Title of Invention

A PROCESS FOR THE ISOLATION OF 5-METHYL-ISOXAZOLE-3-METHYL CARBOXYLATE FROM THE PLANT TRAGIA INVOLUCRATA

Abstract

The invented process pertains to the isolation of a novel antidiabetic (Hypoglycaemic) compound (C H NO ) from the natural plant source, Tragia involucrata having promising lipid (cholesterol) lowering and antioxidant activities. Though this plant has got some folk medicinal uses, the present inventors isolated the claimed active compoune (an isoxazole alkaloid) for the first time from Tragia involucrata and used for the management of diabetes mellitus. The isolation process comprises-extraction of the plant with ethyl alcohol followed by removal of alcohol by controlled distillation. The obtained dried concentrate is passed through column chromatography by using silica gel and different solvents and purified methanol fraction is further purified to collect 98% pure active compound passing through HPLC.

Full Text

The invented process relates to the preparation of a novel isoxazole alkaloid (C6H7NO3 ) having hypoglycaemic activity from natural source. More particularly this invention relates to the isolation of 5-Methyl-isoxazole-3-Methyl-carboxylate from Tragia involucrata of Euphorbiaceae family. It has also been found that this isoxazole compound possesses lipid lowering and antioxidant activities in addition to hypoglycaemic activity and if suitably formulated may find use as a novel oral drug for the treatment of diabetes in humans body. Prior Knowledge: Literature search reveals that similar isoxazole alkaloid (C6H7NO3 ) was reported as Premnazole from separate plants, Premna integrifolia and Gmelina arborea of different families. Also it was reported that Premnazole showed antiinflammatory activities (Barik, B.R.et.al, Fitoterapia, 1992, 63(4), 296-9). There is, however, no report in literature regarding the hypoglycaemic activity of Premnazole. Though a number of oral hypoglycaemic agents are known, during the past few years, intensive attempts have been made to isolate a novel hypoglycaemic compound from the plant source having no and/or less side effects. In the present invention isoxazole alkaloid is iosolated from the plant Tragia involucrata for the first time and for the treatment of diabetes mellitus. The melting point of the active compound (C6H7NO3 ) from Tragia involucrata was found to be 80-90 deg. C. Improvement of impaired glucose tolerance and the hypoglycaemic activity of the crude ethanolic extract of Tragia involucrata (along with a few other hypoglycaemic plants) have been first observed in normal and experimentally produced diabetic albino rats, followed by successful oral application of the suitable formulation in diabetic human volunteers. Also lipid lowering and anti-oxidant activities are noted in triton model of hyperlipaemic rats. Further hypoglycaemic activity with hypolipidaemic and antioxidant activities of the aforesaid purified isoxazole alkaloid have been confirmed in experimental rats . The present invention comprises of a simple, less time consuming and requiring less eluting solvents for the isolation of an active isoxazole alkaloid from Tragia involucrata. The present invention particularly relates to a proces for the isolation of 5-Methyl-isoxazole-3-Methyl-carboxylate from Tragia involucrata which comprises of a) extracting the plant with an aliphatic alcohol followed by b) removal of alcohol by controlled distillation under reduced pressure and temperature. c) The freeze dried concentrate obtained from step (b) is passed through column chromatography using silica gel and different solvents. d) Methanol fraction containing the active compound is collected and the methanol is removal from the product under controlled distillation. e) Finally obtained product is lyophilized and the purified compound of 98% purity is obtained by passing through HPLC using acetonitrile as solvent. According to the present invention there is provided: A process for the isolation of the novel hypoglycaemic compound, 5-Methyl-isoxazole-3-Methyl-carboxylate from the plant Tragia involucrata which comprises: a) extracting the plant with an alcohol followed by b) removal of alcohol by controlled distillation under reduced pressure and temperature below 45 deg. C. c) The freeze dried concentrate obtained from step (b) is passed through column chromatography using silica gel and different solvents. d) Methanol fraction containing active compound is collected and the methanol is removed from the product under controlled distillation as in step (b). e) Finally obtained product is lyophilized and the purified compound of 98% purity is obtained by passing through HPLC using acetonitrile as solvent. The invention is described in details with reference to the accompanying drawings (from X-Ray crytallographic studies). Fig. 1 describes ORTEP diagram showing the molecular structure of the active isoxazole compound from Tragia involucrata. Fig. 2 shows the Packing diagram showing the C-H...0 hydrogen bonding by dashed lines. This invention process is further elaborated with reference to the following experimental steps & example. Example: 1. Full grown matured whole plant (creeper), Tragia involucrata properly cleaned and dried (in shed). 