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Int J Pharm Bio Sci 2016 July; 7(3): (P) 1 - 9Original Research Article Pharmacology International Journal of Pharma and Bio Sciences ISSN 0975-6299 INFLUENCE OF TWO DIFFERENT FORMULATIONS OF ZINCOVIT TABLETS ON SODIUM SELENITE INDUCED CATARACT IN RATS SHAKTA MANI SATYAM AND LAXMINARAYANA BAIRY KURADY* Department of Pharmacology, Melaka Manipal Medical College, Manipal University, Manipal-576104 Department of Pharmacology, Kasturba Medical College, Manipal University, Manipal-576104 ABSTRACTZincovit tablet is a nutritional supplement used to improve general wellbeing of an individual. A study wasundertaken to evaluate the effect of different formulations of Zincovit tablets on sodium selenite induced agerelated cataract. A total of 48 rat pups of either sex were used which were divided into 8 groups of 6 rats each On7th post-natal day a single dose of sodium selenite (20 µM/kg) was administered s.c. to all the groups except forthe normal control group (group l). The rat pups were given two formulations of Zincovit tablets (40, 80 and160mg/kg), one day prior to the administration of selenite and continued till day 20. Eye was examined daily tocheck cataract formation. On day 21st the rat pups were sacrificed lens were separated for estimation ofGlutathione peroxidase (GPx) activity, Superoxide dismutase (SOD) activity, Catalase, Glucose-6-phosphatedehydrogenase (G6PD) activity, Adenosine triphosphate (ATP) level, Aldose reductase (AR) level and Sorbitoldehydrogenase (SDH).Significant reduction in glutathione peroxidase, Adenosine triphosphate level in all selenitetreated group compared to control group. Treatment with Zincovit tablet at different doses was not able tocompletely reverse the selenite induced reduction in glutathione peroxidase. Almost 50% of rats did not developcataract in Zincovit tablet with vitamin A and grape seed treated group. Zincovit tablet (vitamin A: 5000 IU withoutgrape seed extract) was better in delaying the onset of cataract when compared to other groups.KEYWORDS: Nutritional supplement, senile cataract, experimental cataract LAXMINARAYANA BAIRY KURADY Department of Pharmacology, Kasturba Medical College, Manipal University, Manipal-576104 This article can be downloaded from www.ijpbs.net P-1

Int J Pharm Bio Sci 2016 July; 7(3): (P) 1 - 9INTRODUCTION and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (Registration no.Cataract is the leading cause of blindness all over 94/1999/CPCSEA/KMC).Experimental cataract wasthe world. Apart from aging nutritional deficiencies induced by selenite injection. A total of 48 rat pups ofor inadequacies, trace metals, sunlight, smoking, either sex were used whichwere divided into 8 groups ofand certain drugs are known to increase the risk of 6 rats each. On 7th post-natal day a single dose ofcataract1. Oxidative damage to the constituents of sodium selenite (20 µM/kg) was administered s.c. to allthe eye lens is considered to be a major mechanism the groups except for the normal control group (group l).in the initiation and progression of various types of The total volume of drug ranged from 0.1 to 0.15 ml. Thiscataracts, including sodium selenite induced age could easily be given to 7 day old pups by making a smallrelated cataract2.Sodium selenite is considered a pouch of skin.valid model for cataracts caused by oxidativestress3. Similar to human senile cataract, this type Group Iof cataract is accompanied by a decrease in Normal control rats were given 0.1 ml of normal salineactivities of antioxidant enzymes. Several studies subcutaneously from day 7 to day 20 of their post-natalhave suggested that intake of antioxidant-rich foods lifemay slow the progression of cataract3, 4. this studywe have investigated the influence of two different Group IIformulations of Zincovit tablet which contain vitamin Cataract control rats were ???? 0.1 ml of normal salineA: 2000 IU with grape seed extract and vitamin A: subcutaneously from day 7 to day 20 of their post-natal5000 IU without grape seed extract in addition to lifeother vitamins and minerals on sodium seleniteinduced age related cataract in Wistar rats. The Group IIImain objective of this comparison is to see if with Zincovit tablets (vitamin A: 2000 IU with grape seedless vitamin A addition of grape seed extract will be extract 40 mg/kg/day were givensubcutaneously fromas good as 5000 units vitamin A. Yamakoshi et al. day 7 to day 20 of their post-natal lifesuggested the anticataract activity of grape seedextract (GSE, which contains 38.5% procyanidins) Group IVin hereditary cataractous rats (ICR/f rats)5.. Earlier, Sodium selenite andZincovit tablets (vitamin A: 2000 IUwe have reported the effect of Zincovit tablet with grape seed extract) 80 mg/kg/day were given(vitamin A: 5000 IU with grape seed extract) on subcutaneously from day 7 to day 20 of their post-nataldelay of onset and progression of sodium selenite lifeinduced age related cataract in Wistar rats3, 4.According to Silva et al. Zn prevents diabetes- Group Vinduced GSH loss in the retina6. A few studies like Sodium selenite and Zincovit tablets (vitamin A: 2000 IUthe one done by Irina C et al regarding antioxidant with grape seed extract)160 mg/kg/day were giveneffects of grape seed extract suggest that long-term subcutaneously from day 7 to day 20 of their post-nataldaily administration of grape seed extract offers lifeenhanced antioxidant potential and protectionagainst tissue lipid peroxidation and the protein Group VIoxidation7. Sodium selenite and Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 40 mg/kg/day were givenMATERIALS AND METHODS subcutaneously from day 7 to day 20 of their post-natal lifeSeven days old rat pups with mother were housed inpolypropylene cages. They were maintained under Group VIIstandard conditions with temperature (22–240 C), 12- h Sodium selenite and Zincovit tablets (vitamin A: 5000 IUlight/12-h dark cycle and relative air humidity 40–60%. without grape seed extract) 80 mg/kg/day were givenThey had continuous access to normal pellet diet and to subcutaneously from day 7 to day 20 of their post-nataltap water. Animals described as fasted were deprived of lifefood for 16-h but ad libitum water. This study wasconducted in accordance with institutional guidelines and Group VIII Sodium selenite and Zincovit tablets (vitaminwith the Association for Research in Vision and A: 5000 IU without grape seed extract) 160 mg/kg/dayOphthalmology Statement for the Use of Animals in were given subcutaneously from day 7 to day 20 of theirResearch. The experiment was carried out according to post-natal lifethe ethical norms approved by Ministry of Social Justices Blood and Lens Collection and Processing Cataract formation and its prevention weremonitored by examining the eye (cloudy area in the lens) withpen light illumination thereafter till the end of experiment andsubsequent photography of the isolated lenses wasThis article can be downloaded from www.ijpbs.net P-2

