cology, S. N. Medical College, Agra,

INDIGENOUS DRUGS IN EXPERI- MENTAL DIABETES By M. L. GUJRAL N. K. CHOWDHURY* and R. S. SRIVASTAVA (From the Department of Pharmacology, Lucknow University, Lucknow) The number of indigenous drugs used by Vaids and Hakims for the treatment of diabetes in this country is very large. Out of a large number of drugs mentioned in the indigenous * The present address is Department cology, S. N. Medical College, Agra, of Pharma-

142 THE TNDTAN MEDICAL GAZETTE [March, 1954 literature only study. a few were screened in this The first part of our study consisted of deter- mination of normal blood sugar levels in rabbits fed on a standard diet (2 oz. of gram supplemented with green grass). The second part was concerned with the production of experimental diabetes in rabbits. In the third or last part of the study, the effect of these drugs on the blood sugar, urinary sugar and weight was investigated. Normal blood sugar in the rabbit The rabbits selected for the experiments were all males weighing between 1 Kg. and 2 Kg. The marginal vein of the ear was used for taking blood samples. The hair was plucked from the part and the part was properly cleaned. 0.1 ml. of the venous blood was drawn in a tuberculin syringe containing a small amount of sodium fluoride powder to prevent coagulation, impede the enzymatic action and thus prevent the destruction of monosaccharide in the blood. Sodium fluoride powder and not solution was used to prevent any chance in the volume of the blood drawn. The blood sugar was esti- mated according method. Reagents: to the modified colorimetric (?) Cupric sulphate 5 per cent. Crystalline cupric sulphate (50 gm.) was dissolved in distilled water with the aid of heat. The solution was cooled and one drop of con- centrated sulphuric acid was added. It Avas then transferred to a liter volumetric flask with rinsing, diluted to volume with distilled water and mixed. (b) Alkaline tartarate solution Anhydrous sodium carbonate (36 gm.) was dissolved in approximately 400 ml. of distilled water with the aid of heat, 13 gm. of sodium tartarate were added and mixed until dissolved. The solution was transferred to a liter volume- tric flask with rinsings, diluted to volume with distilled water and mixed. On the day of the analysis, a 50 ml. volumetric flask was half filled with solution 'B' and 5 ml. of solution 'A' were added to it. volume with 'B ' and mixed. This was then diluted to (c) Acid molybdate Reagent, Purified. A stock solution of brominated sodium molybdate was prepared by dissolving 300 gm. of sodium molybdate in distilled water in an Erlenmyer flask and 2-3 drops of liquid bromine were added. This was then transferred to a liter volumetric flask with rinsings, diluted to volume with distilled water and mixed. Of this clear, supernatant, brominated sodium molybdate solution, 500 ml. were transferred to an Erlenmyer flask and 225 ml. of 85 per cent Phosphoric acid added. The solution became yellow, because of liberated bromine. Cooled 25 per cent, sulphuric acid, 150 ml., was added and the mixture aerated to remove the bromine. 75 ml. of glacial acetic acid were then added and mixed. The solution was transferred to a liter volumetric flask with rinsings, diluted to volume with distilled water and mixed thoroughly. (d) Acid molybdate solution, Dilute. 20 ml. of purified acid molybdate reagent were added to a 100 ml. of volumetric flask, diluted to volume with distilled water and mixed. Method Normal Blood Sugar.?3.5 ml. of distilled water were transferred to a centrifuge tube, 0.1 ml. of blood (from the ear) was obtained in a tuberculin syringe, and this blood was dis- charged into the lowermost portion of the solution in the centrifuge tube, rinsing the syringe three times with the clear upper fluid. 0.2 ml. of 10 per cent zinc sulphate was added, mixed thoroughly and centrifuged at 2,500 r.p.m. for five minutes. 2 ml. of the clear supernatant fluid were transferred to a Folin sugar tube graduated at 12.5 ml. 2 ml. of distilled water were trans- ferred to a 'second Folin tube for the preparation of the blank. To each tube 2 ml. of freshly prepared alkaline copper tartarate were added. The tubes were then mixed by lateral tapping and heated in an actively boiling water bath for eight minutes. The Folin tubes were cooled in running water. To each 4 ml. of acid molybdate reagent were added. They were kept for one minute to allow the escape of most of the carbon dioxide. Each solution was diluted to 12.5 ml. with diluted acid molybdate solution. The tubes were then stoppered with clean rubber stoppers and mixed by inversion. The stoppers were carefully removed and the Folin tubes were

