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Evaluation

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EvaluationEvalua

    Evaluation

May2008,Volume2,No.5(SerialNo.6)JournalofLifeSciences,ISSN1934-7391,USA

    ;EvaluationofantioxidantactivityofmarineAgrobacteriumSP.1202 ;YANXue-fen,HUANGDanhong,ZHENGZhonghui,SONGSiyang,ZHANGLi

    anru

    ;(SchoolofLifeSciences,XiamenUniversity,XiamenFujian361005,China) ;Abstract:Thethreewater.solubleDolvsaccharides(PI,PIIand ;PIII),theethylacetateextraction(EA)anditsfivefractionsof ;petroleumether(PeF),ethylacetate(EaF),butanol(BuF), ;methanol(MeF)andwater(WtF)frommarineagrobacterium ;1202werepreparedandthensubjectedtoantioxidantactivity ;evaluation.Theantioxidantactivitywastestedusingthe1. ;1-Diphenyl2picrylhydrazyl(DPPH)freeradicalscavenging

    ;assay,thehydrogenperoxideinducedluminot

    ;chemiluminescence(CL)assayandhydroxylfree

    ;radicalinitiatedchemiluminescence(CL)assay.Allthe ;fractionsexe~edsignificantinhibitoryeffectsonhydrogen ;peroxideandhydroxylfleeradica1.TheextractsofEA.EaFand ;BuFcouldalsoshowedinhibitiononDPPH.Amongofthem. ;theextractsofEaFshowedfuUofantioxidantactivityonthe ;threetestedsystem.Theresultsuggestedthatthemarine ;agrobacterium1202isapotentialsourceofnaturalantioxidant ;agent.

    ;Keywords:antioxidantactivities;DPPHf1.

    ;1Diphenyl2picrylhydrazy1);chemiluminescence

    ;1.Introduction

    ;Relativetoterrestrialstrains,marine

    ;microorganismsyieldsignificantlybetterratioof ;structurallynoveltoknownandbiologicallyactive ;secondarymetabolitesinthespecificmarineconditions ;suchashighpressure,highsaltandhighpervasion,et ;al;symbiontsorthoseinhabitingextreme

    ;environments,andthismightbeusefulinthe

    ;developmentofnewpharmaceuticalagentsl-21.

    ;Allaerobicorganismsrequireprotectionagainst ;deleteriousreactiveoxygenspecies(ROS)suchas ;hydrogenperoxideandsuperoxideanions,produced ;fromuncouplingofelectronsatmetabolicand

    ;phOtOsynthetictransfersites,orbytheproductionof ;ZHANGLianru(1964),female,Ph.D.,associateprofessor;

;researchfields:naturalproducts,polysaccharides,proteinand

    ;smallmoleculeinteraction.

    ;singletoxygenandsuper

    ;biOmolecules[-.

    ;Therole

    ;oxidefromphotoexcited

    ;ofexcessivefreeradical

    ;productionandlipidperoxidationisbecoming ;increasinglyrecognizedinthepathogenesisofawide ;numberofhumandiseases,includingarteriosclerosis,

    ;carcinogenesis.diabetesandaging6-.Antioxidants

    ;suchaspolysaccharides,polyphenolsandflavones ;exactedfromplantsandfunguswereexcessively ;studied,howevertheantioxidantactivityderivedfrom ;microbewaslackedinstudy,speciallyonthemarine ;microbe,onlyseveralresearcheswerereportedinthe ;pastyears.

    ;Theobjectiveofthisstudywasmeasurethe

    ;antioxidantpropeaiesofmarineagrobacterium1202 ;isolatedfromthedirtofmarineusingperoxideinduced

    ;andhydroxylfreeradicalinitiatedchemiluminescence ;(CL)assayand1,1-diphenyl2picrylhydrazyl(DPPH)

    ;freeradicalscavengingassay.

    ;2.Materialsandmethods

    ;2.1Materials

    ;MarineAgrobacteriumSP.1202usedinthis

    ;studywasisolatedfromthetidelandzoneofthe ;Xiamencoast.Thecelculturewascarriedoutonagar

    ;platesinamediumcontaining1%peptone.1% ;sodiumchloride.0.2%dextrose.eta1.Theplateswere ;incubatedat28?f0r48h.Tenmillilitersof

    ;ovemightculturecellsweretransferredto20Omlof ;freshmediumcontaining4.4%sucrose,0.1%CaCO3, ;eta1.Incubationwascarriedoutinashakingincubator ;at110rpmand26.5?f0r8d.

