May 1966 The Indian Journal of Pharmacy Vol.28, No.5, pp.129-133

by M. M. Lolitkar, M. R. Rajarama Rao
Charantin, non-nitrogenous, neutral princile, giving colour tests for phytosterolins is isolated in a pure state from the fruits of Momordica…

 

Pharmacology of a Hypoglycaemic Principles Isolated from the Fruits of Momordica charantia Linn

Author: M. M. Lolitkar, M. R. Rajarama Rao

Type of Publication: Pre-Clinical

Date of Publication: May 1966

Publication: The Indian Journal of Pharmacy Vol.28, No.5, pp.129-133, May 1966

Organization: Department of Chemical Technology, University of Bombay

Charantin, non-nitrogenous, neutral princile, giving colour tests for phytosterolins is isolated in a pure state from the fruits of Momordica charantia. Charantin lowers blood sugar in fasting rabbits, the fall being gradual from the 1^st to 4^th hour, and recovering slowly to initial level. Charantin (50 mg./kg.) administered orally lowers blood sugar by 42 per cent at the 4^th hour, the mean fall during 5 hours being 28 per cent. The cumulative hypoglycaemic potency curve is not linear, but tends to flatten out as the dose is increased. Charantin is more potent than tolobutamide in hypoglycaemic activity but the pattern of blood sugar changes is similar to both. The hypoglycaemic activity of charantin is depancreatixed cats is less, but abolished, indicating a pancreatic as well as extra-pancreatic action. It exerts non-specific antispasmodic and mild cholinergic-blocking activity.

The unripe fruit of Momordica charantia (Hindi: karela, Eng.: bitter gourd) is commonly used as vegetable. It is popular as a folk-lore remedy for diabetes mellitus. One to two ounces of the juice of fresh unripe fruits is taken daily to control diabetes. Vad^1,2 reported clinical trial of the fresh juice in 160 cases of diabetes. The juice was found to control diabetes but was not capable of curing it. Rivera³ isolated an alkaloid, momordicine and a glycoside from an alcoholic extract of the fruits. Gaessler⁴ reported hypoglycaemic activity in a crude crystalline substance isolated from the fruits. Sharma et al.,⁵ Kulkarni et al. and Pabrai et al.⁷ gave conflicting reports on the hypoglycaemic activity of the juice of fresh unripe fruits. Lolitkar and Rao⁸ reported hypoglycaemic activity of a non-nitrogenous neutral principle isolated from an alcoholic extract of the dried fruits.

MATERIALS AND METHODS

Isolation of charantin: Fresh unripe fruits of Momordica charantia bought from the market were cut into small bits and dried in a hot air oven below 60⁰. The dried material was broken into a coarse powder and percolated successively with petroleum ether (b.p. 60-80⁰) and 80 per cent ethanol. The petroleum ether extract was rejected and the ethanolic extract was concentrated in vacuo, suspended in 95 per cent ethanol and rendered alkaline with KOH to around pH 10. After 48 hours, the suspension was diluted with water and extracted with ether. The ether extract was washed with water, 5 per cent HCI, with water again, and dried over anhydrous sodium sulphate. The ether was distilled off and the residue recrystallized several times from 95 per cent ethanol. The substance thus obtained will be referred to here as charantin. Yield was 0.035 per cent of the dried fruits.

Charantin is a non-nitrogenous neutral substance, melting at 269-272⁰ with decomposition, giving a play of colours changing from violet to blue to green and yellow with Libermann-Burchard test, decolourising dilute potassium permanganate and bromine water, soluble in ether, benzene and chloroform, sparingly soluble in ethanol and methanol and insoluble in petroleum ether, acetone and water. It answers colour tests for phytosterolins⁹ – a violet ring and greenish orange fluorescence in the bottom layer, on adding saturated solution of thymol to a solution of the compounds in conc. Sulphuric acid. On hydrolysis with HCI it yields glucose and sterol. On analysis it was found to contain C, 74.0; H, 10.4; and O, 15.6 per cent. It forms acetyl and methyl derivatives. Mol. Wt. Of acetyl derivatives is 608, by Rast method, and its elemental analysis is C, 70.7; H, 9.2; and O, 20.1 per cent. Elemental analysis of methyl derivatives is, C, 75.2; H,12.7; and O, 12.1 per cent. Mol wt. Of charantin was not done since it was insoluble in camphor. Determination of acetyl and methoxy groups was not done since the derivatives were not soluble in the solvents usually employed here. Probable mol. Formula of acetyl derivatives is C₃₆ H₅₆O₈. I. R. spectra of charantin and cholesterol have a close resembles, the former having more hydroxyl groups and double bonds. Thin layer chromatography of charantin on silica gel with methanol: benzene mixture (2:8), and spraying with conc. Sulphuric acid gave a single spot with RF value of 0.5.

