MOLECULAR CHARACTERIZATION OF GYMNEMA SYLVESTRE ECOTYPES THROUGH RAPD ANALYSIS

ABSTRACT
Gymnema Sylvester is a large, stout, woody, vine-like plant which climbs on bushes and trees. It is known in Sanskrit- meshashiringi, macho machining (macho=sugar, noshing= destroy), in Telugu- podapatri, in Hindi- guru-mar. The latin name of Gymnema Sylvestre means sugar destroyer and is considered a herbal remedy for high blood sugar. Traditionally it was recommended for stomach problems, constipation, water retention and liver disease but the recent studies conducted in india have further shown that extract of gymnema sylvestre is useful in controlling bloodsugar to treat type-ii diabetes. Gymnema has been clinically proven to reduce excessively high blood sugar levels, perhaps as a result of boosting the production of insulin required to process sugar. The medicinally active parts of the plant are the leaves and the roots. It is known as destroyer of sugar because, in ancient times, Ayurvedic physicians observed that chewing a few leaves of Gymnema sylvestre suppressed the taste of sugar i.e, sweet foods no longer tasted sweet, but rather became almost completely tasteless. In later generations, clinical tests showed that regular use of over a period of three to four months helped to reduce glycosuria, or the appearance of carbohydrates in urine. The present investigation entitled Molecular Characterization of five ecotypes of Gymnema Sylvestre an Anti-diabetic plant. The main objective of the project is to identify the variation between different ecotypes of Gymnema sylvestre. Molecular characterization/genetic diversity of the five different ecotypes of Gymnema Sylvestre was studied using Random Amplified Polymorphic DNA (RAPD) analysis. A preliminary study was carried out using only four primers namely, OPL 01, OPL 02, OPL 06, and OPL 07. Except OPL-01 Primer all the primers gave satisfactory amplification of 63 bands. OPL-01 Primer gave the monomorphic banding pattern. Out of the total 63 bands amplified, 18 (28.57%) were observed to be polymorphic and 5 (7.93%) were unique, i.e specific to a particular ecotype. The preliminary study suggested that existence of genetical differences among five ecotypes of the Gymnema sylvestre and RAPD analysis exhibits the differences in ecotypes.

CONTENTS

1. Introduction 2. Review of Literature 3. Materials & Methods Isolation of DNA Polymerase Chain Reaction Electrophoresis Gel documentation 4. Results & Discussion 5. Conclusions 6. References

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29 37 38

INTRODUCTION

INTRODUCTION:
Gymnema sylvestre is a woody climber which climbs on bushes and trees. It is known as Madhu nashini in Sanskrit, podapatri in Telugu and as Gurmar in Hindi. The Latin name of Gymnema sylvestre means sugar destroyer and is considered as an herbal remedy for high blood sugar. Traditionally it was recommended for stomach problems, and liver disease but the recent studies conducted in India have further shown that extract of Gymnema Sylvestre is useful in controlling bloodsugar to treat Diabetes. The leaves of Gymnema are capable of direct actions on pancreatic cells to increase release of insulin (shanmugasundaram et al. 1990). Gymnema has been clinically proven to reduce excessively high blood sugar levels, perhaps as a result of boosting the production of insulin required to process sugar. The medicinally active parts of the plant are the leaves and the roots. It is known as destroyer of sugar because in ancient times, Ayurvedic physicians observed that chewing a few leaves of Gymnema sylvestre suppressed the taste of sugar i.e, sweet foods no longer tasted sweet, but rather became almost completely tasteless. In later generations, clinical tests showed that regular use of over a period of three to four months helped to reduce glycosuria, or the appearance of carbohydrates in urine. Gymnema sylvestre acts on the taste buds of the oral cavity and on the absorptive surface of the intestine. The recent studies conducted in India as early as 1930 showed that G. sylvestre reduce hyperglycemia, probably by indirectly stimulating the insulin secretion of the pancreas in diabetic patients (Khare etal 1983, Baskaran et al, 1990). Even using experimental organisms like rats the fact that Gymnema leaves reduce hyperglycemia was established (Srivastava et al, 1985). In the present study the Gymnema Sylvestre is used as model plant in the control of Diabetes specially in carrying out the molecular characterization.

GYMNEMA SYLVESTRE GROWN IN FIELD

REVIEW OF LITERATURE

REVIEW OF LITERATURE: Diabetes is a chronic disorder of carbohydrate, fat, and protein metabolism
characterized by fasting elevations of blood sugar (glucose) levels and increased risk of heart and kidney disease, stroke, and loss of nerve function. There are two types of Diabetes in humans. These consist of type I (insulin-dependent) and type II (non-insulin dependent). The two types are different in their mode of action as well as who is affected. Type I diabetes is associated with complete destruction of the Beta cells of the pancreas which manufacture the hormone insulin; therefore, these diabetics rely on insulin regularly to control their blood sugar levels. Type II is associated with obesity, and diet plays an important role in regulating blood sugar. Diabetes is a large disorder, which can be brought on by minute factors. Refined carbohydrates are among the most important contributing factors to diabetes along with reactive hypoglycemia (decreased blood sugar) as well as obesity. Refined sugars are quickly taken up into the body, which leads to hypoglycemia. This puts stress on the adrenal gland, and ultimately, reactive hypoglycemia ensues. This causes the body to become insensitive to insulin or the pancreas to become exhausted, which progresses to diabetes.

The particular concern about the disorder:

Diabetes causes many complications such as troubling blood glucose levels, inadequate insulin production, cardiovascular ailments (or disease), as well as inflammation and damage caused by free radicals. When our body produces ATP, it also releases toxic byproducts (free radicals) that damage our DNA and proteins. Natural antioxidants are produced in our bodies and others can be supplemented in our diets to help further combat damage done by these free radicals.

Researchers have found that oxidative damage plays a role in the damage to tissues caused by Diabetes. It is for this reason that many diabetics would find it useful to explore further ways of gaining antioxidant protection (i.e. through the diet). Diabetes is an auto-immune disease in which the body produces auto antibodies and switches from a cell-mediated response to a humoral response. This refers to a condition in which the body is seeing its own cells as non-self. In the case of Diabetes, the body becomes resistant to its own pancreatic hormone known as Insulin. With the relationship between immunity and diabetes apparent, it is of utmost importance that proper measures be taken to uphold the immune system. With diabetes looming as the seventh leading killer among those in the United States, research is being done vigorously on certain compounds to aid in fighting or reducing its affects. One such compound being meticulously researched may be very familiar in your kitchen, but rarely thought of for medical treatment.

A new substance that suppressed selectively the neural responses of the rat to sweet taste stimuli was isolated from the leaves of Gymnema sylvestre. The substance was proved to be a peptide consisting of 35 amino acids and having a molecular weight of about 4,000. The inhibitory effect on the sweet responses appeared after treating the tongue surface with the peptide at a concentration of more than 1 x 10(-6) M. (Imoto T:Miyasaka A 1991) Across the globe, there are an estimated 150 million people suffering from diabetes mellitus. Each of these people is at increased risk of developing a number of complications, each of which are associated with a reduction in quality of life and an increase in individual morbidity and mortality. However, despite these psychosocial implications, as well as the financial burden associated with the management of the disease, existing treatment options are costly, and have limited, palliative effects. One treatment that is emerging as a potential panacea for the management of diabetes is Gymnema sylvestre.(Matthew J.Leach, 2007) Extracts containing gymnemic acids, which were extracted from the leaves of Gymnema sylvestre (GS) as nine fractions, were evaluated for their effects on a high K(+)-induced contraction of guinea-pig ileal longitudinal muscles, on glucose transport mediated by the difference of glucose-evoked transmural potential difference (delta PD) in the inverted

intestine of guinea-pig and rat, and on blood glucose in rat. Among nine fractions obtained by high performance liquid chromatography from the extract, f-2 and f-4 strongly suppressed the high K(+)-induced contraction of the ileal muscle, f-3 and f-5 did so moderately, and f-8 and f-9 did so weakly, whereas the other fractions did not affect it. The degree of suppression of high K(+)-induced contraction by f-2 at 74% was almost the same as that of f-4 at 67%, at concentrations of 0.1 mg/ml. The suppressed contraction by f-2 or f-4 was recovered by adding 5.5 mM pyruvate. The delta PD increased by 5.5 mM glucose in the inverted intestines of guinea-pig and rat were equally suppressed by 0.1 mg/ml of f-2 or f-4 to 40%. In a rat sucrose tolerance test, f-2 and f-4 suppressed the elevation of blood glucose level. Both f-2 and f-4 suppressed the contraction of guineapig ileal longitudinal muscle, interfered with the increase in delta PD induced by glucose in the inverted intestines of guinea-pig and rat, and inhibited the elevation of blood glucose level. In conclusion, it is suggested that some of the extracts containing gymnemic acids from GS leaves suppress the elevation of blood glucose level by inhibiting glucose uptake in the intestine. (Shimizu K,Yamashita C) Eighteen phenotypically and biochemically distinct Gymnema accessions representing different geographical regions of Kerala were characterized using isozymes and RAPD markers. In the isozyme analysis, three enzyme systems viz., malate dehydrogenase, esterase, and RUBISCO were studied. Five out of the eight resolved loci (62.5%) were polymorphic with the number of alleles expressed at the polymorphic loci being 10. RUBISCO activity was monomorphic across the accessions. In the RAPD assay, 123 amplified products were generated using 15 selected random primers. Of these, 90 bands were polymorphic with the total frequency of polymorphic markers as high as 73.2%. Specific bands were obtained for five accessions. Jaccards coefficient ranged between 0.72 and 0.85 indicating a moderate level of variability. Cluster analysis of RAPD data using Unweighted Pair Group Method of Arithmetic Averages produced two major clusters and three sub-clusters. Overall, molecular fingerprinting revealed the existence of considerable genetic variations in the Gymnema germplasm collection from Kerala. (Smitha Nair, R.Keshavachandran)

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Gastric inhibitory peptide release into the portal vein in response to duodenal infusion of D-glucose was studied in the presence of a leaf extract of Gymnema sylvestre, purified gymnemic acid and inhibitors of some putative glucose sensors and carriers in the intestinal lumen. Intraduodenal infusion of D-glucose significantly increased the portal immunoreactive gastric inhibitory peptide concentration in a dose-dependent manner. The increase in the portal immunoreactive gastric inhibitory peptide induced by glucose was significantly depressed by concomitantly infused leaf extract of Gymnema sylvestre, purified gymnemic acid and phlorizin but not by cytochalasin B. Mannoheptulose, which inhibits glycolysis, and procaine and lidocaine, which inhibit the vagal glucoreceptor in the lumen, did not affect portal immunoreactive gastric inhibitory peptide concentrations. These results suggest that a glucose receptor, which interacts with the leaf extract of Gymnema sylvestre, purified gymnemic acid and phlorizin, exists for the release of immunoreactive gastric inhibitory peptide and that the glucose receptor for gastric inhibitory peptide release is not likely to be identical with a glucose transporter or a vagal glucoreceptor in the lumen.(Tohru Fushiki, Ayako Kojima)

In 1967, Stocklin et al reported that gymnemic acid was the D- glucuronide ofa new hexahydroxy Oleanene triterpene named gymnemagenin , which is esterified with various combinations of formic, acetic, n- butyric , isovaleric and tiglic acids. Koch et al (1973) found that gymnemic acids are inhibitory to the ATPase system from housefly brain and labellum, in addition to fish brain. There fore, they suggested that a portion of the ATPase system might be important biochemical in the sweetness recognition process. Cagan (1974) experimented with gymnemic acid and discovered that it does not inhibit the binding of
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C sucrose to bovine taste papillae, so he proposed that

gymnemic acid might act at a step subsequent to the initial binding of sweet molecules.

