MATERIALS AND METHODS

Extraction of plant material: The plant Trianthema portulacastrum, was collected in the month of October, 2007 and the plant was taxonomically identified and authenticated as Trianthema Portulacastrum Linn. by Prof. Chelladurai, Research Botanist, Palayamkottai, Tamil Nadu, India. The whole plant was dried under shade and ground to a fine powder in a mechanical blender. The powder of the plant was initially extracted in a Soxhlet apparatus with petroleum benzene (60-80°C) to remove the chlorophyll followed by methanol by the method of continuous hot extraction to get the methanol extract of Trianthema portulacastrum (METP). This methanolic extract was stored at 2-8°C and used for subsequent experiments.

Phytochemical screening: The methanolic extract was screened for the presence of various phyto-constituents. Phytochemical properties of the extract were tested using the following chemicals and reagents. Alkaloids with Mayer and Dragendorff’s reagents, terpenoids, glycosides (NaCl and Fehling’s solution A and B), flavanoids (NaCl and HCl), saponins (frothing test), tannins (FeCl₃), phenolic compounds (FeCl₃ and K₃Fe(CN)₆) and carbohydrates (Molisch’s test) (Kokate, 1994; Harborne, 1998).

Acute toxicity studies: Acute toxicity studies were conducted in mice and rats using various doses of methanolic extract of TP up to 4000 mg kg^-1 and observed for the mortality and behavioral changes.

Animals: Wistar albino adult male rats, weighing 200-220 g were selected and housed in polypropylene cages in a room where the congenial temperature was 27±1°C and 12 h light and dark cycles were maintained. The animals were allowed to acclimatize to the environment for 7 days and supplied with a standard pellet diet (Hindustan Lever Ltd., Bangalore) and water ad libitum.

Activity in normoglycemic animals: A total of 30 normal overnight fasted rats were divided into five equal groups. The animals of group I served as an untreated control, group II was treated with glibenclamide (1 mg kg^-1) and group III, IV and V were treated with methanolic extract of T. portulacastrum (METP) at a dose of 100, 200 and 300 mg kg^-1 (single dose day^-1), respectively. Plasma glucose levels were estimated using a commercial kit (GOD-POD kit of Span Diagnostics, India) at 0th, 1st, 3rd and 7th day from the start of treatment and compared with untreated control. At the end of the study i.e., on 7th day, serum lipid levels were estimated in all untreated and treated groups of rats.

Activity in alloxan induced hyperglycemic (diabetic) animals: Diabetes mellitus/hyperglycemia was induced in Wistar rats by single intraperitoneal injection of 120 mg kg^-1 b.wt. of alloxan monohydrate (Sigma, USA), dissolved in normal saline (Ragavan and Krishnakumari, 2006). After 5 days, blood samples were collected by retro-orbital puncture under mild anesthesia and blood glucose levels were monitored. Rats showing serum glucose level above 200 mg dL^-1 were used for the antihyperglycemic evaluations and randomly divided into groups four groups of 6 animals each. Group I served as untreated diabetic control, group II diabetic rats treated with a standard oral hypoglycemic agent, glibenclamide (1 mg kg^-1) while group III, IV and V diabetic rats received 100, 200 and 300 mg kg^-1 of methanolic extract of TP. Blood samples were collected at 0th, 1st, 3rd and 7th day from the start of treatment for the estimation of blood glucose levels in all overnight fasted rats. At the end of the study i.e., on 7th day, serum lipid levels were estimated in all untreated and treated groups of rats.

Collection of blood samples and estimation of glucose and lipid profiles: The blood samples were collected from orbital plexus centrifuge tubes and serum was separated by immediate centrifugation of blood samples using REMI ultra cooling centrifuge at 3000 rpm for 5 min at room temperature and was directly used for estimating glucose, triglyceride, total cholesterol, HDL-cholesterol by using respective span diagnostic kits.

Statistical analysis: The results were expressed as Mean±SD. Statistical analysis were carried out using paired t-test and one-way ANOVA followed by Bonferroni’s test. Differences below p<0.05 implied statistically significance.

RESULTS

Preliminary phytochemical screening of the methanolic extract of T. portulacastrum reveals the presence of alkaloids, flavonoids, saponins, phenolic compounds and terpenoids. Different doses of methanolic extract were screened for their oral toxicity. No mortality was recorded till 4000 mg kg^-1 with methanolic extract, hence, the extracts were found to be safe up to the dose levels of 4000 mg kg^-1.

Table 1:	Effect of METP on plasma glucose levels of normoglycemic rats (mean±SD)
Values are in Mean±SD; n = 6; a: p<0.05, b: p<0.005, c: p<0.0001 vs. 0th day

Table 2:	Effect of administration with T. portulacastrum and glibenclamide on serum lipid parameter level in normal rats (on 7th day)
Values are in Mean±SD; n = 6; a: p<0.01, b: p<0.0001 vs. normal control; (TC: Total Cholesterol; TG: Triglycerides; HDL-C: HDL cholesterol)

Effect of methanolic extract of T. portulacastrum on normal rats: The hypoglycemic effect of methanolic extract of T. portulacastrum in normal rats was shown in Table 1. A significant reduction (p<0.05) in blood glucose levels was observed at 24 h post-administration of methanolic extract (100, 200 and 300 mg kg^-1 b.wt.) of T. portulacastrum in the normal rats; this was further lowered after 3, 5, 7 days of treatment. The maximum reduction in blood glucose level was seen at a dose of 300 mg kg^-1 b.wt. of T. portulacastrum extract administration. However, the hypoglycemic effect of methanolic extract of T. portulacastrum was comparable with that of standard oral hypoglycemic agent, glibenclamide.