Int J Pharm Bio Sci 2016 July; 7(3): (P) 1 - 9done on 21stday. Body weight of each rat pup was 150 µl sodium phosphate buffer (PBS 0.2 M, pH 8.0) andchecked before start andat the end of the experiment. On 25 µl DTNB (0.6mM) were added together in 96-wells of21st day, all the rat pups weresacrificed by administering micro test plate and incubated for 10 minutes at roomoverdose of ketamine (i.p.). The eye lenses were temperature. Absorbancewas read at 412 nm.dissected bythe posterior approach and stored at -70°Cuntil furtheranalysis. Lens weight of each rat pup was Determination of Protein thiol (PT) level15taken. Fasting bloodsamples were drawn on from retro- One hundred eighty microliter disodium edetate (2mMorbital plexus of all theexperimental animals using sodium edetate in0.2 M disodium hydrogen phosphate)capillary tube for the estimation ofblood glucose with the buffer solution, 4 µlDTNB solution (10mM DTNB in 0.2 Mhelp of glucose oxidase-peroxidasereactive strips (Accu- disodium hydrogenphosphate) and 20 µl of lens tissueChek, Roche Diagnostics, USA). Using a Remi C-24 homogenate sample in 96-wells of micro test plate.refrigerated centrifuge lenshomogenate (10% w/v) was Optical density was read at 412 nm.Glutathioneprepared from in 50 mM potassium phosphate buffer (pH peroxidase (GPx) activity, Superoxide dismutase (SOD)7.4). The unbroken cells and cell debriswas removed by activity, Catalase, Glucose-6-phosphate dehydrogenasecentrifugation at 10000 rpm for 20 minutes. The (G6PD) activity, Adenosine triphosphate (ATP) level,resultingsupernatant was stored at -80°C. Aldose reductase (AR) level and Sorbitol dehydrogenase (SDH) level were measured according to the protocolDetermination of Malondialdehyde (MDA) level15 given along with the commercially available assay kitAbout 20 µl lens homogenate sample was taken and 200 from Cayman chemicals, USA.µl 0.67% thiobarbituric acid and 100 µl 20%trichloroacetic acid were added; incubated at 100°C for Data analysis20 minutes. Then it was centrifuged at 12000 rpm for 5 Using SPSS 20.0, normally distributed data (GSH,minutes and hundred microliter of supernatant was Protein thiol, SOD, Catalase, G6PD, SDH, MDA, FBG,transferred to ninety six wells of micro test plate. The Body weight and Isolated lens weight, was analyzed byOptical density of supernatant was read at 540 nm. one way analysis of variance (ANOVA) followed by post hoc Tukey test. Non-parametric K independent sampleDetermination of Reduced glutathione (GSH) level15 test followed by Kruskal Wallis test was employed forAbout hundred microliter of lens tissue homogenate and data which was not normally distributed (Glutathione100 µl of 5% trichloroacetic acid (TCA) solution peroxidase, Aldose reductase and ATP). P value lessweremixed together and centrifuged at 5000 rpm for 5 than 0.05 was considered as statistically significant.minutes. Twenty-five microliter of tissue supernatant, Table 1 Level of Glutathione peroxidase in lens homogenate (nmol/min/ml): Sodium selenite cataract modelGroups Dose Median (Q1, Q3) P value Significance I Normal control-0.1 ml of normal saline 341.38 (220.31, 388.48) 0.012# S II Sodium selenite induced cataract (SSIC) control-0.1 ml of normal saline III SSIC - Zincovit tablets (vitamin A: 2000 IU with grape seed extract) 40 mg/kg/day 87.12 (60.10, 212.30) SSIC - Zincovit tablets (vitamin A: 2000 IU with grape seed extract) 80 mg/kg/day 98.02 (63.04, 126.84) IV 102.50 (62.17, 227.40) V SSIC - Zincovit tablets (vitamin A: 2000 IU with grape seed extract) 160 mg/kg/day 91.34 (46.28, 99.11) VI SSIC - Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 40 mg/kg/day 10.82 (6.44, 21.18) VII SSIC - Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 80 mg/kg/day 28.14 (12.62, 50.23) 24.77 (10.82, 40.55) VIII SSIC - Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 160 mg/kg/day#compared among groups, S- Significant Table 2 Level of Adenosine triphosphate (ATP) in lens homogenate (mol/ml): Sodium selenite cataract modelGroups Dose Median (Q1, Q3) P value Significance I Normal control-0.1 ml of normal saline 3.50 (1.50, 8.50) 0.05* II Sodium selenite induced cataract (SSIC) control-0.1 ml of normal saline 2.50 (0.75, 5.75) III SSIC - Zincovit tablets (vitamin A: 2000 IU with grape seed extract) 40 mg/kg/day 17.50 (3.25, 104.75) SSIC - Zincovit tablets (vitamin A: 2000 IU with grape seed extract) 80 mg/kg/day 11.00 (5.50, 15.25) IV SSIC - Zincovit tablets (vitamin A: 2000 IU with grape seed extract) 160 mg/kg/day 5.00 (2.50, 18.75) V VI SSIC - Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 40 mg/kg/day 16.50 (9.75, 43.75) S VII SSIC - Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 80 mg/kg/day 4.14 (0.00, 9.75) VIII SSIC - Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 160 mg/kg/day 7.50 (3.75, 18.50)*compared among groups, S- Significant This article can be downloaded from www.ijpbs.net P-3

