INTORDUCTION

Hypoglycaemic property of fruits of Momordica Charantia L. (Bitter gourd) has extensively been reviewed and debated (Raman and Lau, 1996)1. A polypeptide-p extracted from fruits and tissue culture of M. Charantia has been reported (Khanna, et al. 19812, Baldwa, et, al, 19773, Khanna, 1985)4. The present study on isolation, identification and anti diabetic property of a novel protein (Polypeptide-k) has been carried out and discussed.

SUMMARY

Polypeptide-k (Gourdin - Trade Name) extracted from seeds of Momordica Charantia L. (Bitter Gourd) is soluble in 10% formic acid. On hydrolysis it shows 18000 mol. wt containing 19 amino acids. A total of 35 amino acids are arranged in four cycles in   interchangeable positions at 12, 13, 15-19, 25-27 and 31-34. Mass elctropectra did not show clear peaks due to noise but gave clear indications of two separate molecules with masses as 11474.32 + 8.77 and 11708.57 + 6.30. The MS/MS search after trypsin digest sample of polypeptide-k showed 229 peptides with no matching with any known NCB nr database.  However, Edman sequencing did show one excellent matching peptide with Cucurbita maxima and another with a mixture of known sequence of this species.

The shelf life of this protein was calculated as 2 years. No toxicity was observed even at doses used (60 mg/Tab). A significant blood sugar lowering effect was observed on clinical trials on diabetic patients.

The polypeptide-k has been patented in India, China, Malaysia, Indonesia, Japan, Australia, Canada, S. Africa and USA after obtaining a search report from PCT. The product has been launched in China, and Malaysia and MOU signed with Japanese company.

METHOD

The dried split seeds of Momordica Charantia L. were washed, dried, powdered, and deoiled thoroughly with hexane (cold extraction). The dried powder so obtained was dissolved in water and this mixture was adjusted to pH 9.5. The upper layer was decanted off and the protein was precipitated at pH. 3 with sulfuric acid. The precipitate thus formed was collected dried, powered and washed with acetone / ethanol again and again till the upper layer was clear of its turbidity. The protein (Polypeptide-K) thus collected was tested for its 100% purity and then subjected to various analytical tests.

OBSERVATION

The polypeptide-k was subjected to HPTLC, which showed a single peak after scanning at wavelength of 254 nm.  (Fig. 1). The protein is insoluble in water but completely soluble in 10% formic acid. On hydrolysis it was found to contain 19 different amino acids with 160 residues and a mol. wt. of 18000. Tryptophan and cystine residues could not be determined (Table 1) ⁵. On further analysis 35 amino acids were found arranged in four cycles with interchangeable amino acids at positions 12, 13, 15-19, 25-27 and 31-34. Mixture of sequences homologous to various legumin and Prunes proteins (Table 2) ⁶ were observed Amino acid mass concentrations of one sample was also determined (Table 3).

Electrospray mass spectra of the sample allowed the detections of two different compounds with molecular masses at 11474.32 + 8.77 and 11708.57 + 6.30 with a complex mixture from which it was difficult to distinguish peaks ((Fig. 2). A. B). When MS/MS ion search on trypsin digest of polypeptide-k was carried out after variable modifications as oxidation with propionamide on ESI-QUAD-TOF instrument, two hundred twenty nine peptides were observed after calculating the probability based on more than 51 Mowse auto score hits out of which only two significant hits resembling fragments of Wolinella succinogenes and Pau troglodytes were observed when Mascot search was carried out (Table 4, 1A-9A). The remaining peptides did not match with any of the known peptides as shown in the Database.

However, amino acid Edman sequencing (Fig. 3) results from peak 81 of polypeptide-k trypsin digest sample (run 2 H P 017) showed very strong sequence (Table. 5, 6) which matched very well with 11S globulin subunit beta precursor from Cucurbita maxima (accession #: P 13744) in the NCBI nr database residues 186-196. The predicted mass (1303.45 Da) was in excellent agreement with the mass observed (1303.8 Da) in the MALDI data. There was a tryptic cleavage site in the known sequence prior to the first residue of this peptide, so a trypsin digest of that protein would generate this peptide. Major mass at 1479.91 and 2101.97 in sample 2HP017-113 and 2HP017 - 126 respectively were observed but these were either unsuitable for Edman sequencing  due to a mixture (Table 7) or did not match with any of the known NCBI nr Database    (Table 8).

The polypeptide-k was tested for heavy metals, total aerobic microbial count (E. Coli and Salmonella sp), hexane and acetone (Gas Chromatography) before formulation and manufacturing into Tablets before animal and clinical trials.       

Animal and Clinical Trials

Toxicity tests were carried out in mice using five times the human dose (60 mg/dose) orally following the method of IP-85 (Chloramphenicol Palmitale Drug and Cosmetic Act, 1940). None of the mice showed undue or abnormal toxicity even after 72 hrs. (Table 9). Shelf life of polypeptide-k was also determined which came to be two years (Table 10).

Clinical trials were carried out in thousands of confirmed diabetic patients from whom only 100 diabetic patients were selected in this study who were on test trials for a long period  (Table 11). Each formulation (60 mg polypeptide-k + 50 mg potato starch / tablet) was administered through tongue four times per day (each dose of one tablet) 10 min. before every meal. Inhaling of the Polypeptide-k was also very effective in lowering the blood sugar level. No side effect has been reported except in rare cases where the patient was allergic to bitter gourd vegetable also allergic to polypeptide-k. Small Scale Industry has been installed in India.

