By Author
  By Title
  By Keywords

November 1988, Volume 38, Issue 11

Original Article

EFFECI OF GRE WIA AS1ATICA, GOSSYPIUM HERBAC1UM AND GYMNEMA SYLVESTRE ON BLOOD GLUCOSE, CHOLESTEROL AND TRIGLYCERIDES LEVELS IN NORMOGLYCAEMIC AND ALLOXAN DIABETIC RABBITS

Iftikhar Ahmad Dogar  ( Department of Biochemistry, Punjab Medical College, Faisalabad. )
Muhammad All  ( Department of Biochemistry, Punjab Medical College, Faisalabad. )
Muhammad Yaqub  ( Department of Biochemistry, University of Agriculture, Faisalabad. )

Abstract

Blood glucose, cholesterol (total) and triglycerides, levels of the normal and alloxan diabetic male rabbits of local strain were determined after oral administration of ground herbal drugs, i.e., Gossypium herbacium (cotton seed), Grewia asiatica (bark phalsa) and Gymnerna sylvestre (gur-mar).
The herbs G. herbacium and G. asiatica produce considerable and significant hypoglycaemic effects in both, normal and diabetic rabbits, but G. sylvestre shows slight hypoglycaemic activity in normal rabbits. Three different doses (20, 10, 5g1 Kg) of two drugs i.e., G. herLacium and G. asiatica produced almost same reduction in blood glucose. The maximum reductions were at 3,6 and 12 hours. It means even minimum dose i.e., 5g/Kg was sufficient to bring about normoglycaemic state in experimental rabbits. In case of blood cholesterol (total) and triglycerides the uniform dose (10 g/Kg) of all the three medicinal herbs was administered daily orally for a period of 10 days; to normal as well as diabetic groups. Significant fall in blood cholesterol and triglycerides was caused by G. asiatica. G. herbacium was second and G. sylvestre was third hypocholesterolaemic and hypotriglyceridemic in nature (JPMA 38:, 1988).

INTRODUCTION

Although considerable developments have been made in the management of diabetes mellitus, yet a wide scope is still left in the search of newer orally applicable and effective antidiahetic agents. G. herbacium, G. asiatica and G. sylvestre are medicinal herbs which are widely used in traditional medicine for various purposes including a metabolic disease like diabetes mellitus which is third commonest cause of death; and 2 % of Pakistani population is suffering from this disease.1 The present study was undertaken to determine the effect of ground herbal drugs on blood glucose levels following oral administration of crude extracts of their selected parts to normoglycaemic and alloxan treated hyperglycaemic male rabbits. As hyperglycaemia is known to produce a rise in the blood lipid contents2 hence we planned to study the effect of these herbal drugs on trigly¬cerides and blood cholesterol (total) levels in normal as well as diabetic rabbits after orally administering each drug for 10 days.

