Beneficial effects of thymoquinone on hepatic key enzymes in streptozotocin–nicotinamide induced diabetic rats

Life Sciences 85 (2009) 830–834

Contents lists available at ScienceDirect

Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Beneficial effects of thymoquinone on hepatic key enzymes in streptozotocin–nicotinamide induced diabetic rats Leelavinothan Pari ⁎, Chandrasekaran Sankaranarayanan Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608 002, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 14 August 2009 Accepted 30 October 2009 Keywords: Diabetes Streptozotocin Carbohydrate metabolic enzymes Thymoquinone

a b s t r a c t Aims: The present study was designed to evaluate the antihyperglycemic potential of thymoquinone (TQ), major constituent of Nigella sativa seeds on the activities of key enzymes of carbohydrate metabolism in streptozotocin (STZ)-nicotinamide (NA)-induced diabetic rats. Main methods: Diabetes was induced in experimental rats weighing 180–220 g, by a single intraperitoneal (i.p) injection of STZ (45 mg/kg b.w), 15 min after the i.p administration of NA (110 mg/kg b.w). Diabetic rats were administered TQ intragastrically at 20, 40, 80 mg/kg b.w for 45 days. The levels of plasma glucose, insulin, glycated hemoglobin (HbA1C) and hemoglobin (Hb) were measured. The activities of hexokinase, glucose 6phosphate dehydrogenase, glucose 6-phosphatase and fructose 1,6-bisphosphatase were assayed in liver homogenates. Key findings: Oral administration of TQ for 45 days, dose dependently improved the glycemic status in STZ-NA induced diabetic rats. The levels of insulin, Hb increased with significant decrease in glucose and HbA1C levels. The altered activities of carbohydrate metabolic enzymes were restored to near normal. No significant changes were noticed in normal rats treated with TQ. Significance: These results show that TQ at 80 mg/kg b.w is associated with beneficial changes in hepatic enzyme activities and thereby exerts potential antihyperglycemic effects. © 2009 Elsevier Inc. All rights reserved.

Introduction Diabetes Mellitus is a serious, complex metabolic disorder of multiple etiologies, characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion (β-cell dysfunction), insulin action (insulin resistance) or both (Kardeşler et al. 2008). Both genetic and environmental factors play an important role in the onset of type 2 diabetes (Lima et al. 2008). It is of particular concern since the disease incidence is expected to increase worldwide by more than 100% between 2000 and 2030 (Wild et al. 2004). Chronic supra physiological glucose concentration affects the secretion of β-cells and brings metabolic imbalances and pathological disturbances in several tissues such as pancreas, eyes, liver, muscle, adipose tissue, kidney and nerves (Brunner et al. 2009). STZ, an antibiotic produced by Streptomyces achromogenes, is a selective pancreatic β-cell genotoxicant used to induce experimental diabetes in model organisms. Several evidences indicate that free radicals may play an essential role in the mechanism of β-cell damage and diabetogenic effect of STZ (Takasu et al. 1991; Ohkuwa et al. 1995). STZ induced diabetic animals exhibit many of the complica-

⁎ Corresponding author. Tel.: + 91 4144 238343; fax: + 91 4144 238145. E-mail address: [email protected] (L. Pari). 0024-3205/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2009.10.021

