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Fitoterapia
journalhomepage:www.elsevier.com/locate/fitote
Structuralcharacterizationandantioxidantactivityofa
low-molecularpolysaccharidefromDendrobiumhuoshanense
Chang-ChengTian,Xue-QiangZha,Li-HuaPan,Jian-PingLuo⁎
SchoolofBiotechnologyandFoodEngineering,HefeiUniversityofTechnology,Hefei230009,PRChina
articleinfoabstract
Thepresentstudyaimedatinvestigatingthestructuralfeaturesandantioxidantactivitiesofapolysaccharidefraction(DHP1A)obtainedfromDendrobiumhuoshanense,apreciousherbmedicineinChina.DHP1Amainlyconsistedofmannose(Man),glucose(Glc)andatraceofgalactose(Gal),withamolecularweightof6700Da.Itsbackbonecontained(1→4)-linkedα-D-Glcp,(1→6)-linkedα-D-Glcpand(1→4)-linkedβ-D-Manp,withabranchofterminalβ-D-Galp.TheinvitroantioxidantevaluationrevealedthatDHP1AhadaremarkableinhibitioneffectontheFeCl2-inducedlipidperoxidation.Furthermore,DHP1Apretreatmentdecreasedtheproductionofmalondialdehyde(MDA),andrestoredtheactivitiesofsuperoxidedismutase(SOD),catalase(CAT)andglutathioneperoxidase(GPx),aswellasthelevelofglutathione(GSH)intheliversofCCl4-treatedmice.TheseresultssuggestedthatDHP1AwasapotentialantioxidantcomponentinD.huoshanense.
©2013ElsevierB.V.Allrightsreserved.
Articlehistory:
Received27July2013
Acceptedinrevisedform24September2013Availableonline3October2013Keywords:
DendrobiumhuoshanensePolysaccharideStructure
Antioxidantactivity
1.Introduction
Naturalpolysaccharides,existinginanimals,plants,fungiandalgae,areatypeofabundantandusefulresource,whichareinvolvedinmaterials,foodsandmedicines.Ithasbeenwelldocumentedthatthepolysaccharidespossesspotentandbeneficialimmunomodulatingfunctions,suchasanti-inflammation,anti-tumorandinnateimmunityenhance-ment[1–3].Somepolysaccharideshavebeenproventobenaturalantioxidantsasevidencedbytheircapacitiesofscaveng-ingfreeradicalsinvitro[4,5]andinhibitioneffectsontheoxidativestressinvivo[6,7].Itiswellknownthattheoxidativestressplaysakeyroleindiversepathogenesis,andthesupplementofantioxidantpolysaccharidesmaybeanidealalternativeforpreventingandcuringsomediseases[8–10].Inrecentyears,investigationsfocusingonthenaturalantioxidantpolysaccharideshaveattractedmoreandmoreattention.
DendrobiumhuoshanenseisaperennialepiphyticorchidspeciesandmainlydistributedinChina,whichhasbeenusedasapreciousfolkmedicinesinceancienttimes.Itsstem,as
⁎Correspondingauthor.Tel./fax:+8655162901539.E-mailaddress:jianpingluo@hfut.edu.cn(J.-P.Luo).
