Antidiabetic effect of secoisolariciresinol diglucoside in streptozotocin-induced diabetic rats

Phytomedicine 20 (2013) 237–245

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Antidiabetic effect of secoisolariciresinol diglucoside in streptozotocin-induced diabetic rats Sadiq S. Moree a,b , G.B. Kavishankar b , J. Rajesha a,∗ a b

Department of Biochemistry, Yuvaraja’s College, University of Mysore, Mysore 570005, Karnataka, India DOS in Biochemistry, Manasagangotri, University of Mysore, Mysore 570006, Karnataka, India

a r t i c l e

i n f o

Keywords: Antidiabetic effect Antioxidant enzymes Synthetic SDG Streptozotocin

a b s t r a c t Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. Its complications such as neuropathy, cardiopathy, nephropathy, and micro and macro vascular diseases are believed to be due to the increase in oxidative stress and decrease in the level of antioxidants. The aim of this study was to determine the antihyperglycemic activity of synthetic Secoisolariciresinol diglucoside (SDG) in streptozotocin (STZ)-induced diabetic rats. The synthetic SDG in a single-dose (20 mg/kg b.w.) two-day study showed dose-dependent reduction in glucose levels with maximum effect of 64.62% at 48 h post drug treatment (p < 0.05), which is comparable to that of the standard drug tolbutamide (20 mg/kg b.w.). In a multi-dose fourteen-day study, lower doses of SDG (5 and 10 mg/kg b.w.) exhibited moderate reduction in glucose levels, lipid profile, restoration of antioxidant enzymes and improvement of the insulin and c-peptide levels which shows the regeneration of ␤-cell which secretes insulin. Altered levels of lipids and enzymatic antioxidants were also restored by the SDG to the considerable levels in diabetic rats. Results of the present investigation suggest that diabetes is associated with an increase in oxidative stress as shown by increase in serum malondialdehyde (MDA), decreased levels of catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH). Also, diabetes is associated with an increase in serum total cholesterol as well as triglycerides levels and decrease in insulin and c-peptide levels. SDG is effective in retarding the development of diabetic complications. We propose that synthetic SDG exerts anti hyperglycemic effect by preventing the liver from peroxidation damage through inhibition of ROS level mediated increased level of enzymatic and non-enzymatic antioxidants. And, also maintaining tissue function which results in improving the sensitivity and response of target cells in STZ-induced diabetic rats to insulin. © 2012 Elsevier GmbH. All rights reserved.

Introduction Diabetes mellitus is a metabolic disorder caused by impaired secretion of insulin from pancreatic ␤-cells and is one of the three leading causes of death worldwide (Arvindkumar et al. 2012; Islam and Choi 2009). It is chronic, multifaceted, dynamic expression of pathological disequilibria, resulting in various micro and macro vascular complications (Marin et al. 2011). The chronic hyperglycemia of diabetes is associated with long term damage, dysfunction and failure of various organs, especially the eyes, kidneys, nerves, heart and blood vessels (ADA 2008). It is also defined as a state in which homeostasis of carbohydrate and lipid metabolism is improperly regulated by insulin, the pancreatic hormone, resulting in an increased blood glucose level. The ostensible reason of diabetes is either decrease in the synthesis of insulin

∗ Corresponding author. E-mail address: [email protected] (J. Rajesha). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.11.011

(Type-I diabetes) or by insulin insensitivity, followed later by death of ␤-cells of islets of Langerhans of pancreas (Type-II diabetes). Diabetes poses a significant health problem worldwide. According to the recent statistics, approximately 285 million people in the age group of 20–79 years, worldwide (6.6%) have diabetes in 2010 and 438 million people (7.8%) among the adult population is expected to have diabetes by 2030. Epidemiological data shown that, diabetes is disproportionately affecting the regions dominated by developing economies (IDF 2009). About 80% of people with diabetes are in developing countries, of which India and China share larger contribution (Ramachandran et al. 2010). There are reports indicating that increase in blood glucose level produces superoxide anions, resulting in peroxidation of membrane lipids and protein glycation. These radicals further damage other important biomolecules including carbohydrates, proteins and DNA (Sato et al. 1979; Muruganandan et al. 2005). Experimental induction of diabetes mellitus in animal models is essential for understanding various aspects of its pathogenesis and ultimately finding new therapies and cure. Several methods have been used to induce diabetes mellitus in laboratory animals with

