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Antioxidant

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Antioxidant

    Antioxidant

Mycosystema

    ;菌物掌报15July2008,27(4):587593

    ;}wxt@im.ac.ciq

    ;lSSN16726472CN11-5180Q

    ;@2008InstituteofMicrobiologyCASallrightsreserved

    ;Antioxidantactivitiesofbioactivecomponentsfrom

    ;Xylariagracillima.nsubmergedcultureXylaclllimaInsubmergeclCUture ;LISaiFei1,2WENHua.An

    ;KeyLaboratoryofSystematicMycology&Lichenology,InstituteofMicrobiology,ChineseAcademyofSciences,Beijing

    ;100101China

    ;.GraduateSchooloftheChineseAcademyofSciences,Beijing100049,China ;Abstract:TheantioxidantpotencyofcomponentsfromXylariagracillimainsubmergedculturewas

    ;investigated,employingvariousestablishedinvitrosystems,suchassuperoxide(O?.)andhydroxyl(‘0H)

    ;radicalscavenging,reducingpower,andferrousionchelatingability.Tocopherol(,,e),butylatedhydroxytoluene

    ;(BHT)andethylenediaminetetraaceticacid(EDTA)wereusedaspositivecontrols.Accordingtotheresults,

    ;componentsfromgracillimainsubmergedcultureshowedsignificanteffectonferrousionchelatingability,

    ;O?.and’OHradicalscavengingabilityattherangeofconcentrationtested,andtheirhighest

    antioxidant

    ;activitiesreached89.72%,70.90%and77.46%respectively.Thecomponentsalsoshowedpositiveresultsof

    ;reducingpower.Theseinvitroresultssuggestedthepossibilitythatcomponentsfromgracillimainsubmerged

    ;culturecouldbeeffectivelyemployedasaningredientinhealthyorfunctionalfood. ;Keywords:antioxidation,ferrousionchelatingability,02.-and’OHradicalscavengingabi

    lity,

    ;freeradicals

    ;1INTRoDUCTIoN

    ;Reactiveoxygenspecies,suchassuperoxideanion(02.3andhydroxylradicals(.0H)can ;readilyreactwithandoxidizemostbiomolecularsincludingproteins,lipidsandDNA,thuscause

    ;variousdiseasesandhealthproblemssuchasaging,coronaryheartdiseases,atherosclerosisand

    ;cancer(Madhavieta1.1996).Antioxidantsareconsideredaspossibleprotectionagentsred

ucing

    ;.Correspondingauthor.Email:wenha@sun.im.ac.cn

    ;Received:19022008,accepted:10?032008

    ;

    ;588

    ;oxidativedamageofhumanbody.Althoughmanysyntheticchemicalssuchasphenoliccompounds

    ;arefoundtobes~ongradicalscavengers,theyusuallyhavesideeffect(Liueta1.1997).Currently,

    ;muchoftheaRentionhasbeenfocusedontheintensiveresearchonnaturalantioxidants,whichmay

    ;serveaspotentcandidatesincombatingcarcinogenesisandagingprocesses.Edibleandmedicinal

    ;fungimaybeoneofthepossiblenaturalsources.

    ;Xylariagracillima(Fr.)Fr.isanedibleandmedicinalfunguswhichbelongstoAscomycota.Itis

    ;foundinwastenestoftermitesandwasreportedtohaveanti-tumoractivities(Dong1998).Sofar,

    ;therehasbeennoreportontheantioxidantactivitiesofgracillima.Ouraimistofindout ;componentshavingexcellentantioxidantabilityfromgracillima,sothattoexploitnewnatural

    ;antioxidantfromedibleandmedicinalfungi.Theantioxidantpropertiesofcomponentsfrom

    ;gracillimaintheformsofmyceliaextract,crudeexo-polysaccharides(cEPS),crudeintracellular

    ;polysaccharides(cIPS)andfermentationbrothfromthesubmergedculturehadbeenstudied.

    ;Antioxidantpropertieswereassayedintermsofchelatingeffectonferrousions,scavengingabilities

    ;on’OHandO’andreducingpower.

