Title of Invention

"A PROCESS OF PREPARING AN EXTRACT OF ANNONA SQUAMOSA FOR TREATMENT OF DIABETES."

Abstract

A process of preparing an extract of annona squamosa comprising collecting leaves of annona squamosa; drying the said leaves; preparing an aqueous extract of the leaves at a temperature of 80-100° C; filtering the extract to remove the solid material, concentrating the extract at temperature range of 30 to 40° C under reduced pressure; subjecting the concentrated extract to the step of liquid-liquid extraction as herein described; subjecting the cluted fractions to thin layer chromatography to obtain semi purified extract.

Full Text

FIELD OF THE INVENTION The invention relates to a process of preparing an extract of Annona squamosa for the treatment of diabetes. BACKGROUND OF THE INVENTION Diabetes mellitus is a very common disease. It is caused due to metabolic disorder produced by insulin ineffectiveness of deficiency. Insulin is required to transfer the sugar from the blood in to the cells for the production of energy. Human insulin is produced by beta cells of the islets of langerhans in pancreas. In a non-diabetic person, the beta cells secretes insulin when blood sugar rises, and when the blood sugar level drops the production of insulin stops. But in a diabetic person, the beta cells produce little or no human insulin (type-I) or cells are unable to utilize it (type-II), when this happens the blood sugar level begins to rise. This disease is called diabetis mellitus. From ancient times until the discovery of insulin in the 1920's nutritional therapy was the only available means of treating diabetes. As early as 2500-1800 BC, there is a mention of curative properties of medicinal plants in Rigveda. Charaka Samhita and Sushrita Samhita give extensive description of various medicinal plants (1). It is interesting to note that till today metafomin is the only ethical drug approved for the treatment of NIDDM patients which is derived from medicinal plant (2). In past there have been many medicinal plants which have been investigated for their antidiabetic properties. The plant annona squamosa of the present invention has not been evaluated till date for it's antidiabetic activity. OBJECTS OF THE INVENTION An object of this invention is to a process of preparing an extract of annona squamosa for the treatment of diabetes. Further object of this invention is to propose an anti-diabetic drug. BRIEF DESCRIPTION OF THE INVENTION According to this invention there is provided a process of preparing an extract of annona squamosa comprising collecting leaves of annona squamosa; drying the said leaves; preparing an aqueous extract of the leaves at a temperature of 80-100° C; filtering the extract to remove the solid material, concentrating the extract at temperature range of 30 to 40° C under reduced pressure; subjecting the concentrated extract to the step of liquid-liquid extraction as herein described; subjecting the cluted fractions to thin layer chromatography to obtain semi purified extract. DETAILED DESCRIPTION OF THE INVENTION Leaves of Annona squamosa were collected. Leaves were washed well with distil water, then air-dried upto 5-6 days in absence of sunlight on blotting paper. Air-dried leaves were crushed to moderately coarse powder and extracted in Millipore water in a ratio of 1:12 preferably, 1:10 using Soxhlet in the temperature range of 80-100°C up to 20 hours, a dark brown extract is obtained. This dark brown extract was cooled and filtered with the help of ordinary filter paper to remove the residue. The extract was concentrated upto 500 ml on rotavapour under reduced pressure in the temperature range of 30 - 40°C. The concentrated extract was subjected to continuous liquid-liquid extraction in a separating funnel of 1 L capacity using 3 x 500ml of Hcxanc, Ether, Ethyl acetate, Chloroform, Dichloromethane and Acetone each. The organic layer was dried on anhydrous sodium sulphate. Activity of all the fractions were checked on alloxan-induced rabbits and no fraction was found to be active. The aqueous portion (left after organic solvents extraction) showed the activity on alloxan-induced rabbits. The aqueous layer was concentrated on rotavapour under reduced pressure at the temperature range of 30-40°C and lyophilized to get a powder weighing 8 gram. On TLC, presence of more than one sport indicated that it is not a pure compound, hence the mixture of compounds were subjected for further studies (called Semipurified extract). The maximum separation of this semipurified extract was done by TLC in chloroform: methanol (7:3) as solvent system showing 7 spots. We have checked the activity of this semi-purified extract on