International Journal of Biological & Medical Research

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1 Int J Biol Med Res. 2010; 1(4): Int J Biol Med Res Volume 3, Issue 4, Sep ISSN: 0976:6685 BioMedSiDiret Pulitions Contents lists ville t BioMedSiDiret Pulitions Interntionl Journl of Biologil & Medil Reserh Journl homepge: Interntionl Journl of BIOLOGICAL AND MEDICAL RESEARCH Originl Artile Antihyperglyemi nd ntilipidperoxidtive effets of flvnoid nringin in streptozotoin-niotinmide indued dieti rts Leelvinothn Pri*, Selvrju Sumn *Deprtment of Biohemistry nd Biotehnology, Fulty of Siene, Annmli University, Annmlingr , Tmilndu, Indi A R T I C L E I N F O Keywords: Dietes Lipid peroxidtion Oxidtive stress Nringin Streptozotoin A B S T R A C T In light of evidene tht some omplitions of dieti mellitus my e used or exerted y n oxidtive stress, the puttive protetive effet of nringin ws investigted in streptozotoin-niotinmide indued dieti rts. Nringin ws given for 42 dys t dily dose equivlent to 80 mg/kg of ody weight. Lipid peroxidtion levels nd non-enzymi ntioxidnts suh s vitmin C, vitmin E nd glutthione were thn mesured in plsm. Under our experimentl onditions, nringin ws found to signifintly redue the lood gluose nd inrese the plsm insulin level in dieti rts. Our results suggest tht the ntihyperglyemi tion of nringin is exerted vi the stimultion of insulin seretion. In ddition, nringin ppers to exert n ntioxidtive property demonstrted y the inrese of vitmin C, vitmin E nd glutthione levels in plsm nd lowering of lipid peroxidtion levels in plsm. In onlusion, nringin possess potentil hypoglyemi effet in streptozotoinniotinmide indued dieti rts. Copyright 2010 BioMedSiDiret Pulitions IJBMR - ISSN: 0976:6685. All rights reserved. 1. Introdution Dietes mellitus is the nme given to group of disorders with different etiologies. It is hrterized y disrrngements in rohydrtes, proteins nd ft metolism used y the omplete or reltive insuffiieny of insulin seretion nd/or insulin tion [1]. The World Helth Orgniztion (WHO) estimtes tht more thn 220 million people worldwide hve dietes nd this numer is likely to more thn doule y 2030 [2]. The ourrene nd onsequenes ssoited with dietes re found to e high in ountries like Indi (31.7%), Chin (20.8%) nd USA (17.7%). The rte is expeted to rise to 79.4%, 42.3% nd 30.3%, respetively, y 2030 in the ove ountries [3]. Oxidtive stress hs een implited in host of dietes relted omplitions inluding premture theroslerosis [4]. However, despite roust experimentl evidene suggesting tht inresed oxidtive lod hs signifint role in the pthogenesis of dietes [5, 6, 7]. Plnts used in trditionl mediine to tret dietes mellitus represent vlule lterntive for the ontrol of this disese. Despite the presene of known ntidieti mediines in the phrmeutil mrket, sreening for new soures from * Corresponding Author : Dr. L. Pri, Professor, Deprtment of Biohemistry nd Biotehnology Fulty of Siene, Annmli University, Annmli Ngr Tmil Ndu, Indi. Tel: , Fx: Emil: pril@gmil.om Copyright 2010 BioMedSiDiret Pulitions. All rights reserved. nturl plnts is still ttrtive euse they ontin sustnes tht re effetive nd sfe in dietes mellitus [8]. The plnt kingdom hs eome trget for the serh for new drugs nd iologilly tive ompounds [9]. Ethnootnil informtion indites tht more thn 800 plnts re used s trditionl remedies for the tretment of dietes, ut only few hve reeived sientifi srutiny. Phytohemils isolted from plnt soures re used for the prevention nd tretment of ner, hert disese, dietes nd high lood pressure. A numer of sustnes hve een identified, whih protet ginst experimentl dietes [10]. Any sustne tht n prevent n ttk or elerte the proess of reovery will hve onsiderle linil pplition. Flvonoids re group of nturlly ourring ompounds uiquitous in the plnt kingdom nd known to hve strong ntioxidnt effets. They re widely distriuted in foods of plnt origin suh s vegetles, fruits, te nd wine. Nringin (4,5,7- trihydroxy flvonone 7-rhmnogluoside) is the predominnt flvonone found in grpe fruit nd relted itrus speies [11]. Currently, there is muh interest in the usefulness of itrus fruits euse of their intke ppers to e ssoited with redued risk of ertin hroni diseses nd inresed survivl [12]. Nringin is responsile for the hrteristi sour flvor of the fruits [13]. Among the nturlly ourring flvonoids, nringin hs een empirilly proven to hve no side effets, s humns hve een ingesting grpes nd itrus fruits for long time [14]. The role of

