Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced diabetes

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Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced diabetes Vinayagam Ramachandran, Ramalingam Saravanan* Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar 608 002, Tamil Nadu, India

A R T I C L E I N F O

A B S T R A C T

Article history:

Oxidative stress is a common pathogenesis of diabetes mellitus and asiatic acid (AA) plays

Received 28 October 2012

an important role in ameliorating those difficulties. The present study was designed the

Received in revised form

protective effects of AA on altered lipid peroxidation products, enzymic and nonenzymic

1 March 2013

antioxidants in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in exper-

Accepted 4 March 2013

imental rats by single dose STZ (40 mg/kg b.w.) injection. Diabetic rats showed significantly

Available online xxxx

increased levels of plasma glucose, thiobarbituric acid reactive substances, lipid hydroperoxides, aspartate aminotransferase, alanine aminotransferase, bilirubin, creatine kinase,

Keywords:

urea, uric acid, creatinine and decreased levels of plasma insulin. The activities of enzy-

Asiatic acid

matic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase and

Streptozotocin

glutathione-S-transferase and the levels of non-enzymatic antioxidants such as vitamin

Antidiabetic Antioxidant Oxidative stress

C, vitamin E and reduced glutathione were decreased in diabetic rats. Oral treatment with AA (20 mg/kg b.w.) showed near normalized levels of plasma glucose, insulin, lipid peroxidation products, enzymatic and nonenzymatic markers in diabetic rats. The results demonstrate that AA possesses potent antioxidant effect comparable with glibenclamide in improving antihyperglycemia and attenuating antioxidant status in diabetic rats. Published by Elsevier Ltd.

1.

Introduction

Diabetes mellitus (DM) is a heterogeneous metabolic disorder characterized by common feature of chronic hyperglycemia with disturbance in carbohydrate, fat and protein metabolism. The World Health Organization estimates that more than 346 million people Worldwide have diabetes mellitus. Without intervention, this number is likely to increase more than twofold by 2030 (WHO, 2012). Various studies have shown that diabetes mellitus is associated with oxidative stress, leading to an increased production of reactive oxygen species (ROS), including superoxide radical (O 2 ), hydrogen peroxide (H2O2), and hydroxyl radical (OH) or reduction of antioxidant defense system (Sklavos et al., 2010). Oxidative

stress results from an imbalance between radical-generating and radical-scavenging systems that are, increased free radical production or reduced activity of antioxidant defenses or both these phenomena. In diabetes, protein glycation and glucose autoxidation may generate free radicals, which in turn catalyze lipid peroxidation (Baynes, 1991). The cellular antioxidant status determines the susceptibility to oxidative damage and is usually altered in response to oxidative stress. Accordingly, interest has recently grown in the role and usage of natural antioxidants as a means to prevent oxidative damage in diabetes with high oxidative stress. The antioxidants such as Vitamins C and E, enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) have been shown to protect the cells against lipid peroxidation, the

* Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 239141. E-mail address: [email protected] (R. Saravanan). 1756-4646/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.jff.2013.03.003

Please cite this article in press as: Ramachandran, V., & Saravanan, R., Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced diabetes, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.03.003

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chemicals used were purchased from standard commercial suppliers and were of analytical grade.

2.2.

Fig. 1 – Structure of asiatic acid.

initial step of many pathological processes (Pandey & Rizvi, 2010). Hypoglycemic sulphonylureas such as glibenclamide can increase pancreatic insulin secretion from the existing b-cells in STZ-induced diabetes by membrane depolarization, and stimulation of Ca2+ influx, an initial key step in insulin secretion. Moreover, glibenclamide has shown a protection effect against oxidative stress in diabetes (Elmai, Altan, & Bukan, 2004). Glibenclamide is often used as a reference drug in STZ-induced moderate diabetic model. Though sulphonylureas are valuable in treatment of diabetes, their use is restricted by their limited action and side effects (hypoglycaemia and liver problems) (Rajalakasmi, Eliza, Cecilia, Nirmala, & Daisy, 2009). The focus has been shifted to treat the various ailments through dietary fruits, green leaves, vegetables and natural plant-derived drugs due to their safety, efficacy, and lesser side effects (Khanal, Howard, Wilkes, Rogers, & Prior, 2010). Triterpenoids receiving considerable attention across the world for the potential health benefits in relation to many diseases including diabetic disorders (Szakiel, Pa˛czkowski, Pensec, & Bertsch., 2012). Thus, antioxidant therapy may be a promising therapeutic approach for controlling diabetes or diabetic complications. Triterpenes are widely available in dietary fruits and vegetables, and are major components in many medicinal plants used in Asian countries. Asiatic acid (AA; 2a, 3b, 23-trihydroxyurs-12-en-28-oic acid; Fig. 1), is a pentacyclic triterpenoid that contributes to the waxy coats on apples, other fruits, and many herbs, including some folkloric herbal medicines for diabetes. Various reports have demonstrated that AA has antioxidant (Lee, Jin, Beak, Lee, & Kim, 2003), hepatoprotective (Ma Zhang, Zhu, & Lou, 2009), anticancer (Liu, Duan, Pan, Zhang, & Yao, 2006), antiinflammation (Huang et al., 2011), neurotoxicity activity (Jew et al., 2000). Recently, we reported that AA administration significantly improved glucose homeostasis through improved activities of key carbohydrate metabolizing enzymes in STZ-induced diabetic rats (Ramachandran & Saravanan, 2013). As yet there is no published study on the role of AA on oxidative stress in STZ induced diabetic rats. Hence, the present study aimed to investigate the ameliorative potential of AA on hyperglycemia mediated oxidative stress in STZ induced diabetic rats and the effect of AA was compared with glibenclamide, an oral antihyperglycemic drug with antioxidant potential.