0367-326X/$–seefrontmatter©2013ElsevierB.V.Allrightsreserved.http://dx.doi.org/10.1016/j.fitote.2013.09.018
afunctionalbeverage,exertsremarkablerestorativeeffects,includingnourishingthestomach,preventingthecataract,relivingthethroatinflammation,promotingthesecretionofbodyfluidandenhancingthebodyimmunity[11].Pharma-cologicalresearcheshaveshownthatthereexistsomeactivecomponentsinD.huoshanense,suchasalkaloids,glycosidesandpolysaccharides[12].Amongthem,thepolysaccharidesareincreasinglyregardedasthemostimportantactivecomponent.SomepolysaccharidefractionsfromD.huoshanensecouldactivatethelymphocytestosecretevariouscytokinesinvitro,exhibitingthepotentimmunomodulatingactivities[13,14].Besides,crudeD.huoshanensepolysaccharides(DHP)hadbeendemonstratedtopossessgreatpotentialasnaturalantioxidantsbytheinvitroexperiments[15].TheoraladministrationofcrudeDHPcouldprotectthediabeticcataractcausedbystreptozotocinthroughdecreasingtheexpressionofNOandiNOS[16],andrestoringtheactivitiesofsystematicantioxidantenzymes[17].However,theseinvestigationsontheantioxidantactivitywerefocusedonthecrudeDHP,andourunderstandingonthespecificantioxidantpolysaccharidefractionwasstilllimited[18].Inthepresentstudy,alow-molecularpolysaccharidefraction(DHP1A)wasscreenedandpurifiedfromthecrudeDHP,anditsstructuralcharacterization
248C.-C.Tianetal./Fitoterapia91(2013)247–255
wascarriedoutbyhighperformanceliquidchromatography(HPLC),gaschromatography–massspectrometry(GC–MS),Fouriertransform-infrared(FT-IR)spectroscopyandnucle-armagneticresonance(NMR).Moreover,theinvitroandinvivoantioxidantactivitiesofDHP1Awereevaluated.2.Materialsandmethods2.1.Materialsandreagents
D.huoshanensewasobtainedbymicropropagationinourlabasdescribedinthepreviousmethods[19].DEAE-celluloseD-52andSephadexG-100werepurchasedfromAmershamPharmaciaBiotech(London,England)andSigma-Aldrich(St.Louis,MO,USA),respectively.Bothdextrans(5.0,25.0,80.0,150.0,470.0,and610.0kDa)andmonosaccharides(D-glucose,D-galactose,D-mannose,D-xylose,L-rhamnose,andD-arabinose)werepurchasedfromFluka(St.Louis,MO,USA).2,2-Diphenyl-1-picrylhydrazyl(DPPH)waspurchasedfromSigma-Aldrich(St.Louis,MO,USA).Totalsuperoxidedismutase(T-SOD),catalase(CAT),glutathioneperoxidase(GPx),malondialdehyde(MDA)andglutathione(GSH)kitswereallpurchasedfromNanjingJianchengBioengineeringInstitute(Nanjing,China).2.2.PreparationofDHP1AfromD.huoshanense
ThedriedD.huoshanensewasgroundtopowderandimmersedin80%ethanol(1:100,g/mL)forthreedaystoremovethepigments,lipidsandsmallmolecularcompounds.Theresultingmaterialwasextractedtwicewithdistilledwater(1:100,g/mL)for2hat65°C.Theextractwasconcentratedusingavacuumrotaryevaporatorandcentrifugedat10,000rpmfor10mintogivethesupernatant,whichwassubsequentlyprecipitatedbyaddingfourvolumesof95%ethanolfor24hatroomtemperature.Aftercentrifugation(10,000rpmfor5min),theprecipitatesweredissolvedindistilledwateragain,anddeproteinizedsixtimesbytheSevagmethod[20].Thepolysaccharidesolutionwasfurtherdialyzedagainsttapwater(molecularweightcutoffof3500Da)andfreeze-driedtoaffordthecrudepolysaccharide,namedDHP.
ThecrudeDHP,dissolvingindistilledwaterataconcentra-tionof10mg/mL,wasloadedonaDEAE-celluloseanionexchangecolumn(2.6×40cm)andelutedwithdistilledwater,0.1,0.2and0.3MNaClsolutionsuccessively,togivefourcorrespondingfractionsofDHP1,DHP2,DHP3andDHP4.Amongthesefractions,DHP1wasfurtherfractionatedusingaSephadexG-100gelchromatographiccolumn(2.6×60cm),whichwaselutedwithdoubledistilledwaterattheflowrateof0.2mL/min.Theeluatewascollectedbyafractioncollector(2mL/tube),andthetotalsugarcontentofeachtubewasmeasuredbythephenol–sulfuricacidmethods[21].Followingtheselectivecollection,concentrationandfreeze-drying,apurifiedfraction(DHP1A)wasprepared.2.3.StructuralcharacterizationofDHP1A
2.3.1.HomogeneityandmolecularweightofDHP1A
ThemolecularweightofDHP1AwasdeterminedbyaHPLCsystem(1260infinity,AgilentTechnologies)equippedwitharefractiveindexdetector(RID).TheseparationactionwasperformedwithTSK-GELcolumnG4000PWXL(7.8×300mm)
andG5000PWXL(7.8×300mm)connectedinseries.Thetemperatureofthecolumnovenwassetat30°Candthecolumnwaselutedwithdoubledistilledwaterattheflowrateof0.5mL/min.Theconcentrationofsamplewas1.0mg/mLandeachinjectionvolumewas20μL.Aseriesofstandarddextrans(5.0,25.0,80.0,150.0,470.0,and610.0kDa)wereanalyzedinthesameconditionandusedforthecalibrationofstandardcurve.
