Antioxidant and free radical scavenging activities of Phellinus merrillii extracts

a_{Theseauthorscontributedequallytothiswork.} *Correspondingauthor:E-mail:[email protected]; Tel:+886-4-22030380;Fax:+886-4-22083362.

INTRODUCTION

Itiscommonlyacceptedthat,inasituationofoxidative stress,reactiveoxygenspecies,suchassuperoxide(O._2), hydroxyl(OH._{)andperoxyl(}._OOH,ROO._{)radicals,are} generated.Thereactiveoxygenspeciesplayanimportant rolerelatedtothedegenerativeorpathologicalprocesses ofvariousseriousdiseases,suchasaging(Burnsetal., 2001),cancer,coronaryheartdisease,Alzheimer’sdisease (Smithetal.,1996;Diazetal.,1997),neurodegenerative disorders,atherosclerosis,cataracts,andinflammation (Aruoma,1998).Traditionalmedicineiswidespreadand plantsstillpresentsalargesourceofnaturalantioxidants thatmightserveasleadsforthedevelopmentofnovel drugs. Several anti-inflammatory, digestive, anti-necrotic, neuroprotective,andhepatoprotectivedrugshaverecently

Antioxidant and free radical scavenging activities of

Phellinus merrillii extracts

Heng-YuanCHANG

1

_,Yu-LingHO

2

_{,Ming-JyhSHEU}

3

_{,Yaw-HueiLIN}

4

_{,Mu-ChuanTSENG}

5

_,

Sheng-HuaWU

6

_{,Guan-JhongHUANG}

1,a

_{,andYuan-ShiunCHANG}

1,7,a,

_*

1_{Institute of Chinese Pharmaceutical Sciences, College of Pharmacy, China Medical University, Taichung 404, Taiwan} 2_{Department of Nursing, Hung Kuang University, Sha Lu, Taichung 433, Taiwan}

3_{Department of Physiology, School of Medicine, College of Medicine, China Medical University, Taichung 404, Taiwan} 4_{Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan}

5_{Bureau of Food and Drug Analysis, Department of Health, Executive Yuan,161-2 Kun-yang St., Nangang District, Taipei}

115, Taiwan

6_{Department of Botany, National Museum of Natural Science, Taichung 404, Taiwan} 7_{Chinese Crude Drug Pharmacy, China Medical University Hospital, Taichung 404, Taiwan}

(ReceivedMay22,2007;AcceptedJuly26,2007)

ABSTRACT. ThisstudyaimedtoinvestigatepossibleantioxidantactivityofvariousextractsofPhellinus

merrillii (PM). The exploreditemsinclude:ABTSfreeradicalscavenging assay,determinationoftotal

phenolicscontents(TPC), ferricreducingantioxidantpowerassay(FRAP),rapidscreeningofantioxidantby dot-blotDPPH(1,1-diphenyl-2-picrylhydrazyl)staining,DPPHradical-scavengingactivitiesandreducing powermeasurement.IntheABTSfreeradicalscavenging assay,then-BuOHfractiondisplayedthehighest totalantioxidantactivity(17.13±0.04mM).Inthedeterminationoftotalphenolicscontents(TPC)andferric reducingantioxidantpowerassay(FRAP),theEtOAcfractionhadthehighestphenolicscontents(46.21± 0.02mM)andreducingantioxidantpower(19.09±0.03mM).Intherapidscreeningofantioxidantbydot-blotDPPHstaining,then-BuOHfractionshowedthehigheststrongdot-blotstaining.Inthereducingpower measurement,thecrudeextracthadthehighestreducingpowerat2mg/mlconcentration.IntheDPPH radical-scavengingactivities,theEtOAcfractionhadthehighestantioxidantactivity(IC50=0.66±0.01mg/ ml). As regard the correlation coefficients among ABTS assay, FRAP assay, and total phenolics contents, it can be seen that correlation coefficients in each case were significant. Among all extracts, the highest amount oftotalphenolicscontentswerefoundintheEtOAcfractiont.ItissuggestedthatthePMmightcontribute itsantioxidantactivitiesonEtOAcandn-BuOHfraction.Inhigh-performanceliquidchromatographytandem mass(LC/MS/MS)analysisforhispolon,thedaughterionscannedchromatogramsofPMwasestablished. BothhispolonandPMshowedsimilardaughterionspectrumattheretentiontimeof4.7minandhadmore lobesinm/z219andm/z135.ThisindicatedthatPMdidcontaintheactiveingredienthispolon.BoththeIC50 of DPPH radical scavenging activity for hispolon and BHT were 42.4 ± 2.9 and 81.2 ± 3.2 μM, respectively. These findings mean that hispolon was most important in antiradical activities. It was suggested that hispolon mightcontributetoitsantioxidantactivitiesinPM.