    ;Lumino(5amino2,3dihydro1,4phtha1azinedio

    ;ne),DPPH(2,2diphenyl1picrylhydrazt1)were

    ;29

    ;EvaluationofantioxidantactivityofmarineAgrobacteriumSP.1202

    ;purchasedfromSigmaChemicals(St.Louis,MO, ;USA).Allotherchemicalsandreagentswere ;analyticalgrade.

    ;2.2Methods

    ;2.2.1Preparationofextractsfrommarine

;agrobacterium1202

    ;Thefermentationsolutions(1000m1)were

    ;centrifugedat7000gfor20rnin,thecellswere ;discarded.Extracellularwater-solublepolysaccharides ;insupernatantwereobtainedwiththemethod ;describedbyInGyuKimwithsomemodification. ;Thesupernatantwasprecipitatedwith1volume ;ethanolandcentrifuged;theresultingsupernatants ;werefurtherprecipitatedbyadditionofethanol(2vol ;and3vo1).Thewatersolublepolysaccharide

    ;precipitates(PI,PII,PIII)werecollectedby ;centrifugationat4?(6000g,18min).Theresulting

    ;supematants(300m1)evaporatedinarotary ;evaporatorunderreducedpressure,theprecipitated ;extractedwithethylacetate,and730mgethylacetate ;extract(Ea)wasobtained.Withfivesolvents ;petroleumether,ethylacetate,butanol,methanoland ;water,theEawasfurtherfractionatedasPeF(1.8mg), ;EaF(131.2rag),BuF(11.2rag),MeF(277.0mg)and ;WtF(54.5mg).Alltheprecipitates(PI.PIIandPIII) ;andthefractions(Ea,PeF,EaF,BeF,MeFandWtF) ;werethensubjectedtoantioxidantanalysis. ;2.2.2Antioxidantactivity

    ;Antioxidantactivityofprecipitatesandextracts ;weretestedbyhydrogenperoxideinducedluminol

    ;chemiluminescence(CL)assay,DPPHflee

    ;radicalscavengingassayandHydroxylfreeradical ;initiatedchemiluminescenceassay.

    ;Thehydrogenperoxideinducedluminol

    ;chemiluminescence(CL)assaywasperformed ;accordingtothereference.’withslightmodification.

    ;Astocksolution(1mgml)ofeachextractwas ;freshlypreparedanddilutedwithmethanoltovarious ;concentrations.Aluminolsolution(2.5xlOM,in

    ;MeOH)andahydrogenperoxidesolution(30mM,in ;distilledwater)werefleshlypreparedbeforethe ;30

    ;experiment.Foreachmeasurement.amixtureof35

    ;1phosphatebuffersolutionatpH7.4,50pltest ;sampleand20BIhydrogenperoxidesolutionwas ;addedtowhite96wellsmicroplate.Afterincubation

    ;for5min,refrigerationfor3min,then40glluminol ;solutionwasaddedtothewellsandinitiatedtheCL. ;TheCLwasthenmeasuredinanabsolutelydark

    ;chamberbytheChemiluminescenceAnalyzing ;System.Totalcountswerecontinuouslyrecordedby ;CLdetectorfor300s.Eachmeasurementwas ;performedatleastintriplicate.Thepercentageof ;scavengingofhydrogenperoxideofboththeextracts ;andpositivecontrol(vitaminC)wascalculatedusing ;thefollowingformula:

    CLO/CLo]xlO0, ;ScavengedH202%=[(CLo

    ;WhereCLoistheluminescenceofthecontro1. ;andeLiisthetestingextractsfromagrobacterium ;l202.

    ;ThescavengingeffectontheDPPHfleeradical ;wasestimatedaccordingtotheproceduredescribedby ;IreneParejowithsomemodifications.Assayswere ;performedin96wellsmicroplate.Inourprocedure,

    ;analiquotof50”lofeachdilutionwasaddedintoa

    ;96wellsmicroplate.AworkingsolutionofDPPH ;(50ruw,inmethano1)wasfleshlypreparedandthen ;analiquotof150glwasaddedtoeachwel1.After ;incubationfor30mintheabsorbancewasmeasuredat ;517nm.Lowerabsorbanceofthereactionmixtureat ;517nmindicatedhigherfreeradicalscavenging ;activity.Eachdilutionwasperformedatleastin ;triplicate.Freeradicalscavengingactivitiesofthetest ;samplesandpositivecontrol(vitaminC)were ;expressedintermsofIC50values.Thecapabilityto ;scavengetheDPPHradicalwascalculatedusingthe ;followingequation:

    ;DPPHscavengingeffect%=[(Ao-Ai)/Ao]x1O0, ;WhereAoistheabsorb?anceofthecontro1

    ;reactionandAiistheabsorbanceinthepresenceof ;theextractsandpositivecontro1.