    ;2MATERIAISANDMETHoDS

    ;2.1Preparationofsamplesforbioassay

    ;ThestrainofX.gracillimawasoriginallyisolatedfromwastenestsoftermites,andmaintained

    ;onPDAmediuminaPetridish.FourmycelialagardiscsrO.5cmdiameter)cutwithasterilizedcu~er

    ;wereinoculatedintoa250mLErlenmeyerflaskcontaininglOOmLofbasicmediumwithinitial

    ;pH6.0,andthenincubatedat25?,150r/minforlweekonarotaryshaker.Thecomponentsof

    basal

    ;mediumwereasfollows(w/V):glucose2%,peptonel%,KH2PO4O.1%,MgSO4O.1%.Themycelia

    ;wereseparatedfromthesupernatantbycentrifugation(4~C,8000r/minfor15min)andthenwashed

;,?

    ithdeionizedwater.Theresidualfermentationbrothwascondensedbyevaporatorandmixedwith

    ;fourvolumesofethanol(95%,v/v)at4~CforcEPSextraction.Myceliaandthecondensed ;fermentationbrothwerelyophilizedtopowder.ThemycelialpowderofX.gracillimawasextracted

    ;withdistilledwaterat100”C,2h,forthreetimes,andthesupernatantwascondensedandlyo

    philized

    ;toconstantweight.ToextractclPS,thefiltrateextractedfromthemycelialpowderwasmixedwith

    ;ethanol(95%,v/v)for24hat4”C.Hotwaterextractofmycelia,fermentationbroth,cEPSandclPS,

    ;fourlyophilizedsampleswereselectedandpreparedforanalysis.

    ;2.2Chemieal’

    ;Ferrozine,potassiumferricyanide,ferricchloride,ethylenediaminetetraaceticacid(EDTA),

    ;hydrogenperoxide(H202),2’-deoxy-Dribose,trichloroaceticacid(TCA),ascorbicaci

    d(Vc),

    ;thiobarbituricacid(TBA).2,6-di-tertbutyl-P-cresol(BHT).Allotherchemicalsandsol

    ventsused

    ;wereofanalyticalgradeavailablecommercially.

    ;2.3Methods

    ;2.3?lFerrousionchelatingactivityassay:Thechelationofferrousionsbythesamplesandstandard

    ;

    ;VOI.27NO.4589

    ;wasestimatedbythemethodofDecker&Welch(1990).1.0mLsample(O.25mg/mL-16mg/mL)was

    ;mixedwith3.7mLofdeionizedwaterand0.1mLofFeC12(2.0mmol/L),thenthemixturewasreacted

    ;with0.2mLofferrozine(5.Ommol/L)for20minafter30s.Ferrozinereactedwiththedivalentironto

    ;formstablemagentacomplexspeciesthatwereverysolubleinwater.Themixturewasshaken

    ;vigorouslyandkeptatroomtemperaturefor10minthenmeasuredat562nmagainstblank. ;Absorbanceofthemixturewasinverselyrelatedtotheferrousionchelatingactivityofthesamples.

    ;EDTAwasusedforcomparison.

    ;ThechelatingactivityofthesamplesforFewascalculatedas:

    ;Chelatingrate(%)=(A0A1)/Aox100%

    ;WhereA0wastheabsorbanceofthecontrol(blank,withoutsamples)andA

    wastheabsorbance

    ;inthepresenceofsamples.

    ;2.3?2Hydroxyl’radical(‘OH)scavengingassay:Thehydroxylradicalscavengingactivit

ywas

    ;measuredbythedeoxyribosemethod(Halliwelleta1.1987).Thereactionmixture,whichcontained

    ;0.4mLofpotassiumphosphatebuffer(20mmoFL,pH7.4),O.1mLofsamplef0.25mg/mL. ;16.Omg/mL),EDTA(1.04mmoYL),FeC13(1.0mmol/L),H2O2(12.0retool/L),2’.deoxy.D.ribose

    ;(60.0mmol/L),andascorbicacid(Vc)(2.0retool/L),wasincubatedinawaterbathat37~Cforlh.

