Antioxidant
Mycosystema
;菌物掌报15July2008,27(4):587—593
;}wxt@im.ac.ciq
;lSSN1672—6472CN11-5180Q
;@2008InstituteofMicrobiologyCASallrightsreserved
;Antioxidantactivitiesofbioactivecomponentsfrom
;Xylariagracillima.nsubmergedcultureXylaclllimaInsubmergeclCUture ;LISai—Fei1,2WENHua.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.E—mail:wenha@sun.im.ac.cn
;Received:19—02—2008,accepted:10?03—2008
;
;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-D—ribose,trichloroaceticacid(TCA),ascorbicaci
d(Vc),
;thiobarbituricacid(TBA).2,6-di-tert—butyl-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(%)=(A0一A1)/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一(As—Asb)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-3,4一
nitrophenyl-5-phenyltetrazoliumchloridewasaddedtoformacoloredcompound ;whichcanbespectrophotometrica1lymeasuredat550nm.Alowerabsorbanceindicatesahigher
;scavengingability.Thescavengingactivityofthesamplesforsuperoxideanionradicalwas ;calculatedas:
;Scavengingactivity(%)=(Ao—A1)/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.
;3.2Hydroxyl—radical(?0H)scavengingactivity
;Thehighlyreactivehydroxyl—radicalinducesseverelydamagetotheadjacentbiomoleculeslike
;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.
;芑
;尝
;0
;专
;.J
;-
;m-Fermentationbroth—cIPScEPS
;士Mycelialextract—EDTA
;00250501248
;Concentration(mg/mL)
;Fig.1Ferrousionchelatingactivityofcomponents
;fromXylariagracillimainsubmergedculture.
;Eachvalueisthemean~SDoftriplicate
;measurements.
;-
;m-Fermentationbroth—*cIPS—CcEPS
;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血
;O025051248
;Concentration『mg/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,
;thUSterminateradica】chainreactions.
;4CoNCLUSIoN
;Sofarasweknow,Ourstudywasthefirstreportontheinvitroantioxidantpropertiesofthe ;componentsfromgracillimainsubmergedculture.Onthebasisofthewholeantioxidantmodels
;studied,itcouldbeconcludedthatcomponentsfromgracillimainsubmergedculturehadstrong
;ferrousionchelatingactivity,freeradicalscavengingabilitylikehydroxyl—radicalandsu
peroxide
;anionradical,andreducingpower,whichindicatedthatthesecomponentshadfairlygoodantioxidant
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;VoI.27No.4593
;abilities.
;Resultsofpresentworkrevealedthatcomponentsfromgracillimainsubmergedculture ;mightbeeffectivelyemployedasaningredientinhealthyorfunctionalfood,toalleviateoxidative
;stress.Clinicalstudiesandresearchregardingthebioavailabilityoftheisolatedbioactivecomponents
;arestillneededinordertOfurtherverifytheirantioxidanteffectinvivoconditions. ;【REFERENCES】
;DongZL,1998.StudyonthecliniceffectforterminalcancercllreofcompoundcapsulefromXylariagracillima.Scienceand
;TechnologyofTermites,15:32—33(inChinese)
;DeckerEA,WelchB,1990.Roleofferritinasalipidoxidationcatalystinmusclefood.JournalofAgricultureandFood
;Chemistry,38:674—677
;GordonMH,1990.Themechanismoftheantioxidantactioninvitro.In:HudsonBJF(ed.)Foodantioxidants.Elsevier
;AppliedScience,London.1-18
;HalliwellB,1991.Reactiveoxygenspeciesinlivingsystems:sollrce,biochemistry,androleinhumandisease.American
;JournalofMedicine,91:14—22
;HalliwellB,GutteridgeJMC,1985.Infreeradicals,ageing,anddisease,freeradicalsinbiologyandmedicine.Clarendron
;Press.Oxford.279—3l5
;HalliwellB,GutteridgeJMC,Arugma0I,1987.Thedeoxyribosemethod:asimple”Test-T
ube’’assayfordeterminationof
;rateconstantsforreactionsofhydroxylradicals.AnalyticalBiochemistry,165:215-219 ;LeeJ,KooN,MinDB,2004.Reactiveoxygenspecies,aging,andanfioxidativenutraceuticals.ComprehensiveReviewsin
;FoodScienceandFoodSafety.3:21—33
;LiuF,OoiVEC,ChangST,1997.Freeradicalscavengingactivitiesofmushroompolys?