Protective role of Zingiber officinale Roscoe on Aceclofenac induced oxidative stress in rat liver

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1 Interntionl Journl of PhrmTeh Reserh CODEN (USA): IJPRIF ISSN : Vol.2, No.1, pp , Jn-Mr 21 Protetive role of Zingier offiinle Rosoe on Aelofen indued oxidtive stress in rt liver Drr S, Bose A, Chttrj T K, Pl T K* Bioequivlene Study Centre, Deprtment of Phrmeutil Tehnology, Jdvpur University, Kolkt-732, West Bengl, Indi * E-mil: tkpl12@gmil.om/drr.soumen@gmil.om Phone: , ABSTRACT: Oxidtive stress due to norml prodution of retive oxygen moleules (ROM) is elieved to e involved in the etiology of toxiities of mny xenoiotis. In this study protetive effet of Zingier offiinle (ZO) ginst Aelofen (ACE) indued oxidtive stress in rt liver ws evluted. Twenty four young wistr rts were divided into four groups s follows: ontrol group, ACE (single dose of 1 mg/kg ody wt., i.p), ACE plus ZO (25 mg/kg) nd ACE plus ZO (5mg/kg). The tivities of ntioxidnt enzymes suh s superoxide dismutse (SOD), tlse (CAT), redued glutthione (GSH), glutthione peroxidse (GPx) nd glutthione S-trnsferse (GST) (p<.1) s well s the onentrtion of mlondildehyde, s n inditor of lipid peroxidtion, were mesured to evlute oxidtive stress in homogentes of the liver. ACE dministrtion inresed mlondildehyde levels (p<.1) nd deresed SOD, CAT, GSH, GPx nd GST tivities. Co-dministrtion of ZO s dose dependent mnner (25 & 5 mg/kg) with ACE injetions used signifintly deresed mlondildehyde levels s well s elevtes SOD, CAT, GSH, GPx nd GST (p<.5 & p<.1) tivities in liver tissue when ompred with ACE lone. It n e onluded tht Zingier offiinle my prevent ACE-indued oxidtive hnges in liver y reduing retive oxygen speies. Key words: Aelofen, Oxidtive stress, Zingier offiinle INTRODUCTION Retive oxygen speies (ROS) re inevitly generted, due to the inomplete redution of O 2 in eletron trnsfer retions, s yproduts of iologil retions. When ROS prodution is greter thn the detoxifition pity of the ell, exessively generted ROS uses extensive dmge to DNA, proteins nd lipids nd ts s meditor of proinflmmtory nd rinogeni events. 1 To void redox imlne nd oxidtive dmge, eroi orgnisms possess effiient iohemil defense systems suh s superoxide dismutse (SOD), tlse (CAT) nd glutthione peroxidse (GPx) 2, though it nnot ompletely protet them from severe oxidtive stress. In this ontext, mny sientists hve tried to otin dietry ntioxidnts suh s sorte, toopherol nd rotenoids from fruits nd vegetles, euse they ould help protet ells from ellulr dmge used y oxidtive stress. Non-steroidl nti-inflmmtory drugs (NSAIDs) re the enterpiee of phrmotherpy for most rheumtologil disorders, nd re used in lrge numers s nlgesis nd ntipyretis, oth s presription drugs nd over the ounter purhses (OCT). 3 They re the most frequently used meditions for the tretment of vriety of ommon hroni nd ute inflmmtory onditions, nd ontinue to e importnt for the pllition of pin nd in deresing inflmmtion nd fever. 4-8 Nerly ll the NSAIDs hve een implited in using liver injury, nd tend to e heptoellulr in nture: the mehnism is thought to e immunologil idiosynrsy. 9,1 Reently, numer of in vitro niml models hve een used to investigte the NSAID-relted heptotoxiity. 11,12 Aelofn is ommon nd well estlished NSAID whih is hemilly designted s [{2-(2, 6 - dihlorophenyl ) mino } phenyletoxyerti id], phenyl eti id derivtive. The mode of tion of elofen hs een reently lrified in tht the ompound ws shown to eliit preferentil inhiition of COX2 s result of limited ut sustined

