Effect of Tectona grandis Linn. on dexamethasone-induced insulin resistance in mice

Journal of Ethnopharmacology 122 (2009) 304–307 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Effect of Tectona grandis Linn. on dexamethasone-induced insulin resistance in mice Mahesh Ghaisas ∗ , Vijay Navghare, Abhijit Takawale, Vinit Zope, Mukesh Tanwar, Avinash Deshpande Department of Pharmacology, Padm. Dr. D.Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune 411018, India a r t i c l e i n f o Article history: Received 5 July 2008 Received in revised form 7 December 2008 Accepted 3 January 2009 Available online 9 January 2009 Keywords: Antioxidant Diabetes Dexamethasone Glucose uptake Insulin resistance Tectona grandis a b s t r a c t Ethnopharmacological relevance: The bark of Tectona grandis Linn. is traditionally used in the treatment of diabetes. Aim: The present study was undertaken to investigate the effect of ethanolic extract of bark of Tectona grandis Linn. (TG) in dexamethasone-induced insulin resistance in mice. Materials and methods: Mice were treated with prestandardised dose of dexamethasone for 22 days and effect of TG at the doses of 50, 100 and 200 mg/kg, p.o. on plasma blood glucose level, serum triglyceride level, glucose uptake in skeletal muscle, levels of hepatic antioxidant enzymes (GSH, SOD, catalase and LPO), and body weight were observed. Results: TG showed significant decrease in plasma glucose and serum triglyceride levels (p < 0.01) at the dose of 100 and 200 mg/kg, p.o. and also stimulated glucose uptake in skeletal muscle. The levels of antioxidant enzymes GSH, SOD, and catalase were significantly increased (p < 0.01) and there was significant decrease (p < 0.01) in level of LPO. Conclusion: Hence it can be concluded that Tectona grandis may prove to be effective in the treatment of Type-II Diabetes mellitus owing to its ability to decrease insulin resistance. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Diabetes is the world’s largest endocrine disorder with deranged carbohydrate, fat and protein metabolism. Hormones such as catecholamine, glucagon, cortisol and thyroxin, either through directly or through their influence on other hormones, affect carbohydrate metabolism to elevate blood glucose level leading to insulin resistance. Insulin resistance has been shown to be present in conditions like Type-II Diabetes, obesity and dyslipidemia. Thus interventions to decrease insulin resistance may postpone the development of Type-II Diabetes and its complications (Gholap and Kar, 2005). Glucocorticoids in excess inhibit insulin secretion from pancreatic beta-cells, decrease glucose utilization, and stimulate glucagon secretion, lipolysis, proteolysis, and hepatic glucose production. Glucocorticoids can modulate the insulin action at both binding sites and postbinding sites and cause decreased glucose utilization in muscles. Glucocorticoids also cause insulin resistance by decreasing hepatic glucose utilization, decreasing glycogen synthe- sis. Free fatty acids may be elevated in insulin resistance because of impaired insulin-dependant down-regulation of lipolysis, hence leading to increase in triglyceride levels in muscles as well as other tissues presumably because of excess of circulating free fatty acids which are then deposited in these organs. The triglycerides are reported to be potent inhibitor of insulin signaling and result in acquired insulin resistance state (Andrews and Walker, 1999; Shalam et al., 2006). Traditionally, Tectona grandis is used in the treatment of diabetes, lipid disorders, inflammation, ulcer, and bronchitis (Warrier, 1994). Tectona grandis Linn. is reported to have antiulcer (Pandey et al., 1982), antimicrobial (Sumthong et al., 2006), wound healing (Majumdar et al., 2007), and anticancer activity (Khan and Miungwana, 1999). Taking into consideration the traditional claims and reported activities, the present study was planned to investigate the effect of Tectona grandis Linn. on dexamethasone-induced insulin resistance in mice. 2. Materials and methods Abbreviations: TG, ethanolic extract of bark of Tectona grandis; GSH, reduced glutathione; SOD, superoxide dismutase; LPO, lipid peroxidation; MDA, malondialdehyde. ∗ Corresponding author. Tel.: +91 9422080072; fax: +91 2027420261. E-mail address: ghaisasmm@yahoo.com (M. Ghaisas). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.01.008 2.1. Plant material and preparation of extract Fresh bark of Tectona grandis Linn. (Verbenaceae) was collected from Nanded, Maharashtra, India. The specimen was authenticated 305 M. Ghaisas et al. / Journal of Ethnopharmacology 122 (2009) 304–307 Table 1 Effect of Tectona grandis Linn. on plasma glucose, serum triglyceride level and body weight in dexamethasone-induced insulin resistance. Sr. no. Groups Plasma glucose (mg/dl) Serum triglyceride (mg/dl) Body weight change (g) (I) (II) (III) (IV) (V) (VI) (VII) (VIII) (IX) (X) Normal control DEXA-control DEXA + PIO DEXA + TG-50 DEXA + TG-100 DEXA + TG-200 PIO TG-50 TG-100 TG-200 54.42 76.40 55.29 73.37 66.73 62.37 53.51 56.81 52.09 50.38 83.68 144.38 85.48 138.45 124.20 110.20 81.87 87.62 87.33 86.44 +0.88 −2.11 +0.88 −1.91 −1.81 +0.91 +0.98 +0.97 +1.26 +1.14 ± ± ± ± ± ± ± ± ± ± 1.15 0.84a ** 0.94b ** 0.68 0.66b ** 1.27b ** 0.51 0.34 0.97 0.53a * ± ± ± ± ± ± ± ± ± ± 1.17 2.42a ** 1.91b ** 1.49 1.90b ** 1.63b ** 1.83 1.04 0.99 0.79 ± ± ± ± ± ± ± ± ± ± 0.05 0.13a ** 0.05b ** 0.12 0.14 0.01b ** 0.07 0.07 0.19 0.18 Values are expressed as mean ± S.E.M., n = 6, DEXA = dexamethasone 1 mg/kg, i.m., PIO = Pioglitazone 2 mg/kg, p.o., TG = Tectona grandis 50, 100, 200 mg/kg, p.o. a *p < 0.05, a **p < 0.01 when compared with normal control. b **p < 0.01 when compared with DEXA-control. (+) and (−) sign indicates increase and decrease in body weight. at Agharkar Research Institute, Pune with voucher specimen no. 08–12 and catalogued. The bark was washed with distilled water and shed dried and latter powdered. This powder was then defatted with petroleum ether and then macerated with ethanol for 72 h with occasional shaking. It was then filtered and the solvent was evaporated under vacuum. The yield of ethanolic extract of bark of Tectona grandis Linn. (TG) was 2.7% (w/w). TG when subjected for phytochemical study showed the presence of beta-sitosterol, terpenoids, phenolic compounds, saponins, glycosides and tannins (Khandelwal, 2005). 2.2. Animals 72 Albino mice weighing 25–30 g were used for study and were kept in animal house at 26 ± 2 ◦ C with relative humidity 44–56% along with light and dark cycles of 12 h. Institution Animal Ethics Committee has approved the experimental protocol (198/99/CPCSEA). Animals were provided with standard diet and water ad libitum. The food was withdrawn 18–24 h before the start of the experiment. 2.3. Experimental design 2.3.1. Acute toxicity study (OECD, 425) The acute toxicity study for ethanolic extract of bark of Tectona grandis Linn. was performed using albino mice. The animals were fasted overnight prior to the experiment and maintained under standard conditions. TG was found safe up to dose of 2000 mg/kg, p.o. 2.3.2. Dexamethasone-induced insulin resistance in mice (Gholap and Kar, 2005) All the mice were weighed before treatment, group I (normal control) received equivalent amount of 1% gum acacia (1 ml/kg, p.o.), and 30 mice were rendered hyperglycemic by daily administration of a prestandardised dose of dexamethasone (1 mg/kg, i.m.) for consecutive 7 days and then divided in to five groups of six each. Group II (DEXA-control) continued to receive only dexamethasone and 1% gum acacia (1 ml/kg, p.o.) for next 15 days, III received Pioglitazone (2 mg/kg, p.o.) along with dexamethasone respectively for 15 days. Groups IV, V, VI were treated with dexamethasone along with three different doses of TG 50, 100, 200 mg/kg, p.o. respectively for 15 days. Simultaneously four other groups (groups VII, VIII, IX, and X), each with six normoglycemic animals, were administered equivalent amount of Pioglitazone and three different doses of TG 50, 100, and 200 mg/kg, p.o. respectively (Table 1). On the last day, after overnight fasting, all the animals were weighed and later sacrificed by cervical dislocation. Blood samples were collected and used for estimation of glucose and triglyceride. Biochemical estimation of plasma glucose and serum triglyceride was done by GOD/POD and GPO/POD method respectively using standard diagnostic kits from Biolabs India Ltd., India. 