Int J Pharm Bio Sci 2016 July; 7(3): (P) 1 - 9 Figure 1 Normal control rat pup: The lens is clear and normal Figure 2Sodium selenite induced cataract: Well developed cataract is seen This article can be downloaded from www.ijpbs.net P-4

Int J Pharm Bio Sci 2016 July; 7(3): (P) 1 - 9 Figure 3 Rat pup treated with Zincovit tablets (vitamin A: 2000 IU with grape seed extract) 40 mg/kg- Sodium selenite cataract model:The lens is clear almost like lens of control rats Figure 4 .Rat pup treated with Zincovit tablets (vitamin A: 2000 IU with grape seed extract)80 mg/kg- Sodium selenite cataract model: The lens is clear almost like lens of control rats This article can be downloaded from www.ijpbs.net P-5

Int J Pharm Bio Sci 2016 July; 7(3): (P) 1 - 9 Figure5 Rat pup treated with Zincovit tablets (vitamin A: 2000 IU with grape seed extract)160 mg/kg- Sodium selenite cataract model: The lens is clear almost like lens of control rats Figure6 Rat pup treated with Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 80 mg/kg- Sodium selenite cataract model: the lens is clear almost like lens of control rats This article can be downloaded from www.ijpbs.net P-6

Int J Pharm Bio Sci 2016 July; 7(3): (P) 1 - 9 Figure7 Rat pup treated with Zincovit tablets (vitamin A: 5000 IU without grape seed extract)80 mg/kg- Sodium selenite cataract model: the lens is clear almost like lens of control rats Figure 8 Rat pup treated with Zincovit tablets (vitamin A: 5000 IU without grape seed extract) 160 mg/kg- Sodium selenite cataract model: The lens is clear like normal control group. This article can be downloaded from www.ijpbs.net P-7