Hypoglycaemic activity of charantin: Rabbits of either sex (Haffkine strain) weighing 1.5-3 kg. were fasted by with drawing food the previous day. Blood sugar was estimated by the method of Hagedron and Jensen.¹⁰ Cahrantin was suspended in 0.3 per cent. Twelve 80, and administered orally through a stomach tube, or intravenously. Alloxan diabetes was produced in rabbits by intravenous injection of alloxan, 200 mg./kg., and 4 out of 20 which survived after 5 days were used. Anaesthetized (pentobarbitone sodium 30 mg./kg. intraperitoneally) cats fasted for a day were also used. Pancreas of cats was removed through a median incision in the abdomen. Removal of samples of blood and administration of charantin was through the femoral vein. Blood pressure of anaesthetized (as above) cats was recorded on a kymographs using a mercury manometer connected to a cannula in the carotid or femoral artery. Respiration was recorded through a tambour connected to a cannula in the trachea.

Contraction of the nictitating membrane of cats was recorded on a kymograph with a frontal lever, the preganglionic cervical sympathetic being stimulated by a 10 volt current from a square-wave stimulator. Isolated hearts of frogs were perfused with frog Ringer, and charantin added to the cannula. Isolated duodenum of rabbit and ileum of guinea pig were suspended in an oxygenated organ bath of 50 ml. capacity at 35⁰ and contractions were recorded through frontal levers. Salivary secretion was induced in rabbits by subcutaneously injection of 15 mg./kh. Pilocapine nitrate. Charantin or atropine sulphate were administered intravenously. Tremors were induced in mice and rabbits by intraperitoneal injection of 10 mg./kg. Tremorine. Toxicity was studied in mice weighing 18-22 g. (Haffkine strain) cahrantin being administered intraperitoneally.

EXPERIMENTAL AND RESULTS

Hypoglycaemic activity. Control rabbits were given 5 ml. of 0.3 per cent Tween 80. Tween 80 had no effect on fasting blood sugar of rabbits. The mean fall in blood sugar was calculated from

IBS – FBS

the formula _________________ X 100 where

IBS

IBS= initial blood sugar, FBS- the mean of five reading taken during five hours following administration of the drug. Peak fall in blood sugar was calculated from the same formula but with FBS as one of the five readings where fall of blood sugar was maximal.Charantin produced a gradual and steady fall of blood sugar for 4 hours after which tended to recover to initial level. The duration of activity was more than five hours. Maximal activity was reached in the 4^th hour. There was no difference in the pattern of blood sugar change between intravenous and oral routes, the peak effect appearing at the 4^th hour in both. However, 15 mg./kg. charantin by intravenous route produced the same effect as 25 mg./kg. orally. Equivalent doses of tolbutamide were less effective than charantin. The pattern of blood sugar changes was similar to both. (Table1). Fig.1 presents cumulative hypoglycaemic potency curve for charantin, where each point indicates the cumulative hypoglycaemic potency for the duration of the testing period, i.e. five hours. There is a clear indication that higher doses of charantin may not produced a proportionate fall in blood sugar. The cumulative hypoglycaemic potency is calculated from the formula, ∑ P₁….P₅ where P₁ = per cent fall in blood sugar at the first hou, P₂ at the second hour and so on. This formula is considered by Radding et al.¹¹ as more informative than a log. dose-response curve, giving some idea on the rate of absorption and metabolic dispersion of the compound.

The course of blood sugar changes in alloxan diabetic rabbits in response to charantin was rather erratic. In two of the rabbits there was a marked rise in the first hour after the drug, while the other two showed marked and steady fall. Besides, only mildly diabetic rabbits had survived 5 days after alloxan.

Charantin produced marked fall (25 per cent for 5 mg./kg.i.v.) of blood sugar in anaesthetized cats. The effect of charantin was considerably less (nearly half) in the depancreatized cat. There was no fall in blood sugar of depancreatized cat not treated with charantin.

Cardiovascular system. In anaesthetized cats 800 g./kg. of charantin lowered the blood sugar pressure by 5-10 per cent, and blocked the ise of blood pressure caused by occlusion for one minute of carotid arteries, but did not affect the changes produced by acetyl choline and adrenaline.

There was no change in the contraction of the nictitating membrane of cat after injection of 400-800 g./kg. of charantin and hence, no ganlionic blockade.

Small doses of charantin (5-10 g.) increased the amplitude of contraction of the isolated perfused heart of frog, and abolished the inhibitory action of acetylcholine (Fig.II).

The vehicle, 0.3 per cent Tween 80, did not have any effect on the blood pressure of cats or the heart of frog.

No significant changes in respiration were observed in anaesthetized cat after the administration of charantin.