2. 2.1.

Materials and methods Chemicals

Asiatic acid, streptozotocin and glibenclamide (99%) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other

Animals

Adult Male albino Wistar rats (9 weeks old; 180–200 g) were obtained from Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University, India, Tamil Nadu and were housed in clean, sterile, polypropylene cages under standard vivarium conditions (12 h light/dark cycle) with ad libitum access to standard rat chow and water. The whole experiment was carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India and approved by the Animal Ethics Committee of Annamalai University, India, Tamil Nadu, Annamalainagar (Reg No.: 160/1999/CPCSEA, Proposal No.: 848).

2.3.

Induction of experimental diabetes

Experimental diabetes was induced in 12 h fasted rats by single i.p. injection of streptozotocin (40 mg/kg b.w.) dissolved in cold citrate buffer (0.1 M, pH 4.5). STZ-injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced hypoglycaemia. STZ-injected animals exhibited hyperglycemia within a few days. Diabetic rats were confirmed by measuring the elevated plasma glucose (by glucose oxidase method) 72 h after injection with STZ. The animals with glucose above 235 mg/dL were selected for the experiment.

2.4.

Experimental design

The rats were randomly segregated into five groups of six rats in each group. AA were dissolved in 5% dimethyl sulfoxide and glibenclamide was diluted in water and administered orally to experimental groups using intragastric tube daily for a period of 45 days: Group Group Group Group Group

1: 2: 3: 4: 5:

normal rats. normal + AA (20 mg/kg b.w.). diabetic control. diabetic + AA (20 mg/kg b.w.). diabetic + glibenclamide (600 lg/kg b.w.).

At the end of the treatment period, the rats were fasted overnight, anesthetized (ketamine, 24 mg/kg b.w. i.p.) and sacrificed by cervical decapitation on 46th day morning and tissues dissected out, washed, weighed, homogenized and centrifuged. The blood was collected with or without anticoagulant for plasma and serum separation, respectively.

2.5.

Biochemical analysis

2.5.1.

Measurement of plasma glucose and insulin

Plasma glucose was estimated using a commercial kit (Sigma Diagnostics Pvt. Ltd., Baroda, India) by the method of Trinder (1969). Insulin in the rat plasma was assayed by the solid phase system amplified sensitivity immunoassay using reagent kits obtained from Medgenix INS-ELISA, Biosource,

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Europe S.A., Nivelles, Belgium by the method of Burgi, Briner, Franken, and Kessler (1988).

2.5.2.

Estimation of lipid peroxidation in liver and kidney

Lipid hydroperoxides as evidenced by formation of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH) were measured by the method of Fraga, Leibovitz, and Tappel (1988) and Jiang, Hunt, and Wolff (1992), respectively. In brief, 0.1 ml of tissue homogenate (Tris–HCl buffer, pH 7.5) was treated with 2.0 ml of (1:1:1, v/ v/v) TBA–TCA–HCl reagent (0.37%, Thiobarbituric acid, 0.25 M HCl and 15% TCA) and mixed thoroughly. The mixture placed in water bath for 15 min, cooled and centrifuged at room temperature for 10 min at 1000g. The absorbance of clear supernatant was measured against reference blank at 535 nm. For hydroperoxides, 0.1 ml of tissue homogenate was treated with 0.9 ml of Fox reagent (88 mg butylated hydroxytoluene (BHT), 7.6 mg xylenol orange and 9.8 ml of methanol and 10 ml 250 mM sulphuric acid) and incubated at 37 °C for 30 min. the color developed was read at 560 nm calorimetrically.

