Preventive effect of Tinospora cordifolia against high-fructose diet-induced insulin resistance and oxidative stress in male Wistar rats Singareddy Sreenivasa Reddy a, Pasurla Ramatholisamma b, Rasineni Karuna a, Desireddy Saralakumari a,* a Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515 003, Andhra Pradesh, India b Department of Biochemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, India a r t i c l e i n f o Article history: Received 6 February 2009 Accepted 5 June 2009 Keywords: Diabetes mellitus Oral glucose tolerance test Medicinal plants Antioxidants Sweetener Metabolic syndrome a b s t r a c t High intake of dietary fructose exerts a number of adverse metabolic effects. The aim of the present study was to investigate whether aqueous extract of Tinospora cordifolia stem (TCAE) alleviates high-fructose diet-induced insulin resistance and oxidative stress in rats. High-fructose diet (66% of fructose) and TCAE (400 mg/kg/day) were given simultaneously for a period of 60 days. Fructose fed rats showed hypergly- cemia, hyperinsulinemia, hypertriglyceridemia, impaired glucose tolerance and impaired insulin sensi- tivity (P 0.05). TCAE treatment prevented the rise in glucose levels by 21.3%, insulin by 51.5%, triglycerides by 54.12% and glucose���insulin index by 59.8% of the fructose fed rats. Regarding liver anti- oxidant status, fructose fed rats showed higher values of lipid peroxidation (91.3%), protein carbonyl groups (44%) and lowered GSH levels (42.1%) and, lowered activities of enzymatic antioxidants, while TCAE treatment prevented all these observed abnormalities. In conclusion, our data indicate the preven- tive role of T. cordifolia against fructose-induced insulin resistance and oxidative stress hence this plant could be used as an adjuvant therapy for the prevention and/or management of chronic diseases charac- terized by hyperinsulinemia, hypertriglyceridemia, insulin resistance and aggravated antioxidant status. �� 2009 Elsevier Ltd. All rights reserved. 1. Introduction The last 25 years have witnessed a marked increase in total per capita fructose intake as a sweetener in the food industry, primar- ily in the form of sucrose (a disaccharide consisting of 50% fruc- tose) and high-fructose corn syrup (HFCS 55���90% fructose content) (Bray et al., 2004). Processed-food manufacturers often prefer HFCS to sucrose because it is inexpensive, sweeter and mixes well in many foods. The increase in HFCS consumption far exceeds the increases in intake of any other food or food group. The disturbing fact is fructose consumption (excluding that which occurs naturally in fruits and vegetables) increased from less than 0.5 g/day in 1970 to more than 40 g/day in 1997 (more than an 80-fold increase) (Gaby, 2005). Concern has arisen because of the realization that fructose, at elevated concentrations, can promote metabolic changes that are actually or potentially deleterious, e.g., hyperlipidemia, hyperinsu- linemia, insulin resistance, hyperuricemia, hypertension, glucose intolerance and non-enzymatic fructosylation of proteins (Thor- burn et al., 1989 Hwang et al., 1987 Reddy et al., 2008 Dills, 1993). In addition, excessive fructose consumption may be respon- sible in part for the increasing prevalence of obesity, diabetes mel- litus, non-alcoholic fatty liver disease and cardiovascular diseases (Jurgens et al., 2005 Reaven, 1988 Reiser, 1985). Rats fed with a high-fructose diet form a model of diet-induced insulin resistance, associated with hyperinsulinemia, hypertriglycer- idemia and glucoseintolerance(Thorburnetal., 1989).Recently,anti- oxidants are found to be effective in preventing a majority of the abnormalities inducedbyhigh-fructosediet(Faure etal., 1997,1999). Tinospora cordifolia (Menispermaceae) is a glabrous, succulent, climbing shrub distributed throughout tropical Indian subcontinent. The plant is commonly known as Guduchi, Giloy or Amritha. This plant has been widely used in the Indian System of Medicine (Ayurv- eda) as Rasayana for the treatment of diabetes, jaundice, rheumatoid arthritis, gout, general weakness, skin diseases and infections (Deva- sagayam and Sainis, 2002). It is known to have hepatoprotective (Nagarkatti et al., 1994), immunostimulatory (Kapil and Sharma, 1997) and hyperlipidemic properties (Prince and Menon, 1999). A variety of constituents have been isolated from this plant belong to different classes such as alkaloids, diterpenoid