*Correspondingauthor:E-mail:wchou@tmu.edu.tw;Fax: 886(2)2378-0134;Tel:886(2)3736-1661ext.6160.
INTRODUCTION
Yam (Dioscorea species) is a member of the monocotyledonousfamilyDioscoreaceaeandisastaple foodinWestAfrica,SoutheastAsia,andtheCaribbean (Akoruda,1984).Yamisrecognizedasanherbalplant sincethetuberdriedsliceshavefreqentlybeenusedas Chineseherbalmedicines.Thetuberstorageproteins of yam, dioscorin, exhibited carbonic anhydrase, trypsininhibitoractivities(Houetal.,1999a;Houet al.,2000),andbothdehydroascorbatereductaseand monodehydroascorbatereductaseactivities(Houetal., 1999b).Changetal.(2004)reportedthatChineseyam(D. alatacv.TainongNo.2)feedinghadantioxidanteffectsin hyperhomocysteinemiarats.However,thecomponentin thefreeze-driedpowderofChineseyamwasresponsible forthisactivitywasnotclear.Wereportedpreviouslythat thestorageproteins(dioscorin)fromJapaneseyam(D. batatas)exhibitedscavengingactivitiesagainstDPPHand hydroxylradicals(Houetal.,2001).Wealsoreported that the crude and purified mucilages from Japanese yam exhibitedantioxidantactivities(Leeetal.,2003).We discoveredthisbytestinganti-DPPHandanti-hydroxyl radicals,reducingpowers,andanti-lipidperoxidation activities(Houetal.,2002). Liaoetal.(2004)usednearinfraredFouriertransform Ramanspectroscopytoanalyzethesecondarystructureof dioscorinfromthreeyamspecies[D. Japonica(Japanese yam), D. alata L., and D. alata L. var. purpurea]. Althoughthedioscorinfromthreespecieshasasimilar molecularmass,theaminoacidcompositionsandthe secondarystructureofdioscorinfromD. alataL.,and D. alataL.var.purpureawereapparentlydifferentfrom thoseofD. Japonica(Japaneseyam).Dioscorinfrom D. JaponicahadlowercontentsofCys,Ile,Lys,and totalessentialaminoacidsthandidtheothertwoyam species.ThedioscorinfromD. alataL.,D. alataL.var. purpurea,andD. Japonicaexhibitedstructuresmostly oftheα-helix,antiparallelβ-sheet,mixedα-helix,and antiparallelβ-sheettypes,respectively.Thesedifferences amongyamspeciesmightresultindifferentbiological activities.Inthisworkweuseddioscorinfromtwo differentyamspecies,Chineseyam(D. alatacv.Tainong No. 1,TN1) and Japanese yam (D. batatas Decne, importedfromJapan,JP),andcomparedtheirantioxidant activity, using DPPH radical and hydroxyl radical scavengingactivityassay,reducingpowertest,anti-lipid peroxidationtest,DNAdamageprotection,andinhibition ofdihydrorhodamine123oxidationbyperoxynitrite. Theresultsshowedthatdioscorinsfromtwospecies exhibiteddifferentscavengingactivitiesevenwithheating 100ºCfor5minagainstDPPHandhydroxylradicals. ThepeptichydrolysatesofTN1dioscorinwereseparated
Comparisons of in vitro antioxidant activities of storage
proteins in tuber of two Dioscorea species
Yuh-HwaLIU
1,Hong-JenLIANG
2,Huey-ChuanCHENG
3,Yen-WennLIU
4,andWen-ChiHOU
4,*
1Division of Gastroenterology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, TAIWAN
2Department of Food Science, Yaunpei University of Science and Technology, Hsinchu 300, TAIWAN
3Mackay Memorial Hospital, Taipei 104, and Mackay Medicine, Nursing and Management College, Taipei 112, TAIWAN 4Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, TAIWAN
(ReceivedNovember8,2005;AcceptedMarch1,2006)
Abstract. Dioscorin was purified by DE-52 ion exchange chromatography from two yam species, Dioscorea alataL.cv.Tainong1(TN1)andDioscorea batatasDecne(importedfromJapan,JP).Bydifferentin vitroantioxidanttests,includingDPPHradicalandhydroxylradicalscavengingactivityassay,areducing powertest,ananti-lipidperoxidationtest,DNAdamageprotection,andinhibitionofdihydrorhodamine123 oxidationbyperoxynitrite,itwasshownthatdioscorinsfromthetwospeciesexhibiteddifferentscavenging activitiesagainstDPPHandhydroxylradicals,evenafterheating100˚Cfor5min.DioscorinsfromTN1were hydrolyzedbypepsinfordifferentdurationsandthepeptichydrolysatesexhibitedDPPHradicalscavenging activities. Peptic hydrolysates separated by Sephadex G-50 (F) gel filtration were tested for anti-DPPH radical activity.Resultsshowedthatfractionsofsmallermolecularweightstillhaveantioxidantactivities.
