Title of Invention

"A HERBAL HYPOGLYCEMIC COMPOUND FOR CONTROLLING DIABETES MELLITUS"

Abstract

This invention relates to a herbal therapeutic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia species preferably Eugenia jambolana comprising an active hypoglycemic compound having four fractions F Ha, F IIb, F IIc and F IId wherein at least one fraction F IIc comprises 90-95% a-hydroxy succinamic acid having the structural formula where -NH2 Amine (primary) group - CONH2 Carbamide group - COOH Carboxylic group - OH Hydroxyl group which is an aliphatic compound having the molecular formula C4H7O4N, low molecular weight 197 and a derivative of succinamic acid and balance being ethyl ene glycol.

Full Text

The present invention relates to a herbal anti-diabetic compound extracted from the fruit-pulp of Eugenia species and the process thereof. More specifically, it relates to the structural composition of the herbal anti-diabetic compound extracted from the fruit pulp of Eugenia jambolana which is having immediate and longer lasting effects in controlling diabetes mellitus and the process thereof. Diabetes is a chronic hereditary disease which is a disorder characterized by presence of abnormally high level of glucose in the blood and in the excretion of urine. There are two types of diabetes- type I and type II. In type I, also called insulin dependent diabetes mellitus (IDDM), insulin levels are less or absent and insulin treatment is needed. However, in type II diabetes mellitus also called non-insulin dependent diabetes (NIDDM), insulin levels are less and there is less sensitivity and /or resistance to insulin so management of diabetes is usually by oral hypoglycemic agents. Since diabetes is primarily a disorder of not only carbohydrate metabolism but also impaired lipid metabolism. Therefore, there is a direct correlation between blood lipid concentration and impaired glucose tolerance. The objective of the present invention is to provide a oral hypoglycemic herbal compound which will be useful for the treatment of both type I and type II diabetes. Another objective of the invention is to produce a herbal anti-diabetic compound having no side effects. An embodiment of the invention resides in the various compounds of the product and their mixture having immediate as well as long lasting effects in controlling diabetes mellitus. Another embodiment of the invention resides in preparing the four active hypoglycemic fractions comprising the product of the subject invention. These four active hypoglycemic fractions individually have varying degrees of hypoglycemic characteristics, however, the second fraction has been found to have the maximum hypoglycemic activity. Still another embodiment of the invention resides in the discontinuity of treatment with such herbal therapeutic product for a predetermined period after controlling the diabetes of the patients as the hypoglycemic compounds of such products are having the property to control the blood glucose level for longer durations. It has been found that the intake of doses can be postponed for 2-3 days after controlling the glucose level because of the long lasting effects of this hypoglycemic compound. In Indian patent application No. 834/DEL/99 (Patent No. 188759) relates to a process for the preparation of herbal therapeutic product for controlling diabetes mellitus comprising at least one hypoglycemic compound extracted from the pulp of fruit Eugenia jambolana. The process for the preparation of the herbal therapeutic product to control the glucose level, comprises cleaning and drying the fruit of a species of genus Eugenia. De-seeding the fruit and soaking the said de-seeded fruit pulp in water under controlled cooled conditions overnight to retain the activity of hypoglycemic compounds in the mixture of water and pulp of de-seeded fruit. It is followed by mixing the contents to achieve a uniform consistency. Kelkar in his article XP-000940531, Phytomedicine Volume 3(4), pages 353-359, 1996/97, teaches a simple two step purification of antidiabetic compounds from Eugenia jambolana fruit-pulp with proteolytic resistance and other properties: It teaches a simple, two-step, procedure for purifying antidiabetic compounds from Eugenia jambolana fruit-pulp. The compounds have been identified as a peptidylglycan and an oligosaccharide with molecular weights of 6.0 and 1.2 kD, respectively. The binding between the sugars and the peptide in the peptidylglycan appears to be covalent. The amino acid and sugar composition of the peptidylglycan have been determined as have the sugars of the oligosaccharide. The intrinsic color of the peptidylglycan was attributed to the compound itself and not to the presence of pigment or metal. The peptidylglycan was resistant to degradation by proteolytic enzymes in vitro, explaining its efficacy of oral feeding. Kelkar further teaches separation of hypoglycemic compound carried by molecular sieving (depending upon the size of two molecules). In short Kelkar's method is not a two step purification process of antidiabetic compounds as it involves ultrafiltration, gel filtration, paper chromatography and SDS polyacrylamide tube gel electrophoresis. Kelkar also teaches that the hypoglycemic activity was found at two distinct positions eluting at 35-45 ml & 60-75 ml, which is not very precise, since vol. of fractions vary with the amount of