2 L. Pri nd S. Sumn / Int J Biol Med Res. 2010; 1(4): nringin hd reently reeived onsiderle ttention s dietry ntioxidnt. Nringin exhiits vrious phrmologil nd therpeuti properties: ntimiroil, ntimutgeni, ntiner, nti-inflmmtory, holesterol lowering, free rdil svenging nd ntioxidnt effets [15, 16, 17]. To our knowledge, iohemil investigtion hs not een rried out on the effet of nringin on the plsm ntioxidnt sttus of experimentl dieti rts. The present study ws undertken in streptozotoin-niotinmide dieti rts to verify the ntihyperglyemi effet, to investigte the effet on lipid peroxidtion nd to evlute the potentil of nonenzymti ntioxidnt in plsm, of nringin. 2. Mterils nd methods 2.1. Animls Adult mle lino Wistr rts (8 weeks), weighing 200 to 200 g red in the Centrl Animl House, Rjh Muthih Medil College, Annmli University, were used. All niml experiments were pproved y the ethil ommittee (Vide. No: 582, 2008), Annmli University nd were in ordne with the guidelines of the Ntionl Institute of Nutrition, Indin Counil of Medil Reserh, Hyderd, Indi. The nimls were housed in polyronte ges in room with 12 h dynight yle, temperture of 24±2 C, humidity of 45% to 64%. During the whole experimentl period, nimls were fed with lned ommeril diet (Hindustn Lever Ltd., Mumi, Indi) nd wter d liitum Drug nd Chemils Nringin, Streptozotoin (STZ) nd ll other iohemils were purhsed from Sigm Chemil Compny, St Louis, MO, USA. All hemils were of nlytil grde Experimentl indution of type 2 dietes Non-insulin dependent dietes mellitus ws indued [18] in overnight fsted rts y single intrperitonel injetion (i.p) of 45 mg/kg ody weight STZ, 15 min fter the i.p dministrtion of 110 mg/kg ody weight of niotinmide. STZ ws dissolved in itrte uffer (ph 4.5) nd niotinmide ws dissolved in norml sline. The elevted gluose levels in plsm determined t 72 h, onfirmed hyperglyemi. The nimls with lood gluose onentrtion more thn 250 mg/dl will e used for the study Experimentl design In the experiment, totl of 24 rts (12 dieti surviving rts, 12 norml rts) were used. The rts were divided into four groups of six eh, fter the indution of STZ dietes. Nringin ws orlly dministered using n intrgstri tue for period of 42 dys. Group 1: Norml rts Group 2: Norml rts + nringin (80 mg/kg.w) Group 3: Dieti rts Group 4: Dieti rts + nringin (80 mg/kg.w) At the end of the experimentl period, the nimls were deprived of food overnight nd srified y depittion. Blood ws olleted in tues ontining potssium oxlte nd sodium fluoride mixture for the estimtion of lood gluose. Plsm ws seprted for the estimtion of insulin nd other iohemil prmeters. Blood gluose ws estimted olorimetrilly using ommeril dignosti kits (Sigm Dignostis (I) Pvt Ltd, Brod, Indi)[19]. Plsm insulin ws ssyed y ELISA using BoehringerMnnheim kit with n ES300 Boehringer nlyzer (Mnnheim, Germny) Estimtion of lipid peroxidtion nd non-enzymi ntioxidnts L i p i d p e rox i d t i o n i n p l s m we re e s t i m ted olorimetrilly y mesuring thiorituri id retive sustnes (TBARS) nd hydroperoxides using the methods of Frg et l. [20] nd Jing et l. [21], respetively. Vitmin C, E nd redued glutthione (GSH) were estimted y the method of Omye et l. [22], Bker et l. [23] nd Ellmn [24], respetively Sttistil nlysis The dt for vrious iohemil prmeters were nlyzed using nlysis of vrine (ANOVA), nd the group mens were ompred y Dunn's multiple rnge test (DMRT) using sttistilly softwre pkge (SPSS for Windows, V.13.0, Chigo, USA). Vlues were presented s men ±S.D p<0.05 were onsidered s sttistilly signifint [25]. 