Antihyperglycemic Activity of Cyamopsis tetragonoloba. Beans on Blood Glucose Levels in Alloxan-Induced Diabetic Rats

Abstract

The effect of administering an ethanol extract of Cyamopsis tetragonoloba. Linn. (Leguminosae) beans on blood glucose levels in normal and alloxan-induced diabetic rats was evaluated in this study. The ethanol extract of beans at 250 mg/kg body weight significantly reduced the blood glucose levels in alloxan-induced diabetic rats within 3 h of administration. Continued administration of the extract at the same dose daily for 10 days produced a statistically significant reduction in the blood glucose levels, whereas marginal activity was seen in normal and glucose-loaded rats.

Keywords:

• Alloxan-induced diabetic rats
• beans
• Cyamopsis tetragonoloba
• hypoglycemic activity
• leguminosae

Introduction

Diabetes mellitus was known to ancient Indian physicians as madumeha.. Many herbal products including several metals and minerals have been described for the care of diabetes mellitus in ancient literature. The Ayurveda and Unani systems are ancient Indian forms of medicine that deal with plants and plant extracts. This indigenous form of medicine uses the active ingredients present in plants for treating diseases. Plant drugs are frequently considered to be less toxic and free from side effects. Many herbs have been shown to have hypoglycemic action in animals and humans (Prince et al., 1998).

Cyamopsis tetragonoloba. Linn. (Leguminosae) is a well-known traditional plant used in folklore medicine of India and is locally known as guar.. It acts as an appetizer, cooling agent, digestive aid, laxative, and galactagogue and is useful in dyspepsia and anorexia (Bakshi et al., 1995; Rastogi & Mehrotra, 1995). Antiulcer, antisecretory, cytoprotective (Rifatullah et al., 1994; Anonymous, 2001), hypoglycemic, and hypolipidemic effects (Bhardwaj et al., 1994; Sadhukhan et al., 1994; Bafna & Baghel, 1998; Frias & Sgarbieri, 1998; Bhandari & Sharma, 1999) are some of its reported biological activities. The pods contain condensed tannins together with p.-coumaric, caffeic, gallic, and gentisic acids, quercetin, kaemferol and its 3-arabinoside, p.-hydroxycinamyl, and coniferyl alcohol (Asolkar et al., 1992).

Because the beans of this plant have been recommended in traditional literature for the treatment of diabetes, it was considered worthwhile to investigate the effect of an ethanol extract of beans of C. tetragonoloba. on blood glucose levels in normal-glucose loaded, normal, and alloxan-induced diabetic rats, and to compare it with gliclazide as a reference drug.

Materials and Methods

Plant material

Cyamopsis tetragonoloba. beans were purchased from a local vegetable market (Okhla, Delhi) and their identity was confirmed at the Taxonomy Division, Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi. The voucher (JH-CT-27) specimen was deposited in the pharmacognosy laboratory for reference.

Preparation of the ethanol extract

The dried and powdered beans (200 g) of C. tetragonoloba. were extracted with ethanol by refluxing in a boiling water bath for 6 h, then concentrated under reduced pressure to yield a dry mass of 17.20 g (8.6%).

Test animals

Wistar rats (180–220 g) of either sex were used in the experiment. They were procured from Central Animal House, Jamia Hamdard, New Delhi (173/CPCSEA), after approval under project no. 135 and housed at standard environmental conditions of temperature, relative humidity, and dark/light cycle. They were fed standard diet (Hindustan Lever, Mumbai, India) and water ad libitum. at the animal house. The animals were fasted for 16 h prior to experiment with water ad libitum..

Experimental design

Effect of test drug on oral glucose tolerance

Fasted normal rats were divided into three groups of five animals each. Group I served as control and received normal saline. Group II received the reference drug, gliclazide, at an oral dose of 25 mg/kg body weight, and group III received ethanol extract at an oral dose of 250 mg/kg body weight. After 30 min of extract administration, the rats of all the groups were orally treated with 2 g/kg body weight of glucose. Blood samples were collected from the tip of the tail just prior to glucose administration and at 30 and 90 min after glucose loading. Serum was separated, and blood glucose levels were measured immediately by the glucose oxidase method (Varley et al., 1980).

Effect of test drug on blood glucose levels in normal fasted rats

Fasted rats were divided into three groups of five animals each. Group I served as control and received only vehicle (i.e., normal saline) orally. Group II served as standard and received the reference drug, gliclazide at an oral dose of 25 mg/kg body weight, and group III served as test and received the extract at an oral dose of 250 mg/kg body weight.

Induction of diabetes

Hyperglycemia was induced by a single i.p. injection of 120 mg/kg of alloxan monohydrate (S. D. fine-chem. Ltd., Mumbai, India) in sterile saline (Joy & Kuttan, 1999). After 5 days of alloxan injection, the hyperglycemic rats (glucose level > 300 mg/dl) were separated and used for the study.

Acute treatment

The hyperglycemic rats were divided into three groups of five diabetic animals each. Group I was previously selected from normal rats and served as normal control and was given normal saline and no alloxan. Group II served as diabetic control and was given normal saline. Group III received reference antidiabetic drug gliclazide at an oral dose of 25 mg/kg body weight (Panacea Biotech Ltd., Chandigarh, India batch no. 01030513). Group IV was treated orally with ethanol extract at a dose of 250 mg/kg body weight; the dose was selected after preliminary behavioral and acute toxicity tests. Blood samples were collected from the tip of tail just prior to and at 1 and 3 h after the extract/reference drug administration.

Subacute treatment

In subacute treatment, the administration of extract/reference drug was continued for 10 days, once daily. Blood samples were collected from the tip of the tail just prior to and on days 1, 3, 7, and 10 of the extract/reference drug administration. The blood glucose levels were determined immediately.

Statistical analysis

Data was subjected to statistical analysis using one-way analysis of variance (ANOVA) followed by Dunnet's t.-test. p < 0.05 indicates significant differences between group means.

Results and Discussion

Effect of ethanol extract of C. tetragonoloba. on glucose tolerance is shown in Table 1. At 30 min after glucose administration, the blood glucose concentration increased rapidly from the fasting value and then attained nearly the same value at the end of the study (i.e., at 90 min). Administration of gliclazide (25 mg/kg body weight) prior to glucose loading, induced a time-dependent and statistically significant hypoglycemic effect.

Table 1 Effect of C. tetragonoloba. beans, ethanol extract (250 mg/kg, p.o.), on oral glucose tolerance in normal rats^a..

	Blood glucose level (mg/dl)
Sample time (min)	Vehicle (Normal saline)	Gliclazide (25 mg/kg)	Ethanol extract (250 mg/kg)
0	65.00 ± 1.00	62.4 ± 2.17	69.60 ± 2.15
30	96.00 ± 2.30	63.4 ± 2.30	82.80 ± 2.33^NS
90	78.40 ± 2.69	48.0 ± 2.15**	81.00 ± 2.44^NS

**

The test extract of C. tetragonoloba. showed a marginal antihyperglycemic effect on blood glucose levels in normal fasted rats (Table 2). Gliclazide exerted a statistically significant hypoglycemic effect in normal fasted rats.

Table 2 Effect of C. tetragonoloba. beans, ethanol extract (250 mg/kg, p.o.), on blood glucose level in normal rats^a..

	Blood glucose level (mg/dl)
Sample time (h)	Vehicle (Normal saline)	Gliclazide (25 mg/kg)	Ethanol extract (250 mg/kg)
0	51.60 ± 2.58	53.40 ± 3.14	57.40 ± 2.29
1	66.40 ± 6.04	38.0 ± 2.70	64.40 ± 0.92^NS
3	71.0 ± 3.60	40.40 ± 2.52	65.60 ± 2.24^NS

*

Oral administration of the ethanol extract led to a significant blood glucose lowering effect in alloxan-induced hyperglycemic rats (Table 3). The fall was seen at 1 h and remained up to 3 h after administration of the extract, whereas the fall in case of gliclazide administration was marginal because alloxan treatment causes permanent destruction of β-cells (Pari & Maheshwari, 1999) and gliclazide requires more than 30% functional pancreas for the effect.