2 Pl T K et l /Int.J. PhrmTeh Res.21,2(1) 496 iotrnsformtion to dilofen. 13 Aelofen indued heptotoxixity is lmost lwys dngerous due to the dignosti onfusion It hs een well known tht elofen hve sever toxi effet on GI trt ut no extensive reserh done till now. Nturl produts nd their tive priniples s soures for new drug disovery nd tretment of diseses hve ttrted ttention in reent yers. Hers nd spies re generlly onsidered sfe nd proved to e effetive ginst vrious humn ilments. Their mediinl use hs een grdully inresing in developed ountries. Zingier offiinle Rosoe, ommonly known s ginger, is one of the ommonly used spies in Indi nd round the world. Ginger is exmple of otnils whih is gining populrity mongst modern physiins nd its underground rhizomes re the mediinlly nd wlinry useful prt. 18 Mny studies were rried out on ginger nd its pungent onstituents, fresh nd dried rhizome. Among the phrmologil effets demonstrted re nti-pltlet, ntioxidnt, nti-tumour, nti- rhinovirl, ntiheptotoxiity nd nti rthriti effet The im of this work ws to estlish the protetive role of Zingier offiinle Rosoe on elofen indued oxidtive stress in rt liver. MATERIALS AND METHODS Drugs nd Chemils Aelofen ws otined from Dey s Medil Stores (Mfg.) Ltd., 62, Bondel rod, Kolkt-719, Indi. All other regents used for the experiments were of nlytil grde. Animls Twenty four dult mle white Wister lino rts, weighing 18-2g were used s experimentl nimls in this study. The nimls were housed in the nimls re entre of fulty of Phrmy, Jdvpur University. They were kept in wire-floored ges under stndrd lortory onditions of 12h/12h light/drk yle, 25 ±2ºC with free ess to food nd wter d liitum. The experiment ws rried out ording to the guidelines of the Committee for the Purpose of Control nd Supervision of Experiments on Animls (CPCSEA), Chenni, Indi nd pproved y the Institutionl Animl Ethis Committee (IAEC) of Jdvpur University. Preprtion of the plnt extrt Rhizome of Z. offiinle ws purhsed from the lol mrket. The rhizome (5 g) were ut into smll piees nd homogenized in kithen mixer using 5% ethnol (w/v). The homogente ws kept on wter th t 7 8 C for 1-15 h with intermittent shking. The homogente ws entrifuged t 15g for 1 min nd the superntnt ws olleted. Solvent in the pooled superntnt ws ompletely evported t low temperture using wter th. The residue thus otined (6.5 g, w/w) ws used for the experiment. 22 Study design Young helthy twenty four mle wistr lino rts weighing 18-2 g were divided into 4 groups eh ontining 6 nimls. Group 1 Norml (supply stndrd lortory food nd wter d liitum ), Group 2 (Control) treted with ACE (single dose of 1 mg/kg, i.p.), Group 3, 4 reeived ZO 25 nd 5 mg/kg/dy orlly long with ACE. Totl Preprtion of tissue homogente After the experimentl period ll the nimls were srified nd liver tissue ws wshed with ie-old sline. The tissue were then ut into frgments nd homogenized with 3 volumes (w/v) of the pproprite uffer using Potter-Elvehjm homogenizer with Teflon pestle nd entrifuged t 12 g for 2 min t 4º C. The superntnt ws used for the iohemil estimtions. Determintion of lipid peroxidtion Lipid peroxidtion ws determined y mesuring thiorituri id retive sustnes (TBARS) in the tissues using the method of Ohkw et l. 23 The pink hromogen produed y the retion of seondry produts of lipid peroxidtion suh s mlondildehyde (MDA) with thiorituri id ws estimted t 532 nm. Determintion of ntioxidnt enzymes SOD ws ssyed ording to the method of Kkkr et l. 24 The ssy ws sed on the 5% inhiition of the formtion of NADH-phenzine methosulfte nitrolue tetrzolium formzn t 52 nm. The tivity of tlse ws ssyed ording to the method of Sinh sed on the onversion of dihromte in eti id to perhromi id nd then to hromi ette, when heted in the presene of H 2 O 2. The hromi ette formed ws mesured t 62 nm. 25 Redued glutthione (GSH) in the tissue ws ssyed ording to the method of Ellmn. 26 GSH estimtion ws sed on the development of yellow olour when 5.5-dithioisnitro enzoi id ws dded to ompounds ontining sulfydryl groups. Hepti GST tivity ws ssyed ording to the method of Hig et. l. (1984) with some modifitions. 27 GPx tivity ws determined y mesuring the derese in GSH ontent fter inuting the smple in the presene of H 2 O 2 nd NN 3 (Hfemnn et. l. 1974) [28]. Protein ws estimted y the method of Lowry et l. using ovine serum lumin s the stndrd. 29 Sttistil nlysis Dt were expressed s men ± SD, nd ll sttistil omprisons were mde y mens of two-wy nlysis of vrine (ANOVA) followed y Dunnett s post ho test. SPSS 1. softwre (SPSS In, 1999) ws used for sttistil nlysis. The Differene showing P level of.5 or lower ws onsidered to sttistilly signifint.

