Rev. bras. farmacogn. vol.25 número1

w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Combined

administration

of

silymarin

and

vitamin

C

stalls

acetaminophen-mediated

hepatic

oxidative

insults

in

Wistar

rats

Saheed

Sabiu

,

Taofik

O.

Sunmonu

,

Emmanuel

O.

Ajani

,

Taofeek

O.

Ajiboye

a_{Phytomedicine,FoodFactorsandToxicologyResearchLaboratory,BiochemistryUnit,DepartmentofBiosciencesandBiotechnology,KwaraStateUniversity,Malete,Ilorin,Nigeria} b_{PhytomedicineandPlantBiochemistryResearchLaboratory,BiochemistryUnit,DepartmentofBiologicalSciences,Al-HikmahUniversity,Ilorin,Nigeria}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14August2014 Accepted28November2014 Availableonline11February2015

Keywords:

Antioxidants Hepatoprotective Reactivemetabolite Therapeutic Lipidperoxidation

a

b

s

t

r

a

c

t

Oxidativeinsultbyfreeradicalshasbeenimplicatedindrug-inducedhepaticdamageandthishasresulted infrequentepisodesofliverdisorders.Therapeuticefficacyofantioxidantsmayprovideapossible solu-tiontothismenace.Thisstudywascarriedouttoinvestigatetheeffectofcombinedadministration ofsilymarinandvitaminCinrescuingacetaminophen-inducedhepatotoxicityinrats.Hepatotoxicrats wereorallyadministeredwithsilymarinandvitaminCat100and200mg/kgbodyweight,respectively. Attheendoftheexperiment,liverfunctionindices,antioxidantparametersandhistologicalanalysis wereevaluated.Weobservedthatthesignificantlyincreased(p<0.05)activitiesofalkalinephosphatase, alanineaminotransferase,aspartateaminotransferase,aswellaslevelsofthiobarbituricacidreactive substancesandserumtotalbilirubin,weremarkedlyreducedfollowingco-administrationofsilymarin andvitaminC.Thecompoundsalsoeffectivelyreversedthereducedactivitiesofsuperoxidedismutase, catalase,glutathioneperoxidase,glutathioneS-transferaseandtotalproteinconcentrationinthe hep-atotoxicrats.Thesefindingsareindicativeofhepatoprotectiveandantioxidantattributesofthetwo compoundswhicharealsosupportedbythehistologicalanalysis.Theavailableevidencesinthisstudy suggestthatthecomplementaryeffectsofsilymarinandvitaminCprovedtobecapableofameliorating acetaminophen-mediatedhepaticoxidativedamageandtheprobablemechanismisviaantioxidative action.

©2014SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Liver,thekeyorganinmaintenanceofhomeostasisaswellas metabolismandexcretion,hasanoverwhelmingtaskof detoxify-ingxenobioticsandchemotherapeuticagents(Ademuyiwaetal., 1994).Itsroleintransformingandclearingchemicalsrendersit susceptibletodamagefromtheseagents.Generally,thisusually goesunnoticedbecauseoftheconsiderablecapacity of hepato-cytestoregenerate.However,overstressedlivercompromisesits detoxificationrolewhichmayexposeittoavarietyofdiseasesand disorders(Amicetal.,2003).Liverinjurycausedbytoxicchemicals andcertaindrugshasbeenrecognizedasatoxicologicalproblem andlinkedtotheoccurrenceofoxidativestress(Wolfetal.,1997). Freeradicals’generation,arisingfromoxidativestress,isa com-monmechanismunderlyinghepatotoxicitycausedbydeleterious effectofdrugsandtoxicants.Oxidativestresshasalsobeen

impli-∗ Correspondingauthor.

E-mail:sabiu.saheed@kwasu.edu.ng(S.Sabiu).

catedinthepathogenesisofcellulardamagecausedbyanumber oftoxicagentsincludingarsenic,diclofenac,carbontetrachloride, rifampicinandacetaminophen(Deavalletal.,2012).

