Original Contribution

ANTIOXIDANT ACTIVITY APPLYING AN IMPROVED ABTS RADICALCATION DECOLORIZATION ASSAY

ROBERTA RE, NICOLETTA PELLEGRINI, ANNA PROTEGGENTE, ANANTH PANNALA , MIN YANG, and

CATHERINE RICE-EVANS

International Antioxidant Research Centre, Guy’s, King’s and St Thomas’ School of Biomedical Sciences, Kings College–Guy’sCampus, London SE1 9RT, UK

(Received4 August1998;Revised29 October1998;Accepted29 October1998)

Abstract—A method for the screening of antioxidant activity is reported as a decolorization assay applicable to bothlipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants.The pre-formed radical monocation of 2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•1) is generated byoxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants.The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cationabsorption are taken into account when determining the antioxidant activity. This assay clearly improves the originalTEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First,the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediaryradical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant testsystems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence theresults obtained with the improved system may not always be directly comparable with those obtained using the originalTEAC assay. Third, it is applicable to both aqueous and lipophilic systems. © 1999 Elsevier Science Inc.

Keywords—ABTS radical cation, Antioxidant activity, Polyphenol, Flavonoid, Hydroxycinnamate, Free radical,Oxidation, TEAC

INTRODUCTION

A number of assays have been introduced for the mea-surement of the total antioxidant activity of body fluids[1–6], food extracts [7–11], and pure compounds [7,12–16]. Each method relates to the generation of a differentradical, acting through a variety of mechanisms and themeasurement of a range of end points at a fixed timepoint or over a range (reviewed in refs 13 and 17). Twotypes of approach have been taken, namely, the inhibi-tion assays in that the extent of the scavenging by hy-drogen- or electron-donation of a pre-formed free radicalis the marker of antioxidant activity, as well as assaysinvolving the presence of antioxidant system during thegeneration of the radical.

Generation of the ABTS [2,29-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid)] radical cation [18]forms the basis of one of the spectrophotometric meth-ods that have been applied to the measurement of thetotal antioxidant activity of solutions of pure sub-stances [12,19,20], aqueous mixtures and beverages[7,8]. The original ABTS•1 assay was based on theactivation of metmyoglobin with hydrogen peroxide inthe presence of ABTS to produce the radical cation, inthe presence or absence of antioxidants. This has beencriticized on the basis that the faster reacting antioxi-dants might also contribute to the reduction of theferryl myoglobin radical. A more appropriate formatfor the assay is a decolorization technique in that theradical is generated directly in a stable form prior toreaction with putative antioxidants.

The improved technique for the generation ofABTS•1 described here involves the direct production ofthe blue/green ABTS•1 chromophore through the reac-tion between ABTS and potassium persulfate. This has

Address correspondence to: Professor Catherine Rice-Evans, Inter-national Antioxidant Research Centre, Guy’s, King’s and St Thomas’School of Biomedical Sciences, Kings College–Guy’s Campus, StThomas’s Street, London SE1 9RT, UK; Tel:144 0171-955-4240;Fax: 144 0171-955-4983.

Free Radical Biology & Medicine, Vol. 26, Nos. 9/10, pp. 1231–1237, 1999Copyright © 1999 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/99/$–see front matter

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absorption maxima at wavelengths 645 nm, 734 nm and815 nm, as reported previously [1,13,17], as well as themore commonly used maximum at 415 nm. Addition ofantioxidants to the pre-formed radical cation reduces itABTS, to an extent and on a time-scale depending on theantioxidant activity, the concentration of the antioxidantand the duration of the reaction. Thus the extent ofdecolorization as percentage inhibition of the ABTS•1

radical cation is determined as a function of concentra-tion and time and calculated relative to the reactivity ofTrolox as a standard, under the same conditions. Themethod is applicable to the study of both water-solubleand lipid-soluble antioxidants, pure compounds, andfood extracts.

MATERIALS AND METHODS

Trolox (Hoffman-La Roche) (6-hydroxy-2,5,7,8-tet-ramethychroman-2-carboxylic acid; Aldrich ChemicalCo., Gillingham, Dorset, UK) was used an antioxidantstandard. Trolox (2.5 mM) was prepared in ethanol or 5mM phosphate buffered saline, pH 7.4, (PBS), for use asa stock standard, as described previously [1]. Freshworking standards were prepared daily on dilution withethanol. ABTS, 2,29-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and potassium persul-fate (di-potassium peroxdisulfate) were obtained fromSigma-Aldrich (Poole, Dorset, UK) and HPLC gradeethanol from Rathburn Chemicals Ltd. (Walkerburn,Peebleshire, Scotland).

Hydroxycinnamates, anthocyanidins, and flavonoidswere obtained from Extrasynthese (Lyon-Nord, France),carotenoids,b-carotene and lycopene, from AOCS (Bit-terne, Hampshire), and ascorbic acid anda-tocopherolfrom Sigma-Aldrich (95% pure). Stock solutions of thecarotenoids were prepared in dichloromethane and con-centrations confirmed using the extinction coefficient.Stock solutions of flavonoids and hydroxycinnamateswere prepared by dissolution in ethanol and subsequentlydiluted in ethanol for introduction into the assay systemat concentrations within the activity range of the assay(1.5 mM to 15 mM final concentration). Anthocyanidinswere diluted in acidic ethanol pH 1.3 to a concentrationof 0.5 mM. Ascorbic acid and uric acid were prepared asstock solutions in 18 MV water to a concentration of 5mM, anda-tocopherol in ethanol at 2 mM. None of thesolvents interfere in the assay.

The antioxidant activity was assessed as describedbelow. Experiments were performed on the Hewlett-Packard spectrophotometer model HP 8453 (CheadleHeath, Stockport Cheshire, UK) fitted with peltier tem-perature control.

Assay protocol—decolorization assay in ethanolicsolution

ABTS was dissolved in water to a 7 mMconcentra-tion. ABTS radical cation (ABTS•1) was produced byreacting ABTS stock solution with 2.45 mM potassiumpersulfate (final concentration) and allowing the mixtureto stand in the dark at room temperature for 12–16 hbefore use (Fig. 1). Because ABTS and potassium per-sulfate react stoichiometrically at a ratio of 1:0.5, thiswill result in incomplete oxidation of the ABTS. Oxida-tion of the ABTS commenced immediately, but the ab-sorbance was not maximal and stable until more than 6 hhad elapsed. The radical was stable in this form for morethan two days when stored in the dark at room temper-ature. For the study of phenolic compounds and foodextracts, the ABTS•1 solution was diluted with ethanoland for plasma antioxidants with PBS, pH 7.4, to anabsorbance of 0.70 (60.02) at 734 nm and equilibrated at30°C. Stock solutions of phenolics in ethanol, carote-noids in dichloromethane and plasma antioxidants inwater were diluted such that, after introduction of a 10-ml aliquot of each dilution into the assay, they producedbetween 20%–80% inhibition of the blank absorbance.After addition of 1.0 ml of diluted ABTS•1 solution(A734nm5 0.7006 0.020) to 10ml of antioxidant com-

Fig. 1. Absorption spectrum of the ABTS radical cation.

1232 R. RE et al.

pounds or Trolox standards (final concentration 0–15mM) in ethanol or PBS the absorbance reading was takenat 30°C exactly 1 min after initial mixing and up to 6min. Appropriate solvent blanks were run in each assay.All determinations were carried out at least three times,and in triplicate, on each occasion and at each separateconcentration of the standard and samples. The percent-age inhibition of absorbance at 734 nm is calculated andplotted as a function of concentration of antioxidants andof Trolox for the standard reference data. The concen-tration-response curve for 5 sequentially and separatelyprepared stock standards of Trolox is illustrated in Fig. 2.

Determination of the molar extinction coefficient (e) ofABTS•1 at 734 nm

Dilutions of ABTS•1 solution, prepared as describedabove, were further diluted in ethanol and in ultra-purewater to give absorbance values of between 0.12 to 0.9 at415 nm (a dilution of between 1/50 and 1/400). The ratiobetween the absorbance at 415 nm and the absorbance at734 nm was calculated at 5 different dilutions. From thisratio and from the molar extinction coefficient ofABTS•1 at 415 nm (e 5 3.6 3 104 mol21l cm21)reported by Forni et al. [22], the extinction coefficient ofABTS•1 at 734 has been calculated in water as 1.53 104

mol21l cm21 6 549 (mean6 SD,n 5 9) and in ethanolas 1.63 104 mol21l cm21 6 606 (mean6 SD, n 5 8).Under the conditions used here for the preparation of theABTS•1, about 60% of the ABTS present was oxidizedto the radical cation form.