Keywords: 1,1-diphenyl-2-picrylhydrazyl(DPPH);Ferricreducingantioxidantpowerassay(FRAP);

Freeradicals;Glutathionereducedform (GSH);High-performanceliquidchromatographytandemmass (LC/MS/MS);Phellinus merrillii (PM);Scavengingeffect;Totalantioxidantcapacity;Totalphenolicscontents (TPC);Troloxequivalentantioxidantcapacity(TEAC).

beenshowntohaveanantioxidantand/oranti-radical scavengingmechanismaspartoftheiractivity(Linand Huang,2002;RepettoandLlesuy,2002).Inthesearch forsourcesofnaturalantioxidantsandcompoundswith radicalscavengingactivityduringrecentyears,somehave beenfound,suchasechinacosideinEchinaceaeroot(Hu andKitts,2000),anthocyanin(Espinetal.,2000),phenolic compounds(Rice-Evansetal.,1997),waterextracts ofroastedCassia tora(YenandChuang,2000),whey proteins(Tongetal.,2000),andthioredoxinhprotein fromsweetpotato(Huangetal.,2004). Theantioxidantspresentindietarymushroomsare ofgreatinterestaspossibleprotectiveagentstohelp thehumanbodyreducesoxidativedamagewithoutany interference(AdamsandWermuth,1999).Nowthey arerecognizedasfunctionalfoodsandasasourceof physiologically beneficial components (Wasser and Weis, 1999).Mushroomshavebeenshowntoboosthearthealth; lowertheriskofcancer;promoteimmunefunction;ward offviruses,bacteria,andfungi;reduceinflammation; combatallergies;andhelpbalancebloodsugarlevels andsupportthebody’sdetoxificationmechanism(Ada et al., 2005). Mushrooms have also been shown to accumulateavarietyofsecondarymetabolitesincluding phenoliccompounds,polypeptides,terpenes,steroids,etc. Mushroomphenolicshavebeenfoundtobeanexcellent antioxidantandsynergist(Lietal.,2005).Furthermore, several companies are developing capsules from combinationsofmushrooms,andthesecapsules,although expensive,havebeenshowntobehealthbeneficial, including fighting against cancer (Mau et al., 2005).

Macrofungi was commonly used as a nutrition supplementstoavarietyofdiseasesinAsia(Jongand Birmingham,1992;Chenetal.,2006a).InTaiwan,several differentspeciesofPhellinus werewidelyappliedfor anticancer,antioxidantpurposesandhepatoprotective effects.Phellinuslinteusdemonstratedanti-tumoractivity inseveralstudies(Linetal.,2003;Kimetal.,2003;Li etal.,2004;Baeetal.,2005).Also,therewereseveral reportsabouttheantioxidanteffectsfrom Phellinus.The methanolicextractofthebasidiocarpsof Phellinus linteus demonstratedantioxidativeeffect(Chungetal.,1998)and antimutagenicactivities(SohnandNam,2001).Studies indicatedthatPhellinus linteuscouldprotectprimary culturedrathepatocytesagainsthepatotoxins(Kimet al.,2004).AlsoPhellinus rimosus(Berk)Pilatpossess antioxidantandantihepatotoxicactivities(Ajithand Janardhanan,2002). Phytochemicals,especiallyphenolicsinfruitsand vegetables,aresuggestedtobethemajorbioactive compoundsforhealthbenefits.Phenolicsareoneofthe groupsofnonessentialdietarycomponentsthathave beenassociatedwiththeinhibitionofatherosclerosisand cancer.Thebioactivityofphenolicsmayberelatedto theirabilitytochelatemetals,inhibitlipoxygenase,and scavengefreeradicals(Mallavadhanietal.,2006;Linet al.,2005). Hispolonandhispolonderivativeswereisolatedfrom thefungusPhellinus igniarius (Moetal.,2004). Hispolon, a yellow pigment was first found in Inonotus hispidusin 1996(Alietal.,1996b).Hispolonhasbeenreportedto exhibitapoptosiseffectonhumanepidermoidKBcells (Chenetal.,2006b)andantivirusactivities(Awadhetal., 2003).Hispolonalsoinhibitchemiluminescenceresponse ofhumanmononuclearcellsandsuppressmitogen-inducedproliferationofspleenlymphocytesofmice(Ali etal.,1996a). NoreportontheantioxidantactivitiesofPMwas presentlyavailable.Inthiswork,wereportedthatPM displayedantioxidantactivitiesinaseriesofin vitro testssuchastotalantioxidantactivity,determination of total phenolics contents (TPC), ferric reducing antioxidantpowerassay(FRAP),DPPH(1,1-diphenyl-2-picrylhydrazyl)staining,DPPHradical-scavengingactivity andreducingpowermethod.

