ZA200108326B

ZA200108326B - Protein/polypeptide-k obtained from momordica charantia and a process for the extraction thereof.

Info

Publication number: ZA200108326B
Application number: ZA200108326A
Authority: ZA
Inventors: Pushpa Khanna
Original assignee: Pushpa Khanna
Priority date: 1999-04-13
Filing date: 2001-10-10
Publication date: 2003-04-29
Also published as: IN188673B

Description

PROTEIN/POLYPEPTIDE-K OBTAINED FROM MOMORDICA

CHARANTIA AND A PROCESS FOR TBE EXTRACTION THEREOF

Field

This invention relates to a highly effective 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 novel hypoglycaemic compositions employing the said extract, and useful in the treatment of diabetes mellitus.

Background

Insulin has hitherto been commercially synthesised from the pancreas of animals and human insulin from E.Coli (Eli Lily, U.S.A). So far there is no commercial extraction of insulin / polypeptide-k 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 are 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.

Co

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 1 oo . ) EE ; AMENDED SHEET 2003 -06- 2 4

PCT/IN99/00052 - the fruits, have been widely used for preparation of hypoglycaemic pharmacological compositions.

The extracts juice of the fruit is known to exhibit hypoglycaemic properties and g often recommended to reduce the blood sugar levels in patients suffering from diabetes mellitus.

Any amount of polypeptide-p can be synthesised from this plant which is so easy to cultivate. :

In Indian Patent No. 136565, the “applicant has disclosed a method for the extraction of 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 and 03 to obtain flocculent precipitates.

This method had the following drawbacks: . 20 : 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 some drawbacks of which a few are given below: :

EE | 2 AMENDED SHEET 2003 -06- 24 iy WO 00/61619 PCT/INYY/uuls2

I. 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 being highly inflammatory and involving high cost. 3. The. presence of pesticides / insecticides / urea and other contaminants : affected the purity of polypeptide-p. 4. The yield was not optimum.

US Patent 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 involves use of solvents and the tedious process of chromotography, 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 polypeptide-k having hypoglycaemic property from Momordica charantia and has also devised a novel process for extraction of the protein from the same source. y : 20 . )

Objects :

It is an object of the invention to provide a novel protein. o5 It is an object of the invention to provide a process for the extraction of protein from the seeds and fruits of Momordica charantia.

Another object is to prepare a novel hypoglyceamic composition using the said protein. | :

Yet another object of the invention is to provide hypoglyceamic composition for treatment of diabetes in human beings and animals. 3

AMENDED SHEE: 2003 -06- 2 4

Summary of the invention

In accordance with the above and other objectives, the invention provides novel polypeptide-k obtained from Momordica charantia, a process for the extraction of the said polypeptide-k and novel hypoglycaemic compositions employing the polypeptide of the invention.

Detailed description : 1 0 - . - .

Accordingly, the invention provides a method for the extraction of proteins fruit seeds from the seeds of Momordica charantia comprising the steps of: 1) grinding the dry seeds to a fine powder in a suitable mill, 1) treating the pulverized seeds with a mixture of non-polar solvents, : 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, to

Vv) treating the supernatant layer with sulfuric acid, og Vi) collecting the flocculent precipitate of polypeptide-k and isolating the protein by selective crystallisation. Thereafter the protein is analysed by chromatography. . ‘

The novel protein polypeptide-k extracted from the seeds of Momordica charantia comprises amino acids including:

Amino acid pn Moles/mg® Molecular number 0.273

Thweomme 0.8

Serine [01% 0.305 19

Proline | 0.1% 10

Glycine | 025 19 [Alanine | 0240 15

Valine | 01m [Cysteine | 00s8 4

AMENDED SHEET p03 -06- 24

Phenylalanine 0.082 5 ee ose 4

NH, 0.431 (27) omit 166

TOTAL residues

Approximate molecular weight 18000 ‘

In one embodiment, the 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 extraction of the protein. Cee -

In another embodiment, the solvents used comprise a mixture of acetone with hexane, taken in the ratio 3:1.

