Title of Invention

HIGHLY EFFECTIVE HYPOGLYCAEMIC POLYPEPTIDE-K FROM A PLANT SOURCE

Abstract

The invention relates to a novel and highly effective hypoglycemic protein called polypeptide-k, extracted from Momordica charantia, provides a method for the extraction of said polypeptide-k from Momordica charantia and provides a novel protein useful in the treatment of diabetes mellitus. A sublingually highly effective hypoglycaemic polypeptide-k, from seeds of Momordica Charantia L. (bitter guard), prepared by a process which comprises, splitting the seeds of Momordica Charantia L (bitter guard) washing thoroughly the splitted seeds with water to remove contaminants, treating the splitted seeds with solvent consisting of hexane and acetone (3:1), grinding the said seeds to obtain powder, treating the said powder of seeds with, hexane and acetone solvents (3:1), dissolving the residual mass in aqueous acetone (water and acetone 80:20), adjusting PH upto 9.5 by adding ammonium hydroxide, separating the supernatant layer from the mixture, treating the supernatant layer with sulphuric acid by adjusting the PH upto 3 to obtain the flocculent precipitate of polypeptide-k.

Full Text

PROTEIN/POLYPEPTIDE-K OBTAINED FROM MOMORDICA CHARANTIA AND A PROCESS FOR THE EXTRACTION THEREOF. 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. 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 cultur-s are separately crushed, homogenize4 in water, ethanol and concentrated sulphuric acid is added for adjusting pH to 3, thereby obtaining flocculent precipitate. This method had the following drawbacks: 1. The use of alcohol 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 hi 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 diethyiether 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' was extracted from the fruits and tissues of Momordica charantia. In the processes described 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 polypeptide-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 V 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 is to provide a novel protein called 'polypeptide-k1, from the dry seeds of Momordica charantia. Yet another object of the invention is to provide a hypoglycaemic protein called '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 seeds of Momordica charantia, from a process for the extraction of the said polypeptide-k and a novel hypoglycaemic protein. Detailed description of the invention Accordingly, the invention provides a sublingually highly effective hypoglycaemic polypeptide-k, from seeds of Momordica Charantia L. (bitter guard) prepared by a process which comprises, splitting the seeds of Momordica Charantia L (bitter guard) washing thoroughly the splitted seeds with water to remove contaminants, treating the splitted seeds with solvent consisting of hexane and acetone (3:1), grinding the said seeds to obtain powder, treating the said powder of seeds with, hexane and acetone solvents (3:1), dissolving the residual mass in aqueous acetone (water and acetone 80:20), adjusting PH upto 9.5 by adding ammonium hydroxide, separating the supernatant layer from the mixture, treating the supernatant layer with sulphuric acid by adjusting the PH upto 3 to obtain the flocculent precipitate of polypeptide-k. 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 caned '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: 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 sulphuric 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. In one embodiment, the dried seeds ofMomordica 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. In another embodiment, the solvents used for removal of the oils from the seeds comprise a mixture of hexane and acetone in the ratio of 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°e. The solution of insulin is applied, the plates developed in n-butanol, acetic acid water (12:5:2) are dried, and single spot nearly 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% ethanol buffered with ammonium hydroxide or 10% of formic acid, filtered, the filtrate dried and pure white needle-like crystals formed. hi yet another embodiment, when the analysis is carried out, the isolated substance is hydrolyzed along with the standard insulin, applied on paper chromatograms separately, developed, yielding 18 amino acids including glutamine. This isolated substance is a protein named as 'polypeptide-k'. hi 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. 