2.3.2.Monosaccharidecompositions
DHP1A(5mg)wasdissolvedin2Mtrifluoroaceticacid(TFA,3mL)andhydrolyzedat120°Cfor4h.Then,thesuperfluousTFAinthehydrolyzatewasremovedviaco-evaporationwithmethanol(3mL)onarotaryevaporatorforatleastfivetimes.TheresultinghydrolyzatewasconvertedintoalditolacetatesthroughreactionwithNaBH4(30mg)atroomtemperature(RT)for3handanhydride–pyridine(1:1,v/v;3mL)at100°Cfor1hsuccessively.Afterdrying,thederivativesweredissolvedinchloroformandanalyzedbyGC(GC–MSQP2010system,Shimadzu)equippedwithaflameionizationdetector(FID)andaHP-5capillarycolumn(30m×0.32mm×0.25μm,Agilent).Theinjectoranddetectortemperatureswere270and300°C,respectively.Theflowrateofcarriergas(N2)wassetat0.3mL/min.Thetemperatureofthecolumnovenwasprogrammedasfollows:(1)150°Cfor1min;(2)increas-ingto180°Cat10°C/min;and(3)increasingto250°Cat4°C/min.
2.3.3.Infraredspectrumanalysis
ThedriedpolysaccharidesamplewaspressedintothepelletswithKBrpowder,andthenscannedwithaFouriertransforminfrared(FT-IR)spectrometer(Nicolet67,ThermoNicolet)intherangeof4000–400cm−1.
2.3.4.Methylationanalysis
DHP1A(5mg)wasmethylatedwithanhydroussodiumhydroxideandmethyliodideindimethylsulfoxideforsixtimesaccordingtothepreviousmethod[22].Theresultingproductswerehydrolyzedwith2MTFA(3mL)for4hat110°C,andfurtherconvertedintothepartiallymethylatedalditolacetatesviareactionwithNaBH4andanhydride–pyridinesuccessively.GC–MS(QP2010system,Shimadzu)equippedwithaHP-5capillarycolumn(0.25μm×0.32mm×30m)wasusedforidentificationoftheresultingderivatives.Thetemperatureofthecolumnovenwasinitiallycooledto50°Cfor1min,andthenraisedto250°Cat10°C/min.Thepartiallymethylatedalditolacetateswereidentifiedbythefragmentions,andtheirmolarratioswerecalculatedbythecorrespondingpeakareas.
2.3.5.Nuclearmagneticresonance(NMR)analysis
DHP1A(30mg)wasdriedinavacuumoven(60°C)for8handthendissolvedinD2O.The1H,13C,heteronuclearsinglequantumcoherence(HSQC)andheteronuclearmultiplebondcorrelation(HMBC)spectraofDHP1Awereperformedandrecordedonanuclearmagneticresonancespectrometer(400.13MHz,Bruker,Switzerland)at50°C.ThechemicalshiftofHOD(δ4.70)wasusedastheinternalreferencefor1HNMRspectrum.
C.-C.Tianetal./Fitoterapia91(2013)247–255249
Fig.1.SpectralanalysisofDHP1A.Highperformanceliquidchromatography(A);infraredspectrum(B);gaschromatogramsofstandardmonosaccharides(C)andDHP1Aderivatives(D).1)Rhamnose;2)arabinose;3)xylose;4)mannose;5)glucose;6)galactose.