3 Pl T K et l /Int.J. PhrmTeh Res.21,2(1) 497 RESULTS Effet of ZO on hepti lipid peroxidtion The lipid peroxidtion (LPO) level in liver ws signifintly inresed (p<.1) in ACE-treted nimls when ompred to norml. Tretment with ZO t 5 mg/kg showed signifint (p<.1) derese in LPO level when ompred to ACE-treted groups. Tretment with ZO t 25 mg/kg showed less signifint (p<.5) derese in LPO level when ompred to ACE-treted groups (Fig. 1). Effet of ZO on ntioxidnt enzymes The SOD nd CAT tivity in the liver tissue of norml nd experimentl nimls were given in Fig. 2 & 3. SOD nd CAT tivity in liver of rts treted with ACE (group 2) were signifintly (p<.1) lowered ompred with norml rts (group 1), wheres dministering ZO to ACE-treted rts (group 3 & 4) signifintly elevted their tivity ompred with ACE tretment lone (group 2). The tivities of GSH nd glutthione-relted enzymes suh s GPx, nd GST in the liver tissue of oth the norml nd experimentl nimls re given in Fig The levels/tivities of GSH, GPx, nd GST were signifintly lowered in the liver of nimls treted with ACE (group 2) ompred with the norml rts (group 1). ZO t dose of 5 mg/kg together with ACE tretment signifintly (p<.1) elevted the tivities of GSH, Gpx, nd GST in liver ompred with those of the unsupplemented ACE-treted rts (group 2). Tretment with ZO t 25 mg/kg showed less signifint (p<.5) inrese in GSH, GPx, nd GST level when ompred to ACE-treted groups. DISCUSSION Liver is the min orgn responsile for drug metolism nd ppers to e sensitive trget site for sustnes modulting iotrnsformtion. 3 During the ourse of eroi metolism, onsiderle mounts of retive oxygen speies (ROS) suh s superoxide nion (O 2 - ) nd hydrogen peroxide (H 2 O 2 ) re generted 31, whih undergo vriety of hin retions nd produe free rdils suh s OH. These highly retive speies ttk polyunsturted ftty ids nd therey initite the proess of lipid peroxidtion. 32 Resulting in oxygen dmge whih n further e mnifested to degrdtion nd intivtion of vrious importnt iomoleules. Lipid peroxidtion, retive oxygen speiesmedited mehnism, hs een implited in the pthogenesis of vrious liver injuries nd susequent liver firogenesis in experimentl nimls nd humns. 33 MDA is mjor retive ldehyde tht ppers during the peroxidtion of iologil memrne polyunsturted ftty id (PUFA). 34 Therefore, the hepti ontent of MDA is used s n inditor of liver tissue dmge involving series of hin retions. 23 In the present study, single dose of ACE t 1 mg/kg, resulted in signifint inrese in the hepti MDA onentrtion, inditing inresed lipid peroxidtion used y dministrtion of ACE (Fig. 1). The signifint dose dependent derese in the hepti MDA onentrtion onfirms tht pretretment with ZO ould effetively protet ginst the hepti lipid peroxidtion indued y ACE. SOD is uiquitous hin-reking ntioxidnt found in ll eroi orgnisms. It is metlloprotein widely distriuted in ll ells nd plys n importnt protetive role ginst oxidtive dmge indued y retive oxygen speies. SOD onverts superoxide ion (O - 2 ) to hydrogen peroxide (H 2 O 2 ) nd the hydrogen peroxide thus formed is degrded y CAT nd GPx. CAT is present in ll mjor ody orgns of nimls nd humns nd is espeilly onentrted in the liver. 