Hypoglycemic effect of aqueous extract of Trichosanthes dioica in normal and diabetic rats

Background: Trichosanthes dioica is used to treat diabetes mellitus, epilepsy, alopecia, and skin disease in folklore medicine. The leaf extract of the plant is used in diabetes mellitus but there have been no scientifi c studies reported. Aims: To study the effect of Trichosanthes dioica on serum glucose level in glucose loaded, normal and hyperglycemic rats. Settings: Kasturba Medical College, Manipal, Karnataka, India. Design: Experimental. Materials and Methods: The aqueous extract of leaves of Trichosanthes dioica are compared with glibeclamide for their infl uence on fasting blood sugar in glucose loaded, normoglycemic and streptozotocin induced (45 mg/ kg ip) hyperglycemic rats. Statistical Analysis: The data was analyzed by one way ANOVA followed by Scheffe’s post hoc test. Results: In glucose loaded rats, normal rats and hyperglycemic rats the aqueous extract at both the doses (800 mg/kg/p.o and 1600 mg/ kg/p.o) reduced blood glucose signifi cantly when compared to control but it was not as effective as glibenclamide. Conclusion: The aqueous extract of Trichosanthes dioica has antihyperglycemic action.


Introduction
In India, Ayurvedic medicine is reported to have been successfully used in the treatment of diabetes mellitus. [5]s an alternative mode of treatment, Ayurvedic medicine has been claimed to be less toxic and more effi cacious.In accordance to the recommendations of World Health Organization (WHO) Expert committ ee on of Mahatma Gandhi Memorial College, Udupi, India aft er botanical identifi cation.The dried leaves were crushed into moderately coarse powder.A kilogram of the coarse powder was immersed in distilled water in a fl ask for seven days.The solid residue obtained by straining the liquid was pressed and fi ltered.The fi ltrate was concentrated on water bath to get a viscous paste. [16]t was fi nally dried in a dessicator.The yield was 4.25%.

Glucose tolerance tests
Fasted rats were divided into four groups of six animals each.Group I served as control and received distilled water.Group II and III received aqueous extract at an oral dose of 800 and 1600 mg/kg respectively.Group IV received the standard drug glibenclamide 0.5 mg/kg orally.
All the groups were loaded with 50% glucose (2 g/kg/p.o)30 minutes aft er drug administration. [17]Blood samples were collected from the tail vein just prior to drug administration and at 30, 60, 120 minutes aft er glucose loading.Serum glucose levels were measured immediately.

Hypoglycemic study in normal fasted rats
The eff ect of aqueous extract on fasting blood glucose was studied in normal rats.Animals were divided into eight groups of six rats each.Four groups received single dose of either distilled water, plant extract (800 mg and 1600 mg/ kg) or glibenclamide 0.5 mg/kg.The blood was collected at 30, 60 and 120 minutes aft er drug administration, to estimate blood sugar.The remaining four groups received the drug as above for 15 days.Fasting blood sample was collected on day 0,7,15 and 30 for estimating blood sugar.

Induction of diabetes
Streptozotocin (STZ) obtained from Sigma chemicals Co, was dissolved in 0.9% ice cold saline immediately before use.The rats were made to fast overnight, administered STZ 45 mg/kg intraperitoneally. [18]Fasting blood glucose levels were determined on day 12 aft er administering STZ to confi rm stable hyperglycemia.

Determination of effi cacy of aqueous extract of T. dioica in diabetic rats
The STZ induced diabetic rats were divided into four groups of six each.Group 1 received distilled water and served as control.Group II and III received aqueous extract of T. dioica at a dose 800 and 1600 mg/kg/p.orespectively.Group IV received the standard drug glibenclamide 0.5 mg/kg.The treatment was continued for 15 days.Fasting blood samples were collected on days 0, 7 and 15 of drug administration.Food and water intake was monitored daily for each rat during 15 days of experimental period.

Statistical analysis
Results are expressed as mean Ϯ SE.The data was analyzed by one way ANOVA followed by Scheff e's post hoc test using SPSS computer package.P Ͻ 0.05 were considered signifi cant.

