AMELIORATIVE EFFECT OF POMENGRATE PEEL WATER EXTRACT AGAINST FOLIC ACID-INDUCED NEPHROTOXICITY IN RATS

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1 Vol. 3. No. 4. July, 2011, II Prt AMELIORATIVE EFFECT OF POMENGRATE PEEL WATER EXTRACT AGAINST FOLIC ACID-INDUCED NEPHROTOXICITY IN RATS Ftm H. Ad El-Rzek, Ens A. Kmel Biochemistry nd Nutrition Deprtment,Women's College,Ain Shms university, Ciro (EGYPT) E-mils: ABSRTACT Recent studies indicte tht free rdicls re importnt meditors of renl dmge induced y high dose folic cid injection. Pomengrte peel ws reported s potent ntioxidnt. Therefore, the im of this study is to evlute the nephroprotective effect of pomengrte peel wter extrct in folic cid induced renl filure. For this purpose, thirty dult mle lino rts were ssigned into 5 groups. group1: norml control group, group 2: experimentlly induced renl filure injected with folic cid (250mg/Kg ody weight), group 3, 4 nd 5 experimentlly induced renl filure injected with folic cid followed y orl dministrtion of pomengrte peel wter extrct( 0.5 ml,1ml nd 1.5 ml /dy, respectively). The results of this study demonstrted tht injection with folic cid (250mg/Kg ody weight) impired renl function judged y significnt increse in serum ure, cretinine, nd uric compred with norml control (p<0.05). Orl dministrtion of pomengrte peel wter extrct significntly ttenuted nephrotoxicity induced y folic cid resulting in significnt decrese in serum ure, cretinine nd uric cid (p<0.05). The results lso showed significnt increse in folic cid injection induced oxidtive stress reflected in significnt increse in serum Mlondildehyde ( MDA) levels. Also, renl MDA significntly incresed with significnt decrese in renl ctlse (CAT) nd superoxide dismutse (SOD) ctivities (p<0.05). Orl dministrtion of pomengrte peel wter extrct restored norml levels of mlondildehyde nd retined control ctivities of renl ntioxidnt enzymes. Qulittive phytochemicl nlysis indicted the presence of nturl ntioxidnts s flvonoid,tnnin,sponin nd sterols.therefore,pomengrte peel extrct hve more potentil s helth supplement nd nephroprotective gent rich in nturl ntioxidnt. Key words: pomengrte peel wter extrct ; folic cid; nephrotoxicity; ntioxidnts; rts. 1. INTRODUCTION Acute renl filure (ARF) is mjor world wide cuse of moridity nd mortlity,. The Most common cuse of ARF is cute tuulr necrosis. The folic cid (FA)-induced renl injury model hs een pplied recently for evlution of epithelil regenertion nd interstitil firosis. Intrperitonel dministrtion of FA with sodium icronte induced renl injury tht showed cute tuulr necrosis with trnsient elevtions of lood ure nitrogen (BUN) nd serum cretinine (Cr) followed y development of interstitil firosis [1]nd [2]Acute dministrtion of FA leds to the genertion of oxidtive stress which induces renl dmge [3]. Therefore, severl compounds with ntioxidnt ctivity cn e successfully used to meliorte folic cid induced nephrotoxiciy. Recently, nturl ntioxidnts hve ecome very populr for medicl nd food pplictions nd re preferred y consumers thn synthesized ntioxidnts Pomegrnte (Punic grntum L., Puniccee) is n importnt source of ioctive compounds nd hs een used for folk medicine for mny centuries. It is widely reported tht pomegrnte exhiits ntivirus, ntioxidnt, ntidietic, ntidirrhel, nti cncer, nd ntiprolifertive ctivities [3] nd [4]. Pomegrnte fruit, juice, nd peel extrcts is rich source of polyphenols nd hence posses potent ntioxidnt properties [6], [7] nd [8] It hs een reported tht the peel in prticulr possesses reltively higher ntioxidnt ctivity thn seed nd pulp nd therefore might e rich sources of nturl ntioxidnts. [9] nd[10] Therefore, the mjor ojective of this study ws to evlute the ntioxidtive nd meliortive effect of pomengrte peel wter extrct on nephrotoxicity nd oxidtive stress induced y intrperitonel FA injection in rts. 2. MATERIAL AND METHODS Animls: Adult mle Wistr lino rts weighing (135g 5 g) were purchsed from Animl House of El-Slm-Frm, Giz, Egypt. Rts were housed individully with constnt environment in controlled stinless steel cges, temperture (25 C 5 C) humidity (50% 10%), nd light cycle were held constnt 12/12 hr. The experimentl period ws 4 weeks on which food nd wter were provided d liitum. Animl's diet The experimentl diet used in the present study ws the lnced diet which ws prepred s descried y [11]. Preprtion of wter extrct of pomengrte peel: Pomegrnte fruits were wshed y distilled wrter then peeled nd their edile portions were crefully seprted. The peels cut into smll pieces nd were ir dried in ventilted oven t 40C for 48 hr nd ground to fine powder.pomengrte peel wter extrct ws prepred s descried y [12].200 ml oiling distilled wter were dded to3gm powder pomegrnte peel, left it for 10 minutes nd filtered. The filterte ws stored t 4 C 554