bySephadexG-50(F)columnandwerethenanalyzed foranti-DPPHactivity.Itwasfoundthatthesmaller molecularweightfractionsstillhadantioxidantactivities.
MATERIALS AND METHODS
Material
Peroxynitrite was obtained from Calbiochem-NovabiochemCo.(Darmstadt,Germany).Calfthymus DNA(activated,25A260units)waspurchasedfrom AmershamBiosciences(Uppsala,Sweden).Hydrogen peroxide(33%)wasfromWakoPureChemicalIndustry (Osaka,Japan).Reducedglutathione,dihydrorhodamine 123(DHR123),1,1-diphenyl-2-picrylhydrazyl(DPPH), 2-deoxy-ribose,andotherchemicalsandreagentswere purchasedfromSigmaChemicalCo.(St.Louis,MO, USA).
Dioscorin extractions and purifications from TN1 and JP yam
Tubersoffreshyams, D. alata L.cv.TainongNo.1 (TN1)andD. batatasDecne(JP),werepurchasedfrom TaipeiAgriculturalProductsMarketingCo.,Wanhua District,Taipei.Afterwashingandpeeling,thetuberswere cutintostripsfordioscorinextractionandpurification. Afterextractionandcentrifugation,dioscorinswere purified from crude extracts successively by a DE-52 ion exchangecolumnaccordingtothemethodsofHouetal. (2001).Theconcentrateddioscorinsolutionwasdialyzed againstdeionizedwaterovernightandlyophilizedfor furtheruse.
DPPH radical scavenging activity of dioscorin TheDPPHradicalscavengingactivityofdioscorin withorwithoutheatingat100ºCfor5minandpeptic hydrolysatesfromTN1andJPyamsweremeasured accordingtothemethodofHouetal.(2001,2002).Every 0.6mLdioscorinsolution(10,20and40nanomoles)was addedto0.05mLof1MTris-HClbuffer(pH7.9)and thenmixedwith0.6mLof50µMDPPHinmethanolfor 20minunderlightprotectionatroomtemperature.The absorbanceat517nmwasmeasured.Deionizedwaterwas usedasablankexperiment.Thescavengingactivityof DPPHradicals(%)wascalculatedfollowingtheequation: (A517blank-A517sample)÷A517blank×100%.