1 filter paper strips developed in n-butanol, acetic acid, water (60:20:20), strips developed sprayed with 0.25% nin-hydrin in acetone. The standard hydrolyzate shows presence of 18 amino acids including glutamine. hi the analysis is carried out, the seeds are extracted in hexane acetone yielding a product which has a 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 most of the plant parts ofMomordica contain the protein disclosed by the invention, in varying degrees. As such, the protein polypeptide-k may be 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-k by removal of undesired oils, flavonoids and sapogenins there from. The Applicant has analyzed the peptide isolated from Momordica charahtia and found that this protein has 18 amino acids. The 18th amino acid is glutamine. The UV spectrum has shown absorption peak at 275nm whereas the peak for polypeptide-p, i.e. another protein found in Momordica charantiais noticed at 250nm. HPLC analyses for the protein polypeptide-'k show'S .a single peak. Thus, the mass spectrum analyses done for polypeptide-k discloses the fact that polypeptide-k consists of two peptide chains as opposed to a single chain present in polypeptide-p. Another notable feature is that polypeptide-p can be isolated from the fruits, fresh seeds and tissues ofMomordica charantia. Whereas, polypeptide-k is obtained from the dried seeds ofMomordica charantia as a storage protein. More importantly, the earlier isolated polypeptide-p has approximate molecular weight of 11,000 kd whereas polypeptide-k of the present invention has an approximate molecular weight of 18,000 kd. Thus, polypeptide-k differs from polypeptide-p in the following respects: 1. The polypeptide-k has 18 amino acids whereas polypeptide-p 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. 4. Polypeptide-k is not water-soluble whereas polypeptide-p is partially water- soluble. 5. Polypeptide-k is not injectible to a patient and can be administered orally only through sub-lingual route whereas polypeptide-p is injectible as described. In other words, polypeptide-p is injectible (intramuscular) and this is inconvenient to patients, however, polypeptide-k is taken sublingually from the above surface of the tongue and its administration and absorption is easy, acceptable and convenient to patients, 6. Polypeptide-k is stable and the life is about 18 months (kept at normal pressure and temperature). On the other hand, polypeptide-p is unstable and its life is hardly 2-3 months when kept at normal pressure and temperature. 7. Polypeptide-k has the combustion point (m.p) of 234°C, whereas polypeptide-p has early combustion point which starts at 228-232°C. 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 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 non-insulin-dependent diabetics (Type n diabetes). Type I diabetes is in the state of lowering of the function of pancreatic beta cells resulting from hereditary cause, viral infection obesity, drug effect, accident etc. wherein insulin is not efficiently secreted, and suddenly attacks mainly in the twenties to thirties. Although it is not sure, onset of type II diabetes hi the forties or in cases with family history of diabetes, obesity, stress, etc. In the case of type II 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 causes the empty stomach to intake excess 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 cycle causing 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 add up as complications. Thus, diabetic complications such as diabetic retinopathy (visual disturbance,: blindness, retinal hemorrhage), 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 noninsulin dependent diabetics. However, majority of these medicines have 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. Since it is very effective, relatively small amounts of the homeopathic medicine need be ingested in order to reduce the blood sugar level. Herbal compositions using the protein of the invention: The protein extracted from Momordica charantia exhibits hypoglyceamic properties and accordingly compositions comprising the protein can be used for the treatment of hypoglycemia 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 to 70 mg of the protein at a time is quite effective. Accordingly, it is, advisable that compositions containing the protein in -single dose should comprise about 12 mg or more of the protein. Hypoglyceamic compositions using the proteins of the invention can be formulated in a variety of physical forms such as tablets, edible products. For preparation of a tablet, about 12 mg to 70 mg of the protein is mixed with pharmacologically acceptable carriers suitable for consumption. The pharmacologically acceptable carrier must be of sufficient purity non-toxicity and should not interfere with the activity/efficacy of polypeptide-k. Edible products like biscuits, chewing gums, losenzes etc., which