brand williams1995

Lebensm.-Wiss. u.-Technol., 28. 25-30 (1995) UseofaFreeRadicalMethodtoEvaluateAntioxidant Activity W.Brand-Wi l l i ams, M.E.CuvelierandC.Berset* LaboratoiredeChimiedesSubstancesNaturelles,DdpartementSciencedel' Aliment,ENSIA1,avenuedesOlympiades, 91305Massy(France) (Recei vedMarch11,1994:accept edJune28,1994) Tileantiradicalactivitiesofvariousantioxidantsweredeterminedusingthe freeradical,2.2-Diphenyl-l-pict3,1hydrazyl(DPPI-I). Inits radicalform,DPPI-Ihasanabsorptionbandat515 nmwhichdisappearsuponreductionbyanantiradicalcompound.Twenty compoundswerereactedwiththeDPPI-Iandshownto followoneofthreepossiblereactionkinetictypes.Ascorbieacid,isoascorbic acidandisoeugenolreactedquicklywiththeDPPI-Ireachingasteadystateimmediately.Rosmarinicacidand6-tocopherolreacteda littleslowerandreachedasteadystatewithin30 rain.Theremainingcompounds reactedmoreprogressivelywiththeDPPHreaching asteadystate f romIto6 h.Caffeicacid,gentisicacidandgallicacidshowedthehighestantiradicalactivitieswithastoichiometo,of4 to6reducedDPPHmoleculespet" moleculeofantioxidant.Vanillin,phenol,y-resort3'licacidandvanillicacidwere foundtobe poor antiradicalcompounds.Thestoichiometry, f ortheother13 phenoliccompoundsvaried fromonetothreereducedDPPHmoleculespet" moleculeofantioxidant.Possiblemechanismsareproposedtoexplainthee.werimentalresults. Introducti on Theoxidationoflipidsinfoodsisresponsibleforthe formationofoff-flavoursandundesirablechemicalcom- poundswhichmaybedetrimentaltohealth.Antioxidants areusedby thefoodindustryto delaytheoxidationprocess. Manydifferentmethods(1)havebeenusedtomeasurethe resistanceofalipidtooxidationwheninthepresenceof potentialantioxidants.Thesetestsaregenerallyperformed ineitheralipidoremulsionmedium.Autoxidationisa slow,radicalprocesswhichproceedsviaachainreaction includinginduction,propagationandterminationsteps. Duringtheinductionperiod,alkylradicalsareformed whichundergoreactionwithoxygenmoleculestoform hydroperoxidesandperoxideradicalsduringthepropaga- tionphase.Terminationproceedsviaassociationoftwo radicalstoformastableadduct. Themajorityoftestsareperformedbyshorteningthe inductionperiodof thechainreaction,eitherbyusinghigh temperatureoranincreasedoxygensupply.Fromthese tests,theantioxidativeactivitiesofanumberofpure compoundsandplantextractshavebeendeterminedby measuringtheoxygen consumptionor productionof hydro- peroxidesorotherdegradationproducts. In our laboratory, anacceleratedtest has been developed(2) whichfollowsthedisappearanceofmethyllinoleateusing gaschromatography. Recently,amethodusingadifferentapproachhasbeen citedintheliterature(3-6). Toevaluatetheantioxidative activityofspecificcompoundsorextracts,thelatterare allowedtoreactwithastableradical,2,2-Diphenyi- l-picrylhydrazyl(DPPH )inamethanolsolution.The * To whom correspondence should be addressed. 0023-6438/95/010025+ 06 $08.00/0 reductionofDPPH asindicatedbelowisfollowedby monitoringthedecreaseinitsabsorbanceatacharacteristic wavelengthduringthereaction.Initsradicalform,DPPH absorbsat515nm,butuponreductionbyanantioxidant (AH)oraradicalspecies(Re),theabsorptiondisappears. DPPH e+AH~DPPH-H+Ae DPPH +Re~DPPH-R Inthispaperwehaveattemptedtoexplaintheresults obtainedusingtheDPPH = methodfor anumber of phenolic compoundsaswellasascorbicandisoascorbicacids.The resultsarecomparedtothoseobtainedusingthemethyl linoleatetest(2). MaterialsandMethods Reagent s Themethanolusedwasofspectrophotometricgrade (990g/L)fromAnalyticalsCarloErba(Milano,Italy). 2,2-Diphenyl-l-picrylhydrazyl(DPPH 950g/kg),BHT (990g/kg),BHA(980g/kg),isoascorbicacid(980g/kg), ~,-resorcylic acid(980 g/kg)andisoeugenol(980 g/kg)were fromAldrich(StQuentinFallavier,France).