Scavenging activity of dioscorin against metal ion-dependent hydroxyl radicals
Thehydroxylradicalscavengingactivityofdioscorin withorwithoutheatingat100ºCfor5minfromTN1 andJPyamswasdeterminedbythedeoxyribosemethod (Halliwelletal.,1987).Every0.5mlsamplecontaining differentamountsofdioscorin(0.357,1.785,3.57,7.14, and17.85nanomole)wereaddedto1.0mlsolutionof 20mMpotassiumphosphatebuffer(pH7.4),2.8mM 2-deoxy-ribose,104µMEDTA,100µMFeCl3,100µM ascorbate,and1mMhydrogenperoxide.Themixtures wereincubatedfor1hat37ºC.Afterincubation,anequal volumeof0.5%thiobarbituricacidin10%trichloroacetic acidwasaddedandthemixtureswereboiledat100ºCfor 15min.Deionizedwaterwasusedasablankexperiment. Theabsorbanceat532nmwasmeasured.Thescavenging activityofhydroxylradicals(%)wascalculatedwiththe equation:(A532blank-A532sample)÷A532blank×100%. The reducing power of dioscorin
Thereducingpowerofdifferentamounts(1.785,3.57, 7.14,and17.85nmole)ofdioscorinfromTN1andJP yamsina1.25mLsamplesolutionwasmeasuredbyferric chloride-potassiumferricyanidemethods(YenandChen, 1995)atawavelengthof700nm.Deionizedwaterwas usedasablankexperiment,andthereducedglutathione (4.69,9.37,10.07,and18.74nmole)wasusedapositive control.Increaseofabsorbanceofthereactionmixtureat awavelengthof700nm(∆A700nm)indicatesanincrease ofreducingpower.
Anti-linoleic acid peroxidation of dioscorin Theantiperoxidationactivityof5µg(0.018nmole) dioscorinfromTN1andJPyamsagainst2.5mLof0.02 Mlinoleicacidemulsionatintervalsafterreactionsat 37ºCwasmeasuredbythethiocyanatemethod(Phamet al.,2000).Atintervalsduringincubation,a0.1-mLaliquot ofthereactionmixturewasmixedwith4.7mLof75% ethanol,0.1mLof30%ammoniumthiocyanate,and0.1 mLof20mMferrouschloridein3.5%hydrochloricacid. Precisely3minaftertheadditionofferrouschloridetothe reactionmixture,theabsorbanceatawavelengthof500 nmwasdetermined.Deionizedwaterwasusedinacontrol experiment.Increaseofabsorbanceatawavelengthof 500nmindicatesadecreaseofantioxidantactivityagainst linoleicacidperoxidation.
Protecting hydroxyl radical-induced damages of calf thymus DNA by TN1 dioscorin
ThehydroxylradicalwasgeneratedbyFentonreaction accordingtothemethodofKohnoetal.(1991).The45µL reactionmixtureincludedTN1dioscorin(20,50,100,and 200µg),15µLofcalfthymusDNA,18mMFeSO4,and 60mMhydroxygenperoxideatroomtemperaturefor15 minor30min.TenµLof10mMEDTAwasaddedtostop thereaction.TheonlycalfthymusDNAwasusedforthe blanktest,andthecontroltestwaswithoutTN1dioscorin additions.Afteragarosegelelectrophoresis,thetreated DNAsolutionswerestainedwithethidiumbromideand observedunderUVlight. Protecting peroxynitrite-mediated DHR 123 oxidation by TN1 dioscorin Theprotectionagainstperoxynitrite-mediatedDHR 123oxidationwasaccordingtothemethodsofKooyet al.(1994).Thetotal175µlreactionmixtureincluded differentamountsofTN1dioscorin(9,12,and16µg), 10µMDHRand10µlperoxynitritein50mMphosphate
buffer(pH7.4),containing90mMNaCl.Aftera10-min reaction,thefluorescentintensitywasmeasuredatthe excitationandemissionwavelengthsof473and520nm, respectively,andatexcitationandemissionslitwidths of2.5nmand3.0nm,respectively.Thecontroltestwas withoutdioscorinadditions.
Determination of the DPPH scavenging activity of peptic hydrolysates of TN1 dioscorin
The7mgTN1dioscorinwasdissolvedin1mL,0.1M KCl-HClbuffer(pH2.0).The0.1mL,14mgpepsinwas addedforhydrolysisat37ºCfor8,12,24and32h.After hydrolysis,0.5mLof0.5MTris-HClbuffer(pH8.3)was addedandheatedat100ºCfor5mintostophydrolysis. Thepepsinwasheatedbeforedioscorinhydrolysisfor zerohourreaction.Eachdioscorinhydrolysatewasused fordeterminationofDPPHscavengingactivity.