are not instantly swallowed, can be prepared. In all such preparations, the content of the protein is about 12 mg to 70 mg: It is found that low salt biscuits prepared using the protein of the invention ~ are very popular with diabetics. It is pertinent to note that the hypoglycemic 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 hypoglycemic composition should only be chewed and should not be swallowed instantaneously. In a feature of the invention, the hypoglycemic composition herein described has no side effects. It can be consumed without restricting the use of other therapies. It has no cross-reaction with insulin. Example (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 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 3:1 for de-oiling the powder and the residual mass was dissolved in 20% acetone. The pH was adjusted to 9.5 by adding- ammonium hydroxide, the supernatant thus obtained was buffered with H2S04 to adjust pH3 for obtaining flocculent precipitate that were collected and crystallised with zinc acetate used of traces. Thin glass plated (20 x 20 em) coated (O.4 mm to O.5 mm thick) with silica gd G (Kieselgel G nach Stahl; E. Merck) were activated at 1OO°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, acetic acid and water (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 nearly 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 /10% formic acid. 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 (234°C) were determined. The melting point of the standard insulin was recorded as 233°C. The standard samples of insulin as well as the isolated were hydrolyzed under reflux with 6N HCI for 20 hours separately. The hydrolyzates were filtered, dried, reconstituted separately in 50% ethanol or 10% formic acid and applied on strips of What man No.l paper. The paper strips were run in an organic solvent mixture of n-butanol, acetic acid and .water (3:1:1). The hydrolyzates of both the isolated and the standard insulin were also applied separately alongwith the known amino acids (including hydroxylysine, methionine, hydroxyproline and trytophan). The various developed chromatograms were sprayed with 0.25% nin-hydrin in acetone. The amino acids of the hydrolyzate of the standard coincided exactly with those of the hydrolyzate of the isolated compound except glutamine being an extra amino acid in isolated polypeptide-k. Hydroxylysine. hydroxyproline 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 protein is marked by the presence of glutamine. Disc electrophoresis was carried out (10% SDS Biophore Gel, run in Tris buffer, operating pH 6. 3% acetic acid hi lower cell: 90 V, mA 2.5 per tube, (Bromophenol blue tracking dye). Samples of the crystallized isolate and bovme in sulin in 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 I of the accompanying drawing. Irnmunoassays of polypeptide-k did not show any cross-reaction when tested with bovine insulin. Sublingual administration of the isolate showed positive and highly effective J-hypoglycemic activity. When five hundred diabetic patients were treated (Table 1) No side effects of the drug were observed. Neuropathy, lethargicity, 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 came down appreciably in one-month time. The results are shown in table 1. 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 10-1.5 minutes before each meal sublingually. In patients with blood sugar level from 355 or more Diaonil in doses of 2 (1 + 1) or 1 (½ +½) was supplemented with the dose of the composition (morning, evening). Diaonil was withdrawn completely after 15 days. Table: Effect of Gourdin (polypeptide-k) on blood sugar level in patients with diabetes mellitus. (Table Removed) The Applicant observed that the present novel Polypeptide-k is highly effective as compared to Polypeptide-p due to its stable nature. It works miracle when used along with Diaonil and it brings the high blood sugar level above 250 mg/dl or above to normal with in 2-3 days. It not only reduces the high blood sugar level but also controls the high blood pressure by controlling the total cholesterol, HDL, LDL, VLDL and triglycerides. It also takes care of neuropathy and makes the patient feel normal. Side effects in diabetics are taken care of by polypeptide-k. It also increases the immunity in the patients against the disease- and hence is helping HIV patients also. It has no cross-reaction with insulin. In many patients, the insulin has been withdrawn gradually. Many patients suffering from diabetics when treated with polypeptide-k showed excellent results in the age group of 50-60 year or above. The patients in the above age group feel normal and full of health. The blood sugar