~-tocopherol (900 g/kg)wasfromSigma(StQuentinFallavier,France). Ascorbicacid(997g/kg)eugenol,gallicacid(990g/kg), phenol(990g/kg)andguaiacolwerefromProlabo(Paris, France).Protocatechuicacid,gentisicacid,rosmarinicacid, ferulicacid,vanillicacid,zingeroneandcaffeicacidwere fromExtrasynth~se(Genay,France).Vanillin(990g/kg) wasaproductof Fluka. Apparat us AllspectrophotometricdatawereacquiredusingaUvikon 810Kontronspectrophotometer.Disposablecuvettes(1 cm (~)1995 AcademicPressLimited 25 l wt / vol . 28( 1995) No. 1 lcmx4. 5cm) fromMul l erRatiolab(Dreieich, Ger- many)wereusedforvisible absorbancemeasurements.Antiradicalactivitiesof potentantioxidantsbyDPPH Theantioxidant activities weredet ermi nedusingDPPHasa freeradical. Foreachantioxidant, differentconcentrations weretested(expressedasthenumberofmolesofanti- oxi dant / mol eDPPHe).Ant i oxi dant solutioninmethanol ( 0. 1mL) wasaddedto3. 9mLofa610--Smol/L methanolDPPH esolution.Thedecreaseinabsorbancewas det ermi nedat 515nmat 0mi n, 1 rainandevery15 minuntil thereaction' reachedaplateau.TheexactinitialDPPH* concentration(CDPPH)inthereactionmedi umwascalcu- latedfromacalibration curvewiththeequation, Abs515n m=12,509(CDPPH)--2.5810 -3.asdeter- minedbylinearregression. Foreachantioxidantconcentrationtested,thereaction kineticswereplotted(Fig. 1).Fromthesegraphs,theper- centageofDPPH eremainingatthesteadystatewasdeter- minedandthevaluestransferredontoanothergraphshow- ingthepercentageof residualDPPH eatthesteadystateasa functionof themolar ratio of antioxidanttoDPPH e(Fi g.2). Ant i radi cal activitywasdefinedastheamountofanti- oxi dant necessarytodecreasetheinitialDPPH econcentra- tionby50%(EfficientConcentration=ECs0((mol/L)AO/(tool/L)DPPHe).Forreasonsofclarity,wewillspeakin termsofI/ECs0ortheantiradicalpower(ARP):thelarger theARP, themoreefficienttheantioxidant.Nineteenpure compoundshavebeentested.Theirformulasaregivenin Fi g. 3. 120 I00! t~ '~80 .E .E =60 E "640 20 (a) moles ascorbicacid/ mole DPPH~ Amm 00.066 0. 099 ~~0. 201 AA0.304 ==- - 0. 49 - - 1I T~ I ,t10203040 Ti me ( mi n)I00 l(b)moles guaiacol/ tmole DPPH 80 "E0.25 20 1.27 Is l ,i 010020~300400 Time (min) Fig.1Examples of thetwoobservedtypes of reactionkinetics. (a)Kineticbehaviourofascorbicacid:(b)kineticbehaviourof guaiacol 100 -- 012 ECs0 Moles of zingerone/mole DPPH Fig. 2The disappearance of DPPH e as a function of the number of moles of zingeronc/mole DPPH" OH Z Comp~,undsXYZ PhenolHHH BHAHC(CHo,OCH~ BHTC(CHo~C(CHo~CH~ p-CoumancactdHHCH=CHCOOH VanillinHOCH~CHO VaBillic acidHOCH, COOH IsoeugenolHOCH 3CH=CH-CH:~ Ferulic acidHOCHaCH=CH-COOH EugenolHOCH 3CH 2CH=CH,ZingeroneHOCH~CH 2-CHzCOCH 3 GuaiacolHOCH ~H Proto,:alechuicacidHOHCOOH CaffetcacidHOHCH=CHCOOH Gallic acidOHOHCOOH Gentisic acidHCOOHOH CH2OHCH2OH H-C- OHHO- C- H !~N==~0H~O0OHCOOHHOOHHOOH ascorbi caci di soascorbi cacidy-resorcylicaci d HO~c OH O- C- CH=CH- ~OHo[ OHtr,l ' l ~HO--~CH2-CH-COOH CH3 5-tocopherolrosmarinicacid Fig. 3Chemical structuresof the tested compounds Resul tsandDi scussi on Antiradicalmeasurements Theevolutionofthedifferentreactionkineticsdependson thenatureoftheant i oxi dant beingtested.Threet ypesof behavi ourwereobserved. InFi g. l ( a) , anexampl eofrapid kineticbehavi ouris shown.Onl ythreeof the20compounds tested,includingascorbicacid,i soascorbi cacidandiso- eugenolreactedrapi dl ywiththeDPPH e,reachingasteady stateinlessthan1 min.Thesecondtypeofbehavi ourwas intermediateandconcernedonlyrosmarinicacidand~-to- copherol. Forthesereactions,thesteadystatewasreached 26 Iwt/vol. 2811995)No.1 afterapproxi mat el y5and30 minforrosmari ni cacidand ~-tocopherol, respectively. The15remai ni