Title of Invention

A PROCESS FOR PREPARATION OF A NOVEL COMPOUND GYMNEMIC TRIACETATE FROM GYMNEMA SYLVESTRE R. Br. LEAVES WITH ANTIDIABETIC ACTIVITY

Abstract Gymnemic triacetate isolated from the leaves of Gymnema sylvestre, was examined for its anti hyperglycemic effect in streptozotocin induced diabetic rats. Oral treatment of I Gymnemic triacetate decreased the plasma glucose concentrations of STZ-diabetic rats.Similar treatment with Gymnemic triacetate increased the plasma insulin levels.
Full Text

FIELD OF INVENTION
This invention relates to a process for preparation of a novel compound Gymnemic triacetate from Gymnema sylvestre R.Br, leaves with antidiabetic property.
PRIOR ART
Diabetes mellitus is one such disease and it is estimated that the number of diabetic patients will continue to increase in the future. Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes. The hypoglycemic activity of a large number of these plants has been evaluated and confirmed in different animal models. Many antidiabetic agents with unique properties have been introduced into the therapy of diabetic patients in the last few years, prevention and treatment of diabetes remains an unresolved problem. Unfortunately, none of the currently used antidiabetic agents provide all the required advantages necessary for successful management including adequate hypoglycaemic activity, modification of insulin secretion, peripheral insulin resistance, and sufficient safety. The search for new antidiabetic agents represents a challenge to the medical profession.
The administration of plant extracts for the treatment of diabetes mellitus is varying in their efficacies because of nutritional status, genetic variation and soil condition of the plant growth. Later treatment of diabetes shifted towards the usage of synthetic oral hypoglycaemic agents. Many oral hypoglycaemic agents, such as biguanides and sulfonylurea are available along with insulin for the treatment of diabetes mellitus, but these synthetic agents can produce serious side effects. Therefore, search for safe and more effective agents has continued to be an important area of active research. Furthermore, after the recommendation made by WHO on diabetes mellitus, investigations on hypoglycaemic agents from medicinal plants have become more important. Many plant-derived phytomedicines such as P-carotene and a-lipoic acid have been reported to have antidiabetic effects. We have isolated antidiabetic compound from Gymnema sylvestre R.Br, leaves, which has been shown to possess antidiabetic activity in streptozotocin (STZ) induced-diabetic rats.

BJECT OF THE INVENTION
> The primary object of the invention is to prepare a novel compound Gymnemic triacetate with antidiabetic property.
> Another object of this invention is to propose a process for preparation of novel compound of with antidiabetic property to overcome the disadvantages of the prior art.
> Further object of this invention is to propose a process for preparation of novel compound of with antidiabetic property, which exhibits antidiabetic activity.
> Still further object of this invention is to propose a process for preparation of a novel compound of with antidiabetic activity, which is not harmful to human beings.
STATEMENT OF THE INVENTION
According to this invention there is provided a process for preparation of novel compound of Gymnemic triacetate with antidiabetic activity comprising steps of: -

Molecular formula: C24H30O7 Molecular weight: 432

a) Gymnema sylvestre Leaves powder (2 Kg) was soaked in hexane and extracted.
b) Residue left after the extraction with hexane was further extracted with acetone.
c) The acetone extract was filtered and concentrated at 40oC by using vacuum rotary evaporator and crude extract was obtained.

d) Acetone crude extract was chromatographed on a silica gel column (Merck 70-230 mesh, 400 gm, 3.5 i.d. x 60 cm) and successively eluted with stepwise gradient of petroleum ether and benzene system.
e) 120 fractions (each 50ml) were collected; each fraction was spotted on a precoated Silica gel 60 F254, 0.25mm thick TLC plate (Merck) and eluted in Petroleum ether: Benzene and fractions with similar Rf values in TLC pattern were pooled together.
f) Fraction 76-90 formed a yellow colored compound and showed a single spot on TLC (Rf- 0.734) (8:2, Petroleum ether: Benzene).
g) The compound was assayed for the plasma glucose reduction level in Streptozotocin (STZ) induced diabetic rats.
h) The compound possessed strong antidiabetic activity.
i) The structure of the isolated compound was determined on the basis of H, C NMR,
MS and FT-IR.
j) Further according to this invention a new compound was provided with antidiabetic
properties, which was shown as Gymnemic triacetate.

DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO ACCOMPANYING DRAWINGS
Fig. 1 shows a flow chart for the isolation of a novel compound of Gymnemic triacetate from Gymnema sylvestre Leaves.


Fig. 2 shows FT-IR spectrum of Gymnemic triacetate.
Fig. 3 shows mass spectrum of Gymnemic triacetate.
Fig. 4. shows 1H spectrum of Gymnemic triacetate.
Fig. 5 shows 13C spectrum of Gymnemic triacetate.
Accordingly Fig 1. Two kg of Gymnema sylvestre leaves powder was completely extracted with Hexane, and the extract was concentrated and ultimately dried. The residue left after the extraction with hexane was further extracted with acetone. The acetone extract was filtered and concentrated at 40°C by using vacuum rotary evaporator and crude extract was obtained. Acetone crude extract was chromatographed on a silica gel column and successively eluted with stepwise gradient of petroleum ether and benzene system. 120 fractions (each 50ml) were collected; each fraction was spotted on a precoated Silica gel 60 F254, 0.25mm thick TLC plate (Merck) and eluted in Petroleum ether: Benzene and fractions with similar Rf values in TLC pattern were combined together. Fraction 76-90 formed a yellow colored compound and showed a single spot on TLC. The single spot was confirmed by TLC (Rf - 0.734; 8:2, Petroleum ether: Benzene). The compound was assayed for the plasma glucose reduction level in Streptozotocin (STZ) induced diabetic rats. The compound possessed strong antidiabetic activity. The structure of the isolated compound was determined on the basis of 1H, 13C NMR, MS and FT-IR. Further according to this invention a new compound was provided with antidiabetic properties, which was shown as Gymnemic triacetate. The novel compound Gymriemic triacetate is applicable for medical and pharmaceutical applications.

EXAMPLE
1. Two kg of Leaf powder of Gymnema sylvestre was soaked in 6L hexane 1:3w/v) and extracted for 72 h and the extract was concentrated and ultimately dried. The residue left after the extraction with hexane was further extracted with 5 L of acetone (1:3 w/v) for 72 h.
2. 40 g of acetone extract was subjected to column chromatography and 120 fractions were collected. The collected fractions were combined based on the TLC spot to get major fractions.
3. Fraction 76-90 formed a new compound, which was studied using spectroscopic techniques and structure of compound was confirmed as Gymnemic triacetate.
It is to be noted that the present invention is susceptible to modification, changes, and adaptations by those skilled in the art. Such modification, changes adaptations are intended to be within the scope of the present invention, which is further, set forth under the following claims.
STRUCTURE ELUCIDATION
1-3 1
The following details were found after subjecting the new molecule to 13X NMR, 1H NMR, MS and FT-IR analysis.
A solid residue was obtained from acetone extract of Gymnema sylvestre leaves, which was characterized by using spectroscopic techniques. The IR spectrum, IR (KBr) (v max cm'l) showed bands at 1737 and 1638 indicating the presence of corresponding functional groups (-CO-) and (-C-0-C-) in its structure (Fig.2). Its molecular ion peak (Fig. 3) at m/z 432 indicated the molecular weight and the elemental analysis showed the molecular formula of the compound as C24H30O7, which showed terpenoid nature of the compound.

1H-NMR, 13C-NMR spectrum
The 1H-NMR and 13C-NMR was recorded as 300MHs and 75MHs (Broker AV 300instrument) respectively. The experiment was performed at 25oC with 5mm 1H-NMR and 13C-NMR dual probe using the residual signal of the solvent CDCL3 and with TMS as an internal standard.

