US20010021401A1

US20010021401A1 - Process for the preparation of an herbal-therapeutic product extracted from the pulp of a species eugenia jambolana

Info

Publication number: US20010021401A1
Application number: US09/776,650
Authority: US
Inventors: Suman Sharma; Pothapragada Murthy; Krishan Prabhu; Afreena Nasir
Original assignee: Indian Council of Medical Research
Current assignee: Indian Council of Medical Research
Priority date: 1999-06-04
Filing date: 2001-02-05
Publication date: 2001-09-13
Anticipated expiration: 2020-05-02
Also published as: WO2000074698A1; AU5844300A; US6428825B2

Abstract

The present invention relates to an herbal therapeutic product for controlling diabetes mellitus comprising at least one hypoglycemic compound extracted from the pulp of fruit Eugenia jambolina. The said product is prepared from the pulp of the fruit Eugenia jambolina comprising cleaning and drying the fruit to remove the extraneous material from the outer layer of the said fruit, deseeding the said fruit, soaking the de-seeded pulp in water, mixing the said mixture of water and pulp and keeping it overnight in controlled cooled conditions to maintain the activity of hypoglycemic compounds, filtering the said mixture, washing the residue with water to extract all the active compounds from the said mixture of water and pulp in the form of clear watery solution, reducing the water content by keeping the said clear watery solution in freezing conditions to get the residues in concentrated form, purifying the said concentrated product in the chromatography by using slurries of different columns. Buffers are added in the said purified mixture present in the said chromatography to elute the mixture of hypoglycemic compounds. The thin layer chromatography is then conducted of the said hypoglycemic compounds in the said mixture to get the said hypoglycemic compounds in the form of bands. The said bands are exposed in the iodine chamber for the clear demarcation of the said hypoglycemic compound bands. The said hypoglycemic compounds are then extracted to get the herbal therapeutic product to control the glucose level.

Description

FIELD OF THE INVENTION

The present invention relates to an herbal therapeutic product for controlling diabetes mellitus comprising at least one hypoglycemic compound extracted from the pulp of a fruit from a species of genus Eugenia specifically Eugenia jambolina and a process for the preparation of the same.

The embodiment of the invention resides in the various compounds of the product and their mixture having immediate as well as longer lasting effects in controlling diabetes mellitus.

The other embodiment of the invention resides in four active hypoglycemic compounds comprising the product of the subject invention. These four active hypoglycemic compounds individually have the varying degrees of hypoglycemic characteristics, however, the combination thereof have been found to be most effective.

Still another embodiment of the invention resides in the discontinuity of 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 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 longer lasting effects of these hypoglycemic compounds

The source of carbohydrates in the diet has a significant influence on lipid (fat) metabolism in human being An amount of sucrose is increased and the quantity of complex carbohydrates is decreased, the concentration of cholesterol and more particularly of triglycerides increases which has a correlation between blood lipid concentration and impaired glucose tolerance

One of the objects of the present invention is that the present invention is a 100% herbal product without any side effects.

Another object of the present invention is to treat the defect in metabolism, by increasing the activity of the glucose utilizing enzymes in liver, muscles and adipose tissues.

The present invention relates to the product obtained from the species of genus Eugenia having hypoglycemic effects in the blood sugar level.

A process for the preparation of the herbal therapeutic product to control the glucose level, comprises cleaning and drying the fruit of a species of family Eugenia to remove extraneous material from the outermost layer of the said fruit. De-seeding the fruit and soaking the said de-seeded fruit in water under controlled cooled conditions overnight to retain the activity of hypoglycemic compounds in the said mixture of water and pulp of de-seeded fruit It is followed by mixing the said mixture to get the mixture of a uniform consistency.

(Prior Art)

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; proteolytic resistance and other properties:

It teaches a simple, two-step, procedure for purify 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 in oral feeding.

Kelkar further teaches separation of hypoglycemic compound carried by molecular sieving (depending upon the size of two molecular). In short Kelkar 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 tells that the hypoglycemic activity was found at two distinct posit ions 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 its review article XP000608379 teaches Hypoglycemic Activity of Eugenia jambolana and Ficus bengalensis:

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention relates to a herbal therapeutic product controlling diabetes mellitus comprising at least one hypoglycemic compound extracted from the pulp of a species of the genus Eugenia, specifically Eugenia jambolina.

The process for the preparation of such herbal product comprising cleaning and drying the fruit of a species of genus Eugenia of family Myrtaceae to remove the extraneous material from the outer layer of the said fruit, deseeding the said fruit, soaking the de-seeded pulp in water, mixing the said mixture of water and pulp and keeping it overnight in a controlled cooled conditions to maintain the activity of hypoglycemic compounds, filtering the said mixture in the conventional manner, washing the residue with water to extract all the active compounds from the said mixture of water and pulp in the form of clear watery solution, reducing the water content by keeping the said clear watery solution in controlled freezing conditions by conventional means to get the residues in a concentrated form, purifying the said concentrated product in a chromatography by using slurries of different columns, adding buffers in the said purified mixture present in the said chromatography to elute the mixture of hypoglycemic compounds

The various steps involved in the aforementioned process are:

(a) The fruit of Eugenia jambolina is cleaned and dried to remove the extraneous material from the outer layer of the said fruit;

(b) The seed is removed from the said fruit in the conventional manner and is kept separately.