ngcompounds reactedmoresl owl ywiththeDPPH e.Anexampl eoftheir behavi our (i.e.guai acol ) is shownin Fi g. l ( b) , Thesesl ower kineticswereallhyperbol i ccurvestakinganywherefrom1 to6 htoreachasteadystate. Asindicatedinthemet hodssection,theantiradicalactivity waseval uat edfromtheplotofthepercentageDPPH remai ni ngwhenthekineticsreachedasteadystateasa functionof themol ar ratios of ant i oxi danttoDPPH(Fig.2). Incontrasttootherresearchers(4,6,7)whodet ermi nedthe ECs0after30minofreactiontime,theantiradicalactivities wereanal ysedatthesteadystate.Forthosecompounds whichreactrapi dl ywiththeDPPH eradicalnodifference wasobservedintheARPs. However, inthecaseofsl ower kineticbehaviour, anARPdet ermi nedat30 minwoul dbe erroneousbecausethereactionwoul dstillbeprogressi ng (i.e.forBHTARP30mi n=1.06andARP240min=5.30;for prot ocat echui cacidARP30mi n=4.0andARPi20min= 7.14).Itwasthereforedeci dedtoanal ysethedataatthe steadystate. Anot herwaytoanal ysetheantiradicalactivitycoul dbeto det ermi netheamount ofant i oxi dant necessarytodecrease theinitialDPPH econcent rat i onby100%(ECIo0).Inthis case,theclassification of theantiradical efficiencywoul dbe differentandcertaincompoundstested(i.e.coumari cacid andvanillin)neverreactwithmorethan75%oftheinitial DPPH e,evenafter7 hofreactiontimeandatveryhigh concentrations. Therefore, theclassificationoftheanti- radicalefficiencieshasbeenest abl i shedfromanECs0 det ermi nedasshowninFi g. 2.InTabl e1,allcompounds areclassifiedinincreasingorderofARPaccordi ngtotheir kineticbehaviour.React i onsstoichiometm.' Thest oi chi omet rywasobt ai nedbymul t i pl yi ngtheECs0of eachant i oxi dant bytwowhichgivesthetheoretical efficient concentrationof eachant i oxi dantneededtoreduce100%of theDPPH e.InTabl e1,thesevaluesarepresentedforall compoundstogetherwiththeirinversevalues(thenumber ofDPPH emolesreducedbyonemol eofantioxidant).Accordi ngtothesedata,thecompoundsthathaverapidor i nt ermedi at ekinetics,havest oi chi omet ri esthatcorrespond approxi mat el ytothenumberofhydrogensavai l abl efor donationonhydroxyl groupsexcept 8-tocopherol. One isoeugenolmol ecul ereducesoneDPPH emolecule. Ascor- bic acid andi soascorbi c acid eachreducenearlytwoDPPH e mol ecul esasisshowninthefol l owi ngreaction(8): CH. OHCH2OHCH2OH I-DPPH oIDPpH I HOCH OtHOCH O(HOCHO HOOHDPPH- HHOO DPPH- HOO I . - a s c or bi c a c i ds e mi -de hydr ode hydr o as c or bi c aci das c or bi c aci d TheARPvaluefoundbyLamai sonetal.(4, 9)forascorbic acidwas4slightlyhigherthanourvalueof3.7.This correspondstotworeducedDPPH emol ecul esper mol ecul e ofantioxidant. Ast oi chi omet ri cvalueof0.3wasdeter- mi nedforrosmarinicacidwhereasLamai sonetal.(4, 9) found0.25.Rosmari ni cacidhasfourhydroxyl groups whichcouldreducefourDPPH emolecules. ~-tocopherol hasast oi chi omet ryof 0.5,reducingtwoDPPH emolecules.However, itonlyhasoneavai l abl ehydroxyl group.A di meri zat i onmayoccurbetweentwotocopheroiradicals.Thenewcompoundformedwoul dthenbeabletoreducea secondDPPH emol ecul e(10). Fortheremai ni ng' sl ower' kineticreactions,thestoichio- metrywasmoredifficult tointerpret.Onl yferulicacid,with onehydroxyl group,reducesoneDPPH emolecule. Phenol, coumari cacid,vanillin,vanillicacidand7-resorcyl i cacid reactverypoorlywiththeDPPH e. Tabl e 1.behaviour Classification of antiradical efficiencies andstoichiometry, according tokinetic Rapid kinetic behaviour Intermediate kinetic behaviour Slowkinetic behaviour CompoundARPStoichiometricNumber of Valuereduced DPPH e Isoeugenol1.941.030.97 Ascorbic acid3.700.541.85 lsoascorbic acid3.700.541.85 ~-tocopherol40.502 Rosmarinic acid6.900.303.33 Phenol0.002270< 1 Coumaricacid0.0298