MATERIALS AND METHODS

Experimental protocol: Healthy eighty male Wistar rats, weighing 225-252 g were provided by the Animal Experimental Unit of King Fahd Medical Research Center, King Abdul Aziz University, Jeddah, Saudi Arabia. The experimental animals were assigned to one of eight groups each of 10 rats. Animals were allocated 5 per cage in a temperature (24±1°C) and light: dark cycle was 12 h: 12 h. Rats of group 1 were intraperitoneally injected with 0.5 mL of sodium citrate buffer solution (pH 4.5), served as controls, fed ad libitum on normal commercial chow and had free access to water. The animals of groups 2, 3, 4 and 5 were intraperitoneally injected with STZ (Sigma Chemical Company, St. Louis, Mo, USA) at a dose of 30 mg kg ^-1 in 0.5 mL sodium citrate buffer solution (pH 4.5). Four days after STZ injection, the blood samples were collected from orbital venous plexus in the fasted rat (two samples from each groups 2, 3, 4, 5), water was not restricted and the level of serum glucose was determined. The serum glucose level of over 277 mg dL^-1 was defined as diabetic model rats. Rats of group 2 were fed with the same diet given in group 1. Groups 3, 4 and 5 were fed with the diets containing 5% ginger oil, 5% clove oil and 2.5% ginger oil plus 2.5 % clove oil, respectively. Groups 6, 7 and 8 received sodium citrate buffer solution (pH 4.5) at the same dose given in group 1 and fed with diets containing 5% ginger oil, 5% clove oil and 2.5% ginger oil plus 2.5% clove oil, respectively. After 2 weeks, the experimental animals were fasted for 8 h, water was not restricted and then anaesthetized with ether. Blood samples were collected from orbital venous plexus in non-heparinized tubes, centrifuged at 2000 rpm for 20 min and blood sera were then collected and stored at 4°C prior immediate determination of glucose, triglycerides, cholesterol, high density lipoprotein HDL-cholesterol (HDL-C), low density lipoprotein LDL-cholesterol (LDL-C), total protein, creatinine, urea, uric acid, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). All of these parameters were measured using an automatic analyzer (Architect c8000 Clinical Chemistry System, USA).

Statistical analysis: All experimental data of haematobiochemical parameters were expressed as mean±SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) using the Statistical Package for Social Sciences (SPSS) version 12.0 and differences between means were detected by Student's t-test. p-values of less than 0.05 were considered significant.

RESULTS

Supplementation effects of ginger, clove and ginger plus clove oils on the haematobiochemical parameters of diabetic and non-diabetic rats are represented in Table 1. In comparison with control, highly significant increases in the values of blood glucose (273.72%), triglycerides (34.97%), cholesterol (65.79%), LDL-cholesterol (201.07%), total protein (21.09%), creatinine (74.31%), urea (82.08%), uric acid (81.23%), ALT (74.36%) and AST (34.99%) were noted in STZ-diabetic rats, while the value of HDL-cholesterol was markedly decreased (21.68%). Administration of ginger oil to diabetic rats resulted in mild increases of the levels of blood glucose (35.05%), triglycerides (9.82%), cholesterol (26.08%), LDL-cholesterol (79.22%), total protein (9.77%), urea (25.83%), uric acid (15.51%) and AST (10.17%), while the value of HDL-cholesterol (7.39%) was significantly decreased. Moreover, the treatment with ginger oil noticeably restored the values of blood creatinine and ALT activity to the control levels. Treatment with clove or ginger plus clove oils induced mild increases in the values of blood glucose, triglycerides, cholesterol, LDL-cholesterol, total protein, creatinine, urea, uric acid, ALT and AST in diabetic rats, while the value of HDL-cholesterol was markedly increased. Supplementation of the tested oils significantly decreased the haematobiochemical changes in STZ-diabetic rats. In comparison with control, supplementation with only ginger oil significantly reduced the level of blood glucose (9.38%) and triglycerides (12.27%) in non-diabetic rats. Lowering effect of ginger plus clove oils on blood glucose (6.47%) was notably observed in non-diabetic rats. From Table 1, it was pronounced that the administration of ginger oil exhibited a notable improvement role, as evidenced by its modulating effects on the studied parameters of diabetic rats.