Anti-sialogogue activity. Subcutaneous injection of pilocarpine nitrate (15 mg./kg.) produced in rabbits copious flow of saliva for over 2 hours. The flow ranged from 1.5-4 ml. per 10 minutes, and the flow was fairly constant during the experimental perion. Intravenous injection of 7.89 mg./kg. of atropine sulphate reduced this flow of saliva by 50 per cent.

Charantin did not have any effect on the amplitude of contractions of isolated duodenum of rabbits, but 500 g./ml. of charantin partially reduced the amplitude of spasm produced by acetyl choline (Fig.III).

Charantin inhibited the spasm produced by acetyl choline histamine and barium chloride on the isolated ileum of guinea-pig. ED₅₀ against acetyl choline was 0.343 g./ml. (atropine sulphate 0.006 g./ml.), against histamine 1.39 g./ml(mepyramine maleate 0.448 g./ml.), and against barium chloride 2.29 g./ml. (papavarine hydrochloride 7.65 g./ml.). Since charantin is insoluble in water, its potency in these experiments is not calculated.

Charantin did not appear to produce any behavioral changes in mice doses up to 400 mg./kg. intraperitoneally. This dose was also not lethal.

Tremorine (10 mg./kg.) injected intraperitoneally produced salivation and tremors in mice within 10 minutes, the tremors lasting over two hours. In the case of rabbits, 10 mg./kg. tremorine produced profuse salivations, and occasional waves of tremors lasting over two hours. Charantin (10-25 mg./kg.) by oral or intravenous route delayed the onset of tremors by 20-40 minutes, but did not affect salivation. Atropine sulphate (2 mg./kg.) given intraperitoneally promptly abolished both the tremors and salivation in these animals.

DISCUSSON

The colour tests and other data of charantin indicate that it is phytosterolin i.e., a sterol glycoside. Since its mode of action is not known, it may be inappropriate to calculate its potency using tolbutamide as a standard. The results however, indicate a similarity in the pattern of blood sugar changes in response to these two agents. On this basis it may be inferred that charantin is more potent than tolbutamide. The cumulative hypoglycaemic potency curve tends to fall from 25 mg./kg. to 50 mg./kg. Since 400 mg./kg. of charantin is not lethal to mice it is possible that higher doses of charantin may not be accompanied by a proportionate fall in blood sugar. This may be due to limited absorption or action or both. The reduced effect of charantin in the depancreatized cat indicates a pancreatic as well as extrapancreatic action. While charantin does not block the fall in blood sugar pressure after acetyl choline it blocks the inhibitory action of the latter on amphibian heart. It also delays the onset tremors due to Tremorine, inhibits the siologogue action of pilocarpine, and exerts antispasmodic activity. It is therefore to be inferred that the cholinergic blocking action of charantin is of a mild nature. The blocking action of charantin on the rise of blood pressure due to occlusion of the carotid arteries is not fully explained by the data collected. It is not, however, due to adrenergic or ganglionic blockage.

It is doubtful if charantin represents all the hypoglycaemic activity present in the fruit of Momordica charantia. For example, it takes more than 1,500 g. of fresh fruits to get 50 mg. of charantin. The beneficial clinical results reported from using daily 50-60 ml. of the juice of fresh fruits could not be entirely due to the few mg. of charantin present in them. Perhaps the whole fruit, instead of the juice, would produce better results clinically, provided it did not exert any toxic effect on prolonged use.

ACKNOWLEDGEMENTS

We are indebted to Dr. T. S. Gore and Dr. M. R. Padhye, for chemical and spectral analysis of charantin, and to Dr. R. K. Richards of Abbott Laboratories, Chicago, for the sample of Tremorine.

We are indebted to the Government of Maharashtra for financial assistance received during the course of this work.

REFERENCES

Vad, B. G., Maharashtra Medical J., 1960, 6, 733.

Vad, B. G., Symposium on Indigenous drugs held at Topiwala Medical College, Bombay, 1961.

Rivera, G., Am. J. Pharm., 1941, 113, 281.

Gaessler, W. G., Am. J. 1942, 72, 114.

Sharma, V. N., Sogani, R. K. and Arora, R. B., Indian J. med Research, 1960, 48, 471.

Kulkarni, R. D. and Gaitonde, B. B., Indian J. Med. Research, 1962, 50, 715.

Pabrai, P. R. and Sehra, K. B., Indian J. Pharm., 1962, 24, 209.

Lolitkar, M. M. and Rajarama Rao, M. R., J. Univ. Bomaby, 1961, 29, 223.

Ambike, S. H. and Rajarama Rao, M. R., unpublished data.

Hagedorn and Jensen, Biochem. Z., 1923, 135, 46.

Radding, R. S., Kern, L. R. and Owens, J. C., metabolism, 1962, 11, 411.