    ;Theabilityofthetestsampleandpositivecontrol ;toscavengehydroxylfreeradicalwasperformed ;EvaluationofantioxidantactivityofmarineAgrobacteriumSP.1202

    ;accordingtothemethod11withslightmodification. ;F1)reachmeasurement.asolutionof20plvitaminC ;(1.8mM)waspreparedindistilledwater;40l ;hydrogenperoxide(30mM)solutionand40l ;CuS04f0.9raM)solutionwereadded.After ;incubationfor2min,60ultestsamplewasadded. ;Incubationfor5min,refrigerationfor3min,andthen ;40plluminol(2.5x10M)solutionwasinjected.Each ;measurementwasperformedatleastintriplicate.The

    ;scavengingactivityofthetestsamplesandpositive ;controlwereexpressedintermsofIC5ovalues.The ;percentageinhibitionofhydroxylfreeradical ;generationwascalculatedusingthefollowingformula: ;Inhibition%=[(CLo-CLi)/CLolx1O0,

    ;WhereCLoistheluminescenceofthecontrol

    ;reactionandCLfistheluminescenceinthepresence ;ofthetestsamplesandpositivecontro1.

    ;3.Resultsanddiscussion

    ;3.1Analysisofantioxidantactivityofthe

    ;polysaccharides(PI,PII,Pil1)invitro

    ;3.1.1ScavengingactivityofPI,PIIandPIIIto ;hydrogenperoxide

    ;Thescavengingabilityofthewatersoluble

    ;polysaccharide(PI,PII,PIII)onhydrogenperoxideis ;showninFig.1.Allofthethreepolysaccharidesshow ;thehydrogenperoxidescavengingactivities,andthe ;inhibitionofhydrogenperoxideinducedCLeffects

    ;wasincreasedwithincreasingconcentration.Butthe ;inhibitioncapacityisnotequalwhichdecreasedinthe ;orderofPIII>PI>PII.Thedifferenceofscavenging ;activitybetweenthepolysaccharidesandvitaminCis ;significantathighadditionquantity,andthe ;polysaccharideshadweakerhydrogenperoxide ;scavengingactivitythanvitaminCofsamedose(Table ;1).

    ;3.1.2ScavengingactivityofPI,PIIandPIIIto ;hydroxylfreeradical

    ;Theinhibitionforhydrogenperoxideofthethree ;polysaccharidesfractionsPI,PIIandPIIIatthe ;differentconcentrationsareshowninFig.2.AIlofthe ;fractionscouldeffectivelyinhibittheCLfo-rmationin ;concentrationdependentmanner.ThePIIandPIII

    ;showgreaterinhibiteffectstohydroxylfreeradical ;thanPI,evenhigherthanvitaminCatthesamedose ;(Table1).

    ;Fig.2Scavengingactivitiesofthepolysaceharideison ;hydroxylfreeradicalwithdifferentconcentration ;Note:TheIC50whichtheconcentrationofinhibitionreach ;50%tohydroxylfreeradicalwascalculated. ;3.2AnalysisofantioxidantactivityofEAand ;EAextractslEaF,BuF,MeF,PeF,WtF)invitro ;3.2.1ScavengingactivityofEA,EaF,BuF,MeF, ;PeFandWtFtohydrogenperoxide

    ;EAanditsfivefractionsandtheinhibitionof ;hydrogenperoxideinducedCLdecreasedintheorder

    ;ofEaF>EA>WtF>BuF>MeF>PeF.Amongofthemthe ;EaFhasthegreatestfiydrogenscavengingactivitywith ;theIC50valueof0.05Pgml,thePeFwiththeIC50

    ;valueof194.9Pgmlisthelowestone.ForEaF,in

    ;lowerconcentration,whenconcentrationchanged0.1 ;gg/m1.theinhibitionincreasednearly20%;andathigh ;concentration.whenconcentrationchangedfrom100 ;gm1..to500ggml,theinhibitiononlyincreased3%

    ;Theresultsindicatedthatethylacetateextractsshow ;thehighestscavengingactivitytohydrogenperoxide, ;whichmeansthescavenginghydrogenperoxide ;componentsmainlyexistinhydrophobicorganic ;phase.