2.5.3.

Activity of enzymatic antioxidants

SOD was determined by the method of Kakkar, Das, and Viswanathan (1984). A single unite of enzyme was expressed as 50% inhibition of NBT (nitroblue tetrazolium) reduction/ min/mg protein. CAT was assayed colorimetrically at 620 nm and expressed as l mol of H2O2 consumed/min/mg protein as described by Sinha (1972). The reaction mixture (1.5 ml) contained 1.0 ml of 0.01 M pH 7.0 phosphate buffer, 0.1 ml of tissue homogenate and 0.4 ml of 2 M H2O2. The reaction supped by the addition of 2.0 ml of dichromate-acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed at a 1:3 ratio). GPx activity was measured by the method described by Rotruck, Pope, Ganther, and Swason (1973). Briefly, reaction mixture contained 0.2 ml of 0.4 M phosphate buffer pH 7.0, 0.1 ml of 10 mM sodium azide, 0.2 ml of tissue homogenate (homogenate on 0.4 M phosphate buffer, pH 7.0), and 0.2 ml glutathione, 0.1 of 0.2 mM H2O2. The content was incubated at 37 °C for 10 min. The reaction was arrested by 0.4 ml of 10% TCA and centrifuged. Supernatant was assayed for glutathione content by using Ellmans reagent. Glutathione-S-transferase (GST) activity was determined spectrophotometrically by the method of Habig, Pabst, and Jakpoly (1974). The reaction mixture contained 1.0 ml of 100 mM phosphate buffer (pH 6.5), 0.1 ml of 30 mM 1-chloro2,4-dinitrobenzene, and 0.7 ml of double distilled water. After pre-incubating the reaction mixture for 5 min at 37 °C, the reaction was started by the addition of 0.1 ml of tissue homogenate and 0.1 ml of glutathione as substrate. After 5 min, the absorbance was read at 340 nm.

2.5.4.

Estimation of nonenzymatic antioxidants

Vitamin E was determined by the method of Baker, Frank, De Angelis, and Feingod (1980). A portion of the sample (0.1 ml), 1.5 ml of ethanol and 2 ml of petroleum ether were added, mixed and centrifuged for 3000g for 10 min. The supernatant was evaporated to dryness at 80 °C then 0.2 ml of 2,2 0 -dipyridyl solution and 0.2 ml of ferric chloride solution was added

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and mixed well. This was kept in dark for 5 min and added 2 ml of butanol. Then the absorbance was read at 520 nm. Ascorbic acid in the tissues was estimated by the method of Omaye, Turbull, and Sauberlich (1979). To 0.5 ml of sample, 1.5 ml of 6% TCA was added and allowed to stand for 5 min and centrifuged. To the supernatant, 0.3 g of acid washed norit was added, shaken vigorously and filtered. This converts ascorbic acid to dehydroascorbic acid. 0.5 ml of the filtrate was taken and 0.5 ml of DNPH was added, stoppered and placed in a water bath at 37 °C for exactly 3 h. Removed, placed in ice-cold water and added 2.5 ml of 85% sulphuric acid drop by drop. The contents of the tubes were mixed well and allowed to stand at room temperature for 30 min. A set of standards containing 20–100 lg of ascorbic acid were taken and processed similarly along with a blank containing 2.0 ml of 4% TCA. The color developed was read at 540 nm. Reduced glutathione (GSH) was determined by the method of Ellman (1959). To the homogenate added 10% TCA, centrifuged. One millilitre of the supernatant was treated with 0.5 ml of Ellmans reagents 19.8 mg of 5,5 0 -dithiobis-(2-nitrobenzoic acid) in 100 ml of 0.1% sodium nitrate) and 3.0 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm.

2.5.5. Activities of serum aspartate transaminase (AST) and alanine transaminase (ALT) Activities of AST and ALT were assayed by the method of Reitman and Frankel (1957). 0.2 ml aliquot of serum with 1 ml of substrate (aspartate and a-ketoglutarate for AST: alanine and a-ketoglutarate for ALT) in phosphate buffer (pH 7.4) was incubated for 1 h for AST and 30 min for ALT. One millilitre aliquot of DNPH solution was added to arrest the reaction and kept for 20 min at room temperature. After incubation, 1 ml of 0.4 M NaOH was added and the absorbance was read at 540 nm.

2.5.6.