2 Vol. 3. No. 4. July, 2011, II Prt Induction of cute renl filure: Acute renl filure ws induced y intrperitonel injection with folic cid (250mg/Kg ody weight) [3]. Experimentl design The nimls were rndomly ssigned to five experimentl groups (ech of 6). All interntionl nd locl rules nd regultion for hndling nimls in experiments were followed. The experimentl groups illustrted s follow: Group1: Helthy rts fed on stndrd diet nd served s norml control. Group2: Fed on stndrd diet nd recevied only single intrperitonelly injection with FA (250mg/Kg ody weight). Group3: Fed on stndrd diet nd recevied only single intrperitonelly injection with FA plus pomengrte peels wter extrct (0.5 ml /dy). Group 4: Fed on stndrd diet nd recevied only single intrperitonelly injections with FA plus pomengrte peel extrct (1 ml /dy). Group5: Fed on stndrd diet nd recevied only single intrperitonelly injection with FA plus pomengrte peel wter extrct (1.5 ml /dy). At the end of the experiment, the nimls were nesthetized with diethyl ether fter 12 hours fsting nd whole lood smples were tken from heptic Portl vein. The lood smples left for 15 minutes t 37 C for serum seprtion, then centrifuged t 3000 rpm for 20 minutes, then ser were seprted nd kept in plstic vils t 20 C until nlyses. Biochemicl ssys Serum ure ws determined ccording method descried y [13] nd uric cid ws determined ccording to method descried y [14] nd serum cretinine ccording to method descried y [15].Alumin ws determined ccording to method descried y [16]. Thiorituric cid (ntioxidnt profile) rective sustnce (TBARS) s Mlondildehyde (MDA) ws determined ccording to the colorimetric method descried y [17]. Preprtion of kidney homogente: The kidneys of the rts from individul group ws dissected out, wshed with ice-cold sline nd weighed nd homogenized in 50 mm phosphte uffer (ph 7.4) using n electronic homogenizer to prepre 10% w/v homogente (ph 7.4). The homogente ws centrifuged t 3000 rpm g for 30 min. The superntnts were used for mesuring ctivity of enzymes- superoxide dismutse (SOD) nd ctlse (CAT) nd thiorituric cid rective sustnces-(tbars) ) s MDA. Superoxide dismutse (SOD) ctivity: The ssy of SOD ws sed on the ility of SOD to inhiit spontneous oxidtion of drenline to drenochrome [18]. To 0.05 ml superntnt, 2.0 ml of cronte uffer nd 0.5 ml of EDTA were dded. The rection ws initited y ddition of 0.5ml of epinephrine nd the utooxidtion of drenline to drenochrome t ph 10.2 ws mesured y following the chnge in OD t 480 nm. The chnge in opticl density every minute ws mesured t 480 nm ginst regent lnk. The results re expressed s units of SOD ctivity (U/g wet tissue). One unit of SOD ctivity induced pproximtely 50% inhiition of drenline. Ctlse (CAT) ctivity: The ctivity ssy ws sed on the ility of CAT to induce the disppernce of hydrogen peroxide [19]. A spectrophotometric method for mesuring the rekdown of hydrogen peroxide y the rection mixture consisted of 2 ml phosphte uffer (ph 7.0) ml of hydrogen peroxide (0.019 M) nd 0.05ml superntnt in finl volume of 3 ml. Asornce ws recorded t 240 nm every10 sec for 1 min. One unit of CAT ws defined, s the mount of enzyme required decomposing 1µmol of peroxide per min, t 25 C nd ph 7.0. The results were expressed s units of CAT ctivity (U/ g wet tissue). Mlondildehyde (MDA) ws determined ccording to the colorimetric method descried y [17]. Phytochemicl nlysis: The queous extrct of the plnt ws sujected to qulittive chemicl screening for the identifiction of the tnnins, flvonoids,sterols nd sponin. using stndrd procedures [20]. Test for Flvonoids: The extrcts were dissolved in lcohol. One piece of mgnesium followed y concentrted hydrochloric cid ws dded drop wise nd heted. Appernce of mgent color demonstrted the presence of flvonoids. Test for Sterols: 10 mg of extrct ws dissolved in 2 ml of chloroform nd 2ml of concentrted sulphuric cid ws dded from the side of the test tue. Test tue ws shken for few minutes. The development of red color in chloroform lyer