MATERIALS AND METHODS

Animals: Albino rats of both sexes, weighing from 150-250 g, were purchased from H.E.J. Research Institute of Chemistry, University of Karachi, Pakistan and were kept under usual management conditions in conventional animal house of Department of Biochemistry, University of Karachi, Pakistan. Rats were given standard labory doratiet and free access to water ad libitum.

Alloxan tetrahydrate (Sigma): Diabetes was induced in experimental rats by freshly prepared doses of alloxan tetrahydrate (120 mg kg^-1) for 3 consecutive days intra-peritoneally (Qureshi et al., 2009).

Chlorpropamide (Sigma): It was used as positive control (20 mg kg^-1) in present study (Cunha et al., 2008).

Reagent kits for biochemical analyses (Human and Randox): Glucose levels were analyzed by glucocard memory 2 (glucometer; Arkray, Inc. Japan). Other parameters viz., triglycerides (GPO-PAP method), total cholesterol (CHOD-PAP method) and alanine aminotransferase (Reitman-Frankel colourimetric method) were determined by commercially available reagent kits (Randox, United Kingdom and QCA, Spain, respectively).

Plant material: Roots of R. serpentina were purchased from Hamdard Dawakana, Sardar, Karachi and identified by experts in Botany Department, University of Karachi, Karachi-75270, Pakistan. The voucher specimen has been kept in our department (KU/BCH/SAQ/02).

Preparation of methanolic root extract: Grinded powder (40 g) of roots of R. serpentina was extracted with methanol (1 L; 95%) overnight and filtered through Whatman No. 1 filter paper twice. The filtrate was then concentrated till dryness in a rotary vacuum evaporator (Eyela-NE) to obtain a brown residue that referred as methanolic root extract (Ju et al., 2008).

Experimental procedure: Experimental rats were divided into 3 groups (6 rats of both sexes/group) according to the treatments (Fig. 1). The rats were deprived of food but not water for over night (12-14 h) before starting the experiment. The rats were kept fasted during the whole experiment. After intra-peritoneal injection of each treatment, glucose was monitored at intervals of 0, 1, 2, and 4 h with the help of glucocard memory 2 (glucometer) by pricking the vein of their tails (Cunha et al., 2008).

Fig. 1:	Grouping of experimental rats according to treatments

Finally rats were decapitated at 4 h to collect the blood, serum was separated that used to analyze other biochemical parameters viz., TC, TG and ALT. Dimethyl sulfoxide (5% DMSO) was used as negative control and vehicle for methanolic root extract (Saleem et al., 1999). The whole experimental research from preparation of methanolic root extract to statistical analysis of biochemical parameters was conducted in Department of Biochemistry, University of Karachi from January to December 2008.

Statistical analysis: The data were analyzed by Student’s t-test (Graphpad Software, Quick Calcs Online calculators for Scientists). Differences were considered significant with p<0.0001, p<0.01 and p<0.05. Values are expressed as Mean±Standard Error Mean (SEM).

RESULTS AND DISCUSSION

Diabetes and hypertension go together (Konzem et al., 2002). Researchers are interested to find out the cure of diabetes which also reduced the risk of chronic complications of this disease (Kim et al., 2006). Studies have already been published related to different medicinal plants/herbs in reducing the blood glucose levels in alloxan-induced diabetic rats (Pari and Maheswari, 1999; Kim et al., 2006; Qureshi et al., 2009). In the present study, methanolic root extract of R. serpentina (30 mg kg^-1) induced a significant decrease (p<0.0001) in serum glucose levels of alloxan-induced diabetic rats as compared to diabetic control group (Table 1). The acute reduction in glucose levels of alloxan-induced diabetic rats after single intra-peritoneal injection of extract showed in a time-dependent manner, which is as significant as decrease in glucose levels observed in chlorpropamide-treated diabetic rats. However, the effect of extract was persists up to 4 h interval of treatment as compared to chlorpropamide-treated group (Table 1). These findings are significant as compared to the previous hypoglycaemic results, reported that different fractions of Rauwolfia alkaloids first induced slight increase and then decrease in blood sugar levels of anesthetized cats (Chatterjee et al., 1960).

Diabetes was induced in experimental rats by alloxan, is known to destroy β-cells of pancreas and inhibit insulin production (Kamanyi et al., 1994). It has been confirmed by observing increase levels of glucose (289-298 mg dL^-1) in diabetic controls rats. On the contrary, it is also interesting to observe decrease glucose levels in chlorprapamide-treated diabetic rats, the drug known to decrease glucose level by stimulating βcells (Rang et al., 2003). Therefore, it might possible that few β-cells were remained alive and their function were improved by chlorpropamide, the work is still in progress to prove this claim.