loading, width & size of column and height of packed material. Saigeeta Achrekar et al. in their review articles XP000608379 teach hypoglycemic activity of Eugenia jambolana and Ficus bengalensis. The present invention relates to the structural composition of the anti-diabetic compound and the process thereof obtained from the species of genus Eugenia having anti-hypoglycemic effects on elevated blood glucose level. Accordingly, the present invention relates to a herbal therapeutic compound for controlling diabetes mellitus extracted from the pulp of species of Eugenia preferably Eugenia jambolana comprising four fractions wherein atleast one fraction II comprises 90-95% a- hydroxy succinamic acid having the structural formula (Formula Removed) where -NH2 Amine (primary) group - CONH2 Carbamide group - COOH Carboxylic group - OH Hydroxyl group which is an aliphatic compound having the molecular formula C4H7O4N, low molecular weight 197 and a derivative of succinamic acid and balance being ethylene glycol. Accordingly, the present invention relates to a process for producing the herbal therapeutic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia jambolana comprising cleaning and drying the fruit, deseeding the fruit to form a deseeded pulp, soaking the deseeded pulp in water, mixing the water and pulp to form a mixture and keeping it overnight at a temperature of 4°C to 10°C for obtaining maximum activity of hypoglycemic compounds present in the pulp of the fruit, filtering the mixture to remove the residue, washing the residue with water to extract all active compounds in the form of clear watery solution, reducing water content of the solution to obtain the filterate in a concentrated form, purifying the filterate by column chromatography using the slurries of column materials to obtain the partially purified four fractions F I to F IV, subjecting the said partially purified hypoglycemic fraction F II to re-column chromatography resulting in four active hypoglycemic fractions wherein fraction F lIe was having maximum activity having 90-95% active hypoglycemic compound and fraction F lla, F lIb and F lId were having 15-30% active hypoglycemic compound. The subject invention relates to the structural composition of herbal anti-diabetic compound which is a hypoglycemic compound to control the glucose level, extracted from the fruit pulp of Eugenia species of family Myrtaceae prepared from the process involving following steps: Cleaning and Drying: the fruit of genus Eugenia of family Myrtaceae is cleaned and dried; Deseeding the said fruit : the seed is removed from the said fruit by conventional manner and is kept separately; Soaking the de-seeded pulp with distilled water, grinding the said mixture of water and pulp to get the uniform consistency of the liquid and keeping it overnight in cooled conditions preferably 4-10°C; Filtering the said mixture with muslin cloth. Washing the residue with water to extract all the active compounds from the said pulp in the form of clear watery solution and mixing with the water extract of the pulp; Reducing the water content by keeping the said clear watery solution in freezing conditions by lyophilization to get the residues in paste form; Purification of the said paste dissolved in minimum volume of water by diethyl amino ethyl cellulose and performing the column chromatography using buffer. Addition of buffers in the chromatography to elute the hypoglycemic compounds from the said mixture. The partially purified hypoglycemic fraction showing maximum activity is further subjected to re-chromatography using above mentioned procedure to get the purified active hypoglycemic compound. The water content from the said mixture can be reduced optionally to get the end product in solid form. The said controlled cooled conditions for keeping overnight the mixture of water and pulp are preferably from4-10°C The water is extracted from the said mixture after the stage of filtration by means of a lyophilizer. The said buffer used in the chromatography is selected preferably from the phosphate buffer, citrate buffer or TRIS buffer. The pH of the said admixture of hypoglycemic compounds is to be maintained neutral. The water used in the subject process is preferably distilled water. After chromatography and lyophilization, different hypoglycemic and hyperglycemic fractions were obtained. Out of these fractions, the fraction II was having the hypoglycemic activity which was partially purified and therefore subjected to re-chromatography producing four active hypoglycemic fractions F Ha, lIb, lIc and lId which were subjected to further studies. The structural elucidation showed fraction F lIc was having 90-95% (w/v) active hypoglycemic compound (herbal anti-diabetic compound) remaining amount being while fractions F lla, lIb and lId were having 15-30% (w/v) active hypoglycemic compound. The active hypoglycemic compound obtained was colored resinous and without any specific odour. It is a water soluble compound and on heating burns with non-smoky flame which shows that the major active component is an aliphatic compound. The elemental analysis of the hypoglycemic compound showed the presence of C, H, O and N with a ratio of C to N 4:1 respectively. Further this compound showed negative Molisch's and Benedict's test and therefore the reducing sugar is absent. The compound also gave faint positive