二氢杨梅素抗氧化作用的研究

二氢杨梅素抗氧化作用的研究

接收日期：。2002-04-22.Foundationitem:Chinapostdoctoralsciencefoundation(2002031285);Guangdongsciencefoundation(020842).*CorrespondingauthorTel:86-20-87114606,E-mail:youshengzhang@263.netDihydromyricetin(3,3′,4′,5,5′,7-hexahydroxy-2,3-dihydroflavanol,DMY),extractedandpurifiedfromthetenderstemandleavesofAmpelopsisgrossedentata(Hand-Mazz)W.T.Wang,isanimportantdihydroflavonolandwasshowntohavemanybioactivefunctions,suchasrelievingcough,removingsputum,protecting-lunginflammation,reducingthedangerofEtOH,protectinglungcancer,etc.ThecontentofDMYwasmorethan27%inthetenderstemandleavesofthisspecies,especially,morethan40%inthecataphyll.However,thereisnoinformationabouttheeffectofDMYonantioxidativeproperties.Inthepresentstudy,theeffectsofDMYontheseaspectswereexamined.MaterialsandmethodsMaterialsThematerialsofAmpelopsisgrossedentatawerecollectedandidentifiedbydoctorZHANGYou-shenginGuangdongprovince.AvoucherspecimenhasbeendepositedattheBotanyHerbariumofSouthChina,theChineseAcademyofSciences.DMY,akindofwhiteneedlecrystals,wasextractedandpurifiedaccordingtothereference.TBHQ(tertiarybutylhydroquinone)werepurchasedfromGuangzhouChemicalFactory.OtherreagentswereofAR.ExtractionandpurificationofDMYDriedtenderstemsandleavesofAmpelopsisgrossedentatawereextractedtwicewith95%ethanolorwateratrefluxconditions,30minuteseachtime.Theextractswerecombinedandevaporatedunderreducedpressure.PurificationofDMYwascarriedoutbyre-crystallizationusingthedifferentsolubilitybetweenboiledwaterandcoldwater.Accordingtothedifferenttimesofre-crystallization,DMYofdifferentpuritieswereobtained.MeasurementofDMYpurityAnHPLCsystem(Beckman,USA)wasusedtomeasurethepurityofDMY.Nova-PakC18column(150mm×3.9mmID)wasusedandthemobilephasewasmethanol-water-phosphoricacid(36∶64∶0.1).Theflowratewas1mL·min-1.Theeffluentwasmonitoredat294nmandtheinjectionvolumewas10μL.StablefreeradicalscavengingpotencyofDMYEthanolsolution(2mL)containingvariousconcentrationofDMYwasaddedtoethanolsolutionof0.5mol·L-11,1-diphenyl-2-picrylhydrazylradical(DPPH)1mL.Afterbeingmixed30min,theabsorbanceat517nminthemixedsolutionwasrecordedinordertoexaminethescavengingpotencyofDPPHradical.ThescavengingpotencyisexpressedasP=[(A0-Ax)/A0]×100%,whereA0andAxaretheabsorbancevaluesofcontrolsandsamples,respectively.DeterminationofantioxidativeactivityofDMYLinoleicacid(2mol·L-1,in95%ethanol)0.1mLwasaddedintoKH2PO4buffer(20mmol·L-1,pH6.8)1.3mLand0.1mLofdifferentsamplesorKH2PO4buffer0.1mLforcontrol.Afterincubatedat37℃for5min,FeSO4-edeticacid0.5mLwasadded.Atintervalsof30minaliquotsweretakenout,20%trichloroaceticacid0.5mLand0.67%TBA1.0mLwereaddedandsimultaneously2%butylatedhydroxytoluene0.05mLaddedtopreventfurtherperoxidationoflinoleicacidduringheating.Themixturewasheatedfor10mininaboilingwaterbathandthetubeswerethencooledandcentrifuged(3500r·min-1,15min).Theabsorptionofsupernatantwasreadat532nmforthedeterminationofmalondiadehyde(MDA).Theantioxidativeactivitywasexpressedbyreactionrate(A532·min-1),thebiggerthevaluesofA532·min-1are,theweakertheantioxidativeactivitiesare.MeasurementofchelatingactivityofDMYChelatingactivityforFe2+byDMYwasmeasuredusingUV-spectrum.Firstly,theUVabsorptionspectrumofDMYinbuffersolutionwasmeasured,then,FeSO4wasaddedintoDMYsolutionandspectrumwasrecordedatthesamecondition.Results1u3000各specificagranct.u3000e-racketity.u3000.u3000e-roindex,wratching.u300和tratch................3.3.4和tratch...