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As for the mode of action of anti-sweet active principles obtained from Gymnema Sylvester, Warren et al. (1969) and Yackzan ( 1969) have independently suggested that the compounds might act at the taste receptor surface, and chemically and physically alter it so that originally stimulating sweet molecules provoke lesser degrees of stimulation. Hooper was the first investigator who attempted to isolate the active compounds from the leaves of Gymnema Sylvester. He noted that the anti sweet principle could be precipitated from a crude alcoholic extract of the leaves as a brownish- black aggregate of resinous substances gymnemic acid and reported that it occurred in the potassium salt form in the plant.

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BRIEF LITERATURE ON GYMNEMA SYLVESTRE

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BRIEF LITERATURE ON GYMNEMA SYLVESTRE:

DISTRIBUTION:
Gymnema sylvestre is distributed in Deccan peninsula, extending to parts of northern and western India, wild in the forests of India. These plants grown in central and southern India and also widely distributed thoughout Africa. Gymnema sylvestre belongs to ASCLEPIADACAE family, LATICIFEROUS genus. About 10 species are found in India.They include G. hirsutasm, G. montanum, G. tingens, G. acuminatum. Gymnema is tropical and native to central and western India and can also be found growing in tropical Africa and Australia. It is a large or more pubescent shrub with young stems and branches. Leaves sub-coriaceous, 2.5-6 cm long, ovate or elliptical, acute.

CLASSIFICATION:

Kingdom: Plantae Division: Angiospermae Class: Dicotyledoneae Order: Contortae Family: Asclepiadaceae Genus: Gymea Species: sylvestre

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Botanical Synonyms:
Periploca sylvestris Willd Gymnema melicida Edgew. Asclepias geminata Roxb.

Vernacular names: English Names:

Sugar destroyer.,Periploca of the the woods.

Sanskrit names:
Ajaballi, Ajagandini, Ajashringi, Bahalchakshu, Chakshurabahala, Grihadruma, Karnika, Kshinavartta, Madhunasini, Medhasingi, Meshashringi, Meshavishanika, Netaushadhi, Putrashringi, Sarpadanshtrika, Tiktadughdha, Vishani.

Local Names in India:

Hindi- Gur-mar, merasingi; Bengali- Mera-singi; Marathi- Kavali, kalikardori, vakundi; Gujarati- Dhuleti, mardashingi; Telugu- Podapatri; Tamil- Adigam, cherukurinja;

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Kannada- Sannager-asehambu; Malyalam- Cakkarakkolli, Madhunashini;

Part used: leaves Taxonomic Description:

Extensive, much-branched, twining shrubs. Leaves 3-6 x 2-3 cm, 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. Follicles solitary, upto 8 x 0.7 cm, terete, lanceolate, straight or slightly curved, glabrous. Seeds ovate-oblong, glabrous, winged, brown. Flowering: August-March; Fruiting: Winter.

Traditional uses :
Sushruta describes Gymnema sylvester, as a destroyer of madhumeha (glycosuria) and other urinary disorders. On account of its property of abolishing the taste of sugar it has been given the name of gur-mar meaning sugar destroying and it is believed therefore that it might neutralize the excess of sugar present in the body in Diabetes mellitus. The plant is also reported to be bitter, astringent, acrid, thermogenic, anti-inflammatory, anodyne,digestive, liver tonic emetic, diuretic, stomachic, stimulant, anthelmenthics, laxative, cardiotonic, expectorant, antipyretic and uterine tonic. It is useful in dyspepsia, constipation, jaundice, haemorrhoids, renal and vesical calculi, cardiopathy, asthma, bronchitis, amenorrhoea, conjuctivitis and leucoderma.

Habitat:
Grows wild in forest as a climber also found in the plains from the coast, in scrub jungles and in thickets; wild. Through out India, in dry forests upto 600m, common throughout the district from January to November. Distributed in Asia, Tropical Africa, Malaysia and Srilanka.

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Distribution in India:
It is occurring in Bihar, Central India, Western Ghats, and Konkan.

Distribution in Patalkot:
Gaildubba, Harra-ka-Char, Kareyam, Raja khoh, Sajkui etc.

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PHARMACOLOGY

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Pharmacology :
i) Antidiabetic activity : Gymnema sylvestre a plant used in the Ayurvedic medicine of India for the treatment of Diabetes mellitus has been known from antiquity also to have an antisaccharin taste effect. The active principles are glycosides (several Gymnemic acid) which shows selective anaesthetic effect. In a study conducted on rats the feeding of powdered leaves of Gymnema sylvestris in 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 seen in rats receiving beryllium nitrate alone. The feeding of the leaves for 25 days to normal rats did not alter blood glucose significantly. The exact mode of protective action against beryllium toxicity was not established. Experimental studies were conducted on rats fed on high carbohydrate diet for 15 days and later rendered hyperglycaemic by injecting anterior pituitary extract 100mg/kg subcutaneously daily for ten days. These animals were treated with ethanol extract of Gymnema sylvestre at a dosage of 100mg/kg orally. Results indicated insignificant reduction in blood sugar in normal rats, whereas significant reduction in anterior pituitary treated hyperglycaemic rats. Effect of the drug was comparable to that of tolbutamide (50mg/kg) in the hyperglycaemic rats. The drug influenced the disturbed carbohydrate metabolism in hyperglycaemic animals by limiting the carbohydrate turnover and thus inhibiting the vicious cycle of hyperglycaemia. Further studies were conducted on albino rats to establish the antidiabetic activity of Gymnema sylvestre, which was compared with other conventional indigenous oral antidiabetic drugs like Coccinia indica, Pterocarpus marsupium, Momordia charantia. The animals were subcutaneously injected with 100mg/kg dose of the anterior pituitary extract, these animals were then fed with alcoholic extract of Gymnema sylvestre and Coccinia indica (100mg/kg each), aqueous infusion of Pterocarpus marsupium (20ml/kg), extract of Momordia charantia (5ml/kg) and tolbutamide (50mg/kg) orally. Inhibition of the hyperglycaemic response of the anterior pituitary extract at 6, 12 and 24 hours was most marked in tolbutamide & Gymnema sylvestre. The inhibitory effect was highly significant in Gymnema sylvestre when compared with Pterocarpus marsupium and Momordia charantia. The usefulness of Gymnema sylvestre therapy in alloxan induced diabetic rabbits in correcting the abnormal accumulation of lipids, glycogen and protein depletion in the liver, kidney and muscle were investigated by feeding crude leaf powder at a dosage of 250mg/kg body weight once a day. Gymnema sylvestre therapy not only produced blood glucose homeostasis but also increased the activities of the enzymes affording the utilization of glucose by insulin dependent pathways, it controlled phosphorylase levels, gluconeogenic enzymes and sorbitol dehydrogenase. The uptakeand incorporation of glucose into the glycogen and protein are increased in the liver, kidney and muscle in Gymnema sylvestre administered diabetic animal when compared to the untreated diabetic animals.

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The inhibitory effects of an extract of Gymnema sylvestre and purified gymnemic acid on Gastic Inhibitory Peptide (GIP) release was studied in rats. The GIP release into the portal vein in response to duodenal infusion of D-glucose in presence of leaf extract of Gymnema sylvestre at a dosage of 0.5ml/kg. The results suggested that a glucose receptor which interacted with the leaf extracts ofGymnema sylvestre and purified Gymnemic acid. The inhibition of GIP release by Gymnemic acid observed was attributed to the interaction with the glucose receptor for GIP release which was similar in specificity to the active glucose transport system. Two water soluble extracts GS3 and GS4 obtained from the leaves of Gymnema sylvestre, were tested in streptozotocin treated rats for their effects on blood glucose homeostasis and pancreatic endocrine tissue. In the diabetic rats, fasting blood glucose levels returned to normal after 60 days of GS3 and after 20 days of GS4 oral administration. In diabetic rat pancreas, both therapy led to a rise in serum insulin to levels close to normal testing levels. In diabetic rats pancreas both GS3 and GS4 doubled the islet number and beta cell number. This herbal therapy appeared to bring about blood glucose homeostasis through increased serum insulin levels provided by repair/regeneration of the endocrine pancreas. Clinical trials: In a clinical trial conducted on 27 patients with insulin-dependent Diabetes mellitus (IDDM) on insulin therapy, GS4 a water soluble extract of the leaves of Gymnema sylvestre was administered at a dosage of 400 mg/day. Insulin requirements came down together with fasting blood glucose and glycosylated haemoglobin(HBA1c) and glycosylated plasma protein levels, while serum lipids returned to near normal level with GS4 therapy, glycosylated haemoglobin and glycosylated plasma protein levels remained higher than controls. IDDM pateints on insulin therapy showed no significant reduction in serum lipids, HBA1c or glycosylated plasma proteins. GS4 therapy appeared to enhance endogenous insulin , possibly by regeneration/revitalization of the residual beta cells in insulin-dependent Diabetes mellitus. Further clinical studies were conducted to test the effectiveness of GS4 therapy an extract from the leaves of Gymnema sylvestre in controlling hyperglycaemia in 22 patients with Type2 Diabetes (NIDDM -Non-insulin dependent Diabetes mellitus) on conventional oral anti-hyperglycaemic agents. GS4 (400mg/day) was administered for 18 - 20 months as a supplement to the conventional oral drugs. During GS4 supplementation, the patients showed a significant reduction in blood glucose, glycosylated haemoglobin and glycosylated plasma proteins and conventional drug dosage could be reduced. Five of the 22 diabetic patients were able to discontinue their conventional drug and maintain their blood glucose homeostatis with GS4 alone. These data suggested the beta cells regeneration/repair in Type 2 diabetic patients on GS4 supplementation, which is further, supported by the appearance of raised insulin levels in the serum of patients after GS4 supplementation. In another clinical trial hypoglycaemic activity of indigenous drug (Gymnema sylvestre) was evaluated in ten normal and six diabetic patients. They were subjected to glucose tolerance test and venous blood samples were collected at 30 minutes intervals upto 2 hours. Aqueous decoction of the leaves was given at a dosage of 2g thrice daily for a period of 10 days in healthy volunteers and in diabetic patients with mild to moderate