2.4.AntioxidantactivitiesofDHP1Ainvitro
2.4.1.DPPHradicalscavengingactivityofDHP1A
TheDPPHradicalscavengingactivityofDHP1Awasdeter-minedaccordingtothepreviousmethod[23]withsomemodifications.Thereactionmixturecontained2.0mLofDPPHmethanolsolution(0.1mM)and1.0mLofDHP1Asolution(dissolvingindoublewaterat0.3,0.6,1.5,3.0and6.0mg/mL).Aftershakingwell,themixturewaskeptatroomtemperaturefor30min,andtheabsorbanceat517nmwasmeasured.Doubledistilledwaterwasusedinthecontrolgroup.VitaminCanddextran(20.0kDa,Shanghai,China)wereusedaspositiveandnegativereferencesubstances,respectively.Themea-surementofeachsamplewascarriedoutintriplicate.Thescavengingratewascalculatedbythefollowingformula:
Scavengingrateð%Þ¼Acontrol−Asample=AcontrolÂ100whereAcontrolistheabsorbancevalueofthecontrolgroup,andAsampleistheabsorbancevalueofthegrouptreatedwithsample.
2.4.2.InhibitionactivityofDHP1Aonlipidperoxidation
TheinhibitionactivityofDHP1Aonlipidperoxidationwasevaluatedaccordingtothiobarbituricacidmethodasde-scribedintheliterature[24]withsomemodifications.Thereactionmixture,including1.0mLofDHP1Asolution(0.5–10.0mg/mL),1.0mLofliverhomogenate(1%,w/v),50μLof
FeCl2solution(0.5mM)and50μLofH2O2(0.5mM),wasincubatedat37°Cfor60min,andthenwasterminatedbyadding1.5mLoftrichloroaceticacid(20%,w/v).Theresultingmixturewasmixedwith1.5mLofthiobarbituricacid(0.8%,w/v)andincubatedforanother60minat95°C.Aftercentrifugationat4000rpmfor10min,thesupernatantwasmeasuredat532nm.Theinhibitioneffectwascalculat-edbytheformulaasdescribedinSection2.4.1.2.5.EffectsofDHP1Aonoxidativestressinvivo
2.5.1.Experimentdesign
SixtymaleKunmingmice(23±2g)werepurchasedfromtheExperimentalAnimalCenterofAnhuiMedicalUniversity,China.Theywerehousedinanair-conditionedroom(25±2°C)withanormalday/nightlightcycle,andfedwithrodentchowandtapwater.AnimalcareandprocedurewerecarriedoutinaccordancewiththeGuidelineforanimalexperimentationofHefeiUniversityofTechnology(Hefei,China).
Afteracclimatizingforaweek,themiceweredividedinto6groupsrandomly(10mice/group):group1,controlgroup;group2,CCl4group;group3,CCl4plussilymarin(25mg/kgbodyweight,BW);group4,CCl4plusdextran(200mg/kgBW);group5,CCl4plusDHP1A(100mg/kgBW)andgroup6,CCl4+DHP1A(200mg/kgBW).AllsamplesincludingDHP1A,silymarinanddextranweredissolvedindistilledwater.Groups1and2wereadministeredorallywithdistilledwaterat
250C.-C.Tianetal./Fitoterapia91(2013)247–255
Table1
MethylationanalysisofDHP1AfromDendrobiumhuoshanense.Methylatedsugar
Massfraction
MolarLinkagetyperatio
Terminal→4)-Glcp-(1→→6)-Glcp-(1→→4,6)-Glcp-(1→→4)-Manp-(1→
0.92,3,4,6-Me4-Galp43,71,87,101,117,129,
145,161,205
43,58,71,87,101,117,12.502,3,6-Me3-Glcp
127,143,161,173,23343,58,71,87,99,101,2.002,3,4-Me2-Glcp
117,129,161,189,23343,85,87,101,117,127,1.102,3-Me3-Glcp
142,159,161,201,261
2.602,3,6-Me3-Manp43,45,71,87,99,101,113,
117,129,161,173,233
2.5.2.EffectsofDHP1AonMDA,T-SOD,CAT,GPxandGSH
ThesupernatantobtainedfromthelivertissuehomogenatewasusedtoevaluatethelevelsofT-SOD,MDA,CAT,GPxandGSHbythecorrespondingcommercialclinicalkitsaccordingtothemanufacturer'sinstructions.2.6.Dataanalysis
Theresultswereexpressedasmeans±standarddeviations(SD).Statisticaldifferenceamongthegroupswasevaluatedbyone-wayanalysisofvariance.Thedifferencewasconsideredassignificantwhenpvalueb0.01.3.Results
3.1.IsolationandpurificationofDHP1AfromD.huoshanense
10mL/kgBW.Group3wasadministeredwithsilymarinatthedoseof25mg/kgBW.Group4wasadministeredwithdextranatthedoseof200mg/kgBWandtheothergroupswereadministeredwithDHP1A(100and200mg/kgBW).Allmiceweretreatedoncedailyfor14days.Twohoursafterthelasttreatment,themiceincontrolgroupwereinjectedintraperitoneallywiththeoliveoilatthedoseof10mL/kgBW,whiletherestofthemicewereinjectedintraperitoneallywith0.15%CCl4solution(dissolvingintheoliveoil,w/v)atthesamedose.