3. Results 3.1. Chnges in lood gluose nd plsm insulin The levels of lood gluose nd plsm insulin in norml nd experimentl rts re shown in Tle 1. The experimentl rts showed signifint inrese in the level of lood gluose with signifint derese in the level of plsm insulin. Tretment with nringin (80 mg/kg wt) for period of 42 dys to experimentl rts signifintly retined the ove iohemil hnges. In our previous study, we reported tht nringin t 80 mg/kg wt, showed etter effet thn 20 nd 40 mg/kg wt, therefore the 80 mg/kg wt ws used in this study. The dministrtion of nringin to norml rts did not show ny signifint hnges in lood gluose nd plsm insulin levels. Tle 1. Chnges in the levels of lood gluose nd plsm insulin in norml nd experimentl nimls Norml Norml + nringin Dieti Dieti + nringin Blood gluose ± ± ± ± Vlues re given s men ± S.D for 6 rts in eh group. Vlues not shring ommon supersript letter differ signifintly t p< Effet of nringin on plsm lipid peroxidtion Plsm insulin (µu/ml) ± ± ± ± 0.74 Tle 2. Shows the levels of TBARS nd lipid peroxide in plsm in norml nd dieti rts. Untreted dieti rts showed signifint inrese in plsm lipid peroxidtion levels when ompred to norml rts. Nringin treted with experimentl rts hd signifintly lower plsm lipid peroxide nd TBARS levels s ompred with the untreted dieti rts. 2.5.Biohemil ssys Estimtion of lood gluose nd plsm insulin

3 208 L. Pri nd S. Sumn / Int J Biol Med Res. 2010; 1(4): Tle 2. Chnges in the levels of plsm TBARS nd hydroperoxides in norml nd experimentl nimls Norml Norml + nringin Dieti Dieti + nringin Vlues re given s men ± S.D for 6 rts in eh group. Vlues not shring ommon supersript letter differ signifintly t p< Effet of nringin on plsm ntioxidnts Tle 3. Shows the levels of plsm ntioxidnts in norml nd experimentl rts. Plsm GSH, vitmin C nd vitmin E levels were signifintly lower in dieti rts thn in norml rts. Experimentl rts treted with nringin hd ner norml levels of plsm ntioxidnts. Tle 3. Chnges in the levels of vitmin C, vitmin E, nd redued glutthione (GSH) in plsm of norml nd experimentl nimls Norml Norml + nringin Dieti Dieti + nringin Vlues re given s men ± S.D for 6 rts in eh group. Vlues not shring ommon supersript letter differ signifintly t p< Disussion TBARS (mmoles/dl) Vitmin C 1.83 ± ± ± ± ± ± ± ± 0.01 Vitmin E 1.64 ± ± ± ± 0.09 Hydroperoxides -5 (x 10 mm/100ml) ± ± ± ± 0.84 Redued glutthione ± ± ± ± 1.36 Dietes is hrterized y defets in oth metoli nd vsulr domins; this disese represents privileged sitution for oxidtive stress exerting hrmful effets [26]. The tthment of free rdils in dietes nd the responsiility of these toxi speies in lipid peroxidtion nd the ntioxidnt defense system hve een studied. Streptozotoin-indued dietes is welldoumented model of experimentl dietes. Streptozotoindietes provides relevnt exmple of endogenous hroni oxidtive stress due to the resulting hyperglyemi [27]. Hyperglyemi n stimulte oxidtive stress is the utoxidtion of gluose in the presene of trnsition metls s well s the genertion of ROS during the proess of glytion [28]. The roles of oxidtive stress nd ntioxidnts in orgns nd tissues dmge hve een studied extensively in experimentl dietes nd dieti ptients [29]. STZ diretly genertes oxygen free rdils indued lipid peroxidtion [30]. This study ws therefore undertken to ssess ntiperoxidtive properties of nringin in STZ dieti rts. We found tht 42 dys tretment with nringin t 80 mg/kg ody weight redues lood gluose levels in dieti nimls. The possile mehnism(s) y whih nringin initites ntihyperglyemi tion ould e due to possile pnreti seretion of insulin from existing â-ells, with n intensifition of gluose uptke y peripherl tissues or redued gluose sorption from the gstrointestinl trt [31]. Hyperglyemi, the primry linil