Estimation of bilirubin

Serum bilirubin was estimated by the method of Malloy and Evelyn (1937). Diazotised sulphonilic acid (0.5 ml) reacts with bilirubin in diluted serum (0.2 ml serum + 1.8 ml distilled water) to form a purple-colored azobilirubin, which was measured at 540 nm.

2.5.7.

Estimation of creatine kinase

The activity of creatine kinase was estimated by the method of Okinaka et al. (1961). This reaction involves the conversion of creatine to creatine phosphate. The amount of phosphorous liberated was estimated at 640 nm.

2.5.8.

Estimation of urea

Urea in the plasma was estimated by using the diagnostic kit based on the method of Fawcett and Scott (1960). One millilitre of buffered enzyme (phosphate buffer, urease, sodium nitroprusside and ethylenediaminetetraacetic acid), 10 ll of sample added, mixed well and kept at 37 °C for 5 min. Ten microlitres of standard and 10 ll of distilled water (blank) were also processed simultaneously. To all the tubes, 1.0 ml of color developing reagent was added and mixed will. Exactly after 5 min of incubation at 37 °C, 1.0 ml of distilled water was added and the color developed was read at 600 nm.

Please cite this article in press as: Ramachandran, V., & Saravanan, R., Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced diabetes, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.03.003

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2.5.9.

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Estimation of uric acid

Uric acid in the plasma was estimated by using the diagnostic kit based on the enzymic method described by Caraway (1955). To 1 ml of the enzyme reagent, 25 ll of sample were added and mixed by inversion. Twenty-five microlitres of standard and 25 ll of distilled water (blank) were also processed simultaneously. The tubes were incubated at 37 °C for 5 min and the color developed was read at 510 nm.

2.5.10. Estimation of creatinine Creatinine in the plasma was estimated using the diagnostic kit based on the method of Tietz (1987) using Jaffe (1886) color reaction. A portion of the sample (0.1 ml) was added to a reagent mixture containing 0.5 ml picric acid solution and 0.5 ml of sodium hydroxide. The tubes were mixed well and incubated for 20 s. With the spectrophotometer adjusted to zero absorbance with distilled water, reading was taken at 510 nm.

2.6.

Statistical analysis

The results were expressed as mean ± SD of six rats per group and statistical significance was evaluated by one-way ANOVA using SPSS (version 16.0) program followed by the post hoc test, least significant difference. Values were considered statistically significant when p < 0.05.

3.

Results

Fig. 2 depicts the values of the initial and final body weights of the normal and experimental rats. Body weight significantly (p < 0.05) decreased in diabetic rats compared to normal control rats. Oral administration of AA to diabetic rats protects the loss of body weight compared to diabetic control rats. Table 1 epitomizes the levels of plasma glucose and insulin in normal and experimental rats. Diabetic rats were significantly (p < 0.05) increase in the level of plasma glucose and a significantly (p < 0.05) decrease in plasma insulin in diabetic rats compared with control rats. Oral administration of AA as well as glibenclamide to diabetic rats significantly (p < 0.05) normalized the altered levels of plasma glucose and plasma insulin when compared with diabetic rats. Fig. 3 shows the levels of TBARS and LOOH in liver and kidney of normal and experimental rats. Diabetic rats exhibited increased levels of TBARS and LOOH when compared to normal control. Administration of AA and glibenclamide to diabetic rats significantly decreased lipid peroxidation markers in liver and kidney when compared to diabetic rats. Table 2 represents the activities of antioxidant enzymes (SOD, CAT, GPx and GST) in the liver and kidney of normal and experimental rats. A fall in the activities of antioxidants enzymes was observed in diabetic rats when compared to normal control. AA and glibenclamide administration to diabetic rats significantly improved the activities of the above enzymes. The levels of liver and kidney non-enzymatic antioxidants such as vitamin C, vitamin E and GSH are represented in Table 3. Diabetic rats showed a significantly (p < 0.05) decrease in these levels when compared with control rats. Conversely,

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administration of AA as well as glibenclamide to diabetic rats significantly (p < 0.05) increased the levels to near control values. The levels of serum ALT, AST, bilirubin, CK, urea, uric acid and creatinine in normal and experimental rats are represented in Table 4. The activities of ALT, AST, bilirubin, CK, urea, uric acid and creatinine were significantly (p < 0.05) increased in diabetic rats. These values were brought back to near normal levels after treatment with AA and glibenclamide.

4.