In one embodiment, thin glass plated (20 x 20) coated (0.4 mm to 0.5 mm thick) with silica gel G are activated at 100°C, the solution of insulin applied, the plates developed in n-butanol, water, acetic acid (12:5:2) dried, the single spot corresponding to standard insulin visualized by spraying nin-hydrin (0.25%) in acetone, isolated along with silica gel G from unsprayed plates, extracted in 50% i ethanol] buffered with ammonium hydroxide, filtered, the filtrate dried and pure white needle-like crystals formed. :

In yet another embodiment, when the analysis 1s carried out, the isolated substance is hydrolyzed alongwith the standard insulin, applied on paper chromatograms separately, developed, yielding same amino acids except glutamine being an extra amino acid in the polypeptide -k. : 5

AMENDED SHEET 5003 -06- 24

In another feature. the isolated substance and the standard insulin are hydrolyzed separately by 6 N HCI for 20 hours, dried. reconstituted in 50% ethanol, applied on Whatman No. | filter paper strips developed in /i-butanol, acetic acid, water ) (60:320:20), strips developed sprayed with 0.25% nin-hydrin in acetone, amino acids same as of the standard hydrolyzate except glutamine being extra intra in or polypeptide-k.

In the analysis is carried out, the seeds are extracted in hexaneacetone yielding a product which has the same melting point (234°C), Gel electrophoretic pattern of the accompanying drawings and number of amino acids of the standard insulin except glutamine being extra in polypeptide-k. ’

It may be noted that although most of the plant parts of Momordica contain the protein disclosed by the invention, in varying degrees the protein polypeptide-k is extracted from - the dry seeds.

The dried seeds are processed using hexane (food grade) along with acetone instead of ether as used in the process described in earlier Patent No. 176040. The process has resulted in high yield, improved purity and high efficacy of polypeptide by removal of undesired oils, flavonoids and sapogenins therefrom. *

Diabetes is a disease wherein glucose is not utilized as an energy source in the body such glucose remains at a high levels in the blood and eventually gets og excreted through urine. In some conditions, insulin secreted from beta cells of pancreas is insufficient or does not sufficiently fulfill its function.

Diabetes is generally classified into insulin-dependent diabetes (Type I diabetes) and noninsulin-dependent diabetcs (Type II diabetes). Type 1 diabetes is in the q0 state of lowering of the function of pancreatic beta cells resulting from hereditary cause, viral infection -etc. wherein insulin is substantially not secreted, and suddenly attacks mainly in the twenties to thirties. Although it is not sure. onset of type 11 diabetes in the forties or in cases with family history of diabetes, obesity, 6

AMENDED SHEET 2003 -06- 2 4

I stress. etc. In the case of type Il diabetes, since insulin is sufficiently secreted from pancreas but insulin resistance and glucose utilization are different from those of normal person, blood sugar is not returned to normal level in spite of hyperinsulinemia.

Diabetes is accompanied with numerous symptoms. Typical examples of such symptoms are polyuria, excessive drinking and polyphagia. That is, diabetic patients exhibit polyuria which is caused by excretion of glucose and excessive water through urine by the action of osmotic pressure originated from high blood glucose level, and therefore, complain of thirst caused by dehydration, which induces excessive drinking, and feels the empty of stomach to cause excessive intake of food. Diabetic patients cannot efficiently utilize glucose as an energy source and, instead, utilize protein and fat as preserved in the body, and this phenomenon is caught in a vicious circle to cause the reduction in body weight.

However, such phenomena are merely acute symptoms observed in the primary stage of diabetes. If diabetes becomes chronic by delay of treatment, chronic vascular diseases are induced as a complication. Thus, diabetic complications such as diabetic retinopathy (visual disturbance, blindness, retinal hemorrhage), oo diabetic nephropathy, diabetic peripheral neuropathy, etc. reduce general metabolic and sensory function of human body.

A number of medicines have been produced and tested act to lower blood sugar of a non-insulin dependent diabetics. However, the majority of these medicines have og one or more undesirable features, some of them have significant side effects for a large portion of the population, or a large dosage is necessary. Also, some of them reduce the blood sugar level too much so that they can only be used sporadically or they can be a threat to health, and others have possible toxicity. At present, there is no natural antidiabetic drug which is highly effective at lowering blood sugar, yet does not lower it to an unsafe level, and has no significant side effects.

