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ARTICLE Potentiation of antioxidant effect of dietary tender cluster beans (Cyamopsis tetragonoloba) by garlic (Allium sativum) in high-cholesterol-fed rats Shubhra Pande and Krishnapura Srinivasan

Abstract: The antioxidant role of tender cluster beans (Cyamopsis tetragonoloba, CB), a rich source of soluble fibre, was investigated in a hypercholesterolemia-induced oxidative stress situation in rats. In the context of dietary garlic (Allium sativa) potentiating the hypocholesterolemic influence of CB, we also examined if dietary garlic enhances the antioxidant potential of CB. Groups of Wistar rats were rendered hypercholesterolemic by feeding them a 0.5% cholesterol diet for 8 weeks. Dietary interventions were made by inclusion of 15% tender CB powder or 1% garlic powder or their combination in a high-cholesterol diet. Concentrations of antioxidant molecules and activities of antioxidant enzymes in blood and liver were examined. Dietary CB displayed an antioxidant influence in terms of elevating ascorbic acid and glutathione concentrations and stimulating the activities of antioxidant enzymes both in blood and liver. The antioxidant effect of dietary CB was generally potentiated by co-administration of garlic. Thus, consumption of tender CB and garlic together could form a strategy for improving the body’s antioxidant status. Key words: antioxidant enzymes, antioxidant molecules, garlic, hypercholesterolemia, tender cluster beans. Résumé : Le rôle antioxydant du haricot de Guar (Cyamopsis tetragonolaba, HG), une riche source de fibres solubles, a été examiné en situation de stress oxydant induit par l’hypercholestérolémie chez les rats. Nous avons aussi examiné si l’ail (Allium sativa) accroissait le potentiel antioxydant du HG, dans un contexte de potentialisation du pouvoir hypocholestérolémiant du HG par l’ail. L’hypercholestérolémie a été induite chez des groupes de rats Wistar par une alimentation comportant 0,5 % de cholestérol pendant 8 semaines. Des interventions diététiques ont été réalisées en incluant 15 % de poudre de HG ou 1 % de poudre d’ail, ou la combinaison des deux, dans la diète riche en cholestérol. Les concentrations de molécules antioxydantes et les activités des enzymes antioxydantes dans le sang et le foie ont été examinées. Le HG alimentaire exerçait une influence antioxydante en termes d’augmentation des concentrations d’acide ascorbique et de glutathion et de stimulation des activités des enzymes antioxydantes du sang et du foie. L’effet antioxydant du HG alimentaire était généralement potentialisé par l’administration conjointe d’ail. Ainsi, la consommation conjointe de haricot de Guar et d’ail pourrait constituer une stratégie d’amélioration de l’état antioxydant du corps. [Traduit par la Rédaction] Mots-clés : enzymes antioxydantes, molécules antioxydantes, ail, hypercholestérolémie, haricot de Guar.

Introduction Oxidative damage by free radicals has been implicated in the pathogenesis of vascular diseases, diabetes, and hypertension (Drobiova et al. 2011). Free radicals are implicated in the etiology of cancer, Alzheimer’s disease, inflammatory diseases, ischemicreperfusion injury, and a myriad of other disease conditions. Organisms possess a natural defense against free radicals in the form of antioxidant enzymes and antioxidant molecules. Dietary antioxidants, such as ascorbic acid, tocopherols, ␤-carotene, polyphenols, and other phytochemicals, provided by fruits and vegetables and grains are understood to augment the inherent antioxidant system. Dietary approaches for the prevention of oxidative stress are more cost-effective (Anderson 2003). Dietary soluble fibre-rich tender cluster bean (Cyamopsis tetragonoloba, CB) pods are understood to interfere with the reabsorption of bile acids by enterohepatic circulation from the colon (Redard et al. 1992) and thus exert the desirable antihypercholesterolemic influence (Pande et al. 2012; Pande and Srinivasan 2012). The gastrointestinal protective effect of dietary tender CB with

respect to activities of antioxidant enzymes and concentration of antioxidant molecules in Wistar rats has been recently reported by us (Pande and Srinivasan 2013). Dietary intervention with tender CB (15% freeze-dried powder in the diet for 8 weeks) significantly enhanced the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GPX)) and the concentrations of antioxidant molecules in both gastric and intestinal mucosa. The soluble fibre-rich tender CB were also found to alleviate the diminished activities of antioxidant enzymes and antioxidant molecules in gastric and intestinal mucosa under conditions of ethanol-induced oxidative stress (Pande and Srinivasan 2013). The gastroprotective effect of CB was also reflected in its positive effect on gastric mucosal glycoproteins, thereby lowering mucosal injury. Incidentally, serum and liver also showed an elevated antioxidant status, thus suggesting a desirable lowered oxidative stress by consuming tender CB. The bioactive spice compound capsaicin, the pungent principle of red pepper, was generally found to potentiate the protective

Received 2 March 2013. Accepted 7 May 2013. Abbreviations: ANOVA, analysis of variance; CAT, catalase; CB, cluster beans; CDNB, 2,3-dichloro-4-nitrobenzene; GPX, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione (reduced); GST, glutathione-S-transferase; HCD, hypercholesterolemic diet; LPO, lipid peroxides; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); SOD, superoxide dismutase. S. Pande and K. Srinivasan. Department of Biochemistry and Nutrition, CSIR – Central Food Technological Research Institute, Mysore 570020, India. Corresponding author: Krishnapura Srinivasan (e-mail: [email protected]).

Can. J. Physiol. Pharmacol. 91: 818–822 (2013) dx.doi.org/10.1139/cjpp-2013-0082

Published at www.nrcresearchpress.com/cjpp on 4 June 2013.

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by ACQ Service/Serials (A) on 10/27/13 For personal use only.