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4-dinitrobenzene (CDNB), reduced glutathione (GSH), superoxide dismutase (SOD), nitroblue tetrazolium (NBT), butylated hydroxytoluene (BHT), and trichloroacetic acid (TCA) were purchased form Sigma Chemical Co., USA. Mannitol was procured from SD Fine Chemicals, Mumbai, India. Trolox and phenazine methosulphate (PMS) were procured from Himedia, Mumbai, India. UV visible spectrum measurements were carried out using Shimadzu 160A Spectrophotometer, Shimadzu Instrumentation Co., USA. All solvents and other chemicals used in the studies were of analytical grade and purchased from Ranbaxy Fine Chemicals Ltd., India. Drug preparation

Fig. 1. Chemical structure of SDG.

variable success. Majority of studies done during 1996–2006 in ethno-pharmacology employed chemical induced model like streptozotocin (STZ) (69%) and alloxan (31%). The former is the most frequently used and well studied drug and has been useful for the study of multiple aspects of the disease (Rydgren et al. 2007). Streptozotocin is known to act as alkylating and oxidantine in pancreatic islets (Lenzen 2008). Phytoestrogens are naturally occurring plant based diphenolic compounds that are similar in structure and function to estradiol. They have gained increasing attention because of their protective roles against numerous chronic diseases including cancer, cardiovascular disease, dyslipidaemia and diabetes (Duncan et al. 2003; Bhathena and Velasquez 2002). The well-studied phytoestrogens are isoflavones and lignans, known as dietary lignans which are of plant origin. Dietary lignans are broadly available in plant-based foods, and is particularly concentrated in flaxseed (Duncan et al. 2003). It is reported that daily supplementation of lignan results in significant improvements in glycemic control in diabetic patients without apparently affecting fasting glucose levels, lipid profiles and insulin sensitivity (Pan et al. 2007). Several studies reported that SDG (Fig. 1), the major lignan in flaxseed (Kurzer and Xu 1997), significantly reduced total and LDL cholesterol (LDL-C) concentrations in rabbits (Prasad 1999, 2005). Earlier study in our laboratory and other in vivo and in vitro studies have showed that flaxseed SDG could be a potent antioxidant (Sadiq and Rajesh 2012; Rajesha et al. 2006; Prasad 1997) and cardiovascular disease preventing agent (Zanwar et al. 2011). It is hypothesized that SDG with its anti-platelet-activating factor (PAF) and antioxidant activity would prevent the consequence of diabetes in diabetic rats through inhibiting the production of ROS and eliminating the ROS produced, which is an antagonist of PAF-receptor (Cox and Wood 1987). Supplementation of lignan, SDG to diabetic rats significantly prevented or delayed the onset of diabetes and improved glycemic control in rats with Type-I and Type-II diabetes which are mediated through oxidative stress (Prasad 2000, 2001). However, there are no reports on antidiabetic potential of synthetic SDG and its association with ROS generated pancreas. Therefore, the purpose of this investigation was to assess the effect of synthetic SDG on serum glucose, insulin, c-peptide, lipid profile, protein content and enzymatic and non-enzymatic antioxidant levels in liver and pancreas in streptozotocin-induced diabetic rats. Materials and methods Chemicals Streptozotocin (STZ), thiobarbituric acid (TBA), 5,5 -dithiobis-2-nitrobenzoic acid (DTNB, Ellman’s reagent), 1-chloro-2,