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hyperglycaemic the above doses were given for 15 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 hypoglycaemic activity. The hypoglycaemic effect of Gymnema sylvestre was studied in 16 normal subjects and in 43 milddiabetic patients. All the subjects were administered with leaf powder 10g/day for 7 days. The results indicate that Gymnema sylvestre leaf powder has a hypoglycaemic 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. ii) Hypolipidaemic activity : Leaf extract at a dosage of 25-100 mg/kg administered orally to experimentally induced hyperlipidaemic rats for two weeks reduced the elevated serum trigylceride (TG), total cholesterol (TC), very low density lipoprotein(VLDL) and low density lipoprotein(LDL)cholesterol in a dose dependent manner. The ability of the extract at 100mg/kg to lower TG and TC in serum and its antiantheroscelrotic potential were almost similar to that of a standard lipid lowering agent clifibrate. (i) Toxicity: The LD50 of ethanol and water extract of Gymnema administered intraperitoneally inmice was found to be 375mg/kg. (j) Phytochemistry : The major bioactive constituents of Gymnema sylvestris are a group of oleanane type triterpenoid saponins known as gymnemic acids1-2. The latter contain several acylated (tigloyl, methylbutyroyl etc.,) derivatives of deacylgymnemic acid (DAGA) which is 3-O-- glucuronide of gymnemagenin (3, 16, 21, 22, 23, 28hexahydroxy-olean-12-ene)2. The individual gymnemic acids (saponins) include gymnemic acids I-VII, gymnemosides A-F, gymnemasaponins1-3 etc.

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GYMNEMA SYLVESTRE AND ITS COMPOUNDS

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GYMNEMA SYLVESTRE AND ITS COMPOUNDS: ACTIVE COMPOUNDS:

The medicinally active parts of the plant are the leaves and roots. The important active ingredient of Gymnema sylvestre is an organic acid called gymnemic acid. The Gymnemic acid is made up of molecules whose atom arrangement is similar to that of glucose molecules. Those molecules fill the receptor locations on the taste buds for a period of one to two hours, there by preventing the taste buds from being activated by any sugar molecules in the food (Yoshikawa.R, Arihara.S). Similarly, the glucoselike molecules in the gymnemic acid fills the receptor locations in the absorptive external layers of the intestine, there by preventing the intestine from absorbing the sugar molecules.

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STRUCTURE OF GYMNEMIC ACID

GYMNEMIC ACID
The active principle of this plant suppresses the sweet taste not only of sucrose, but also the sweetness effects of sodium saccharin, cyclamate, glycine, D- alanine, D- tryptophan, D- leucine, beryllium chloride and lead acetate, but not that of chloroform. As for the mode of action of anti-sweet active \ principles obtained from Gymnema sylvestre, Warren et al. (1969) and Yackzan (1969) have independently suggested that the compounds might act at the taste receptor surface, by chemically and

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physically altering it so that originally stimulating sweet molecules provoke lesser degrees of stimulation.

Mechanism of Action of Gymnemic acid:

Gymnemic acid formulations have also been found useful against obesity, according to recent reports. This is attributed to the ability of gymnemic acids to delay the glucose absorption in the blood. The atomic arrangement of gymnemic acid molecules is similar to that of glucose molecules. These molecules fill the receptor locations on the taste buds thereby preventing its activation by sugar molecules present in the food, thereby curbing the sugar craving. Similarly, Gymnemic acid molecules fill the receptor location in the absorptive external layers of the intestine thereby preventing the sugar molecules absorption by the intestine, which results in low blood sugar level . G. sylvestre leaves have been found to cause hypoglycemia in laboratory animals and have found a use in herbal medicine to help treat adult onset non-insulin dependent diabetes mellitus (NIDDM). When Gymnema leaf extract is administered to a diabetic patient, there is stimulation of the pancreas by virtue of which there is an increase in insulin release . These compounds have also been found to increase fecal excretion of cholesterol, but further studies to prove clinical significance in treating hypercholesterolemia (high serum cholesterol) are required. Other uses for Gymnema leaf extract are its ability to act as a laxative, diuretic, and cough suppressant. These other actions would be considered adverse reactions when Gymnema is used for its glucose lowering effect in diabetes. Gymnema leaf extract, notably the peptide Gurmarin, has been found to interfere with the ability of the taste buds on the tongue to taste sweet and bitter. Gymnemic acid has a similar effect. It is believed that by inhibiting the sweet taste sensation, people taking it will limit their intake of sweet foods, and this activity may be partially responsible for its hypoglycemic effect. There are some possible mechanisms by which the leaves and especially Gymnemic acids from G. Sylvestre exert its hypoglycemic effects are: 1) It increases secretion of insulin, 2) It promotes regeneration of islet cells,

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3) It increases utilization of glucose: it is shown to increase the activities of enzymes responsible for utilization of glucose by insulin-dependant pathways, an increase in phosphorylase activity, decrease in gluconeogenic enzymes and sorbitol dehydrogenase, and 4) It causes inhibition of glucose absorption from intestine. The gymnemic acid components are believed to block the absorption of glucose in the small intestine, the exact action being unknown. It could be involve one or more mechanisms. One of the mechanisms responsible for adult onset diabetes mellitus is a form of insulin resistance, which is attributed to the inability of insulin to enter cells via the insulin receptor. Gymnema may overcome this resistance, but require further studies to confirm its validity and also whether the effect is clinically relevant. Should this effect be proven, Gymnema may prove useful in both adult onset non-insulin dependent diabetis mellitus(NIDDM) and juvenile onset insulin dependent diabetes mellitus (IDDM) to help insulin enter cells. In the case of IDDM, the insulin is injected by syringe and is not secreted from the pancreas. The leaves are also noted for lowering serum cholesterol and triglycerides. The primary chemical constituents of Gymnema include gymnemic acid, tartaric acid, gurmarin, calcium oxalate, glucose, stigmasterol, betaine, and choline. While the water-soluble acidic fractions reportedly provide the hypoglycemic action, it is not yet clear what specific constituent in the leaves is responsible for the same. Some researchers have suggested gymnemic acid as one possible candidate, although further research is needed. Both gurmarin (another constituent of the leaves) and gymnemic acid have been shown to block sweet taste in humans. The major constituents of the plant material 3B glucuronides of different acetylated gymnemagenins, gymnemic acid a complex mixture of at least 9 closely related acidic glucosides. The following figure could provide a diagrammatic representation for explaining the action of gymnemic acids on the intestinal receptors. The basic function of the acids is to bind to the receptor on the intestine, and stop the glucose molecule from binding to the receptor. Thus, gymnemic acids prevent the absorption of excess glucose.

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Schematic representation of competitive inhibition of receptor site on intestine by Gymnemic acid

Physiological Functions of Gymnemic acid:

Gymnemic acid combined with the recognized site of sugar, and so it prevents sugar from combining with the site. It have suppressive activity of absorption of sugar. The following effects have been reported.

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Gymnemic acid help controlling sweet taste. Gymnemic acid help controlling absorption of sugar in the body. Gymnemic acid depress appetite and cause weight reduction. Gymnemic acid restoring pancreas functions. Gymnemic acid also show anti-tooth decaying effect.

Applications of Gymnemic acid and Gymnema Sylvestre Extracts:

For health food for diet. Prevention of diabetes. For protection of decayed tooth processed in foods. Research indicates this amazing herb has positive benefits on blood sugar control, helps with sugar cravings and to regenerate the pancreas. In tests on diabetic rats, the pancreas doubled in size and grew new insulin-producing cells. In clinical studies of animals with diabetes, gymnema sylvestre also appeared to reduce body weight, blood cholesterol, and triglyceride levels. Although the exact reasons are not clear, it is believed that gymnema sylvestre blocks the absorption of dietary fats into the bloodstream. Possibly, more fats are then eliminated instead of being stored. Some individuals taking gymnema sylvestre for diabetes have also seen a reduction in cholesterol and/or weight, but no human studies of gymnema sylvestre potential cholesterol-lowering or anti-obesity effects have been reported. All the potential uses of gymnema sylvestre need more study before it can be recommended for medical use.

Gymnema Sylvestre extract, 260 mg, 120 Tablets - Source Naturals:

Gymnema Sylvestre extract may help to maintain healthy blood sugar levels when used as part of your diet. Gymnema has been used traditionally in India for centuries and has been shown in research to support healthy glucose metabolism by mediation of insulin release and activity and enhancement of healthy pancreatic function. Source Naturals Gymnema Sylvestre extract is standardized to 25% gymnemic acids, the same concentration used in clinical research. 28

Gymnema Sylvestre extract Supplement Facts:

Serving Size: 1 Tablet Gymnema Leaf Extract 25% - 260 mg(Yielding 65 mg Gymnemic Acids) Suggested Use: 2 gymnema tablets daily. Take 1 tablet after breakfast, and 1 after dinner, or as recommended by your health care professional . * Gymnema sylvestre leaf extract daily value not established.

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GYMNEMA SYLVESTRE IN DETAIL

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Gymnema Description: Gymnema sylvestre is a woody, vine-like plant which climbs on bushes and trees in the Western Ghats in South India, and to the west of those mountains in the territory around the coastal city of Goa. It came to be known as destroyer of sugar because, in ancient times, Ayurvedic physicians observed that chewing a few leaves of Gymnema suppressed the taste of sugar. Recent clinical trials conducted in India have shown that an extract of Gymnema sylvestre is useful in both insulin-dependent diabetes mellitus (IDDM) and in certain types of non-insulin-dependent diabetes mellitus (NIDDM). As a result of these clinical tests and years of successful treatments, Gymnema is used today all over India for treating diabetes mellitus. Gymnema Hypoglycemia: Studies conducted in India as early as 1930 showed that the leaves of Gymnema sylvestre cause hypoglycemia in experimental animals. This state of hypoglycemia is explained on the assumption that the drug indirectly stimulates insulin secretion of the pancreas, since it has no direct effect on carbohydrate metabolism. Gymnema: Gymnemic Acid: Recent pharmacological and clinical studies have shown that Gymnema sylvestre acts on two sites, the taste buds in the oral cavity and the absorptive surface to the intestines. The important active ingredient of Gymnema sylvestre is an organic acid called Gymnemic

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acid. The Gymnemic acid is made up of molecules whose arrangement is similar to that two hours, thereby preventing the taste buds from being activated by any sugar molecules present in the food. Gymnema in Taste: It hs also been noted that Gymnema sylvestre takes away the bitter taste of bitter substances, such as quinine, in much the same way that it affects the sense of sweetness associated with candies and other swwet foods. Therefore, if you are eating an orange within two hours after chewing Gymnema sylvestre leaves, for instance, you would taste the sourness of it but not the sweetness. Benefits of Gymnema Sylvestre : * By suppressing the taste of sweet foods, the desire to eat them is also suppressed. Picture a luscious-looking large piece of chocolate candy, which you know, despite the tempting look, is not sweet. Why bother to eat it? It is important to remember that this effect of Gymnema sylvestre's will last for only one to two hours. If you are using the herb to break the sugar habit, then it would be wise to take some Gymnema sylvestre before social events or other times when you might be tempted to dive into the tray of sweets. * Gymnema sylvestre significantly reduces the metabolic effects of sugar by preventing the intestines from absorbing the sugar molecules during the process of digestion. Because there is a change in the absorption of sugar, there is a consequent change in the blood sugar level.