A Juvenile diabetic patient (blood sugar level 550mg/dl) working in the unit for the last 6 years is now having considerably reduced blood sugar level (128 mg/dl as PP and fasting as 90 mg/dl). He has been feeling normal and the dose of Polypeptide-k is reduced from four to two.

Patents of polypeptide-k have been obtained from India 7, South Africa 8, U.S.A. 9, Australia 10, China 11, Japan 12, Canada 14, and Indonesia 15 after getting search report from PCT 16. and European Union 17 . The Patent from Malaysia is awaited. The product has been launched in China and Malaysia and is being formulated and sold in Japan as well. The product is registered for sale in some parts of South East Asia. The polypeptide-k is also being sold world wide on Internet. A feedback from some of the selective patients is also given (Table 12).Toxicity tests were carried out in mice using five times the human dose (60 mg/dose) orally following the method of IP-85 (Chloramphenicol Palmitale Drug and Cosmetic Act, 1940). None of the mice showed undue or abnormal toxicity even after 72 hrs. (Table 9). Shelf life of polypeptide-k was also determined which came to be two years (Table 10).

Clinical trials were carried out in thousands of confirmed diabetic patients from whom only 100 diabetic patients were selected in this study who were on test trials for a long period  (Table 11). Each formulation (60 mg polypeptide-k + 50 mg potato starch / tablet) was administered through tongue four times per day (each dose of one tablet) 10 min. before every meal. Inhaling of the Polypeptide-k was also very effective in lowering the blood sugar level. No side effect has been reported except in rare cases where the patient was allergic to bitter gourd vegetable also allergic to polypeptide-k. Small Scale Industry has been installed in India.

A Juvenile diabetic patient (blood sugar level 550mg/dl) working in the unit for the last 6 years is now having considerably reduced blood sugar level (128 mg/dl as PP and fasting as 90 mg/dl). He has been feeling normal and the dose of Polypeptide-k is reduced from four to two.

Patents of polypeptide-k have been obtained from India 7, South Africa 8, U.S.A. 9, Australia 10, China 11, Japan 12, Canada 14, and Indonesia 15 after getting search report from PCT 16. and European Union 17 . The Patent from Malaysia is awaited. The product has been launched in China and Malaysia and is being formulated and sold in Japan as well. The product is registered for sale in some parts of South East Asia. The polypeptide-k is also being sold world wide on Internet.

DISCUSSION

Polypeptide-k has interchangeable/floating amino acids at positions 12, 13, 15-19, 25-27 and 31-34. The human insulin secreted by pancrease become inactive due to chemical effects, obesity, accident or genetic causes and thus the disease Diabetes Millitus is caused. According to my hypothesis the Polypeptide-k is broken in to smaller peptides when taken by tongue and absorbed which goes into the blood stream. The damaged insulin is repaired in the blood by peptides having free floating amino acids or active peptide through receptors/acceptors at the inactive site making the human insulin active. This rejuvenating effect is supported by the following facts:

• The patients who are given chemical drugs/insulin, this requirement is reduced when Polypeptide-k doses are administered and ultimately withdrawn completely after some time, the time factor depends on the chronicity of the disease.
• The intake of four doses of Polypeptide-k is reduced from four doses to two doses in the patients who have the disease due to obesity, chemical or drug effect or accidents. In some such cases it comes to one and ultimately to nil. But those who have genetic causes, intake continues and the repairement sometimes reduces the doses.
•  Polypeptide-k intake creates a feeling of normalcy with no side effects. It also increases the immunity in the patients, which is caused due to side effects of the disease. It also acts as a preventive in those persons who have hereditary history of this disease. The patients who are allergic to bitter gourd vegetable should not take polypeptide-k. However such cases are rare.



CONCLUSION

The number of traditional medicinal plants is enormous in tropics. Many of them are known for their medicinal uses and have yielded medicines being used in various ailments. There are many plants, which are known for their wholesome uses and have sustained our lives for centuries but many generic drugs have been isolated. In some instances, we have isolated the active principle but economically they are not viable. I suggest that more work should be done with a team of scientists belonging to Botany, biochemistry, pharmacology and medical doctors so that we in true sense can serve the suffering humanity of dreaded disease like Cancer, HIV etc., with co-ordination of different groups the time factor which normally takes 16 years of a new product can be reduced. The benefit of plant products if effective have no side effects in many instances, as they are natural and can fit in well in the biosynthetic human system biochemical pathways. The Mascot search in the present study has shown two peptides matching with Cucurbita maxima confirming thereby the database search NBCI nr.   The product study clearly concludes an antidiabetic natural product with hardly any side effects (except in those patients who are allergic to bitter gourd vegetable).

Patents for Polypeptide-k have been obtained after search through PCT from India, S. Africa, U.S.A., Australia, China, Malaysia, Japan, Canada  and   Indonesia  whereas patent in European Union  is pending. The Technology has been transferred or MOU signed with China, Malaysia and Japan. The product has been launched in India, China and Malaysia. The product is being manufactured in India, China and Malaysia and is being sold in Japan as a 'Health Product'.

ACKNOWLEDGEMENT

I thank Dr. Pravesh Kumar, Professor, South California University, U.S.A. for his help. My grateful thanks are to Director, Smithonian Institute, HHMI Biopolymer/W. M. Keck Foundation, Biotechnology Resource Laboratory, Yale University, New Haven, Con; U.S.A. for help in analysis. My thanks are also to Arbro Pharmaceuticals Ltd., New Delhi India, for carrying out some of the tests.

GOURDIN ( POLYPEPTIDE -K )

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