MATERIALS AND METHODS

Plant Material
Fresh ground (selected) parts of the Gossypium herbacium, Grewia asiatica and G. sylvestre plants locally known as cotton seed or binola, bark phalsa and gur-mar were obtained in sufficient amounts each from the local fields and market. They were carefully washed with tap water, dried, milled in an electric grinder and stored in well closed cellophane bags at 4°C in refrigerator.
Chemicals
Alloxan-monohydrate, alpha D~glucose (an. hydrous) (BD.H.) Glacial acetic acid, benzoic acid (sublimed), O-toluidine, thiourea and tricho. racetic acid and carboxymethyl cellulose was purchased from the local market, all other chemi­cals and reagents used were of analytical grades, prepared either by F. Merk or BD.H. laboratories. Tolbutamide was obtained from Hoechst (Pakis­tan). Kits were used for the estimation of choles­terôl and triglycerides (Boehringer).
Animals
Male, adult, healthy rabbits of local strain weighing between 1000 — 1700 g were used in these experiments. The animals were kept in an air cooled animal room. They were offered a balance rabbit feed and allowed tap water ad libitum. The effects of Gossypium herbacium, Grewia asiatica and G. sylvestre were studied on blood glucose levels of the normal and alloxan diabetic rabbits, in addition, separate experiments were performed to study their effects on blood cholesterol and triglycerides levels of normal and diabetic rabbits. Toxicity and behavioural studies showed no disturbance following oral adminis­tration of any herbal drug.
A group of rabbits weighing 1000-1700 g were made diabetic by injecting intravenously 150mg/kg body weight of alloxan-monohydrate which causes permanent necrosis of B cells. Eight days after injection the blood glucose levels of all surviving rabbits were determined by the 0-toluidine method3. Rabbits with blood glucose levels of more than 200 mg/100 ml were consi­dered as diabetic and were employed for further study as already used by Sharma et al.4 in their experiments.
Grouping of Rabbits
Rabbits were randomly divided into 8 different groups of six animals each, group I (diabetic) for each drug was served as control and received orally 20 ml of 1.5% carboxyrnethyl cellulose solution in water only. Group second (normal) control for each drug was treated with carboxymethyl cellulose 1.5% solution (20 ml) in water only. Animals, of group III were treated orally with 10 g/kg body weight of G. herbacium, G. asiatica and G. sylvestre in 1.5 percent CMC solution, respectively. The animals of groups W, V and VI were treated with all the three drugs at doses 20, 10 and 5 g/kg body weight respecti­vely. Gluconil at 100 mg/kg dose, tolbutamide 250 mg/kg body weight respectively were also administered to diabetic rabbits of groups VII and VIII for each drug. Three groups of 6 animals (diabetic) and other three groups of normal rabbits were treated with each drug at uniform dose of 10 g/kg for 10 days to find the compara­tive effect of these drugs on blood cholesterol and triglycerides, levels of normal and diabetic rabbits; while keeping all the conditions of feed and environment constant.
Preparation and administration of drug suspen­sions
The amount of G. herbacium, G. asiatica and G. sylvestre required for each animal was calculated on body weight basis and the required amount of drug was well triturated with about 10 ml of 1.5% CMC solution and final volume made upto 20 ml. The drugs were then administered to each animal using a stainless steel feeding needle attached to a standard syringe containing 20 ml of drug suspension. The needle was inserted into the stomach through the oesophagus and the plunger was pressed slowly and steadily. Immediate sneezing and coughing indicated injection into the lungs and in such a condition the syringe was withdrawn and after some time again inserted properly.
After administration of drug, the animal was held in a proper way and immediately 0.1 ml of blood was collected from saphenous vein. Similar samples of 0.1 ml were collected at 3,6, 12 and 24th hours. After collecting blood, abso. lute alcohol was applied at the pricked site to protect against infection. The detailed methodo­logy has already been reported5-6
Determination of blood glucose, triglycerides and cholesterol
Blood glucose was determined using the 0-toluidine reagent. The blood cholesterol and triglycerides were determined at zero hour and then after 10 days of oral administration of each drug using Boehringer (West Germany) Kits with the method of Watson7, Fredrickson et a18. Statistical analysis
Mean blood glucose, cholesterol and tri­glycerides levels were expressed in mg/l00 ml in all experiments and analysis of variance technique was applied along with Factorial Method and Duncan’s Multiple Range Test. Data was fed into the computer at Meteorology Department, Univer­sity of Agriculture, Faisalabad.

RESULTS

The mean blood glucose concentrations of control and drug treated animals after oral administration of different doses of G. herbacium, G. asiatica and Gymnema sylvestre at various time intervals are summarised in Table I, II and III.



The blood glucose levels of diabetic animals treated with 1.5% Carboxymethyl cellulose (CMC) solution at zero hour were found to be 393.00, 365.50 and 386.17 mg/100 ml respectively. And there was no change in blood glucose level upto 24 hours. The mean blood glucose levels of normal rabbits after (CMC) were 10333, 105.33 and 96.33 mg/lOU ml respectively at zero hour, which were not changed during further intervals. The (CMC) did not affect the blood glucose levels of diabetic or the normal rabbits. The levels were statistically same at 0, 3,6, 12 and 24 hours.
Gossypiuni herbaciuin (Binola)
The blood glucose level of normal rabbits treated with 10 g/Kg of G. herbacium at zero hour was 111.17 mg/100 ml. It was slightly lowered at 3, 6 and 12 and 24 hours. Statistically these reductions were non-significant as compared with zero level. Mean blood glucose level of diabetic animals treated at the dose of 20g/Kg at time interval 0 was 385.83 mg/100 ml. This was lowered at further intervals and statistically these lowerings were highly significant. The mean blood glucose level of diabetic animals treated with dose of log/Kg at time interval 0 hour was 390.83 mg/100 ml which lowered at further hours and the reductions were significant at 3, 6, 12 and 24 hours. Mean blood glucose level of diabetic animals treated at the dose of 5 g/Kg at 0 hour was 359.67 mg/100 ml which further lowered at further time intervals (Figure 1)

