April 2005
Notes
Biol. Pharm. Bull. 28(4) 729—732 (2005)
729
Hypoglycemic and Antihyperglycemic Effect of Gmelina asiatica LINN. in Normal and in Alloxan Induced Diabetic Rats Routhu KASIVISWANATH,* Alluri RAMESH, and Kilari Eswar KUMAR a
Pharmacology Division, Department of Pharmaceutical Sciences, Andhra University; Visakhapatnam-530 003, Andhra Pradesh, India. Received June 26, 2004; accepted January 21, 2005 The hypoglycemic and antihyperglycemic effect of alcoholic extract of root of Gmelina asiatica LINN. (G. asiatica) was investigated in normal and in alloxan induced diabetic rats. The blood glucose levels were measured at 0 h and 1, 2, 4, 6, 8 and 16 h after the treatment. The alcoholic extract of G. asiatica showed significant (p0.05) dose dependent percentage blood glucose reduction in normal (25.8% at 100 mg/kg, 28.9% at 250 mg/kg and 32.4% at 500 mg/kg body weight) and in diabetic rats (26.6% at 100 mg/kg, 32.1% at 250 mg/kg and 48.2% at 500 mg/kg body weight) respectively at 6 h. The antihyperglycemic effect of G. asiatica was compared with the reference standard drug tolbutamide (40 mg/kg). Key words
Gmelina asiatica; diabetes; alloxan; nicorandil
Diabetes mellitus is the most important disease involving the endocrine pancreas. Type 2 diabetes is a heterogeneous disease with both genetic and environmental contributory factors, involved multiple defects in insulin action and insulin secretion leads to hyperglycemia and effecting nearly 10% of the population all over the world.1) In modern medicine, the beneficial effects on glycemic levels are well documented; the preventing activity of these drugs against progressive nature of diabetes and its micro and macrovascular complications was modest and not always effective. Insulin therapy affords effective glycemic control, yet its short comings such as ineffectiveness on oral administration, short shelf life, requirement of constant refrigeration, and in the event of excess dosage—fatal hypoglycemia—limits its usage. Treatment with sulfonylureas and biguanides is also associated with side effects.2) For various reasons in recent years, the popularity of complementary medicine has increased. Dietary measures and traditional plant therapies as prescribed by Ayurvedic and other indigenous systems of medicine were used commonly in India.3) Surveys conducted in Australia and US indicate that almost 48.5 and 34% respondents had used at least one form of unconventional therapy including herbal medicine.4) Indian figures are not available. WHO (1980) has also recommended the evaluation of the plants effective and in conditions where we lack safe modern drugs.5) Since time immemorial, patients with diabetes mellitus have been treated orally by folklore with a variety of plant extracts. In the indigenous system of medicine (Ayurveda), a mention was made on good number of plants for the cure of diabetes or ‘Madhumeha’ and some of them have been experimentally evaluated and the active principles were isolated.6—11) However, search for newer antidiabetic drugs continues. Gmelina asiatica LINN. (syn. Gmelina parvifolia) (Family, Verbenaceae) is commonly known as ‘Nilakkumil’ in Tamil and ‘Gopabhandra’ in Sanskrit. The root of the plant is used in gonorrhoea, catarrh of the bladder, rheumatism and as a blood purifier.12) Flavonoids and lignans13,14) were reported from G. asiatica. It has been subjected to various phytochemical studies.15,16) Since the plant is claimed to be useful in rheumatism, and also possess anti-inflammatory effect.17) The present study focused to evaluate the hypoglycemic and ∗ To whom correspondence should be addressed.