Twenty-fourhoursaftertheCCl4treatment,thebloodwascollectedfrominnercanthusandcentrifugedat3000rpmfor10mintogivetheserum,whichwasstoredat−80°Cuntilanalysis.Afterthat,themiceweresacrificedbycervicaldislocationandtheliverswereremovedquickly.Apartofthelivertissue(0.2g)washomogenizedwithninevolumesof0.9%NaClsolution(1.8mL)usingahomogenizer.Aftercentrifugationat3000rpmfor10min,thegeneratedsuper-natantwascollectedforthesubsequentanalysis.
ThecrudeDHPwasisolatedfromthedriedD.huoshanensebywaterextractionandethanolprecipitation,anditsyieldwasabout5.1%ofthedriedmaterial.Afterthat,DHPwasfractionatedontheDEAE-cellulosecolumntogivefoursub-fractions,DHP1(78%ofthecrudeDHP),DHP2(12%ofthecrudeDHP),DHP3(5%ofthecrudeDHP)andDHP4(3%ofthecrudeDHP).Amongthem,DHP1wasfurtherpurifiedbyagelfiltrationchromatography(SephadexG-100)toaffordapolysaccharidefractionofDHP1A(yield:50%ofDHP1).DriedDHP1Awaswhitepowder,andnoabsorbancepeakswerefoundintheUVspectrum.Accordingtothephenol–sulfuricacidmethods,itscarbohydratecontentreachedto98.9%,andnoproteinsweredetectedbasedontheLowrymethod[25].3.2.StructuralcharacterizationofDHP1A
AsshowninFig.1A,theHPLCprofileofDHP1Apresentedanarrowandsymmetricalpeak,suggestingthatitwasa
Fig.2.1Hand
13CNMRspectraofDHP1A.
C.-C.Tianetal./Fitoterapia91(2013)247–255251
Fig.3.HSQCandHMBCspectraofDHP1A.
homogenouspolysaccharidefraction.Itsmolecularweightwasabout6700Daaccordingtothecalibrationofstandarddextrans.TheFT-IRspectrumofDHP1AwasshowninFig.1B.Thebroadcharacteristicpeakat3388cm−1wasattributedtothestretchingvibrationofhydroxylgroup.Thepeaksat2929cm−1and1644cm−1wereassignedtothevibrationofC\\Handboundwater,respectively.Thepeakat930cm−1suggestedtheexistenceofα-configurationglucose,andthepeakat870cm−1wasthecharacteristicabsorptionpeakofmannose[26].ThemonosaccharidecompositionanalysisshowedthatDHP1Awasmainlycomposedofmannose,glucoseandalittleofgalactoseinamolarratioof2.5:16.0:1.0(Fig.1C,D).Accordingtotheresultsofmethylationanalysis,fivemethylatedalditolacetateswerefoundinDHP1AaslistedinTable1.Amongthem,(1→4)-linkedD-Glcpand(1→4)-linkedβ-Manpwererelativelypredominant.Besides,thebranchstructurewasfoundinDHP1Aduetotheexistenceof(1→4,6)-linkedGlcp,whichwaspossiblysubstitutedbyasingleD-GalpatC-6position.
