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Lebensm.-Wiss. u.-Technol., 28.25-30 (1995) Use of a Free Radical Method to Evaluate Antioxidant Activity W. Brand-Williams, M. E. Cuvelier and C. Berset* Laboratoire de Chimie des Substances Naturelles, Ddpartement Science de l'Aliment, ENSIA 1, avenue des Olympiades, 91305 Massy (France) (Received March 11, 1994: accepted June 28, 1994) Tile antiradical activities of various antioxidants were determined using the free radical, 2.2-Diphenyl-l-pict3,1hydrazyl (DPPI-I°). In its radical form, DPPI-I ° has an absorption band at 515 nm which disappears upon reduction by an antiradical compound. Twenty compounds were reacted with the DPPI-I ° and shown to follow one of three possible reaction kinetic types. Ascorbie acid, isoascorbic acid and isoeugenol reacted quickly with the DPPI-I ° reaching a steady state immediately. Rosmarinic acid and 6-tocopherol reacted a little slower and reached a steady state within 30 rain. The remaining compounds reacted more progressively with the DPPH ° reaching a steady state from I to 6 h. Caffeic acid, gentisic acid and gallic acid showed the highest antiradical activities with a stoichiometo, of 4 to 6 reduced DPPH ° molecules pet" molecule of antioxidant. Vanillin, phenol, y-resort3'lic acid and vanillic acid were found to be poor antiradical compounds. The stoichiometry, for the other 13 phenolic compounds varied from one to three reduced DPPH ° molecules pet" molecule of antioxidant. Possible mechanisms are proposed to explain the e.werimental results. Introduction The oxidation of lipids in foods is responsible for the formation of off-flavours and undesirable chemical com- pounds which may be detrimental to health. Antioxidants are used by the food industry to delay the oxidation process. Many different methods (1) have been used to measure the resistance of a lipid to oxidation when in the presence of potential antioxidants. These tests are generally performed in either a lipid or emulsion medium. Autoxidation is a slow, radical process which proceeds via a chain reaction including induction, propagation and termination steps. During the induction period, alkyl radicals are formed which undergo reaction with oxygen molecules to form hydroperoxides and peroxide radicals during the propaga- tion phase. Termination proceeds via association of two radicals to form a stable adduct. The majority of tests are performed by shortening the induction period of the chain reaction, either by using high temperature or an increased oxygen supply. From these tests, the antioxidative activities of a number of pure compounds and plant extracts have been determined by measuring the oxygen consumption or production of hydro- peroxides or other degradation products. In our laboratory, an accelerated test has been developed (2) which follows the disappearance of methyl linoleate using gas chromatography. Recently, a method using a different approach has been cited in the literature (3-6). To evaluate the antioxidative activity of specific compounds or extracts, the latter are allowed to react with a stable radical, 2,2-Diphenyi- l-picrylhydrazyl (DPPH °) in a methanol solution. The * To whom correspondenceshould be addressed. 0023-6438/95/010025 + 06 $08.00/0 reduction of DPPH ° as indicated below is followed by monitoring the decrease in its absorbance at a characteristic wavelength during the reaction. In its radical form, DPPH ° absorbs at 515 nm, but upon reduction by an antioxidant (AH) or a radical species (Re), the absorption disappears. DPPH e + AH ~ DPPH-H + Ae DPPH ° + Re ~ DPPH-R In this paper we have attempted to explain the results obtained using the DPPH = method for a number of phenolic compounds as well as ascorbic and isoascorbic acids. The results are compared to those obtained using the methyl linoleate test (2). Materials and Methods Reagents The methanol used was of spectrophotometric grade (990g/L) from Analyticals Carlo Erba (Milano, Italy). 2,2-Diphenyl-l-picrylhydrazyl (DPPH ° 950g/kg), BHT (990g/kg), BHA (980g/kg), isoascorbic acid (980g/kg), ~,-resorcylic acid (980 g/kg) and isoeugenol (980 g/kg) were from Aldrich (St Quentin Fallavier, France). ~-tocopherol (900 g/kg) was from Sigma (St Quentin Fallavier, France). Ascorbic acid (997g/kg) eugenol, gallic acid (990g/kg), phenol (990g/kg) and guaiacol were from Prolabo (Paris, France). Protocatechuic acid, gentisic acid, rosmarinic acid, ferulic acid, vanillic acid, zingerone and caffeic acid were from Extrasynth~se (Genay, France). Vanillin (990g/kg) was a product of Fluka. Apparatus All spectrophotometric data were acquired using a Uvikon 810 Kontron spectrophotometer. Disposable cuvettes (1 cm (~) 1995 Academic Press Limited 25

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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


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