indicted the presence of sterols. Test for Tnnins: The queous extrct (1ml) ws mixed with10 ml of Distilled wter nd filtered.ferric chloride regent (3 drops ) ws dded to the filtrte.a lue-lck or green precipitte confirmed the presence of gllic tnnin or ctechol tnnins, respectively. Test for Sponins: 1ml extrct ws treted with 1% led cette solution. Formtion of white precipittes indictes the presence of sponins. Sttisticl nlysis The dt were presented s mens ±SD. One wy nlysis of vrince (ANOVA) followed y post hoc-lest significnt difference nlysis (LSD) t (p< 0.05) ws performed using the sttisticl pckge for socil science (SPSS) version 9 to compre ll treted groups. Differences were considered to e significnt when (p< 0.05). 3. RESULTS The results in Tle 1 nd Fig.1 reveled tht intrperitonel injection with folic cid led to significnt increse in serum ure, cretinine nd uric cid level in untreted induced renl filure group compred with the B k u, A z e r i j n 555

3 Vol. 3. No. 4. July, 2011, II Prt corresponding control group (P< 0.05). Following orl dministrtion of pomengrte peel wter extrct, these ltered levels reverted ck to ner norml level. Orl dministrtion of pomengrte wter peel extrct cused significnt decrese in these elevted levels. Serum ure, cretinine nd uric cid decresed y 18 %, 76.59% nd %, respectively s compred to untreted induced renl filure group. Tle 1. Serum ure, cretinine,uric cid (mg/dl) nd lumin (g/l) in different experimentl Prmeters Rt groups Group 1 Group 2 Group 3 Group 4 Group 5 ure Cretinine Uric cid Significnt difference (P < 0.05): compred to: (): to group 1, (): to group 2, (c): to group 3, (d) compred to group 4 ure cretinine uric cid Group5 Group4 Group3 Group2 Group Fig. 1. Serum ure, cretinine,uric cid (mg/dl) nd lumin (g/l) in different experimentl Induction of renl filure y folic cid injection cused significnt increse in serum MDA level. As shown in Tle 2 nd Fig.2 serum MDA level mrkedly incresed in untreted induced renl filure group (6.39 Vs 4.37 μ mol/l). Due to pomengrte peel wter extrct tretment these levels were rought close to tht in the control group. Tle 2. Alumin(g/dl) nd mlondildhyde levels(mda) in serum ( mol/l) in different experimentl groups Prmeters Rt groups Group 1 Group 2 Group 3 Group 4 Group 5 Alumin(g/dl) MDA( mol/l) Significnt difference (P < 0.05): compred to: (): to group 1, (): to group 2, (c): to group 3, (d) compred to group 4 Alumin MDA Group5 Group4 Group3 Group2 Group Fig. 2. Alumin (g/dl) nd mlondildhyde levels(mda) in serum ( mol/l) in different experimentl groups

4 Vol. 3. No. 4. July, 2011, II Prt The results illustrted in Tle 3 nd Fig. 3 demonstrted tht folic cid injection induced prooxidnt stte in kidney. Antioxidnt enzymes (CAT), nd (SOD), ctivities were significntly decresed nd LPO levels s MDA incresed following FA tretment. Orl dministrtion of pomengrte peel wter extrct incresed renl ctlse nd SOD ctivities to e ner the norml levels. Also, renl MDA significntly decresed fter pomengrte peel wter extrct wter dministrtion. Tle 3. Ctlse, SOD, (U/g of kidney homogente), mlondildhyde level kidney( mol/g) in different experimentl groups. Prmeters Rt groups Group 1 Group 2 Group 3 Group 4 Group 5 Ctlse ctivity(u/g of kidney homogente) c c SOD ctivity(u/g of kidney homogente) MDA level in Kidney( mol/g) c c Significnt difference (P < 0.05): compred to: (): to group 1, (): to group 2, (c): to group 3, (d) compred to group 4 Ctlse SOD MDA Group5 Group4 Group3 Group2 Group Fig. 3. Ctlse, SOD, (U/g of kidney homogente), mlondildhyde kidney ( mol/g) in different experimentl groups. Phytochemicl nlysis of some pomengrte peels extrct components re summrized in Tle 4. The results showed tht pomengrte peel extrct contins nturl ntioxidnts such s flvonoids, tnnin, sponin nd sterols. Tle 4. Chemicl constituents of queous extrct of pomengrte peel Chemicl constituents Flvonoid Tnnins Sponin Sterols Aqueous extrct 4. DISCUSSION Oxidtive stress, which involves the production of excessive levels of rective oxygen spieces (ROS) is closely relted to the progression of renl filure [21].. Under norml circumstnce, the kidney genertes rective oxygen species (ROS), including superoxide nions, hydrogen peroxide, peroxynitrite nd hydroxyl rdicl,which re efficiently eliminted y enzymtic (SOD),(CAT), gultthione peroxidse (GPX)- nd non enzymtic systems (glutthione,vitmins C nd E). It hs een shown tht