U.S. patent application number 09/776650 was filed with the patent office on 2001-09-13 for process for the preparation of an herbal-therapeutic product extracted from the pulp of a species eugenia jambolana.
This patent application is currently assigned to INDIAN COUNCIL OF MEDICAL RESEARCH. Invention is credited to Murthy, Pothapragada Suryanarayana, Nasir, Afreena, Prabhu, Krishan Madhav, Sharma, Suman Bala.
Application Number | 20010021401 09/776650 |
Document ID | / |
Family ID | 26324672 |
Filed Date | 2001-09-13 |
United States Patent
Application |
20010021401 |
Kind Code |
A1 |
Sharma, Suman Bala ; et
al. |
September 13, 2001 |
Process for the preparation of an herbal-therapeutic product
extracted from the pulp of a species eugenia jambolana
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.
Inventors: |
Sharma, Suman Bala; (Delhi,
IN) ; Murthy, Pothapragada Suryanarayana; (Noida,
IN) ; Prabhu, Krishan Madhav; (Delhi, IN) ;
Nasir, Afreena; (Delhi, IN) |
Correspondence
Address: |
Ladas & Parry
26 West 61St Street
New York
NY
10023
US
|
Assignee: |
INDIAN COUNCIL OF MEDICAL
RESEARCH
|
Family ID: |
26324672 |
Appl. No.: |
09/776650 |
Filed: |
February 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09776650 |
Feb 5, 2001 |
|
|
|
PCT/IN00/00052 |
May 2, 2000 |
|
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Current U.S.
Class: |
424/769 ;
424/777 |
Current CPC
Class: |
A61K 36/61 20130101;
Y10S 514/866 20130101; A61P 5/50 20180101 |
Class at
Publication: |
424/769 ;
424/777 |
International
Class: |
A61K 035/78 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 4, 1999 |
IN |
834/DEL/1999 |
May 1, 2000 |
IN |
475/DEL/2000 |
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.
Description
FIELD OF THE INVENTION
[0001] 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.
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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
[0006] 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
[0007] One of the objects of the present invention is that the
present invention is a 100% herbal product without any side
effects.
[0008] 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.
[0009] The present invention relates to the product obtained from
the species of genus Eugenia having hypoglycemic effects in the
blood sugar level.
[0010] 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.
[0011] (Prior Art)
[0012] 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:
[0013] 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.
[0014] 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.
[0015] 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
[0016] Saigeeta Achrekar et al. in its review article XP000608379
teaches Hypoglycemic Activity of Eugenia jambolana and Ficus
bengalensis:
DETAILED DESCRIPTION OF THE INVENTION
[0017] 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.
[0018] 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
[0019] The various steps involved in the aforementioned process
are:
[0020] (a) The fruit of Eugenia jambolina is cleaned and dried to
remove the extraneous material from the outer layer of the said
fruit;
[0021] (b) The seed is removed from the said fruit in the
conventional manner and is kept separately.
[0022] (c) The de-seeded pulp is soaked in (distilled) water and
the same is mixed to get the uniform consistency of the liquid;
[0023] (d) The said mixture is kept overnight in a controlled
cooled conditions preferably between 4-10 degrees centigrade;
[0024] (e) The said mixture is filtered in the conventional
manner
[0025] (f) The residue are washed with water to extract all the
active compounds from the said pulp in the form of clear watery
solution
[0026] (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;
[0027] (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;
[0028] (i) Buffers are added in the said purified mixture present
in the chromatography to elute the hypoglycemic compounds from the
said mixture.
[0029] 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.
[0030] 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.
[0031] The said hypoglycemic fraction is subjected to thin layer
chromatography using silica get to form at least 2 visible bands .
The R.sub.f value of all the four bands is between 0.1-1.0, wherein
the R.sub.f value of one visible band which is having highly active
hypoglycemic compound is between 0.5 to 0.7 and the R.sub.f value
of less active hypoglycemic compounds is between 0.1-0.2 and
0.9-1.0.
[0032] 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.
[0033] The said buffer used in the chromatography is selected
preferably from the phosphate buffer citrate buffer
[0034] The pH of the said mixture of hypoglycemic compounds is to
be maintained neutral.
[0035] 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.
[0036] 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.
[0037] The water used in the subject process is preferably
distilled water
[0038] 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
[0039] Various tests were conducted to analyze the effect of said
product on testing the blood glucose level in fasting and in
glucose tolerance test.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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
[0044] 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.degree. 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.
Example 2
[0045] An herbal therapeutic product of present invention is
prepared by taking 500 grams of pulp of the fruit Eugenia Jambolina
and mixing it in 200 ml of distilled water which is kept overnight
in cooled conditions at 4-10.degree. 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.
* * * * *