These loweririgs were highly significant uptil 12 hours but was significant at 24 hours.The mean blood glucose levels of gluconil (100 mg/Kg) treated diabetic animals were 364.17 mg/ 100 ml at 0 hour which further lowered with time. All reductions were statistically significant but after 24 hours the zero level was reached again. Tolbutamide (250 mg/Kg) treated diabetic animals showed no alteration of blood glucose level (Table 1).
Grewia asiatica (bark phalsa)
In case of normal rabbits treated at the dose of lOg/Kg the mean blood glucose level was 116.17 mg/ 100 ml at zero hour which lowered with time. The level at 24 hour was statistically significant.
The mean blood glucose level of diabetic group treated with the dose of 20 gm/Kg were recorded as 368.67 mg/100 ml at zero hour which was lowered at further hours. The reduction was significant at 24 hour and highly significant at 3, 6, and 12 hours. Diabetic group treated at the dose of lOg/Kg had blood glucose level 383.67mg/lOO ml at zero which was lowered at further intervals. The significant decreases were at 3, 6. 12 and 24 hours (Figure 2).

The mean blood glucose level of diabetic rabbits tEeated at the dose of 5 gm/Kg were recorded to be 357.00 at zero hour which was further decreased with time. All these lowerings of blood glucose were statistically significant. Diabetic group treated with gluconil at 100 mg/Kg gave the mean of blood glucose levels to be 364.83 mg/100 ml which was lowered at further time intervals. All these lowerings were significant uptil 12th hour only (Table II).
Gymnema sylvestre (gur-inar)
When the decoction of leaves of this herb was administered to normal as well as diabetic animals, it produced no effect on blood glucose level of diabetic rabbits while it reduced the blood glucose level of nonnal rabbits. These values were non-significant at 3,12 and 24hours but the values after 6 hours were significantly lower as compared to zero level (Table III and Figure 3).


Mean of Triglycerides in mg/100 ml of normal group (I) were reduced non-significantly after oral administration of 10 gm/Kg G. herba­cium for 10 days. Mean triglycerides of diabetic rabbits (II) treated with this drug at a dose of 10 gm/Kg were reduced within the same period but the reduction was statistically not significant. In case of G. asiatica treated normal (III) decrease of blood triglycerides was not significant. However, significant decrease was caused in diabetic group (IV) when level was 219.09 mg/lOO ml, which was reduced to 159.01 mg/l00 ml after 10 days.
In case of Gymnema sylvestre treated normal (V) the level was 154.84 mg/lOU ml which was lowered to 142.93 mg/lOU ml, as a non-signi­ficant decrease. In diabetics (VI) the level was 176.67 and then after 10 days it was 163.66, again a non-significant lowering was recorded (Table IV).


The mean blood cholesterol of G. her­bacium treated rabbits were lowered from 80.47 to 71.57 mg/100 ml in nonnal group (I) and from 97.89 to 85.77 mg/100 in diabetic group (II). Both alterations were not-significant. In G. asiatica treated normal animals (III), the level was reduced from 78.87 to 61.83 mg/100 ml and in diabetics (N), the level was reduced from 106.92 to 90.30 mg/lOU ml both the reductions were statistically significant.
G. sylvestre treated normal (V) reduced blood cholesterol from 83.24 to 76.00 mg/ 100 ml and then in diabetics (VI), the level was 103.86 which was raised to 106.21 mg/l00 ml. The lowering in normal and increase in diabetics were statistically not-significant (Table V).