Int J Pharm Bio Sci 2016 July; 7(3): (P) 1 - 9RESULTS oxidant followed by opening ofcalcium channel or inhibition of calcium ATPase2. This leads toThere is a significant reduction in glutathione accumulation of calcium, activation ofcalpain, proteolysisperoxidase, Adenosine triphosphate level in all selenite of beta crystalline polypeptides that finally form insolubletreated group compared to control group (Table 1 &2). pellets which scatter light2.Although the rate ofTreatment with Zincovit tablet at different doses was not opacification in the selenite model is much more rapidable to completely reverse the selenite induced than in human cataract, ithas many general similaritiesreduction in glutathione peroxidase. There was no to human cataract such as increased calcium, proteinsignificant difference in aldose reductase, ATP, glucose- aggregation,decreased water-soluble proteins, and6-phosphate dehydrogenase, reduced glutathione, in reduced glutathione levels3.We found that there was abody weight, isolated lens weight, fasting blood glucose, decrease in percent of rats that developed cataractMDA, sorbitol dehydrogenase, catalase, protein thiol, compared to selenite alone treated rats. This shows thatsuperoxide dismutase level in the lens homogenate of the vitamins (Vitamin C and vitamin E present in Zincovitrats which were treated with both the formulations of tablet must have protected against cataract induction byZincovit tablet when compared to the selenite control selenite. The potential role of vitamins in preventinganimals respectively.In age related cataract model, all cataract is well documented, especially vitamin Clenses in normal control group were clear and normal orascorbic acid which plays an important part in lensbut in sodium selenite induced cataractous rat pups, biology, both as an antioxidant and as a UV filter13.90%, 62%, 57%, 53%, 42%, 51%, 50%, and of lenses Dietary deficiency of vitamin C led to reduction in lensdeveloped cataract in sodium selenite induced concentrations of ascorbate7. Vitamin E also hascataractous control, Zincovit tablets (vitamin A: 2000 IU animportant part to play in lenticular antioxidant status. Awith grape seed extract) and Zincovit tablets (vitamin A: number of studies have evaluated theanticataract5000 IU without grape seed extract) 40 mg/kg, 80 mg/kg potential of vitamin E and the found it to be effectiveand 160 mg/kg treatment groups respectively(Figures 1- against galactose, steroid and UVradiation-induced8). cataract14. Plasma levels of zinc and copper were found to besignificantly low in cataract patients3. Selenium isDISCUSSIONS an integral part of the enzyme, glutathioneperoxidase14. A decrease in glutathione peroxidase activity has beenLoss of transparency during human cataract formation found in the lenses of selenium deficientrats14.Theresults from a variety of complex metabolic and therapeutic benefit for delay the onset and progressionphysiological mechanisms, which act in combination to of senile cataract was found to be highest with Zincovitchange the refractive index3. Sulfhydryl oxidation is tablet (vitamin A: 5000 IU with grape seed extractthought to be one of the main pathological events followed by Zincovit tablet (vitamin A: 5000 IU withoutleading, through disulfide cross-linking and molecular grape seed extract) and Zincovit tablet (vitamin A: 2000aggregation, to protein precipitation and lens IU with grape seed extract) respectively.opacification10-12.The selenite experimental model wasselected because of its rapid, effective, and reproducible ACKNOWLEDGEMENTScataractformation2. It shows a number of generalsimilarities to human cataract in addition tovesicle The authors hereby acknowledge the support given byformation, such as increased calcium, insoluble protein, Apex Laboratories Private Ltd, Chennai and Manipalproteolysis, and decreased glutathionelevels. Selenite University,damages lens epithelial cells by acting as a sulfhydryl Manipal for carrying out this project.REFERENCES1. Congdon NG, Friedman DS, Lietman T. Important 4. Satyam SM, Bairy KL, Pirasanthan R, Vaishnav causes of visual impairment in the world RL. Grape seed extract and Zinc containing today.JAMA. 2003Oct 15; 290(15):2057–60 nutritional food supplement, delays progression and maturation of Streptozocin- induced diabetic2. Spector A. Oxidative stress and disease. J Ocul cataract in Wistar rats. IFST 2015; 52(5):2824-32. Pharmacol Ther. 2000 April; 16:193–201 5. Yamakoshi J, Makoto S, Shigehiro K, Shoichi T.3. Satyam SM, Bairy KL, Pirasanthan R, Vaishnav Procyanidin Rich Extract from Grape Seeds RL. Prevention of Selenite- induced senile Prevents Cataract Formation in Hereditary cataractogenesis by Grape seed extract and Zinc Cataractous (ICR/f) Rats. Agric. Food Chem2002; containing nutritional food supplement in Wistar 50 (17): 4983–88. rats. J Nutr Health Aging. 2014; 18(5):524-30. This article can be downloaded from www.ijpbs.net P-8

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