mg of

HNQ was used to analyze the genotoxicity effect in animal model. At higher

concentrations the plant based chemical induced significant level of genotoxicity

(Kirkland and Marzin, 2003). Similarly a natural food colour yielding annatto (Bixa

orellana) was also tested for mutagenic and antimutagenic properties. The results

concluded that based on the test in bone marrow cells of mouse, the dye was neither

mutagenic nor antimutagenic at lower concentrations. However, it has been cautioned

to have genotoxicity effects in higher concentrations (Lima et al., 2003).

The occurrence of micronucleus during cell division has been recognized as a

genotoxic assay. Chromosomal fragments and lagging chromosomes lead to the

formation of small nuclei covered by a membrane known as micronuclei (Schmid,

1975). Bisht and Uma Devi (1994) have reported the genotoxic effect and formation

of micronucleus due to Misonidazole – a radio sensitizer.

In the past two decades, the single-cell gel electrophoresis (comet) assay has become

a method of high esteem (Collins, 2004; Folkmann et al., 2008). This was a technique

originally developed for the measurement of DNA strand breaks, but further

modifications have increased the range of lesions that can be measured. This is a

simple, rapid, visual and reliable technique for the assessment of DNA damage in

organisms. This method was developed to measure low levels of strand breaks with

high sensitivity (Forcehhammer et al., 2008). The mutagenic, chromosomal damaging

and carcinogenic properties of several chemicals and mutagens were determined using

the comet assay employing blood cells of animal models (Collins et al., 1997; Rank

and Jensen, 2003). Induction of comet by certain genotoxic substances like hydrogen

peroxide, mitomycin, cycloheximide, ethyl methane sulphonate, nitroquinoline, 9 –

aminoacridine, N – nitroso-N-ethylurea, trypsin and 4-nitroquinoline oxide were

35

investigated. The assay was able to detect DNA damage induced by an alkylating

agent, an intercalating agent and oxidative damage (Henderson et al., 1998).

CHROMOSOMAL ABERRATIONS

The analysis of chromosomal aberrations (CA) was exclusively restricted to mitotic

cells arrested in metaphase, because only in this phase of the cell cycle the DNA is so

condensed that it can be observed as chromosomes under the light microscope

(Johnson and Rao, 1970). Chromosomal aberrations have long been recognized as an

important biomarker of genotoxic chemicals. Chromosomal changes leading to

mutations were first described in Oenothera by De Vries (1922). In earlier studies in

Drosophila, relatively a few translocations but numerous paracentric inversions were

reported (Morgan, 1922; Bridges, 1923). Both structural and numerical aberrations

have been associated with human chromosomes. The spontaneous frequency of

chromosome aberrations was about 0.6 % in live births. Chromosome analyses of

spontaneous abortions indicate that about 50 % of them are chromosomally abnormal

(Natarajan, 2002).

Most of the known mutagenic agents induce chromosomal aberrations. Thus, it is

important to understand the mechanism by which the chromosomal aberrations are

induced and exhibited. It has been generally agreed that DNA is the main target for

the action of the chromosome breaking agents. Several different types of lesions are

introduced in the DNA such as DNA strand breaks, base damages and DNA cross

links. The damage depends on the type of mutagenic agent employed (Natarajan and

Boei, 2003).

There are two main theories on the mechanism of chromosome aberration formation,

namely the breakage and reunion theory (Sax, 1941). According to breakage theory

the primary event caused by any mutagenic substance or agent is a chromatid or

36

chromosome break. The types of aberrations observed during mitosis depend on the

fate of chromosomal breaks. Either they restitute or rejoin (illegitimate fusion) with

another break if two breaks are close enough in space and time. If two lesions are

close together in space and time they may enter a more stable stage called exchange

initiation stage (Revell, 1959). The effect of a toxicant on chromosomes may be large

enough to be visible microscopically, and manifest as structural aberrations or as

changes in their number. Aberrations include deletions, duplications, and

translocations which result in the chromosomal anomalies such as gap, break,

fragment, bridge, polarity changes, abortiveness, lag and C-metaphase (Boei et al.,

1996).

Movement of nucleus towards the poles in different directions during division is

termed as affected polarity. The nucleus about to abort after division is called abortive

anaphase. Segregation of chromosomes in the middle is C-metaphase. A small gap

which appears in one arm of the chromosome is termed as chromatid gap. An

achromatic region from one of the chromatids, larger than the width of chromatid can

be called as chromatid break. A gap which is found in both the arms of a chromosome

which may be equal or smaller in size than the arm length of chromosome is called

chromosome gap. A chromosome break is a large gap found in both the arms of

chromosomes. A part of a material getting deleted from one of the chromatid is a

deletion. A single piece of a chromatid in the absence of an evident centromere is

called as a fragment. The two chromatids aligned parallel without a prominent

centromere is called as acentric fragment (Gaiani et al., 2006)

However, chemical mutagens which do not induce directly DNA strand breaks but

cause other lesions were shown to induce only chromatid type of aberrations

irrespective of the DNA synthesis stage treated. An intervening ‘S’ phase is necessary

to visualize the aberrations (Evans, 1969).

37

Genetic damage at the chromosomal level entails an alteration in chromosome

number or chromosome structure and such alterations can be measured as CA or MN

frequency in both plants and animals. Micronuclei are acentric chromosomal

fragments or whole chromosomes left behind during mitotic cellular division and

appear in the cytoplasm of interphase cells as small additional nuclei. In contrast to

the CA evaluation, the scoring of micronuclei in lymphocytes is simple and fast

(Bolognesi, 2003). Aneuploidy involves a decrease or increase in the number of the

chromosomes. Some of the effects are heritable. The mode of action underlying these

effects may involve molecular cross-linkage, which may cause an arrest of the

synthesis of DNA, thereby leaving a gap in the chromosome. An unsuccessful repair

of the DNA damage may also be responsible for the above mentioned anomalies.