Based on the spectral data (5/ppm, JH-H) of the compound indicated that the molecule should be symmetrical and points towards the Gymnemic triacetate.
Gymnemic triacetate
FT-IR= 1737(-CO-) and 1638 (-C-0-C-); MS: m/z = 432 [M]+ [M+1]. The above spectral and analytical data led to the structure of compound Gymnemic triacetate.

ANTIDIABETIC ACTIVITY OF GYMNEMIC TRIACETATE FROM GYMNEMMA SYLVESTRE LEAVES IN STZ-INDUCED DIABETIC RATS
1.0. Experimental animals
Male albino rats of Wistar strain with the body weight ranging from 180-200g, were procured from Central Animal House, King's institute. Department of Animal and Preventive medicine, Guindy, Chennai and were maintained in an air conditioned room (25 ± 1 °C) with a 12 h light: 12 h dark cycle. Feed and water were provided ad libitum. Studies were carried out in accordance with Indian National Law on Animal Care and Use, and Committee for the Purpose of Control and Supervision of Experiments on Animals of Holly Cross College, Trichy.
1.1. Source of chemicals
Streptozotocin was purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals were of analytical grade obtained from E. MERCK and HIMEDLA, Mumbai, India.
1.2 Experimental induction of diabetes
The animals were rendered diabetes by a single intraperitonial injection of STZ (45 mg/kg body weight) in freshly prepared citrate buffer (0.IM, pH 4.5) after an overnight fast. STZ injected animals were given 20 % glucose solution for 24 h to prevent initial drug-induced hypoglycemic mortality, STZ injected animals exhibited massive glycosuria (determined by Benedict's qualitative test) and hyperglycaemia (by glucose oxidase method) within a few days. Diabetes in STZ rats was confirmed by measuring the fasting blood glucose concentration, 72 h after injection with STZ. The animals with plasma glucose above 350 mg/dL were considered to be diabetic and used for the experiment.

1.3 Experimental design
1.3.1 Effect of Gymnema sylvestre Leaf acetone extract {GSLAExt) in normoglycaemic and STZ-induced hyperglycaemic rats (daily for 45 days).
The animals were randomly divided into 5 groups of six animals each as given below. The insulin was administered intraperitonially and GSLAExt was administered orally using vehicle solution (Water).
Group I: Normal control
Group II: Normal + GSLAExt (600 mg/kg b.wt.)
Group IE: Diabetic control Group IV: Diabetic + GSLAExt (600 mg/kg b.wt. in)
Group V: Diabetic + Insulin (6 U/kg b.wt.)
1.3.2 Effect of Gymnemic triacetate on plasma glucose and plasma insulin in normoglycaemic and STZ-induced hyperglycaemic rats (daily for 30 days).
The animals were randomly divided into 5 groups of six animals each as given below. The insulin was administered intraperitonially and Gymnemic triacetate was administered orally using vehicle solution (Water).
Group I: Normal control (water only)
Group II: Normal + Gymnemic triacetate (20 mg/kg b.wt.)
Group III: Diabetic control (water only)
Group IV: Diabetic + Gymnemic triacetate (20 mg/kg b.wt.)
Group V: Diabetic + Insulin (6 U/kg b.wt.)

1.3.3. Determination of the plasma glucose
Blood samples were collected through the tail vein just prior to and after 15th, 30th and 45 day after STZ injection. The following biochemical investigation was carried out in our laboratory. Plasma glucose was estimated by the enzymatic method (Trinder, 1969) by using Boehringer Mannheim Kit.
1.3.4 Estimation of plasma insulin
Plasma insulin concentrations were determined by radioimmunoassay kit (Pharmacia, Uppsala, Sweden) with a beta metric coimter (Cronex, Dupont, France). The kit included human insulin as standard and I-labeled human insulin antibody, which cross-reacts similarly with rat insulin.
1.4. Data and statistical analysis
Data were expressed as means ± SD, One-way analysis of variance (ANOVA) was used to determine the significant difference between the groups. Differences were considered to be significant at P Table 1 Effect of Gymnema sylvestre Leaf acetone extract (GSLAExt) in normoglycaemic and STZ-induced hyperglycemic rats (daily for 45 days).