Ninhydrin test showing the presence of primary -NH2 group. The one dimensional 1H NMR (nuclear magnetic resonance) spectra of all the four fractions obtained after lyophilization showed persistent four signals at 82.4(dd), 62.66 (dd), 83.66(s) and at 8 4.38 (dd). On comparing of all these four fractions, fraction He was found to be very dominant of the above signals. The integration of the 1H NMR indicated that the singlet at 83.66 is not a part of the molecule of the closed AMX spin system (a type of coupling constant system) as the integration ratio was 1:0.5 with the AMX spin system. Type 13C NMR showed five signals at 845.5, 865.3, 873.0, 8182.7 and 8184.0. The signals at 8182.7 and 8184.0 was described to be carbonyl containing carbon. The presence of bond in the IR spectrum at 1656.0 confirmed the presence of amide and at 1590.2 for acid moiety respectively. The DEPT (distortion enhancement by polarization transfer)135° showed the presence of two methylene, one methine and two quaternary carbons. The 2D HMQC (heteronuclear multiple quantum coherence) spectrum provided the 1H, 13C correlation of the 1H signals at 52.40 and 2.66 to 13C at 545.5, 1H at 53.66 to 13C at 565.3 and at 64.28 to 13C at 573.0 respectively. The long range correlation of the 1H signals with its attached carbon as confirmed in the HMQC along with its methylenic nature and the chemical shifts confirmed it to be ethylene glycol. The long range heteronuclear correlation of the closed AMX spin system suggested it to be moiety having a linkage as shown below: (Formula Removed) On the basis of the above information, active hypoglycemic compound contains considerable amount of a-hydroxy succinamic acid and with some signals of ethylene glycol. A one month toxicity study was conducted to determine the effect of hypoglycemic compounds prepared from the pulp extracted from the genus Eugenia on the kidney, liver, aorta, pancreas and heart. The results showed that there were no side effects which is the main embodiment-of the present invention. Various tests were conducted to analyze the effect of said product on testing the blood glucose level in fasting and in GTT(glucose tolerance test ) The active compound at a dose of 7 mg/Kg by weight showed 23.8 % fall in fasting blood glucose and 34% fall in peak blood glucose during GTT in streptozotocin mild diabetic and 29.6% in fasting blood glucose after 90 min of oral administration in streptozotocin induced severe diabetic rats. The ability of active compound to bring down the blood glucose levels in mild (in which some beta cells are still functional) and severe diabetic (in which the beta cells are almost destroyed) in one day as well as 7 or 15 days studies clearly indicated that it probably has pancreatic and extra-pancreatic mechanism of action. It could also stimulate beta cells like tolbutamide and causes significant insulin release as evident from in-vivo and in-vitro studies (pancreatic action) The in-vitro studies suggested that the active hypoglycemic compound stimulates release of insulin secretion from beta cells. The in-vivo studies also suggested that the purified compound increases insulin levels significantly may be due to partial regeneration of beta cells as evident from histo-morphological studies. The extra-pancreatic mechanism of action of active hypoglycemic compound is due to increase in activity of key enzymes of glycolysis, on one hand and decreased activity of key enzymes of gluconeogenesis on the other hand. These observed changes could be either due to the peripheral effect of the compound or ability to increase sensitivity or decrease resistance of insulin on the peripheral tissues. The active hypoglycemic compound is also found to decrease glycosylated hemoglobin in diabetic rabbits. The said product was found to increase the activity of enzymes in liver, muscle and adipose tissue, which use glucose and thereby reduces the blood glucose level. The total lipids were significantly decreased and the activity of HMG CoA reductase (3-hydroxy 3 methyl glutaryl CoA reductase) was also decreased which is a key enzyme for cholesterol bio-synthesis by the use of the said product. The total cholesterol, low density lipoprotein cholesterol and serum triglycerides which were increased in alloxan induced diabetic rabbits returned to normal after 15 days oral administration of active hypoglycemic compound. The liver and muscle glycogen were also increased after oral administration of active hypoglycemic compound. The said active hypoglycemic compound when orally administered for one month did not show any toxic effect on the functions of the liver and kidney as judged by appropriate function tests. The active hypoglycemic compound which is of vegetable origin seems to be useful in both moderate and severe diabetic condition in rabbits without any side effects. Due to its hypoglycemic and hypolipidemic effect, the compound isolated from fruit-pulp of E. jambolana may be beneficial for the treatment of NIDDM patients (Non-insulin dependent diabetes mellitus) with cardiovascular complications. While particular embodiments of the subject invention have been described, it would be obvious to those skilled in the art that various changes and modifications to the subject invention can be made without departing from the spirit and scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of the invention. EXAMPLES