DISCUSSION

The present data shows that the adminis­tration of various doses of Gossypium herbacium caused a decrease in blood glucose level of normal and alloxan diabetic rabbits. For the comparison of hypoglycaemic activity tolbutamide and gluconil were also administered. Tolbutamide produced no change in blood glucose level of diabetic animals, but gluconil produced a signi­ficant decrease in blood glucose level of diabetic animals. It has been reported that sulphonyl ureas (Tolbutamide) produce hypoglycaemia in normal and non-insulin dependent animals, by stimulating the pancreatic B-cells to produce more insUlin and by increasing the glycogen deposition in the liver. These drugs, however, do not decrease blood glucose in alloxan diabetic animals. But gluconil does not produce hypoglycaemia in normal animals, because the peripheral utilization of glucose is compensated by hepatic synthesis of glucose9. Grewia asiatica also reduced blood glucose levels in normal and alloxan treated rabbits.
It was noted that the duration of hypogly­caemic action of the drug was nearly the same in normal as well as diabetic rabbits. This can be said with certainty that there are some active principles which are functioning in normal as well as diabetic rabbits.
In contrast to the oral antidiabetic agents the exogenous administration of insulin is well known to produce hypoglycaemiâ in both normal and alloxan diabetic subjects10-11 O,j 1. It is, there­fore, conceivable that hypoglycaemic principle in the Gossypium herbacium seeds and Grewia asiatica bark exert a direct effect on the diabetic rabbits probably bya mechanism similar to insulin. The drugs do not seem to act indirectly i.e., by stimulating the release of insulin as the alloxan treatment causes permanent destruction of B-cells12. In fact it may be supposed that in the normal rabbits these substances act not only by a direct insulin like action but also by stimulating the release of insulin. It must however be accepted that the possibility of some other factors producing this alteration cannot be excluded at present. It may be hypothesized that G. herbacium and G. asiatica contain more than one active principles which are potent in normal as well as in diabetic rabbits. This view is similar to that reported by others5-6 who reported that Momordica charantia possesses more than one hypoglycaemic principle Le., functioning in normal and diabetic rabbits both.
G. sylvestre leaves caused a significant decrease of blood glucose level in normal rabbits. This decrease in blood glucose is most probably due to its action as an insulinotropic agent i.e., to stimulate the release of insulin from B-cells. This view is strengthened by the work of Sun­daram et al who have reported that this medicinal herb is used in controlling diabetes mellitus. However, G. sylvestre did not cause and decrease in blood glucose level of diabetic rabbits. It means this drug can act as hypoglycaemic agent only through insulin.
G. asiatica has shown hopeful results to­wards decrease of blood cholesterol and trigly­cerides in normal as well as dIabetic rabbits. Secondly G. herbacium has precluded some hope of being hypoiypaemic whereas decoction of G. sylvestre leaves, has shoWn somewhat promising results in nOrmal animals, but in diabetic animals, there is seen a zero effect. This reduction of lipids is most probably due to capability of the herbal drugs towards glycaemic control of experi­mental animals because when diabetes mellitus is controlled lipids are also lowered13-14

REFERENCES

1. Khan, N. Problems of diabetes mellitus. Specia­list, 1985; 2:7.
2. Adlersberg, D. Biochemistry and Pathophyso­logy with special reference to lipid metabolism. Diabetes Mellitus: JU. Cong. Inst. Diab. Fed. Dusseldorf, 1958; P. 61.
3. Fings, C.S., Totliff, C.R. and Dunn, R.T. Glucose determination by 0-toluidine  method using acetic acid. Clinical chemistry by Tore and P.G. Ackerman. Boston, Little Brown, 1970; p. 115.
4. Sharma, A.L., Sapru, H.N. and Chowdhury, N.K. Hypoglycaemic action of Cryptostegia grandi­flora. R. Br. in rabbits. Indian j. Med. Res., 1967; 55:1277.
5. Yaqub, M., Akhtar, M.S. and Athar,MA. Karela anti diabetic agent. JPMA., 1980; 30:181.
6. Akhtax, M.S., Athar, M.A. and Yaqub, M. Effect of Momoradica charantia on blood glucose level of normal and alloxan-diabetic rabbits. Planta Med., 1981; 42:205.
7. Watson, D. A simple method for the deter­mination of serum cholesterol. Clin, Chim. Acta,1960; 5:637.
8. Fredzickson, D.S., Levy, R.l. and Lees, R.S. Fat transport in lipoproteins, an integrated approach to mechanisms and disorders. N. Engi. J. Med., 1967; 276:34.
9. Goth, A. Medical pharmacology. 9th ed. Saint Louis,Mosby, 1978; p.421.
10. Guyton, M.D. Insulin, glucagon, and diabetes mellitus, in textbook of medical physiology, 4th ed. Philadelphia, Saunders, 1971~ p. 915.
11. Lamer, J. Insulin and oral hypoglycaemic drug; glucagon, in the pharmacological  basis of therapeutics. Editors: Alfred Goodman Gilman et al. 6th ed. New York, Macmillan, l98O;p. 1497.
12. Kapalan, M.H. and Timmons, E.M. The rabbit. A Model for the principle of Mammalian physio­logy and surgery. New York, 1979, p. 122.
13. Keiding, N.R., Root, H.F. and Marble, A. Impor­tance of control of diabetes in prevention of vascular complications. JAMA., 1952; 150:964.
14. •Wolf, O.H. and Salt, H.B. Serum-lipid and blood-sugar levels in childhood  diabetes. Lancet,1958; 1:707.

Journal of the Pakistan Medical Association has agreed to receive and publish manuscripts in accordance with the principles of the following committees: