Nov-Dec 1981 Journal of Natural Products Vol.44, No.6, pp.65

by Pesupa Khanna, S. C. Jain, A. Pangaria, V. P. Dixit
Insulin used in the treatment of diabetes mellitus has usually been obtained in very low yield from animal pancreas, i.e…

Hypoglycemic Activity of Polypeptide-p from A plant Source

Author: Pesupa Khanna, S. C. Jain, A. Pangaria, V. P. Dixit

Type of Publication: Pre-Clinical

Date of Publication: Nov-Dec 1981

Publication: Journal of Natural Products Vol.44, No.6, pp.65, Nov-Dec 1981

Organization: University of Rajasthan

Abstract: A hypoglycemic peptide, Ploypeptide-p, has been isolated from fruit, seeds, and tissue of Momordica charantia Linn (bitter gourd). Amino acid analysis indicates a minimum molecular weight of approximately 11,000 (166 residues). Polypeptide-p is a very effective hypoglycemic agent when administered subcutaneously to gerbils, langurs, and humans.

Insulin used in the treatment of diabetes mellitus has usually been obtained in very low yield from animal pancreas, i.e., one pound of pure insulin per 10,000 animals. Side effects of the animal insulin are well known. Recently, insulin has been synthesized by genetic manipulation in Escherichia coli, which is a significant scientific achievement.

A number of indedigenous drugs have been tried in the past for the treatment of diabetes mellitus. In the tropical world, fruits of Momordica charantia (biiter gourd) have been successfully used by diabetic patients; crude extracts have shown hypoglycaemic activity in rabbits (1-3). Khanna et al. (4,50 were able to isolate an active principle earlier called p-insulin of v-insulin from fruits, seeds and tissue culture of this plant species (6,7). When administered subcutaneously to human patients, v-insulin showed a significant blood sugar lowering effect (6).

MATERIALS AND METHODS

TISSUE CULTURE

Seed coats of Momordica charantia were removed, and the seeds were inoculated on revised Murashinge and Skoog’s medium (8,9) supplemented with 1 ppm of 2,4-dichlorophenoxyacetic acid (2,4-1) and 1% agar. The seeds took 5-6 days to germinate and form seedlings. Organized tissue was established (10) from the seedlings and maintained for 12-18 months by frequent subculturing of 6-8 weeks in fresh RT medium. This tissue was harvested after the transfer age of 6 weeks and extracted for its polypeptide-p content.

Fruits, soaked seeds, and tissue samples (100 g each) were crushed separately and then frozen. Each of the frozen samples was dissolved in 10 ml of distilled water, 45 ml of 955 ethanol and 3.6 ml of sulfuric acid 99.5%). The mixture was stirred vigorously (5,11) for 15-20 min at 25-28⁰ and then homogenized by the addition of 60 ml distilled water and 250 ml of 95% ethanol separately. After each of the mixtures was filtered enough ammonium hydroxide (28%) was added to adjust the pH of the filtrate to 3. To each of the filtrates, 1.5 liters of acetone was added till a while flocculent precipitate was formed. These mixtures were kept at 5⁰ for 8-10 hour.

The supernatant from each of the containers was decanted off, and the precipitate was dialyzed in a dialysis membrane (36 DIABETES MELLITUS, Union Carbide Corporation, Chicago, U.S.A.; molecular weight cut-off was 6000): distilled water was usd to removed the last traces of salt and other dialyzable impurities until the outside water gave a negative test with barium carbonate. The non-dialyzed fraction was collected, dried and crystallized in a 0.0001% solution of zinc acetate in water (12). The excess was removed by washing with ethylenediame tetracetic acid (EDTA) solution.

Crsytallized metrial was applied to silica gel coated and activated glass plates along with a standard sample of bovine insulin. The plates were deloped in a solvent mixture of n-butanol acetic acid-water 12:5:2. When the plates were sprayed with ninhydrin (0.25% in acetone) and heated, a single yellow spot (Rf 0.19), which nearly coincided with that of the standard sample of bovine insulin, was observed.

Disc electrophoresis was carried out (10% SDS Biophore Gel, run in tris buffer, operating pH 6.1; 3% acetic acid in lower cell; 90 V, 2.5 per tube; Bromophenol blue tracking dye). Samples of the crystallized isolate and bovine insulin were separately with SDS biophore buffer containing dithiothreitol EDTA, injected, and run for 7 hour. Gels collected from the tubes were stained (0.05% Coomassie Brilliant Blue R-250 in 7% aqueous acetic acid) and washed with 10% acetic acid.

The isolate (25 mg) from each of the samples was hydrolyzed with 6N HCI at 100⁰ for 24 hour and filtered. The filtrate was dried and the residue taken up in 50% ethanol. Two dimensional tlc was carried out (silica gel C; solvent system first: n-butanol-acetic acid-water, 5:1:1; secondly phenol saturated with water; 0.25% ninhydrin in acetone as spraying reagent), and seventeen amino acids were resolved. The isolates were also run in an automatic amino acid analyzer separately (Table 1).

A derivative of the crystallized material (polypeptide-p ZnCI₂) was prepared in the same manner (12) as bovine insulin-ZnCI₂. Doses of polypertide-p and polypeptide-p ZNCI₂ inn 0.9% NaCI as the vehicle were prepared (1.8 mg/ml equivalent to 40 units) as used in the case of bovine insulin. Immunoassays also carried out.