Tolbutamide tablets were purchased from a local pharmaceutical company in Mysore, Karnataka, India. The tablets were finely powdered and suspended in distilled water and administrated to animals at 20 mg/kg body weight of tolbutamide substance. SDG synthesis SDG was synthesized via a novel five-step synthesis sequence beginning from the bromination of the commercially available compound 3,4-dimethoxy toluene with N-bromo succunimide in presence of carbon tetrachloride to achieve 1,2-dimethoxy4-bromomethyl benzene (1). Compound, 2,3-bis (3,4-dimethoxy benzyl) butane-1,4-diol (2) was afforded by stirring compound1 with 1,4-butanediol in presence of n-butyl lithium and DMF (15 ml). Sequentially condensation of compound-2 with 2,3,4,6 tetra-o-acetyl ␣-d glucopyranosyl bromide in presence of 2 N hydrochloric acid and ethanol gave 2,3-bis (3,4-dimethoxybenzyl) butane-1,4-O-tetra acetyl glucose (3). Compound, 2,3-bis (3,4dimethoxybenzyl)butane-1,4-O-glucose (4) was achieved in excellent yield by deacetylation of compound-3 with 0.75 g sodium hydroxide in presence of ethanol. Partial regioselective demethylation of compound-4 using Lewis acid, stannous chloride, dichloromethane and 2 N hydrochloric acid was carried out to attain SDG (Fig. 1) (Sadiq et al. in press). Experimental animals Male Wistar albino rats weighing (150–175 g) were obtained from the animal house, Department of Studies in Zoology, University of Mysore, Mysore, India. The animals were maintained under controlled conditions of temperature (24 ± 2 ◦ C), humidity (40 ± 5%) and 12-h light–dark cycles and provided with a standard pellet diet (Hindustan Lever, Bangalore, India). The animals were randomized into experimental and control groups and housed six animals each in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free access to standard pellets as basal diet and water ad libitum. All animal studies conducted were approved by the Institutional Animal Ethics Committee, University of Mysore [Approval No. UOM/IAEC/01/2011], Mysore, as stated by prescribed guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Induction of experimental diabetes mellitus Animals were fasted overnight before injecting with streptozotocin. Diabetes mellitus was induced by single intraperitoneal injection of freshly prepared solution of STZ (60 mg/kg b.w.) in 0.1 M citrate buffer of pH 4.5, after overnight fasting for 12 h. Control normal rats received equivalent amounts of vehicle (Olive oil) intraperitoneally. Animals were treated with 5% glucose solution orally to combat the early phase of drug-induced hypoglycemia. The blood glucose levels of animals were measured 48 h after STZ

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administration through tail tipping using glucometer (One touch, life scan Europe, Switzerland). Those rats showing fasting blood glucose levels of above 250 mg/dl with the indication of glycosuria were considered diabetic and included in the study. Control and STZ-treated rats were given food and water ad libitum. The non-diabetic rats were randomly divided into two groups (I and III) and diabetic rats were randomized into four groups (II, IV–VII) each group of six rats. Group-I (normal rats) received equal volume of vehicle. Group-II (diabetic rats) served as diabetic control. Group-III (normal rats) received synthetic-SDG (10 mg/kg b.w. P.O.). Group-IV (diabetic rats) received antidiabetic agent tolbutamide (20 mg/kg b.w. I.P.), and treated as positive control. Group-V, VI and VII (diabetic rats) received synthetic SDG at 5, 10 and 20 mg/kg b.w. P.O. respectively. Protocol 1: The effect of single oral dose of synthetic SDG (5, 10 and 20 mg/kg b.w.) on hyperglycemia at different time intervals was studied in 12 h fasted STZ-induced diabetic animals. Blood glucose level was measured at different time intervals (0, 2, 6, 12, 24 and 48 h) through tail tipping by using glucometer (one touch, life scan Europe, Switzerland). Glycemia reduction percentage was calculated with respect to the initial (0 h) level according to: Glycemia reduction % =

 (G − G )  t i Gi

× 100

where Gi is initial glycemia and Gt is glycemia at t h (Yanardag and Colak 1998). Protocol 2: The above groups of animals were further treated with single daily doses for further 14 days in order to evaluate the chronic effect of synthetic SDG treatment on hyperglycemia (Multiple-dose fourteen-day study). At the end of 14 days of treatment, the animals were fasted overnight and blood samples were collected into heparinised tubes by cardiac puncture. Serum was separated and analyzed for the quantitative measurement of the level of glucose, insulin by solid phase competitive enzyme linked immunosorbent assay (ELISA), C-peptide by standard radioimmunoassay and triglyceride (TG), total cholesterol (TC), and HDL-cholesterol (HDL-c), using diagnostic reagent kit (Nicholas Piramal India Ltd., Mumbai). VLDL-cholesterol (VLDL-c) and LDL-cholesterol (LDL-c) in serum calcu were  ; LDL − c = lated as per Friedewald’s equation.VLDL − c = TG 5



Total cholesterol −

 TG  5



− HDL − c .

