MATERIALS AND METHODS

Plant material: Aerial part of Hibiscus rosasinensis collected from the botanical garden of S.N. Institute of Pharmacy, Pusad, India. Identification and authentication of the samples was done by using standard botanical monographs. They were further confirmed with the Department of Botany, R.S.T.M University Nagpur.

Preparation of crude extract and fractionation: The plant material was cleaned off adulterants; shade dried and was coarsely grounded. The powdered material (1 kg) was soaked in 80% aqueous-ethanol for 3 days with occasional shaking. It was filtered through a muslin cloth and then through a filter paper. This procedure was repeated thrice and the combined filtrate was evaporated on a rotary evaporator under reduced pressure (-760 mmHg) to a thick, semi-solid mass of dark brown color; i.e., the crude extract (Hr.Cr) with a yielding of approximately 10% (Gilani et al., 2005).

For the purpose of fractionation, 20 g of the crude extract was dissolved in a minimum amount of 80% aqueous-ethanol and loaded on silica gel as inert support in the proportion of 1:20. Dried silica gel was packed in a chromatographic column and successively eluded with solvents of increasing polarity to get petroleum ether, ethyl acetate and aqueous fractions. Individually collected fractions were evaporated on rotary evaporator to give the fractions with yield of 9.2, 6.1 and 66%, respectively.

Phytochemical investigation: The preliminary phytochemical studies (Rathi et al., 2003) were conducted for the above extracts of H. rosasinensis to find out the presence of Sterols, Carbohydrates and glycosides, Tannins, Flavonoid was carried out using standard test (Gupta et al., 2009).

Selection of animals: Wistar albino rats of either sex weighing between 160-180 gm were used. Institutional Animal Ethics Committee approved the experimental protocol; animals were maintained under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). Albino rats were used in this project was obtained from the Animal House of S.N.Institute of Pharmacy, Pusad. The animals were housed in Poly propylene cages and maintained at 24±2°C under 12 h light/dark cycle and were feed ad libitum with standard pellet diet and had free access to water.

Experimental procedure: The animals, irrespective of sex, between 2-3 months of age with body weight ranging between 160 to 180 g were distributed into four groups (with eight animals in each group) as follows: I control group, II diabetic control group and III diabetic group treated with 500 mg kg^-1 body weight extract of H. rosasinensis IV diabetic group treated with Insulin 6 units/kg body weight of rats/day. Animals of group II, III and IV were rendered diabetic by a single intraperitoneal (i.p.) injection of 65 mg kg^-1 of Streptozotocin (STZ) (Al-Attar and Zari, 2007) freshly prepared in 0.1M of citrate buffer (pH 4.5). Group I was injected with buffer alone. (Sharma et al., 2006). After 72 h, of STZ injection blood was drawn from the tail of conscious rats and the glucose content was estimated with glucometer. Only those rats with blood glucose above 250 mg dL^-1 were selected for the study. 15 days after the STZ injection, animals of group III and IV received extract of H. rosasinensis (500 mg kg^-1, respectively) and insulin (6 unit kg^-1) for 4 weeks.

At the end of the experiment, blood was collected into heparinised tubes and the plasma and serum were separated by centrifugation. The liver and kidney were quickly removed, washed in ice-cold, isotonic saline and blotted individually on ash-free filter paper and the organ weights were measured (Atangwho et al., 2007). The tissues were then homogenised in 0.1 M Tris-HCl buffer, pH 7.4. The homogenate was used for the estimations of proteins, enzymes and other parameters. Blood glucose, urea, uric acid and creatinine were estimated using a commercial diagnostic kit (Ranbaxy Laboratories, New Delhi, India). The albumin and globulin contents were estimated by the method described by Reinhold (1980).

The enzymes, AST, ALT and ALP, were assayed by the method of King and Armstrong (1988) and γ-glutamyl transpeptidase (γ-GT) was assayed by the method of Rosalki and Rau (1972). The protein content in the plasma, liver and kidney were estimated by the method of Lowry et al. (1951). All spectrophotometric measurements were carried out in a UV-visible spectrophotometer.

Hypothesis testing methods included one way Analysis of Variance (ANOVA) followed by least significant differences test. P-values of less than 0.05 were considered to indicate statistical significance. All the results were expressed as Mean±SD for eight animals in each group.