“

According to the present invention, a pharmacologically active hypoglycaemic agent is produced in a simple and straightforward way using only the protein of the invention. It is relatively small amounts of the homeopathic medicine need be ingested in order to perform the desired function.

Herbal composition using the protein of the invention:

The protein extracted from Momordica charantia exhibits hypoglyceamic properties and accordingly compositions comprising the protein can be for the treatment of hypoglyceamia treatment in mammals. The protein obtained from

Momordica is in the form of an amorphous powder. The protein activates the inactive insulin and, thus, it can rejuvenate the pancreas depending upon the chronicity of the pathological condition of the individual. In fact, in course of time, it may act as a cure for diabetes. The applicant has conducted more than 500 experiments and confirmed that the single dose to about 12 mg of the protein at a time is quite effective. Accordingly, it is advisable that compositions containing the protein in single does should comprise about 12 mg of the protein.

Hypoglyceamic compositions using the proteins of the invention can be 9g formulated in a variety of physical forms such as tablets, injections, edible products. For preparation of a tablet, about 12 mg of the protein is mixed with pharmacologically acceptable carriers suitable for consumption. The pharmacologically acceptable carrier must be of sufficient purity and low toxicity.

Edible products like biscuits, chewing gums etc, which are not instantly 55 swallowed can be prepared. In all such preparations, the content of the protein is about 12 mg. It is found that salt biscuits prepared using the protein of the invention are very popular with diabetics.

It is pertinent to note that the hypoglyceamic composition of the invention is to be consumed 10 minutes before meals, at least 4 times a day. The most important aspect is that the tablet or the hypoglyceamic composition should only be chewed and should not be swallowed instantaneously.

EN

In a feature of the invention, the hypoglyceamic composition herein described has no side effects. It can be consumed without restricting the use of other therapies.

The invention is described in detail with reference to the following illustrative examples. Several modifications that may be apparent to those in the art are deemed to be included within the scope of the invention.

Example 1 (Protein preparative example)

Extraction of protein from Momordica charantia L: 100 gms of dry seeds were taken from the ripe fruits of Momordica charantia L. The seeds were split manually. The split seeds were then thoroughly washed with water 3-4 times to render them substantially free of all impurities. The split seeds were then dried "15 under vacuum and pulverized to a fine powder using a milling device. Any other conventional device may also be used.

The fine powder was then treated with acetone hexane solvent mixed in the ratio 1:2 and the residual mass was dissolved in 20% acetone. The pH was adjusted to 50 9.5 by adding ammonium hydroxide / organic buffer, the supernatant thus obtained was buffered with H,SO,4 to adjust pH3 for obtaining flocculent precipitate which were collected and crystallised with zinc acetate.

Thin glass plated (20 x 20 cm) coated (0.4 mm to 0.5 mm thick) with silica gel G (Kieselgel G nach Stahl; E. Merck) were activated at 100°C for half an hour. The solution containing the isolated substance was applied 1 cm above the edge of the plates were run in an organic solvent mixture of n-butanol, water and acetic acid (12:5:2). The developed plates were dried at room temperature and sprayed with 0.25% ninhydrin in acetone. The ninhydrin positive spots (R = 0.19) of the isolate corresponding to insulin were collected from about 200 unsprayed plates along with the silica gel G and extracted with 50% ethanol buffered with ammonium hydroxide. The extract was filtered and dried in vacuo. Pure colorless crystals thus obtained were weighed (3 g/100 gram dry weight of seeds).