PHARMACOLOGICAL TRIALS

Pharmacological studies involved Meriones Hurianne Jerdon (gerbils), males and females, and Presbytis entillus entillus Dufsne (male langurs). 105 gerbils weighing 63-7 g were used in the present work. The animals were divided into groups of five each and all were tested for 12 hour before the beginning of the experiment. These animals were provided with water and libitum. Polypeptide-p-ZnCI₂ (0.5 unit/kg in 0.9% NaCI) was administered subcutaneously. Thirty-five animals were injected with an equal amount og 0.9% saline vehicle. The blood samples were obtained, through eardias, and the total blood sugar was estimated (13) at different time intervals (0, ½,1,2,4,8,12 hour). These results were compared with those of the vehicle-treated controls and statistically analyzed (14; Table 2).

A total of six healthy adult male langurs of different age groups with large eannines, a well developed pinkish oedematous band, and the sexual skin on the rump were used as experimental non-human primate models. The animals were fed with wheat chapaty (unleavened bread), banana, onion, carrot, potatoes, and soaked Bengal grams were provide with water ad libitum. Continous veterinary supervision was maintained.

Polypeptide-p-ZnCI₂ (0.5 unit/kg in saline) was administered subcutaneously. Fasting blood sugar samples of each of the animals were taken before any dose of drug was given. Blood samples were taken at different time intervals. As shown in Table 3. Food was given after 4 hour blood samples were taken. An equal number of male langurs were kept fasting and injected with saline (0.9% NaCI in water); their blood sugar samples were taken according to the schedule in Table 3.

CLINICAL TRIALS

A total of nineteen patients (15 males and 4 females) suffering from primary idiopathic 915) diabetes mellitus (15-56 hour age group) for a period of three months to eight years were selected for clinical trials. Out of the nineteen patients selected 11 cases were of juvenile diabetes and 8 were of maturity onset diabetes. Diabetic patients suffering from ketoacidosis, cerebrovascular accidents, acute myocardial infarction and renal failure were excluded from this study.

All patients were admitted to medical wards of S.m.S. Hospital, Jaipur, 4-5 days prior to the commencement of the study. Long-lasting insulin was withdrawn from patients 72 hour prior to the test, and plain insulin was withdrawn 12-18 hour before the test. Oral hypoglycemics were withdrawn 48 hour preceding the study. A blood sugar sample after the overnight fast was taken at 7 a.m. Polypeptide-p- preparations in saline solution was administered subcutaneously in a dose depending on the severity of diabetes mellitus (less than 180 mg/100 ml blood sugar level, 10 units; 180 250 mg/ml blood sugar level, 20 units; 250 mg/ml of blood sugar or above, 30 units).

After administration of the polypeptide-p preparation, the first three samples were taken at half-hour intervals to record the onset of the hypoglycemic effect. Subsequent samples were taken at different time interval, as shown in Table 4, to show the peak effect and duration of the action of this polypeptide. The blood samples were withdrawn from the medial cubital vein. The subjects were kept fasting during the study: only plain lemon water was given, if desired by the patients. Supervision was maintained for administration of glucose upon development of hypoglycemic symptoms. Blood sugar determination were performed by the method of Nelson-Somogyi (16).

The control group consisted of eight of the original nineteen patients with diabetes mellitus. Control blood samples were withdrawn at the same time intervals without the polypeptide-p being administered (Table 4). Polypeptide-p-ZnCI₂ was administered s.c. to three juvenile patients. These patients required smaller doses of this drug than on bovine insulin.

RESULTS

A single electrophoretic band of dialyzed and crystallized substance (Rf 0.41) was observed which, however, did not coincide (Rf 0.47) with that of bovine insulin (Fig.1). On scanning, a single main peak (Rf 0.41) of pure polypeptide-p was observed (Fig.2).

Two-dimensional tlc and the amino acid analysis (automatic amino acid analyzer) of the polypeptide-p hydroyzate showed 17 amino acids with a total of 166 residues and a minimum molecular weight of approximately 11000 (Table 1). Methionine was the extra amino acid observed in the unknown samples when compared with that of the known bovine insulin. Bio-immunoassays of this polypeptide were found to be negative against bovine insulin.

The pharmacological study revealed that the polypeptide-p-ZnCI₂ was long acting in gerbis and langurs and showed a significant blood-sugar-lowering effect (Table 2,3).

Clinical trials showed a hypoglycemic effect of polypeptide-p in juvenile and maturity-onset diabetic patients (Table 4). The peak effect in the juvenile diabetic may be between 4-8 hour as compared with 2 hour for crystalline bovine insulin. The peak response in maturity-onset diabetics as readily determined as in juvenile diabetics (Table 4).

No complaints of any side effects followed administration of polypeptide-p-ZnCl₂ to the three juvenile patients. One juvenile patients who expressed frequent heaviness of the head, a swollen face, apin in the stomch, and recurrent episodes of hypoglycemia when kept on crystalline bovine insulin was free of the these side effects when maintained continuously on polypeptide-p-ZnCI₂ for a period of five months. Immunoassays did not show cross reaction wheb tested with bovine insulin.

DISCUSSION

No apparent side effects were observed when the p-insulin was screened in diabetic patients. Bovine insulin so far is the only remedy against diabetes mellitus. With these new data, a new horizon in the treatment of diabetes mellitus. With these new data, a new horizon in the treatment of diabetes mellitus amy have been opened. Since the active principles is from a plant source, it is likely to be antigenic. More clinical trials of action, antigenecity, and various effects of intermediary metabolism in human beings are in progress and shall be reported later.

ACKNOWLEDGEMENTS

We gratefully acknowledge the financial support from the Indian Council of Medical Research, New Delhi, and the Department of Science and Technology, New Delhi, for carrying of Eli Lilly & Company, Indianapolis, Indiana, U.S.A., for the help extended for further confirmation of this polypeptide.

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