Discussion

In the present study, we evaluated the antidiabetic and antioxidant effects of AA in STZ diabetic rats. In the past few years, natural substances have been shown to have the potential to treat DM. Attention has been notably focused on plants rich in triterpenoid, which generally have shown antioxidant and antidiabetic effects (Ardiles et al., 2012; Manna, Sinha, & Sil, 2009). The plant Centella asiatica contains huge amount of AA and its alcoholic extracts in humans predicts a detectable concentration of AA in plasma. AA which is a metabolite of asiaticoside and by the hydrolytic cleavage of the sugar moiety it becomes AA which is responsible for the therapeutic effects and it clearly delineates the pharmacokinetic nature of AA (Grimaldi et al., 1990; Nair, Menon, & Shailajan, 2012). Moreover, AA is a non-toxic compound with a LD50 value of 980 mg/kg when administered to rats. AA administration 5, 10 and 20 mg/kg body weight gave significant reduction of plasma glucose in STZ-diabetic rats. Since AA at 20 mg dose gave a maximum improvement on body weight, and decreased plasma glucose level, it was fixed as the optimum dose (Ramachandran & Saravanan, 2013). From the results of the present study, it is evident that the AA, a triterpenoids significantly attenuated hyperglycemia and improved antioxidant in experimentally induced diabetic rats. STZ causes depletion in the secretion of insulin by partial destroying pancreatic b-cells (Frode & Medeiros, 2008). Reduction in insulin production results in enhancement of blood glucose level that inturn causes protein glycosylation. Oxidation of enhanced glucose triggers overproduction in ROS that leads to diabetic complications. Current antidiabetic treatment strictly focuses on the management of glycaemia along with reduction of associated diabetic complications. Some bioactive compounds isolated from plants like terpenoids was reported to stimulate insulin secretion with numerous mechanisms such as exertion distal to K+-ATP channels and Ca2+ channels (Hoa et al., 2007). Since oxidative stress and free radicals injure or destroy pancreatic b-cells in diabetes, AA is able to increase the secretion of insulin via its antioxidant actions. The level of LPO is a measure of membrane damage and alterations in structure and function of cellular membranes. The level of thiobarbituric acid reactive substance is an indirect measurement of lipid peroxidation (Halliwell, Aeschbach, Loligger, & Aruoma, 1995). Free radical-induced lipid peroxidation has been associated with a number of disease processes including diabetes mellitus. Increased endogenous peroxides may initiate uncontrolled lipid peroxidation, thus

Please cite this article in press as: Ramachandran, V., & Saravanan, R., Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced diabetes, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.03.003

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Fig. 2 – Effect of AA on body weight in control and experimental rats. NC – normal control; AA – asiatic acid; DC – diabetic control. Values are means ± SD for six rats. Values not sharing a common marking (a, b, c) differ significantly at p < 0.05 (DMRT).

Table 1 – Effect of AA on plasma glucose and insulin in normal and experimental rats. Groups Normal control Normal + AA (20 mg/kg b.w.) Diabetic control Diabetic + AA (20 mg/kg b.w.) Diabetic + glibenclamide (600 lg/kg b.w.)

Plasma glucose (mg/dL) a

80.02 ± 5.24 82.39 ± 6.21a 248.36 ± 12.47b 105.11 ± 7.89c 98.74 ± 5.76c

Insulin (lU/mL) 14.02 ± 1.01a 14.54 ± 1.05a 6.74 ± 0.47b 12.35 ± 1.22c 13.87 ± 1.43c

AA – asiatic acid. Values are means ± SD for six rats. Values not sharing a common marking (a, b, c) differ significantly at p < 0.05 (DMRT).

leading to cellular in filtration and islet cell damage (Pasupathi, Chandrasekar, & Senthil kiumar, 2009). In diabetes, it is thought that hypoinsulinemia increases the activity of the enzyme, fatty acyl coenzyme A oxidase, which initiates beta-oxidation of fatty acids, resulting in LPO (Rahimi, Nikfar, Larijani, & Abdollahi, 2005). Increased LPO impairs membrane function by decreasing membrane fluidity and changing the activities of membrane-bound enzymes and receptors. STZinduced diabetic rats showed an increased concentration of lipid peroxidation products such as TBARS and LOOH in the tissues, an indirect evidence of intensified free radical production (Maritim, Sanders, & Watkins, 2003). The accumulation of free radical observed in diabetic rats is attributed to

chronic hyperglycemia that alters antioxidant defense system as demonstrated by previous studies (Hong et al., 2004). Free radicals may also be formed via the auto-oxidation of unsaturated lipids in plasma and membrane lipids. They may react with polyunsaturated fatty acids in cell membrane leading to lipid peroxidation (Lery, Zaltzber, Ben-Amotz, Kanter, & Aviram, 1999). Recent studies have shown that the supplementation of food triterpenoid with antioxidant potential is significantly associated with a reduction in the level of lipid peroxidation (Manna, Ghosh, Das, & Si, 2010). Oral treatment of AA to diabetic rats prevented the lipid peroxidation markers enzymes to near normal levels which could be as a result of improved glycemic control and antioxidants status.

