MATERIALS AND METHODS

Plant Material and Preparation of Extract
Fresh, mature M. koenigii leaves (Rutaceae) were collected from a plant in Attur, Tamil Nadu, India, after identification and authentication by Dr. V. Kaviyarasan, Centre for Advanced Studies in Botany, University of Madras and a voucher specimen (685) was deposited at the herbarium of Botany Department, University of Madras. The leaves were dried at room temperature, powdered and stored at 5°C until when needed. A 100 g of the powder was defatted with petroleum ether (60-80°C) overnight and re-extracted with 95% ethanol using soxhlet apparatus. Ethanol was evaporated in-vacuo using a rotary evaporator to give a 5.4% w/w yield.

Animals
Male albino rats of Wistar strain (160-180 g) obtained from Tamil Nadu Veterinary and Animals Sciences University, Chennai, India, acclimatized to animal house conditions. They were fed with commercial pelleted rat chow (Hindustan Lever Ltd., Bangalore, India) and had free access to water. All the rat experiments were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (IAEC 01/013/06).

Experimental
The rats were fasted overnight and diabetes was induced in rats with a single intraperitoneal injection of a freshly prepared solution of streptozotocin (55 mg kg^-1) in 0.1 M cold citrate buffer of pH 4.5 (Rakieten et al., 1963). Rats were supplied with 5% glucose solution for 48 h after STZ injection in order to prevent severe hypoglycemia. After one week of allowing for the development and aggravation of diabetes, the rats with moderate diabetes having persistent glycosuria and hyperglycemia (Blood glucose range of above 250 mg dL^-1) were used for the experiment. The treatment was started on the 8th day after STZ injection and this was considered as 1st day of treatment. The rats were grouped into four, comprising of six rats in each group as follows.

At the end of the experimental period of 37 days and after an overnight fasting, the rats were anaesthetized and sacrificed by cervical dislocation and blood was collected using EDTA as anticoagulant for the estimation of glucose level (Sasaki et al., 1972), glycosylated haemoglobin were estimated according to methods of Nayak and Pattabiraman (Nayak and Pattabiraman, 1981). The plasma was separated and used for the assay of insulin using RIA assay kit (for rats) supplied by Linco Research Inc., USA.

For enzymes and glycogen assay, a portion of the liver and kidney tissues were dissected out, washed with ice-cold saline immediately and the tissues were homogenized in 0.1 M Tris HCl buffer (pH 7.4). The homogenate was centrifuged at 10000 rpm to remove the debris and the supernatant was used as the enzyme source for the assays of hexokinase (Brandstrup et al., 1957), glucose-6-phosphatase(Koide and Oda, 1959), fructose 1,6-diphosphatase (Gancedo and Gancedo, 1971), lactate dehydrogenase (King, 1959), glycogen synthase (Leloir and Goldenberg, 1979), glycogen phosphorylase (Carnblath et al., 1963), glucose-6-phosphate dehydrogenase (Koide and Oda, 1959) and pyruvate kinase (Pogson and Denton, 1967), Another portion of wet liver tissue was used for the estimation of glycogen content (Carnblath et al., 1963).

Statistical Analysis
All the grouped data were statistically evaluated with SPSS/10.00 software. Hypothesis testing methods included one-way analysis of variance followed by Least Significant Difference (LSD) test. p<0.05 was considered to indicate statistical significance. All the results were expressed as mean±S.D for six rats in each group.

RESULTS

Levels of Blood Glucose, Glycosylated Hemoglobin and Insulin of Normal and Streptozotocin-diabetic Groups of Rats
There were significant increases in the levels of blood glucose, glycosylated hemoglobin and a concomitant decrease in the level of insulin in STZ-induced diabetic rats. The oral administration of the leaf extract and glibenclamide tended to bring these values back to those of the normal (control) rats (Table 1).