    ;1.0mLofTCA(2.8%)and1.0mLofTBA(1.O%)wereaddedtothemixtureandheatedinawater

    ;bathat100~Cfor15min.Cooledinice,andthentheabsorbanceoftheresultingsolutionwas ;measuredspectrophotometricallyat532nm.BHTwasusedasapositivecontro1.Thescavenging

    ;activityofthesamplesforhydroxylradicalwascalculatedas:

    ;Scavengingactivity(%)=[Ab(AsAsb)1/Ab×100%

    ;WhereAbwastheabsorbanceofthecontrol(blank,withoutsamples),Aswastheabsorbancein

    ;thepresenceofsamplesandAsbwastheabsorbancewithout2’.deoxy.D.ribose

    ;2.3?3Superoxideanion(O2.-)radicalscavengingassay:Thesuperoxideanionscavengingactivity

    ;wasmeasuredusingacommercialkit(JianchengBioengineeringInstitute,Nanjing.China).The

    ;superoxideradicalsweregeneratedbythexanthine/xanthineoxidasesystemand ;4-iodiphenyl-3,4

    nitrophenyl-5-phenyltetrazoliumchloridewasaddedtoformacoloredcompound ;whichcanbespectrophotometrica1lymeasuredat550nm.Alowerabsorbanceindicatesahigher

    ;scavengingability.Thescavengingactivityofthesamplesforsuperoxideanionradicalwas ;calculatedas:

    ;Scavengingactivity(%)=(AoA1)/Ao×100%

    ;WhereA0wastheabsorbanceofthecontrol(blank,withoutsamples)andA1wastheabsorbance

    ;inthepresenceofsamples.

    ;2?3?4Reducingpowerassay:Measurementofreducingpowerwasquantifiedbythemethod

    ;describedearlierbyYen&Chen(1995)withslightmodifications.1.0mLofeachsample

    ;(O.25mg/mL-8.Omg/mL)inphosphatebuffer(O.2mol/L,pH6.6),wasincubatedwithpotassium

    ;hccp://joumafsim.accn/jwxtcn

    ;

    ;590Mycosystema

    ;ferricyanide(1.0%)at50~Cfor20min.Thereactionwasstoppedbyadding1.0mLofTCAsolution

    ;(10.0%1andthencentrifugedat3000r/minfor10min.2.5mLofthesupematantwasmixedwith

    ;2.5mLofdistilledwaterand0.5mLofFeC13solution(0.1%)for10min,andtheabsorbancewas

    ;measuredat700nm.Ahigherabsorbanceindicatesahigherreducingpower.VeandBHTwereused

    ;forcomparison.

    ;2.4Statisticalanalysis

    ;Theexperimentaldatawereexpressedasmeans4-S.D.(n:3)ofthreeparallelmeasurements. ;ThedatawereanalyzedbytheFisher’sstatisticaltestforanalysisofvariance(ANOVA).Testsof

    ;significantdifferencesweredeterminedbyDuncan’smultiplerangetestsatp<0.05usin

    gSPSS11.5

    ;(SPSSInc.,Chicago,IL,USA).

    ;3RESULTSANDDISCUSSION

    ;3.1Chelatingeffectonferrousions

    ;ThedataobtainedfromFig.1revealsthatcomponentsfromgraciHimahaveeffective ;capacitiesforferrousironchelating.Chelatingabilityimprovedastheconcentrationofsamples

    ;increased,whiletheimprovingratewasslowdownwiththeincreasingconcentration.Thewhole

    ;trendpresentedan”S”figure.cEPS,clPS,fermentationbroth,andmyceliaextractwereallgood

    ;chelatorsforferrousions,andtheirhi.ghestchelatingabilitiesreached81.54%,89.72%,89.47%,and

    ;79.92%respectively.Attheconcentrationof0.5mg/mL-4.0mg/mL.thechelatingabilitycompared

    ;wasclPS>fermentationbroth>cEPS>myceliaextract<0.05).Asfermentationbrothalso

    ;containsexo-polysaccharides,thehigherchelatingeffectoffermentationbroththancEPStellsusthat

    ;theremaybesomecomponentsintheexo-biopolymerthathasbetterchelatingeffectthan ;exo-polysaccharide.

    ;FerrousionscouldstimulatelipidperoxidationbyFentonreactionandaccelerateperoxidation

    ;bydecomposinglipidhydroperoxidesintoperoxylandalkoxylradicals,whichcanthemselves,drive

    ;thechainreactionoflipidperoxidation(Halliwell1991).Ithasbeenreportedthatchelatingagents.

