Title of Invention

A NOVEL PROCESS FOR ISOLATING A FLAVONOID GLYCOSIDE COMPOUND CONTAINING ARGYROLOBIUM ROSEUM EXTRACT

Abstract

The present invention relates to a process for isolating plant Argyrobium roseum extract that contains flavonoid glycoside, wherein the extract possesses hypoglycaemic activity. The present invention also contemplates a composition containing the extract and a method of treating various hyperglycaemic conditions including non-insulin dependent diabetes mellitus disease condition by administering the extract.

Full Text

A method of treating diabetes using plant Argyrolobium roseum extract, and a process for the isolation of extract from the said plant Field of the present invention The present invention relates to antidiabetic activity and the process for the isolation of a standardised fraction, and its composition possessing antidiabetic activity, from Argyrolobium roseum. Background and prior art references Argyrolobium roseum (Cambers) Jaub & Spauch (Fabaceae) was collected from the hilly area of Udhampur district of Jammu and Kashmir, India. It is available in the matured state during the month of March/April. The plant was collected and shade dried, subjected to phytochemical investigations to isolate the active fraction. The A. roseum is an herb or shrub. Leaves digitately 3-foliodate; stipules free. Flowers yellow terminal or leaf-opposed racemes or umbels. Calyx 2-lipped, slit nearly to the base, with distinct teeth (in Indian species), 2 to the upper, 3 to the lower lip. Corolla scarcely if at all exerted, standard roundish, wings oblong. Keel broad joined down the back to the obtruse incurved tip. Stamens monadelphous, anthers dimorphous. Ovary linear, sessile, ovuled; style elongate, filiform. Curved gradually upwards inside the keel, stigma minute oblique. Pod linear, straight, continuous within, compressed [Flora Of British India, Vol. 11, Page 63 , By Hooker]. Flowering & fruiting time of Argyrolobium roseum is from April to June. (Kangra District, Himachal Pradesh State, India). It is distributed in the tropical & sub-temperate tracts of north-western India [Flora of Himachal Pradesh by H. J. Chowdhary & B.M. Wadhwa. Page 181-1821. No prior art is available on the antidiabetic activity of Argyrolobium roseum (Cambers) Jaub & Spauch (Fabaceae). The literature survey conducted revealed that so far the plant has not been used in Folklore or indigenous system of medicine nor reported for any biological activity. However the plant finds its mention in the following texts: [Dictionary of Natural Products, Vol. 7, 414, 1994. Vol. 1, 457, 1994 By Chapman & Hail] [Index Kewensis,Vol. 1,184, 1895],[Wild & Cultivated Plants of J&K & Ladakh, Page 23, 1997, By Om. Prakash Vidyarthil [Flora of West Pakistan, Page 385, volume 100, By E.Nasirl [Flora of the Himalaya, Page 94, Plate No. 323, d 460, By Oleg Polunin, Adam Staintonj [Flora of Jammu and Plants of Neighbourhood Volume II, Year 1983, Plate 64, Page-180-1821 [Flora of Ihmachal Pradesh, Page 181-182. By H.J. Chowdhary & B.M. Wadhwal [Flora of Bashahr Itmalayas, Page 69, 1997, By N.C. Nairl [Flora of British India, Vol. II, Page 63, By Hooker], Argyrolobium megharizum and Argyrolobium flaccidum are two other species of Argyrolobium (Dictionery of Natural Products, Vol. 1,457, 1994, By Chapman & Hall). Objects of the present invention The main object of the present invention is to develop a method of treating hyperglycemia and non-insulin dependent diabetes mellitus (NIDDM) in animals including humans, using extract from the plant Argyrolobium roseum, containing compound flavnoid glycoside. Another object of the present invention is to develop a dosage schedule for treating hyperglycemic disease conditions including diabetes. Yet another object of the present invention is to understand about side effect (side effect) on the animals including humans. Still another object of the present invention is to develop a composition useful for treating hyperglycemia and non-insulin dependent diabetes mellitus (NIDDM) in animals including humans, said composition comprising extract from the plant Argyrolobium roseum, containing compound flavnoid glycoside, optionally along with other hypoglycemic agents and additives. Another main object of the present invention is to develop a process for isolation of an extract from plant Argyrolobium roseum containing compound flavnoid glycoside having hypoglycemic activity. Summary of the present invention The present invention relates to a process for isolating plant Argyrobium roseum extract, containing flavonoid glycoside, with said having extract showing hypoglycemic activity and a composition thereof, and also, a method of treating various hyperglycemic conditions including non-insulin dependent diabetes mellitus (NIDDM) disease condition. Detailed description of the present invention Accordingly, the present invention relates to a method of treating hyperglycemia and noninsulin dependent diabetes mellitus (NIDDM) in animals including humans, by administering an effective amount of extract from plant Argyrolobium roseum containing compound flavnoid glycoside, or compound flavnoid glycoside per se, optionally along with pharmaceutically acceptable additives. In an embodiment of the present invention, wherein