tions observed in human diabetes. Therefore, the beneficial effects of antidiabetic principles are well studied in these animal models. Experimental diabetes is characterized by derangement in carbohydrate metabolism resulting from altered activities of enzymes that control glycolysis, gluconeogenesis in liver and muscle and produces hyperglycemia. Maintenance of normoglycemia involves the integration and coordinated regulation of several metabolic pathways including gluconeogensis and glycolysis (Ashokkumar and Pari 2005). Renewed attention to alternative medicines and natural therapies has stimulated new wave of research to look for more efficacious agents with lesser side effects (Kim et al. 2006). Naturally occurring phytochemicals with antidiabetic activities are relatively nontoxic, inexpensive and available in an ingestive form. Therefore, they are commonly used to prevent morbidity and mortality from chronic diseases in countries where low or middle-income populations are important (Gazioano et al. 2007). Among the beneficial medicinal plants Nigella sativa, a dicotyledon of the Ranunculacea family is an amazing herb with a rich historical and religious background. The seeds of this plant are called black cumin or black seeds. Thymoquinone (TQ) (2- isopropyl-5-methyl1,4-benzoquinone) (Fig. 1 ), an active principle of the volatile oil of black cumin seeds possess diverse pharmacological activities such as antioxidant (Erkan et al. 2008), hepatoprotective (Daba and AbdelRahman 1998), neuroprotective (Abdulhakeem et al. 2006), antidiabetic (El-Mahmoudy et al. 2005), anti-inflammatory (El Gazzar

L. Pari, C. Sankaranarayanan / Life Sciences 85 (2009) 830–834

Fig. 1. Structure of thymoquinone.

831

Group I: Normal control rats (vehicle treated) Group II: Normal rats received intra gastrically TQ (80 mg/kg b.w) dissolved in 1 ml of corn oil for 45 days Group III: Diabetic control rats (vehicle treated) Group IV: Diabetic rats received intra gastrically TQ (20 mg/kg b.w) dissolved in 1 ml of corn oil for 45 days Group V: Diabetic rats received intra gastrically TQ (40 mg/kg b.w) dissolved in 1 ml of corn oil for 45 days Group VI: Diabetic rats received intra gastrically TQ (80 mg/kg b.w) dissolved in 1 ml of corn oil for 45 days Sample collection

et al. 2006), nephroprotective (Fouda et al. 2008), anti-mutagenic (Badary et al. 2007), anti-carcinogenic (Gali Muhtasib et al. 2006) and anti-convulsant (Hosseinzadeh and Parvardeh 2004). TQ is of low toxicity (LD50 2.4 g/kg) and is well tolerated when given sub chronically till 90 mg/kg/day for 90 days (Badary et al. 1998). Though previous studies on TQ show its glucose lowering effects, the mechanisms are not delineated. Blood glucose reduction can be achieved by several ways. The present study was designed to investigate the role of TQ on glucose utilization pathways and on hepatic glucose production since both of them contributes significantly to plasma glucose levels. For this, the activities of key enzymes of carbohydrate metabolism (glucose utilization/production) are measured in STZ-NA induced diabetic rats.

The initial and final body weights of the various groups were recorded. At the end of the experimental period, the animals were fasted overnight, anesthetized using ketamine (24 mg/kg b.w intramuscular injection), and sacrificed by cervical decapitation. Blood samples were collected in tubes containing potassium oxalate and sodium fluoride (3:1) mixture for the estimation of plasma glucose and insulin. Hb and HbA1C levels were estimated in whole blood samples. Liver was immediately dissected, washed in ice-cold saline, homogenized in Tris-HCl buffer (0.1 M, pH 7.5), centrifuged (3000 rpm/min), and the supernatant was collected. Biochemical estimations were carried out in the homogenates.