    ;whichform~6-bondswithametalareeffectiveassecondaryantioxidantsbecausetheyreducethe

    ;redoxpotential,therebystabilizingtheoxidizedformofthemetalion(Gordon1990).Sinceferrous

    ;ionsarethemosteffectiveprooxidantsinthefoodsystem,highchelatingabilitiesofextracts

fromX.

    ;gracillimawouldbebeneficialforhealthyfoodadditive.

    ;3.2Hydroxylradical(?0H)scavengingactivity

    ;Thehighlyreactivehydroxylradicalinducesseverelydamagetotheadjacentbiomoleculeslike

    ;DNA,lipidsandproteins(Spencereta1.1994).Thecomponentsextractedfromgracillima ;exhibitedadose-dependentscavengingactivitytowardshydroxyl-radicals(Fig.2).Thesefour

    ;samplescouldscavengehydroxyl-radicalbutwerenotaseffectiveasBHTwithaconcentrationrange

    ;of0-16mg/mL.Fermentationbrothreachedthehighestscavengingabilityof70.90%at16.Omg/mL

    ;comparedwithothersamples,whileBHTreacheditshighestabilityof97.37%at8.0mg/mL.The

    ;

    ;VOI.27NO.4591

    ;scavengingabilityoffermentationbrothwassignificantlyhigherthanmycelialextractandcEPSat

    ;alltheconcentrationdetermined<0.05).ThefactthatfermentationbrothexceededcEPSindicated

    ;thattheremaybeothercomponentsinthefermentationbroththathadstronger’OHscavenging

    ;activitythancEPS.Athigherconcentrationof4.Omg/mL-16mg/mL,thescavengingactivityofthese

    ;foursamplesincreasedsharplywiththeincreasingconcentration.

    ;

    ;

    ;0

    ;

    ;.J

    ;-

    ;m-FermentationbrothcIPScEPS

    ;MycelialextractEDTA

    ;00250501248

    ;Concentration(mg/mL)

    ;Fig.1Ferrousionchelatingactivityofcomponents

    ;fromXylariagracillimainsubmergedculture.

    ;Eachvalueisthemean~SDoftriplicate

    ;measurements.

    ;-

    ;m-Fermentationbroth*cIPSCcEPS

    ;00250.51248

    ;Concentration(mg/mL)

    ;Fig.2Hydroxylradicalscavengingactivityof

;componentsfromXylariagracillimainsubmerged

    ;culture.Eachvalueisthemean~SDoftriplicate

    ;measurements.

    ;3.3Superoxideanion(o’.)radicalscavengingactivity

    ;Superoxideanionisareducedformofmolecularoxygencreatedbyreceivingoneelectron.Itis

    ;aninitialfreeradicalformedfrommitochondrialelectrontransportsystemsandplaysanimportant

    ;roleintheformationofotherreactiveoxygenspecies,suchashydrogenperoxide,hydroxyl-radical,

    ;orsingleoxygeninlivingsystems(Leeeta1.2004).Itisalsoveryharmfultothecellularcomponents

    ;inabiologicalsystem(Halliwell&Gutteridge1985).

    ;ThescavengingeffectofthesamplesonO?.generatedbyphotochemicalreactionwere ;determinedandtheresultswereshowninFig.3.Allofthesampleshadascavengingactivityina

    ;dosedependentmanneratthetestedconcentrations.Attheconcentrationof8.Omg/mL,the ;scavengingabilityofcEPS,cIPS,fermentationbrothandmyceliaextractreachedto70.95%,50.32%,

    ;77.46%and63.97%respectively.Amongwhich,fermentationbrothshowedmostnearpowertothe

    ;comparisonBHT,whosehighestabilitywas80.04%.Astheconcentrationincreased.thescavenging

    ;abilityofmycelialextractandcEPStogetherwithBHTpresentedanalmostparallelincreasingtrend.

    ;cEPSshowedsignificanthigherscavengingabilitythancIPSandmycelialextractatallthe ;concentrationtested<0.05).

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    ;~Fermentationbroth*clPScEPS

    ;00120.2505l248

    ;Concentration(mg/mL)

    ;Fig.3Superoxideanionradicalscavengingactivityof

    ;componentsfromXylariagracillimainsubmergedculture.