TheNMRspectra,including1H,13C,HSQCandHMBC,wereusedtodelineatethestructuralfeaturesofDHP1Afurther.In
the1HNMRspectrum(Fig.2A),thestronganomericsignalsaroundδ5.37–5.29wereassignedto(1→4)-linkedand(1→4,6)-linkedα-D-Glcp.Thesignalsatδ4.95,4.73and4.49wereassignedto(1→6)-linkedα-D-Glcp,(1→4)-linkedβ-D-Manpandterminalβ-D-Galp,respectively.Moreover,thesignalatδ2.16showedthepresenceofacetylgroup.The13CNMRspectrumofDHP1Apresentedfiveanomericsignalsatδ103.56–99.50.Thesignalsatδ103.39,100.82,100.58and99.5belongedtoterminalβ-D-Galp,(1→4)-linkedα-D-Glcp,(1→4,6)-linkedα-D-Glcpand(1→6)-linkedα-D-Glcp,respectively.Theanomericsignalof(1→4)-linkedβ-D-Manpwasatδ100.47,whichwasoverlappedwiththesignalof(1→4)-linkedα-D-Glcp.Thesignalatδ77.80indicatedthesubstitutionofC-4ofα-D-Glcp.PresenceofacetylgroupsinDHP1Awasfurtherconfirmedbythesignalsatδ173.94and21.48(Fig.2B).
TheHSQCspectrumofDHP1Ashowedfourdominantcouplingsignalsatδ5.37/100.82,4.95/99.50,4.73/100.58and4.49/103.39(Fig.3A),whichwereassignedto(1→4)-linkedGlcp,(1→6)-linkedGlcp,(1→4)-linkedManpandterminal
252C.-C.Tianetal./Fitoterapia91(2013)247–255
Galp,respectively.IntheHMBCspectrumofDHP1A(Fig.3B),theglycosidiclinkagesofthesugarresidueswereproposedaccordingtothecouplingbetweenprotonandcarbon.Thecouplingsignalsatδ5.37/77.80indicateda(1→4)linkageexistingamongtheglucopyranose.H-1ofGlcplinkingtoC-6ofGlcpshowedaweakcouplingsignalatδ4.95/71.47.Moreover,thesignalatδ4.73/76.89representedthelinkagebetweenH-1ofβ-ManpandC-4ofα-Glcp[13,14,26–30].
Basedonthechemicalandspectralanalysisabove,apossiblestructuralunitofDHP1AwasproposedasshowninFig.4.
3.3.InvitroantioxidantactivitiesofDHP1A
3.3.1.ScavengingactivityonDPPHradicalofDHP1A
TheDPPHscavengingeffectofDHP1AwasshowninFig.5A,anditsscavengingrateswereincreasedfrom31.4%to38.7%withincreasingthepolysaccharideconcentrationfrom0.5to2.0mg/mL.AscomparedwithvitaminC,awell-knownlowmolecularanti-oxidant,thescavengingrateofDHP1Awaslower.However,itseffectwasproventobebetterthandextran(pb0.01),areferencepolysaccharide.
3.3.2.InhibitioneffectonlipidperoxidationofDHP1A
AsshowninFig.5B,DHP1AshowedasignificantinhibitioneffectontheFeCl2-inducedlipidperoxidation.Therewasanobviousdose-dependentrelationshipastheconcentrationsofDHP1Arangingfrom0.1to1.0mg/mL.At2.0mg/mL,theinhibitionrateofDHP1Awasincreasedto56.5%,whichwashigherthanthatofdextran(pb0.01),andclosetothatofvitaminC.
3.4.EffectofDHP1AonoxidativestresscausedbyCCl4inmice3.4.1.EffectofDHP1AonlevelofMDA
Theinvivoanti-lipidperoxidationofDHP1AwasalsoevaluatedintheCCl4-inducedliverinjurymodel(Fig.6A).ItwasconfirmedthatCCl4administrationsignificantlyin-creasedthelevelofMDA(2.71nmol/mgprot)ascomparedtothecontrolgroup(1.42nmol/mgprot),whichsuggestedtheoccurrenceoflipidperoxidation.ThemicepretreatedwithDHP1AshowedaneffectiveinhibitioneffectontheincreaseofMDA.At200mg/kgBW,thelevelofMDAintheliverwas
Fig.4.PredictedstructuralunitofDHP1AfromD.huoshanense.
Fig.5.AntioxidantactivitiesofDHP1Ainvitro.ScavengingcapacityforDPPHradical(A);inhibitioneffectonthelipidperoxidation(B).Valuesaremeans±SD(n=3).
decreasedto1.56nmol/mgprot,whichwasclosetothenormallevel.