lactones, glyco- sides, steroids, sesquiterpenoid, phenolics, aliphatic compounds and polysaccharides (Singh et al., 2003). Although, the antidiabetic and antioxidant activities of this plant in experimentally diabetic rats has been well documented in scientific literature (Prince and Menon, 1999 Grover et al., 2001), studies regarding its efficacy in 0278-6915/$ - see front matter �� 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.06.008 Abbreviations: AUC, area under the curve CAT, catalase GPx, glutathione peroxidase GR, glutathione reductase GSH, reduced glutathione GST, glutathione- S-transferase HFCS, high-fructose corn syrup OGTT, oral glucose tolerance test ROS, reactive oxygen species SOD, superoxide dismutase TC, Tinospora cordifolia TCAE, aqueous extract of Tinospora cordifolia stem. * Corresponding author. Tel.: +91 08554 255879 fax: +91 08554 255805. E-mail address: skumari1@yahoo.co.in (D. Saralakumari). Food and Chemical Toxicology 47 (2009) 2224���2229 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

preventing insulin resistance which plays a role in pathophysiology of type 2 diabetes mellitus have not been undertaken. The anti-hyperglycemic, anti-hyperlipidemic and antioxidant properties of T. cordifolia (TC) (in diabetic rat model) prompted us to design the present study to investigate whether management with TC has any preventive effect on plasma glucose, insulin, tri- glycerides, hepatic lipid peroxidation and activities of both enzy- matic and non-enzymatic antioxidant status in fructose fed rat model of insulin resistance. 2. Materials and methods 2.1. Chemicals Thiobarbituric acid and pyrogallol were obtained from the Sigma Chemical Co., St. Louis, MO, USA. All other chemicals and solvents were of analytical grade and were from Sisco Research Laboratories (P) Ltd., Mumbai, India. 2.2. Plant extract An aqueous extract of T. cordifolia stem (TCAE brown, dry powder with Lot No. L5111031) was procured from the manufacturers and exporters of herbal extracts, Ms. Plantex Pvt. Ltd., Vijayawada, Andhra Pradesh, India. Procedure followed by the firm for the preparation of extract is as follows: The plant was identified by Dr. K. Narasimha Reddy, Taxonomist, Laila Impex R&D Center, Vijayawada. The collected plant sample (stem) was washed thoroughly with tap water, dried at room temper- ature away from sun light, cut into small pieces and then powdered. The aqueous extract was prepared by cold maceration of stem powder in drinking water for 7 days. The extract was filtered, concentrated under reduced pressure and finally dried in a vacuum desiccator. Herb-to-product ratio was 10:1. A voucher specimen has been deposited in the Department of Biochemistry, Sri Krishnadevaraya Univer- sity, Anantapur, under number SK-TC-08. 2.3. Control and fructose diet The control diet for the rats contained 66% starch, 15% protein, 8% fat, 4% cellu- lose, 1% of each mineral and vitamin mix. The fructose diet contained 66% of fruc- tose instead of starch and remaining composition is same as that of the control diet. Both the diets were obtained from National Centre for Laboratory Animal Sciences, National Institute of Nutrition (Hyderabad, India). 2.4. Animals Male Albino Wistar rats (140���160 g) used for the present study were procured from Sri Venkateswara Enterprises (Bangalore, India). The animals were acclima- tized for 7 days in our animal house (Regd. No. 470/01/a/CPCSEA) before dietary manipulation. They were housed two per cage in an air-conditioned room (22 �� 2 ��C) with 12 h light/dark cycle and had free access to standard pellet diet and water. All the procedures were performed in accordance with the Institutional Animal Ethics Committee. 2.5. Experimental design All the animals were 6 weeks of age, weighing around 200 g at the time of die- tary manipulation. Animals were randomly assigned into four groups of eight each as given below: Group-C: normal control rats, received tap water and control diet, Group-F: fructose fed rats, received tap water and fructose diet, Group-F+TC: TCAE treated fructose fed rats, received TCAE (400 mg/kg/day) and fructose diet, Group-C+TC: TCAE treated normal rats, received TCAE (400 mg/kg/day) and con- trol diet. Vehicle (tap water for group-C and -F) and TCAE (dissolved in tap water) were administered orally by gastric intubation. The animals were maintained in their respective groups for 60 days. The dose of TCAE used in the current study was based on the earlier report on the anti-hyperglycemic effect of this plant in experimental diabetic rats (Grover et al., 2000) and our previous dose fixation studies for anti- hyperglycemic effect of TCAE in alloxan-induced diabetic rats (data not shown). The body weight, fasting plasma glucose, insulin and triglycerides of all animals were measured on initial, 15, 30, 45 and 60th day of experiment. 