Tissue preparation After the experimental regimen (Protocol 2), animals were sacrificed by cervical dislocation. Liver and pancreas were excised, cleaned of gross adventitial tissue, blotted dry and processed for biochemical measurements (CAT, SOD, GSH and MDA). Tissues were homogenized in 5.0% (w/v) 0.15 M KCl and centrifuged at 800 × g for 10 min. The cell-free resultant supernatant was used for the measurement of enzymatic and non-enzymatic antioxidants (CAT, GSH and MDA). The other 5.0% (w/v) homogenate prepared using phosphate buffer (5.0 M) containing 0.25% sucrose (w/v) was used for SOD assay. All protocols were followed as per Ethical Committee Guidelines after clearance for experiment. Estimation of catalase The CAT assay was carried out as described (Aebi 1984). Briefly, 1 ml of liver/pancreas homogenate was taken with 1.9 ml of

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phosphate buffer in different test tubes (10 mM, pH 7.4). Hydrogen peroxide (H2 O2 , 1 ml, 30 mM) was added to initiate the reaction. Blank was prepared with 2.9 ml of phosphate buffer and 1 ml of H2 O2 without liver/pancreas homogenate. The decrease in optical density due to decomposition of H2 O2 was measured at the end of 1 min using the blank at 240 nm. Enzyme activity was defined in terms of units of catalase required to decompose 1 ␮M of H2 O2 at 25 ◦ C. The specific activity was expressed in terms of units/mg protein. Estimation of SOD The SOD assay was based on the reduction of nitroblue tetrazolium (NBT) to water insoluble blue formazan as described by Beuchamp and Fedovich (1976). Liver or pancreas homogenate (0.5 ml) was taken and 1 ml of 10 mM sodium carbonate, 0.4 ml of 24 ␮M NBT, and 0.2 ml of 0.1 mM EDTA were added. The reaction was initiated by adding 0.4 ml of 1 mM hydroxylamine hydrochloride. Absorbance was read at 560 nm after 5 min at 25 ◦ C. Control was simultaneously run without liver or pancreas homogenate. Unit of SOD activity was defined as the amount of enzyme required to inhibit the reduction of NBT by 50.0%. The specific activity was expressed in terms of units/mg protein. Estimation of reduced glutathione (GSH) The GSH level was estimated according to the method of Moron et al. (1979). The protein free filtrate after precipitation with metaphosphoric acid was reacted with 5-5 dithiobis (2nitrobenzoic acid) (CDNB). The CDNB and sulphydryl groups form a relatively stable yellow color which can be read at 420 nm against the blank. Lipid peroxidation activity Thiobarbituric acid (TBA) reacts with malondialdehyde (MDA) to form a diadduct, a pink chromogen, which can be detected spectrophotometrically at 532 nm as reported by Buege and Aust (1978). Liver or pancreas homogenate (0.5 ml) and 1 ml of 0.15 M KCl were taken. Peroxidation was initiated by adding 250 ␮l of 0.2 mM ferric chloride. The reaction was run at 37 ◦ C for 30 min and stopped by adding 2 ml of an ice-cold mixture of 0.25 N HCl containing 15% TCA, 0.30% TBA and 0.05% BHT. The reactants were further heated at 80 ◦ C for 60 min and cooled, and results were expressed as MDA an equivalent, which was calculated by using an extinction coefficient of 1.56 × 105 M−1 cm−1 . Unit of lipid peroxidation activity was defined as the amount of TBA that gets converted into thiobarbituric acid reactive substances (TBARS). The specific activity was expressed in terms of units/mg protein. Estimation of protein Protein was determined using bovine serum albumin (BSA) as standard following the method of Lowry et al. (1951). Statistical analysis The experimental data are expressed as mean ± SD. Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Student’s t-test to determine the significant difference between samples within 95% confidence interval. The results were considered statistically significant at p < 0.05. The numbers of samples used in each experiment are indicated in appropriate places.