Materials and methods

Analytical procedures

Chemicals

Plasma glucose levels were estimated using a commercial kit (Sigma Diagnostics Pvt. Ltd., Baroda, India) by the method of Trinder (1969). Plasma insulin was assayed by ELISA kit (BoeheringerManneheim Kit, Manneheim, Germany). Hemoglobin (Hb) was estimated by the cyanmethemoglobin method (Drabkin and Austin 1932). Glycated hemoglobin (HbA1C) was estimated by the method of Sudhakar Nayak and Pattabiraman (1981) with modifications according to Bannon (1982). Hepatic hexokinase was assayed by the method of Brandstrup et al (1957). The reaction mixture in a total volume of 5.3 ml contained the following: 1 ml of glucose (0.005 M), 0.5 ml of ATP (0.072 M), 0.1 ml of magnesium chloride (0.050 M), 0.4 ml of potassium dihydrogen phosphate (0.0125 M), 0.4 ml of potassium chloride (0.10 M), 0.4 ml of sodium fluoride (0.50 M) and 2.5 ml of Tris-HCl buffer (0.01 M, pH 8.0). The mixture was pre-incubated at 37 °C for 5 min. The reaction was initiated by the addition of 2 ml of tissue homogenate. 1 ml of the reaction mixture was immediately transferred to the tubes containing 1 ml of 10% TCA that was considered as zero time. A second aliquot was removed and deproteinised after 30 min incubation at 37°C. The protein precipitate was removed by centrifugation and the residual glucose in the supernatant was estimated by the method of Trinder (1969). Glucose 6-phosphate dehydrogenase was assayed by the method of Ellis and Kirkman (1961). The incubation mixture contained 1 ml of Tris-HCl buffer (0.05 M, pH 7.5), 0.1 ml of magnesium chloride, 0.1 ml of NADP+, 0.5 ml of phenazine methosulphate, 0.4 ml of 2,6-dichlorophenol indo phenol dye solution and 0.5 ml of liver homogenate. The contents were incubated at 37 °C for 10 min. The reaction was initiated by the addition of 0.5 ml of glucose 6-phosphate. The absorbance was read spectrophotometrically at 640 nm against water blank at 1-min intervals for 3 to 5 min. Glucose 6-phosphatase was assayed by the method of Koida and Oda (1959). Incubation mixture contained 0.7 ml of citrate buffer (0.1 M, pH 6.5), 0.3 ml of substrate (0.01 M) and 0.1 ml of tissue homogenate. The reaction mixture was incubated at 37°C for 1 h. Addition of 1 ml of 10% TCA to the reaction tubes terminated the reaction. The suspension was centrifuged and the supernatant was made up to known volume. The inorganic phosphorus content in the supernatant was estimated by adding 1 ml of ammonium molybdate

Thymoquinone, STZ, phenazine methosulphate and NADP+ were purchased from Sigma Chemical Co (St. Louis, Mo. USA) and NA was obtained from Ranbaxy Chemicals Ltd., Mumbai, India. All the other chemicals used in the present study were of analytical grade. Animals Thirty-Six healthy male albino rats of Wistar strain, weighing 180–220 g, bred in the Central Animal House, Rajah Muthiah Medical College, Annamalai University, were used in this study. They were maintained in a controlled environment (12 h light/dark cycle) and temperature (28 ± 2 °C). The animals were fed on pellet diet from Karnataka Agro Food Corporation Limited, Bangalore and water ad libitum. The animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the Institutional Animal Ethical Committee, Annamalai University (Vide. No. 564, 2008). Experimental induction of diabetes Diabetes was induced in overnight fasted experimental groups by a single intra-peritoneal (i.p) injection of freshly prepared STZ (45 mg/kg b.w) dissolved in 0.1 M citrate buffer (pH 4.5), 15 min after the i.p administration of NA (110 mg/kg b.w) (Masiello et al. 1998). The animals were allowed to drink 20% glucose solution overnight to overcome drug-induced hypoglycemy. Control rats were injected with the same volume of isotonic saline. After 72 h, plasma glucose was determined and those rats with fasting glucose levels greater than 250 mg/dl were used in the present study. Experimental design The animals were randomly divided into six groups of six animals in each group (24 diabetic surviving and 12 normal). TQ, a lipid soluble compound is dissolved in corn oil and administered to experimental rats.