mnifesttion of dietes, is responsile for the development of dieti omplitions. Severl mehnisms my led to inresed oxidtive stress in dietes. Firstly, hyperglyemi my inrese the genertion of free rdils through the ility of gluose to enolize nd yield oxidizing intermedites suh s superoxide nion, hydroxyl rdil, hydrogen peroxide nd nitri oxide [32, 33]. Seondly, ntioxidnt defenses re redued in dietes [34]. Oxidtive stress nd the oxidtive modifition of iomoleules re involved in numer of physiologil nd pthophysiologil proesses. Oxidtive stress ours s onsequene of imlne etween prooxidnts nd ntioxidnts. If produed in exess, retive oxygen speies (ROS) led to dmge of mromoleules suh s DNA, proteins nd lipids. The elevted level of lipid peroxides in the plsm of dieti rts nd lipid peroxidtion is one of the hrteristi fetures of hroni dietes [35]. The inresed level of plsm lipid peroxide in STZ-indued dietes is generlly through to e due to pthologil hnges to tissues tht inrese the prodution nd liertion of lipid peroxides into the irultion [36]. Previous studies hve reported inresed levels of lipid peroxidtion in plsm of dieti rts [37]. Flvonoids hve shown to inhiit lipid peroxidtion formtion in rt nd lso inhiit the free rdil prodution in the ells t vrious stges. Our study lerly shows tht the tretment of nringin signifintly redued the levels of TBARS nd hydroperoxides in plsm of dieti rts. In this ontext, Ali nd Ad El Kdr hve reported tht nringin tretment redue the levels of TBARS nd hydroperoxides in STZ-indued lipid peroxidtion [31]. Antioxidnts ply mjor role in proteting iologil systems ginst retive oxygen speies nd reflet the ntioxidnt pity of the system [38]. Vitmin E is the most nient ntioxidnt in the lipid phse [39]. Aprt from enzymi ntioxidnts, non-enzymi ntioxidnts ply vitl role in proteting ells from oxidtive hnges. Vitmin E neutrlizes the free rdils, preventing the hin retion tht ontriutes to oxidtive dmge [40]. It hs the potentil to quenh lipid peroxidtion nd protets ellulr struture from the ttk of free rdils [41]. The deresed level of vitmin E oserved in the plsm of dieti rt [37]. Asori id is the most widely ited form of wter-solule ntioxidnt; it prevents oxidtive dmge to the ell memrne indued y queous rdils. Vitmin C n t s o-oxidnt y regenerting á-toopherol from the lph-toopheroxyl rdil produed during svenging of free rdils. It is lso possile tht the orretion of hyperglyemi hs spring effet on vitmin C, nd this inreses the potentil to reyle vitmin E [42, 43]. We lso oserved signifint derese in the levels of plsm vitmin C in dieti rts ould e due to inresed utiliztion of vitmin C s n ntioxidnt defense ginst retive oxygen speies [44]. GSH, non-enzymti ntioxidnt is known to ord protetion ginst ROS y effetively svenging free rdils nd other ROS diretly nd indiretly through enzymti retions [45]. GSH is n importnt ntioxidnt, whih lso mintins the

4 id in their tive redued forms, nd synergisti intertion exists etween á-toopherol nd sori id in suppressing the peroxidtion. Deresed GSH levels in the irultion might e due to inresed utiliztion in proteting 'SH' ontining proteins from lipid peroxides [46]. The elevted level of GSH on tretment with nringin protets ellulr proteins ginst oxidtion through glutthione redox yle nd detoxifies the free rdils generted. Nringin my exhiit ntioxidnt tivity pity sed on inresing the upregultion of gene expressions in the ntioxidnts, with onsequently enhnement the svenging of retive oxygen speies ompnying with signifintly lowering of lipid peroxidtion suggests tht dministrtion of nringin inrese the ntioxidtive potentil. In ddition like ll the flvonoid ompounds, presene of hydrogen donting sustituents tthed to romti rings my enle nringin to svenge free rdils. [47]. Orl dministrtion of nringin my revel their tion on gluose through redution of oxidtive stress intensity in hyperglyemi. 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