Activities of Glycolytic Enzymes in Normal and Streptozotocin-diabetic Groups of Rats
The significant decreases in the activities of hexokinase, pyruvate kinase and increase in lactate dehydrogenase in livers of STZ-diabetic rats over those of normal were reversed by the extract and glibenclamide as shown in Table 2. Similarly, the increased activities of these enzymes in the kidneys of the diabetic rats were significantly lowered by the extract and glibenclamide to values close to those of the normoglycaemic rats (Table 3).

Activities of Gluconeogenic and Glycogen Metabolic Enzymes in Normal and Streptozotocin-diabetic Groups of Rats
Significantly increased activities of hepatic glucose-6-phosphatase, fructose 1,6-diphosphatase and glycogen phosphorylase as well as concomitant decreases in the levels of glycogen and activities of lucose-6-phosphate dehydrogenase and glycogen synthase were observed in diabetic rats. Administration of the leaf extract, similar to that of glibenclamide, tended to revert the levels of glycogen and activities in the diabetic rats to those of normal rats (Table 4 and 5). The activities of the gluconeogenic enzymes such as glucose-6-phosphatase and fructose-1, 6-diphosphatase and glucose-6-phosphate dehydrogenase that were significantly increased in the kidneys of the diabetic rats were brought back to values of the normal rats after treatment with the extract and glibenclamide (Fig. 1).

Table 1:	Levels of blood glucose, plasma insulin and glycosylated haemoglobin in normal and streptozotocin-diabetic groups of rats
Values are given as mean±SD for groups of six rats in each. Values are statistically significant at *p<0.05 and #p<0.05. Statistical significance determined by ANOVA was compared within the groups as follows: *Diabetic control rats were compared with normal rats; #Experimental groups of rats were compared with diabetic control

Table 2:	Activities of hexokinase, pyruvate kinase and lactate dehydrogenase in livers of normal and streptozotocin-diabetic groups of rats
The enzyme activities expressed as: Hexokinase-μ moles glucose-6-phosphate formed/h/mg of protein. Pyruvate kinase (μ moles of pyruvate formed/min/mg of protein). Lactate dehydrogenase-μ moles pyruvate formed/h/mg of protein. Values are given as mean±SD for groups of six rats in each. Values are statistically significant at *p<0.05 and #p<0.05. Statistical significance determined by ANOVA was compared within the groups as follows: *Diabetic control rats were compared with normal rats; #Experimental groups of rats were compared with diabetic control

Table 3:	Activities of hexokinase, pyruvate kinase and lactate dehydrogenase in kidneys of normal and streptozotocin-diabetic groups of rats
The enzyme activities expressed as: Hexokinase-μ moles glucose-6-phosphate formed/h/mg of protein. Pyruvate kinase (μ moles of pyruvate formed/min/mg of protein). Lactate dehydrogenase-μ moles of pyruvate formed/h/mg of protein.Values are given as mean±SD for groups of six rats in each. Values are statistically significant at *p<0.05 and #p<0.05.Statistical significance determined by ANOVA was compared within the groups as follows: *Diabetic control rats were compared with normal rats; #Experimental groups of rats were compared with diabetic control

Table 4:	Activities of Glucose-6-phosphatase, fructose1, 6-di phosphatase and glucose-6 phosphate dehydrogenase in livers of normal and streptozotocin-diabetic groups of rats
Activities are expressed as: Glucose-6-phosphatase, fructose 1,6 di-phosphatase- μ moles of phosphate liberated/h/mg of protein; Glucose-6-phosphate dehydrogenase-μmoles of NADPH formed/min/g tissue. Values are given as mean±SD for groups of six rats in each. Values are statistically significant at *p<0.05 and #p<0.05. Statistical significance determined by ANOVA was compared within the groups as follows: *Diabetic control rats were compared with normal rats; #Experimental groups of rats were compared with diabetic control