832

L. Pari, C. Sankaranarayanan / Life Sciences 85 (2009) 830–834

and 0.4 ml of ANSA. The blue colour developed after 20 min was read at 680 nm (Fiske and Subbarrow 1925). Fructose 1,6-bisphosphatase activity was measured by Gancedo and Gancedo (1971). The assay mixture in a final volume of 2 ml contained 1.2 ml of Tris-HCl buffer (0.1 M, pH 7.0), 0.1 ml of substrate (0.05 M), 0.25 ml of magnesium chloride (0.1 M), 0.1 ml of potassium chloride (0.1 M), 0.25 ml of EDTA (0.001 M) and 0.1 ml of liver homogenate. The incubation was carried out at 37°C for 15 min. The reaction was terminated by adding 1 ml of 10% TCA. The suspension was centrifuged and the supernatant was used for phosphorus estimation by the method of Fiske and Subbarrow (1925) as described previously. Oral glucose tolerance test (OGTT) was performed according to the method of Du vigneaud and Karr (1925). After overnight fasting, ‘0’ minute blood sample (0.2 ml) was taken from control and experimental rats. Without delay, a glucose solution (2 g/kg body weight) was administered by oral gavage. Blood samples were taken at 30, 60, 90 and 120 min after glucose administration. Blood samples were collected with potassium oxalate and sodium fluoride and glucose levels were determined by the kit method of Trinder (1969) as described previously.

Fig. 2. Effect of TQ on oral glucose tolerance test. Values are mean ± SD for 6 rats in each group. p b 0.05.

observed in diabetic rats. Oral administration of TQ to diabetic rats significantly reversed the activities of the above enzymes to near normal. Determination of Hb and HbA1C

Statistical analysis All data were expressed as mean± SD for six rats in each group. The statistical analysis was done by one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT) using SPSS software package, version 11. p b 0.05 were considered as significant and included in the study (Duncan 1957). Results Estimation of plasma glucose, insulin and glucose tolerance Table 1 shows the levels of plasma glucose, insulin in normal and experimental rats. A significant increase in plasma glucose and a decrease in insulin levels were observed in diabetic rats. Treatment with TQ significantly improved the values towards normal. There was no significant change in normal rats treated with TQ. Results of OGTT conducted on control and different experimental groups are shown in Fig. 2. After the oral dose of glucose in normal control rats the blood glucose reached the fasting levels at 2 h. In diabetic control rats, the blood glucose levels remained higher even after 2 h. Treatment with TQ showed a significant decrease in blood glucose levels. The maximum glucose lowering effect of TQ was observed at a dose of 80 mg/kg b.w than the other two doses. Therefore, further studies were carried out with this dose. Activities of hepatic gluconeogenic and glycolytic enzymes Table 2 depicts the activities of carbohydrate metabolic enzymes in liver of experimental rats. Decreased activities of hexokinase, glucose 6phosphate dehydrogenase and increased activities of gluconeogenic enzymes glucose 6-phosphatase and fructose 1, 6-bisphosphatase were

Table 1 Changes in the levels of plasma glucose and insulin in control and experimental rats. Groups

Plasma glucose (mg/dl)

Plasma insulin (μU/ml)

Normal control Normal + Thymoquinone (80 mg/kg) Diabetic control Diabetic + Thymoquinone (20 mg/kg) Diabetic + Thymoquinone (40 mg/kg) Diabetic + Thymoquinone (80 mg/kg)

93.36 ± 7.15a 94.30 ± 7.22a 283.17 ± 21.68b 226.18 ± 17.31c 163.32 ± 12.57d 110.24 ± 8.31ea

16.98 ± 1.30a 17.44 ± 1.34a 6.46 ± 0.49b 7.95 ± 0.61c 10.25 ± 0.78d 14.95 ± 1.15e

Values are mean ± SD for 6 rats in each group. a–dIn each column, different superscript letters mean significant differences at p b 0.05 (DMRT).