MATERIALS AND METHODS

Materials

1,1-Diphenyl-2-picrylhydrazyl(DPPH),potassium peroxodisulfate (K2S2O8), tris (hydroxylmethyl) aminomethane, glutathione reduced form (GSH), potassiumferricyanide(K3Fe(CN)6),trichloroacetic acid (TCA), ferric chloride (FeCl3), (+)-catechin, aluminumchloridehexahydrate(AlCl3·6H2O),rutin, sodium bicarbonate (Na2CO3), sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4),2,4,6-tris(2-pyridyl)-s-triazine(TPTZ)were purchasedfromSigmaChemicalCo.(St.Louis,MO USA).Folin-Ciocalteusolution,methanolandethanol werepurchasedfromMerck.Trolox(6-hydroxy-2,5, 7,8-tetramethychroman-2-carboxylicacid),ABTS[2, 2´-azinobis(3-ethylbenzothiazoline)-6-sulfonicacid] diammoniumsaltwerepurchasedfromRoche.Hispolon waspurchasedfromBJYMPharm.&Chem.Co.Ltd. (Beijing,China).Phellinus merrilliiwaspurchasedfrom the Ji Pin mushroom store (Nantou, Taiwan) and identified byDr.Yu-ChengDai (InstituteofAppliedEcology, ChineseAcademyofScience,China).

Extraction of Phellinus merrillii

1.5kgofPMwassoakedwith5lof70%ethanolat roomtemperature.Thesampleswerefilteredwithfilter paper(AdvantecNo.1,Japan)whiletheresiduewas furtherextractedunderthesameconditionsthreetimes. Thefiltratescollectedfromtheseseparateextratcions werecombinedandevaporatedtodrynessundervacuum. Thecrudeextract(60g)wasdissolvedintowaterand formedassuspension.Hexane250mlwasaddedintothe suspensionandmixedthoroughly.Then,hexaneandwater layerswereseparated.EtOAcaddedintothewaterlayer wasmixedwellandwereseparated.Finally,n-BuOHwas mixedwithwaterlayerandformedn-BuOH,watersoluble andwaterinsolublefractions(Figure1).

ABTS free radical scavenging assay TotalantioxidantstatusofthePMwasmeasuredusing 2,2´-azinobis[3-ethylbenzthiazoline]-6-sulfonicacid (ABTS)assay(Reetal.,1999).ABTSwasdissolvedin deionizedwaterto7mMconcentration,andpotassium persulphateaddedtoaconcentrationof2.45mM.The reactionmixturewaslefttostandatroomtemperature overnight(12~16h)inthedarkbeforeuse.Theresultant intensely-colouredABTS•+_{radicalcationwasdiluted} with0.01MPBS(phosphatebufferedsaline),pH7.4,to giveanabsorbancevalueof~0.70at734nm.Thetest compoundwasdiluted100×withtheABTSsolution toatotalvolumeof1ml.Absorbancewasmeasured spectrophotometricallyattimeintervalsof1minafter additionofeachextract.Theassaywasperformedatleast in triplicate. Controls containing 990 μl of PBS, to replace ABTS,wereusedtomeasureabsorbanceoftheextract themselves.Theassayreliesontheantioxidantcapability of the samples to inhibit the oxidation ofABTS to ABTS•+_{radicalcation.Thetotalantioxidantactivitieswere} expressedasmMtroloxequivalentantioxidantcapacity (TEAC).

Determination of total phenolics contents (TPC) Totalphenolicscontentsweredeterminedusingthe Folin-Ciocalteumethod(RagazziandVeronese,1973). OnemLoftheextractwasaddedto10.0mldistilledwater and2.0mlofFolin-Ciocalteuphenolreagent(Merck-Schuchardt,Hohenbrun,Germany).Themixturewas allowedtostandatroomtemperaturefor5minandthen 2.0mlsodiumcarbonatewasaddedtothemixture.The resultingbluecomplexwasthenmeasuredat680nm. ThecontentsofphenolicscontentswereexpressedasmM troloxequivalentantioxidantcapacity(TEAC)(Houetal., 2004).

Ferric reducing antioxidant power assay (FRAP) Theferricreducingantioxidantpowerassay(FRAP) ofeachstandardsolutionwasmeasuredaccordingtoa modified protocol developed by Benzie and Strain, 1996. TopreparetheFRAPreagent,amixtureof0.1Macetate buffer(pH3.6),10mMTPTZ,and20mMferricchloride (10:1:1,v/v/v)wasmade.To1.9mlofreagentwasadded 0.1mlofextract.Readingsattheabsorptionmaximum (593nm)weretakenevery15susingaShimadzuUV-visible2501spectrophotometer,andthereactionwas monitoredforupto10min.Troloxsolutionwasusedto performthecalibrationcurves.Thereducingantioxidant powerwereexpressedbythemethodofBenzieand Strain,1996,asmMtroloxequivalentantioxidantcapacity (TEAC).

Rapid screening of antioxidant by dot-blot and DPPH staining EachdilutedsampleofthePMwascarefullyloaded ontoa20cm×20cmTLClayer(silicagel60F254; Merck)andallowedtodry(3min).Dropsofeachsample wereloaded,inorderofdecreasingconcentration(2,1, 0.5,0.25,and0.125mg/ml),alongtherow.Thestaining ofthesilicaplatewasbasedontheprocedureofSoler-Rivasetal.,2000.Thesheetbearingthedryspotswas placedupsidedownfor10sina0.4mMDPPHsolution. Thentheexcessofsolutionwasremovedwithatissue paperandthelayerwasdriedwithahair-dryerblowing coldair.Stainedsilicalayerrevealedapurplebackground withwhitespotsatthelocationwhereradical-scavenger capacitypresented.Theintensityofthewhitecolor dependsupontheamountandnatureofradicalscavenger presentinthesample(Huangetal.,2004).