35,36 The tivities of SOD nd CAT were signifintly lowered in ACE treted rts s ompred with those of the norml rts. ZO supplementtion to the ACE-treted group elevted the SOD nd CAT tivity in the liver emphsizing the ntioxidnt nd heptoprotetive tivities of Z. offiinle. GSH is tripeptide (L-γ-glutmylysteinylglyine), n ntioxidnt nd powerful nuleophile, ritil for ellulr protetion suh s detoxifition from retive oxygen speies, onjugtion nd exretion of toxi moleules, nd ontrol of the inflmmtory ytokine sde. 37 Depletion of GSH in tissues leds to impirment of the ellulr defene ginst retive oxygen speies, nd my result in peroxidtive injury. The levels of GSH were signifintly deresed in ACE-treted rts. Moreover, in ddition to eing diret free rdil svenger, GSH is known to funtion s sustrte for GPx nd GST. The tivities of GPx nd GST in this study were redued on ACE tretment, whih my e ttriuted to the unvilility of GSH. Administrtion of ZO to ACEtreted rts inresed the levels of GSH nd the tivities of GPx nd GST. CONCLUSION On the whole, it n e onluded tht Z. offiinle my protet from free rdil indued oxidtive injury used y elofen indued heptotoxiity in niml model.

4 Pl T K et l /Int.J. PhrmTeh Res.21,2(1) M DA (m iro m o le/m g protein ) Norml ACE ACE & ZO 25 ACE & ZO 5 Fig. 1. Effet of ethnol extrt Z. offiinle (ZO) on hepti MDA level in rt treted with Aelofen (ACE). Vlues re men ± S.D., n=6 nimls. p<.1 signifintly different from norml group. p<.5, p<.1 signifintly different from ACE group. 1.6 SOD (unit/mg protein) Norml ACE ACE & ZO 25 ACE & ZO 5 Fig. 2. Effet of ethnol extrt Z. offiinle (ZO) on hepti SOD tivity in rt treted with Aelofen (ACE). Vlues re men ± S.D., n=6 nimls. p<.1 signifintly different from norml group. p<.5, p<.1 signifintly different from ACE group. 6 CAT (unit/mg protein) Norml ACE ACE & ZO 25 ACE & ZO 5 Fig. 3. Effet of ethnol extrt Z. offiinle (ZO) on hepti CAT tivity in rt treted with Aelofen (ACE). Vlues re men ± S.D., n=6 nimls. p<.1 signifintly different from norml group. p<.5, p<.1 signifintly different from ACE group.

5 Pl T K et l /Int.J. PhrmTeh Res.21,2(1) 499 G SH (m iro m ole/m g p rotein) Norml ACE ACE & ZO 25 ACE & ZO 5 Fig. 4. Effet of ethnol extrt Z. offiinle (ZO) on hepti GSH onentrtion in rt treted with Aelofen (ACE). Vlues re men ± S.D., n=6 nimls. p<.1 signifintly different from norml group. p<.5, p<.1 signifintly different from ACE group. 5 GPx (Units/mg protein) Norml ACE ACE & ZO 25 ACE & ZO 5 Fig. 5. Effet of ethnol extrt Z. offiinle (ZO) on hepti GPx tivity in rt treted with Aelofen (ACE). Vlues re men ± S.D., n=6 nimls. p<.1 signifintly different from norml group. p<.5, p<.1 signifintly different from ACE group. 4 GST (miromole/mg protein) Norml ACE ACE & ZO 25 ACE & ZO 5 Fig. 6. Effet of ethnol extrt Z. offiinle (ZO) on hepti GST tivity in rt treted with Aelofen (ACE). Vlues re men ± S.D., n=6 nimls. p<.1 signifintly different from norml group. p<.5, p<.1 signifintly different from ACE group.

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