Fig. 3 shows the levels of Hb and HbA1C in normal and diabetic rats. Decreased Hb and an increase in HbA1C were observed in diabetic rats. These values improved towards near normal on treatment with TQ. Changes in body weight, food and water intake The food and water intake of experimental rats are shown in Fig. 4. The increased intake of food and water in diabetic rats were significantly decreased on treatment with TQ. Fig. 5 represents the body weight of normal and diabetic rats. Decreases in the body weight of diabetic control rats were observed during the experimental period, which significantly improved on treatment with TQ. Discussion The incidence of type 2 diabetes mellitus is increasing worldwide (Wild et al. 2004). Over-production (excessive hepatic glycogenolysis and gluconeogenesis) and decreased utilization of glucose by the tissues are the fundamental basis of hyperglycemia in diabetes mellitus (Shirwaikar et al. 2006). Normal glucose homeostasis represents the balance between intake (glucose absorption from the gut), tissue utilization (glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, glycogen synthesis) and endogenous production (glycogenolysis and gluconeogenesis) (Meyer et al. 2002). By this way, the body tries to keep a constant supply of glucose for cells by maintaining a constant glucose concentration in the blood. In the present study we investigated the effect of TQ, the main active principle in the volatile oil of Nigella sativa seeds on the activities of carbohydrate metabolic enzymes and on hyperglycemic status in STZ-NA induced diabetic rats. We found that oral administration of TQ for 6 weeks resulted in a significant reduction in plasma glucose concentrations and an increase in insulin levels in diabetic rats. A consistent and a dose dependent decrease in plasma glucose and an increase in insulin levels by TQ were observed in the present study. These results are in agreement with Mehmet Kanter (2008) who studied the actions of TQ on sciatic nerves in experimental diabetic neuropathy. In the present study the protective and insulinotrophic action of TQ on β-cells was well documented at higher doses. Hyperglycemia in type 2 diabetes is in part due to the lack of suppression of hepatic glucose production in the absorptive state and excessive glucose production in the post absorptive state. Enzymes that regulates hepatic glucose metabolism are potential targets for controlling hepatic glucose balance and thereby blood glucose levels in type 2 diabetes.

L. Pari, C. Sankaranarayanan / Life Sciences 85 (2009) 830–834

833

Table 2 Changes in the activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, hexokinase and Glucose-6-phosphate dehydrogenase in control and experimental rats. Groups

Normal control

Gluconeogenic enzymes Glucose-6-phosphatase (μmol of Pi liberated/min/mg protein) Fructose-1, 6-bisphosphatase (μmol of Pi liberated/h/mg protein) Glycolytic enzyme Hexokinase (μmol of glucose phosphorylated/min/g protein) Lipogenic enzyme (Ub/mg protein) Glucose-6-phosphate dehydrogenase Values are mean ± SD for 6 rats in each group.

Normal + Thymoquinone (80 mg/kg)

0.178 ± 0.016a 0.354 ± 0.030a

0.174 ± 0.014a 0.338 ± 0.030a

149.34 ± 12.59a

152.28 ± 12.65a

4.83 ± 0.15a

4.78 ± 0.14a

Diabetic control

0.270 ± 0.024b 0.602 ± 0.050b

106.76 ± 7.56b

2.17 ± 0.12b

Diabetic + Thymoquinone (80 mg/kg) 0.213 ± 0.020c 0.402 ± 0.030c

134.52 ± 11.02c

3.34 ± 0.24c

a–c

In each column, different superscript letters mean significant differences at p b 0.05 (DMRT).