level in various cases has shown 50-55%, lowering effect after administration of polypeptide-k and when compared with polypeptide-p, such effect was observed to be 25-30% and that too was fluctuating. In fact, no patient complained of any side effects when treated with polypeptide-k. Diaonil combinations with polypeptide-k have shown excellent results. In some cases, the doses of polypeptide-k have to be reduced from 4-3-2 showing that the pancreases in the long run get rejuvenated and hence, it can be concluded that polypeptide-k activates the inactive insulin present in the blood. After all insulin is an enzyme and attachment of a small peptide of polypeptide-k at any point can make the insulin activated. When the other blood tests were performed, the creatinine, uric acid and blood urea levels were found to be normal in diabetic patients treated with polypeptide-k. Now, a larger population is responding positively internationally. The most important property of polypeptide-k is that it brings the pH of the body (blood) to 6. If one is feeling giddy or throwing bile, one tablet can regulate the acidity or alkalinity respectively to normal level pH 6 and the person feels just normal and healthy and thus it can control the functioning of liver as well. By controlling the neuropathy the patient has no nerve problems. Being from a vegetable source it is easily acceptable. In case of polypeptide-p such a response has not been observed because of the unstable condition of the protein. 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. 2. Polypeptide-k was found to be highly sublingually effective hypoglycemic drug. 3. The cholesterol level including total cholesterol, HDL, LDL, VLDL and triglyceride go down to normal using this drug as an antidiabetic remedy. 4. Symptoms as leg pain, lethargicity did not appear when more than 500 patients of 24 years of duration with this drug were treated. 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 hypoglycemic composition and its effects 1. It is the protein extract of "Karela"/ Bittergourd/ Momordica charantia L. 2. Combustion point/ mp of Gourdin was found to be 234°C. 3. When analyzed with amino acid analyzer the hydrolyzate showed 18 amino acids. 4. A single electrophoretic band was observed which on scanning showed a single main peak of pure Gourdin. 5. 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 insoluble in water and partially soluble at pH 9.5 and fully in 10% formic acid. 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-k fraction, the first terminal was found to be free. 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. 1 1 If hereditary, a single dose by a normal person acts as preventive dose. 12. If cholesterol level is high, its intake reduces the level. 13. High Triglyceride level is also reduced. 14. Pain and inflammation of the joints is either eliminated or reduced. 15. Its intake gives a feeling of normalcy to the diabetic patient. 16. No other side effects were observed. CLAIM 1. A sublingually highly effective hypoglycaemic polypeptide-k, from seeds of v Momordica Charantia L. (bitter guard), prepared by a process which comprises, splitting the seeds of Momordica Charantia L (bitter guard) washing thoroughly the splitted seeds with water to remove contaminants, treating the splitted seeds with solvent consisting of hexane and acetone (3:1), grinding the said seeds to obtain powder, treating the said powder of seeds with, hexane and acetone solvents (3:1), dissolving the residual mass in aqueous acetone (water and acetone 80:20), adjusting PH upto 9.5 by adding ammonium hydroxide, separating the supernatant kyer from the mixture, treating the supernatant layer with sulphuric acid by adjusting the PH upto 3 to obtain the flocculent precipitate of polypeptide-k. 2. A hypoglycaemic polypeptide-k as claimed in claim 1 where the peeled seeds treated dried at room temperature before grinding. 3. A hypoglycaemic polypeptide-k as claimed in claim 1 and 2 wherein the residual mass is preferably dissolved in 80 % of acetone. 4. A hypoglycaemic polypeptide-k as claimed in any of preceding claim wherein the supernatant layer in treated with 6 N sulphuric acid. 5. A sublingually highly effective hypoglycaemic polypeptide-k substantially as herein before described with reference to any of the forgoing examples.

Documents:

561-del-1999-abstract.pdf

561-del-1999-claims.pdf

561-del-1999-correspondence-others.pdf

561-del-1999-correspondence-po.pdf

561-del-1999-description (complete).pdf

561-del-1999-drawings.pdf

561-del-1999-form-1.pdf

561-del-1999-form-18.pdf

561-del-1999-form-2.pdf

561-del-1999-form-3.pdf

561-del-1999-pct-409.pdf

561-del-1999-petition-138.pdf