2.3.3. Hepatic antioxidant enzymes assay (estimation of MDA, GSH, SOD, and CAT) Liver samples were dissected out and washed immediately with ice cold saline to remove as much blood as possible. Liver homogenates (5%, w/v) were prepared in cold 50 mM Tris buffer (pH 7.4) using Remi homogenizer. The unbroken cells and cell debris were removed by centrifugation at 5000 rpm for 10 min using a Remi refrigerated centrifuge. The supernatant was used for the estimation of GSH (Ellaman, 1959), malondialdehyde (MDA) (Slater and Sawyer, 1971), superoxide dismutase (SOD) (Mishra and Fridovich, 1972) and catalase (Aebi, 1974; Colowick et al., 1984) levels (Table 2). 2.3.4. Effect on glucose uptake in isolated mice hemidiaphragm (Chattopadhyay et al., 1992; Sabu and Subburaju, 2002; Ghosh et al., 2004). Glucose uptake in mice hemidiaphragm was estimated by the method described by Chattopadhyay et al. (1992) with some modification. Twelve sets, containing graduated test tubes (n = 6) each, were used for study of non-insulin assisted and insulin assisted glucose uptake. The diaphragms were taken out quickly avoiding traumas and divided into two halves. The hemidiaphragms were rinsed in cold Tyrode solution (without glucose) to remove any blood clots. In non-insulin assisted glucose uptake study, one hemidiaphragm of each animal from groups I to VI was exposed to 2 ml Tyrode solution with glucose (2000 mg/l) in respective graduated test tubes. In insulin assisted glucose uptake study, the remaining hemidiaphragm of each animal from groups I to VI was exposed to 2 ml Tyrode solution with glucose (2000 mg/l) + insulin (0.25 IU/ml) in respective graduated test tubes. All the graduated test tubes were incubated for 30 min at 37 ◦ C in an atmosphere of 95% O2 –5% CO2 with shaking at 140 cycles per minutes. Following incubation, the hemidiaphragm was taken out and weighed. The glucose content of the incubated medium was measured by GOD/POD, enzymatic method. Glucose uptake was calculated as the difference between the initial and final glucose content in the incubation medium (Table 3). 2.4. Statistical analysis The results were expressed as mean ± S.E.M. and statistically analyzed by ANOVA followed by Dunnett test, with level of significance set at p < 0.05. 306 M. Ghaisas et al. / Journal of Ethnopharmacology 122 (2009) 304–307 Table 2 Effect of Tectona grandis Linn. on different antioxidant enzyme levels. Sr. no. Groups GSH (␮g of GSH/g of tissue) SOD (units/mg of tissue) Catalase (␮M of H2 O2 /g of tissue/min) LPO (nM of MDA/g of tissue) (I) (II) (III) (IV) (V) (VI) (VII) (VIII) (IX) (X) Normal control DEXA-control DEXA + PIO DEXA + TG-50 DEXA + TG-100 DEXA + TG-200 PIO TG-50 TG-100 TG-200 24.42 14.77 18.58 16.60 18.21 22.31 25.73 23.41 25.61 24.80 73.65 22.98 72.55 45.81 58.04 68.55 72.37 71.66 71.80 71.61 6.83 3.07 5.83 3.53 4.14 5.79 6.81 7.06 6.91 6.51 10.58 20.94 10.29 17.63 12.72 11.76 10.95 10.83 10.80 10.90 ± ± ± ± ± ± ± ± ± ± 2.43 1.67a ** 1.76b ** 1.35 1.57b ** 2.08b ** 2.31 1.87 2.10 2.04 ± ± ± ± ± ± ± ± ± ± 2.95 1.02a ** 3.62b ** 2.12b ** 2.79b ** 3.98b ** 3.21 2.87 3.07 2.11 ± ± ± ± ± ± ± ± ± ± 0.03 0.02a ** 0.03b ** 0.02 0.03 0.03b ** 0.04 0.05 0.07 0.05 ± ± ± ± ± ± ± ± ± ± 0.94 1.27a ** 0.87b ** 1.20b ** 0.64b ** 0.75b ** 1.10 0.83 1.02 0.79 Values are expressed as mean ± S.E.M., n = 6, DEXA = dexamethasone 1 mg/kg, i.m., PIO = Pioglitazone 2 mg/kg, p.o., TG = Tectona grandis 50, 100, 200 mg/kg, p.o. a **p < 0.01 when compared with normal control. b **p < 0.01 when compared with DEXA-control. 3. Results 3.1. Effects of TG on plasma glucose, serum triglyceride and body weight In DEXA-control group there was significant increase in plasma glucose level (p < 0.01) and serum triglyceride level (p < 0.01) when compared with normal control. All mice treated with DEXA and TG showed significant decrease (p < 0.01) in the levels of plasma glucose and serum triglyceride when compared with DEXA-control. The mice treated with DEXA and Pioglitazone showed significant decrease in plasma glucose level (p < 0.01) and serum triglyceride level (p < 0.01) when compared with DEXA-control. TG at the dose of 200 mg/kg, p.o. showed marginal hypoglycemia (p < 0.05) when compared with normal control. Significant reduction (p < 0.01) in body weight was observed in DEXA-control group when compared with normal control. TG and Pioglitazone treatment significantly inhibited the dexamethasone induced decrease in body weight (p < 0.01) when compared DEXA-control. 3.2. Effects of TG on MDA, GSH, SOD and CAT levels In DEXA-control group there was increase in the levels of MDA (p < 0.01) when compared with normal control, treatment with TG significantly prevented this rise (p < 0.01). DEXA-control group showed significant decrease (p < 0.01) in GSH, SOD and CAT when compared with normal control, where as significant increase (p < 0.01) in the levels of these enzymes was observed in the TG treated groups when compared with DEXA-control group. 