Consequences of chromosomal aberrations, DNA Damage and Repair

Organisms exposed constantly to exogenous and endogenous mutagens are subjects

for DNA–damage in due course of time. In such cases DNA repair mechanisms have

evolved to repair different types of DNA damage and to maintain genomic integrity

(Ron, 1998). Multiple repair mechanisms have evolved in all organisms to minimize

the consequences of cellular exposure to endogenous and environmental agents that

inflict deleterious alterations in DNA. The versatile process of nucleotide excision

repair (NER) removes many structurally unrelated DNA lesions that can cause

mutations, abnormal differentiation patterns, cancer or cell death (Hanawalt et al.,

2003). Among the many types of damage that arise in DNA, the most dangerous are

the double strand DNA breaks (DSBs), where both phosphate back bones are not

repaired. Such damage can result in chromosomal defects and lead to inborn diseases

or cancer (Hartwell and Weinert, 1989). DNA repair is not merely an extraordinary

scheme needed by organisms that are exposed to DNA damaging ultraviolet

38

wavelength in sun light. Rather, it is an essential set of mechanism required to

maintain genomic stability in the face of a plethora of threats, deriving from

endogenous exposure to radiation and noxious chemicals (Friedberg et al., 1995).

The early research and discovery in DNA was on restriction endonucleases, enzymes

that protect various bacterial species from invading foreign DNA. The fundamental

research that led to the discovery of excision repair in E. coli in the early 1960s has

now developed to the point that the DNA repair is ubiquitous and essential for life.

Further more it has been learnt that DNA repair interferes in some manner with each

of the other cellular DNA transactions (Hanawalt, 1995).

Most chromosomal aberrations are believed to result in lethal events. It is known, how

ever, that some aberrations persist in cycling cells as stable events. Reciprocal

translocations have been found in some tumor types adjacent to known oncogenes and

in the case of colon cancer may compromise distinct to colon cancer (Vogelstein et

al., 1988). Increased levels of CA have been associated with higher cancer risk

(Hagmar et al., 1994). The fate of aberrant cells depends on the type of aberration

induced. Cells carrying unstable chromosomal aberrations (such as dicentrics and

fragments) are eliminated during subsequent cell divisions, whereas cells containing

stable aberrations such as translocations might proliferate for a long period (Boei et

al., 1996).

Impact of Mutations

Knowledge about mutation rates can shed light on issues relating to the mechanism

and basis of germ line mutation (Ellegren et al., 2003). Mutations in somatic cells or

germ cells of mammals often lead to a decrease in the fitness of the organism or its

offspring. Mutations in dividing somatic cells may cause cancer, whereas mutations

39

in germ cells may result in defective offspring. Human beings experience both

spontaneous mutations and induced mutations and are exposed to mutagens

throughout life. Mutagenesis can occur as a result of interaction between mutagenic

agents and the genetic materials of organisms. Source of mutagen exposure include

toxicants from nature, dietary constituents, industrial chemicals, medicines and drugs

(Malling, 2004).

The eventual effects of human exposure to these mutagenic substances cannot be

predicted at present. However, some of the spontaneous abortions, stillbirths, and

heritable diseases have been shown to be related to changes in DNA molecules and to

chromosomal aberrations. In earlier studies, mutagenic activity was demonstrated

mainly in fruit flies and onion root tips because of the simpler techniques involved.

More recently, many new test systems have been developed, which range from

complex microorganisms to intact mammals. The use of such widely different

organisms is based on the fact that all double – stranded DNA share the same

biochemical characteristics (Rank and Neilson, 1993; Grant, 1978; Niida and

Nakanishi, 2006). An increase in somatic mutations has been documented in aged

cells and tissues of both humans and mice (Harman, 1956; Ames et al., 1993). A

major issue in the field of genetic toxicology has been the question whether general

relationships can be established for adverse effects of genotoxic chemicals and cancer

and hereditary damage (Vogel and Natarajan, 1995.)

RATIONALE AND OBJECTIVES OF THE PRESENT STUDY

Gymnema sylvestre, Syzygium cumini and Terminalia chebula are some of the most

commonly used medicinal plants for treating diabetes in south India. The extracts and

crude powders are either prescribed individually or in combinations to combat

diabetes by the traditional systems of medicine and local medicinal practitioners.

40

Though, these three plants are known to have antidiabetic properties in traditional and

modern systems of medicine, their efficacy and safety have not been tested and

proved scientifically. Further, the dosages of individual or combined prescriptions

vary among practitioners and standardized doses are lacking by and large. The basis

of suggesting G. sylvestre in traditional practice may be attributed to transient

inactivity of taste buds. The other two plants are being used in treating diabetes for

their astringent properties.

Prior to commencing the experimental study, a random survey (Questionnaire

Enclosed in Annexure) was conducted among the diabetic patients to evaluate the

frequent usage of antidiabetic plants as well as the mode of uptake. Nearly 70 % of

the diabetic patients rely on the above mentioned three plants for controlling sugar

level following prescriptions given by traditional healers and physicians. Hence, the

present study was envisaged to determine the genotoxic properties of above

mentioned antidiabetic plants.

The objectives of the investigation are as follows:

• To asses the genotoxicity of selected antidiabetic medicinal plants

• To determine the chromosomal aberrations through mitotic analysis in

Allium cepa

• To analyze the chromosomal aberrations and micronucleus induced by

plant extracts in bone marrow and blood cells of Wistar rats

respectively

• To establish the ideal concentration of plant extracts for safe usage

• To understand the DNA damage due to plant extracts through comet

assay

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• To detect the occurrence of colchicine like compounds if any among

the plant extracts tested