Determination of antioxidant activity by reducing power measurement

ThereducingpowersofthePMandglutathionewere determinedaccordingtothemethodofYenandChen, 1995.ThePM(0,0.125,0.25,0.5,1.0,and2.0mg/ml) andglutathione(0,0.125,0.25,0.5,1.0,and2.0mg/ml) weremixedwithanequalvolumeof0.2Mphosphate buffer,pH6.6,and1%potassiumferricyanide.The mixturewasincubatedat50°Cfor20min,duringwhich periodferricyanidewasreducedtoferrocyanide.Then anequalvolumeof1%trichloroaceticacidwasaddedto themixture,whichwasthencentrifugedat5,000gfor 10min.Theupperlayerofthesolutionwasmixedwith distilledwaterand0.1%FeCl3ataratioof1:1:2,andthe absorbanceat700nmwasmeasuredtodeterminethe amountofferricferrocyanide(prussianblue)formed. Increasedabsorbanceofthereactionmixtureindicated increasedreducingpowerofthesample(Huangetal., 2005).

Scavenging activity against DPPH radical

TheeffectofcrudeextractsontheDPPHradical wasestimatedaccordingtothemethodofYamaguchi et al., 1998. An aliquot of crude extract (30 μl) and Figure 1.ExtractionandfractionationschemeofPhellinus

glutathione (GSH) (0.5 mg/ml, 30 μl) were mixed with 100 mM Tris-HCl buffer (120 μl, pH 7.4) and then with 150 μl of the DPPH in ethanol to a final concentration of 250 μM. The mixture was shaken vigorously and left tostandatroomtemperaturefor20mininthedark. Theabsorbanceat517nmofthereactionsolutionwas measuredspectrophometrically.ThepercentageofDPPH decolourizationofthesamplewascalculatedaccordingto thefollowingequationas:%decolourization=[1-ABS sample/ABScontrol]×100.The50%inhibition(IC50)of antioxidantactivitywascalculatedastheconcentrations ofsamplesthatinhibited50%ofscavengingactivityof DPPHradicalsactivityundertheseconditions(Huanget al.,2007).

Analysis of hispolon and PM by LC/MS/MS LC/MS/MSwasconductedtoanalyzeboththestandard (hispolon)andthe70%ethanolcrudeextractofPM accordingtoChenetal.,2006a,b;Laietal.,2006. Its puritywasmorethan95%basedonreversedphaseLC/ MS/MSanalysis(Instrument:MicromassQuattroUltima tandemmass;HPLC:Waters2695AllianceLC&996 PDAwithAutomaticLiquidSamplerandInjector;Data processor:MassLynxNTQuattroDataAcquisition). Analyseswerecarriedouttousingnegativemode electrospray ionization of LC/MS/MS analysis. ChromatographicseparationwasperformedonaC18 column(Cosmosil5-C18, 150 × 4.6 mm, i.d., 5 μm) underanisocraticelutionofamixedsolventsystem inacompositionofeach70%ofmethanoland30%of water at flow rate of 0.5 ml/min. A full UV spectrum was scannedfrom200to400nm.Thesourceanddesolvation temperaturewassetat120°Cand350°C,respectively. Allprocessesofthecapillaryvoltagesetting3kV,cone voltagesetting100Vandcollisionenergysetting15eVfor hispolon[M-H]-_{fragment(m/z=219.8)daughterionscan.} Thedaughterionspectrumobtainedwillbecomparedwith theLC/MS/MSlibrarydatabase. Statistical analysis

Data are expressed as mean ± S.E.M. Means of triplicateanalyseswerecalculated.TheStudent’sttestwas usedforcomparisonbetweentwotreatments.Adifference wasconsideredtobestatisticallysignificantwhenp< 0.05.

RESULTS

Extraction and fractionation of Phellinus merrillii From1.5kgofPM,60gof70%ethanolextractwas obtained.Theyieldwas4%.Thecrudeextract(60g)was suspendedinwaterandpartitionedwithhexane,EtOAc, n-BuOHsequentiallytoyield3ghexanefraction(5%), 20gEtOAcfraction(33%),23gn-BuOHfraction(39%), 13gwatersolublefraction(21%)and1gwaterinsoluble fraction(2%)(Figure1).