Table 3 Effect of acute treatment of C. tetragonoloba. beans, ethanol extract (250 mg/kg, p.o.), on blood glucose level in alloxan-induced diabetic rats^a..

	Blood glucose level (mg/dl)
Sample time (h)	Vehicle (Normal saline)	Gliclazide (25 mg/kg)	Ethanol extract (250 mg/kg)
0	353.80 ± 2.30	329.00 ± 2.57	357.80 ± 3.61
1	352.60 ± 2.13	315.60 ± 2.70^NS	219.60 ± 2.31
3	349.20 ± 2.08	308.20 ± 2.52^NS	159.40 ± 2.44

*

Subacute treatment with the ethanol extract of C. tetragonoloba. on alloxan-induced hyperglycemic rats produced consistent reduction in the blood glucose levels (Table 4). Again, the hypoglycemia shown by gliclazide was marginal, as observed in the acute treatment.

Table 4 Effect of subacute treatment of C. tetragonoloba. beans, ethanol extract (250 mg/kg, p.o., once daily), on blood glucose level in alloxan-induced diabetic rats^a..

	Blood glucose level (mg/dl)
Sample time (Day)	Vehicle (Normal saline)	Gliclazide (25 mg/kg)	Ethanol extract (250 mg/kg)
0	353.80 ± 2.30	329.00 ± 2.57	357.80 ± 3.61
1	352.80 ± 2.13	303.80 ± 2.70^NS	119.40 ± 3.45
3	349.20 ± 2.08	306.40 ± 2.82^NS	175.00 ± 2.09
7	354.40 ± 2.52	308.50 ± 1.89^NS	164.60 ± 2.53
10	356.80 ± 2.06	304.60 ± 2.17^NS	152.40 ± 2.93

*

It is generally accepted that sulfonylureas, including gliclazide, produce hypoglycemia in normal animals by stimulating the pancreatic β-cells to release more insulin. These drugs, however, do not release blood glucose in alloxan diabetic animals. In contrast to the oral antidiabetic agents, the exogenous administration of insulin is known to produce hypoglycemia in both normal and alloxan-induced rats. It is, therefore, conceivable that the hypoglycemic principle(s) in the ethanol extract of C. tetragonoloba. exert a direct effect in diabetic rats. In diabetic rats the ethanol extract cannot act indirectly by stimulating the release of insulin because alloxan treatment causes permanent destruction of β.-cells (Pari & Maheshwari, 1999). The antihyperglycemic effect in the alloxan-diabetic rats suggests that its primary mechanism may not be due to potentiation of insulin release from pancreatic cells, and, thus, the drug may be effective in insulin-independent diabetes.

In conclusion, our observations suggest that the antihyperglycemic effect of C. tetragonoloba. beans, ethanol extract, may be due to presence of tannins, coumarins, or flavonoids, which are present in the beans. Further detailed studies are needed to identify the active principle(s) responsible for the antihyperglycemic effect and to understand the mechanism of action involved.

Acknowledgments

The authors wish to thank the UGC, New Delhi, for providing financial support and Panacea Biotech Ltd. for providing a complimentary sample of gliclazide.

Notes

^a.Values are means ±SE; n = 5, *p < 0.001, **p < 0.01, NS, no significant difference relative to the vehicle.

^a.Values are means ±SE; n = 5*p < 0.001, NS, no significant difference relative to the vehicle.

^a.Values are means ±SE; n = 5*p < 0.001, NS, no significant difference relative to the vehicle.

^a.Values are means ±SE; n = 5*p < 0.001, NS, no significant difference relative to the vehicle.