Table 1:	Effect of methanolic root extract of R.serpentina on serum glucose levels in alloxan-induced diabetic rats
Values are expressed as Mean±SEM (n = 6). Diabetic Test (DT) and Positive Control (PC) compared with their respective diabetic control (***p<0.0001 and *p<0.05)

Table 2:	Serum Total cholesterol (TC), triglycerides (TG) and alanine aminotransferase (ALT) in alloxan-induced diabetic rats. Rats were decapitated after 4 h of intra-peritoneal injection of each treatment (n = 6)
Diabetic test (DT) and Positive control (PC) compared with their respective diabetic control (*p<0.05 and **p<0.01)

The following mechanisms have been proposed for hypoglycemic effect of extract in test rats viz., to inhibit hepatic glucose production, to inhibit intestinal glucose absorption, to enhance glucose absorption in muscles and adipose tissues or to correct insulin resistance (Kamanyi et al., 1994; Ju et al., 2008; Qureshi et al., 2009). The possibility of β-cells regeneration has not been ruled out as many medicinal plants were reported to regenerate β-cells leading to an increased insulin production and secretion (Daisy et al., 2004).

Alloxan-induced hyperglycaemia is also accompanied with hypertriglyceridemia and hypercholesterolemia (Ju et al., 2008). In the present study, TC (p<0.05) and TG (p<0.01) were significantly decreased in test rats by methanolic extract as compared to diabetic controls (Table 2). While a significant decrease (p<0.01) was only observed in serum TG level of chlorpropamide-treated rats (Table 2). The significant effect of methanolic extract on diabetic hypertriglyceridemia could be due to its effect on glycemic control. It has also been confirmed by observing the decrease in TG levels of chlorpropamide-treated rats. The improved reduction in glucose levels by sulfonylureas accompanied with decrease serum VLDL and TG levels has already been reported by Roa et al. (1999). On the other hand, the reduction in cholesterol level in test rats may be due the inhibitory effect of methanolic extract on 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG CoA reductase), the rate-regulatory enzyme of cholesterol biosynthesis (Sharma et al., 2003; Ju et al., 2008) or by stimulating effect on glucose utilization by peripheral tissues (Kamanyi et al., 1994).

Rauwolfia is used to treat numbers of brain disorders where it known to depletes catecholamines from nerves in central nervous system (Craig et al., 1999).

Catecholamines are not only hypertensive but also hyperglycaemic in action by enhancing glycogenolysis in liver and muscles, gluconeogenesis, lipolysis in adipose tissues, stimulating glucogen secretion while inhibiting hepatic glycogen formation and insulin secretion (Rang et al., 2003). Therefore, it might be possible that methanolic root extract has also one indirect effect in reducing glucose and lipid contents in alloxan-induced diabetic rats. No depression or sedation was observed in test rats during the experiment. Alanine aminotransferase (ALT) is the liver-specific enzyme and its high concentration in blood represents the damage of hepatocytes (Bishop et al., 2005). Chronic mild elevation of aminotransferases is frequently found in type 2 diabetic patients (Harris, 2005). In the present study, methanolic root extract-treated rats were showed normal or decreased levels of ALT as compared to diabetic control and chlorpropamide-treated rats (p<0.05).

In conclusion, the present investigation proved that the methanolic root extract of R. serpentina was found to have hypoglycaemic and hypolipidemic effects (both hypotriglyceridemic and hypocholesterolemic) in alloxan-induced diabetic rats. In addition, the extract has hepato-protective effect by observing normal serum levels of ALT. Due to the significant effect of methanolic extract on alloxan-induced diabetic hyperglycaemia and hyperlipidemia, work is still going on in lab to investigate the same effects in different fractions of methanolic extract in order to isolate an active principle having same activity with no side effects followed by establishing the most probable mechanism of action of reducing serum glucose and lipids.

ACKNOWLEDGMENTS

Authors are highly thankful to University of Karachi for providing research grant for conducting this study and Professor Dr. Viqar Sultana, Biotechnology and Drug Development Laboratory, Department of Biochemistry, of the same university for providing facility of rotary vacuum evaporator for the preparation of methanolic root extract. Special thanks also go to Ms. Tooba Lateef, Lecturer of Biochemistry department, Jinnah University for Women, Karachi-74600 for providing help in finding out literature related to the research topic.