Effect of the Phellinus merrillii on the total antioxidant capacity Theantioxidantcapacityofcrudeextract,hexane, EtOAc,n-BuOH,solublewaterandwaterinsoluble fractionsofPMwereevaluatedaccordingtotheABTS decolorationmethodandtheFRAPassay.Theirtotal phenoplicscontentswerealsodetermined.Theresults were shown in Figure 2. Inhibition of generation of the ABTS˙+_{ radical cation was the basis of the} spectrophotometricmethodsthathadbeenappliedtothe measurementofthetotalantioxidantactivitiesofsolutions ofpuresubstances,aqueousmixturesandbeverages.Total antioxidantactivitiesofseveraldifferentfractionsofPM (0.1 mg/ml) were measured using the ABTS˙+_method. Troloxwasusedaspositivecontrol.Thetotalantioxidant activitywasexpressedasTroloxmMbyreferencetothe Troloxstandardcalibrationcurve.Then-BuOHfraction displayedthehighesttotalantioxidantactivity(17.13± 0.04mMTEAC)(Figure2).Totalantioxidantcapacityof crudeandsubfractionsofPMwereevaluatedas16.29± 0.35mMforcrudeextract,16.10±0.31mMforEtOAc fraction,13.88±0.19mMforwaterinsolublefractionand 13.78±0.39mMforwatersolublefraction.Thehexane fractionhadthelowesttotalantioxidantcapacityof12.36 ±0.11mM.

Total Phenolics contents of fractions of Phellinus merrillii

Thetotalphenolicscontentsofcrudeextractand hexane, EtOAc, n-BuOH, soluble water and water insolublefractionswereexpressedasmMoftrolox equivalent.TheEtOAcfractionhadthehighestphenolics contentsof46.21±0.02mM,crudeextractandn-BuOH fractionshadthephenolicscontentsof36.32±0.01mM and44.95±0.01mM,waterinsolublefractionandhexane fractionhadthephenolicscontentsof8.19±0.01mM and9.71±0.01mM.Thewaterfractionhadthelowest phenolicscontentsof2.26±0.01mM (Figure2). Figure 2. TPC,ABTS,andFRAPassaysofvariousfractions includingcrudeextrat,hexane,EtOAc,n-BuOH,watersoluble andwaterinsolublefractions.Eachabsorbancevaluerepresented theaverageoftriplicatesofdifferentsamplesanalyzed. ABTS assay FRAP assay Total phenolics

Crude Hexane EtOAC n-BuOH H2O H2O insoluble

TEA

Ferric reducing antioxidant power assay (FRAP) Theferricreducingantioxidantpowerofcrudeextract andhexane,EtOAc,n-BuOH,watersolubleandwater insolublefractionswereexpressedasmMoftrolox equivalent.TheEtOAcfractionhadthehighestreducing antioxidantpowerof19.09±0.02mM,crudeextractand n-BuOHfractionhadthereducingantioxidantpowerof 12.00±0.01mMand13.98±0.02mM,waterinsoluble fractionandwaterfractionhadthereducingantioxidant powerof3.37±0.01mMand1.01±0.01mM.The hexanefractionhadthelowestreducingantioxidantpower of0.68±0.01mM(Figure2).

Relationship between total antioxidant activity (TEAC), total phenolics contents (TPC) and FRAP assay

The Correlation cofficients (R2_{)ofantioxidantcapacity} (TEAC),totalphenolicscontents,andFRAPassayof crudeextract,hexane,EtOAc,n-BuOH,watersolubleand waterinsolublefractionsofPMwereshowninFigure3A and3B. RelationshipbetweenABTSassay(TEAC)andTPC incrudeextract,hexane,EtOAc,n-BuOH,solublewater andwaterinsolublefractionswereshowninFigure3A(R2 =0.8145,p <0.05).RelationshipbetweenABTSassay (TEAC)andFRAPassayincrudeextract,hexane,EtOAc, n-BuOH,solublewaterandinsolublewaterfractionswere showninFigure3B(R2_{=0.7878,p <0.05).} Thecorrelationcoefficients(R2_{)valuesofTEAC} andtotalphenolicscontentsshowedhighercorrelation. Thehigherthetotalphenolicscontents,thehigherthe antioxidantactivitythefractionsexhibited.

Rapid screening of antioxidant by dot-blot and DPPH staining AntioxidantcapacityofthePMwaseye-detectedsemi-quantitativelybyarapidDPPHstainingTLCmethod. Eachdilutedcrudeextractandhexane,EtOAc,n-BuOH, watersolubleandwaterinsolublefractionswereapplied asadotonaTLClayerthatwasthenstainedwithDPPH solution(Figure4).Thismethodwastypicallybasedon theinhibitionoftheaccumulationofoxidizedproducts. Thegenerationoffreeradicalswasinhibitedbythe additionofantioxidantsandscavengingofthefreeradicals shiftedtheendpoint.Thereducedglutathionewasused asapositivecontrol.Initialfaintspotsappeared,and1h laterweakspotscouldbeobservedinsamplerow.This whitespotswithstrongintensityappearedquicklyatthe concentrationof0.25mg/mlofPMperapplication,and downtothedilutionat0.125mg/mlofthePM(Figure 4).IntheDPPHstaining,then-BuOHfractonshowedthe higheststrongdot-blotstaining. Figure 4.Dotblotassayofthecrudeandvariousfractionsof Phellinus merrilliionasilicasheetstainedwithaDPPHsolution inmethanol.ThecrudeandfractionsofPM(2,1,0.5,0.25,and 0.125mg/ml)wereappliedfromlefttorightinsamplerow; GSH(2,1,0.5,0.25,and0.125mg/ml)wereappliedfromleftto rightincontrolrow. Figure 3. Theantioxidantcapacityofvariousextractsincluding crudeextract,hexane,EtOAc,n-BuOH,watersolubleand waterinsolublefractionsofPhellinus merrilliiwereevaluated

accordingtotheABTSdecolorationmethodandtheFRAP assay. (A) correlation coefficient between TPC and ABTS assay; (B) correlation coefficient between FRAP assay and ABTS.