(c) The de-seeded pulp is soaked in (distilled) water and the same is mixed to get the uniform consistency of the liquid;

(d) The said mixture is kept overnight in a controlled cooled conditions preferably between 4-10 degrees centigrade;

(e) The said mixture is filtered in the conventional manner

(f) The residue are washed with water to extract all the active compounds from the said pulp in the form of clear watery solution

(g) The water content are reduced by keeping the said clear watery solution in freezing conditions by conventional means to get the residues in concentrated form;

(h) The said concentrated product is purified by treating the same with slurries of solvents selected preferably from diethyl amino ethyl cellulose, anion exchange resins or silica gel and distilled water in the chromatography;

(i) Buffers are added in the said purified mixture present in the chromatography to elute the hypoglycemic compounds from the said mixture.

The water content from said mixture can be reduced optionally to get the end product in solid form. The water is extracted from the said mixture after the stage of filtration by means of lypholizer.

The said pulp of Eugenia jambolina, is subjected to column chromatography for a period of 15 minutes-2 hours to obtain a hypoglycemic fraction at a slightly acidic pH of 5-7 preferably 6.0 to 6.5.

The said hypoglycemic fraction is subjected to thin layer chromatography using silica get to form at least 2 visible bands . The R _fvalue of all the four bands is between 0.1-1.0, wherein the R_fvalue of one visible band which is having highly active hypoglycemic compound is between 0.5 to 0.7 and the R_fvalue of less active hypoglycemic compounds is between 0.1-0.2 and 0.9-1.0.

The pH of the column containing mixture of chemical compounds is maintained at pH of 6.0-8.0 preferably 6.5 to 7.5.

The said buffer used in the chromatography is selected preferably from the phosphate buffer citrate buffer

The pH of the said mixture of hypoglycemic compounds is to be maintained neutral.

The said mixture of hypoglycemic compounds were further analyzed to separate the active hypoglycemic compounds by means of thin layer chromatography to separate the different compounds in the form of separate bands. Such bands were further exposed to the iodine chamber for the clear demarcation of the individual compound. It has been found that out of 4 such hypoglycemic compounds present in the mixture, two compounds are having immediate effect in controlling and maintaining the blood sugar level, while other two hypoglycemic compounds have their effect after certain time.

In the thin layer chromatography, the said mixture of hypoglycemic compounds are dropped in the lower most end of a silica gel coated plate to get the deposition of said hypoglycemic compounds in the form of bands on the said silica gel coated plate by running with a mixture of solvents for 2-3 hours in the ascending manner. The said solvents are n-butanol, acetic acid and water in the ratio of 5:1:4. The silica gel coated plate with bands of hypoglycemic compounds is dried in cooled conditions and is exposed to iodine chamber to get the clear demarcation of the different hypoglycemic compounds bands which are extracted from the silica gel coated plate along with the fractions of silica gel. The extracted hypoglycemic compound along with silica gel is mixed with water for the separation of said silica gel by centrifugation.

The water used in the subject process is preferably distilled water

A study was conducted to determine the effect of a herbal therapeutic product prepared from the pulp extracted from the genus Eugenia on the blood glucose, liver, aorta, pancreas and heart. The results were found very effective with 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 glucose tolerance test.

The test was conducted on alloxan induced diabetic rabbit where the said product prepared from the process of subject invention was given to alloxan induced diabetic rabbit after drawing the blood from fasting rabbits. After giving the dose of said product, the blood was withdrawn again from said rabbit after 90 minutes. After that 2 grams of glucose in a water solution was given to that rabbit. The blood was again withdrawn after two hours of glucose feed. It was found that said mixture reduced the blood glucose level even after the glucose feed, which was due to the rise in insulin level in the blood.

The said product was found to increase the activity of enzymes in liver, muscle and adipose tissue, which use glucose and thereby reduces blood glucose level. The said product was further found to reduce the level of lipids in blood which are responsible for heart diseases like total cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol and triglycerides. At the same time it increases the level of favourable lipids for health such as high density lipoprotein cholesterol (HDLC). The said mixture of hypoglycemic compounds were found to be having no adverse effects on the liver and kidney functions.

No abnormalities were found after conducting the histopathology of liver, aorta, pancreas and heart, when the patient was treated with the product of subject invention.

Hence, the subject invention relates to a herbal therapeutic product to control the glucose level in diabetes mellitus and also minimizes the risk of heart diseases extracted from the pulp of a species from the genus Eugenia prepared from the process as herein before described, comprising said hypoglycemic compound or a group of said hypoglycemic compounds in the form of a product.