antihyperglycemic activity of alcoholic extract of G. asiatica roots at various doses in normal and alloxan induced diabetic rats. MATERIALS AND METHODS Plant Material The bark of Gmelina asiatica L. was collected from Annavaram hills of East Godavari district, Andhra Pradesh, South India. Dr. K. Hemadri, taxonamist, Regional Research Institute, Botanical Survey of India, Vijayawada, identified the herb. The specimens were preserved in the herbarium of our department. Alcoholic Extraction Shade dried powdered bark (500 g) of G. asiatica was extracted with alcohol (95% v/v) by soxhlation until there is no compound left over in solvent. The crude extract was evaporated to dryness in a rotary film evaporator (15.5% w/w). Toxicity Evaluation in Mice The alcohol extract was tested for its acute and short-term toxicity (if any) in mice. To determine acute toxicity of a single oral administration of the herbal drug, different doses of the drug (0.25, 0.5, 0.75, 1.0 g/kg) were administrated to different groups of mice (2 mice were used for each group, control mice received Tween 80). Mortality and general behavior of the animals were observed periodically for 48 h. The animals were observed continuously for the initial 4 h and intermittently for the next 6 h and then again at 24 h and 48 h following drug administration. The parameters observed were grooming, hyperactivity, sedation, loss of righting reflex, respiratory rate and convulsion. To study short-term toxicity, 3 groups of mice each containing 6 male mice (20—25 g, body weight) were used. Group I was kept as control and group II and III received 200 and 400 mg/kg alcohol extract respectively in 5% Tween 80. The drug was administrated daily for 14 d (p.o.). Control group received 5% Tween 80 in an identical manner. The behavior of the animals was observed daily for 1 h in the forenoon (10 to 11 a.m.) for 14 d. Initial and final body weights, water and food intake, state of stool and body temperatures were observed. Drugs Alloxan monohydrate was purchased from Sigma Chemicals (St. Louis, U.S.A.). All other chemicals used for this study were analytical grade.
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© 2005 Pharmaceutical Society of Japan
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Animals Laboratory bred Sprague Dawley rats of either sex weighing 150—200 g were employed for the study. All animals were procured from National institute of Nutrition, Hyderabad. The rats were maintained under standard laboratory conditions at 252 °C, relative humidity 5015% and normal photo period [12 h dark/12 h light] were used for the experiment. Commercial pellet diet [Ratan Brothers, India] and water were provided ad libitum. The experimental protocol has been approved by the Institutional Animal Ethics committee and by the Regulatory body of the government [Regd no. 516/01/A/CPCSEA]. Induction of Diabetes Animals were allowed to fast 18 h and were injected with alloxan monohydrate dissolved in sterile normal saline at a dose of 150 mg/kg body weight intraperitoneally.18) After 2 week, diabetic rats (250— 350 mg/dl) were used for the experiment. Experimental Design In the experiment a total number of 54 rats (24 normal and 30 diabetic surviving rats) were used. The rats were divided into nine groups (4 normal and 5 diabetic), each group consisting of 6 animals. Group 1 normal rats treated with vehicle (5% gum acacia) and served as control, and Group 2, 3 and 4 normal rats were treated with alcoholic extract of G. asiatica at a doses of 100, 250, and 500 mg/kg respectively, Group 5 diabetic rats treated with vehicle served as diabetic control, Group 6, 7 and 8 diabetic rats were treated with alcoholic extract of G. asiatica at a doses of 100, 250 and 500 mg/kg respectively, Group 9 diabetic rats were treated with 40 mg/kg dose of Tolbutamide. All the doses were administered orally. For the interaction of nicorandil with G. asiatica and tolbutamide in normal and diabetic rats, animals were divided into eight groups, each group consisting of five animals. Group A, B, C, and D were normal and Group E, F, G and H were diabetic rats. Group A and E were treated with alcoholic extracts of G. asiatica (500 mg/kg), Group B and F were treated with nicorandil (10 mg/kg), Group C and G were treated with alcoholic extract of root of G. asiatica (500 mg/kg) and nicorandil (10 mg/kg). Group D and H were treated with tolbutamide (40 mg/kg) and nicorandil (10 mg/kg). The rats were fasted for 18 h and blood samples were collected by puncture of retro-orbital plexus immediately with capillary tube under ether anesthesia into glass vials containing a small quantity of a mixture of potassium oxalate and sodium fluoride as an anticoagulant at 0 h (before treatment) and 1, 2, 4, 6, 8 and 16 h (after treatment). The plasma blood Table 1.
glucose levels were determined by using GOD–POD method.19) Oral Glucose Tolerance Test After overnight fasting, an 0-min blood sample (0.2 ml) was taken from the rats in normal, diabetic control, normalplant extract treated groups, diabeticplant extract treated groups by orbital sinus puncture.20) Glucose solution (2 g/kg) was administered orally immediately. Four more samples were taken at 30, 60, 90 and 120 min after glucose administration.21) Statistical Analysis All values were expressed as meanS.E.M. The differences were compared using oneway analysis of variance (ANOVA) followed by Dunnet’s test. p values 0.05 were considered as significant. RESULTS Effect on Normal Rats The effect of different doses of alcoholic extract of G. asiatica on fasting blood sugar levels in normal rats were assessed at different time interval. The percentage blood glucose reduction with 100, 250 and 500 mg/kg doses of G. asiatica at 6 h were 25.8%, 28.9% and 32.4% respectively and the results are shown in Table 1. Effect on Alloxan Induced Diabetic Rats The antihyperglycemic effect of different doses of alcoholic extract of G. asiatica on fasting blood glucose levels in diabetic rats were assessed at different time intervals. The percentage blood glucose reduction with 100, 250 and 500 mg/kg dose of G. asiatica at 6 h were 26.6%, 32.1% and 48.2% respectively. Tolbutamide 40 mg/kg dose produced 41.2% blood glucose reduction and the results are shown in Table 2. Effect on Oral Glucose Tolerance Test Table 3 shows the changes in the levels of blood glucose in normal, diabetic control and experimental groups after oral administration of glucose (2 g/kg). The diabetic rats showed that significant increase in the blood glucose at 60 min and 90 min. In G. asiatica treated animals blood glucose concentration was significantly (p0.05) decreased after 60 min and 90 min. G. asiatica treated animals tend to bring the values to near normal. G. asiatica (500 mg/kg) were more effective then Tolbutamide. Interaction of Nicorandil with Plant Extract In G. asiatica treated animals produced maximum percentage blood glucose reduction were found to be 32.45% in normal and 48.24% in diabetic rats at 6 h hour respectively. Nicorandil a K ATP channel activator produced percentage increase in blood glucose level at 6 h were found to be 18.74% and
Effect of Alcoholic Extract of Gmelina asiatica LINN. on Fasting Blood Glucose Levels of Normal Rats
Group
Dose mg/kg
Blood glucose levels (mg/dl) at different hours after the treatment 0h
1h
Control
—
93.51.1
92.71.1
Extract
100
99.71.4
90.70.3
Extract
250
97.30.9
87.80.5
Extract
500
96.50.7
88.10.2
2h
4h
6h
8h
16 h
91.41.3 (2.1) 80.60.5 (19.1)* 74.10.8 (23.7)* 79.30.6 (17.8)*
90.41.3 (3.4) 76.00.7 (23.7)* 72.10.7 (25.8)* 71.30.4 (26.1)*
89.11.5 (4.6) 74.00.1 (25.7)* 69.20.6 (28.8)* 65.20.6 (32.4)*
87.81.5 (6.1) 80.20.6 (21.7)* 76.40.4 (21.4)* 67.70.4 (29.9)*
85.61.5 (8.40) 87.30.5 (12.4)* 83.50.9 (14.6)* 73.90.4 (23.4)*
All values are expressed as meanS.E.M. Values given in the parenthesis are percent blood glucose reduction. ∗ Statistically significant p0.05 compared to 0 h of their respective group.
April 2005 Table 2.
731 Effect of Alcoholic Extract of G. asiatica on Fasting Blood Glucose Levels of Alloxan Induced Diabetic Rats Dose mg/kg
Group
Blood glucose levels (mg/dl) at different hours after the treatment 0h
1h
2h
Diabetic Control
—
277.415.1
273.414.9
270.714.6
Extract
100
259.64.73
244.34.73
230.42.2
Extract
250
274.73.9
262.83.1
242.31.7
Extract
500
334.823.9
316.322.3
286.023.4
40
270.16.0
237.83.7
222.33.5
Tolbutamide
4h
6h
8h
16 h
268.514.7 (0.2) 204.19.9 (21.3)* 212.62.8 (22.6)* 213.914.4 (36.1)* 214.95.2 (34.8)*
265.215.3 (4.3) 190.43.4 (26.6)* 186.35.3 (32.1)* 223.37.5 (48.2)* 158.65.1 (41.2)*
265.515.1 (4.2) 208.34.4 (19.7)* 218.65.5 (20.4)* 218.018.1 (34.8)* 174.93.3 (35.3)*
270.615.5 (2.4) 224.16.7 (13.6)* 235.84.3 (14.1)* 263.24.7 (21.3)* 224.14.8 (17.0)*
All values are expressed as meanS.E.M. Values given in the parenthesis are percent blood glucose reduction. ∗ Statistically significant p0.05 compared to 0 h of their respective group.
Table 3.
Oral Glucose Tolerance Test in Normal and Experimental Animals Blood glucose levels (mg/dl) Groups 0 min
Normal Diabetic control Diabeticextract Diabetictolbutamide
89.122.88 247.844.46 120.643.05 122.121.88
30 min
60 min
90 min
120 min
172.386.02* 322.245.47* 207.025.49* 207.14.62*
153.725.82* 377.0110.59* 183.667.66* 187.345.93*
124.923.14* 349.283.77* 149.065.11* 148.924.95*
95.383.28 316.663.57* 128.163.64 129.621.92
All values are expressed as meanS.E.M. ∗ Statistically significant p0.05 compared to 0 min of their respective group.
Table 4.
Effect of Alcoholic Extract of G. asiatica Alone and in Combination with Nicorandil in Normal Rats Percentage increase or decrease in blood glucose levels at different time intervals Treatment
G. asiatica Nicorandil G. asiaticanicorandil Tolbutamidenicorandil
Dose
500 mg/kg 10 mg/kg 500 mg/kg10 mg/kg 40 mg/kg10 mg/kg
1h
2h
4h
6h
8h
16 h
8.72 0.77 4.58 2.53
17.80 0.93↑ 2.18 5.78
26.12 6.68↑ 1.71* 5.48
32.45 18.74↑ 3.01* 8.33
26.5 11.01↑ 4.92* 6.16
16.5 1.25↑ 9.73 8.84
Significant difference with extract identical times ∗ p0.05. ↑ Percentage increase in blood glucose levels.
Table 5.
Effect of Alcoholic Extract of G. asiatica Alone and in Combination with Nicorandil in Diabetic Rats Percentage increase or decrease in blood glucose levels at different time intervals Treatment
Dose 1h
G. asiatica Nicorandil G. asiaticanicorandil Tolbutamidenicorandil
500 mg/kg 10 mg/kg 500 mg/kg10 mg/kg 40 mg/kg10 mg/kg
5.52 1.16↑ 0.48 0.77
2h
4h
6h
8h
16 h
14.58 7.09↑ 3.29 1.87
36.11 18.71↑ 4.38* 3.89
48.24 14.98↑ 10.74* 6.05
34.88 10.00↑ 11.22* 7.54
18.37 5.32↑ 14.98 7.70
Significant difference with extract identical times ∗ p0.05. ↑ Percentage increase in blood glucose levels.