Acetaminophen(paracetamol)isawidelyusedantipyreticand analgesic which produces acute liver damage if overdoses are ingested.Thedrugismainlymetabolizedinthelivertoexcretable glucuronideandsulphate conjugates(Jollowet al.,1974;Wong etal.,1981).Hepatotoxicitycausedbyparacetamolingestionhas beenattributedtotheformationofahighlyreactivemetabolite,

N-acetyl-p-benzoquinoneimine(NAPQI),bytheactionofhepatic cytochromeP-450(Savidesand Oehne,1983).NAPQIisinitially detoxifiedbyconjugationwithreducedglutathione(GSH)toform mercapturicacid(Mooreetal.,1985).However,whentherateof NAPQIformationexceedstherateofdetoxificationbyGSH,it oxi-dizestissuemacromoleculessuchaslipidsorsulfylhydride(SH) groupofproteinandaltersthehomeostasisofcalciumafter deplet-ingGSH.

Hepatotoxicityisoneofverycommonailmentsresultinginto seriousdebilitiesrangingfromseveremetabolicdisordersto mor-tality(Pateletal.,2008).Agoodnumberofantioxidantshavebeen

http://dx.doi.org/10.1016/j.bjp.2014.11.012

exploitedinthemanagementofliverdisorders.Theseagentsplay animportantroleinscavengingfreeradicalstherebyproviding pro-tectionagainstinfectionsanddegenerativediseases(Subramaniam etal.,2000).SilymarinandvitaminChavereceivedconsiderable attentionovertheyearsduetotheirdiverseantioxidantand hep-atoprotectiveactivities(McDowell,1989;Siesetal.,1992;Burtis andAshwood,1994;Shakeretal.,2010;Tongetal.,2011).

Silymarin(1)isaflavonoidextractedfromtheseedofSilybum marianum(L.)Gaertn.(milkthistleplant)whichisa memberof theasterfamily (Asteraceae).Silybin (silibinin),silychristinand silydininhavebeenidentifiedasitsotheractiveprinciples(Rui, 1991). S. marianum is one of the oldest and most thoroughly researchedplantsinthetreatmentofliverdiseases(Morazzoniand Bombardelli,1995).Seedsoftheplanthavebeenusedformorethan 2000yearstotreatarangeofliverandgallbladderdisorders includ-inghepatitis,cirrhosis andicterus (Morazzoniand Bombardelli, 1995).Itisalsocommonlyusedasinherbaltherapyespeciallyfor treatingliverdiseasespartlyduetoitsantioxidantactivity(Tong etal.,2011).Indrug-andchemically-inducedoxidativestress, sily-marinhasbeenreportedastheprimarytherapeuticmodalityof choice(Ferencietal.,1989;BlázovicsandFehér,2001;Féherand Lengvel,2012).

HO

OH

OH OH

O

1 O

O CH₂OH

OCH₃ O

VitaminCisasix-carboncompoundstructurallyrelatedto glu-cose.Itconsistsoftwo inter-convertiblecompounds:l-ascorbic acid,whichisastrongreducingagent,anditsoxidizedderivative,l -dehydroascorbicacid(EliasandOputiri,2013).VitaminCisfound incitrus,softfruitsandleafygreenvegetableswhilekidneyand liveraregoodanimal-derivedsourcesofthecompound(Stangeland etal.,2008).VitaminC ishydrophilicandexertsitsantioxidant actionbyinhibitinglipidperoxidationandoxidativecelldamage (Xavieretal.,2007).Pathogenicdysfunctionoftissuesowingtocell deathviaapoptosisisoneoftheimportantoutcomesofoxidative stressthatcouldbeamelioratedbyvitaminC(Santosetal.,2009). Erguletal.(2010)havealsoreportedtheeffectofvitaminCon oxidativeliverinjuryinducedbyisoniazidinrats.Theyfoundthat isoniazid-mediatedhepaticonslaughtwasassociatedwith oxida-tivestressandtreatmentwithvitaminCamelioratedliverdamage appreciably.