EXAMPLES

Example 1

To prepare an herbal therapeutic product of present invention, 1 Kg 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 conventional manner, 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 is done by diethyl amino ethyl cellulose, a phosphate buffer is then added to elute the mixture of hypoglycemic compounds. The said hypoglycemic compounds were given in the form of solution mixed with water. [0044]

Example 2

An herbal therapeutic product of present invention is prepared by taking 500 grams of pulp of the fruit [0045] Eugenia Jambolina and mixing it in 200 ml of distilled water which is kept overnight in cooled conditions at 4-10° C., the said mixture is filtered by conventional manner, and the residue is washed twice to extract all the active compounds from the said mixture, the water from the resultant mixture is extracted by keeping the said resultant mixture in a lypholizer to get the residues in paste form, the chromatography of the said paste is done a phosphate buffer is then added to elute the mixture of hypoglycemic compounds, the said hypoglycemic compounds are dropped in very small quantity on the lower most end of a glass plate having silica coating on it, the said plate is placed in a glass chamber having solvents n-butanol, acetic acid and water in the ratio of 4:1:5, the bands of hypoglycemic compounds are deposited on the silica coated plate by the capillary action, the plate with the deposition of hypoglycemic compounds is removed from the said glass chamber and is dried in cooled condition, then the plate is exposed to the iodine chamber to get the clear demarcation of the different hypoglycemic compounds bands which are extracted by scratching them off from the plate in the form of powder along with silica gel abstracts, the same is mixed with water for the separation of said silica gel by centrifugation.

Claims

1. A process for the preparation of an herbal therapeutic product extracted from the pulp of a species Eugenia jambolina, comprising, cleaning and drying the fruit to remove the extraneous material from the outer layer of the said fruit, deseeding the said fruit, soaking the de-seeded pulp in water, mixing the said mixture of water and pulp and keeping it overnight in controlled cooled conditions to maintain the activity of hypoglycemic compounds, filtering the said mixture by conventional manner, washing the residue with water to extract all the active compounds from the said mixture of water and pulp in the form of clear watery solution, reducing the water content by keeping the said clear watery solution in controlled freezing conditions by conventional means to get the residues in concentrated form, purifying the said concentrated product in a chromatography by using slurries of different columns, adding buffers in the said purified mixture present in the said chromatography to elute the mixture of hypoglycemic compounds, conducting thin layer chromatography of the said hypoglycemic compounds in the said mixture to get the said hypoglycemic compounds in the form of bands, exposing the said bands of the said hypoglycemic compounds in an iodine chamber for the clear demarcation of the said hypoglycemic compound bands and extracting such hypoglycemic compounds to get the herbal therapeutic product to control the glucose level.

2. A process for the preparation of an herbal therapeutic product as claimed in

claim 1

where the said controlled cooled conditions for keeping overnight the mixture of water and pulp are from 4-10 degrees C.

3. A process for the preparation of an herbal therapeutic product as claimed in

claim 1

, wherein the water is extracted from the said mixture after the stage of filtration by means of lypholizer.

4. A process for the preparation of an herbal therapeutic product as claimed in

claim 1

, wherein the said slurries used in the chromatography are selected from diethyl amino ethyl cellulose anion exchange resins.

5. A process for the preparation of an herbal therapeutic product as claimed in

claim 1

, wherein the said buffer used in the chromatography is selected from the phosphate buffer citrate buffer.

6. A process for the preparation of an herbal therapeutic product as claimed in

claim 1

, where in the pH of the column containing mixture of chemical compounds is maintained at pH of 6.0-8.0 preferably 6.5-7.5.

7. A process for the preparation of an herbal therapeutic product as claimed in

claim 1

, where in the thin layer chromatography, the said mixture of hypoglycemic compounds are applied in the lower most end of a silica gel coated plate to get the deposition of said hypoglycemic compounds in the form of bands on the said silica gel coated plate by running with a mixture of solvents for 2-3 hours in the ascending manner.

8. A process for the preparation of an herbal therapeutic product as claimed in

claim 1

, wherein the said solvents are n-butanol, acetic acid and water in the ratio of 4:1:5 to 5:1:4.

9. A process for the preparation of an herbal therapeutic product as claimed in

claim 8

, wherein said silica gel coated plate with bands of hypoglycemic compounds is dried at cooled temperature.

10. A process for the preparation of an herbal therapeutic product as claimed in

claim 9

, wherein said plate is exposed to iodine in a closed chamber to get the clear demarcation of the different hypoglycemic compounds bands and extracting the same from said plate.

11. A process for the preparation of an herbal therapeutic product s claimed in

claim 1

, wherein the water used in any of the preceding claims is distilled water.

12. An herbal therapeutic product extracted from the pulp of Eugenia jambolana whenever prepared by the process s claimed in any of the preceding claims.

13. An herbal therapeutic product extracted from the pulp of Eugenia jambolana as prepared by the process as claimed in any of the preceding claims, used for controlling the diabetes mellitus.