March, 1954] INDIGENOUS DRUGS IN DIABETES : GUJRAL & OTHERS 143 allowed to stand for twelve minutes. The solution was then transferred to 5 ml. absorption cell and readings were taken with Hellige? Diller Bio-Photo Colorimeter using filter B. The corresponding values for the metre readingobtained were determined from the 'glucose and metre reading' table which were previously made with known concentrations of glucose. The value of the blank was subtracted from that of the unknown, for mg. glucose per ICO ml. of whole blood. A catheter specimen of urine was drawn from each animal and was tested for the presence of sugar and acctone bodies. The normal range of blood sugar was found to be from 90 to 108 mg. per cent, with standard deviation (s) equal to, =, 4.4, as shown in table no. 1. The blood sugar estimation was made after keeping the animal for a week on the standard diet. Three readings on alternate days were made for each animal. As Eugenea jambolana is reported in indigenous literature to be a useful antidiabetic agent, a suspension of powdered seed in water was administered orally to three rabbits and blood sugar estimations were again made. I'he dose used was 720 mg./kg. daily, divided inio the equal doses and was given morning and evening. This dose is much higher than the therapeutic dose recommended. No measurable effect on the blood sugar level was produced by the powdered Eugenea jambolana seeds after administration over a period of one week. Production of alloxan diabetes.?alloxan dia- betes was produced in methods. the rabbit by two (A) In the acute method in which a -single dose of alloxan was injected intravenously into the marginal vein of the ear, the dose used m the first experiment was 400 mg./kg. Three animals were used. All of them died within 24 hours due to convulsions. In the second series of six rabbits 200 mg./kg. dose was used. All the animals with the exception of one died within a period of 24 to 48 hours. The blood sugar of the surviving animal after 72 hours was found to be 386 mg. per cent. This rabbit lost rapidly in weight and within three days the weight came down from 1.46 to 1.26 Kg. The animal was treated with Eugenea jambolana seed powder as described above, but in spite of treatment its weight continued to decline. The animal died on the 8th day after the injection of alloxan i.e. 5th day after the treatment was instituted. During this time its weight had come down to 1.03 Kg. and the blood sugar was unchanged. In the third set of six rabbits, single intravenous dose of 100 mg./kg. was administered and after 72 hours the blood sugar was estimated and found to range from 260 mg. to 350 mg. per cent. They all recovered in sugar content and after a week they came to the normal. The acute method of production of diabetes was, therefore, regarded as unsatisfactory and resort was made to the second method. (B) Chronic diabetes was produced by re- peated intravenous as well as intraperitoneal injections of small dose of alloxan. In this method 100 mg./kg. alloxan was injected on the first day and 50 mg./kg. daily for two subsequent days by intravenous route. Simultaneously every day 100 mg./kg. alloxan was also given intraperitoneally on three subsequent days. The rabbits were fed orally each day with 20 gm. of glucose administered as a 50 per cent solution in water. Twenty animals were used in the experiment. Of this four animals died within 72 hours after the last dose. In the remaining 16 animals blood sugar estimations were made and urine qualitatively tested for sugar and acetone. The result of blood sugar estimation after 72 hours are given in table no. II. Out of sixteen surviving animals, six (nos. 2, 3, 7, 8 and 9) were rejected as they showed automatic recovery as evidenced by gain in weight or fall in blood sugar. The remaining ten animals were used for further tests. A set of two animals was used for each of the following agents? (1).Tamun (Eugenea jambolana). (2) Bargad (Fiats bengalensis). (3) Gular (Ficus glomerata). (4) Pipal (Ficus religiosa). (5) Neem (Melia azadirachta). Gular and Pipal were used in the form of bark infusion and neem in the form of juice obtained from the fresh leaves. All the preparations were fed orally.

144 THE INDIAN MEDICAL GAZETTE [March, 1954 Table I Showing the normal blood sugar content in rabbits No. Wt. of rabbit in Kg. Glucose in mg./loo ml. 3rd day 5th day 7th day 1 1.31 105 103 104 2 1.30 93 92 93 3 1.00 99 98 98 4 1.41 100 98 99 5 1.12 107 108 108 6 1.46 92 93 92 7 1.21 92 89 90 8 1.21 100 103 101 9 1.30 98 101 100 10 1.41 98 97 98 11 1.60 99 101 100 12 1 70 98 96 97 13 1.50 96 97 96 14 1.40 101 99 100 15 1,30 95 98 96 Showing the evidence of diabetes in rabbits Table II 72 hours after 7.7. and intraperitoneal alloxan No. Wt. in Kg. Blood sugar after 72 hours in Urine sugar Urine acetone mg. 100 ml. 1 1.22 164 2 1.57 380 3 1.22 254 4 1.70 336 5 1.30 342 6 1.31 324 7 1.75 212 Traces. 8 1.70 324 9 1.42 400 10 1.29 died before 72 hours 11 2.09 290 12 1.32 358 13 1.38 298 14 1.55 370 15 1.55 324 16 1.00 324 17 1.16 died before 72 hours 18 1.17 19 1.00? 20 1.57 370