Contemporary uses of gymnema:

Currently, gymnema is known primarily for its sugar-blocking properties. It is used to treat high blood sugar in diabetics and has been promoted as a weight loss remedy. In India, gymnema has been used by both Type I and Type II diabetics, but is used mainly to treat Type II diabetics. Some clinical trials in India indicated that gymnema could help with both types of diabetes. During the 1990s, Type II diabetics in India were studied, and gymnema proved successful for lowering blood sugar with continuous use for 18 to 24 months. In another

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study, people diagnosed as juvenile diabetics took gymnema along with insulin. In some cases, people were able to reduce their dosages of insulin. In mid-2002, a U.S. clinical trial was reported to further support gymnema's use in managing Type I diabetes. Of those participating in the trial, about 16% were able to decrease usage of their prescription medication usage. The same research group also found gymnema beneficial for non-insulin dependent diabetics. While those results appeared promising, medical professionals caution that more research is still needed. That research would include double-blind studies and involve more people. Gymnemas therapeutic value Gymnema is an herb that reduces blood sugar levels after sugar consumption. It has a molecular structure similar to that of sugar that can block absorption of up to 50% of dietary sugar calories. Gymnema can be taken for Blood Sugar, Hypoglycemia, Weight Management, Cholesterol, Digestion Gymnema Therapeutic Uses

Suppress the taste of sweet foods, and consequently the desire to eat Reduce metabolic effect of sugar by preventing the intestines from absorbing sugar molecules during digestion Treatment of diabetes Snakebite treated by powder or paste of the root applied to the wound Fever treated with oral administration of half announce to an ounce ( one part in 10) of leaves Swollen glands treated with an external application of triturated leaves mixed with castor oil Gymnema helps to normal blood sugar Gymnema controls sugar level Gymnema controls and regulate weight Gymnema controls sugar craving Gymneam reduces the taste of sugar when it is on the mouth Gymnema curb sweet tooth Gymnema for blood glucose Gymnema for Cholesterol Gymnema gurmar is the natural way to help control blood sugar

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Therapeutic Action: 1 . Gymnema neutralized the craving for sweets by abolishing the taste of sugar. Gymnema promotes regeneration of beta cells responsible for releasing insulin in the pancreas. 2. Gymnema prevents adrenaline from stimulating the liver to produce glucose. Gymnema lowers serum cholesterol and triglycerides. 3. Overall, Gymnema normalizes blood sugar levels without the use of insulin or oral compounds.

Gymnema : Toxicity Cautions and Contra indications For most people using Gymnema sylvestre, blood sugar goes down toward but not below normal blood sugar levels. This can happen in a small number of patients, however, because the mechanisms of the diabetic syndrome vary with different patients. Remarkably, unlike insulin or oral hypoglycemic sulfonylurea compounds, the hypoglycemic effects of Gymnema sylvestre are seen in only a small percentage of diabetic patients. Safety of Gymnema sylvestre The safety of Gymnema sylvestre has been demonstrated by the fact that it has been safety and successfully used for more than 2,000 years in traditional Ayurvedic medicine. Gymnema who can take it: Gymnema is typically recommended for people who have trouble taming their sweet tooth and who are either borderline diabetic or waging a war against Type II diabetes. When there a high level of blood sugar, the body produces more insulin, which is the hormone that helps shuttle blood sugar into the cells of the body. But as too much insulin is produced, the cells become resistant to it a condition known as insulin resistance. When that happens, blood sugar remains high. Many people who have insulin resistance gain weight, no matter what their diet. In such cases, Gymnema may help by improving the cells uptake of blood sugar and helping the body utilize it. Gymnema addictive? Gymnema does not contain any addictive components and can be taken without fear of addiction for extended periods of time. Gymnemas Side Effect?

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No side effects have been reported, but researchers have not established whether this herb is absolutely safe for pregnant women or during lactation. Gymnema be taken along with other pharmaceutical medication or alternative health products. It is advisable to use caution when taking Gymnema along with other pharmaceutical medication (oral hypoglycemics or insulin) for diabetes. The combination of gymnema and these medications can lower blood-sugar levels to potentially risky levels. If you are already taking diabetes medication, it is extremely important to work with a physician before supplementing with gymnema because the physician needs to closely monitor blood-sugar levels. In many instances, your doctor will need to lower the dosage of the pharmaceutical medication you are on. Gymnemas action on the body Gymnema has the ability to lower blood sugar levels, improve the bodys ability to metabolize and respond to insulin and regenerate the pancreas, although results depend on how advanced the condition is the extent to which medical management and monitoring is essential. Gymnema decreases insulin needs by 45-75% and helps lower blood sugar and triglycerides. Further, gymnema reduces craving for sugar and blocks the ability to taste sweets for 3 hours, although it does not completely destroy all sense of taste. In type II diabetes, gymnema improves insulins effects and can even substitute for oral sugar-lowering drugs

How can we test if Gymnema really blocks ones ability to taste sweets? A taste test can show how Gymnema works. First taste something sweet. Then, take one dosage of Gymnema. Now, taste something sweet again. You will not be able to taste the sugar because Gymnema has blocked the taste of the sugar in your mouth in the same way it blocks sugar in digestion. How long do I have to take Gymnema before I see any results? Herbal medicines provide progressive, long-term benefits. Certain classes of herbs work well in acute conditions to provide quick relief, however herbs like Gymnema need to be taken for at least 3-4 weeks before the benefits of taking it can be experienced. Optimally, it can take anywhere between 6 months and 2 years to experience the full range of benefits. Individual results can vary, depending on the nature and seriousness of the condition for which Gymnema is being consumed. 35

Gymnema: Clinical Studies and Validation: In one controlled study, a standardized gymnema extract was given to 50 people with Type I diabetes, all receiving a dosage of 400 mg daily for periods ranging from 6 months to 2 and years. 50 of the other people in the study continued to take the usual insulin therapy without the addition of gymnema. Among those who took the herbal extract, researchers found that insulin requirements fell off dramatically. In addition, there was a statistically significant decrease in the blood sugar markers that tests longterm blood sugar. Those in the control group showed no significant decreases in blood sugar or insulin requirements. In a second study, 55 people with Type II diabetes were given 400mg of gymnema extract every day for 18 to 20 months while they also continued to get their usual medication for hypoglycemia. Average blood sugar levels improved significantly in the group, along with another sugar related factor (glycosylated hemoglobin). Results also showed that there was an increase in pancreatic release of insulin among the people who got gymnema. People in the study were able to reduce their medication, and five were able to discontinue their drugs completely Gymnema has been scientifically noted by various clinical data. Its formula has been subjected to the modern controlled studies of clinical testing to prove its therapeutics in gently addressing your health and lifestyle and its quality is controlled by the most sophisticated chromatographic methods of analysis. Note of Gymnema Gymnema Ayurvedic herbal for diabetes, Gymnema ayurvedic nutrient for blood sugar, Gymnema ayurvedic for blood sugar, Gymnema ayurvedic natural herb nutrient for diabetes mellitus, Gymnema for glycosuria, Gymnemic acid for weight control, Gymnemic acid controls glucose molecules, Gymnemic acid controls taste buds, Gymnemic acid controls surface of intestines, Gymnema for people predisposed to diabetes, Gymnema for athletes needing to develop lean muscle, Gymnema acts as a substitute for chromium, Gymnema acts for the hormone insulin, Gymnema regulates metabolism of protein, Gymnema regulates fats, Gymnema regulatesd carbohydrates, Gymnema increase HDL levels

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Pharmacological actions:
Blood Sugar Balance is made of the root and leaf of the plant Gymnema or its extraction. Its functions can explain as follow:

1) The Gymnema molecular structure is similar to the dextrose molecular structure. Its affinity with sugar molecular acceptor is 20 times more than dextrose. The acceptor that can absorb sugar in the small intestine cannot absorb sugar when occupied by Gymnema. As the result, the blood sugar consistency plays down. 2) As same reason, Gymnema can act on the taste buds in the tongue. The tongue feels no sweet taste when chewing Gymnema and candy at the same time. 3) The blood sugar is controlled by insulin, and the insulin is produced by b-cells in the pancreas. Usually, in the adult diabetic, the b-cell had been damaged and the Gymnema can help the b-cell regenerate and thus lesson the diabetic symptoms. Therefore Blood Sugar Balance and Blood Sugar Balance II are effective for both dependent (infant) and adult diabetics.

MEDICINAL VALUES:
Gymnema sylvestre commonly referred to as sugar killer. Gymnema leaves comprised of gymnemic acids which suppress the absorption of glucose molecules, and it reduces the sensation of sweet ness of foods. Gymnema sylvestre reduces the blood sugar levels, lower blood cholesterol levels and balance insulin levels. It is used as Astringent, stomachinic, tonic, ant diabetic. Leaves have a peculiar property neutralizing temporarily the sensation for sugar and are used in diabetes. Liver disorders, cardiac Amenorrhoea, , Cough and Asthma. Besides all these values, Gymnema also has hypolipidaemic & antiatherosclerotic effects (Bishayee.A, Chatterjee) Several companies were using Gymnema for preparing as Herbal tea and Capsules.

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1. DIA-TEA

Gymnema sylvestre DIA-TEA is made from Gymnema and Camellia Sinensis. An excellent health beverage, DIA-TEA prepared from the Gymnema herb is considered to be the best from as it has an effect in the mouth to reduce sugar craving as well in the intestine to reduce absorption of sugar. DIA-TEA is completely caeffine free. Having regularly can reduce the appetite for sweet tasting food, which can result in substantial weight loss, promoting balanced blood sugar levels and healthy pancreatic activity. Ingredients: Gymnema sylvestre Camellia sinensis Coffen 60% 40%

2. Gymnema Herbal Tea:

Best Nutrition has introduced the beneficent powers of Gymnema sylvestre in two forms. Herbal tea and capsules, both are equally effective. An extract of ayurvedic herb, effects the: Taste buds in the oral cavity, the acid prevents the taste buds from being activated by any sugar molecules in the food. Absorptive surface of the intestines. The acid prevents the intestine from absorbing sugar molecules. Gymnema sylvestre (Ayurvedic Herbal extract) Ayurvedic physicians observed that chewing a few leaves of Gymnema sylvestre suppressed the taste of sugar.