75 gms. of the dried plant sample is so extracted with 450 ml., 95% ethanol at controlled temperature. The extract collected and concentrated under reduced pressure (using vacuum pump) and temperature below 45 deg.C through rotary evaporator. Complete removal of the alcohol is ensured and dried ethanolic extract is stored at 4-5 deg C for isolation of the active compound (isoxazole alkaloid). 2. Dried ethanolic extract of the plant sample of step 1 is next passed through column chromatography loading the dried and impregnated column of silica gel (60-120 mesh) prepared in hexane, eluting first with hexane and then with benzene, chloroform, ethyl acetate successively and finally eluting with methanol. Methanol fraction is collected carefully for complete extraction of the aforesaid active compound as mentioned in step 1. 3. Total methanol fraction of step 2 is concentrated under reduced pressure and temperature below 45 deg. C using rotary evaporator. Complete removal of methanol is ensured. 4. Crude dry extrct containing the desired active compound (isoxazole alkaloid) is next transfered to lyiophilizer and further purified (98%) passing through HPLC using acetonitrile as solvent. 5. Also for the elucidation of structure, the said purified isoxazole alkaloid of Tragia involucrata (as in step3) routine IR, NMR, Mass spectra etc. have been recorded and final structure of the said isoxazole alkaloid of Tragia involucrata has been confirmed through X-ray Crystallography. We claim: 1. A process for the isolation of hypoglycaemic compound, 5-Methyl-isoxazole-3-Methyl-carboxylate, from the plant Tragia involucrata which comprises: a) extracting the plant with an alcohol followed by b) removal of alcohol by controlled distillation under reduced pressure and temperature below 45 deg. C. c) The freeze dried concentrate obtained from step (b) is passed through column chromatography using silica gel and different solvents. d) Methanol fraction containing active compound is collected and the methanol is removed from the product under controlled distillation as in step (b). e) Finally obtained product is lyophilized and the purified compound of 98% purity is obtained by passing through HPLC using acetonitrile as solvent. 2. A process for the isolation of 5-Methyl-isoxazole-3-Methyl- carboxylate from the plant source, Tragia involucrata as claimed in claim 1 wherein an aliphatic alcohol and preferably ethanol is used. Methanol fraction from column chromatography showing maximum activity. 3. A process for the isolation of novel 5-Methyl-isoxazone-3- Methyl-carboxylate from the plant Tragia involucrata substantially as herein before described with referance to the accompanying drawings. The invented process pertains to the isolation of a novel antidiabetic (Hypoglycaemic) compound (C H NO ) from the natural plant source, Tragia involucrata having promising lipid (cholesterol) lowering and antioxidant activities. Though this plant has got some folk medicinal uses, the present inventors isolated the claimed active compoune (an isoxazole alkaloid) for the first time from Tragia involucrata and used for the management of diabetes mellitus. The isolation process comprises-extraction of the plant with ethyl alcohol followed by removal of alcohol by controlled distillation. The obtained dried concentrate is passed through column chromatography by using silica gel and different solvents and purified methanol fraction is further purified to collect 98% pure active compound passing through HPLC.

Documents:

410-KOL-2004-(15-03-2012)-CORRESPONDENCE.pdf

410-KOL-2004-(15-03-2012)-FORM-27.pdf

410-KOL-2004-FORM 27.pdf

410-kol-2004-granted-abstract.pdf

410-kol-2004-granted-claims.pdf

410-kol-2004-granted-correspondence.pdf

410-kol-2004-granted-description (complete).pdf

410-kol-2004-granted-drawings.pdf

410-kol-2004-granted-examination report.pdf

410-kol-2004-granted-form 1.pdf

410-kol-2004-granted-form 18.pdf

410-kol-2004-granted-form 2.pdf

410-kol-2004-granted-form 26.pdf

410-kol-2004-granted-form 3.pdf

410-kol-2004-granted-form 5.pdf

410-kol-2004-granted-reply to examination report.pdf

410-kol-2004-granted-specification.pdf