    ;Eachvalueisthemean~SDoftriplicatemeasurements.

    ;3.5

    ;3

    ;2.5

    ;2

;15

    ;1

    ;0.5

    ;0

    *clPScEPS ;~Fermentationbro

    ;O025051248

    ;Concentrationmg/mL)

    ;Fig.4ReducingpowerofcomponentsfromXylaria

    ;gracillimainsubmergedculture.Eachvalueisthe

    ;mean+SDoftriplicatemeasurements.

    ;3.4Reducingpower

    ;Thereducingpropertiesaregenerallyassociatedwiththepresenceofreductonewhichhave ;beenshowntoexertantioxidantactivitiesbybreakingthefreeradicalchainthroughdonatinga

    ;hydrogenatom(Rongeeta1.2007).Ourdataonthereducingpowerofcomponentsfrom ;gracillimasuggestedthattheywerelikelytocontributesignificantlytowardstheobserved ;antioxidant.Fig.4depictsthatthereducingpowerofthesamplescorrelatedwellwiththeincreasing

    ;ofconcentrations.VeandBHTshowedexcellentreducingpowerattheconcentrationsobserved.

    ;MycelialextractandcEPSexceededBHTatahigherconcentrationf8.Omg/mL)andwerealmostthe

    ;sanlepowerwithVe.cIPSandfermentationbrothshowedthelowestlevelamongthesamples.Asa

    ;whole,thereducingpowerofthesampleswas(Ve,BHT)>(mycelialextract,cEPS)>(clPS,

    ;fermentationbroth)<0.05).MycelialextractandcEPShadalmostthesamereducingpower,as

    ;wellascIPSandfermentationbroth.Thisphenomenonindicatesthattheremighthaveequalcontent

    ;ofreductoneorothercomponentsinthesamplesthathavethesameabilitytoreactwithfreeradicals,

    ;thUSterminateradicachainreactions.

    ;4CoNCLUSIoN

    ;Sofarasweknow,Ourstudywasthefirstreportontheinvitroantioxidantpropertiesofthe ;componentsfromgracillimainsubmergedculture.Onthebasisofthewholeantioxidantmodels

    ;studied,itcouldbeconcludedthatcomponentsfromgracillimainsubmergedculturehadstrong

    ;ferrousionchelatingactivity,freeradicalscavengingabilitylikehydroxylradicalandsu

    peroxide

    ;anionradical,andreducingpower,whichindicatedthatthesecomponentshadfairlygoodantioxidant

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    ;VoI.27No.4593

    ;abilities.

    ;Resultsofpresentworkrevealedthatcomponentsfromgracillimainsubmergedculture ;mightbeeffectivelyemployedasaningredientinhealthyorfunctionalfood,toalleviateoxidative

    ;stress.Clinicalstudiesandresearchregardingthebioavailabilityoftheisolatedbioactivecomponents

    ;arestillneededinordertOfurtherverifytheirantioxidanteffectinvivoconditions. ;REFERENCES

    ;DongZL,1998.StudyonthecliniceffectforterminalcancercllreofcompoundcapsulefromXylariagracillima.Scienceand

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    ;GordonMH,1990.Themechanismoftheantioxidantactioninvitro.In:HudsonBJF(ed.)Foodantioxidants.Elsevier

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    ;HalliwellB,1991.Reactiveoxygenspeciesinlivingsystems:sollrce,biochemistry,androleinhumandisease.American

    ;JournalofMedicine,91:1422

    ;HalliwellB,GutteridgeJMC,1985.Infreeradicals,ageing,anddisease,freeradicalsinbiologyandmedicine.Clarendron

    ;Press.Oxford.2793l5

    ;HalliwellB,GutteridgeJMC,Arugma0I,1987.Thedeoxyribosemethod:asimple”Test-T

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    ;rateconstantsforreactionsofhydroxylradicals.AnalyticalBiochemistry,165:215-219 ;LeeJ,KooN,MinDB,2004.Reactiveoxygenspecies,aging,andanfioxidativenutraceuticals.ComprehensiveReviewsin

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    ;LiuF,OoiVEC,ChangST,1997.Freeradicalscavengingactivitiesofmushroompolys?

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