Total phenolic contents (Trolox mM)

Ferric reducing ability (Trolox mM)

TEA C (mM) TEA C (mM) R2 _{= 0.8145 (p < 0.05)} R2 _{= 0.7878 (p < 0.05)} Crude extract Hexane EtOAc n-BuOH H2O soluble H2O insoluble GSH 2 1 0.5 0.25 0.125 (mg/ml)

Measurement of reducing power

WeinvestigatedtheFe3+_-Fe2+_{transitiontomeasure} thePM’sreducingcapacity.Thereducingcapacityof acompoundmayserveasasignificantindicatorofits potentialantioxidantactivity(Meiretal.,1995).The antioxidantactivitiesofputativeantioxidantshavebeen attributedtovariousmechanisms,amongwhichare preventionofchaininitiation,bindingoftransitionmetal ioncatalysts,decompositionofperoxides,preventionof continuedhydrogenabstraction,andradicalscavenging (Diplock,1997).ThereducingpowerofPMwasshownin Figure5,withGSHasapositivecontrol.PMexhibiteda dosedependentreducingpoweractivitywithintheapplied concentrations(0,0.125,0.25,0.5,1.0,and2.0mg/ml). Thereducingpowerofcrudeextractand hexane,EtOAc, n-BuOH,watersoluble,waterinsolublefractionandGSH werecompared. Thecrudeextracthadthehighestreducing powerat2mg/mlconcentration.However,hexanefraction showedlowestreducingpower(Figure5).

Scavenging activity against DPPH radical

The DPPH radical had been used widely in the modelsystemtoinvestigatethescavengingactivitiesof severalnaturalcompoundssuchasphenoliccompounds, anthocyanins,orcrudeextractsofplants.DPPHradical wasscavengedbyantioxidantsthroughthedonation ofhydrogen,formingthereducedDPPH-H•_.Thecolor changedfrompurpletoyellowafterreduction,whichcan be quantified by its decrease of absorbance at wavelength 517nm.Table1showedtheradical-scavengingactivityof thedifferentfractionsofPMandGSH,usingtheDPPH coloringmethod.Itwasfoundin0.5mg/mlofEtOAc fractionofPMhadthehighestradical-scavengingactivity (IC50=0.66±0.01mg/ml),followedbyn-BuOHfraction (0.78±0.01mg/ml),crudeextract(0.81±0.02mg/ml), waterfraction(0.83±0.05mg/ml)andwaterinsoluble fraction(0.93±0.06mg/ml).Hexanefractionhadthe lowestradical-scavengingactivity(IC50=3.79±0.01mg/ ml).

Compositional analysis of hispolon and PM by LC/MS/MS Inhigh-performanceliquidchromatographytandem mass(LC/MS/MS)analysisforhispolon,thedaughter ionscannedchromatogramsofPMwasestablished.Both hispolonandPMshowedsimilardaughterionspectrum attheretentiontimeof4.7minandhadmorelobesinm/z 219andm/z135(Figure6).ThisindicatedthatPMdid containtheactiveingredientofhispolon.

Determination of the antioxidative activity of hispolon Weusedhispolontomeasureantioxidantactivityand BHTwasusedaspositivecontrol.BothIC50valuesof hispolonandBHTwere42.4±2.9and81.2±3.2mM, respectively,whenscavengingactivityofDPPHradicals (%)wasmeasured.Theseresultsdemonstratedthat hispolonexhibitedbetterantioxidativeactivitythanBHT (Table2).

DISCUSSION

Mushroomsarenutritionallyfunctionalfoodsand important sources of physiologically beneficial medicines. Theyproducevariousclassesofsecondarymetabolites withinterestingbiologicalactivitiesand,thus,havethe potentialtobeusedasvaluablechemicalresourcesfor drugdiscovery(Zjawiony,2004).Severalmushrooms belongingtothegeneraInonotusandPhellinus,such Table 1.DPPHradicalscavengingactivityofvariousfractions

ofPhellinus merrillii.

DPPHradicalscavengingactivityIC50(mg/ml) GSH 0.42±0.04 H2O 0.83±0.05 Crude 0.81±0.02 Hexane 3.79±0.01 EtOAc 0.66±0.01 n-BuOH 0.78±0.01 H2OInsoluble 0.93±0.06 Table 2.Antioxidantactivityofhispolon. ScavengingactivityofDPPHradicals(%), IC50 (μM) BHT(control) 81.2±3.2 Hispolon 42.4±2.9 Figure 5.Antioxidativeactivitiesofthecrudeextractand

variousfractionsofPhellinus merrillii(0,0.125,0.25,0.5,

1.0,and2.0mg/ml)weremeasuredbythereducingpower method.GSHwasusedasapositivecontrol.Eachabsorbance valuerepresentedtheaverageoftriplicatesofdifferentsamples analyzed. GSH Crude Hexane Et0Ac n-BuOH H2O H2O Insoluble

Δ 700 nm

Concentration(mg/ml)

asInonotus obliquua,Phellinus linteus,Phellinus ribis andPhellinus igniariushavebeenusedastraditional medicinesforthetreatmentofgastrointestinalcancer, cardiovasculardisease,tuberculosis,liverorheartdiseases, fester,bellyache,bloodygonorrhea,stomachailments, anddiabetes(Nakamuraetal.,2004).Polysaccharides, especially β-glucan, are considered to be responsible for theirbiologicalactivity.Theisolationofpolysaccharides derivedfrommedicinalmushroomsandtheirbiological activityhadbeenreported(VinogradovandWasser,2005). Interestingly,thesemushroomsoftencontainabundle ofyellowantioxidantpigmentswhicharecomposedof hispidinderivativesandpolyphenols. Theantioxidantactivityofmushroomextractswith strongerinhibitionoflipidperoxidationoccurringathigh concentrationsoftheextractsinmostcases(Cheungand Peter,2005).Thepossiblemechanismofantioxidant activityofmushroomextractsincludesscavengingoffree radicalspossiblythroughhydrogen-holdingcapacityand oxidationbyperoxyradicals(Alietal.,1996b).

Inthispaper,theresultsfromin vitro experiments demonstratedthatcrudeextract,n-BuOHandEtOAc fractionshadbetterantioxidantactivityinreducingpower method.Thecrudeextracthadthehighestreducingpower at2mg/mlconcentration.IntheDPPHstaining,the EtOAcandn-BuOHfractionssharedthesamepatterns withthecrudeextractandappearedaswhitespotswhen theyweredilutedfrom2.0to0.125mg/ml.IntheDPPH staining,then-BuOHfractionshowedthehigheststrong dot-blotstaining.IntheDPPHcolorimetricmethod,itwas foundthatEtOAcfractionofPMhadthehighestradical-scavengingactivities.IntheABTSfreeradicalscavenging assay,then-BuOHfractiondisplayedthehighesttotal antioxidantactivity.Totalphenolicscontentsdemonstrated thattheEtOAcfractionhadthehighestamountsoftotal phenoliccontentsthanotherfractions.Inferricreducing antioxidantpowerassay(FRAP),theEtOAcfraction exhibitedthehighestreducingpower.ABTSwithhigh correlationwereestablishedbetweentotalphenolics contentsandtotalantioxidantactivity(TEAC)assay.This data indicated that the correlation coefficients (R2_)values ofTEACandtotalphenolicscontentsshowedhigher correlation.Thehigherthetotalphenolicscontentsthe highertheantioxidantactivityofthefractions.Theex vivo orin vivoantioxidantactivityofPM shouldbestudiedin thenearfuture.

In LC/MS/MS analysis, the finger print chromatogram ofPMwasestablished.BothPMandhispolonshowed similarpeakattheretentiontimeof4.7minandhadmore lobesinm/z219andm/z135.ThisimpliedthatPMdid containtheactiveingredienthispolon.Hispolonwasbetter thanBHTintheIC50ofDPPHradicalscavengingactivity. These findings imply that hispolon was most important in antiradicalactivities.Itwassuggestedthathispolonmight contributetotheantioxidantactivitiesofPM. TherelativelyhighercontentsofphenolicsinPMmight explainthehighantioxidantproperty (BimlaandPunita, 2006).Positivecorrelationswereestablishedbetween totalphenolicscontentsinthemushroomextractandtheir antioxidantactivities,similartothoseobservedinLentinus

edodes andVolvariella volvacea (Cheungetal.,2003).

Polyphenoliccompoundshaveanimportantrole instabilizinglipidoxidationandareassociatedwith antioxidantactivity(Yenetal.,1993).Thephenolic compoundsmaycontributedirectlytoantioxidative action(Duhetal.,1999).Itissuggestedthatpolyphenolic compoundsmayexhibitinhibitoryeffectsonmutagenesis and carcinogenesis in humans, when up to 1.0 g is ingesteddailyfromadietrichinfruitsandvegetables sources(Tanakaetal.,1998).Theantioxidativeactivities couldbeascribedtothedifferentmechanismsexertedby differentphenoliccompoundsandduetothesynergistic effectsofdifferentcompounds.Theantioxidantspresent insixdifferentfractionsmayhavedifferentfunctional properties,suchasreactiveoxygenspeciesscavenging (quercetinandcatechin)(Hatanoetal.,1989),inhibitionof thegenerationoffreeradicalsandchain-breakingactivity, Figure 6.Comparisonofdaughterionscanned(ES-D219.8) chromatograms(A&B)ofhispolonstandardvs.sampleand eachofdaughterionspectrum(C&D),itindicatedthatsample containshispolon.