3.3. Effect of TG on glucose uptake in mice isolated hemidiaphragm Hemidiaphragm of the mice treated with dexamethasone showed significant decrease (p < 0.01) in glucose uptake when Table 3 Effect of Tectona grandis Linn. on glucose uptake in mice isolated hemidiaphragm. Sr. no. Group Non-insulin assisted glucose uptake mg/g/30 min Insulin assisted glucose uptake mg/g/30 min I II III IV V VI Normal DEXA-control DEXA + PIO DEXA + TG-50 DEXA + TG-100 DEXA + TG-200 8.89 5.12 7.44 7.18 12.34 18.66 14.47 8.74 23.46 14.50 17.15 22.41 ± ± ± ± ± ± 0.31 0.33a ** 0.40 0.46 0.37b ** 0.54b ** ± ± ± ± ± ± 0.77 0.34a ** 1.23c ** 0.38c ** 0.93c ** 1.02c ** Values are expressed as mean ± S.E.M., n = 6, DEXA = dexamethasone 1 mg/kg, i.m., PIO = Pioglitazone 2 mg/kg, p.o., TG = Tectona grandis 50, 100, 200 mg/kg, p.o. a **p < 0.01 when compared with normal control or normal + insulin. b **p < 0.01 when compared with DEXA-control. c **p < 0.01 when compared with DEXA + insulin group. compared with normal control. Pioglitazone did not show any significant increase in glucose uptake by mice isolated hemidiaphragm. TG at higher doses i.e. 100, 200 mg/kg, p.o. showed significant increase (p < 0.01) in glucose uptake when compared with DEXA-control. In insulin assisted glucose uptake, Pioglitazone showed significant increase (p < 0.01) in glucose uptake when compared with DEXA + insulin group. Higher doses TG 100, 200 mg/kg, p.o. along with insulin showed significant increase (p < 0.01) in glucose uptake when compared with DEXA + insulin group. 4. Discussion In the present study, dexamethasone administration resulted in significant increase in blood glucose and triglyceride level. TG showed dose dependent decrease in elevated plasma glucose and triglyceride levels caused by dexamethasone, <beta> lapachone, one of the chemical constituents of Tectona grandis was reported to possess glucocorticoid antagonistic action (Schmidt et al., 1984). The effect of TG on the plasma glucose level and triglyceride level may be due to the glucocorticoid antagonism and the presence of other chemical constituents like terpenoids and tannins which are reported to have antihyperglycemic action (Matsudha et al., 2002). TG showed significant increase in insulin assisted glucose uptake, which indicates that there was increase in the insulin sensitivity. Glucocorticoid treatment is known to induce insulin resistance and catabolic states in rats (Harber and Weinstein, 1992). Pharmacological doses of glucocorticoids induce ob gene expression in rat adipocyte tissues within 24 h which is followed by complex metabolic changes like hyperleptinemia, resulting in decrease in food consumption, reduction in body weight with enhanced blood glucose and triglyceride levels (Kim et al., 2002; Shalam et al., 2006). As expected, in the present study, dexamethasone group showed reduction in body weight, while TG and Pioglitazone treatment inhibited dexamethasone-induced reduction in body weight and showed marginal increase in body weight. The effect of TG on the body weight may be attributed to the increase in the sensitivity to insulin and the subsequent increase in the glucose uptake. Oxidative stress can be generated by hyperglycemia and for a long time it has been accused to cause insulin resistance. Insulin resistance induces release of cytokines like TNF-alpha, IL-8 which leads to development of oxidative stress in liver by reducing the mitochondrial levels of Cu/Zn SOD, glutathione and producing H2 O2 radicals and leads to increase in lipid peroxidation. Mitochondria are significant source for generation of superoxide radicals (Marfella et al., 2001). Insulin resistance is associated with increase in fat accumulation and mitochondrial oxidative stress. In present study, TG showed significant increase in the concentration of various antioxidant enzymes, which could be beneficial in countering, the hyperglycemia induced oxidative stress. Ethanolic extract of Tectona grandis contains tannins, terpenoids and saponins (Pandey M. Ghaisas et al. / Journal of Ethnopharmacology 122 (2009) 304–307 et al., 1982; Majumdar et al., 2007). 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