Please cite this article in press as: Ramachandran, V., & Saravanan, R., Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced diabetes, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.03.003

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Fig. 3 – Effect of AA on TBARS and hydroperoxides in normal and experimental rats. NC – normal control; AA – asiatic acid; DC – diabetic control. Values are means ± SD for six rats. Values not sharing a common marking (a, b, c) differ significantly at p < 0.05 (DMRT).

Cytosolic free radicals are either removed non-enzymatically or by antioxidant enzymes such as SOD, CAT. SOD one of the first antioxidant enzymes in the line of defense against the deleterious effects of oxygen radicals in the cells, scavenges ROS by catalyzing the dismutation of superoxide to H2O2 (McCord, Keele, & Fridovich, 1976), while CAT is an enzymic antioxidant, which decomposes hydroxyl radicals and is widely distributed in all animal tissues with the highest activity in the red blood cells and liver (Maritim et al., 2003). Reduction in these enzyme activities results in various deleterious effects due to accumulation of superoxide and hydroxyl radicals. In the present study, a reduced activity of CAT has been observed. In diabetic conditions, the uncontrolled production

of hydrogen peroxide due to the auto-oxidation of glucose, protein glycation and lipid oxidation led to a marked decline in the CAT activity (Rajasekaran, Sivagnanam, & Subramanian, 2005). GPx, a selenium containing tetrameric glycoprotein, present in significant concentrations, detoxifies hydrogen peroxide into water and molecular oxygen through the oxidation of reduced glutathione (Ewis & Abdel-Rahman, 1995). GPx has been shown to be an important adaptive response to condition of increased peroxidative stress. During diabetic conditions, the activity of glutathione peroxidase is decreased as a result of radical-induced inactivation and glycation of the enzyme (Zhang & Tan, 2000). The low activity of GPx could be

Please cite this article in press as: Ramachandran, V., & Saravanan, R., Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced diabetes, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.03.003

Groups

Normal control

Normal + AA (20 mg/kg b.w.)

Diabetic control

Diabetic + AA (20 mg/kg b.w.)

Diabetic + glibenclamide (600 lg/kg b.w.)

SOD (U*/mg of protein)

Liver Kidney

8.73 ± 0.51a 13.91 ± 0.96a

8.95 ± 0.68a 14.15 ± 1.06a

4.65 ± 0.27b 7.21 ± 0.48b

7.40 ± 0.59c 11.75 ± 1.00c

7.93 ± 0.66c 12.46 ± 1.01c

CAT (U**/mg of protein)

Liver Kidney

82.36 ± 6.18a 41.71 ± 3.10a

85.73 ± 6.02a 44.86 ± 2.93a

47.91 ± 3.67b 20.99 ± 1.36b

68.91 ± 4.95c 34.15 ± 2.74c

71.24 ± 4.81c 36.48 ± 3.42c

GPx (U@/mg of protein)

Liver Kidney

10.86 ± 0.82a 7.93 ± 0.68a

10.68 ± 0.74a 8.06 ± 0.57a

5.12 ± 0.25b 4.61 ± 0.37b

8.64 ± 0.68c 5.98 ± 0.41c

9.52 ± 0.55c 6.34 ± 0.57c

GST (U$/mg of protein)

Liver Kidney

7.05 ± 0.52a 5.71 ± 0.48a

7.23 ± 0.61a 5.79 ± 0.51a

3.59 ± 0.24b 3.15 ± 0.7b

5.81 ± 0.47c 4.68 ± 0.39c

5.21 ± 0.38c 5.18 ± 0.394c JOURNAL OF FUNCTIONAL FOODS

AA – asiatic acid. Values are means ± SD for six rats. Values not sharing a common marking (a, b, c) differ significantly at p < 0.05 (DMRT). U* = enzyme concentration required to inhibit the chromogen produced by 50% in 1 m in under standard condition. U** = lmol of hydrogen peroxide decomposed/min. U@ = lmol of GSH utilized/min. U$ = lg of CDNB conjugate formed/min.

Table 3 – Effect of AA on nonenzymatic antioxidant in normal and experimental rats. Groups Liver (lg/mg of protein)

Normal control Normal + AA (20 mg/kg b.w.) Diabetic control Diabetic + AA (20 mg/kg b.w.) Diabetic + glibenclamide (600 lg/kg b.w.) Vitamin C Vitamin E GSH

Kidney (lg/mg of protein) Vitamin C Vitamin E GSH

0.91 ± 0.05a 0.73 ± 0.06a 13.68 ± 10.98a

0.95 ± 0.04a 0.76 ± 0.04a 13.93 ± 1.21a

0.51 ± 0.03b 0.29 ± 0.02b 6.84 ± 0.38b

0.74 ± 0.06c 0.48 ± 0.03c 9.38 ± 0.75c

0.79 ± 0.05c 0.53 ± 0.03c 10.12 ± 0.91c

0.85 ± 0.07a 0.65 ± 0.040a 12.84 ± 1.12a

0.88 ± 0.05a 0.63 ± 0.05a 12.90 ± 0.87a

0.54 ± 0.03b 0.25 ± 0.02b 7.46 ± 0.34b

0.68 ± 0.04c 0.39 ± 0.02c 10.12 ± 0.48c

0.71 ± 0.05c 0.42 ± 0.03c 11.25 ± 0.95c

x x x ( 2 0 1 3 ) x x x –x x x

AA – asiatic acid. Values are means ± SD for six rats. Values not sharing a common marking (a, b, c) differ significantly at p < 0.05 (DMRT).

7

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Table 2 – Effect of AA on enzymatic antioxidant in normal and experimental rats.

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29.45 ± 1.36c 81.28 ± 7.03c 0.61 ± 0.03c 160.13 ± 6.21c 28.35 ± 2.24c 1.57 ± 0.09c 0.98 ± 0.05c 32.17 ± 1.45c 84.01 ± 4.97c 0.65 ± 0.04c 165.68 ± 7.18c 30.23 ± 2.61c 1.65 ± 0.15c 1.13 ± 0.08c AA – asiatic acid. Values are means ± SD for six rats. Values not sharing a common marking (a, b, c) differ significantly at p < 0.05 (DMRT).

22.71 ± 1.25a 72.07 ± 4.12a 0.49 ± 0.02a 139.79 ± 10.06a 23.86 ± 1.45a 1.35 ± 0.07a 0.80 ± 0.06a ALT (IU/L) AST (IU/L) Bilirubin (mg/dL) CK (IU/L) Urea (mg/dL) Uric acid (mg/dL) Creatinine (mg/dL)

25.02 ± 1.40a 75.05 ± 5.01a 0.52 ± 0.02a 140.11 ± 9.12a 24.41 ± 1.87a 1.38 ± 0.08a 0.85 ± 0.04a

58.33 ± 4.26b 115.87 ± 9.06b 1.18 ± 0.08b 215.12 ± 13.73b 37.08 ± 2.48b 2.30 ± 0.16b 2.10 ± 0.11b

Diabetic + AA (20 mg/kg b.w.) Diabetic control Normal + AA (20 mg/kg b.w.) Normal control Groups

Table 4 – Effect of AA on ALT, AST, bilirubin, CK, urea, uric acid and creatinine in normal and experimental rats.

Diabetic + glibenclamide (600 lg/kg b.w.)

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directly explained by the low content of glutathione found in diabetic state, since glutathione is a substrate and cofactor of GPx. AA augmented the activities of antioxidant enzymes in STZ-treated rats by inhibiting lipid peroxidation. Hence, a compound that could prevent the generation of these oxygen free radicals or increase the free radical scavenging enzymes may be effective in STZ-diabetes. In our study the enzymatic antioxidant activities such as SOD, CAT and GPx decreased in diabetic rats, and diabetic rats treated with AA possess free radical scavenging activity. GST, a glutathione-dependent enzyme, protects cell s from ROS by utilizing a wide variety of products of oxidative stress as substrates. The present investigation revealed significant decrease in liver GPx and GST activities in the STZ-induced diabetic rats, as compared to the control group. In accordance with our results, Schettler et al. (1994) demonstrated that the reduced antioxidant production may be due to the increase in oxygen metabolites that causes a decrease in the activity of the antioxidant defense system. Moreover, Kennedy and Baynes (1984) reported that the decrease in antioxidant enzyme activity in diabetes mellitus may be due to non-enzymatic glycosylation of the enzymes. According by Al-Wabel, Mousa, Omer, and Abdel-Salam (2008) suggested that the depletion of GSH content also may lower GST enzyme, because GSH is required as a substrate for GST activity. In this context, other workers also reported a diminished activity of enzymatic antioxidants in diabetic rats (Karthikesan, Pari, & Menon, 2010). However, oral administration of AA to diabetic rats significantly ameliorates the activities of enzymatic antioxidants, which in turn reflects the antioxidant property of AA. Earlier research has shown that diabetics have low levels of vitamin C, vitamin E and GSH. Vitamin E supplementation can help prevent the development of diabetes. Ascorbic acid is a major antioxidant that is essential for the scavenging of toxic free radicals in both blood and tissues. Vitamin E, a lipophilic antioxidant, transfers its phenolic hydrogen to a peroxyl free radical of peroxidized polyunsaturated fatty acids, thereby breaking the radical chain reaction and averting the peroxidation of membrane lipids (Opara, 2002). GSH is a tripeptide (L-c-glutamyl cysteinyl glycine), an antioxidant and a powerful nucleophile, critical for cellular protection, such as detoxification of ROS, conjugation and excretion of toxic molecules and control of inflammatory cytokine cascade (Brown, Harris, Ping, & Gauthier, 2004). Depletion of tissues GSH levels demonstrated among diabetic rats clearly suggests the increased utilization by the hepatic cells which could be the result of decreased synthesis or increased degradation of GSH by oxidative stress in diabetes (Furfaro et al., 2012). In earlier published reports, which shows that the GSH concentration decreases in the diabetic rats (Sayed, 2012). It has been observed that oral treatment of AA and glibenclaimaide significantly elevates the vitamin C, vitamin E and GSH levels in diabetic rats. AA may act by reducing hyperglycemia-mediated oxidative stress probably by decreasing the consumption of free radical scavengers. STZ induced DM results in abnormal values for kidney and liver enzymes. This phenomenon is attributed to free radical production that causes membrane damage especially in the liver and kidney tissues. In DM, raised activities of

Please cite this article in press as: Ramachandran, V., & Saravanan, R., Asiatic acid prevents lipid peroxidation and improves antioxidant status in rats with streptozotocin-induced diabetes, Journal of Functional Foods (2013), http://dx.doi.org/10.1016/j.jff.2013.03.003

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transaminase enzymes (ALT and AST) are employed as the evidence of liver injury. These enzymes pour out of liver cells in greater quantities when liver is damaged, thus their levels in the blood were raised. The serum urea, creatinine, creatinine kinase and bilirubin levels are elevated in the diabetics due to increased protein catabolism, glomerular injury and renal dysfunction (Prangthip et al., 2012). The damage to the renal cells is mainly due to glucose mediated osmotic diuresis, reactive oxygen species and glucose overload. Uric acid is the end product of purine catabolism, a potent antioxidant in blood. The increased concentration of serum uric acid in diabetes is associated with accumulation of purine bases due to oxidative stress induced cellular necrosis. Renal dysfunction, insulin resistance and increased cellular turnover also lead to increased production of uric acid (Anwar & Meki, 2003). The reversal of ALT, AST, bilirubin, creatinine kinase, urea, uric acid and creatinine activities in AA treated diabetic rats towards near normalcy indicate the liver and kidney protective nature. These results are in agreement with, Ma Zhang et al. (2009) who reported that AA improved hepatic enzymes in hepatotoxicity. AA is a natural triterpenoid that is also derived from medicinal plants, fruits, green leaves and vegetables possessing a C2a-OH function exhibited more potent glycogen phosphorylase inhibitory activity, making it an interesting compound for the treatment of diseases caused by abnormalities in glycogen metabolism, such as diabetes (Zhang et al., 2009). AA has a powerful antioxidant property mainly possessing a number of hydroxyl groups at a position of C2a, C3b, C23-trihydroxyl within their structure, which is favorable for the antioxidative, anti-inflammatory (Lee et al., 2003) and ester formation with the C(28) carboxylic acid, are relatively important to enhance the wound healing activity. C-2 position on AA showed the most potent hepatoprotective activity against carbon tetrachloride-induced hepatotoxicity. AA has the ability to trigger the proinsulin synthesis and also insulin release, which might be helpful to reduce the plasma glucose and increase insulin during diabetes (Liu et al., 2010). Likewise, the antioxidant potential of AA are due to the hydroxyl groups present at C2, C3 and C28th positions and thus the oral administration of AA is an essential trigger for the liver and kidney to revert its normal homeostasis during experimental diabetes.

5.

Conclusion

In conclusion, the current results indicate that AA has the ability to ameliorate oxidative stress in tissues of STZinduced diabetic rats as evidenced by improved glycemic and antioxidant status along with decreased lipid peroxidation. Therefore, further studies are necessary to elucidate the exact mechanism by which AA elicits its modulatory effects.

Acknowledgement The authors thank the Indian Council of Medical Research (ICMR), New Delhi, India for providing financial support for this research project, in the form of Senior Research Fellowship (SRF) to Mr. V. Ramachandran.

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