Pande and Srinivasan

antioxidant influence of dietary CB in the gastrointestinal tract under an alcohol-induced oxidative stress condition when coadministered with CB (Pande and Srinivasan 2013). The Allium spice garlic contains highly unstable thiosulfonate allicin, which is transformed into a variety of organosulfur compounds that impart health beneficial effects. The antioxidant potential of dietary garlic has been well documented (Srinivasan 2012). In the present animal study we investigated the antioxidant role, if any, of dietary tender CB as a source of soluble fibre under hypercholesterolemic-condition-induced oxidative stress by measuring the concentrations of antioxidant molecules in blood and liver and the activities of endogenous antioxidant enzymes in blood and liver. In the context of dietary garlic potentiating the hypocholesterolemic influence of dietary CB (Pande and Srinivasan 2012), we also examined if dietary garlic would enhance the antioxidant potential of CB in this regard.

Material and methods Materials Cholesterol, bile salts, glutathione, nicotinamide adenine dinucleotide phosphate (NADPH), tert-butyl hydroxyperoxide, GR, xanthine oxidase, cytochrome c, sodium dodecyl sulfate, and thiobarbituric acid were purchased from Sigma Chemical Co., (St. Louis, Missouri, USA). Phosphotungstic acid, 1-chloro-2dinitrobenzoic acid, ethylenediaminetetraacetate, trichloroacetic acid, m-phosphoric acid, and sodium citrate were obtained from SISCO Research Laboratory (Mumbai, India). Casein was purchased from Nimesh Corporation (Mumbai, India). Corn starch and cane sugar powder were locally purchased. All other chemicals and solvents were of analytical grade and solvents were distilled prior to use. Tender CB and garlic were purchased from the local market. The edible portions of CB were freeze-dried and powdered. Garlic bulbs were ground to a fine paste and then freeze-dried into a dry powder. Animal treatment This animal experiment was carried out taking required measures to minimize pain and discomfort in accordance with the standard guidelines laid down regarding the care and use of animals for experiments and with due clearance from the Institute’s Animal Ethics Committee. Groups of male Wistar rats, 8 weeks of age and weighing between 90 and 100 g were used here. The animals (8 per group) were fed ad libitum with control and various test diets for 8 weeks and had free access to water throughout the experiment. The animals were fed with AIN-76 semipurified diet. The basal control diet consisted of starch, 54%; casein, 21%; sugar, 10%; refined groundnut oil, 9.9%; AIN-76 mineral mix, 4%; AIN-76 vitaminised starch, 1%; and vitaminised oil, 0.1%. Hypercholesterolemic diet (HCD) was made by supplementing the AIN-76 diet with 0.5% cholesterol and 0.125% bile salts and substituting the same quantity of starch and employing saturated fat instead of refined groundnut oil. The test diets were prepared by incorporating freeze-dried tender CB at 15% and freeze-dried garlic at 1% level of incorporation, alone and in combination (Pande and Srinivasan 2012), and by substituting the same amount of starch. Body masses were recorded at weekly intervals, and feed intake was monitored daily. At the end of the feeding trial, animals were sacrificed under light ether anesthesia. Blood was collected by cardiac puncture, and serum was separated by low-speed centrifugation. Liver was excised, washed with ice cold saline, blotted dry, weighed, and stored at −20 °C until further analysis. Lipid peroxides and antioxidant molecules Serum lipid peroxides (LPO) were estimated as thiobarbituric acid reactive substances by employing the method of Yagi (1984). The assay was carried out by fluorimetric measurement at an excitation wavelength of 515 nm and an emission wavelength of 553 nm, and results were compared with the standard pre-

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pared by reacting 0.5 nmol of 1,1,3,3-tetraethoxypropane with thiobarbituric acid reagent. LPO in liver tissue was determined by the method described by Ohkawa et al. (1979), involving photometric measurement of the thiobarbituric acid complex extracted into n-butanol, and the absorbance of butanol was measured at 532 nm and compared with that of standard tetraethoxypropane treated similarly. Total thiols were measured by spectrophotometric method according to the method of Sedlak and Lindsay (1968). Glutathione in serum and liver was quantitated by using Ellman’s reagent according to the method of Beutler et al. (1963). Ascorbic acid in serum and liver was estimated spectrophotometrically by measuring the 2,4-dinitrophenolhydrazene derivative of dehydroascorbic acid employing the method of Omaye et al. (1973). Antioxidant enzymes GR activity was monitored in serum and liver by measuring oxidation of NADPH at 340 nm by oxidized glutathione as described by Carlberg and Mannervik (1985). GST was assayed by measuring 2,3dichloro-4-nitrobenzene (CDNB)–GSH conjugate formed by using 1-chloro-2,4-dinitrobenzene as substrate using the method of Warholm et al. (1985). GPX in serum and liver was estimated using NADPH oxidation in a coupled reduction system of hydrogen peroxide and oxidized glutathione as described by Flohe and Gunzler (1984). CAT activity was assayed by the method of Aebi (1984) following the decomposition of hydrogen peroxide at 240 nm. SOD activity was estimated using cytochrome c and xanthine oxidase as per the method given by Flohe and Otting (1984). Statistical analysis Statistical analysis was carried by using Grad prism statistical software. Results are analyzed by one-way ANOVA, and the significance level was calculated using Tukey–Kramer multiple comparison test. Differences were considered significant when p was

Potentiation of antioxidant effect of dietary tender cluster beans (Cyamopsis tetragonoloba) by garlic (Allium sativum) in high-cholesterol-fed rats.

The antioxidant role of tender cluster beans (Cyamopsis tetragonoloba, CB), a rich source of soluble fibre, was investigated in a hypercholesterolemia...
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