The melting point of the purified compound (232° - 235°C) as well as the mmp (254°C) were determined. The melting point of the standard insulin was recorded as 233°C. :

The standard sample of insulin as well as the isolated polypeptide-k were hydrolyzed under reflux with 6N HCI for 20 hours separately. The hydrolyzates were filtered, dried, reconstituted separately in 50% ethanol and applied on strips of Whatman No. | paper. The paper strips were run in an organic solvent mixture of n-butanol, water and acetic acid (60:20:20). The hydrolyzates of both the isolated , and the standard insulin were also applied separately along with the known amino acids (hydroxylysine, methione, hydroxyproline and trytophan). The various developed chromatograms were sprayed with 0.25% ninhydrin in acetone. The isolated : compound had 18 amino acids; glutamine is the extra amino acid in polypeptide-k. _

Hydroxylysine, hydroxy-proline and tryptophan were found to be absent from the hydrolyzate of the isolated polypeptide-p as well as of the standard. hydrolyzate which gave an indication that the isolated polypeptide-p is nearly identical with that of the insulin except glutamine being an extra-amino acid in polypeptide-k. Eighteen amino- acids were observed in polypeptide-k as compared to 17 amino acids in polypeptide-p.

Disc eletrophoresis was carried out (10% SDS Biophore Gel, run in tris buffer, operating pH 6:1, 3% acetic acid in lower cell; 90 V, mA 2.5 per tube; Bromophenol blue tracking dye). Samples of the crystallized isolate and bovine insulin containing dithiothreltol and EDTA, injected and run for 7 hr. Gel collected from the tubes were stained (0.05% coomassie Brilliant Blue R-250 in 7% aqueous acetic acid) and washed with 10% acetic acid. Electrophoretic pattern of both the isolate and the bovine insulin were nearly identical as shown in figure 1 of the: accompanying drawings. Immuno assays of popypeptide-p did not show any cross reaction when tested with bovine insulin. 10 AMENDED SHEET 2003 -05- 24

EK WO 00/61619 PUT/INYY100052

Sublingual administration of the isolate showed positive and highly effective hypoglycaemic activity. When five hundred diabetic patients were treated (Table 1) no side effects of the drug were observed. Neuropathy, lethargieity, hypoglycaemia were not reported in these patients even when the drug was administered for a period of 2-4 years. At the same time, sugar level in the blood come down appreciably in one month time. The results are shown in table I.

Example 2 (Extra ction of protein from Momordica charantia L.) 200 gms of dry seeds were taken from the ripe fruits of Momordica charantia L.

The seeds were spilt manually. The split seeds were then thoroughly washed with water 3-4 times to render them substantially free of all impurities. These seeds were then dried under vacuum and pulverized to a fine powder using a milling device. Any other conventional devices may also be used. - The fine powder was then treated with acetone hexane solvent mixed in the ratio 1:2. Thin glass plated (20 x 20 cm) coated (0.4 mm to 0.5 mm thick) with silica Co

NB gel G (Kieselgel G nach Stahl; E. Merck) were activated at 100°C for half an hour.

The solution containing the isolated substance was applied 1 cm above the edge of the plates were run in an organic solvent mixture of n-butanol, water and acetic acid (12:5:2). The developed plates were dried at room temperature and sprayed with 0.25% ninhydrin in acetone. The ninhydrin ‘positive spots (R = 0.19) of the isolate corresponding to insulin were collected from about 200 unsprayed plates along with the silica gel G and extracted with 50% ethanol! buffered with ammonium hydroxide. The extract was filtered and dried in vacuo. Pure colorless crystals thus obtained were weighed (3 g/100 gram dry weight of seeds).

The melting point of the purified compound (232° - 235°C) as well as the mp (234°C) were determined. The melting point of the standard insulin was recorded as 233°C. ; AMENDED SHEET. . 2003 -06- 24