Values are means ± SD from six rats in each group.
Table 3. Effect of Gymnemic triacetate on plasma insulin in normoglycaemic and STZ-induced hyperglycemic rats (daily for 30 days).

Values are means ± SD from six rats in each group.











ADVANTAGES OF THIS INVENTION
Administration of Gymnemic triacetate showed a significant hypoglycemic effect and the maximum effect on blood glucose level was observed, over a period of 2 h with the dose of 20 mg/kg b.wt. Chronic administration of Gymnemic triacetate was found to significantly decrease the plasma glucose levels on 30th day and also increased the levels of insulin.
The Gymnemic triacetate isolated from Gymnema sylvestre leaves is having good glycaemic control properties in STZ-induced diabetic rats. The effect of Gymnemic triacetate is comparable with insulin. Gymnemic triacetate will be useful to revitalize treatment of diabetes mellitus. Hence, further studies are in progress for the assessment of Gymnemic triacetate on various biochemical changes like carbohydrate metabolic enzymes, lipid peroxidative markers, non-enzymic antioxidants, enzymic antioxidants, hepatic markers, lipid profiles, nephritic markers and histopathological changes.
WE CLAIM
1. A process for preparation of novel compound of Gymnemic triacetate with antidiabetic activity comprising steps of; -

Molecular formula: C24H30O7 Molecular weight: 432

a) Gymnema sylvestre Leaves powder (2 kg) was completely extracted with hexane.
b) Residue left after the extraction with hexane was further extracted with acetone.
c) 40 g of acetone extract was subjected to column chromatography and 120 fractions were collected. The collected fractions were combined based on the TLC spot to get major fractions.
d) Fraction 76-90 formed a new compound, which was studied using spectroscopic techniques
and structure of compound was confirmed as Gymnemic triacetate.
2. A process as claimed in claim 1, wherein the ratio between the plant powder and hexane was
1:3 (WAV) and acetone 1:4 (W/V) ratio.
3. A process for preparing a novel compound Gymnemic triacetate substantially herein described
and exemplified.
4. A compound substantially herein described and exemplified.


Documents:

1653-CHE-2007 EXAMINATION REPORT REPLY RECEIVED 13-08-2010.pdf

1653-che-2007 form 1 13-08-2010.pdf

1653-che-2007 form 3 13-08-2010.pdf

1653-CHE-2007 AMENDED CLAIMS 13-08-2010.pdf

1653-CHE-2007 AMENDED PAGES OF SPECIFICATION 13-08-2010.pdf

1653-che-2007-abstract.pdf

1653-che-2007-claims.pdf

1653-che-2007-correspondnece-others.pdf

1653-che-2007-correspondnece-po.pdf

1653-che-2007-description(complete).pdf

1653-che-2007-drawings.pdf

1653-che-2007-form 1.pdf

1653-che-2007-form 3.pdf

1653-che-2007-others.pdf


Patent Number 246537
Indian Patent Application Number 1653/CHE/2007
PG Journal Number 10/2011
Publication Date 11-Mar-2011
Grant Date 03-Mar-2011
Date of Filing 30-Jul-2007
Name of Patentee PITCHAI DAISY
Applicant Address DEPRATMENT OF BIOTECHNOLOGY, HOLY CROSS COLLEGE, TIRUCHIRAPALLI-620002
Inventors:
# Inventor's Name Inventor's Address
1 SAVARIMUTHU IGNACIMUTHU ENTOMOLOGY RESEARCH INSTITUTE, LOYOLA COLLEGE, NUNGAMBAKKAM, CHENNAI-600 034
2 PITCHAI DAISY DEPRATMENT OF BIOTECHNOLOGY, HOLY CROSS COLLEGE, TIRUCHIRAPALLI-620002
3 MOHAMMED FAROOK DR.ZAKIR HUSSAIN COLLEGE, ILAYANGUDI-630702
PCT International Classification Number A61K 35/78
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA