Chemistry: natural resins or derivatives; peptides or proteins; – Lignins or derivatives – Aldehyde – ketone or ozone reacted with a lignin or lignin...
Reexamination Certificate
2001-06-14
2004-12-14
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Lignins or derivatives
Aldehyde, ketone or ozone reacted with a lignin or lignin...
Reexamination Certificate
active
06831162
ABSTRACT:
FIELD
This invention relates to a highly effective hypoglycaemic protein called polypeptide-k, extracted from
Momordica charantia
. This invention also provides a method for the extraction of said polypeptide-k from
Momordica charantia
. Further, the invention provides a novel hypoglycaemic composition employing the said protein, and useful in the treatment of diabetes mellitus.
BACKGROUND
Insulin has hitherto been commercially synthesized from the pancreas of animals and human insulin from
E. coli
(Eli Lily, U.S.A.). So far there is no report of commercial extraction of insulin like polypeptide from plant source.
Isolation of insulin from animal pancreas is open to objection due to the following reasons:
1. By killing 10,000 animals only one pound of pure insulin is obtained.
2. It is not being sublingually administered.
3. If the pancreas is infected by some diseases there is always a probability of its being carried (if it is a virus) along with the insulin.
4. Human insulin can be synthesised from
E. coli
which is expensive.
Hence, to obviate these and other drawbacks in conventional insulin extraction methods, scientists focussed on plant based products.
Momordica charantia
is a perennial herb of the family Cucurbitaceae, widely grown in Asia. The herb is endemic to tropical regions like India, S. Africa, Philippines, China and Burma. The species of Momordica found in western countries are different from the tropical species in that, the plants differ in morphological and organoleptic properties.
Various parts of this plant, especially the fruits, have been widely used for preparation of hypoglycaemic pharmacological compositions.
In Indian Patent No. 136565, the applicant has disclosed a method for the extraction of a protein called ‘polypeptide-p’ from
Momordica charantia
. The dried and pulverized fruits and tissue cultures of Momordica are separately extracted in ethanol and then mixed with cold ethanol and diethyl ether. Thereafter, needle-like crystals are formed by adding zinc in traces after 18 hr. The fruits and cultures are separately crushed, homogenized in water, ethanol and concentrated sulfuric acid is added for adjusting pH to 3, thereby obtaining flocculent precipitates.
This method had the following drawbacks:
1. The use of alcohol in the extraction procedure was not practical due to its unavailability in large amount and the impurities present in it.
2. The use of raw material as fruits and tissue culture creates problems in handling, uneconomically viable and the yield was very poor.
The drawbacks of this patent were obviated in another Indian Patent No. 176040. This patent discloses a process for extraction of a highly effective polypeptide-p by using hexane along with diethylether. Although the process developed and disclosed in above referred patent resulted in good yield, improved purity and high efficacy of the drug by removal of oil and sapogenins and other contaminants therefrom, yet, it had a few drawbacks, some of which a few are given below:
1. The purification of polypeptide-p was a cumbersome method due to the presence of interfering radicals as oil and sapogenins.
2. Use of diethylether in the extraction procedure was not practical due to its highly inflammatory nature and high cost.