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3. DIA-BOTICA Capsules :

Dia-Botica Capsules contain 4 most powerful anti sugar absorption herbal standardized extracts which helps for not absorbing sugar into our blood stream during digestion. G. sylvestre is one of the main herbal standardized extract.

4. Gymnema sylvestre Extract: (Standardized 25% gymnemic acid)

Research indicates this amazing herb has positive benefits on blood sugar control, helps with sugar cravings and to regenerate the pancreas. In tests on diabetic rats, the pancreas doubled in size and grew new insulin- producing cells.

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GENERAL DISCOVERY

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GYMNEMA SYLVESTRE - DIABETES TREATMENT STRONG EVIDENCE IT RESTORES BETA CELLS Gymnema Sylvestre is an herb from India. It has been used as a treatment for diabetes mellitus since ancient times. The first scientific studies were about 70 years ago and showed a beneficial effect on the pancreas. Despite the promising initial results, scientific investigation into the effect of Gymnema Sylvestre on diabetes wasn't resumed until 1981. Then, because of a number of drugs which already had similar results for diabetes, interest in the herb again became almost non-existent. Of course, the drugs do nothing to prevent the continued deterioration of pancreatic function. In 1988 research was resumed and carried a step further by using Gymnema Sylvestre Extract (GSE). This concentrated the herb and brought strong evidence of a miracle in store for diabetics. In 1990 several published research studies on Gymnema Sylvestre Extract changed this herb from being interesting to revolutionary. It was discovered that GSE resulted not only in improved glucose homeostasis but that there was also a regeneration of beta cells in the pancreas (not conclusive proof as yet, but strong EVIDENCE OF A MIRACLE). You often hear a Type 1 diabetic (also called child-onset diabetes) say they cannot repair their diabetes but can only control it because beta cells in the pancreas were destroyed. There is also some evidence now that the beta cells were destroyed in these youngsters by drinking cow's milk and eating milk products such as cheese and butter. The strong possibility that the beta cells are being repaired or regenerated by taking Gymnema Sylvestre Extract will give lots and lots of hope to diabetics, especially the Type 1 diabetics. Taking GSE is not a short term deal. For example, after the research patients were taking it for two years is when the scientists found the strong evidence that the beta cells were regenerating. In the meantime, the patients were able to reduce their insulin or pills, their cholesterol and triglycerides came down, and their free fatty acid levels reduced. Type 2 diabetics often are able to get off their drugs entirely early on, especially if their diet is right. Significant damage could occur before a person recognizes they have diabetes. Gymnema Sylvestre Extract may be of use to anyone concerned about preventing diabetes as well as elderly persons who are at high risk for developing it. Overweight people are also at risk for developing it. GSE has no side effects. If you do not have diabetes, you may not realize what a deadly disease this can be and what constant care and vigilance it takes to keep it under control -- otherwise various body systems deteriorate. A person who is diabetic will have to monitor their blood glucose levels on a daily basis and adjust their medication as there is usually improved insulin production and release during GSE supplementation, even early on. Regeneration of the beta cells is the important discovery in this newest research. Regeneration of beta cells was thought impossible before. It is shocking that Gymnema Sylvestre remains unknown in most of the world because its usefulness was first reported almost three-quarters of a century ago.

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NATURAL REMEDIES FOR DIABETIC TREATMENT USING GYMNEMA

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Natural Remedies for Diabetes Management Natural Remedies for Diabetes Management: Gymea is proven a very useful remedy in diabetes management. Studies Show that Gymnema sylvestre is a Natural Remedy for Diabetes Management. Studies show that Gymnema Sylvestre helps control both type 1 diabetes and type 2 diabetes. Gymnema is a well-proven natural treatment for diabetis, and it has been used for this purpose for over 2,000 years and has proved to be very effective in type 1 diabetes. In human studies, the most common doses of Gymnema sylvestre used for blood sugar control were 400 mg to 600 mg per day. Gymnema sylvestre is commonly added to many different herbal combination products, but the majority of studies used GS4, a standardized product that contains only gymnema sylvestre. Standardization by the manufacturer should assure the same amount of active ingredient in every batch of the commercial preparation. (DrugDigest). Doses of 400 mg to 600 mg per day correlates to about 8 to 12 grams per day of the dried herb in tablet form. Therefore taking 4 grams in tablet form two to three times a day would give a total daily dose of 8 to 12 grams. Some practitioners find Gymnema works best when taken before meals. A WebMD Medical News article (9 October 2000) related the story of a 71-year-old woman whose doctor had prescribed a widely used drug glyburide (Micronase) used to stimulate the pancreas to make insulin, which can help control blood sugar in a person with type 2 diabetes. According to the woman she was frequently hitting blood sugar highs and lows. She had been using the drug for six years but fours years prior to the abovementioned report she had dumped glyburide and started taking the herbal product Beta Fast GXR which contains the herb Gymnema without her doctors knowledge for three of those years. She said her doctor may not have noticed the change because her blood sugar checks always turned out so well. The woman also watches her diet and tests her blood sugar daily. (WebMD Medical News 9/10/2000).

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what is Gymnema? Gymnema (Gymnema sylvestre) is a climbing plant that grows in open woods and bushland at an altitude of m in India, China, Indonesia, Japan, Malaysia, Sri Lanka, Vietnam and South Africa. Gymnema is also known as gurmar, gurmabooti, periploca of the woods, and meshasringi (rams horn). The Hindu word gurmar best describes the primary use of gymnema. Gurmar translates in English as sugar destroyer. The translation is literal: When Gymnema is applied to the mouth, it prevents the taste buds from perceiving sweet tastes. Chewing chocolate is like chewing butter. Grains of sugar are like grains of sand. Gymnema is a well-proven treatment for diabetes, and it has been used for this purpose for over 2,000 years. In my own clinical experience, I have found Gymnema to be the best herb available for treating this condition and controlling excessive blood sugar levels. (Kerry Bone, FNIMH, FNHAA, Nutrition and Healing Newsletter, January 2002). Gymnema is best known for its apparent ability to lower blood sugar levels. Results from case reports and studies in humans and animals suggest that it may work in several ways to help control both type 1 and type 2 diabetes. First, the acids contained in gymnema sylvestre seem to decrease the amounts of sugar that are absorbed from foods. As a result, blood sugar levels may not increase as much as usual after meals. Secondly, gymnema sylvestre may promote the production of insulin by the body. It is possible that gymnema sylvestre may even prompt the pancreas to develop more beta cells - the source of insulin. It may also make body cells more responsive to the insulin that is available. (DrugDigest). The first scientific confirmation of Gymnemas effects on glucose in human diabetics was in 1926 when it was demonstrated that the leaves of Gymnema reduced urinary glucose. (K.G. Gharpurey, Indian Medical Gazette 1926; 61: 155). Four years later it was shown that GS (Gymnema sylvestre) had a blood glucose lowering effect when there was residual pancreatic function, but was without effect in animals lacking pancreatic function, suggesting a direct effect on the pancreas. Despite the promise of these initial results, scientific inquiry into the effect of GS on diabetics was not seriously resumed until 1981 when it was again demonstrated that oral administration of the dried leaves of GS brings down blood glucose and raises serum insulin levels, recorded during an oral glucose tolerance test in diabetic animals and healthy human volunteers. (Parenting Naturally).

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An unusual attribute of Gymnema sylvestre of great interest is its ability to lower blood sugar by increasing insulin output by apparently regenerating beta cells which are the cells that manufacture and secrete insulin. As abnormalities in beta cell number and/or function are directly related to both Type 1 and Type 2 diabetes, it appeared that Gymnema sylvestre was a major discovery in the battle against one of the most common disorders in the world. In 1990 researchers at the University of Madras in India orally administered Gymnema extract to on group of diabetic rats while another group acted as controls and were not given Gymnema. A toxic agent was used to chemically destroy the insulin producing beta cells in the pancreas of each rat thereby causing diabetes. In the group of rats receiving Gymnema leaves in any form, whole or extracted, their fasting blood glucose levels returned to normal within 20 to 60 days. In addition, their insulin levels rose toward normal levels and the number of beta cells in the pancreas (the cells that manufacture and secrete insulin) increased. Their findings were published in the Journal of Ethnopharmacology. (J Ethnopharmacol. 1990 Oct;30(3):265-79). Another 1990 study by researchers at Kobe University School of Medicine, Japan, and published in the journal Diabetes Research and Clinical Practice, concluded that gymnema is useful in the treatment of type 2 diabetes. Diabetic rats were divided into two groups and fed either a normal diet or one supplemented with an extract of Gymnema. After four weeks the rats supplemented with the Gymnema extract showed reduced glucose levels in the fed state and an improvement in glucose tolerance. (Diabetes Res Clin Pract. 1990 May-Jun; 9(2): 1438). Of course, what works well in test animals may not work in the same way or at all in humans, but this is not the case with Gymnema sylvestre. There have been numerous human clinical trials that have all had positive results. In a 1990 study, researchers at the University of Madras in India, 22 patients with type 2 diabetes who were non-insulin-dependent were given 400 milligrams of Gymnema extract daily, in two divided doses, in addition to their normal dose of oral hypoglycemics for 18 to 20 months. Ages ranged from 40 to 62 years and disease duration from 1 to 12 years. Over the duration of treatment, Gymnema significantly lowered fasting blood glucose levels (average of 174mg/dl to 124mg/dl).