The standard sample of insulin as well as the isolated polypeptide-p were hydrolyzed under reflux with 6N HCI for 20 hours separately. The hydrolyzates were filtered, dried, reconstituted separately in 50% ethanol and applied on Strips of Whatman No. 1 paper. The paper strips were run in an organic solvent mixture of n-butanol, water and acetic acid (60:20:20). The hydrolyzates of both the isolated and the standard insulin were also applied separately along with the known amino acids (hydroxylysine, methione, hydroxyproline and trytophan). The various developed chromatograms were sprayed with 0.25% ninhydrin in acetone. The amino acids of the hydrolyzate of the standard coincided exactly with those of the hydrolyzate of the isolated compound except methionine and glutamine being extra amino acid in isolated polypeptide-k. Hydroxylysine, hydroxy-proline and tryptophan were found to be absent from the “hydrolyzate of the isolated polypeptide-k as well as of the standard hydrolyzate which gave an indication that . the isolated polypeptide-k is nearly identical with that of the insulin. The polypeptide-k showed 18 amino acids. oo Amino acid 1 Moles/mg? Molecular number 0273 | 17 |" [Threonine [Tous | 87 [Serine 0793 0.305 (Proline 35 [0

Glycine 055 | 1g

Alanine 0550 [1s

Valine oa 11] [VaCysteine 058 0.031

Tsolewine 1 g11

Lene 3

Tyrosine 001g |]

Phenylalanine 0.082 5

Histidine | o.6

Lysine op 5 (Arginine 0061 | 0

Glutamine 0.182 TT 12

AMENDED SHEET 79p3 -06- 74

- PCT/IN9Y/00052

TOTAL residues

Approximate molecular weight 18000 : Disc eletrophoresis was carried out (10% SDS Biophore Gel, run in tris buffer, operating pH 6.1, 3% acetic acid in lower cell; 90 V, mA 2.5 per tube;

Bromophenol blue tracking dye). Samples of the crystallized isolate and bovine insulin containing dithiothreltol and EDTA, injected and run for 7 hr. Gel collected from the tubes were stained (0.05% coomassie Brilliant Blue R-250 in 79 aqueous acetic acid) and washed with 10% acetic acid. Electrophoretic pattern of both the isolate and the bovine insulin were nearly identical as shown in figure 1 of the accompanying drawings. Immuno assays of popypeptide-k did not show any cross reaction when tested with bovine insulin. ‘Below are representative examples of case studies, wherein patients have been administered the hypoglyceamic composition of the invention. All these patients were afflicted with diabetes mellitus, though the chronicity varied from case to case. The patients were advised to consume good quality food substantially free from starch, and containing at least 1 fruit. The patients were also advised exercise besides use of the composition of the invention. Most of the patients took drugs like Diaonil/ Glyciphate/ Glynase/ DBI/ Euglucon/ Dimicron or combination of 8lyciphage and glynase before commencement of the treatment.

Gradual fall in blood sugar level of the patients was observed after one week to 40 days and then it came to normal. Continued intake of the composition of the invention varied from 6 months to 3 years as four doses 10-15 minutes before each meal sublingually. In patients with blood sugar level from 355 or more diaoni] in doses of 2 (1+1) or 1 (% + /2) was supplemented with the dose of the composition (morning, evening). Diaoni] was withdrawn completely after 15 days. 13 «ENDED SHEET 2003 -06- 24

Case studies . I. Mr. Vivek Mukherjee. aged 18, complained of leg pain, excessive thirst for

N walter, frequent urination and blurring vision. Upon examination, his blood sugar

N 5 level was found to be 425 mg/dl as pp and fasting as 209 mg/dl. No oral drug worked effectively. The patient was advised to consume the hypoglycaemic composition of the invention (12 mg/dl/dose and 4 doses per day. Each dose was consumed 10 minutes before every meal without any liquid or solid) along with - : diaonil (1+1+1). This reduced the blood sugar level considerably. Diaonil was 10 reduced to half after one month and later withdrawn completely and surprisingly the blood sugar level was reduced to 114 mg/dl (pp) after 2 months. The patient is now completely dependant on the composition of the invention and is maintaining normal blood sugar level with no side effects. 3 2. Another patient, Prof. K.P. Mishra, aged 71 years, was a chronic diabetic with a blood sugar level of 305 mg/dl. He was on insulin (18 units/day). The patient was advised to consume the composition of the invention 4 times. He is maintaining normal blood sugar for the last two years. 3. Mr. D.P. Gaur, aged 60 years, was a chronic diabetic since last 10 years. He developed neuropathy and lethargicity in the body even on taking 60 units of insulin. There were symptoms of high blood pressure and high triglyceride levels.