和tratch..........4和tratch.........................DPPHisastablenitrogen-centeredfreeradicalagent.ThechangeofDPPHinabsorbancewasusedtoevaluatetheabilityoffreeradicalscavengers(testcompounds).Themoretheabsorbancereduced,thestrongerthescavengingeffectsonhydroxylandsuperoxygenfreeradicalsthetestcompoundsexhibited.FromTable1,itcanbeseenthatthespecificabsorptionofDPPHradicalsolutionat517nmwassignificantlyreducedby73.3%-91.5%whenDMYwasaddedintothereactionsolutionintheconcentrationrangefrom0.01%to0.04%,althoughTBHQreducedtheabsorptionby75.5%-82.7%intheconcentrationrange0.01%-0.02%(thebiggestconcentrationallowedforTBHQis0.02%,so0.03%and0.04%treatmentswerenotmeasured).ThereductivepotencyofDMYorTBHQagainstthespecificabsorptionofDPPHradicalwasinaconcentration-dependentmanner.ThereductivepotencyofDMYwaslessthanthatofTBHQwhentheconcentrationwas0.01%or0.02%weight,butthereductivepotencyofDMYwasbetterthanthatofTBHQwhentheconcentrationwas0.02%or0.04%weight.ThisshowedthatDMYwouldbeagoodscavengertointeractwiththenitrogen-centeredstablefreeradical.ThecontentofDMYwasmorethan99%2回归结果lhaceFe2+-depedentlipidperoxidationisthoughttoplayacentralroleinpathologicallyrelevantoxy-radical-inducedtissuedamageinvitro.ReagentsthatinhibitFe2+-dependentlipidperoxidationmayrepresentarationalapproachtothemanagementofoxy-radical-diseases.Table2showsthatDMYcangreatlyinhibittheincreaseofLPOvaluesinlinoleiacidsystemcatalyzedbyFeSO4-edeticacid,thereactionrate(A532·min-1)was0.0021-0.0004,especially,withtheincreaseoftheconcentrationofDMY(0.01%-0.04%)added,theantioxidativeactivitiesofDMYwereshowntobeinaconcentration-dependentmanner.TBHQ,acommonandeffectiveantioxidant,wasusedinthesamesystemasDMYandshowedstrongerinhibitionactivitiesthanDMYinthesameconcentrations.But,theantioxidativeactivitiesofDMYwerestrongerthanthoseofTBHQ,whileDMYreached0.04%andTBQHwasequalto0.02%.ThecontentofDMYwasmorethan99%3u3000esrafe-关于sc浆Accordingtothereference,neitherDMYnorFeSO4showedabsorptionpeakat355nm,butwhenFeSO4wasaddedtoDMYsolution,thenewsolutionshowedanabsorptionpeakat355nm.ThepeakshouldbeaFe2+-DMYchelatingcompoundaccordingtothereference(Figurewasnotgiven).DiscussionFreeradicalsarereactivechemicalspeciesthatdifferfromothercompoundsinthattheyhaveunpairedelectronsintheirouterorbital.Theyarecapableofdamagingcellularcomponentsandleadtovariousdiseases,suchasinflammation,cancer,aging,ischemia-reperfusion,etc.Inordertoscavengetheexcessivefreeradicals,searchingforsafeandeffectiveantioxidantsfrombotanicalresourceshasbecomethefocusofattentioninrecentyears.Moreandmoreinvestigationindicatedthatmostoftheflavonoidsexistinginvariousplantspossessantioxidativefunction,whicharerelatedtotheirspecialchemicalstructureincludingthenumberandpositionofthephenolichydroxygroupsintheirmolecule.Ingeneral,ThephenolichydroxygroupsatC-3oftheC-ringandC-5,C-7oftheA-ringareimportantforthescavengingofhydroxylfreeradicals,whiletheortho-phenolichydroxygroupsatC-3′andC-4′oftheB-ringareimportantforscavengingofsuperoxygenanionfreeradicals.DMY,possesessixphenolichydroxygroups,especially,possesesOHgroupsatC-3intheC-ring,andatC-5andC-7intheA-ringandortho-phenolichydroxylsintheB-ring,possesavailablechemicalstructurewithhighscavengingactivityofhydroxylandsuperoxidefreeradicals(Figure1).So,itexpresseshighscav