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Results and discussion Diabetes mellitus is often linked with abnormal lipid metabolism. The impairment of insulin secretion results in enhanced metabolism of lipids from adipose tissue to the plasma (Ananthan et al. 2004). It has been demonstrated that insulin deficiency in diabetes leads to a variety of disruption in metabolic and regulatory processes, which in turn lead to accumulation of lipids (Goldberg 1981). Diabetes induced by STZ is characterized by apoptosis of ␤-cells of pancreas, attenuation of gene expression of insulin and reduced synthesis of insulin. Usually, ␤-cells of pancreas normally maintain blood glucose concentrations within a narrow range by modulating their insulin secretion rate in response to the blood glucose concentration (Patel et al. 2006; Jiang et al. 2007). Apoptosis of pancreatic ␤-cells is believed to be the primary factor which ultimately results in hyperglycemia. The various symptoms observed in STZ-induced diabetes include polydipsia, polyphagia, and great loss in body weight, which results from structural proteins degradation/loss (Rajkumar et al. 1991). In the present study, we investigated the effects of synthetic SDG on a series of biochemical parameters in STZ-induced diabetic rats. Treatment of diabetic rats with selected anitidiabetic agents produced varying levels of blood glucose reduction. The choice of tolbutamide was to reflect the two commonly used major groups of hypoglycemic agents namely sulphonylureas and biquanides, which have different mechanism of action. The hypoglycemic action of sulfonylurea is due to stimulation of pancreatic islets cells, which results in an increase in insulin secretion (Chika and Bello 2010). Tolbutamide is one of sulfonylureas applied for non-insulin-dependent, Type-II diabetes, and its metabolism is dependent mainly upon the CYP2C9 activity in the liver (Proks et al. 2002; Lee et al. 2003). It is reported that tolbutamide is a potent oral blood-glucose-lowering drug of the sulfonylurea class and it acutely relies on stimulating the release of insulin from the pancreas, an effect depending upon functioning of beta cells in the pancreatic islets to reduce blood glucose level in diabetes mellitus (Federiuk et al. 2004). Tolbutamide appears to lower the blood glucose acutely by stimulating the release of insulin from the pancreas, an effect depending upon functioning of beta cells in the pancreatic islets, and its absorption is relatively rapid, and peak levels are achieved after 3–4 h. The drug is 95% protein bound and extensively metabolized to hydroxytolbutamide and carboxytolbutamide by microsomal oxidation in the liver, and is excreted by the kidneys. The elimination of half-life varies between 3 and 27 h, and the effective duration of action is 6–10 h (Sartor et al. 1980). The present study measures series of biochemical indicators including glucose, insulin and c-peptide levels. The c-peptide is considered to be a marker for pancreatic insulin secretion that has a longer half-life than insulin itself and, therefore, reflects more accurately the level of circulating insulin. In addition, the levels of enzymatic and non-enzymatic antioxidant were estimated in liver and pancreas of examined animals and the results are discussed below. Administration of STZ (60 mg/kg b.w. I.P.) induced hyperglycemia (blood glucose level ≥150 mg/dl) in almost all treated rats. The blood glucose level was monitored for 48 h at intervals (0, 2, 6, 12, 24 and 48) as per the protocol-1 for all the seven groups. The blood glucose level remained fairly stable at about 98 and 305 mg/dl in the normal control plus SDG 10 mg/kg (Group-III) and diabetic control (Group-II) rats, respectively during the 2 h observation period (Fig. 2), and it dramatically decreased to 268 mg/dl at end of the 48 h in diabetic control group. Whereas, no such change was noticed in normal control with SDG (10 mg/kg) fed group. Moderate activities (43.42 and 51.56%) were observed after 48 h in diabetic rats group treated with single dose of synthetic SDG (5 and 10 mg/kg) respectively.

Fig. 2. Effect of synthetic SDG on serum glucose levels in STZ-induced diabetic rats [single dose two-day study]. Each point represent mean ± SD; N = 6 in each group.

Rats treated with single dose of synthetic SDG at 20 mg/kg significantly (p < 0.05) exhibited high hypoglycemic activity (64.62%) in terms of reduction of blood glucose levels as compared to diabetic control (Group-II) in two days. The hypoglycemic activity of synthetic SDG showed almost an equal to that of the standard drug tolbutamide (65.20%) in reducing the glucose level at 20 mg/kg in STZ-induced diabetic rats (Fig. 3). Repetitive administration of synthetic SDG for 14 days as in protocol-2, showed significant reduction (p < 0.01) in blood glucose levels in Groups-V, VI and VII when compared to the diabetic control (Group-II). SDG treated diabetic rats showed higher hypoglycemic activity, where it reduced the serum glucose level to almost normal range which is comparable to tolbutamide treated animals (Group-IV). Concentrations of insulin and c-peptide were also determined as per the protocol2. The levels of insulin and c-peptide were found to be restored gradually in synthetic SDG treated groups (Table 1). Diabetic control animals (Group-II) showed 56.41 and 61.96% reduction of insulin and c-peptide levels respectively, when compared to normal control animals (Group-I). These results indicate the damage and death of pancreatic ␤-cells of diabetic animals. The reductions of insulin and c-peptide levels in diabetic animals were restored in tolbutamide treated animals (Group-IV) by (157.8 and 180.6%) and in SDG treated animals (Groups-V, VI and VII) by (18.6 and 9.7%), (79.4 and 56.5%) and (109.8 and 150%) respectively. It was also found that supplementation of synthetic SDG to STZ-induced diabetic rats resulted in dose-dependent fall in serum glucose level. Serum protein level was also significantly (p < 0.001) restored totally to the normal range in the synthetic SDG (20 mg/kg) in STZ-induced diabetic rats. The restoration of serum protein level was higher in Group-VII than that of the standard drug

Fig. 3. Effect of synthetic SDG on serum glucose levels in STZ-induced diabetic rats [single dose two-day study]. Bar graph represents the percentage reduction in glycemia with respect to the initial (0 h) level. Each bar represent mean ± SD; N = 6 in each group.