alloxan-induced diabetic rabbits and Streptozotocin induced diabetic rat. This is found to be as active fraction and the whole study was done using this extract only. Reference is now made to a working example for the process for preparation of the extract of Annoxia squamosa. Examples: Leaves of Annona squamosa were collected from the garden of Indian Agricultural Institute in the month of March and April. Leaves were washed well with distilled water, then air-dried upto 5-6 days in absence of sunlight on blotting paper. Air-dried leaves were reduced to moderately coarse powder and weighed 250 grams powder were extracted in 3.0 litre Millipore water in a Soxhlct apparatus at boiling temperature 100°C for 20 hours, a dark brown extract was obtained. This dark brown extract was cooled and filtered with the help of ordinary filter paper to remove the residue. The extract was concentrated on rotavapour under reduced pressure at 40°C, up to 500 ml. The concentrated extract was subjected to continuous liquid-liquid extraction in a separating funnel of 1 litre capacity the solvents used are Hexane, Ether, Ethyl acetate, Chloroform, Dichloro methane, Acetone as solvents. All solvents used in liquid-liquid extraction was dried on anhydrous sodium sulphate. Activity of all the fractions was checked on alloxan-induced rabbits no fraction was found to be active. The mother liquor shows the activity on alloxan-induced rabbits. The mother liquid was concentrated on rotavapour under reduced pressure and solidified under high vaccum. This solid, weight 8 gram was loaded on 500 gram silica gel (Mesh 60-120, purchased from Merck) and eluted with methanol and water as solvents. The eluted fractions were concentrated and employed for thin layer chromatography using glass plates coated with Silica gel (for thin layer chromatography, purchased from Merck). In TLC, presence of more man one spot indicated that it is not a pure compound, but the mixture of compounds called semi purified extract. The maximum separation of this semipurified extract takes place on TLC, in chloroform methanol (7:3) are clucnt having 7 spots. The activity of this semi-purified extract on alloxan-induced diabetes in rabbits was checked. RESULTS: INDUCTION OF DIABETES IN RABBITS AND RATS The experimental animals used are rabbit and Wister rats. Alloxan is used for inducing diabetes in rabbits and streptozotocin in rats. (Alloxan and streptozotocin are purchased from Aldrich Chem. Co. USA). Intravenous injections of alloxan in rabbits and intrapehtoneal injections of streptozotocin in rats are administered at a dose of 80 mg/kg and 50 mg/kg of body weight respectively to overnight starved animals. Fasting blood glucose (FBG) levels are estimated by commercial kits based on glucose oxidase method (Ranbaxy Laboratories, Delhi). Diabetes was confirmed by testing FBG and postprandial blood glucose levels, depending on their glucose levels the animals are divided in to three groups, namely sub, mild and severe diabetic. All animals has free access to tap water and laboratory food (Ad-libitum) during the period of treatment. Alloxan induced Rabbits: Studies were carried out using Glucose Tolerance Test (GTT) on Sub and mild diabetic animals, the protocol employed is as follows: Animals were fasted overnight (12-14 hours) and their Fasting blood glucose level was estimated using one touch glucose sensor (Accu-check) drug administered orally by gastric incubation and after 90 minute, glucose level in blood is again estimated; this gives the '0 hour' value for GTT. Following this 2 g/kg body weight of glucose administered by gastric incubation as an aqueous solution and blood glucose level was estimated at the intervals of 1 hour upto 3 hours viz. 1, 2 and 3 hour values for GTT. Improvement in the glucose tolerance was assessed by comparing reduction in peak blood glucose levels seen at' 1, 2 & 3 hour values and also the area under the curve of GTT obtained with drug and without drug. Details of the animals used in experiments are given in Table-1 and GTT curve is shown in Graph-1. Table-I (Table Removed) (Graph Removed) Graph-l, Showing the Effect of different Doses (200, 300,350 mg/kg boby weight)of extract On Alloxan induced diabetic Rabbits.it has been observed that a dose of 350 mg/kg body weight showing maximum fall in the blood glucose level and no further more fall in blood glucose level on increasing the dose. Streptozotocin induced Diabetes in Rats : Final testing was done on streptozotocine induced diabetes in rats, same protocol were employed as in case of alloxan induced diabetes. Details of the animals used in the experiments is given in the Table-Ill and results obtained, shown in the