Hypoglycemic and Hypolipidemic Potential of Nigella sativa L. Seed Extract in Streptozotocin (STZ)-Induced Diabetic Rats

Abstract

The different extracts obtained were subjected to phytochemical screening for the presence of flavanoids, tannins, alkaloids, carbohydrates, phytosterols, triterpinoids, saponins according to standard procedures [9]. Experimental animals care Experimental animals were collected from International Cholera and Dysentery Disease Research, Bangladesh (ICDDR). Ethical permission was provided by Institute of Biological science (IBSC), Rajshahi University. Male albino mice weighing ranged (25-27 g) were used for the study. They were individually housed in polypropylene cages in well-ventilated rooms under hygienic conditions, allowed free access to food (standard pellet diet) and water ad libitum and kept under maintained day night cycle. They were adapted for one week before the experiment. Induction of diabetics The overnight fasted animals were induced diabetes by a single administration of STZ (65 mg/kg b.w. i.p.) in ice-cold citrate buffer (pH 7.4). The threshold level of fasting serum glucose to diagnose diabetes was taken as >150 mg/dl (11.5 mmol/L) and only those animals were included in the study, rest are excluded from the study. The animals were divided into six groups of six in each group. Blood collection Blood samples from all groups were collected on days 1, 7, 14, 21 and 28 in a fasting state from rat's marginal ear vein by 26 G needle and syringe. Blood glucose levels, plasma cholesterol levels, triglyceride levels, LDL and HDL levels were determined by "Humylazer 2000" analyser (Human, Germany). The values were expressed as mean ± SEM, Statistical analyses were performed by SPSS-16 one-way analysis of variance (ANOVA), followed by post-hoc Tukey's test for multiple comparisons. P<0.05 was considered as significant. Experimental animals grouping and treatment The animals were randomly divided into six groups. Each group contain six rats (n=6). The treatment of animals began on the initial day after STZ injection and this was considered as 1st day of treatment. The animals were treated for 4 weeks as follows: Group-1: Control rats feed with standard pellet diet and water. Group-2: The rats were made diabetic by an intra-peritoneal injection of single dose of 110 mg/kg body weight followed by 65 mg/ kg body weight Streptozotocin. Animals whose blood glucose level exceeded 11.0 mmol/L at 72 h after treatment were considered diabetic. These animals served as untreated diabetic control. Group-3: The diabetic rats treated with ethanol extract of NS (Nigella sativa) at a dose of 300 mg/kg body weight for 28 days. Group-4: Hyper Cholesterol rats were given cholesterol (1.5%) and cholic acid (0.5%) mix with diet. Group-5: Hyper cholesterol rats were treated with ethanol extract of NS (Nigella sativa) at a dose of 300 mg/kg body weight for 28 days. Group-6: Diabetic rats were treated by Glibenclamide at a dose of 0.5 mg/kg b.wt. Measurement of blood parameters Plasma concentrations of triglyceride (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), VLDL, SGPT, SGOT and CRP were measured using a quantification kit (Linear chemicals, Barcelona, Spain) by automatic Bioanalyzer (Hitachi 7180, Hitachi, Tokyo, Japan). Statistical Analysis The assays were carried out in triplicate, and the results were expressed as mean values and the standard deviation (SD). Results were analyzed by using Scientific Package of Social Science (SPSS) version 17.0. Two different set of statistics, which is descriptive and analytical statistics was applied. The descriptive statistic was used to analyse mean, standard deviation (SD) whereby analytical statistics, one-way ANOVA was used to determine statistical significance (p<0.05, p<0.001) among the groups. Results Comparing the blood sugar level in Streptozotocin induced diabetic rats, NS (Nigella sativa) administered subject showed significant reduction of blood glucose level which is as near as glibenclamide administered subject at (P<0.001) (Table 1). Day 1, day 7, day 14, day 21 and day 28 NS (Nigella sativa) supplementation group's glucose levels maintained 32.58%-75.38% lower than the diabetic control group where as in case of glibenclamide it was 21.71%-87.63% lower than significantly diabetic control group (P<0.001). The Table 2 shows the serum levels of Total cholesterol (TC), Triglycerides (TG), LDL, VLDL, HDL and hypercholesterol of control and streptozotocin-induced diabetic rats. Reduction of Total Cholesterol (TC) level was 14.38%-23.05% observed by NS (Nigella sativa) treatment respectively in diabetic rats whereas hyper cholesterol reduces 19.04%-46.55%. The 3 to 28 days Total Cholesterol levels in the NS (Nigella sativa) treatment groups showed significant decrease compared with the diabetic control group and hyper cholesterol control group (P<0.05). The effect of NS (Nigella sativa) treatment on serum triglyceride (TG) content in diabetic rats is illustrated in Table 2. The Levels of triglyceride in the diabetic and hyper cholesterol group on the 28 days increased. Compare with the diabetic and hyper cholesterol control group by the treatment of NS (Nigella sativa) serum triglyceride was considerably lower 6.30%-11.73% whereas reduction of hypercholesterol group was 52.30%-82.31%. During the course of the experiment the significantly decrease diabetic and hypercholesterol group (P<0.05). The diabetic and hypercholesterol group shows an increase in LDL levels higher than the (normal) control group. LDL level was significantly reduced (P<0.05) in diabetic group received NS (Nigella sativa) treatment 15.7%-27.30% and hypercholesterol 19.72%-38.03%. VLDL level was significantly reduced (P<0.05) for due to received NS (Nigella sativa) treatment 9.2%-12.35% whereas hypercholesterol 63.00%-82.80%. The HDL level was increased significantly (P<0.001) at diabetic 13.4%-30.43% whereas hypercholesterol increased 12.25%-37.40% (Table 2). The Table 3 showed the phytochemical investigation revealed the fact that the hypoglycemic and hypolipidemic activity could be due to the presence of flavonoids, terpenoids or alkaloids. Increasing of SGPT and SGOT level after diabetes induction which was compensated by NS (Nigella sativa) significantly (P<0.05). The reduction of SGPT by NS (Nigella sativa) was 32.75% respectively whereas 25.23% for glibenclamide. The reduction of SGOT level was highly significant for NS (Nigella sativa) 29.84% than glibenclamide