Table 1:	Effects of ginger, clove and ginger plus clove oils supplementation on the levels of haematobiochemical parameters (mean±SD) in the experimental diabetic and non-diabetic rats (p<0.05), n = 5
a: Indicates a significant difference between control and treated groups, b: Indicates a significant difference between STZ group and groups treated with STZ+ginger, STZ+clove or STZ+ginger plus clove oils, c: Indicates a significant difference between group treated with STZ+ginger and groups treated with STZ+clove or STZ+ginger plus clove oils, d: Indicates a significant difference between group treated with STZ+clove oil and group treated with STZ+ginger plus clove oils, e: Indicates a significant difference between group treated with STZ+ginger and group treated with ginger oil only, f: Indicates a significant difference between group treated with STZ+ clove oil and group treated with clove oil only, g: Indicates a significant difference between group treated with STZ+ginger plus clove oils and group treated with ginger plus clove oils only, h: Indicates a significant difference between group treated with ginger oil only and groups treated with clove oil only or ginger plus clove oils only and I: Indicates a significant difference between group treated with clove oil only and groups treated with ginger plus clove oils only

DISCUSSION

The present investigation showed that the supplementation of ginger, clove or ginger plus clove oils significantly inhibited the haematobiochemical changes in STZ-diabetic rats. The most changes were pronounced in diabetic rat treated with ginger oil. Generally, it is obviously from the present data that the STZ-induced several disturbances on carbohydrate, lipid and protein metabolism in experimental rats. Bolkent et al. (2004), Prakasam et al. (2004), Ravi et al. (2005), Singh et al. (2005), Yanardag et al. (2005) and Rajasekaran et al. (2006) reported that in the STZ-diabetic rats, the levels of blood glucose, total lipid, cholesterol, triglycerides, LDL-cholesterol, creatinine, urea, uric acid, ALT and AST activities were significantly increased, while the levels of HDL-cholesterol were markedly decreased. In the study of Shinde and Goyal (2003), histopathological investigations of kidney and liver showed several changes included the increases in the intensity and incidence of vacuolations, cellular infiltration and hypertrophy in STZ-diabetic rats. Additionally, they showed that STZ-induced an elevation of serum creatinine and urea levels as well as an elevation of serum level of hepatic enzymes in diabetic rats. Moreover, Murali et al. (2003), Sato et al. (2005) and Yoshida et al. (2005) reported that kidney damage in STZ-induced diabetic rats includes glomerular expansion, renal hypertophy, glycogen degeneration of distal tubules, fatty degeneration of glomerular endothelium. It is worth to mention that the above previous studies demonstrated that the administrations of several herbal extracts could restore the alterations in the levels of blood and tissue parameters, morphological and histological structure. In the present investigation, it can not excluded that the possibility that diabetes-induced liver and kidney damage. However, the increases of serum ALT, AST, creatinine, urea and uric acid levels are considered as obvious indicators for liver and kidney damage and dysfunctions. ALT and AST are directly associated with the conversion of amino acids to keto acids and the increased protein catabolism accompanying gluconeogenesis and urea formation that are seen in diabetic state might be responsible for the elevation of these aminotranferases. The diabetic hyperglycaemia induces elevations of the blood levels of creatinine, urea, uric acid which are considered as significant markers of renal dysfunction (Almdal and Vilsturp, 1988). It has been documented that several medicinal plants show their hypoglycaemic effects associated with a significant alteration in the activity of liver hexokinase (Bopanna et al., 1997; Santhakumari et al., 2006), glucokinase (Kumari et al., 1995; Lee et al., 1997). It has been reported that treatment with the herbs caused an improvement in the activities of liver glucose- 6-phosphatase, glycogen synthetase, glycogen phosphorylase, glucose-6-phosphate dehydrogenase and phospho-fructokinase. Diabetes mellitus is also grossly reflected by profound changes in protein metabolism and by a negative nitrogen balance and loss of nitrogen from most organs (Almdal and Vilstrup, 1987). Increased urea nitrogen production in diabetes may be accounted by enhanced catabolism of both liver and blood proteins (Jorda et al., 1981, 1982). The effect of diabetes mellitus on lipid metabolism is well established. The association of hyperglycaemia with an alteration of lipid parameters presents a major risk for cardiovascular complications in diabetes. Many secondary plant metabolites have been reported to possess lipid-lowering properties (Rajasekaran et al., 2006). The serum cholesterol and triglycerides were significantly decreased in diabetic rats supplemented with ginger, clove and ginger plus clove oils. These oils supplementation also result the significant attenuation in the levels of HDL-cholesterol and LDL-cholesterol in serum toward the control level which again strengthen the hypolipidaemic influence of these oils. A variety of derangements in metabolic and regulatory mechanisms, due to insulin deficiency, is responsible for the observed accumulation of lipids (Rajalingam et al., 1993; Sharma et al., 2003). The impairment of insulin secretion results in enhanced metabolism of lipids from the adipose tissue to the plasma. Further, it has been reported that diabetic rats treated with insulin show normalized lipid levels (Pathak et al., 1981). We suggest that the present effects of these oils-treated diabetic rats may be due to its role in normalization of insulin secretion, lowering activity of lipid biosynthesis enzymes, especially cholesterol and or lowering level of lipolysis.