Chromatograms of peptic hydrolysates of TN1 dioscorin on Sephadex G-50 column and their DPPH scavenging activities ThepeptichydrolysatesofTN1dioscorinat8,12,24 and32hwerelyophilizedandseparatedbySephadex G-50(F)chromatography(1×75cm).Thecolumnwas elutedwith20mMTris-HClbuffer(pH7.9).Flowrate was30mL/h,andeachfractioncontained2mL.Each fractionwasdeterminedattheabsorbanceof210nmfor peptidecontentsandforDPPHscavengingactivity(570 nm). Statistical analysis Student’st-testwasusedforcomparisonsbetween controlandeachexperimentaltest.Adifferencewas considered statistically significant when P<0.05(*)orP< 0.01(**).
RESULTS AND DISCUSSION
Owingtothedifferentaminoacidcompositionsandthe secondarystructuresofdioscorinfromChineseyamand Japaneseyam(Liaoetal.,2004),theantioxidantactivity ofthedioscorinfromtwodifferentyamspecies,Chinese yam(D. alatacv.TainongNo.1,TN1)andJapanese yam(D. batatasDecne,importedfromJapan,JP),was compared.OurresultsshowthatdioscorinfromChinese yam(TN1yam)hadhigherantioxidantorscavenging activitiesthandidJapaneseyam(JPyam)accordingtothe DPPHradicalandhydroxylradicalscavengingactivity assay,reducingpowertest,anti-lipidperoxidationtest. Figure1showsthedioscorinscavengingactivity againstDPPHradicalfromTN1andJPwithorwithout heatingat100ºCfor5min.WhenDPPHradicalwas scavenged,thecolorofthereactionmixturechanged frompurpletoyellowwiththedecreaseofabsorbance atwavelength517nm.Itwasfoundthatthedioscorin fromTN1andJPexhibiteddose-dependentDPPHradical scavengingactivity(Figure1).Underthesame40nmole ofdioscorin(28kDa),thescavengingactivityofJP dioscorindecreasedfrom46%to34%afterheatingat 100ºCfor5min.However,anactivationofscavenging activity(from49%to56%)wasfoundinTN1dioscorin. DioscorinfromD. Japonicawasfoundtohaveacontent ofCysroughlytenfoldlowerthantheothertwoTaiwanese yamspecies(Liaoetal.,2004).TheCysinpatatin(Liu etal.,2003)andinsweetpotatoTIs(Houetal.,2005) wasreportedtocontributetoDPPHscavengingactivities. Itwasproposedthattheheatingprocesscouldaffect JPdioscorinstabilityandreducetheDPPHscavenging activity.However,itmightalsoexposeotherinnerCys residuesinTN1dioscorinandelevateTN1dioscorin scavengingactivity. Figure2showsthedioscorinscavengingactivity againsthydroxylradicalfromTN1andJPwithorwithout heatingat100ºCfor5min.ThedioscorinfromTN1and JPwasalsofoundtoexhibitdose-dependenthydroxyl radicalscavengingactivity,evenafterheatingat100ºC for5min(Figure2).Underthesame17.85nmoleof dioscorin(28kDa),thescavengingactivityofTN1(62%) washigherthanthatofJP(48%).Theheatingprocessing affectedthehydroxylradicalscavengingactivityin dioscorinsfromtwoyamspecies.Thehigherhydroxyl radicalscavengingactivityofthedioscorinfromTN1 mightbeduepartlytothedifferentcontentsofTrpsince dioscorinfromD. JaponicahadlessTrp(intensityratio ofRamanspectra,I878/759)thantheothertwoTaiwanese yamspecies(Liaoetal.,2004).TheTrpinpatatin(Liu etal.,2003)andinsweetpotatoTIs(Houetal.,2005) wasreportedtocontributetohydroxylradicalscavenging activities.Waterspinachconstituentswerealsoreportedto haveantioxidantactivity(Huangetal.,2005).Theheating processcouldaffectdioscorinstabilityandreduced hydroxylradicalscavengingactivity. Figure 1.ThescavengingactivityagainstDPPHradicalof dioscorinfromTN1andJPyamswithorwithoutheatingat 100ºCfor5min.Meansoftriplicatesweremeasured.Deionized waterwasusedasablankexperiment.Thescavengingactivity ofDPPHradical(%)wascalculatedaccordingtothefollowing equation:(A517blank-A517sample)÷A517blank×100%.