3.4.2.EffectofDHP1AonlevelsofT-SOD,CAT,GPxandGSH
Toevaluatetheinvivoanti-oxidativestress,theeffectsofDHP1Aonthesystematicantioxidantenzymes(substance)wereinvestigated(Fig.6,B–E).Silymarin,atraditionalhepatoprotectivedrug,wasusedasapositiveagentinthisstudy[31],whiledextran(20.0kDa,Shanghai,China)wasregardedasaparallelnegativecontrol.FollowingCCl4treatment,thelevelofGSHwasdecreasedto4.33nmol/mgprotascomparedwiththatinthenormalmice(7.8nmol/mgprot).PretreatmentwithDHP1A(100or200mg/kgBW)remark-ablyalleviatedthedepletionofGSHascomparedwiththemicetreatedwithCCl4alone(pb0.01).Silymarinpretreat-mentalsodecreasedthedepletionofGSH,butdextranseemedtobeinvalid.Moreover,theactivitiesofT-SOD,CATandGPxweresignificantlydecreasedintheCCl4-treatedmiceascomparedwiththecontrolgroup(pb0.01),whileDHP1Apretreatmentshowedthesignificantprotectiveeffectsontheseantioxidantenzymes.Inparticular,theactivitiesofT-SOD,CATandGPxinthemicepretreatedwithDHP1Aat200mg/kgBWwereincreasedto248.18U/mgprot,140.08U/gprotand340.13U/mgprot,respectively,whichweresignifi-cantlyhigherthanthemicetreatedwithCCl4alone(pb0.01).
C.-C.Tianetal./Fitoterapia91(2013)247–255253
Fig.6.EffectsofDHP1AonCCl4-inducedoxidativestressinliver.MDA(A);GSH(B);T-SOD(C);CAT(D);GPx(E).Datawereexpressedasmean±SD(n=9–10).*,pb0.01ascomparedtothemodelgroup.
SilymarinpretreatmentalsoexhibitedobviousprotectiveeffectsonT-SODandGPx,buttheeffectsofdextranwerenotsignificant.4.Discussion
Duringthepastyears,theantioxidantactivitiesofnaturalpolysaccharideshadbeenreportedinmanyliteratures[32–34].Unlikethesyntheticantioxidants,mostofnaturalpolysaccha-rideswerelowtoxicity,thereforetheymightbeexploredasidealantioxidantagents.However,therealsoexistedafewoppositeviews.Wangetal.indicatedthattheradicalscavengingpotentialofthecrudeteapolysaccharides(TPS)wasattributedtoteapolyphenolsratherthanteapolysaccharides[35].Inthecurrentstudy,ouraimwastoelucidatewhetherapurifiedpolysaccharidefraction(DHP1A)obtainedfromD.huoshanensepossessedtheantioxidantactivities.Compositionanalysisindi-catedthatthetotalsugarcontentinDHP1Awasabout98.9%,andnoproteinexisted,soitsantioxidantactivityshouldbeattributedtothepolysaccharidecomponent.Moreover,DHP1Awasproventobeanovellow-molecularpolysaccharideaccordingtoitsstructuralcharacterization,whichwasdistinguishedfromthepolysaccharidesfoundinD.huoshanensepreviously.
Itiswellknownthattheantioxidantactivityofpolysaccha-rideisdependentonitsstructuralfeatures,suchasmolecularweight,glycosyllinkage,monosaccharidecompositionandconfiguration.Amongthem,themolecularweightwasproventobeanimportantparameter,andthepolysaccharidewitharelativelylowermolecularweightusuallyshowedthehigher
254C.-C.Tianetal./Fitoterapia91(2013)247–255
antioxidantactivity[36].Besides,somesubstitutedgroups,suchassulfate,acetylandphosphategroups,couldimprovetheantioxidantactivitiesofpolysaccharidesinvitro[37].Ontheotherhand,apolysaccharideusuallyshowedthedifferentantioxidanteffectswhenevaluatedindifferentexperimentalmodels.Luoetal.foundthatawater-solublepolysaccharidederivedfromDendrobiumnobilelindl.(DNP)hadahighscavengingeffectonABTSradicalsandanappropriatescavengingeffectonhydroxylradicals,butaweakscavengingeffectonDPPHradicals[38].Thisphenomenonpossiblyreflecteditsspecialantioxidantmechanism.Inthepresentstudy,theinvitroantioxidantactivitiesofDHP1Aweresignificantlyhigherthandextraninthesameconditions,possiblyduetoitsspecialstructuralfeatures.Moreover,thescavengingeffectofDHP1AonDPPHradicalswasrelativelyweakascomparedwithvitaminC,butithadaremarkableinhibitioneffectontheFeCl2-inducedlipidperoxidation.Theseresultsimpliedthattheanti-lipidperoxidationplayedacrucialroleintheantioxidantmecha-nismofDHP1A.