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Participants in the Gymnema group also had a significant reduction in hemoglobin A1c. (Hemoglobin A1C is tested to monitor the long-term control of diabetes and is increased in the red blood cells of persons with poorly controlled diabetes. From this test clinicians can estimate the average blood glucose level during the preceding two to four months. The target for most people is below 7). Twenty-one of the 22 diabetic patients were able to reduce their intake of drugs, while five patients were able to stop their conventional drugs completely, maintaining normal glucose levels with the Gymnema supplements alone. Also, their Insulin levels increased significantly compared to those on drugs alone. The authors suggested that this increase in insulin levels was probably due to regeneration or repair of beta cells facilitated by Gymnema. All of the above contrasts significantly to the diabetic group on drugs alone. Their fasting glucose and hemoglobin A1c had elevated slightly and their drug doses either stayed the same or rose over the trial period. (J. Ethnopharmacol. 1990 Oct; 30(3): 295-300). The above study demonstrates that the use of Gymnema may result in the need for smaller doses of oral diabetes drugs to control the disease. However, it is important that people with this disease dont abandon proven ways to manage it, from a healthy diet to regular exercise and medications when needed. In another study in 1990 carried out by the same research group at the University of Madras, India, 27 participants with insulin-dependent type 1 diabetes, ages from 10 to 50 years and with varying disease duration, were supplemented with 400 milligrams of Gymnema extract daily in two divided doses. All were using daily insulin injections. After 6 to 8 months of treatment with the Gymnema extract the average insulin requirements were decreased from 60 to 45 units per day and fasting blood glucose levels were lowered. In fact, one patient with a type 1 diabetes duration of 10 years and another with a lesser duration were able to discontinue insulin usage completely. These improvements continued through out the trial till its completion. One has to find it remarkable that, compared to conventional treatment, patients with type 1 diabetes for 25 years responded equally as well as those who had been diagnosed for only one year when given the Gymnema extract supplement. So, there appears to be no time limitation on Gymnema use to still receive benefits. This contrasts significantly to the diabetic group not taking Gymnema and receiving insulin alone for a year. Their insulin requirements either remained the same or increased and their average insulin usage was almost double that of the Gymnema group. Also, the drop in fasting blood glucose was statistically insignificant in this group.

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In addition, cholestrol levels also returned to near normal levels and triglycerides were lowered in the group taking Gymnema. (J. Ethnopharmacol. 1990 Oct; 30(3): 281-94). In 1997 Japanese researchers at the Nippon Veterinary and Animal Science University, Tokyo, Japan, decided to test Gymnema inodorum, since it has an advantage that it does not suppress sweetness nor is it bitter in taste. Their conclusions, published in The Journal of Veterinary Medical Science, was that their studies suggested Gymnema inodorum inhibits the increase in the blood glucose levels by interfering with the intestinal glucose absorption process. (J Vet Med Sci. 1997 Sep; 59(9): 753-7). In a study, published in the Diabetes In Control Newsletter (30/10/2001), conducted by Diabetes Educators, under the protocols established by Diabetes in Control and Informulab of Omaha, NE, the makers of Beta Fast GXR, Gymnema Sylvestre was found to lower HbA1c from 10.1% to 9.3%. HbA1c or Hemoglobin A1C is tested to monitor the long-term control of diabetes and is increased in the red blood cells of persons with poorly controlled diabetes. From this test clinicians can estimate the average blood glucose level during the preceding two to four months. The target for most people is below 7. The study included 65 participants with type 2 diabetes, some diet controlled others insulin dependent, ages 18 to 73 and was conducted for a ninety day period. Patients were given Beta Fast GXR brand of Gymnema Sylvestre containing 400mg leaf extract per tablet twice daily. An interesting observation made by the researchers was that the higher the participants initial HbA1c values (and therefore the less controlled their diabetes) the more significant the results. In the group of participants that started with an HbA1c above 10% the Gymnema Sylvestre supplementation lowered HbA1c from 11.1% to 9.9% (1.2% decrease) compared with the group that started at 9% or above, HbA1c was lowered from 10.1% to 9.3% (0.8% decrease). (Diabetes In Control Newsletter, Issue 76 (1) : 30 Oct 2001). The reduction of HbA1c in this study is of great importance as an improvement in glycemic control reduces the risk of complications from diabetes. Glycemic control is a medical term referring to the typical levels of blood sugar (glucose) in a person with diabetes type 2. Much evidence suggests that many of the long-term complications of diabetes, especially the microvascular (smaller blood vessels) complications, result from many years of hyperglycemia (elevated levels of glucose in the blood). Good glycemic control, in the sense of a target for treatment, has become an important goal of diabetes care.

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In 2003 researchers, reporting in the journal Pharmacological Research, found that Oral administration of 200 mg of Gymnema extract per kg of body weight to diabetic rats for 3 weeks resulted in a significant reduction in blood glucose levels and an increase in insulin levels. There was also a decrease in free radical formation in the blood of the diabetic rats. These effects were compared with glibenclamide, an anti-diabetic drug (Diabeta, Glynase Micronase Daonil, Semi-Daonil Euglucon. Also sold in combination with metformin under the trade name Glucovance.) The researchers concluded that the results suggest that Gymnema extract was more effective than the drug glibenclamide. (Pharmacological Research. 2003 Dec; 48(6): 551-6). Antioxidant activity of Gymnema in diabetes management The above study shows that Gymnema appears to recycle available vitamin C and E. The prevalent long term depletion of vitamin C, common in diabetes, may contribute to depressed immune function, compromised wound healing ability and reduced blood vessel integrity. These and other related aliments can possibly be arrested and reversed by vitamin C supplementation. (Dr. Brian Jakes, Jr. Diabetes In Control July 2002). C-reactive protein is linked to cardiovascular disease in people with diabetes. Having both diabetes and an elevated C-reactive protein (CRP) level compounds ones risk of developing cardiovascular disease, for which people with diabetes are at particularly high risk, scientists writing in the American Journal of Nursing report in their review. (Life Extension Foundation Daily News 10/04/06). Vitamin C supplements can reduce levels of C-reactive protein, a marker of inflammation and chronic disease risk in humans, according to a new study led by researchers at the University of California, Berkeley. Participants who took about 500 milligrams of vitamin C supplements per day saw a 24 percent drop in plasma C-reactive protein (CRP) levels after two months. C-reactive protein is a marker of inflammation, and there is a growing body of evidence that chronic inflammation is linked to an increased risk of heart disease, diabetes and even Alzheimers disease, said Gladys Block, UC Berkeley professor of epidemiology and public health nutrition and lead author of the study. The researchers say that long-term adverse health effects occur when inflammation persists at low levels. This chronic inflammation, with persistent elevated levels of CRP, has been found among smokers and Type 2 diabetics, as well as among overweight or obese persons.

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The researchers noted that in other studies, higher doses of vitamin E produced lower CRP levels among Type 2 diabetics and healthy individuals. (Sarah Yang, Media Relations, University of California, Berkeley, 12/4/2004). Recent clinical trials confirm the uses of typical antioxidants alone or in combination with other natural supplements may delay or even prevent the normal progression of diabetic complications. Importantly, none of the participants in these studies reported any adverse side effects, although many patients developed hypoglycemia (low blood glucose) and required a lowering of their dose of conventional oral medication or insulin. Therefore, any diabetic that uses GSE (Gymnema Sylvestre Extract) must carefully monitor blood glucose levels and adjust their medication, in consultation with their physician, to maintain desired blood glucose levels. This is because improved insulin production and release during GSE supplementation may result in over-medication, and therefore low blood glucose levels, unless the dosage of conventional oral medication or insulin is lowered. Since most diabetics monitor their blood glucose levels on a daily basis, this shouldnt present a problem. Japanese researchers conducted a study in 2004 using both male and female rats to determine Gymnema sylvestre toxicity. One group of rats was fed a normal diet, the other group supplemented with Gymnema at increasing doses for 52 weeks. The researchers concluded there were no toxic effects in rats treated with Gymnema sylvestre at up to 1.00% in the diet for 52 weeks. This equated to an average of 504 mg/kg/day for male rats and 563 mg/kg/day for female rats daily intake over the period. (Shokuhin Eiseigaku Zasshi. 2004; 45(1):8-18). Dose size of Gymnema required to reduce blood glucose in humans efficiently: This can vary widely depending on numerous factors including the specific type of herb used and the patient being treated. Gymnema can work quite quickly to control blood sugar levels. Used on its own, it will not drop blood sugar so far as to cause hypoglycemia. However, there is the risk this could happen if it is used together with insulin or anti-diabetic drugs. So in these cases, Gymnema should only be taken under professional supervision. (Kerry Bone, FNIMH, FNHAA, Nutrition and Healing Newsletter, January 2002). Aside from lowered blood sugar and increased effects of anti-diabetic drugs following chronic use of gymnema, no significant adverse effects have been reported with the herb, in several studies up to 20 months long.

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Caution: It is advised in patients with diabetes or low blood sugar, and in those taking drugs, herbs, or supplements that affect blood sugar. Serum glucose levels may need to be monitored by a qualified healthcare professional, and medication adjustments may be necessary. (MedlinePlus). Signs that blood sugar may be too low include shakiness, sweating, confusion, distorted speech, and loss of muscle control.

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MATERIALS & METHODS

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Over the last three decades DNA markers are widely being used for genetic mapping, molecular taxonomical studies and for the detection of genetic changes in organisms caused due to mutations or genetic engineering. Arbitrary primed PCR has been used to detect dominant polymorphic markers in genetic mapping experiments (Williams et al, 1990). DNA markers have been widely used in genome mapping in a wide range of plant species and are now being increasingly employed for studies of genetic relatedness among species. New source of high quality genetic markers, based on the identification of polymorphisms in DNA and protein was developed in the last three decades. They have been termed as Molecular markers. Linkage maps of many plant species were limited in size until the advent of molecular mapping. The primary difficulty with developing linkage maps was the inability to incorporate many markers into a single stock to be used for genetic analysis. This inability occurred because of the deleterious effects of the expression of all mutant phenotypes in the single stock. Because normal DNA or protein molecules are used to score the genetic material, molecular markers are phenotypically neutral. This is a significant advantage compared to traditional phenotypic markers.

The three most common types of markers used today are RFLP, RAPD and isozymes. Of the three marker types, RFLPs have been used the most extensively. RFLP markers have several advantages in comparison with the RAPD and isozyme markers: 1) they are codominant and unaffected by the environment; 2) any source DNA can be used for the analysis; and 3) many markers can be mapped in a population that is not stressed by the effects of phenotypic mutations.

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The primary drawback to RAPD markers is that they are dominant and do not permit the scoring of heterozygous individuals. The weakness of isozyme markers is that each of the proteins that are being scored may not be expressed in the same tissue and at the same time in development. Therefore several samplings of the genetic population need to be made.

RFLP -

Restriction Fragment Length Polymorphism; a molecular marker based on the

differential hybridization of cloned DNA to DNA fragments in a sample of restriction enzyme digested DNAs; the marker is specific to a single clone/restriction enzyme combination

RAPD sequences

Randomly Amplified Polymorphic DNA; a molecular marker based on the

differential PCR amplification of a sample of DNAs from short oligonucleotide

AFLP -

Amplified Fragment Length Polymorphism; a molecular marker generated by

a combination of restriciton digestion and PCR amplification

Isozyme - a molecular marker system based on the staining of proteins with identical
function, but different electrophoretic mobilities<

Selection with markers depends mainly on the quality of the polymorphism used. Williams et al (1990) proposed the use of single short random primers (usually 10-mers) in polymerase chain reaction (PCR) as a method of generating polymorphic markers (RAPDs). DNA polymorphism result either from differences in the DNA sequence at primer binding sites or by chromosomal changes affecting the amplified regions. After electrophorasis the amplified fragments can be visualized by florescence and the resulting patterns of bands can interpreted genetically The present investigation entitled Molecular Characterization of five ecotypes of Gymnema Sylvester an Anti-diabetic plant was conducted during the year 2009 in the Molecular biology laboratory of Prof. G.M. Reddy Research Foundation, Hyderabad.