Example 1 To prepare an herbal therapeutic product of present invention 450 gms of pulp of the fruit from the genus Eugenia is mixed in 500 ml of distilled water and kept overnight in cooled conditions at 4°C, the said mixture is filtered by muslin cloth, and the residue is washed twice or thrice to extract all the active compounds from the said mixture, the water from the resultant mixture is reduced by keeping the said resultant mixture in a lypholizer to get the residues in paste form, the chromatography of the said paste dissolved in water is done by diethyl amino ethyl cellulose, a phosphate buffer is then added to elute the mixture of partially purified hypoglycemic fractions F I to F IV. The said partially purified hypoglycemic fraction II having the maximum hypoglycemic activity was further subjected to re-column chromatography resulting in four hypoglycemic fractions wherein fraction lie was having maximum activity having 95% active hypoglycemic compound and fraction F Ha, lib, lid were having 18%, 25% and 24% active hypoglycemic compound respectively. Example 2 A herbal therapeutic product of present invention is prepared by taking 100 mg of pulp of the fruit Eugenia jambotana and mixing it in 2 ml of distilled water which is kept overnight in cooled conditions at 4-10°C, the said mixture is filtered by muslin cloth, and the residue is washed twice to extract the active compound from the said mixture, the water from the resultant mixture is extracted by keeping the said resultant mixture in a lyophilizer to get the residues in paste form, the chromatography of the said paste is done and a phosphate bufer is then added to elute the different fractions of hypoglycemic compounds, subjecting the said partially purified fraction having the maximum hypoglycemic activity F II to re-column chromatography resulting in four hypoglycemic fractions wherein fraction F lIe was having maximum activity having 95% active hypoglycemic compound and fraction F IIa, lIb, lId were having 18%, 25% and 24% active hypoglycemic compound respectively. We claim: 1. A herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia species preferably Eugenia jambolana comprising alpha hydroxy succinamic acid having the structural formula : (Formula Removed) said compound being an aliphatic compound having low molecular weight 197 and is a derivative of succinamic acid; 2. A process for producing the herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of Eugenia jambolana as claimed in claim 1 comprising cleaning and drying the fruit, deseeding the fruit to form a deseeded pulp, soaking the deseeded pulp in water, mixing the water and pulp to form a mixture and keeping it overnight at a temperature of 4°C to 10°C for obtaining maximum activity of hypoglycemic compounds present in the pulp of the fruit, filtering the mixture to form a residue, washing the residue with water to extract all active compounds in the form of clear watery solution, reducing water content of the solution to obtain the filterate in a concentrated form, purifying the filterate by column chromatography using the slurries of column materials at neutral pH to obtain the partially purified four fractions F I to F IV, subjecting the partially purified hypoglycemic fraction F II to re-column chromatography using buffers resulting in four hypoglycemic fractions wherein fraction F IIc is separated to obtain the hypoglycemic compound. 3. A process for producing the herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia jambolana as claimed in claim 2 wherein the said water content is extracted from the said watery solution after the stage of filteration by means of a lyophilizer. 4. A process for producing the herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia jambolana as claimed in claim 2 wherein the said column materials are selected from the group consisting of diethyl amino ethyl cellulose and anion exchange resins or mixtures thereof. 5. A process for producing the herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia jambolana as claimed in claim 2 wherein the said buffers are selected from the group consisting of phosphate buffers and citrate buffers or mixtures thereof. 6. A process for producing the herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia jambolana as claimed in claim 2 wherein the pH of the said buffer is maintained at 6. 7. A process for producing the herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia jambolana as claimed in claim 2 wherein the said buffer is maintained at pH 6 neutral in the re-chromatography. 8. A herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of species of Eugenia preferably Eugenia jambolana substantially as herein before described with reference to the foregoing examples. 9. A process for producing the herbal hypoglycemic compound for controlling diabetes mellitus extracted from the pulp of fruit of Eugenia jambolana substantially as hereinbefore described with reference to the forgoing examples.

Documents:

838-del-2003-abstract.pdf

838-del-2003-claims.pdf

838-del-2003-correspondence-others.pdf

838-del-2003-correspondence-po.pdf

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

838-del-2003-form-1.pdf

838-del-2003-form-13.pdf

838-del-2003-form-18.pdf

838-del-2003-form-2.pdf

838-del-2003-form-3.pdf

838-del-2003-gpa.pdf

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