Accordingly,sincetheattentionoftoday’sclinicalpracticeis focusedonantioxidantconsumptionagainstdrug-induced oxida-tive stress, the present study examined the combined effects ofsilymarinandvitaminConacetaminophen-mediatedhepatic oxidativeinsultinrats.

Materialsandmethods

Chemicalsandreagents

Silymarinwasprocured fromSigma-AldrichChemicals Com-pany(St.Louis,Mo,USA).ParacetamolandvitaminCwereproducts of EmzorPharmaceuticals, Lagos,Nigeria. Assay kitswere pur-chased fromRandox Laboratories limited,United Kingdom and Sigma-AldrichChemicalsCompany(St.Louis,Mo,USA).Distilled waterwasobtainedfromBiochemistryLaboratory,KwaraState University,Malete,Ilorin,Nigeria.Otherchemicalsandreagents wereofanalyticalgrade.

Experimentalanimals

Wistarstrainalbinoratswithameanweightof180.00±2.33g wereobtainedfromtheAnimalHouseofAl-HikmahUniversity, Ilorin,Nigeria. The animalswerekeptin clean metaboliccages placedinawell-ventilatedroomwithoptimumcondition (tem-perature:23±1◦_{C,photoperiod:12hnaturallightand12hdark,} relative humidity: 45–50%). They were acclimatized to animal house conditionsfor ten daysand were allowedfree accessto foodandwateradlibitum.Theresearchwascarriedoutfollowing approvalfromtheEthicalcommitteeontheuseofLaboratory Ani-malsofAl-HikmahUniversity,Ilorin,Nigeria.Aclearancenumber HUI/ECULA/014/04/002wasassignedandissuedfortheresearch.

Inductionofliverdamage

Hepatotoxicity(liver damage)wasinduced inratsaccording totheproceduredescribed byKanchanaand Mohamed(2011). Briefly,theanimalswereorallyadministeredwith400mg/kgbody weight(b.w.)ofacetaminophenoncedailyforsevendays.Feedand waterweremadeavailabletotheanimalsadlibitumthroughoutthe inductionperiod.

Animalgroupingandtreatments

Thirtyalbinoratswererandomizedintofivegroupsofsixrats each.Group1animalsservedasnormalcontrolandweregiven distilledwater.Group2comprisedanimalsinducedwithliver dam-agebutnottreated.Animalsingroups3–5werehepatotoxicrats administeredwiththerapeuticdosesofsilymarin(200mg/kgb.w.), vitaminC(200mg/kgb.w.)andbothcompoundsco-administered (100mg/kgb.w.each)respectively(Mongietal.,2011;Jánosand Gabriella,2012;SabzevarizadehandNajafzadeh,2012;Santhrani etal.,2012).Alladministrationsweredoneoncedailyforsevendays usingoralintubatorwithadlibitumprovisionoffoodandwater throughouttheexperimentalperiod.

Preparationofserumandexcisionofliver

Twenty-four hours after the last treatment, the rats were humanelysacrificedbydiethyletheranaesthetization.Bloodwas collectedbycardiacpunctureintocentrifugetubesandallowedto stayfor20minbeforecentrifugingat3000×gfor15minusinga benchcentrifuge(BeckmanandHirsch,Burlington,IO,USA).Serum wascarefullyaspiratedandusedforliverfunctiontests.Theliver wasexcised,cleanedoffatandslicedintotwoportions.Aportionof theliverwashomogenizedinTris–HClbuffer(0.05mol/lTris–HCl and1.15%KCl,pH7.4)forantioxidantanalyses,whiletheother portionwasfixedinsalineformaldehydesolutionforhistological examination.