This patient started taking the formulation of the invention 4 times a day alongwith the insulin which was reduced to Yy. The blood sugar level was reduced. The insuling dose was gradually reduced and finally withdrawn and now the patient is maintaining normal blood sugar level and keeping fit with 4 doses of formulations of invention. 4. Mrs. Rashmi Geha, 46 years old, developed symptoms, which on examination of blood profile confirmed (haemoglobin-glycosilation test) the diabetes (320 mg/dl). She started taking the powder of the invention and has been maintaining normal blood sugar levels She has not taken any other oral drug. a d WO 00/61619 PCT/IN99/00052 5. Mr. Banerjee. 70 years old, was a chronic patient (15-20 years) and with a high blood pressure. high triglyceride and cholesterol levels. He took the powder for 3 years, four doses per day of formulation of the invention. This kept normal blood sugar levels and controlled levels of blood pressure and cholesterol levels.

Tablel:Effect of Gourdin (polypeptide-k) on blood sugar level ‘in patients with diabetes mellitus.

No. of subjects Diabetes duration “Range of blood "Polypeptide-k (yrs.) | sugar level mg/dl | effect Mean mg/dl (post prantl) Sfall in blood sugar level (after 207 days). : 160-200 120-110 250 [610 210350 | 150090 100 [Ws 355450 | 250270

Ls [1520 | 460500 | am

The following are the advantages 1. The polypeptide-k was found to be more effective than polypeptide-p isolated by the process claimed in earlier patent no. 176040. The extraction procedure for polypeptide-k was improved and made more effective. 5 1 2. The polypeptide-k was highly effective hypoglyceamic drug when administered sublingually. . 3. The cholesterol level including total cholesterol, HDL, LDL, VLDL and triglyceride iE go down to normal using this drug as an anti-diabetic remedy. 4, Symptoms as leg pain, lethargy did not appear when more than 500 patients of 2-4 years of duration with this drug was given. This invention as described in the example is merely illustrative in nature and not intended to restrict the scope of the invention.

The origin of the hypoglycaemic composition and its effects : 1. It is the protein extract of "Karela" / Bittergourd / Momordica charantia L. 2. Combustion point /mp of Gourdin or polypeptide-k was found to be 232° C (£)2° C. 1s } .

AMENDED SHEET po oc,

Sap 3. When analyzed with amino acid analyzer the hydrolyzate showed 18 amino acids (Fig. 1). 4. A single electrophoretic band was observed which on scanning showed a single main peak of pure gourdin (Fig. 2).

OS. Bio-immunoassays of polypeptide-k were found to be negative against insulin. 6. Pharmacological study revealed a significant blood-sugar-lowering,. 7. Polypeptide-k is completely insoluble in water. 8. It physically can be tested with nin-hydrin which on heating the soluble fraction turned yellow in colour turning purple later. 9. On sequencing the polypeptide fraction, the first terminal was found to be free (Fig. 3). 10. The hypoglycaemic composition of the invention activates the inactive insulin present in the blood and hence, it cures the disease, the time factor _ depends on the chronicity of the illness. 11. If hereditary, a single dose by a normal person acts as preventive dose.

B 12... If cholestrol level is high, its intake reduces the level. 13. High Triglyceride level is also reduced. } EE 14. ) Pain and inflammation of the joints is either eliminated or reduced. oo 15." Its intake gives a feeling of normalcy to the diabetic patient. 16. No other side effects were observed. . 16

AMENDED SHEET 2003 -06- 24 oY

Claims: 1. A novel protein (polypeptide-k)extracted from Momordica charantia comprising amino acids including;

Amino acid u Moles/mg? Molecular number 0.138 0.195 0.159 [| 10 0.225 0.240 0.174 0.058 0.031 0.116 0.207 0.016

Phenylalanine 0.082 0.066 0.209 0.161

NH; 0.431

TOTAL

Aapproximate molecular weight 11000 2. A process for the extraction of a novel protein (polypeptide-k) from