FA exerts its dverse renl effects y genertion of (ROS) which is resulted in sever tissue dmge [3]..Therefore,pomengrte peel with its ntioxidnt nd rective oxygen species scvenger properties my hve the cpcity to prtilly reduce or eleminte the deleterious effects cused y folic cid injection. The result of the current study showed tht intrperitonel injection with folic cid induced cute renl filure in rts. These results re in ccordnce with [22], [23], [24] nd [2]. Folic cid induced renl filure is chrcterized y severe reduction in glomerulr filtertion rte (GFR) which is monitrted y significnt in increse in serum ure,cretinine nd uric cid in folic cid treted group.on the other hnd,dministrtion of pomengrte peel wter extrct y gvge recovered these ltered levels. B k u, A z e r i j n 557

5 Vol. 3. No. 4. July, 2011, II Prt The results of [25] supported the results of current study who reported tht injection with folic cid incresed serum cretinine level to 3.32±0.69 compred to men vlue of 0.28±0.06 mg/dl in control group. Also, the results of [26]. showed tht, intrperitonel injection with FA induced significnt increse in serum ure level. Moreover, [27]. nd [1]. reported tht FA injection cused sttisticlly significnt elevtion of ure nd cretinine. Orl dministrtion of pomengrte peel wter extrct restored serum ure, cretinine nd uric cid ner to control levels indicting their meliorting effect ginst cute renl filure. The present study confirm the pervious finding of [28] reporting tht pomengrte peel extrct reduced the elevted levels of serum ure, cretinine tht re mrker of kidney toxicity. In the present study, the role of ROS in folic cid nephrotoxicity ws ssessed y evlution of ltertions in the iochemicl indictors of oxidtive stress induced y FA or comintion of folic cid plus pomengrte wter peel extrct. These oxidtive stress prmeters include MDA levels, SOD nd CAT ctivities. The results of the current study reveled tht there ws significnt increse in lipid peroxide levels (MDA) in oth serum nd kidney tissue in folic cid treted group compred to control group. On the other hnd, the ctivities of (SOD) nd (CAT) enzymes were gretly reduced in kidney tissue of folic cid treted group compred with control group. This decline in the ntioxidnt sttus suggesting tht oxidtive stress is one of the cuses of folic cid -induced renl dmge. The results this study re coherent with the results of [3] who demonstrted tht the ctivities of ntioxidnt enzymes SOD nd CAT were significntly decresed nd lipid peroxide levels significntly incresed in kidney tissue fter folic cid tretment. Supplementtion with pomengrte peel wter extrct mrkedly enhnced the ctivities of SOD nd CAT enzymes nd reduced the elevted level s of MDA. This improvement in oxidtive stress prmeters indicting tht pomengrte peel extrct countercted the oxidtive stress induced y folic cid injection through its ntioxidnt properties. Consistent with the results otined from the study of [28] suggested tht pomegrte peel extrct improved renl ntioxidnt enzymes ctivities nd decresed renl MDA level in ferric nitrilotricette induced renl filure rts. The results of [29] re similr to the results of this study who confirmed the ntioxidtive ctivity of pomengrte peel extrct in chloropyrofos-ethyl induced oxidtive stress in rts kidney tissue.in ddtion, [30] reported tht dministrtion with different levels of pomegrnte peel powder or pomegrnte peel extrct (1, 2 nd 3%) cused significnt decrese in lipid peroxidtion levels. Phytochemicl nlysis supported these results s the pomengrte peel extrct gve positive tests for tnnins, flvonoid, sponin nd sterols which posses potent ntioxidnt properties. These results re in ccordnce with those reported y [5] which confirmed tht the extrct gve positive result for tnnins, nd flvonoid. Previous studies of [6], [7] nd [8] confirmed the potent ntioxidnt ctivity of pomengrte peel. Moreover,, [31] found tht pomengrte peel hd the highest ntioxidnt ctivity mong the peel, pulp nd seed frctions of 28 kinds of fruits. Also, the results of [32] nd, [10] reported tht the peel in prticulr possesess reltively higher ntioxidnt ctivity thn seeds nd pulp nd therefore might e rich sources of nturl ntioxidnts. 5. CONCLUSION From the results of the current study, it is cler tht pomengrte peel wter extrct ttenuted the renl dmge cused y oxidtive chllenges. 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