Liverfunctionindices,antioxidantanalysesandhistopathological examination

lipidperoxidationmeasuredintermsofthiobarbituricacid reac-tivesubstances(TBARS)wasdeterminedintheliverhomogenate. Histopathologicalexaminationoftheliverwascarriedoutusing themethodofBancroftandStevens(1990).

Statisticalanalysis

All data were subjected to one-way analysis of variance (ANOVA)usingSPSSsoftwarepackageforwindows(Version16) andexpressedasmean(X)±standarderrorofmean(SEM)(n=6). Significantdifferencebetweenthetreatmentmeanswas deter-minedat5%confidencelevelusingDuncan’sMultipleRangeTest.

Results

Liverfunctiontests

Table1shows theeffectsof silymarinand vitaminC onthe activitiesofserumALP,ALT,ASTaswellasconcentrationsoftotal bilirubinandtotalproteinintheexperimentalrats.Oral adminis-trationof400mg/kgb.w.ofacetaminophenforsevendayscaused asignificant(p<0.05)increaseintheactivitiesoftheseenzymes andleveloftotalbilirubinaswellassignificantreduction(p<0.05) in the concentrationof total protein when compared withthe normal control. Theelevatedactivities of theassayedenzymes andtotalbilirubinconcentrationinducedbyacetaminophenwere significantlyattenuated(p<0.05)followingtreatmentwitheither silymarin/vitaminCor both.Thereduced concentrationof total proteinwasalsosignificantlyincreased(p<0.05)aftertreatment witheithersilymarin/vitaminC orbothcompounds.Theeffects werehowevermorepronouncedintheratswhollytreatedwith vitaminCandthoseco-administeredwithbothsilymarinand vita-minC.

Antioxidantanalyses

TheeffectsofsilymarinandvitaminContheantioxidant sta-tusofratliverarepresentedinTable2.TBARSwassignificantly increased(p<0.05)inthehepatotoxicrats.Separateandcombined treatments with silymarin and vitamin C significantly reduced (p<0.05)thelevelofTBARScomparabletonormal.Asignificant reduction(p<0.05)wasalsoobservedintheactivitiesofSOD,CAT, GPxandGSTinliverofhepatotoxicratswhencomparedwith nor-malcontrol.Co-treatmentwithsilymarinandvitaminCresulted insignificantincrease(p<0.05)intheactivitiesoftheseenzymes whichwascomparabletonormal.

Histopathologicalanalysis

ThelivermicrographsoftheratsarepresentedinFig.1.The liverofthecontrol ratsshowednormal architecturewith well-preservedcordsofhepatocytes,well-demarcatedsinusoidsandno areaofinfiltrationbyinflammatorycells(Fig.1A).Thisisin con-trasttothefeaturesobservedintheliverofhepatotoxicrats.There weredrasticalterationsinliverarchitecturerangingfromextensive fattychange,distendedhepatocytes,vacuolatedcytoplasm, com-pressedsinusoids,fattydegenerationareaofnecrosis,toinfiltration byinflammatorycells(Fig.1B).However,thelivermicrographsof ratstreatedwithsilymarin/vitaminCorbothshoweddistinctand essentiallynormalcordsofhepatocytewithnon-prominentfatty change(Fig.1C–E).