Concerning the previous studies on the role of ginger and clove extracts, but not their oils, in diabetic status, Al-Amin et al. (2006) and Ojewole (2006) stated that the extracts of ginger possess hypoglycaemic, hypocholesterolaemic and hypolipidaemic potential in STZ-induced diabetic rats and mice. Additionally, Al-Amin et al. (2006) demonstrated that ginger is effective in reversing the diabetic proteinuria observed in the diabetic rats. Thus, ginger may be of great value in managing the effects of diabetic complications in human subjects. Bhandari et al. (2005) reported that the extract of Zingiber officinale produced significant antihyperglycaemic effect in STZ-induced diabetic rats. Further, the extract treatment also lowered serum total cholesterol, triglycerides and increased the HDL-cholesterol levels when compared with pathogenic diabetic rats. Moreover, they mentioned that STZ-treatment also induced a statistically significant increase in liver and pancreas lipid peroxide levels as compared to normal healthy control rats and the extract of ginger can protect the tissues from lipid peroxidation. Akhani et al. (2004) showed that STZ-diabetes produced a significant increase in fasting glucose levels that was associated with a significant decrease in serum insulin levels. Treatment with ginger juice produced a significant increase in insulin levels and a decrease in fasting glucose levels in diabetic rats. In an oral glucose tolerance test, treatment with ginger juice was found to decrease significantly the area under the curve of glucose and to increase the area under the curve of insulin in STZ-diabetic rats. Treatment with ginger juice also caused a decrease in serum cholesterol, triglycerides and blood pressure in diabetic rats. They suggested a potential antidiabetic activity of the juice of Zingiber officinale in diabetic rats, possibly involving serotonin (5-hydroxytryptamine; 5-HT) receptors. In addition, Kadnur and Goyal (2005) reported that the treatment with extract of rhizomes of Zingiber officinale produced a significant reduction in fructose-induced hyperglycaemia and elevations in lipid levels. Prasad et al. (2005) demonstrated that Syzygium aromaticum extract acts like insulin in hepatocytes and hepatoma cells by reducing phosphoenolpyruvate carboxykinase and glucose-6-phosphatase gene expression. Much like insulin, clove-mediated repression is reversed by phosphatidylinositol 3-kinase inhibitors and N-acetylcysteine. A more global analysis of gene expression by DNA microarray analysis reveals that clove and insulin regulate the expression of many of the same genes in a similar manner. These results demonstrate that consumption of certain plant-based diets may have beneficial effects for the treatment of diabetes and indicate a potential role for compounds derived from clove as insulin-mimetic agents. Recently, several studies reported that increased oxidative stress was shown to play an important role in the etiology and pathogenesis of diabetes mellitus and its complications which especially induced by STZ (Babu et al., 2006; Barbosa et al., 2006; Kim et al., 2006; Nangle et al., 2006). Additionally, several investigations demonstrated that ginger, clove and their constituents increased the activities of tissue antioxidant enzymes. The protective effects ginger and clove are, therefore, suggested to be mediated by their potent antioxidant activities (Ippouchi et al., 2005; Shin et al., 2005; Haskar et al., 2006; Jirovetz et al., 2006). Finally, we suggest that ginger, clove and ginger plus clove oils supplementation may act as antioxidant agents and these oils could be an excellent adjuvant support in the therapy of diabetic mellitus and its complications.