ThereducingpowerofdioscorinfromTN1andJP isshowninFigure3.Thereducedglutathionewasused asapositivecontrol.TN1dioscorinexhibitedadose-dependentreducingpoweractivitywithintheapplied concentrations(1.785,3.57,7.14,and17.85nmole)and hadhigheractivitiesthanglutathioneunderthesame nmoleconcentrationbasis.ThereducingpowerofTN1 dioscorinwasabouttenfoldthatofJP(Figure3),which wascomparabletothereportedratioofCyscontentin eachyamspecies(1.18±0.13vs0.13±0.07mg/gprotein, Liaoetal.,2004).
The products of lipid peroxidation (such as malondialdehyde)couldcausedamagetoproteinsand DNA(Esterbaueretal.,1991).Theanti-lipidperoxidation of5µgdioscorinfromTN1andJPisshowninFigure4. BoththedioscorinfromTN1andJPcouldretardlinoleic acidperoxidationduringtheintervalsof8hat37ºC comparedtothatofthecontrol.DioscorinfromTN1 andJPwereabletoeffectretardationsoflinoleicacid peroxidationthatwereabout15.60and8.3-foldofcontrol, respectively,inthe12hreaction. Liaoetal.(2004)reportedtheCyscontentofdioscorin inChineseyamwasabouttenfoldthatofJapaneseyam. ThesecondarystructuresofdioscorinfromD. alataL. (Chineseyam)andD. Japonica(Japaneseyam)were mostlyα-helix,amixedα-helixtype,andanantiparallel β-sheettype,respectively.Fromourpresentresults, underthesameweightbasis,dioscorinfromChineseyam (TN1)hadhigherantioxidantorscavengingactivities thanJapaneseyam(JPyam)basedonDPPHradicaland hydroxylradicalscavengingactivityassay,areducing powertest,andananti-lipidperoxidationtest.These resultsmightbeattributabletothedifferentaminoacid compositionsandproteinconformations.Therefore, TN1dioscorinwasfurtherstudiedinthefollowing Figure 3.ThereducingpowerofdioscorinfromTN1andJP yam(1.785,3.57,7.14,and17.85nmole).Theglutathione(4.69, 9.37,14.07,and18.74nmole)wasusedasapositivecontrol. Meansoftriplicateweredeterminedattheabsorbanceof700 nm.