Momordica charantia comprising the steps of: 1. grinding the dry seeds of Momordica charantia, il. treating the pulverized seeds with a mixture of non-polar solvents, iil. 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

V. treating the supernatant layer with sulfuric acid after adjusting the pH to 3, and vi. collecting the flocculent precipitate of polypeptide-k and isolating the protein by selective crystallization. 3. A process as claimed in claim 2 wherein the protein is extracted from the fruit = and/or seeds of Momordica charantia 4. A process as claimed in claim 2, wherein the 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 extraction of the protein. 5. A process as claimed in claim 2 wherein the non-polar solvents used comprise a mixture of acetone with an aromatic or aliphatic hydrocarbon selected from the group of benzene, hexane, petroleum ether and ethyl ether. 6. A process as claimed in claim 2 wherein the ratio of the aliphatic or aromatic hydrocarbon in the non-polar solvent mixture is 2:1. 7. Use of the protein extracted from Momordica charantia to manufacture a hypoglycemic composition useful in the treatment of diabetes mellitus.

Claims

vy AMENDED CLAIMS [received by the International Bureau on 13 June 2000 (13.06.00); original claims 1,3 and 7 amended; remaining claims unchanged (3 pages)]

2. A process for the extraction of a novel protein (polypeptide-k) from Momordica charantia comprising the steps of: 19 AMENDED SHEET (ARTICLE 19)

i. grinding the dry seeds of Momordica charantia, w ii. treating the pulverized seeds with a mixture of non-polar solvents,

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,

Vv. treating the supernatant layer with sulfuric acid after adjusting the pH to 3, and vi. collecting the flocculent precipitate of polypeptide-k and isolating the protein by selective crystallization.

3. A process as claimed in claim 2 wherein the protein is extracted from the fruit and seeds of Momordica charantia.

4. A process as claimed in claim 2, wherein the 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 extraction of the protein.

5. A process as claimed in claim 2 wherein the non-polar solvents used comprise a mixture of acetone with an aromatic or aliphatic hydrocarbon selected from the AMENDED SHEET (ARTICLE 19)

‘ of group of benzene, hexane, petroleum ether and ethyl ether.

6. A process as claimed in claim 2 wherein the ratio of the aliphatic or aromatic hydrocarbon in the non-polar solvent mixture is 2:1.

7. Use of the protein polypeptide-k extracted from Momordica charantia for curing or treating diabetes mellitus by sub-lingual administration of polypeptide- k to a patient. +21 AMENDED SHEET (ARTICLE 19)

he STATEMENT UNDER ARTICLE 19 Khanna P et al: "Hypoglycaemic activity polypeptide-p from a plant source" Journal of Natural products, xx xx Vol 44, pages 648-655. This citation is the inventor's own previous investigation and work. Inadvertently, at the time of filing, the details of polypeptide-p which is referred in the citation has been repeated in the claim. In fact, the Applicant has isolated a novel protein called "polypeptide-k". This polypeptide-k differs from polypeptide-p in the following respects:

1. The polypeptide-k has 18 amino acids whereas polypeptide-k has only 17 amino acids. The extra amino acid present in polypeptide-k is glutamine.

2. The approximate molecular weight of polypeptide-k is 18,000 units whereas the weight of polypeptide-p is 11,000.

3. Polypeptide-k has a free N-terminal whereas polypeptide-p has no such terminal.

4. Polypeptide-k is not water soluble whereas polypeptide-p is water soluble.

h WO 00/61619 PCT/IN99/00052

- 5. Polypeptide-k is not injectible to a patient and can be administered only through sub-lingual route whereas polypeptide-p is injectible to a patient. Polypeptide-k is a different protein than polypeptide-p. Polypeptide-k has been identified recently by the inventor and has different properties. It is possible that a plant may have different proteins of which the applicant has now isolated and characterized a novel protein named as polypeptide-k. The process claimed in claim 2 has not been envisaged in the citation. This process has been specifically devised to . extract polypeptide-k which is partially soluble at pH 9.5. The sequence of steps and the ingredients used in the . present invention are different from the process disclosed in the citation. The other citations are graded "A" as being relevant to the technological background of the invention.