Discussion

Drug-inducedliverdisorders occurredfrequentlyand canbe life threatening. Oxidative stress occasioned by highly reactive intermediates(freeradicals)hasbeenlinkedto acetaminophen-mediated hepatotoxicity in rats (Balamurugan, 2007). The catastrophic freeradical eventssuchas lipidperoxidation, pro-tein oxidation and DNA oxidation are rarely the cause of cell deathinrealisticinvivocondition.Thisisbecausetheantioxidant defensearsenal inlivercellsiscapableofdetoxifyingfree radi-calsandrepairdamageresultingfromhighlyreactivemetabolites (Jaeschke etal., 2003).However, whenthe antioxidantdefense systemisoverwhelmed,freeradicalsmayinflictdirectoxidative damagetocellularmacromolecules,leadingtocelldeath(Sabiu etal.,2014).Timelyinterventionwithexogenousantioxidants aug-mentsthecellulardefensesystemtopreventtheseilleffectson cellularmacromolecules.Studieshavereportedtheantioxidants andcytoprotectiveactivitiesofsilymarinandvitaminC(Crocenzi et al., 2003; Santos et al., 2009; Mor and Ozmen, 2010). This study,thus, demonstratestheantioxidant andhepatoprotective potentialsofsilymarinandvitaminCinacetaminophen-mediated hepaticoxidativedamageinrats.

IncreasedactivitiesofserumAST,ALTandALPareindicativeof cellularleakageandlossoffunctionalintegrityoflivercell mem-brane.Theextentofdrug-inducedhepatotoxicityisassessedbythe release oftheseintracellularenzymesviathehepatocyte mem-braneintocirculation (Sabiuetal.,2014).Specifically,increased activityofASTisindicativeofliverdamageduetoviralhepatitis, cardiacinfarctionandmuscleinjury.ALTismorespecifictotheliver andthusabetterparameterforassessingliverinjury(Willianson etal.,1996)whileserumALPactivitygivesacluetothe function-ality ofthehepatocytes.ElevatedactivityofserumALP maybe attributedtoincreasedsynthesisinthefaceofincreasingbiliary pressure(GiniandMuraleedhara,2010).Inthepresentstudy, ele-vatedactivitiesofAST,ALTandALPinacetaminophen-treatedrats

Table1

EffectsofsilymarinandvitaminConliverfunctionindicesofacetaminophen-inducedhepatotoxicrats(n=6,X±SEM).

Treatments AST(U/L) ALT(U/L) ALP(U/L) Totalbilirubin(mg/dl) Totalprotein(g/dl)

Control 85.36±4.51a _47.15±2.35a _75.23±5.31a _0.92±0.06a _9.32±0.32a

Acetaminophen induced

147.81±9.20b _160.28±4.52b _126.21±3.36b _1.98±0.09b _5.46±0.06b

Acetaminophen induced+silymarin

110.17±5.32c _63.65

±2.25c _90.32

±2.63c _0.70

±0.04c _7.75

±0.32c

Acetaminophen

induced+vitamin

C

100.36±4.63a _60.87

±3.19a _70.25

±9.61a _0.80

±0.05a _8.99

±0.15a

Acetaminophen

induced+silymarin+vitamin C

90.04±4.68a _55.39±2.86a _73.61.±2.92a _0.88±0.02a _9.25±0.35a

Table2

EffectsofsilymarinandvitaminConantioxidantstatusofacetaminophen-inducedhepatotoxicrats(n=6,X±SEM).

Treatments TBARS(mM/100gtissue) SOD(U/mgprotein) CAT(U/mgprotein) GPX(U/mgprotein) GST(U/mgprotein)

Control 14.64±1.75a _7.25

±0.90a _3.98

±0.35a _18.56

±0.31a _1.02

±0.05a

Acetaminopheninduced 18.80±1.55b _5.65±0.93b _2.18±0.25b _16.42±0.79b _0.59±0.03b

Acetaminophen induced+silymarin

15.30±1.75a _6.69±0.52a _2.99±0.23a _17.48±0.30a _0.82±0.02a

Acetaminophen

induced+vitaminC

14.01±1.63a _6.99

±0.32a _3.55

±0.97a _18.27

±0.08a _0.92

±0.02a

Acetaminophen induced+silymarin

+vitaminC

14.52±1.09a _7.20

±0.59a _3.81

±0.05a _18.48

±0.21a _0.99

±0.02a

Valueswithdifferentsuperscriptsalongthesamecolumnforeachparameteraresignificantlydifferent(p<0.05).