graph-Ill. Table-II (Table Removed) Graph-II (Graph Removed) Treatment of severely Diabetic Animals : We have treated a group of severely Diabetic rats (n=4) and rabbits. Stable hyperglycemia was produced after one week of giving Streptozotocin, confirmed by regular checking of post parandial blood glucose level.The control animal was die during the experiment, Detail of animals given in Table-Ill and result explained in graph-Ill,shown below, Table-Ill (A) (Table Removed) Graph-Ill (A) (Graph Removed) Results obtained after 15 days treatment of the alloxan induced diabetic rabbits. Table-lll(B) (Table Removed) Graph-lll(B) (Graph Removed) Results obtained after 15 days treatment of streptozotocin induced rats The result obtained in the case of one week treatment of severely diabetic rats are shown in the graph-Ill, the treated animal shows 40 % fall in post parandial blood glucose level the control animal were died during the course of experiment due to hyperglycemia. Effect on Lipid profile of Diabetic animals: To see the effect of extract on lipid profile we have treated a group of severely diabetic rabbits, having high cholesterol level, after a 15 days treatment we observe a remarkable fall in total cholesterol, the results obtained are shown in the table-IV, Table-IV (Table Removed) LD50 Experiments: No mortality have been found up to 15 times of effective dose in experiment. Animals are normal, only the consumption of food is more up to 24 hour. Fecal and urine is normal. RESULTS AND OBSERVATION Results indicate a significant decrease in FBG (Fasting Blood Glucose), as well as PP (Post Parandial) blood glucose level during drug treatment. FBG fall by 20% and PPBG Fall by 40%. In the case of 15 days treatment of severe animals the reduction of 40.63% in the level of BGL have been observed and 25% reduction in cholesterol level was also found by annona extract treatment in the animals. A significant lowering in blood sugar level and improved glucose tolerance was observed. The findings from this study suggest that semipurified compounds from annona leaves may be prescribed as adjunct to dietary and drug treatment in mild and severe cases. To explore further studies the effect of treatment on glycemic albino rats is in progress, and effort are being made isolate pure compound which is responsible for antidiabetic property. WE CLAIM: 1. A process of preparing an extract of annona squamosa comprising collecting leaves of annona squamosa; drying the said leaves; preparing an aqueous extract of the leaves at a temperature of 80- 100° C; filtering the extract to remove the solid material, concentrating the extract at temperature range of 30 to 40° C under reduced pressure; subjecting the concentrated extract to the step of liquid-liquid extraction as herein described; subjecting the eluted fractions to thin layer chromatography to obtain semi purified extract. 2. The process as claimed in claim 1, wherein the leaves are air dried upto 5 to 6 days in absence of sunlight on blotting paper. 3. The process as claimed in claim 1, wherein the said aqueous extract of the leaves is prepared by separating the powder of the leaves to the steps of extraction in distilled water in a ratio of 1:12 in a Soxhlet apparatus at the temperature range of 80-100° C upto 20 hours. 4. The process as claimed in claim 3, wherein the ratio of the dried leaves and water is preferably 1:10. 5. The process as claimed in claim 3, wherein the time required is 20 hours. 6. The process as claimed in claim 1, wherein ,the extract was concentrated on rotavapour under reduced vapour pressure upto 500 ml. 7. The process as claimed in claim 1, wherein the liquid-liquid extraction is performed using the solvents such as Hexane, Ether, Ethyl acetate, Chloroform, Dichloromethane and Acetone. 8. The process as claimed in claim 7, wherein the solvents used in liquid-liquid extraction is dried on anhydrous sodium sulphate. 9. The process as claimed in claim 1, wherein the eluted fractions are concentrated as herein described and then employed for thin layer chromatography using glass plates coated with silica gel. 10. The process as claimed in claim 9, wherein the said thin layer chromatography is performed in solvents like chloroform methanol in a ratio of 7: 3. 11. A process for preparing an extract of Anona squamosa as herein described and illustrated.

Documents:

21-del-2003-abstract.pdf

21-del-2003-claims.pdf

21-del-2003-correspondence-others.pdf

21-del-2003-correspondence-po.pdf

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

21-del-2003-form-1.pdf

21-del-2003-form-19.pdf

21-del-2003-form-2.pdf

21-del-2003-form-26.pdf

21-del-2003-form-3.pdf

21-del-2003-form-5.pdf