Figure 5.TheeffectsofTN1dioscorinontheFe2+-mediated
DNAoxidation.LaneBwasnativecalfthymusDNA;laneC wasmetal-mediatedoxidizedDNA;andlanes1to4weremetal-mediatedoxidizedDNAwith20,50,100,and200µgTN1 dioscorinadditions.Thereactionwasstoppedafter15minor30 minbyadding10mMEDTA.Afterelectrophoresis,thegelwas stainedwithethidiumbromideandobservedunderUVlight. Figure 4. TheeffectsofdioscorinfromTN1andJPyamon theanti-linoleicacidperoxidation.Eachof5µgdioscorinwas addedtotheemulsionandincubatedat37ºCfor0,2,4,and8h. Ateachtimeinterval,0.1mLwaspickedandwasdetermined bythiocyanidemethod(A500nm). Figure 2.Thescavengingactivityagainsthydroxylradical ofdioscorinfromTN1andJPyamwithorwithoutheatingat 100ºCfor5min.Meansoftriplicatesweremeasured.Deionized waterwasusedasablankexperiment.Theabsorbanceat532 nmwasmeasured.Thescavengingactivityofhydroxylradicals (%)wascalculatedwiththeequation:(A532blank-A532sample)÷
Figure 7. TheDPPHscavengingactivity(A)andchromatogramsonSephadexG-50(F)column(B)ofpeptichydrolysatesofTN1 dioscorin.TN1dioscorinwashydrolyzedbypepsinat37ºCfor8,12,24and32h.Afterhydrolysis,0.5mLof0.5MTris-HClbuffer (pH8.3)wasaddedandheatedat100ºCfor5mintostophydrolysis.Thepepsinwasheatedbeforedioscorinhydrolysisforzero hourreaction.EachdioscorinhydrolysatewasusedfordeterminationsofDPPHscavengingactivityandwasseparatedbySephadex G-50(F). Figure 6. TheTN1dioscorin(9,12,and16µg)protected peroxynitrite-mediated dihydrorhodamine123oxiadtion.The total175µlreactionmixtureincludeddifferentamountsof TN1dioscorin,10µMDHR,and10µlperoxynitritein50mM phosphatebuffer(pH7.4)containing90mMNaCl.After10min reaction, the fluorescent intensity was measured at the excitation andemissionwavelengthsof473and520nm,respectively, andexcitationandemissionslitwidthsof2.5nmand3.0nm, respectively.Thecontroltestwaswithoutdioscorinsadditions. Adifferencebetweenthecontrolandtheexperimentaltestwas considered statistically significant when p<0.05(*)orp<0.01 (**).
experimentofDNAdamageprotection,inhibitionof dihydrorhodamine 123 oxidation by peroxynitrite, and DPPH scavenging activity of dioscorin peptic hydroxylates. FreeradicalscandamagemacromoleculesinDNA, proteinandthelipidcellsinmembranes(Halliwell,1999). Figure5showsTN1dioscorinprotectedagainsthydroxyl radical-inducedcalfthymusDNAdamagein15or30 min.TheonlycalfthymusDNAwasusedforablank test(laneB),andthecontroltest(laneC)waswithout TN1dioscorinadditions.Comparedtotheblanktestand controltest,itwasfoundthattheaddedTN1dioscorin above50µg(lane2)couldpreventhydroxylradical-inducedcalfthymusDNAdamageinboth15minor30 minreactions.
Peroxynitrite is formed from a nearly diffusion-limitedreactionbetweennitricoxideandsuperoxide anionandasaninitiatorofpotentiallyharmfuloxidation reaction(Brannanetal.,2001).TheresultsofFigure6 demonstratethattheprotectiveeffectofperoxynitrite- mediatedDHRoxidationofTN1dioscorinwasdose-dependent. Significant variation was observed among the peroxynitrite,peroxnitrite+12µgTN1dioscorin(p< 0.05),andperoxynitrite+16µgTN1dioscorin(p<0.01).
Figure7(A)showstheDPPHscavengingactivity ofTN1 dioscorin hydrolysates at different pepsin hydrolysistimes.AstheresultsofFigure7makeclear, thescavengingactivityagainstDPPHradicalincreased from7.1%(0h)toabout21%(32h).Figure7(B)shows thechromatogramsofpepticdioscorinhydrolysatesof8, 12,24and32honSephadexG-50chromatography.The smallerpeptideswerefoundtoincreasewithincreasing pepsinhydrolytictimeandalsoexhibitedDPPHradical scavengingactivities.Thepurificationsofantioxidant peptideswillbeinvestigatedinthefuture.
Acknowledgments.The authors want to thank the
financial support (SKH-TMU-94-03) from Shin Kong Wu Ho-SuMemorialHospital,Taipei,Taiwan.
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