Table 5:	Level of glycogen and activities of glycogen synthase and glycogen phosphorylase in livers of normal and streptozotocin-diabetic groups of rats
Values are given as mean±SD for groups of six rats in each. Values are statistically significant at *p<0.05 and #p<0.05.Statistical significance determined by ANOVA was compared within the groups as follows: *Diabetic control rats were compared with normal rats; #Experimental groups of rats were compared with diabetic control

Fig. 1:

Activities of glucose-6-phosphatase, fructose 1, 6-diphosphatase and glucose-6-phosphate dehydrogenase in kidneys of normal and streptozotocin-diabetic groups of rats. Activities are expressed as: Glucose-6-phosphatase, fructose 1, 6-diphosphatase-μ moles of phosphate liberated/h/mg of protein; Glucose-6-phosphate dehydrogenase-μmoles of NADPH formed/min/g tissue. Values are given as mean±SD for groups of six rats in each. Values are statistically significant at *p<0.05 and #p<0.05. Statistical significance determined by ANOVA was compared within the groups as follows: *Diabetic control rats were compared with normal rats; #Experimental groups of rats were compared with diabetic control

DISCUSSION

Streptozotocin is well known for its selective pancreatic islet β-cell cytotoxicity and has been widely used to induce diabetes mellitus in experimental rats. It interferes with cellular metabolic oxidative mechanisms (Papaccio et al., 2000).

Based on the histological observations made on the pancreatic tissue, the prevalence of diabetes was confirmed and the dose (200 mg kg^-1) was selected after preliminary behavioral and acute toxicity tests. The extract of the drug was found to be safe for biological studies as no lethality was observed at 1000 mg kg^-1 b.w in rats. Assay of pathophysiological enzymes such as asparatate aminotransferase, alanine aminotransferase, alkaline phosphatase and histological observations made on vital organs revealed the non-toxic nature of the plant extract. Similar results were also reported for normal rats by Adebajo et al. (2006). The report that sub-chronic administration of 200 mg kg^-1 of the extract for 14 days gave a better reduction in blood glucose and the enzymes studied (Adebajo et al., 2006), agreed with our choice of same dose and duration of treatment used in this present study.

Glibenclamide is often used as a standard drug in STZ-induced moderate diabetic models to compare antidiabetic properties of a variety of compounds and its effectiveness in insulin stimulation and by extra pancreatic effects is also reported (Andrade-Cetto et al., 2000). The elevated blood glucose concentration in the STZ induced diabetic rats clearly indicated persistent hyperglycemia in the rats used. However, treatment with M. koenigii leaf extract markedly lowered the blood glucose concentration in diabetic rats. The increase in plasma insulin stimulated by both the extract and glibenclamide suggested that the extract probably exerted its effect by stimulating insulin secretion from the remnant β-cells or from regenerated β-cells (Table 1). A number of other plants have also been reported to elicit their antihyperglycemic effects by a stimulatory effect on insulin release (Latha and Pari, 2003).

A significant increase in the level of glycosylated hemoglobin was reported in STZ-induced diabetes. The rate of glycosylation is proportional to the concentration of blood glucose. The peak of the glucose tolerance curve correlates with glycosylation and with the improvement of glycemic control, glycosylated hemoglobin also decreases (Koenig et al., 1976). The estimation of glycosylation of hemoglobin is a well accepted parameter useful in the management and prognosis of the diabetes (Alyassin and Ibrahim, 1981; Sheela and Augusti, 1992). The significant increase in the level of glycosylated haemoglobin observed in STZ-induced diabetic rats was lowered b y the extract and glibenclamide (Table 1). This further demonstrated the antihyperglycaemic effect of the plant (Koenig et al., 1976).

The administration of leaf the extract treatment to STZ-induced diabetic rats modulated key carbohydrate metabolizing enzymes in liver and kidney, resulting in normal blood glucose homeostasis. Hexokinase, that is responsible for the first phosphorylation step of glucose in glycolysis, was significantly reduced in the liver of diabetic rats resulting in the diminished utilization of glucose and the increased amount of blood glucose (Vestergaard, 1999). Enhanced hexokinase activity in M. koenigii treated rats suggested activation of glycolysis leading to greater uptake of glucose from blood by the liver cells and attendant increase in the utilization of glucose for energy production (Table 2).