additives are selected from a group of nutrients comprising proteins, carbohydrates, sugar, talc, magnesium sterate, cellulose, calcium carbonate, starch-gelatin paste, and/or pharmaceutically acceptable carrier, excipient, diluent, solvent and other hypoglycemic agents. In another embodiment of the present invention, wherein the dosage is ranging between 1 to 3000-mg/kg p.o./day for about 2-4 weeks. In yet another embodiment of the present invention, wherein the preferred dosage is ranging between 200 to 1000 mg/kg p.o./day for about 2-4 weeks. In still another embodiment of the present invention, wherein the effect lasts for about 8 hours. In still another embodiment of the present invention, wherein the said compound is about 20 times more effective as compared to the whole extract. In still another embodiment of the present invention, wherein said method shows no side effect on the animals including humans. In another embodiment of the present invention, wherein a composition useful for treating hyperglycemia and non-insulin dependent diabetes mellitus (NIDDM) in animals including humans, said composition comprising an effective amount of extract from the plant Argyrolobium roseum, containing compound flavnoid glycoside or compound flavnoid glycoside per se, optionally along with additives. In another embodiment of the present invention, wherein additives are selected from a group of nutrients comprising proteins, carbohydrates, sugar, talc, magnesium sterate, cellulose, calcium carbonate, starch-gelatin paste, and/or pharmaceutically acceptable carrier, excipient, diluent, solvent, and other hypoglycemic agents. In yet another embodiment of the present invention, wherein said composition is administered orally in the physical state of capsule, tablet, syrup, concentrate, powder, granules, aerosol, or beads. In still another embodiment of the present invention, wherein the additives do not interfere with the functioning of the extract. In an embodiment of the present invention, wherein a process for isolation of an extract from plant Argyrolobium roseum containing compound flavnoid glycoside having hypoglycemic activity, said method comprising steps of: (a), drying the whole plant in powder form, (b). percolating the powder about 3-5 times with polar or non-polar solvents, (c). macerating the extract, (d). obtaining the macerated extract of yield ranging between 7-10%, (e). drying the extract in vacuum, (f). isolating the pure compound flavnoid glycoside by chromatography, and (g). obtaining the compound flavnoid glycoside in the range of 0.015- 0019%. In another embodiment of the present invention, wherein the said compound is isolated by column chromatography on silica gel 60-120 microns mesh size. In still another embodiment of the present invention, wherein macerating the extract with compound selected from a group comprising hexane, ethyl acetate, acetone, chloroform. In still another embodiment of the present invention, wherein preferred solvents for percolation are alcohols and acetones. In still another embodiment of the present invention, wherein An embodiment of the present invention, wherein flavonoid glycoside is used for preparing a pharmaceutical composition for treating non-insulin dependent diabetes in mammals and human beings. In still another embodiment of the present invention, wherein said compound flavnoid glycoside having significant hypoglycaemic activity. In still another embodiment of the present invention, wherein said compound alcoholic extract having the activity of regeneration of p-cells of pancreas in streptozotocin treated rats. In still another embodiment of the present invention, wherein flavonoid glycoside isolated from Argyrolobium roseum with 20 times less dose i.e. lOmg as compared to 200mg/kg p. o. dose of alcoholic or acetone fraction of the plant showed significant hypoglycaemic activity. In still another embodiment of the present invention, wherein the aqueous extract, alkaloidal & non-alkaloidal fractions have not shown any significant hypoglycaemic activity. The invention is described in the examples given below which are provided by a way of illustrations only and should not be construed to limit the scope of the present invention. Example - 1 The dose of streptozotocin is used to induce diabetes mimicking to non-insulin dependent diabetes mellitus (NIDDM), encountered clinically in Majority of patients. The diabetic rats (NIDDM) when treated with alcoholic extract of the plant Argyrolobium roseum, for 2-3 weeks recovered to normal state, whereas the rats of control diabetic group (NIDDM) continued to have diabetes and died in due course of time. Example - 2 1 kg ofA.roseum (whole plant) is shade dried and powdered. The powdered plant material was extracted with ethyl alcohol (5 litres) for 20 hours. The alcohol extract was vacuum dried. The yield was 91 gms. The dried alcoholic extract was macerated successively with hexane and chloroform to give 8.2gms. and 39.3 gms. of the extracts respectively. A flavonoid glycoside (230mgs) was isolated from the chloroform extract by repeated column chromatography over silica gel of mesh size 60-120 micron. The extract (200mg/kg p.o.) showed significant hypoglycaemic activity as tested on normal, 18h fasting, glucose