53

Experimental materials:
Different ecotypes of Gymnema plants present in and around the lab (Fig. 2) were used as source of experimental material for collection of explants.

54

55

Glass and plastic ware:

All the glassware used were of Borosil make for various studies, test tubes, Flasks, Bottles, beakers, MicroPipettes etc were used for preparations of various chemicals. All these glass and plastic ware were autoclaved at 121o C for 15 min. at 15 lb pressure. The pipette tips and the micro pipettes used were of Qualigen make.

Chemicals: Chemicals required for DNA isolation:

1) DNA extraction buffer 2) Chloroform: Isoamyl alcohol (24:1) 3) Chilled ethanol / Propanol. 4) 70% Ethanol. 5) RNase 6) TE buffer (T10 E1) 7) Phenol :Chloroform : Isoamyl alcohol. (25:24:1)

Chemicals required for PCR (Polymerase Chain Reaction).

1) Thermostable DNA polymerase 2) A pair of synthetic Oligonucleotides to prime DNA synthesis 3) Deoxynucleoside triphosphates (dNTPs) 4) Mg +2 Buffer 5) 10X Buffer 6) Template DNA.

Chemicals required for Agarose gel electrophoresis.

1) TAE (50X) buffer 2) Agarose 3) Ethidium bromide 4) Running buffer (1X) 5) Loading dye.

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Isolation of DNA (Deoxy ribonucleic acid):

Genomic DNA is the blue print of life, which is inherited and passed on from generation to generation directing the development of cells, organelles and organisms. Isolation of DNA from RNA and proteins is essential for all molecular biology investigations. For the isolation of DNA, the cell wall must be broken or digested in order to release cellular constituents, which is done by grinding tissue in dry ice or liquid nitrogen. Cell membrane is disrupted by using detergents such as CTAB (Cetyl Trimethyl Ammonium Bromide) or SDS (Sodium Dodecyl Sulphate). For the protection of DNA from endogenous nucleases, EDTA (Ethylene Diamine Tetra Acetic Acid.) is used. EDTA chellates Mg++ ions, which is essential co-factor for most of the nucleases. The tissue mixture is emulsified with chloroform and phenol to denature proteins from DNA. The DNA isolation from plant tissues is very difficult due the presence of various secondary plant products. A protocol works with one plant group and may fail with the others. Hence a number of DNA isolation methods have been developed for different target plant groups. But two methods originally developed by Dellaporta et al (1983) and Murray and Thompson (1980) respectively are being commonly used with certain modifications for isolating high molecular weight DNA from small amounts of tissue.

57

PREPARATION OF CHEMICALS INVOLVED IN ISOLATION OF DNA I) DNA extraction buffer (DEB) stock preparation.
Stock solution. Working solution for Preparation of 100ml DEB. 35 ml 4 ml 10 ml 30 ml 2 ml.

4M NaCl 0.5M EDTA (pH 8.0) 1M Tris Cl (pH 8.0) 10% CTAB Mercapto Ethanol

Make up the volume to 100 ml with distilled water and it is autoclaved.

II). 24:1 Chloroform Isoamyl alcohol.

24 ml of chloroform is taken in a flask and 1 ml of isoamyl alcohol is added to it.

III) Phenol: Chloroform: Isoamyl alcohol. (25:24:1)

25 ml of Phenol is taken in a clean conical flask and to it 24 ml of chloroform and I ml of isoamyl alcohol are added.

IV). TE Buffer
1ml of 1M Tris (pH 8.0) is taken in a clean conical flask and to it o,2 ml of EDTA (pH- 8.0) is added and the volume is made up to 100 ml with distilled water. The conical flask is then autoclaved.

V). 70% Ethanol

70 ml of alcohol is taken in a clean conical flask and the volume is made up to 100ml with distilled water.

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METHODS:
Steps involved in extraction of DNA .
1) One gram of gymnema leaf was taken from each different four ecotypes, washed under running tap water and then dried on filter paper. 2) These leaves were taken in different Motor and pestles and were made to fine powder with the help of liquid N2. 3) The powder was then transferred to different centrifuge tubes and 5 ml of DEB was added to each tube. 4) The tubes were shaken gently for about 10 minutes and then placed in a water bath for about 1 hour at 60 C. 5) After 1 hour the tubes were removed and equal volume of (24:1) Chloroform : Isoamyl alcohol is added and were gently shaked for about 5 10 minutes. 6) The tubes were then centrifuged at 15,000 rpm for about 10 minutes at room temperature. 7) These tubes were carefully removed and the supernatant was collected using a micropipette. 8) To the above supernatant equal volume of chilled ethanol was added and gently shaked. 9) A pellet of white fibrous structure, DNA was observed in the tube. 10) This DNA was then separated from the tube and washed twice or thrice with 70% Ethanol to remove the impurities. 11) Latter the DNA is taken in a small MCT(Micro Centrifuge Tube) tube, the mouth wrapped with a paraffin wax with a hole in the centre is dried overnight. 12) Next day, after the DNA is dried, 150 300microlitres of TE was added to the DNA until it is dissolved. 13) To the dissolved DNA, RNase is added and is stored in cool conditions for future use.

PROTOCOL FOR ISOLATION OF DNA FROM GYMNEMA SYLVESTRE LEAVES USING CTAB METHOD

59

1gm of young Gymnema leaves were ground in liquid nitrogen until a fine powder was obtained

The fine powder was transferred into a 20ml capacity centrifuge tube

To the leaf powder 5ml of CTAB-DNA extraction buffer (pre-heated to 60C) has been added and mixed thoroughly

The mixture was incubated for 1hour at 65C in a water bath

After incubation, the mixture was emulsified with an equal volume of chloroform: Isoamyl alcohol (24:1)

The mixture was centrifuged at 10,000 rpm for 20min at room temperature (25C)

After centrifugation the upper aqueous phase was pipetted out with the help of micropipette

To the aqueous phase add equal volume of chilled isopropanol by quick inversion

DNA, like a mass of cotton threads was precipitated

The precipitated DNA was spooled out and transferred to micro centrifuge tube and washed twice with 70% ethanol and kept for air-dried.

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Precautions:
1) Time between thawing of frozen pulverized tissue and exposure to extraction buffer should be minimized to avoid nucleotide degradation of DNA. 2) DEB stock is light sensitive; hence it must be always covered with a black plastic cover/with aluminum foil. 3) The DEB should be preheated before using it. 4) The Ethanol used for precipitating DNA should be very chilled.

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POLYMERASE CHAIN REACTION:

The method of PCR was first proposed by H. Gobind Khorana in early 1970.The technique was put into practice by Kary Millis at Cetus Corporation in 1985 which described invitro amplification of single copy mammalian genes using the Klenow fragment of E. Coli DNA Polymerase I. The first description of PCR precise, laconic, impersonal was published by Kleppe et al in 1971.

Thermal cycler for PCR

62

Principle:

PCR involves synthesizing multiple copies of a region of a DNA which bind to opposite strands flanking the target sequences.

Essential components/Reagents of PCR are1) Thermostable DNA polymerase (Taq polymerase). 2) A pair of synthetic oligonucleotides to prime DNA synthesis (Primers). 3) Deoxynucleoside triphosphates.(dNTPs) 4) Template DNA. 5) 10X buffer 6) Mg+2 buffer.

63

Temperature cycles involved in PCR are :

1) Pre denaturation 94C for 4 min. 2) Denaturation 94o C for 1 min. In Both 1and 2, i.e Predenaturation and denaturation the two strands of target DNA are separated. 3) Annealing It depends upon the primers used. Upon the primer bottles the nucleotide sequence will be given from 3 to 5 end. Depending on the nucleotide sequence the annealing temperature is calculated using the two formulas: 4(G+C) 2(A+T)

A = Adenine T = Thymine G = Guanine C = Cytosine. This annealing temperature anneals two complementary primers to ends of separated single stranded DNA strands of targeted DNA. 4) Primer extension 72C for 2 min. This temperature allows thermostable Taq DNA polymerase to use single standard DNA strands of target and primers to synthesize new strands. 5) Final extension 72C for 5 min.

64

Schematic drawing of the PCR cycle

(1) Denaturing at 94-96C. (2) Annealing at ~65C (3) Elongation at 72C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses.

65

Figure: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96C. (2) Annealing at (eg) 68C. (3) Extension at 72C (P=Polymerase). (4) The first cycle is complete. The two resulting DNA strands make up the template DNA

66

for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.

Protocol:
Preparation of 20 lit. PCR reaction mixture.
1) 20 different sterile thin walled autoclaved PCR tubes were taken and numbered serially from 1-20. 2) In these tubes 1lit of DNA sample and 1lit. of the respective primers are taken and stored in Ice. 3) In sterile autoclaved tubes the cocktail mixture is prepared by adding the below contents as follows. For 1 tube DNTPs 10X buffer Mg+2 buffer Taq polymerase Dist. Water 2lit. 2lit. 2lit. 0.5l. 11.5lit. * * * * * 20 20 20 20 20 For 20 tubes = = = = = 40lit. 40lit 40lit. 10lit. 230lit.

4) Then the tube of the cocktail reaction mixture is maintained at cool temperatures. 5) With the help of micropipette, 18lit. of the cocktail mixture from the tube is pippetted into the PCR tubes. 6) Then the tubes are gently tapped and then they are placed in the PCR machine. 67

7) Program the PCR machine to the following conditions. Predenaturation Denaturation Annealing Primer l extension Final extension No. of cycles Hold temperature 94C for 4 min. 94C for 1 min. Depending on the Primer sequence. 72 for 2 min. 72C for 5 min 40 cycles. 4C.

8) After the 40 cycles are over the PCR machine comes to a hold temperature of 4 C. Then the samples are removed and stored at 4C.

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TABLE SHOWING DISTRIBUTION OF PRIMERS TO SAMPLES

Primers Ecotype I Ecotype II Ecotype III Ecotype IV Ecotype V

OPL-01 1 5 9 13 17

OPL-02 2 6 10 14 18

OPL-06 3 7 11 15 19

OPL-07 4 8 12 16 20

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ELECTROPHORESIS:
The idea of using Electrophorasis to analyze DNA came from Vin Thorne 1960. The first modern electrophoresis was developed by Walter schffner. Electrophoresis is used to identify, separate and purify DNA fragements.The technique is simple, rapid to perform and can separate DNA fragments that can not be separated adequately by other procedures such as gradient centrifugation. Generally two types of gels are being used. The choice depends mainly on the sizes of the fragments being separated. 1). Agarose gels. 2). Poly acrylamide gels. Polyacrylamide gels are most effective for separating small fragments of DNA (5 500bp) and have high resolving power. But the polyacrylamide gels are difficult to handle and prepare. Agarose gels are used for separating larger double stranded molecules and have low resolving power and have grater range of separation. DNAs from 50bp to several megabases in length can be separated in agarose gels.