As the blood glucose-lowering effect of METP was observed with both 100, 200 and 300 mg kg^-1, the effect of these doses on the lipid profile of normal rats were estimated and the results were shown in Table 2.

Similar to glibenclamide, administration of the methanolic extract led to a significant fall in the level of triglycerides, total cholesterol and improved the HDL levels in normal rats after 7 days of METP treatment.

Table 3:	Effect of METP on plasma glucose levels of hyperglycemic rats (Mean±SD)
Values are in Mean±SD; n = 6; a: p<0.005, b: p<0.0001 vs. 0th day

Table 4:	Effect of administration with T. portulacastrum and glibenclamide on serum lipid parameter level in diabetic rats (on 7th day)
Values are in Mean±SD; n = 6; a: p<0.0001 vs. normal control; (TC: Total Cholesterol; TG: Triglycerides; HDL-C: HDL cholesterol)

Effect of methanolic extract of T. portulacastrum on alloxan induced diabetic rats: In alloxan induced diabetic rats, the methanolic extract (METP) and glibenclamide produced significant antihyperglycemic effects (p<0.05) from day 1 onwards of their continuous administration and this antihyperglycemic effect was pronounced after 3, 5, 7 days of treatment (Table 3). In untreated diabetic rats, the plasma glucose levels on different days were significantly more (p<0.0001) than that of treated groups (group III, IV and V).

In alloxan-induced diabetic rats, there was a significant increase in the level of triglycerides (TG), total cholesterol (TG) and decreased HDL-C was observed. Continuous administration of the methanolic extract (300 mg kg^-1) of T. portulacastrum led to significant decrease in the level of triglycerides, total cholesterol in the diabetic rats, while increasing the level of HDL-C (Table 4).

DISCUSSION

Diabetes mellitus is possibly the world’s largest growing metabolic disease and as the knowledge on the heterogeneity of this disorder is advanced, the need for more appropriate therapy increases (Bailey and Day, 1989). Considerably, large number of hypoglycemic/antidiabetic plants and herbs are known through folklore but their introduction into modern therapy waits pharmacological testing by modern methods. The Study of such medicines might offer a natural key to unlock a diabetologist’s pharmacy for the future.

The present study results suggest that the methanolic extract of TP whole plant exhibited significant hypoglycemic, antihyperglycemic and hypolipidemic activities in normal and alloxan induced diabetic rats. Fasting blood glucose level in diabetic rats is an important basal parameter for monitoring diabetes (Rajkumar et al., 2005) and it has shown that the METP causes the hypoglycemic and antihyperglycemic effect by reducing the fasting blood glucose level (Table 1, 2). The significant decrease in the levels of fasting blood glucose in diabetic rats treated with the METP may be by stimulation of the residual pancreatic mechanism, probably by increasing peripheral utilization of glucose (Erah et al., 1996).

Prolonged administration of methanolic extract of T. portulacastrum lead to significant reduction in blood glucose levels. The hypoglycemic activity of the METP was due to the regeneration of pancreatic cells that were partially destroyed by alloxan and potentiation of insulin secretion from surviving b-cells of the islets of Langerhans (Suba et al., 2004).

The methanolic extract may increased secretion of insulin from b-cells of pancreas and results in of this increased secretion of insulin stimulates fatty acid biosynthesis and also the incorporation of fatty acids into triglycerides in the liver and adipose tissue (Best and Taylor, 1989).

Diabetes is associated with hyperlipidemia (De-Sereday et al., 2004). Alteration in the serum lipid profile is known to occur in diabetes and this is likely to increase the risk for coronary heart disease. A reduction in serum lipids, particularly of the LDL and VLDL fraction and triglycerides, should be considered as being beneficial for the long-term prognosis of these patients (Chattopadhay and Bandyopadhyay, 2005). Lowering of blood glucose and plasma lipid levels through dietary modification and drug therapy seems to be associated with a decrease in the risk of vascular disease.

Moreover, supplementation of methanolic extract of T. portulacastrum produced significant beneficial effects in the lipid profile in normal and diabetic rats by reducing triglycerides, total cholesterol and increasing HDL levels significantly (Table 2, 4). This effect may be due to low activity of cholesterol biosynthesis enzymes and or low level of lipolysis which are under the control of insulin (Sharma et al., 2003).

It is widely accepted that reduction in plasma HDL is a risk factor for developing atherosclerosis. HDL facilitates the translocation of cholesterol from the peripheral tissue, such as arterial walls to liver for catabolism. The increase in HDL may slow down the atherosclerotic process (Nofer et al., 2002). Increased levels of HDL (cardio protective lipid) after administration of TP extract concluded the cardio protective effect of METP.

The higher lipid levels seen in diabetic rats was due to increased mobilization of free fatty acids from peripheral depots and also due to lipolysis caused by hormones (Ei-Soud et al., 2007; Nikkila and Kekki, 1973). The methanolic extract may cause the regeneration of the b-cells of the pancreas and potentiation of insulin secretion from surviving b-cells; the increase in insulin secretion and the consequent decrease in blood glucose level may lead to inhibition of lipid peroxidation and control of lipolytic hormones. In this context, a number of other plants have also been reported to have antihyperglycemic, antihyperlipidemic and insulin stimulatory effects (Fernandes et al., 2007; Ramalingam and Pari, 2005).

In conclusion, the methanolic extract of T. portulacastrum produced significant hypoglycemic, antihyperglycemic and antihyperlipidemic activities in normal and alloxan induced diabetic rats. Further studies, are needed to identify the chemical constituents of the methanolic extract of T. portulacastrum that may be responsible for the hypoglycemic and hypolipidemic activity.