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Table 1 Effect of synthetic SDG on serum protein, glucose, insulin, c-peptide and body weight in experimental diabetic animals [multi-dose fourteen-day study]. Groups

Treatment

Serum

Body weight reduction %

Protein (mg/dl) I. II. III. IV. V. VI. VII.

Normal control Diabetic control Control + SDG (10 mg/kg b.w.) Diabetic + tolbutamide (20 mg/kg b.w.) Diabetic + SDG (5 mg/kg b.w.) Diabetic + SDG (10 mg/kg b.w.) Diabetic + SDG (20 mg/kg b.w.)

8.62 6.32 7.92 7.97 6.92 7.52 8.12

± ± ± ± ± ± ±

b

1.1 1.5 2.1a 1.3 1.1a 0.9a 0.7b

Glucose (mg/dl) 98 223 102 105 129 120 96

± ± ± ± ± ± ±

b

12.3 20.3 14.2b 6.5a 7.6 12.4a 10.5b

Insulin (␮mol/ml) 23.4 10.2 25.7 26.3 12.1 18.3 21.4

± ± ± ± ± ± ±

b

1.3 2.1 1.9a 1.1 1.6a 1.2b 1.8b

C-peptide (pM/ml) 163 62 152 174 68 97 155

± ± ± ± ± ± ±

09.3b 12.8 08.2a 05.9b 10.6 14.1b 11.8b

0 16% 1.1% 3.4% 8.5% 6.4% 2.3%

Each value represents mean ± SD (n = 6). a p < 0.05 compared to diabetic control. b p < 0.01 compared to diabetic control.

tolbutamide treated Group-IV and it was found to be dose dependent restoration. The increase in serum glucose and decrease in insulin and cpeptide levels of diabetic rats (Group-II) indicates the death of pancreatic ␤-cells of tested animals. However, the increase in serum insulin and c-peptide shows that some regeneration of pancreatic ␤-cells has occurred in Groups-IV, V, VI and VII with the use of tolbutamide and SDG. This regeneration of pancreatic ␤-cells has occurred slowly and was maximal after a period of 14 days. Cpeptide, a cleavage product of the pro-insulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. But the level of insulin is not proportionate to c-peptide because insulin is metabolized by the liver and has a short half-life of 4–5 min. Although the stimulation of insulin secretion might have contributed to the effects of SDG, the other extra pancreatic effects are also additional possibilities for the large effects seen with SDG. Especially, when the high dose level of streptozotocin (60 mg/kg) used in the present study can effectively destroy the pancreatic ␤-cells, the effectiveness of drugs to treat this condition might rely on the actions other than pancreatic cells insulin release (Muruganandan et al. 2005). The effect of SDG on serum glucose, insulin and c-peptide in normal animals (Group-III) is found to be not significant whereas, in diabetic animals (Groups-V, VI, VII) these parameters were found to be changed significantly (p < 0.05). Consequently, the administration of SDG at several doses and tolbutamide tended to bring serum glucose, insulin and c-peptide significantly toward normal values, while normal rats did not exhibit any significant alterations in the studied parameters duration of the experiment. The body weights of all treated animals were measured before and after the completion of the experiment. The results showed substantial body weight reduction in SDG 20 mg/kg (Group-VII) treated animals