14.98% respectively. Where as animals treated with nicorandil and G. asiatica produced 3.01% and 10.74% decreased in blood glucose level in normal and diabetic rats at 6 h hour respectively. In animals treated with tolbutamide and nicorandil produced 8.33% and 6.05% decrease in blood glucose level in normal and diabetic rats at 6 h respectively. The results are shown in Tables 4 and 5. Toxicity Evaluation In the acute toxicity study, the alcohol extract of G. asiatica did not show any mortality up to a
dose of 1 g/kg body weight in male mice. Even at this high dose there was no gross behavioral changes. Daily feeding for 14 d with the alcohol extract (200, 400 mg/kg) did not result in any change in general behavior of the animals. Body temperature and state of the stool were also not influenced by the drug treatment. Body weight, weight of liver, kidneys and spleen and food and water intake were not significantly altered by the drug administration.
732
DISCUSSION Alloxan has been observed to cause a massive reduction of the b -cells of the islets of Langerhans and induce hyperglycaemia.22) In our study we have found that G. asiatica decreases blood glucose in alloxan diabetic rats. The possible mechanism by which G. asiatica brings about its hypoglycaemic action may be potentiating the insulin effect of plasma by increasing either the pancreatic secretion of insulin from the b -cells of islets of Langerhans or its release from bound insulin. In this context a number of other plants have also been observed to have hypoglycaemic effects.23,24) The present study was conducted to evaluate the hypoglycemic and antidiabetic activity of G. asiatica, a very new herbal drug first time identified by us to get a berth in the group of antidiabetic herbal drugs. In this study the alcoholic extract of G. asiatica produced a dose dependent, percentage blood glucose reduction in normal and in diabetic groups. The percentage blood glucose reduction produced by the extract in diabetic group is greater than the percentage reduction observed in normal treated groups and G. asiatica extract 500 mg/kg produced better percentage blood glucose reduction than the tolbutamide in diabetic group. Nicorandil, a K ATP channel activator produce hyperglycemic by activating K ATP channels in pancreatic b cells. In the activated state the channels open much more frequently than in the ground state. The activation of K ATP channels lowers the plasma insulin level. There is evidence that ATP dependent K channel activator diazoxide lowers the plasma insulin level and this effect could be dose dependently counteracted by glibenclamide administration.25) Hence nicorandil produced hyperglycemic may be due to the reduction of plasma insulin level. Combined treatment with nicorandil, plant extracts and tolbutamide in normal and diabetic rats, the hypoglycemic and antihyperglycemic effects produced by plant extract and tolbutamide was inhibited. Tolbutamide by inhibiting K ATP channels in the pancreatic b cells, open much more frequently resulted in frequent depolarization. Depolarization results in opening voltage gated Ca channels and there by enhances the secretion of insulin from pancreatic b -cells. Nicorandil by activating the K ATP channels counteracted the tolbutamide induced hypoglycemic and antihyperglycemic effects; similarly nicorandil also counteracted the hypoglycemic and antihyperglycemic effects produced by alcoholic extract of G. asiatica. The results are clearly indicating the hypoglycemic and antihyperglycemic activities of G. asiatica and mechanism of action may be due to inhibition of K ATPase channels in pancreatic b -cell and subsequent increased insulin secretion or by enhanced glucose utilization by peripheral tissue. Further work is in progress to explain the possible mechanisms of action for its antihyperglycemic activity. Although it is the normal practice to determine the LD50 value, now it is acceptable to limit the study with an acute
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toxicity test using several doses including reasonably high doses of the drugs. In the present study, acute toxicity was tested up to a high concentration of 1 g/kg (two times more than the therapeutic dose). Even at this dose the herbal extract did not exhibit any sign of toxicity. Since the main purpose of the preliminary acute toxicity study is to get some idea on conspicuous behavioral changes and death, if any, and the alcoholic extract of G. asiatica did not exhibit any toxic symptoms in the limited toxicity evaluation in male mice. A perusal of literature shows that studies were not carried out on this plant with regard to other pharmacological properties and phytochemistry. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10)
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