TBARS,thiobarbituricacidreactivesubstances;SOD,superoxidedismutase;CAT,catalase;GPX,glutathioneperoxidase;GST,glutathioneS-transferase.

AST,aspartateaminotransferase;ALT,alanineaminotransferase;ALP,alkalinephosphatase.

maybeanindicationofliverdamageandcellnecrosisresulting fromformationofNAPQIinexcessofGSHdetoxificationcapacity. ThisagreeswithearlierreportsbyBalamurugan(2007),Giniand Muraleedhara(2010),andKanchanaandMohamed(2011).These authorsopinedthatoverdoseofacetaminophencouldbetoxicto thehepatocytes.Conversely,thesignificantreductioninenzyme activitiesofratstreatedwithsilymarinandvitaminCsuggeststhat bothcompoundswereabletoamelioratethedeleteriouseffectsof acetaminophen.Theeffectsweremorepronouncedinthevitamin C-treatedratsaswellasthoseco-treatedwithbothsilymarinand vitaminC.

The serum levels of total protein and bilirubin may indi-catethestateoftheliverand thetype ofdamage (Sabiu etal., 2014).Hypoproteinemiaisafeatureofliverdamagewhichmay be attributed to a decrease in protein synthesis (Oloyede and Sunmonu, 2009). In this study, hypoproteinemia was observed in the acetaminophen-treated rats and may be a consequence ofimpairedhepato-cellular functions.In addition,theobserved hyperbilirubinemia may be due to excessive heme destruction andblockageofbiliarytractinacetaminophen-treatedrats.This obstructionmighthaveresultedtomassinhibitionofconjugation reactionandreleaseofunconjugatedbilirubinfromdamagedand deadhepatocytes(Wolfetal.,1997).Thisagreeswiththeearlier reportsbyAjiboyeetal.(2010),KanchanaandMohamed(2011),

andChinnasamyetal.(2011)whereacetaminophenwasreported tohavecausedalterationinserumconcentrationsoftotal biliru-binandtotalprotein.Co-treatmentswithsilymarinandvitamin C,however,reducedthelevelofbilirubin andincreasedprotein concentrationsuggestingthattheyofferedconsiderablelevelof hepatoprotectionatthetestedregimen.

Oxidativeinsulttoacellinducesperoxidationof membrane-bound lipids whose toxic products cause damage of macro-molecules.In thepresent study,the increasedconcentrationof TBARSintheliverofhepatotoxicratsissuggestiveoffacilitatedlipid peroxidationleadingtotissuedamageandfailureofbody’s antiox-idantdefensemechanismstopreventformationofexcessivefree radicals.Ithasbeenreportedthatacetaminophencaused signifi-cantincreaseinhepaticlipidperoxidationduetofreeradicalinjury innecroticliversofrats(GiniandMuraleedhara,2010).Reactive oxygenspeciesinconjunctionwithNAPQIarerequiredtoinitiate lipidperoxidationwhichhasbeenopinedasanimportant initia-tioneventinthetoxicitymechanismofacetaminophen(Thabrew etal.,1987).ThesignificantlyreducedconcentrationofTBARSinthe liverofsilymarinandvitaminC-treatedratsindicatestheirpossible antiperoxidativeattributeandthusantioxidativepotential.

Thebodyisendowedwithaneffectivemechanismtocounter theravagingeffectoffreeradical-induceddamage.Thisisattained

viaendogenousantioxidantenzymes,suchasSOD,CAT,GPXand

a

b

c

d

e

GST.Animbalancebetweenfreeradicalsproductionand antiox-idant defense system resultsin oxidative stress which further deregulatescellularfunctionsleadingtovariouspathological con-ditions.Inthepresentstudy,thereducedactivityofantioxidant enzymesinacetaminophen-treatedratsisan obviousreflection ofexcessiveformationoffreeradicalsresultingintissuedamage. However,thesignificantincreaseintheiractivitiesfollowing treat-mentwithsilymarinandvitaminCisanindicationofantioxidant effect.