Liver plays a central role in blood sugar homeostasis. In diabetic state the activities of hexokinase and glucose 6-phosphate dehydrogenase are decreased which is due to the total absence or insufficiency of insulin. TQ up regulates the activities of both these enzymes in hepatic tissues through insulin release and thereby it enhances the utilization of glucose for cellular biosynthesis, which is marked by the significant decrease in plasma glucose levels. Hepatic glucose production through gluconeogenesis significantly contributes to hyperglycemia in diabetes mellitus (Ishikawa et al. 1998). It has been demonstrated that in diabetes mellitus, the increased rate of gluconeogenesis is related to the increased expression of key gluconeogenic enzymes such as phosphoenol pyruvatecarboxykinase (PEPCK), glucose 6-phosphatase, fructose 1,6-bisphosphatase in hepatic tissues (Van de Werve et al. 2000). Studying the effect of TQ on gluconeogenesis helps to clarify part of its hypoglycemic action. In the present study TQ decreased the activities of glucose 6-phosphatase and fructose 1,6bisphosphatase in diabetic rats. These results are in harmony with Fararh et al. (2005) who reported that TQ reduces hepatic glucose production in isolated hepatocytes in diabetic hamsters. These results conclusively prove that TQ normalizes disturbed carbohydrate metabolism by enhancing glucose utilization and by decreasing hepatic glucose production through insulin release and indicates its beneficial effect in the treatment of diabetes mellitus. Glucose tolerance test performed 6 weeks after oral administration of TQ showed improvement in the glucose utilization ability of diabetic rats by bringing blood glucose to the basal level after the oral dose. Glucose tolerance in the diabetic control group remains impaired. Glucose lowering effect was markedly increased at higher dose, which clearly shows the insulinotrophic property of TQ. Advanced glycation occurs during normal aging but to a greater degree in diabetes in which it plays a major role in the development of diabetic complications (Ahmed 2005). There are several studies, which

report that serum advanced glycation end products (s-AGEs) increase in senile diabetic patients. Total HbA1C is an important parameter used to monitor response to glucose-lowering therapy and long-term blood sugar control, as it reflects the average blood sugar concentration over an extended period of time and remains unaffected by short-term fluctuations in blood glucose levels. Our results showed that treatment with TQ significantly reduced total HbA1C compared with the control

Fig. 3. Changes in the levels of hemoglobin and glycosylated hemoglobin in control and experimental rats. Values are mean ± SD for 6 rats in each group. a–cIn each column, different superscript letters mean significant differences at p b 0.05 (DMRT).

Fig. 4. Effect of TQ on food and water intake in normal and diabetic rats. Values are mean ± SD for 6 rats in each group. a–dIn each column, different superscript letters mean significant differences at p b 0.05 (DMRT).

834

L. Pari, C. Sankaranarayanan / Life Sciences 85 (2009) 830–834

Fig. 5. Changes in the body weight of normal and experimental rats. Values are mean ± SD for 6 rats in each group. a–dIn each column, different superscript letters mean significant differences at p b 0.05 (DMRT).