TheCCl4-inducedtissueinjuryisarepresentativeoxidativemodelthatcanbeusedtoevaluatetheantioxidantactivityofnaturalproducts[6,7].InthepathogenesisofCCl4,theexcessiveproductionoffreeradicals,suchasthetrichloromethylradical(•CCl3)andtrichloromethylperoxylradical(•CCl3O2•)derivedfromCCl4bythecytochromeP450system,leadstoarapidlipidperoxidationanddisturbsthesystemicredoxbalance[39].Inthisstudy,MDA,atypeofimportantlipidperoxide,obviouslyincreasedintheliversfollowingCCl4administration.Meanwhile,CCl4injectionloweredtheactivitiesofSOD,CATandGPx,aswellasthelevelofGSH,indicatingtheoccurrenceofoxidativestress.
Previousstudieshaddemonstratedthatsomepolysaccha-ridescouldalleviatetheCCl4-inducedoxidativestressasnaturalantioxidants.However,theunderlyingmechanism,sofar,wasstillnotclear.Xiaoetal.foundthatthepretreatmentwithLyciumbarbarumpolysaccharides(LBP)couldrestorethemRNAexpressionlevelsofGPx,CATandCu/ZnSODintheCCl4-treatedmice[7].Panaxginsengpolysaccharides(Ginsan)showedashort-termpotentiationeffectontheactivityofhemeoxygen-ase(HO),whichwasanimportantantioxidantenzymeinlivers[40].Shimetal.alsodemonstratedthatthepretreatmentwithginsanenhancedthelevelofGSH,andtheproteinexpressionsofMn-SOD,CATandGPxinlivers[6].Therefore,theanti-oxidativestressofpolysaccharidesmaybeassociatedwiththeirmodula-tioneffectsonthesystemicantioxidantdefense.Inthisstudy,themicepretreatedwithDHP1AshowedasignificantresistanceagainsttheoxidativestresscausedbyCCl4,inwhichtheantioxidantenzymes(substance)remainedinarelativelyhigherlevel.
Basedontheseresults,DHP1A,wesupposed,shouldbeconsideredasapolysaccharidefractionwithantioxidantactiv-ities,anditpossiblyplaysanimportantroleinthetherapeuticeffectsofD.huoshanense.Furthermore,itisofgreatinterestforustoimprovetheisolationyieldofDHP1Aviaoptimizingtheextractionprocess,andinvestigateitshepatoprotectiveeffectsinournextresearches.5.Conclusion
Insummary,apurifiedpolysaccharidefraction(DHP1A)wasobtainedfromD.huoshanense,anditsstructuralcharac-teristicwasdelineatedbythediversespectralmethods.DHP1A
wasmainlycomposedofMan,GlcandatraceofGal,withamolecularweightof6700Da.Moreover,DHP1Awasproventopossessaremarkableinhibitioneffectonthelipidperoxidationinvitro,anditcouldalleviatethehepaticoxidativestresscausedbyCCl4inmice,viarestoringthesystemicantioxidantdefense.Conflictofinterest
Wedeclarethatwehavenoconflictsofinteresttothispapersubmitted.Acknowledgments
Wefirstlythankourcolleaguesfortheirassistanceinthecourseoftheresearch.ThisstudywasfinanciallysupportedbytheNationalNaturalScienceFoundationinChina(GrantNos.21006019and20872024)andtheProjectforScienceandTechnologyResearchPlanfromAnhuiProvinceofChina(12010402088).References
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