70

Gel electrophoresis apparatus - An agarose gel is placed in this buffer-filled box and electrical current is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera.

Theory/Principle of DNA migration:

Nucleic acids being negatively charged will migrate towards the +ve pole i.e anode. The electrophoratic mobility of a macromolecule is determined by the volume fraction of pores within the gel that the macromolecule can enter. Since small DNA fragments can fit into more pores than large DNA fragments, small DNA fragments will migrate through the matrix faster than large DNA fragments. Larger molecules migrate more slowly because of greater frictional drag and because they worn their way though the pores of the gel less efficiently than the smaller molecules.

The velocity of the DNA fragments decreases as their length increases and is proportional to electric field strength. The greater the pore size of the gel, the larger the DNA that can 71

be sieved. Agarose gels with low concentration of agarose (0.1 0.2% w/v) are capable of resolving larger DNA molecules, but are very fragile and must be run for several days and cannot resolve linear DNA molecules larger then 750kb in length. The PCR DNA samples should be subjected to electrophoresis for observing the amplification patterns of the DNA.

PREPARATION OF CHEMICALS INVOLVED IN AGAROSE GEL ELECTROPHORESIS: 1) Buffers:

Two types of running buffers are commonly used for DNA gel electrophoresis.

A) 50X TAE buffer (Tris Acetate EDTA) stock solution.

242 g. of Tris and 37.2 g. of Na2 EDTA (2H2O) are dissolved in 900ml of water. To the above solution 57.1 ml of glacial acetic acid is added and the final volume is made up to 1 lit. Stored at 4 degrees.

B) TBE buffer (Tris Borate EDTA).

108g. of tris base and 55g. of Boric acid are dissolved in 900ml of water. To the above solution 40ml of 0.5 M EDTA (pH 8.0) is added and the final volume is made to 1 liter with the help of distilled water.

2) Ethidium bromide Available readymade. 3) Agarose Available readymade as powder. 4) Preparation of Tank buffer.

72

2ml of 50X TE is taken in a clean conical flask and is made upto 100ml with distilled water.

Steps involved in Electrophoresis of crude DNA. Casting the gel.

1) 2ml of the 50X TAE was taken and was made up to 100ml with distilled water. 2) 1g of Agarose powder was added to the buffer solution. 3) The solution was then heated till all the agarose is completely dissolved. 4) The solution was then cooled on the bench and Ethidium bromide is added and is mixed gently with out formation of air bubbles. 5) Meanwhile the casting tray along with the comb was set on the bench and the above solution was poured carefully in the tray with out formation of air bubbles. 6) The tray is left as such for about half an hour with out disturbing it to solidify. 7) Meanwhile the tank buffer is prepared and poured in the electrophoresis tank up to the level. 8) After the gel gets solidified, the gel is mounted in the electrophoresis tank carefully. 9) The DNA samples were taken out from the fridge and were gently shaked for uniformity. 10) 8 microlit of the DNA sample was taken and to it 2 micro lit. of loading dye was added and mixed properly. 11) Then the solution is taken into a pipette carefully and loaded in the wells.

73

12) The electrophoresis is switched on and the voltage is adjusted in-between 40 80 volts. 13) It takes atleast 3 4 hours for the DNA to run completely. 14) After the electrophoresis is completed the gel is carefully removed from the electrophoresis tank and is kept in the gel documentation unit for viewing the DNA bands under U.V. light. (Fig.3) In gel Documentation the software used is alpha imager-2200.

74

4 5 GENOMIC DNA OF DIFFERENT ECOTYPES OF GYMNEMA SYLVESTRE 1 1 2 2 3 3 4 4 5 5

Fig: 3 1 ----- Ecotype I 2 ----- Ecotype II 3 ----- Ecotype III 4 ----- Ecotype IV 5 ----- Ecotype V

75

RESULTS & DISCUSSION

76

RESULTS
Molecular characterization/genetic diversity of the five different ecotypes of Gymnema sylvestre was studied using Random Amplified Polymorphic DNA (RAPD) analysis. A preliminary study was carried out using only four primers namely, OPL 01, OPL 02, OPL 06, and OPL 07. Except OPL-01 Primer all the primers gave satisfactory amplification of 63 bands.

OPL-01:
OPL-01 Primer gave the monomorphic banding pattern .Out of the total 63 bands amplified, 18 (28.57%) were observed to be polymorphic and 5 (7.93%) were unique, i.e specific to a particular ecotype.

OPL-02:
The primer OPL 02 resulted with a total number of 21 amplicons. Ten bands were found to be polymorphic and 3 were specific to the ecotypes Ecotype III & Ecotype V. Ecotype I,

OPL-06:
The primer OPL 06 gave bands with the fragment sizes ranging from approximately 500 bp to 1500 bp. Eight out of these 19 bands were polymorphic and two, with fragment size approximately 1500 bp were specific to the Ecotype II & Ecotype III respectively.

77

OPL-07:
Eighteen amplicons were observed with the primer OPL 07. All the bands were monomorphic except one, which is specific for the ecotype Il. The preliminary study suggested the existence of genetic differences among five ecotypes of the Gymnema sylvestre and RAPD analysis exhibits the differences in ecotypes.

78

RAPD PROFILES OF DIFFERENT ECOTYPES OF GYMNEMA SYLVESTRE WITH OPL-01 PRIMER

L 1 2 3 4 5

4500 4000 3500 3000 2500 2000

1500

1000

500

Fig: 4 L----- 500 bp Ladder 1 ----- Ecotype I 2 ----- Ecotype II 3 ----- Ecotype III 4 ----- Ecotype IV 5 ----- Ecotype V PRIMER OPL-01 GAVE THE MONOMORPHIC BANDS AND THE AMPLICONS RANGED FROM 500 TO 1000 BP

79

RAPD PROFILES OF DIFFERENT ECOTYPES OF GYMNEMA SYLVESTRE WITH OPL-06 PRIMER L 1

L
4000 4000 3500 3000 2500 2000 1500

2
1 2

3
3

4
4

5
5

1000 500

Fig: 6 L ----- 500 bp Ladder 1 ----- Ecotype I 2 ----- Ecotype II 3 ----- Ecotype III 4 ----- Ecotype IV 5 ----- Ecotype V PRIMER OPL_06 GAVE MAXIMUM NO OF BANDS. THE AMPLICONS RANGED FROM 500BP TO 2500BP.

80

RAPD PROFILES OF DIFFERENT ECOTYPES OF GYMNEMA SYLVESTRE WITH OPL-07 PRIMER

L
4500 4000 3500 3000 2500 2000 1500

11 2 3 4 25

1000

500

Fig: 7 L----- 500 BP LADDER 1 ----- Ecotype I 2 ----- Ecotype II 3 ----- Ecotype III 4 ----- Ecotype IV 5 ----- Ecotype V PRIMER OPL-07 GAVE THE ALL MONOMORPHIC BANDS EXCEPT ONE i.e., SPECIFIC TO THE ECOTYPE 2. THE AMPLICONS RANGED FROM 500 BP TO 3500 BP

81

DISCUSSION
The main objectives of the project are acquisition of different ecotypes of Gymnema Sylvester and their molecular characterization. Different ecotypes of Gymnema Sylvester were collected from various places of Andhra Pradesh including Tropical Botanical Garden Research Institute. Pallad, Kerala and were maintained in the net houses at the Foundation. The material required for the research work is taken from the plants grown in the net houses. The DNA was isolated from the leaves of Gymnema Sylvester using CTAB method and is dissolved in approximate quantity of TE and stored at 4 C for future use. This dissolved DNA is subjected to electrophoresis along with lambda DNA marker for estimating the concentration of the isolated DNA which is essential for further work. Depending on the estimated concentration of DNA, it is subjected to PCR (Polymerase Chain Reaction). The amount of DNA required for PCR depends upon the concentration of DNA. The DNA sample, Primers, and the cocktail mixture are taken in a sterile PCR tube and made upto 20 lit. With doubled distilled water. During the work a temperature of 4 C is maintained, else all the primers and cocktail mixture gets deactivated. Then the PCR tubes were subjected to PCR. The various temperatures associated in the PCR cycles are as follows. Predenaturation Denaturation Annealing Extension Final extension No. of cycles Hold temperature 94C for 4 min. 94C for 1 min.

36C for 1 min. 72C for 2 min. 72 for 5 min. 4C. 82 40 cycles.

After the 40 cycles are over the PCR machine comes to a hold temperature of 4 C. Then remove the samples and store them at 4C.

A preliminary study was carried out using only five primers namely, OPL 01, OPL 02, OPL 06, and OPL 07. Except OPL-01 Primer all the primers gave satisfactory amplification of 63 bands. Out of the total 63 bands amplified, 18 (28.57%) were observed to be polymorphic and 5 (7.93%) were unique, i.e specific to a particular ecotype. The primer OPL-01 gave the monomorphic banding pattern. The primer OPL 02 resulted with a total number of 21 amplicons. Ten bands were found to be polymorphic and 3 were specific to the ecotypes I, ecotype III and ecotype V. A total number of 19 bands were amplified with the primer OPL 06 with the fragment sizes ranging from approximately 500 bp to 1500 bp. Eight out of these 12 bands were polymorphic and two, with fragment size approximately 1500 bp were specific to the ecotype II and ecotype III and ecotype respectively. Eight amplicons were observed with the primer OPL 07. All the bands were monomorphic except one, which is specific for the ecotype II. The preliminary study suggested that existence of genetical differences among six ecotypes of the Gymnema Sylvester and RAPD analysis exhibits the differences in ecotypes.

83

CONCLUSION
Molecular characterization of different ecotypes was carried out using RAPD technique. Based on differences in the banding pattern the genetic variations existing between the different ecotypes of gymnema sylvestre can be identified.

After the electrophoresis the banding pattern is obtained with 4 different primers. The variations in the banding pattern indicate the genetic variations in the ecotypes. Among the existing ecotypes, ecotype-2 has high percentage of Gymnemic acid

(48%) & this ecotype gave a specific banding pattern with opl-07 primer, which was confirmed by using the rapd technique.

So by analyzing the banding pattern and on carrying out further studies the gene sequence responsible for high percentage of gymnemic acid can be identified.Further it can be cloned to generate the transgenic plants that can be further used for medicinal purposes.

The preliminary study suggested that existence of genetical differences among ecotypes of the Gymnema sylvestre and RAPD analysis exhibits the differences in ecotypes.

84

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85

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89