animals. This reflects the effective action of TQ in long-term control of hyperglycemia through insulin secretion. Diabetes is characterized by a severe loss in body weight, which might be the result of protein wasting due to unavailability of carbohydrate as an energy source (Al-Shamaony et al. 1994). In the present study initial and final body weight of experimental animals were measured, whereas food and water intake were evaluated on a daily basis. Though the food and water intake of diabetic rats were increased during the experimental period, the weight gain was significantly reduced when compared to control animals. Oral administration of TQ resulted in marked improvements in the above parameters. From the above studies, it is evident that TQ administration causes improvement in glycemic status of diabetic rats, which is attributed to the modification of activities of key enzymes of carbohydrate metabolism through enhanced insulin secretion. Thus, TQ normalizes the disturbed carbohydrate metabolism in diabetic rats and exerts significant antidiabetic effects. Conclusions From the above findings, we conclude that TQ administration significantly increased insulin secretion and normalized the deranged carbohydrate metabolism in diabetic rats by enhancing glucose utilization and decreasing hepatic glucose production, thereby exhibiting significant antidiabetic effects. Further studies are needed to explore the molecular mechanisms of TQ in altering hormone secretion. References Abdulhakeem Al-Maje A, Fadhel Al-Omar A, Mahmoud Nagi N. Neuroprotective effects of thymoquinone against transient forebrain ischemia in the rat hippocampus. European Journal of Pharmacology 543 (1-3), 40–47, 2006. Ahmed N. Advanced glycation end products-role in pathology of diabetic complications. Diabetes Research and Clinical Practice 67 (1), 3–21, 2005. Al-Shamaony L, Al-Khazraji SM, Twaiji HA. Hypoglycemic effect of Artemisia herba alba II: effect of a valuable extract on some blood parameters in diabetic animals. Journal of Ethnopharmacology 43 (3), 167–171, 1994. Ashokkumar N, Pari L. Effect of N-benzoyl-D-Phenylalanine and metformin on carbohydrate metabolic enzymes in neonatal streptozotocin diabetic rats. Clinica Chimica Acta 351, 105–113, 2005. Badary OA, Abd-Ellah MF, El-Mahdy MA, Salama SA, Hamada FM. Anticlastogenic activity of thymoquinone against benzo(a)pyrene in mice. Food and Chemical Toxicology 45 (1), 88–92, 2007. Badary OA, Al-Shabanah OA, Nagi MN, Al-Bekairi AM, Elmazar MMA. Acute and subchronic toxicity of thymoquinone in mice. Drug Development Research 44, 56–61, 1998. Bannon P. Effect of pH on the elimination of the labile fraction of glycosylated hemoglobin. Clinical Chemistry 28 (10), 21–83, 1982. Brandstrup N, Kirk JE, Bruni C. The hexokinase and phosphoglucoisomerase activities of aortic and pulmonary artery tissue in individuals of various ages. Journal of Gerontology 12 (2), 166–171, 1957.