by 2.3% whereas it was 3.4% in tolbutamide (Group-IV) treated animals. The hyperglycemia in diabetes mellitus mechanism involves overproduction (excessive hepatic glycogenolysis and gluconeogenesis) and decreased in glucose utilization by the tissues, and the diabetes pathogenesis associated with disturbances in carbohydrate, fat, and protein metabolism. These complex multifactorial changes of metabolic often lead to functional impairment damage of various organs in both types of diabetes (Momose et al. 2002), and the associated disturbances are usually characterized by hyperglycemia, hypertriglyceridemia combined with low level of insulin, c-peptide and HDL-C (Valcheva-Kuzmanova et al. 2007; Tunali and Yanardag 2006). However, the results of the present investigation indicate that SDG significantly reduced (p < 0.05) the TG, TC, VLDL-c and LDL-c levels in diabetic rats and significantly increased (p < 0.05) plasma insulin, c-peptide and HDL-C. The biochemical results showed that serum TG, TC, VLDL-c and LDL-c levels were significantly increased (p < 0.05) whereas HDL-c was reduced in diabetic rats when compared to normal rats. Treatment of diabetic rats with synthetic SDG (5, 10 and 20 mg/kg) for 14 days resulted in marked decrease in serum TG, TC, VLDL-c and LDL-c levels and increase in HDL-c levels when compared to the diabetic control group (Fig. 4). It has also been reported that insulin significantly normalizes lipid levels in diabetic rats (Pathak et al. 1981). Supplementation of synthetic SDG to Group-V, VI and VII resulted in significant and dose dependent attenuation in serum TG, TC, VLDL-c, and LDL-c. These effects may be due to low activity of cholesterol biosynthesis enzymes or low levels of lipolysis. The well-known dyslipidaemia markers like TC/HDL-c and LDL-c/HDL-c ratios in STZ-induced diabetic rats (Rajkumar et al. 2005) were significantly elevated (p < 0.05) in diabetic group as compared to normal control

Fig. 4. Effect of oral administration of synthetic SDG at doses of 5, 10 and 20 mg/kg b.w. on serum lipid profiles in experimental diabetic animals [multi-dose fourteen-day study]. Each column represent mean ± SD; N = 6 in each group. *p < 0.05, # p < 0.01 different from control diabetic group.

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Fig. 5. Effect of oral administration of synthetic SDG at doses of 5, 10 and 20 mg/kg b.w. on liver and pancreas homogenates CAT in experimental diabetic animals [multi-dose fourteen-day study]. Each column represent mean ± SD; N = 6 in each group. *p < 0.05, # p < 0.01 different from control diabetic group.

group. However, supplementation of SDG (20 mg/kg) for 14 days caused a significant fall (p < 0.05) in these dyslipidemic markers, which were restored to near-normal values (Fig. 4). Therefore, normalization of lipids in diabetic rats treated with synthetic SDG can be partly due to its stimulatory effect on insulin secretion from pancreatic ␤-cells. There are reports suggesting that enzymatic antioxidants such CAT, SOD and hydroxyl radical scavengers (mannitol, dimethylsulfoxide) prevent the damage to isolated pancreatic cells exposed to the diabetogenic agent (Grankvist et al. 1979). Asplund et al. (1984) found that SOD coupled to polyethylene glycol (long acting SOD) reduced the hyperglycemic response in mice injected with a single dose of STZ. Evaluation of the endogenous enzymatic and non-enzymatic antioxidants in the hepatic and pancreatic tissues for all the studied groups indicated the possible evidence for the antihyperglycemic activity of the synthetic SDG. The findings of our study are in agreement with other studies which reported decreased levels of lipid peroxidation in pancreas of diabetic rats after treatment with SDG isolated from flaxseed. Particularly in pancreatic cells, increased stress level in the form of lipid peroxidation, nitrite, SOD and GSH can alter the structure of cell membrane lipids and compromising the cell viability (Prasad 2000). The results showed that the antioxidant enzymes such as CAT, SOD were significantly (p < 0.05, p < 0.01) decreased in diabetic rats compared to normal rats in both liver and pancreas tissues respectively as shown in Figs. 5 and 6. Similarly, the non-enzymatic

antioxidants such as GSH was significantly lower (p < 0.01) whereas MDA levels increased significantly (p < 0.01) in untreated diabetic rats compared to normal rats (Figs. 7 and 8). Diabetic groups rat treated with synthetic SDG (5, 10 and 20 mg/kg) exhibited significantly higher (p < 0.05) CAT enzyme level. Similarly, the SOD levels were also elevated significantly (p < 0.05) in the liver and pancreas homogenates compared to diabetic rats. Tolblutamide (20 mg/kg) diabetic treated group animals showed increased levels of liver and pancreas antioxidant enzymes CAT and SOD as compared to diabetic rats (Figs. 5 and 6). Lowered levels of CAT and SOD have been noticed in the diabetic untreated groups as displayed in Figs. 5 and 6. Treatment with synthetic SDG combats the decreased levels of these enzymes, thereby indicating the protective effect of synthetic SDG. Reduced glutathione (GSH) plays a significant role in the generation of cellular redox state and consequently, the imbalance in reduced glutathione to oxidized glutathione ratio is a putative indicator of cellular oxidative stress. Our investigation indicated that, SDG possessed antihyperglycemic activity as well as protective effect against lipid peroxidation. Hyperglycemia is one of the main leading factors to increase the lipid peroxidation due to its role in elevating free radical concentration in the cell. According to Randle’s glucose-fatty acid hypothesis, oxidation of excessive free fatty acid causes production of reactive oxygen species (ROS) including hydrogen peroxide, which inhibit glucose utilization by the tissues, damage cellular structures and ruin glucose metabolism (Balasubashini et al. 2004). The decrease