    ;31

    ;EvaluationofantioxidantactivityofmarineAgrobacteriumSP.1202

    ;Fig.3Inhibitoryactivitiesoftheextrac~of ;1202O11hydrogenperoxide

    ;3.2.2ScavengingactivityofEA,EaF,BuF,MeF, ;PeFandWtFtoDPPHfreeradical

    ;Theeffectofanti0xidantsonDPPHradical

    ;scavengingwasthoughttobeduetothe

    ;hydrogendonatingability.Thescavengingpercentage ;inthepresenceoftheEaanditsfractionsatdifferent ;concentrationsonDPPHfreeradicalareshowninFig. ;3.TheresultsindicatedthattheEAandEaFcould ;effectivelyscavengetheDPPHfreeradical,while ;otherfractionshaveslightlyeffects.Inorderto ;quantifytheantioxidantactivity,theIC5o,whichisthe ;concentrationofsamplerequiretodecreasethe ;absorbanceat517nm,isalsocalculated(Table11. ;TheEaFhasthehighestDPPHscavengingactivities ;followedbvEAandBuF’andalotherfractions

    ;includepositivecontrolvitaminChaveslighteffect. ;32

    ;Fig.4Scavengingactivityofthe

    ;extractsfrom1202onDPPH

    ;3.2.3ScavengingactivityofEA,EaF,BuF’MeF’

    ;PeFandWtFtohydroxylfreeradical

    ;TheinhibitionofhydrogenperoxideinducedCL

    ;oftheEAanditsfivefractionsatdifferent

    ;concentrationsareshowninFig.5.Allofthefractions ;couldeffectivelyinhibittheCLformationin

;concentrationdependentmanner.

    ;Theactivityshowintheorderof

    ;EaFl>EA>BuF>WtF>MeF>PeF,EaFshowthe ;greatestinhibiteffects.

    ;Fig.5Inhibitoryactivitiesoftheextracts ;of1202onhydroxylfreeradical

    ;3.3TheICsovaluesofthetestsamplesfrom

    ;agrobacterium1202

    ;Theantioxidantactivityofalltestsamplesis ;evaluatedbythevalueofIC5o.Table1givesallthe ;IC5ovaluesoftheprecipitatesandextractsfrom ;agrobacterium1202.Comparewithpositivecontrol ;vitaminC,thetestsamplesofEA,EAF,BuFandWcF ;showgoodantioxidantactivitytohydroxylfree ;radical,andtohydrogenperoxide,a”thetestsamples

    ;showhigherinhibitionthanvitaminC.Thetest ;samplesofEA,EaFandBuFallshowsome

    ;scavengingactivitytoDPPHfreeradicalsandthe ;activityhigherthanpositivecontrolvitaminC,the ;scavengingeffectofPI,PIIandPII1werealsostudied, ;butlittleDPPHfreeradicalscavengingactivitywas ;found.

    ;EvaluationofantioxidantactivityofmarineAgrobacteriumSP.1202

    ;Table1TheIC5ovaluesoftheprecipitatesandextractsfromagrobacterium1202

    ;Notes:and.Theantioxidantactivitieswasevaluatedastheconcentrationofthetestsamplen

    eededtodecrease,thehydrogen

    ;peroxide-inducedandhydroxylfreeradicalinitiatedCLby50%incomparisontothecontrolresponse.TheIC5o,whichisthe

    ;concentrationofthesamplerequiredtodecreasedtheabsorbanceat517nmby50%incomp

    arisontothecontrolresponse,wasto

    ;evaluatetheantioxidantactivities.

    ;Chemiluminescence(CL)istheemissionoflight ;throughanoxidizingreactionofluminolbyhydrogen ;peroxideandhydroxylfreeradical,aReaction ;OxygenSpecies(ROS)andanintermediateduring ;endogenoxidativeprocesses.Sinceitdoesnotmake ;useofenzymes,theCLassayforthestudyof

    ;hydroxylfreeradicalandhydrogenscavenging ;activitieswassimpler,andbecauseofthespeedof ;response,theinstrumentissimple.Indeed,hydroxyl ;radicalisthemostreactivechemicalspeciesknown. ;Thehydroxylfreeradicalinducessomeoxidative ;damagetobiomolecule,andthisdamagecausesaging, ;cancer,andseveraldiseases.Theremovingof

    ;hydrogenperoxideandhydroxylfleeradicalarevery ;importantofantioxidantdefenceincellororganism. ;DPPH,astableradical,withthestrongpointofahigh ;reproducibilityandsimplywaswidelyusedto ;determinationtheantioxidantactivitiesofcompounds ;orextracts.AscomparedwithDPPHassay,theCL ;assayishighersensitiveformonitoringthefree ;radicals,withtheIC50ofmeasuredpositivecontrol ;vitaminCof73.8gg/ml,whileDPPHwiththeIC50of ;567.5gg/ml(Table1).Ourresearchsupportthatthe ;water-solublepolysaccharidesandethyleacetate ;extractsfrommarineagrobacterium1202aregood ;scavengingagentstohydroxylfreeradica1.Further ;studiesareinprogresstoidentifytheactive ;compoundspresentinthesecondarymetabolitesof ;AgrobacteriumSP.bacterium1202.

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