RESULTS

A significant increase in the level of blood glucose, a decrease in plasma insulin and C-peptide were observed in diabetic rats when compared to control rats. Administration of H. rosasinensis (500 mg kg^-1) and insulin to diabetic rats significantly decreased the level of blood glucose to near control level. Table 1 demonstrates the levels of protein, plasma albumin and albumin/globulin ratio in control and STZ-diabetic rats. The level of protein in plasma was found to be reduced in diabetic animals (p<0.05) when compared to control animals. The lowered level of protein, after H. rosasinensis treatment, increased to near control. The levels of albumin and albumin/globulin ratio in plasma were decreased in diabetic animals. These lowered levels of plasma albumin and albumin/globulin ratio level were restored significantly in H. rosasinensis-treated diabetic rats.

Urea, uric acid and creatinine levels were significantly elevated in STZ-DM in rats (p<0.05) when compared to control animals. Oral administration of H. rosasinensis extract for 4 weeks significantly lowered urea, uric acid and creatinine levels in STZ-diabetic rats. Table 2 shows the activities of AST, ALT, ALP and γ-GT in plasma, liver and kidney of control and STZ-diabetic rats. The activities of these enzymes were found to be significantly increased (p<0.05) in the plasma and liver of diabetic rats. In the kidney of diabetic animals, the activities of ALP and γ-GT were increased while the activities of AST and ALT were not altered. Oral administration of H. rosasinensis for 30 days resulted in the near normalisation of the activities of AST, ALT, ALP and γ-GT in the plasma, liver and kidney of diabetic rats.

Table 1:	Different levels ratio in control and STZ diabetic
Values are given as Mean±SD for groups of eight animals each. Values are statistically significant at *p<0.05. Diabetic rats were compared with control rats, H. rosasinensis-treated diabetic rats were compared with diabetic rats, insulin-treated diabetic rats were compared with diabetic rats

Table 2:	The activities of plasma, kidney and liver
Values are given as Mean±SD for groups of eight animals each. Values are statistically significant at *p<0.05. Diabetic rats were compared with control rats, H. rosasinensis-treated diabetic rats were compared with diabetic rats, insulin-treated diabetic rats were compared with diabetic rats. Units of measurement (per L) for AST and ALT: μmol of pyruvate liberated/hr, ALP: μmol of phenol liberated/min, γ-GT: mol of pnitroaniline liberated/min

Fig. 1:	Photomicrograph of liver section of rats, control (a) and STZ (b), H. rosasinensis extract treated (c). The specimens were stained with Hematoxylin and Eosin

Histological results
Liver: By light microscopy, liver of the STZ treated diabetic rats showed 2-3 foci of interlobular lymphocytes predominant inflammatory cells infiltration per x100 magnifications as compared by necrosis and apoptosis of few hepatocytes. Mild lymphocytic infiltration and congested vessel intlemajority of portal spaces were noted. Histological examination of livers of the diabetic rats treated by H. rosasinensis extract, showed gradual significant reduction in parenchymal and portal inflammation and lymphocytes were replaced by few eosinophils, the hepatic tissue appeared somewhat like the control and the Insulin treated groups (Fig. 1a-c).

Fig. 2:	Photomicrograph of Kidney section of rats, control (a) and STZ (b), H. rosasinensis extract treated (c). The specimens were stained with Hematoxylin and Eosin

Kidney: Histological examination of the STZ-induced diabetic rats’ renal tissue compared to the controls groups revealed mild increase in mesangial cells and matrix of glumeroli. Hyaline thickening of some arteriole wall was noted. By H. rosasinensis extract these pathologic changes improved toward to the Insulin treated groups (Fig. 2a-c).

DISCUSSION

The present investigation indicates the hypoglycaemic and protective effects of H. rosasinensis leaves in the liver and kidney of STZ-diabetic rats. We have observed a significant decrease in blood glucose in H. rosasinensis-treated diabetic rats, when compared with diabetic control rats. The optimum dosage (500 mg kg^-1) was standardized and confirmed by a previous study with significant hypoglycaemic activity. The possible mechanism of H. rosasinensis hypoglycaemic action may be through potentiation of pancreatic secretion of insulin from β-cell of islets or due to enhanced transport of blood glucose to the peripheral tissue (Saravanan and Pari, 2008). This was clearly evidenced by the increased level of insulin in diabetic rats treated with H. rosasinensis.