Fig. 6. Effect of oral administration of synthetic SDG at doses of 5, 10 and 20 mg/kg b.w. on liver and pancreas homogenates SOD in experimental diabetic animals [multi-dose fourteen-day study]. Each column represent mean ± SD; N = 6 in each group. *p < 0.05, # p < 0.01 different from control diabetic group.

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Fig. 7. Effect of oral administration of synthetic SDG at doses of 5, 10 and 20 mg/kg b.w. on liver and pancreas homogenates GSH in experimental diabetic animals [multi-dose fourteen-day study]. Each column represent mean ± SD; N = 6 in each group. *p < 0.05, # p < 0.01 different from control diabetic group.

Fig. 8. Effect of oral administration of synthetic SDG at doses of 5, 10 and 20 mg/kg b.w. on liver and pancreas homogenates MDA in experimental diabetic animals [multi-dose fourteen-day study]. Each column represent mean ± SD; N = 6 in each group. *p < 0.05, # p < 0.01 different from control diabetic group.

in the antioxidant defence mechanism in the biological systems may be due to the elevated free radical concentration and lipid peroxidation in tissues. The untreated diabetic rats have exhibited a great decrease in the non-enzymatic level of GSH and increase in the MDA level in both liver and pancreas tissues. Administration of synthetic SDG (5, 10 and 20 mg/kg) to diabetic rats showed significant (p < 0.01) increase in GSH and reduced MDA compared to untreated diabetic rats (Figs. 7 and 8) in liver and pancreas tissues. Depletion of GSH tissue was caused by the generation of oxygen radicals by increased levels of glucose and as a result of GSH attempts to overcome the deleterious effects of lipid peroxidation decrease. Glutathione peroxidase utilizes GSH as its co-substrate while scavenging H2 O2 formed by the action of SOD (Mahesh and Venugopal 2004). Therefore, diminished levels of GSH in both hepatic and pancreatic tissues were observed in untreated diabetic rats and this low level of GSH was restored by the administration of synthetic SDG. This effect is comparable to that of drug tolbutamide. Kakkar et al. (1995) reported that MDA level in pancreas has markedly increased as compared to that in the liver, kidney and heart in diabetic rats. Similar trend was observed in our study and the increase in MDA level was diminished significantly by supplementation of tolbutamide as well as synthetic SDG as compared with normal and untreated diabetic groups. These findings were consistent with other studies which reported decreased levels of lipid peroxidation in pancreas of diabetic rats after treatment with synthetic SDG (Prasad 2000).

Defence mechanisms of each tissue against lipid peroxidation are presented by several enzymatic and non-enzymatic antioxidants in addition to lipid soluble vitamins. Control of diabetes by SDG is associated with a decrease in liver and pancreatic-MDA and an increase in enzymatic and non-enzymatic antioxidants (CAT, SOD and GSH). Increase in lipid peroxidation was observed in streptozotocin-induced diabetic rats which could be due to an increase in ROS levels. Increased ROS are a part of the generalized defect present prior to the onset of diabetes. Kakkar et al. (1998, 1995) have stated that selective damage of islet cells in Type I diabetes could be probably due to lowest levels of antioxidant enzymes (SOD, CAT and glutathione peroxidase) in pancreas as compared to other organs such as liver, kidney and heart. The accumulating evidences suggest that, the possible mechanism by which SDG brings about its hypoglycemic action may be by induction of pancreatic insulin secretion from ␤-cells of islets of Langerhans or due to enhanced transport of blood glucose to peripheral tissue. This is a clear evidence from the data with significant increase (p < 0.01) in plasma insulin and c-peptide level of diabetic rats treated with SDG.

Conclusion It is concluded that diabetes, in general is mediated through oxidative stress and synthetic SDG can scavenge reactive oxygen species, thereby protecting liver tissue from peroxidation damage

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