3. The presence of pesticides/insecticides/urea and other contaminants affected the purity of polypeptide-p.
4. The yield was not optimum.
This protein called ‘polypeptide-p’ (SEQ ID NO: 1) was extracted from the fruits and tissues of
Momoridca charantia
. This protein comprised amino acids as shown in Table 1 below:
TABLE 1
Molecular
Amino acid
&mgr; Moles/mgs
number
Aspartic acid
0.273
17
Threonine
0.138
8.7
Serine
0.195
12
Glutamic acid
0.305
19
Proline
0.159
10
Glycine
0.225
19
Alanine
0.240
15
Valine
0.174
11
½ Cysteine
0.058
3.6
Methionine
0.031
2
Isoleucine
0.116
7
Leucine
0.207
13
Tyrosine
0.016
1
Phenylalanine
0.082
5
Histidine
0.066
4
Lysine
0.209
13
Arginine
0.161
10
NH
3
0.431
(27) omit 166
TOTAL
residues
Approximate molecular weight 11,000
U.S. Pat. No. 5484889 describes a plant protein useful for treatment of tumors and HIV infection. The protein has been obtained from the seeds of
Momordica charantia
. It is pertinent to note that this protein isolated and purified is a ribosome inactivating protein and hence, useful in tumor therapy. The processes described for the extraction of the protein involve use of solvents and the tedious process of chromatography, dialysis etc. In the processes described in this patent as well as in Indian patent No. 176040, the yield, purity were low and had several contaminants. Accordingly, to obviate these and other drawbacks, the applicant has isolated a novel protein called ‘poplypeptide-k’ having hypoglycaemic property from
Momordica charantia
and has also devised a novel process for extraction of the protein from the same source. The letter ‘k’ is derived from the term ‘karela’ which means ‘bitter gourd’ or
Momordica charantia
in a main Indian language.
OBJECTS OF THE INVENTION
The main object of the invention to provide a novel protein called ‘polypeptide-k’.
It is an object of the invention to provide a process for the extraction of a protein called ‘polypeptide-k’ from the dry seeds of
Momordica charantia.
Another object is to prepare a novel hypoglycaemic composition using the said protein.
Yet another object of the invention is to provide a hypoglycaemic composition containing ‘polypeptide-k’ for treatment of diabetes in human beings and animals.
Still another object of the invention is to provide a novel protein called polypeptide-k which is capable of reducing high blood pressure and increasing immunity in human beings and animals.
One more object of the invention is to provide a novel protein namely polypeptide-k which takes care of neuropathy and makes the patient feel normal.
SUMMARY OF THE INVENTION
In accordance with the above and other objectives, the invention provides a novel protein ‘polypeptide-k’ extracted from
Momordica charantia
, a process for the extraction of the said polypeptide-k and a novel hypoglycaemic composition employing the novel polypeptide of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the invention provides a method for the extraction of proteins from dry seeds of
Momordica charantia
, said process comprising the steps of:
i) grinding the dry seeds to a fine powder in a suitable mill,
ii) treating the pulverized seeds with a mixture of hexane-acetone,
iii) dissolving the residual mass in about 80% aqueous acetone,
iv) adjusting the pH upto 9.5 by adding suitable organic buffer like ammonium hydroxide,
v) treating the supernatant layer with sulfuric acid,
vi) collecting the flocculent precipitate of polypeptide-k and isolating the protein by selective crystallisation. Thereafter, the protein is analysed by chromatography.
The protein isolated in the present invention i.e. ‘polypeptide k’ is different as compared to the protein in the prior art. To describe in detail, the protein isolated in the present invention is a protein having 18 amino acids, and is called ‘polypeptide k’. The process for extraction of the protein consists of de-oiling of the washed seeds of
Momordica charantia
, using hexane and a little acetone in the ratio of 3:1. The dry seeds are used because polypeptide-k is a storage protein and it gets accumulated in large quantities when the seed is dried. After de-oiling, the seeds are dried, powdered and dissolved in water and acetone taken in the ratio of 3:1. A mixture is made and then the pH is adjusted to 9.5 by adding ammonium hydroxide. Supernatant was remove and the pH was adjusted to 3 by adding sulfuric acid. The flocculent precipitate was collected and dried. The dried mass was powdered and washed with water and acetone to remove oil, salts and other undesirable material, till it gives a single spot in TLC and HPLC.
In one embodiment, the dried seeds of
Momordica charantia
are split, washed thoroughly with water 2-3 times to render it substantially free from impurities and dried under vacuum, before ext
Carlson Karen Cochrane
Ladas & Parry
Snedden Sheridan K
LandOfFree
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