loaded & streptozotocin induced hyperglycaemic rats, The onset of effect within l/2h and the effect lasted for more than five hours. The fall of blood sugar recorded with above dose was 26mg/dl (n=24) as compared to fall of 17 & 23 mg/dl recorded with tolbutamide, 50mg/kg p.o. (n=9) and glipizide, 0.5mg/kg p.o. (n=8) respectively in 18h fasting rats. The hypoglycaemic activity was also recorded with plant powder (2.5g/kg p.o.) & flavonoid glycoside isolated from the plant. Example - 3 1 kg of A.rosewn (whole plant) is dried and powdered. The powdered plant material was percolated four times with 95% methyl alcohol (3lit. each time). The alcohol extract was vacuum dried 85gms. The alcohol extract was macerated successively with hexane and acetone to give 6.7 gms. and 49.6 gms. of the extracts respectively. A flavonoid glycoside (170mgs) was isolated from the acetone extract by repeated column chromatography over silica gel of mesh size 60-120 micron. There was highly significant recovery of rats from non-insulin dependent diabetes mellitus treated with the extract (200mg/kg p.o./day) for 3 weeks as per with following parameters: i) Blood glucose level, ii) Effect on body weight, iii) Survival of the animals, iv) Water intake, urine output & presence of glucose in urine, v) General condition of the animals and vi) Visit of ants to the voided urine of the rats. Example - 4 1 kg of A.roseum (whole plant) was shade dried and powdered. The powdered plant material was percolated four times with 95% acetone (3 lit each time). The acetone extract was vacuum dried. The yield was 87 gms. The alcoholic extract was macerated successively with hexane and ethyl acetate to give 7.2 grns and 42.8gms of the extracts respectively. A flavonoid glycoside (210mgs) was isolated from ethyl acetate extracts by repeated column chromatography over silica gel of 60-120^ mesh size. There was highly significant recovery of rats form non-insulin dependent diabetes mellitus treated with KA (200mglkg p. o. /day) for 2-3 weeks as per with following parameters Blood glucose level: i.) Blood sugar level, ii) Effect on body weight, iii) Survival of the animals, iv) Water intake, urine output & presence of glucose in urine, v) General condition of the animals and vi) Visit of ants to the voided urine of the rats. Advantages The plant Argyrolobium roseum has been for the first times experimented for its potential use in the treatment of diabetes. The standardised extract can be used for the treatment of diabetes mellitus, afflicting all strata of society. Plant based non-toxic herbal preparations can be made for the treatment of diabetes mellitus. A process for the isolation of a fraction by percolating the plant powder with solvents and then vacuum dried, possessing antidiabetic activity from Argyrolobium roseum. A fraction isolated from the plant using polar or non-polar solvents preferably aliphatic alcohol. Anti diabetic activity of the plant extract in which the extraction was tested upto 2000mg/kg p.o. dose in rats was found to be free from any undesirable effect. Anti diabetic activity of the extract form the plant extract above in which the extraction can be put to therapeutic use without any undesirable effect for controlling hyperglycemia and for radical cure from non-insulin dependent diabetes mellitus (NIDDM). We claim: 1. A novel process for isolating a flavonoid glycoside compound-containing Argyrolobium roseum extract, comprising (a) drying a Argyrolobium roseum plant into powder form; (b) percolating the powder about 3-5 times with polar or non-polar solvents, (c) macerating the extract; (d) drying the extract in vacuum; and (e) isolating the flavnoid glycoside compound by chromatography, wherein said flavnoid glycoside compound has hypoglycemic activity. 2. A process as claimed in claim 1, wherein the flavonoid glycoside compound is isolated using a silica gel of 60-120 microns mesh size in a chromatography column. 3. A process as claimed in claim 1, wherein maceration of the extract is performed in the presence of hexane, ethyl acetate, acetone, or chloroform. 4. A process as claimed in claim 1, wherein percolation is performed using an alcohol or an acetone. 5. A composition comprising an effective amount of flavonoid glycoside compound containing Argyrolobium roseum extract as prepared by the process of claim 1, wherein the said extract comprises a flavonoid glycoside compound along with pharmaceutically acceptable excipients.

Documents:

174-DEL-2003-Abstract-(14-10-2008).pdf

174-del-2003-abstract.pdf

174-DEL-2003-Claims-(14-10-2008).pdf

174-DEL-2003-Claims-(23-10-2008).pdf

174-del-2003-claims.pdf

174-DEL-2003-Correspondence-Others-(14-10-2008).pdf

174-DEL-2003-Correspondence-Others-(23-10-2008).pdf

174-del-2003-correspondence-others.pdf

174-del-2003-correspondence-po.pdf

174-del-2003-description (complete).pdf

174-DEL-2003-Form-1-(14-10-2008).pdf

174-DEL-2003-Form-1-(23-10-2008).pdf

174-del-2003-form-1.pdf

174-del-2003-form-18.pdf

174-DEL-2003-Form-2-(14-10-2008).pdf

174-DEL-2003-Form-2-(23-10-2008).pdf

174-del-2003-form-2.pdf

174-DEL-2003-Form-3-(14-10-2008).pdf

174-del-2003-form-3.pdf

174-DEL-2003-Form-5-(14-10-2008).pdf

174-DEL-2003-Others-Document-(14-10-2008).pdf

174-DEL-2003-Petition-137-(14-10-2008).pdf