e.g.p-coumaricacids(Laranjinhaetal.,1995)andmetal chelation(Van-Ackeretal.,1998).Thesecompounds werenormallyphenoliccompounds,whichwereeffective hydrogendonors,suchastocopherols,flavonoids,and derivativesofcinnamicacid,phosphatidicandother organicacids. AlthoughethanolextractofPhellinus linteuswas showntoscavengedirectlythestableDPPHradical overaconcentrationrangeof10µg/ml(30.1±2.72% inhibition)to300µg/ml(85.5±4.2%inhibition). It scavengedthestableradicalDPPHinaconcentraction-dependentmanner.TheEC50valueofPhellinus linteus wascalculatedtobe22.07µg/ml,whereasthatofVitamin C,usedasapositivecontrol,was5.11 µg/ml(Songetal., 2003).TheydemonstratedthatethylacetateextractofP.

rimosus exhibited significant in vitroantioxidantactivity.

TheethylacetateextractofP. rimosusalsoshowedpotent anthepatotoxicactivityagainstCCl4-inducedacutetoxicity inratliver(AjithandJanardhanan,2002).Ourprevious studiesalsoshowedthesimilarresults. Inconclusion,PMpossessedantioxidantandfree radicalscavengingactivities.Aboveall,therearemany totalphenoliccompoundsinPMincludinghispolon. PMmightbeanagentwithanti-cancer,hepatoprotective andant-inflammatorypotentialsandshouldbefurther investigatedinthefuture. Acknowledgement.Theauthorsgratefullyappreciated

the support by Dr.Yu-Cheng Dai from Institute of AppliedEcology,ChineseAcademyofScience,China. Dr.DaihelpedusidentifyingtheauthenticityofPM. TheauthorsalsoliketothankChinaMedicalUniversity andCommitteeofChineseMedicineandPharmacy, DepartmentofHealth,ExecutiveYuan,TAIWANforthe grantsupport(CMU94-020;CCMP93-RD-037).

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梅里爾針層孔菌抽出物之抗氧化與自由基清除活性

張恆源

1

　何玉鈴

2

　許明志

3

　林耀輝

4

　曾木全

5

_

吳聲華

6

　黃冠中

1

　張永勳

1,7 1

_{中國醫藥大學藥學院中國藥學研究所}

2

_{弘光科技大學}

_護理系

3

_{中國醫藥大學醫學院醫學系生理學科}

4

_{中央研究院植物暨微生物學研究所}

5

_{行政院衛生署}

_{藥物食品檢驗局}

6

_{國立自然科學博物館植物組}

7

_{中國醫藥大學附設醫院藥劑部中藥局}

本研究主要的目的是研究梅里爾針層孔菌不同溶劑分層的萃取物之抗氧化活性。探討的方法如下：

_

ABTS自由基清除測定、測定總酚類含量、FRAP測試、快速DPPH（1,1二苯基2picrylhydrazyl）呈

色反應、

_{DPPH清除自由基活性和還原力等幾項。ABTS自由基清除測定中，在n-BuOH分層中，表現}

出最高的總抗氧化活性（

17.13±0.04mM）。測定總酚類含量和FRAP分析中，在EtOAc分層中，有

最高的酚類含量（

_{46.21±0.02mM）和還原性抗氧化力（19.09±0.03mM）。在快速DPPH呈色反應}

中，在

n-BuOH分層中，表現出最強的呈色反應。在DPPH清除自由基活性中，EtOAc分層中，有最強

的抗氧化活性，其

IC

50

為（

0.66±0.01mg/ml）。在還原力的測試中，粗抽出物中，在2mg/ml濃度中

有最高的還原力。在

ABTS測試、FRAP測試和總酚類含量相互之間的比較時其相關係數是有意義的。

在所有不同溶劑分層之萃取物之中，在

 EtOAc 有最高的總酚類含量。由結果可顯示梅里爾針層孔菌在

EtOAc、n-BuOH分層中具有較高的抗氧化活性。在高效液相層析質譜儀分析法中，梅里爾針層孔菌的

抽出物的指紋圖譜被建立，

hispolon與梅里爾針層孔菌的抽出物顯示出有相似的波峰與相同的4.7分鐘

的滯留時間，均獲得

_{m/z219與m/z135較大的裂片，這表示梅里爾針層孔菌的乙醇抽出物含有活性成}

份

hispolon。而hispolon和BHT的DPPH自由基清除之IC

50

為42.4±2.9和81.2±3.2µM，由此發現

hispolon對於抗氧化活性是非常重要的，因此表示hispolon在梅里爾針層孔菌中有助於抗氧化活性。

關鍵詞：

1,1二苯基2picrylhydrazyl(DPPH)；三價鐵還原性抗氧化力測試；自由基；谷胱甘肽；高效液

相層析串聯式質譜分析法；梅里爾針層孔菌；清除活性；總抗氧化力；總酚類含量；

_{Trolox相等性的抗}

氧化力。