Results
Eff ects of the extract on glucose loaded rats are shown in Table 1.It is observed that the treatment of T. dioica leaf extract at both the doses of 800 mg/kg and 1600 mg/ kg reduced blood sugar signifi cantly as observed at 30, 60 and 120 minutes when compared to normal control rats.Similarly, Glibenclamide treated rats also showed signifi cant blood glucose lowering effi cacy at all the time intervals.
Administration of the plant extract was found to reduce blood glucose level in normoglycemic rats both in single dose and repeated dose administration for 15 days [Tables 2 and 3].In a single dose study the maximum reduction in blood glucose was noted at one hour for 800 mg/kg, 30 minutes for 1600 mg/kg and the standard drug, glibenclamide.In the multidose study, the treated rats (extract/drug) showed significantly reduced blood glucose levels on days 7, 15 and the values returned to the pretreatment levels by day 30.
The Glibenclamide treated diabetic rats also showed a signifi cant reduction from 255 mg/dl to 184.33 on day 7 and to 113.66 on day 15.Though the hypoglycemic activity of the extract treated rats were signifi cant when compared to control it was not as eff ective as glibenclamide.
The eff ects of T. dioica extract on the body weight changes of diabetic rats are shown in Table 5.During the two weeks of observation of the treated diabetic rats there was weight loss, relative to day 0, i.e. before the start of the treatment.The untreated diabetic rats lost 16.38% of their body weight.The loss was 9.75% and 8% (P Ͻ 0.05) for 800 mg/kg and1600 mg/kg respectively, signifi cantly lower as compared to control.The diabetic rats treated with Glibenclamide also showed a bodyweight reduction of 5.5%, which is signifi cantly much lesser than control diabetic rats.The untreated diabetic rats had severe polyphagia and polydipsia by the end of week 2 of experiment with respective increase in food and fl uid intakes of 43 and 20%.However, in the presence of extract the food intake was reduced signifi cantly to 35% by day 7 and 51% by day 15(P Ͻ 0.05) with 800 mg/kg and 34% in week 1, 41% (P Ͻ 0.05) in week 2 with 1600 mg/kg as compared with diabetic control rats.A similar reduction of 37% in week 1 and 57% in week 2 was observed for glibenclamide, the standard drug [Table 6].
Fluid intake decreased by 32% (P Ͻ 0.05) in rats treated with both doses of T. dioica.Diabetic rats treated with glibenclamide also showed a signifi cant lower water intake of 48% (P Ͻ 0.05) as compared to control [Table 7].

Discussion
Diabetes is a chronic disease aff ecting millions of people world wide.The WHO expert committee has aptly suggested that research should be aimed at investigating the traditional methods of treatment for refractory    diseases like diabetes. [19]r study has detected the antidiabetic activity of aqueous extract of T. dioica in STZ induced type 2 diabetic rats.STZ is a valuable tool in the experimental production of non insulin dependent diabetes mellitus (NIDDM) model in lab animals.The mechanisms by which STZ brings about its diabetic state include selective destruction of pancreatic insulin secreting beta cells, which make cells less active. [20]The use of lower dose of STZ (45 mg/kg) produced an incomplete destruction of pancreatic beta cells even though rats became permanently diabetic. [21]The glucose loaded model indicates the kinetic functional changes induced by any antidiabetic drug.
When T. dioica aqueous leaves extract was administered to glucose-loaded normal rats, hypoglycemia was observed after 30 minutes, with maximum effects being seen at two hours.Our results also indicate the effi cacy of aqueous extract in the maintenance of glucose levels in normal and streptozotocin induced diabetic rats.However, it was less efficacious than glibenclamide.It is well established that sulphonylureas cause hypoglycemia by stimulating insulin release from pancreatic beta cells. [22]The comparable eff ect of the extract with glibenclamide suggests the possibility of a similar mode of action.
Induction of diabetes with streptozotocin is associated with a characteristic loss of body weight, which is probably due to muscle wasting. [23]In our study there was a signifi cant weight loss in the vehicle treated diabetic rats, where as treatment with the aqueous extract of leaves of T. dioica at both the doses showed improvement in their body weight, indicating that the aqueous extract had benefi cial eff ect in preventing loss of body weight of diabetic rats [Table 5].The probable mechanism of this benefi t is due to its eff ect in controlling muscle wasting, i.e., by reversal of antagonism. [24]The metabolic disturbances were corrected aft er the plant extract was administered for two weeks as shown by a reduction in polyphagia, polyuria and polydipsia in diabetic rats treated with plant extract.

Conclusion
The aqueous extract of the leaves of T. dioica has antidiabetic activity as it lowers serum glucose levels in diabetic rats and significantly increases glucose tolerance.It also increases body weight of diabetic rats.Further studies are necessary to substantiate the above observation and to work out the exact mechanism of action involved in the antidiabetic activity of this plant.