Pyruvate kinase is an insulin dependent enzyme and its activity is regulated at the mRNA levels (Noguchi et al., 1985). The observed diminished levels of the plasma insulin and pyruvate kinase in Table 1 and 2, respectively, confirmed the diabetic states of the rats used. The increased activities of pyruvate kinase obtained with the diabetic rats fed with the extract (Table 2) might have led to enhanced glucose metabolism (utilization) in the rats.

Pozzilli et al.(1997) have shown increased activity of LDH in diabetes mellitus. Normal LDH activity is indicative of improved channeling of glucose (pyruvate) for mitochondrial oxidation and an abnormally high LDH activity showed a blockage in the glucose metabolism (Table 2). Hence, the reversal effects of M. koenigii, similar to glibencalmide, on the values of hexokinase, pyruvate kinase and LDH confirmed the ability of the plant to induce better glucose utilization in the diabetic rats.

In STZ-diabetic rats, the renal hexokinase, pyruvate kinase and LDH activities were significantly increased (Table 3) and were consistent with the raised renal glucose-6-phosphatease, fructose-1, 6-diphosphatase and glucose-6-phosphate dehydrogenase levels (Fig. 1). These results were further confirmed by the reduction in the glycogen content and glycogen synthase activity in the liver (Table 4). The extract and glibenclamide however successfully reversed this trend. Diabetic nephropathy is a serious microvascular renal complication in Type II diabetes and involves a series of metabolic events in its pathogenesis (Bolli, 1999).

Glucose-6-phosphatase catalyses the final step of glucose production in liver and kidney. Glucose-6-phosphatase and fructose-1,6-diphosphatase are regulatory enzymes of gluconeogenic pathway. The activities of both glucose-6-phosphatase and fructose-1,6-diphosphatase were increased in the liver and kidney tissues during diabetic condition resulting in a decrease of glycolytic flux. Administration of the leaf extract and glibenclamide significantly reduced the increased activities glucose-6-phosphatase and fructose 1,6-diphosphatase in these two organs during diabetic conditions (Table 4) similar to insulin treatment or pancreas transplantation (Makoff et al., 1983).Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of the Hexose Mono Phosphate (HMP) shunt pathway producing NADPH. The reversal of the changes in this enzyme activity achieved by the extract and glibenclamide in the diabetic rats, revealed improvement in the formation of NADPH, favouring lipogenesis and the use of an alternative channel to dispose excess glucose via the HMP pathway (39). These findings are similar to that of Ramachandran et al. (2003).

Diabetes mellitus is known to impair the normal capacity of the liver to synthesize glycogen (Roesler and Khandelwal, 1986). Glycogen synthase and glycogen phosphorylase are the two key regulatory enzymes that catalyze glycogen synthesis (glycogenesis) and degradation (glycogenolysis), respectively. An increased glycogen phosphorylase, a decreased glycogen synthase with the resultant decrease in the glycogen content was observed in STZ-diabetic rats. However, in the rats treated with the extract and glibenclamide, these effects were effectively reversed (Table 5). Hence, the M. koenigii leaf extract maintained the glucose homeostasis in STZ-induced diabetic rats additionally by altering the activities of carbohydrate metabolizing enzymes.

The findings of our study suggests that the administration of M. koenigii leaf extract to diabetic rats gives good control over key glycolytic enzymes. The changes in the activities of the enzymes effected by this extract suggest that a normal glucose metabolism, in peripheral tissues such as liver and kidney, is critical in achieving normoglycemia. Hence, the extract has anti-hyperglycaemic activity elicited by stimulation of insulin production from the pancreas and extra-pancreatic effects.