2.6. Oral glucose tolerance test (OGTT) At the end of experimental period (60 days), the 12-h fasted animals were sub- jected to oral glucose tolerance test. For this, a 40% glucose solution was introduced directly into the stomach through a fine gastric catheter at a dose of 2 g/kg body weight to conscious rats. Plasma glucose and insulin levels were determined at 0 (before glucose administration), 30, 60 and 120 min after glucose administration. 2.7. Measurement of glucose���insulin index The action of insulin on glucose disposal rate was measured using the glucose��� insulin index, which is the product of the areas under the curve (AUC) of glucose and insulin during the glucose tolerance test. 2.8. Sample collection Blood was collected from 12-h fasted rats with capillary tube from retino-orbi- tal plexus of the animals in fresh vials containing EDTA (10 mg/ml) as anticoagu- lant. The samples were centrifuged at 3000 rpm for 5 min (MSE Micro Centaur, UK) and the plasma obtained was aliquoted and frozen for insulin assay. Plasma glucose and triglycerides were determined immediately. The blood sample col- lected during oral glucose tolerance test was from the tail vein of animals. After the experimental period the animals were fasted overnight and killed by cervical decapitation. The body was cut open and liver was dissected out into ice- cold saline and then thoroughly rinsed. 2.9. Biochemical measurements The concentration of plasma glucose was measured by the glucose oxidase method, using Span Diagnostic Kit (Surath, India). Plasma triglyceride level was estimated by GPO���POD enzymatic method using the Monozyme Diagnostic kit (Secunderabad, India). Insulin was determined by radioimmunoassay kit (RIAK-1) provided by Bhabha Atomic Research Center (Mumbai, India) according to the method of Yalow and Berson (1961). Human insulin was used for the preparation of standard curve of insulin. The concentration of lipid peroxidation intermediates, liver thiobarbituric acid reactive substances (TBARS) was measured by following the method of Utley et al. (1967), using 10% liver homogenate in 0.15 M KCl and expressed as nmol MDA formed/15 min/mg protein. The extent of protein carbonyl groups (Levine et al., 1990) and reduced glutathione (GSH) levels (Ellman, 1959) in liver were deter- mined. Protein content in the liver homogenate was measured by the method of Lowry et al. (1951). 2.10. Enzyme assays Ten percent liver homogenate was prepared in ice-cold 0.15 M KCl, centrifuged at 12,000 rpm for 45 min in Sigma Laboratory centrifuge 3K18 model, rotor No. 12150. The clear supernatant thus obtained was used for the assay of superoxide dismutase (SOD E.C.1.15.1.1 Soon and Tan, 2002), catalase (CAT E.C.1.11.1.6 Beers and Sizer, 1952), glutathione peroxidase (GPx E.C.1.15.1.9 Rotsruck et al., 1973), glutathione-S-transferase (GST E.C.2.5.1.14 Habig et al., 1974) and glutathi- one reductase (GR E.C.1.8.1.7 Pinto and Bartley, 1969). 2.11. Statistical analysis All results were expressed as means �� SEM for the number, n = 8 of animals in the group as indicated in the figures and table. To determine the statistical signifi- cance of clinical and laboratory findings Duncan Multiple Range test (DMRT) was used. P values of less than 0.05 were regarded as significant. 3. Results 3.1. Effect of TCAE on body weight The body weights of four groups of animals during experimen- tal period are represented in Fig. 1A. No significant variation in body weights of groups C+TC and F+TC was observed when com- pared with group-C. Whereas, group-F also showed no significant variation up to 15 days but a significant (P 0.05) increase was observed from 30 days onwards till the end of experimental period when compared with group-C. 3.2. Fasting plasma glucose There was no significant variation in the plasma glucose con- centrations of group-C and C+TC throughout the experimental per- iod (Fig. 1B). Group-F showed a gradual and significant increase in plasma glucose levels from 30 days onwards till the end of exper- S.S. Reddy et al. / Food and Chemical Toxicology 47 (2009) 2224���2229 2225