Reduction in plasma total protein and albumin level was observed in diabetic rats and this is consistent with the results obtained by Bakris (1993) and Tuvemo et al. (1997). The decrease in protein and albumin may be due to microproteinuria and albuminuria which are important clinical markers of diabetic nephropathy (Mauer et al., 1981) and/or may be due to increased protein catabolism (Almdal and Vilstrup, 1988). The results of the present study demonstrated that the treatment of diabetic rats with the extract of H. rosasinensis caused a noticeable elevation in the plasma total protein and albumin levels as compared with their normal levels (Safiyeh et al., 2007). Such improvement of serum protein and albumin was previously observed after the oral administration of Balanites aegyptiaca (B. aegyptiaca) to experimentally diabetic rats (Mansour and Newairy, 2000). It has been established that insulin stimulates the incorporation of amino acids into proteins (Almdal and Vilstrup, 1988).

C-peptide and insulin are the products of the enzymatic cleavage of proinsulin and secreted into the circulation in equimolar concentrations. The measurement of both C-peptide and insulin levels have been reported to be a valuable index of insulin secretion rather than insulin alone. In this study, the plasma C-peptide and insulin levels were significantly higher in the H. rosasinensis than in the DM group.

The plasma levels of urea, uric acid and creatinine levels were measured, as DM also causes renal damage due to abnormal glucose regulation, including elevated glucose and glycosylated protein tissue levels, haemodynamic changes within the kidney tissue and increased oxidative stress (Aurell and Bjorck, 1992). The STZ-induced diabetic rats exhibited significantly higher plasma urea, uric acid and creatinine levels compared to the DM group. However, the H. rosasinensis supplement lowered these plasma values to a control range. A significant elevation in serum creatinine and urea levels indicate an impaired renal function of diabetic animals (Shinde and Goyal, 2003). Thus, it would appear that the H. rosasinensis leaves supplement lowered the plasma urea, uric acid and creatinine levels by enhancing the renal function that is generally impaired in diabetic rats. These results are in agreement with other previous studies on the mesocarp extract of B. aegyptiaca (Saeed et al., 1995) and herbal formulation D-400 (Dubey et al., 1994).

The increase in the activities of plasma AST, ALT and ALP indicated that DM may induce hepatic dysfunction. The enzymes directly associated with the conversion of amino acids to keto acids are AST and ALT and are increased in the diabetic condition. Begum and Shanmugasundaram (1978) also reported an increase in the activities of AST and ALT in the liver of diabetic animals. Treatment with H. rosasinensis or insulin normalised these enzyme activities. Similarly, increased activities of AST and ALT in the diabetic liver were also reported by Jorda et al. (1982). The increased protein catabolism accompanying gluconeogenesis and urea formation that are seen in the diabetic state might be responsible for the elevation of these tissue transaminases. The rise in the activity of ALT is due to hepatocellular damage and is usually accompanied by a rise in AST (Rao et al., 1989). This might be the reason for the elevated activities of these enzymes which were brought back to near normal value by H. rosasinensis treatment. This result shows the normalizing effects of H. rosasinensis on hepatocellular damage and suppression of gluconeogenesis. Elevated activity of ALP was observed in Elevated activity of ALP was observed in STZ-diabetic rats. Prince et al. (1997) have also reported increased ALP activity in experimentally diabetic rats. The increased activity of this enzyme in plasma may be a result of diabetes-induced damage to the tissues. H. rosasinensis treatment restored the activity of this enzyme to near normal by reducing its induction in DM. γ-GT catalyses the transfer of the γ-glutamyl group from γ-glutamyl peptides to another peptide or L-amino acids or to water. The assay of γ-GT is a helpful adjunct in detecting hepatic damage. A highly significant elevation in the activity of γ-GT was observed in plasma, liver and kidney of STZ-induced diabetic rats. This is in accord with earlier investigations (McLennan et al., 1991), where in a dramatic increase in γ-GT expression was found in the liver of diabetic rats. Elevated activity of γ-GT in plasma takes place as a result of hepatic induction of the enzyme. In addition, hepatocellular damage or cholestasis may also contribute to the elevation in the activity. Increased activity of γ-GT in STZ-induced diabetic rats was lowered to near normal by H. rosasinensis treatment that indicates the possible prevention of necrosis by H. rosasinensis treatment.

In conclusion, H. Rosasinensis Aerial part extract lowered blood glucose with a simultaneous increase in the plasma insulin and C-peptide levels. In addition, H. Rosasinensis extract could influence protein metabolism and marker enzymes in STZ-induced diabetic rats. Extract also protect liver and Kidney from damage due to diabetes (Zanna et al., 2008).