Inthepresentstudy,theprotectionofferedbysilymarinagainst acetaminophen-inducedhepatotoxicity maybegenerallylinked toitsbeneficialattributesasrevealedbyValenzuelaandGarride (1994).Theseincludeitsabilitytoscavengefreeradicals,increase cellularGSHcontentand regulate membranepermeability. The authorsalsoreportedthatsilymarinhasthecapacitytoregulate nuclearexpressionbymeansofasteroid-likeeffect.

Similarly,theabilityofvitaminCtotrapfreeradicals,protect biomembranes fromperoxide damage and effectively scavenge reactive oxygen species as reported by Sminorff and Wheeler (2000)maybesuggestiveofitseffectexhibitedinthetreatment groups.Bendich(1990)hasalsoreportedvitaminCasanexcellent electrondonortofreeradicalswhichsubsequentlyquenchtheir deleteriousactivityoncellularmacromolecules,thusplayingarole inantioxidantmechanism.Inaddition,the200mg/kgb.w.of vita-minCexploitedinthisstudymaybesufficientenoughtocontribute toitstherapeuticpotentialexhibitedinthetreatedrats.Thisagrees withthereportofMongietal.(2011)wherevitaminCwasreported tohaveofferedprotectionagainstbiochemicaltoxicityinducedby deltamethrininmaleWistarrats.This,perhaps,couldalsobea ten-ablefactfortheratswhollyadministeredwithvitaminCandthose co-administeredwithsilymarinforhavingpronounced hepatopro-tectiveandantioxidantpotentialscomparedtoratstreatedwith onlysilymarin.Thissubmissionisinconformitywiththereportof SabzevarizadehandNajafzadeh(2012)thatvitaminCmodulates myoglobinurichepaticfailurebetterthansilymarinbybindingto variousharmfulsubstancesinrats.

Anotherimportantconsideration in assessing theefficacyof potentialtherapeuticagentsagainsthepaticinjuryistheireffect onhistology.Theeffectsaremanifestationsofinflammatoryinsult ontheliverandoftencomplementenzymeanalysis(Adesokanand Akanji,2007).Theapparentlyannulleddegenerativethreatsposed byacetaminophenonthearchitecturalfeaturesofhepatocytesin theratswhollytreatedwithvitaminCandincombinationwith silymarinsuggestthatthetwocompoundsconferredareasonable levelofintegrityontheliver.Infact,architecturalorganizationof someofthehepatocyteswasalmostcompletelyrestoredto nor-mal.Theeffects noticedwereinconsonancewiththeresultsof biochemical analysisobtainedand in agreementwiththe find-ingofBalamurugan(2007),whererecoverytowardsnormalization ofserum enzymesand liverhistologicalarchitecturecaused by acetaminophenwereattributedtoantioxidantagents.

Conclusively,therestoration ofdegenerative insultsinflicted by 400mg/kg b.w. acetaminophen by co-administration with silymarinandvitaminCisanindicationoftheirinherent hepato-protectiveandantioxidantattributesinrats.Though,theeffects wereprominentlyexhibited byvitaminC, theircomplementary efficacy is formidable and thus recommended against hepatic oxidativedamage.

Authors’contributions

SS and SOT designed the study and were involved in the manuscript preparation. AOT evaluated the compounds in acetaminophen-treated rats. SS, AOE and SOT performed

biochemicalestimationsandhistopathology.Allauthorsreadand approvedthefinalmanuscript.

Conflictofinterest

Allauthorshavenothingtodeclare.

Acknowledgements

TheauthorsareimmenselythankfultotheBiochemistryUnits ofKwaraStateUniversity,Malete,Ilorin,NigeriaandAl-Hikmah University,Ilorin,Nigeriaforprovidingamplesupportandaccess toresearchfacilities.

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