Brunner Y, Schvartz D, Priego-Capote F, Couté Y, Sanchez JC. Glucotoxicity and pancreatic proteomics. Journal of Proteomics 71 (6), 576–591, 2009. Daba MH, Abdel-Rahman MS. Hepatoprotective activity of thymoquinone in isolated rat hepatocytes. Toxicology Letters 95 (1), 23–29, 1998. Drabkin DL, Austin JM. Spectrophotometric constants for common haemoglobin derivatives in human, dog and rabbit blood. Journal of Biological Chemistry 98, 719–733, 1932. Du Vigneaud V, Karr WG. Carbohydrates utilization rate of disappearance of D-glucose from the blood. Journal of Biological Chemistry 66, 281–300, 1925. Duncan BD. Multiple range tests for correlated and heteroscedastic means. Biometrics 13, 359–364, 1957. El Gazzar M, El Mezayen R, Marecki JC, Nicolls MR, Canastar A, Dreskin SC. Antiinflammatory effect of thymoquinone in a mouse model of allergic lung inflammation. International Immunopharmacology 6 (7), 1135–1142, 2006. Ellis HA, Kirkman HN. A colorimetric method for assay of erythrocytic glucose 6-phosphate dehydrogenase. Proceedings of the Society for Experimental Biology and Medicine 106, 607–609, 1961. El-Mahmoudy A, Shimizu Y, Shiina T, Matsuyama H, El-Sayed M, Takewaki T. Successful abrogation by thymoquinone against induction of diabetes mellitus with streptozotocin via nitric oxide inhibitory mechanism. International Immunopharmacology 5 (1), 195–207, 2005. Erkan N, Ayranci G, Ayranci E. Antioxidant activities of rosemary (Rosmarinus officinalis L.) extract, black seed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol. Food Chemistry 110, 76–82, 2008. Fararh KM, Shimizu Y, Shiina T, Nikami H, Ghanem MM, Takewaki T. Thymoquinone reduces hepatic glucose production in diabetic hamsters. Research in Veterinary Science 79 (3), 219–223, 2005. Fiske GH, Subbarrow Y. The colorimetric determination of phosphorus. Journal of Biological Chemistry 66, 375–400, 1925. Fouda AM, Daba MH, Dahab GM. Sharaf El-Din OA. Thymoquinone ameliorates renal oxidative damage and proliferative response induced by mercuric chloride in rats. Basic and Clinical Pharmacology and Toxicology 103 (2), 109–118, 2008. Gali Muhtasib H, Roessner A, Schneider-Stock R. Thymoquinone: a promising anticancer drug from natural sources. The International Journal of Biochemistry and Cell Biology 38 (8), 1249–1253, 2006. Gancedo JM, Gancedo C. Fructose-1, 6-diphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from fermenting and non fermenting yeasts. Archives of Microbiology 76 (2), 132–138, 1971. Gazioano TA, Galea G, Reddy KS. Scaling up interventions for chronic disease prevention: the evidence. Lancet 370 (9603), 1939–1946, 2007. Hosseinzadeh H, Parvardeh S. Anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine 11 (1), 56–64, 2004. Ishikawa Y, Saito MN, Ikemoto T, Takeno H, Watanabe K, Tani T. Actions of the novel oral antidiabetic agent HQL-975 in insulin-resistant non-insulin-dependent diabetes mellitus model animals. Diabetes Research and Clinical Practice 41 (2), 101–111, 1998. Kanter M. Effects of Nigella sativa and its major constituent, thymoquinone on sciatic nerves in experimental diabetic neuropathy. Neurochemical Research 33 (1), 87–96, 2008. Kardeşler L, Buduneli N, Bıyıkoğlu B, Çetinkalp Ş, Kütükçüler N. Gingival crevicular fluid PGE2, IL-1ß, t-PA, PAI-2 levels in type 2 diabetes and relationship with periodontal disease. Clinical Biochemistry 41 (10–11), 863–868, 2008. Kim SH, Hyun SH, Choung SY. Antidiabetic effect of cinnamon extract on blood glucose in db/db mice. Journal of Ethnopharmacology 104 (1–2), 119–123, 2006. Koida H, Oda T. Pathological occurrence of glucose 6-phosphatase in serum in liver diseases. Clinica Chimica Acta 4, 554–561, 1959. Lima LC, Assis GV, Hiyane W, Almeida WS, Arsa G, Baldissera V, Campbell CS, Simões HG. Hypotensive effects of exercise performed around anaerobic threshold in type 2 diabetic patients. Diabetes Research and Clinical Practice 81 (2), 216–222, 2008. Masiello P, Broca C, Gross R, Roye M, Manteghetti M, Hillaire-Buys D, Novelli M, Ribes G. Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes 47 (2), 224–229, 1998. Meyer C, Dostou JM, Welle SL, Gerich JE. Role of human liver, kidney, and skeletal muscle in postprandial glucose homeostasis. American Journal of Physiology. Endocrinology and Metabolism 282 (2), E419–E427, 2002. Nayak SS, Pattabiraman TN. A new colorimetric method for the estimation of glycosylated hemoglobin. Clinica Chimica Acta 109 (3), 267–274, 1981. Ohkuwa T, Sato Y, Naoi M. Hydroxyl radical formation in diabetic rat induced by streptozotocin. Life Sciences 56 (21), 1789–1798, 1995. Shirwaikar A, Rajendran K, Barik R. Effect of aqueous bark extract of Garuga pinnata Roxb. in streptozotocin–nicotinamide induced type-II diabetes mellitus. Journal of Ethnopharmacology 107 (2), 285–290, 2006. Takasu N, Komiya I, Asawa T, Nagasawa Y, Yamada T. Streptozocin and alloxan-induced H2O2 generation and DNA fragmentation in pancreatic islets. H2O2 as mediator for DNA fragmentation. Diabetes 40 (9), 1141–1145, 1991. Trinder P. Plasma glucose was measured by standard methods. Annals of Clinical Biochemistry 6, 24–27, 1969. Van de Werve G, Lange A, Newgard C, Mechin MC, Li Y, Berteloot A. New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system. European Journal of Biochemistry 267 (6), 1533–1549, 2000. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27 (5), 1047–1053, 2004.