March, 1954] INDIGENOUS DRUGS IN DIABETES : GUJRAL & OTHERS i45 Dosage* and Results The powdered Jamun seeds were given in a dose of 0.8 gin. daily in two divided doses. The dose for Bargad, Gular and Pipal (F. Religiosa) was 4 gm. daily in two divided doses morning and evening. The Neem was used as fresh juice of the leaves and the daily dose of 2.5 ml. was administered in two divided doses morning and evening. Blood sugar estimations were made at the end of the first, second and third weeks and no appreciable difference from the untreated levels was found after use of any of the drugs. There was, however, one important difference between the rabbits treated with Jamun, Bargad, Pipal and Gular and those treated with the juice of the fresh Neem leaves. Whereas the former continued to lose weight after the pro- duction of chronic diabetes and lost consider- ably at the end of three weeks, the rabbits treated with juice of fresh Neem leaves began putting on some weight after the initial loss at the end of 72 hours and after treatment with the juice was commenced. At the end of three weeks the weight of the Neem treated rabbits was almost the same as before the production of diabetes. These results are shown in table No. III. Discussion From time to time one hears of wonder cures of diabetes by use of indigenous herbs. The herbs examined by us have been used for a long time in the treatment of diabetes by indigenous practitioners in this country. None of the herbs studied show any evidence of anti- diabetic action in rabbits. Table III Showing the effects on weight and change in blood sugar levels with indigenous drugs in experimental diabetes No. of Wt. of Wt. of Wt. of r>aily dose rabbits Name of drug. rabbit rabbit rabbit in two di- Blood sugar in mg./kg. as indie- before after 72 after 3 vided doses??* ated in alloxan hours of weeks. after 72 1st week 2nd week 3rd week Table II alloxanis. hours 1 E. Jambolana 1.22 M8 1.10 0.8 Gm. 165 1G4 162 164 4 >. 1.70 1-62 1.50 336 332 325 330 5 F. Bengalensis 1.30 1-26 1.18 4 Gm. 342 336 338 335 6? 1.31 L28 X? 324 316 320 died 14th day. 12 F. Glomerata 1.32 1.18 x 4 Gm. 358 350 355 died 15th day 13? l-38 J-34 1-24? 298 302 290 300 16 F. Religiosa J.00 0.9 x 4 Gm. 324 320 324 died 16th day. 14? 1.55 1-48 1.38? 370 372 366 368 15 Melia azadirachla 1.55 1-45 1.60 2.5 ml. 324 322 334 316 20? 1.57 1.49 1.58? 372 368 360 364! 1. Fresh neem leaves were washed with Water and put in a Waring Homogettiser, After they had been thoroughly homogenised, they were expressed through cloth. 2. The barks of Bargad, Gular and Pipal trees were used in a dry form. They were powdered and eight Gm. of the powdered bark (of each) was boiled with 100 cc. of water till the total volume was reduced to 25 cc. This amount was given in two divided doses, in the morning and the evening. 3. The specification of the powdered seeds of E. jambolcma is between meshes No. 60-90 B.P. It is difficult to explain that although all the rabbits were kept on a standard diet throughout the experiment, the group treated by fresh juice of Neem leaves, in spite of no diminution in the blood sugar level, continued to gain weight. Summary 1. Normal blood sugar of rabbits kept on a standard diet was estimated. No effect on the level of blood sugar could be de-

146 THE INDIAN MEDICAL GAZETTE [March, 1954 monstrated after oral administration of Eugenea jambolana seed powder in these animals. 2. Chronic alloxan diabetes was produced by giving repeated small doses of alloxan intravenously and intraperitoneally. 3. The treatment of the animals by Eugenea jambolana, F. bengalensis, F. glomerata, F. religiosa and Melia azadirachta proved ineffective. SELECTED BIBLIOGRAPHY Dymock, W. (1883).. Folin, 0. (1929).. Pharmacographia Indica, 2, 26, 29. Education Society's Press, Byculla, Bombay. J. Biol. Chan., 82, 83. Khory, R. N., and Materia Medico, of India, 2, Katrak, N. N. (1903). 370. Times of India Press, Bombay. Morris, M. (1946).. Manual oj Quantitative Biochemical Analysis. Hellige. Nadkarni, K. M. (1927). The Indian Materia Medica. Presidency Printing Press, Bombay, p. 367. Somogyi, M. (1926).. J. Biol. Chem., 79, 599. Idem (1929).. Ibid., 86, 655. Sushrut (1940).. Section on Treatment. Chowkhamba Sanskrit Publishers. Banaras.