RESULTS AND DISCUSSION

The method described gives a measure of the antiox-idant activity of the range of carotenoids, phenolics, and

some plasma antioxidants, determined by the decoloriza-tion of the ABTS•1, through measuring the reduction ofthe radical cation as the percentage inhibition of absor-bance at 734 nm. Figure 3 illustrates the effects of theduration of interaction of specific antioxidants on thesuppression of the absorbance of the ABTS•1 radicalcation at 734 nm for Trolox, the standard referencecompound, compared with glutathione, uric acid, ascor-bic acid,a-tocopherol, and the flavonoid aglycone anti-oxidants, kaempferol, and cyanidin. The results demon-strate that the reaction with ABTS•1 is complete by 1min, except for cyanidin and glutathione that show afurther small inhibitory effect up to 4 min reaction.

The extent of inhibition of the absorbance of theABTS•1 is plotted as a function of concentration in orderto determine the TEAC, that can be assessed as a func-tion of time. The dose-response curve obtained by anal-ysis of a range of concentrations of antioxidant com-pounds, Trolox standards and selected food extracts, atselected time points in the reaction, 1, 4 and 6 min, insome cases, was plotted as the percentage inhibition ofthe absorbance of the ABTS•1 solution as a function ofconcentration of antioxidant (Fig. 4). The concentrationof antioxidant giving the same percentage inhibition ofabsorbance of the radical cation at 734 nm as 1 mMTrolox was calculated in terms of the Trolox equivalentantioxidant activity at each specific time-point. To cal-culate the TEAC, the gradient of the plot of the percent-age inhibition of absorbance vs. concentration plot forthe antioxidant in question is divided by the gradient ofthe plot for Trolox. This gives the TEAC at the specifictime point and the calculated results for the flavonoids,carotenoids, some plasma antioxidants, and a represen-tative fruit and beverage sample are given in Table 1.

The antioxidant activity can also be expressed interms of the total contribution to the antioxidant activity

Fig. 2. Concentration-response curve for the absorbance at 734 nm forABTS•1 as a function of concentration of standard Trolox solution.(Five separately prepared stock standard solutions6 SD.)

Fig. 3. The effects of time on the suppression of the absorbance of theABTS•1. Control ABTS•1 radical cation (}), Trolox 10 mM (1),vitamin C 12mM (2), a-tocopherol 15mM (F), kaempferol 6mM (■),cyanidin 5mM (Œ), reduced glutathione 12mM (✳), uric acid 6mM(✖).

1233ABTS•1 decolorization assay

over the time range studied by calculating the area underthe curve, derived from plotting the gradient of thepercentage inhibition / concentration plots as a functionof time of reaction. The ratio between the area under thecurve for the reaction of the specific antioxidant and thatfor Trolox gives the relative antioxidant activity (AUC),as in Fig. 5.

The comparison between the antioxidant activity de-termined from the AUC, and the TEAC values derivedfrom the decolorization assay at individual 1-min, 4-min,and 6-min time-points are tabulated relative to the orig-inal TEAC value obtained from the ferryl myoglobin/TEAC assay. All the selected phenolics (except del-phindin) demonstrate lower TEAC values with thedecolorization assay at the individual time-points of 1and 4 min reaction than those obtained with the originalmyoglobin/ABTS assay at 6 min. At 6 min the values areclose, excepting quercetin and cyanidin, among the most

reducing of the flavonoids [23], for which the values donot attain the levels as in the myoglobin/ABTS assaysystem. This is likely to be accounted for by the possi-bility that some interaction occurred in the previousassay of the polyphenols with ferryl myoglobin, prior tothe latter’s reaction with ABTS, and the complex natureof the procedure of the ferryl myoglobin assay in that theformation of the radical cation and its inhibition wereoccurring in the same time frame. Strube et al. [24]previously proposed this explanation for the higher val-ues obtained for quercetin in the ferryl myoglobin/ABTSassay. It should be noted that quercetin has a lower halfoxidation potential than luteolin, that is itself lower thankaempferol, due to the importance of the catechol struc-ture in the B ring as well as the reducing 3-hydroxylgroup on the unsaturated C ring adjacent to a carbonylgroup [23].

The results demonstrate the time-dependency of the

Fig. 4. The effects of concentration of the antioxidant on the inhibition of the ABTS•1. (A) Kaempferol (r2 5 0.966); (B) ascorbic acid (r2 5 1); (C)a-tocopherol (r2 5 0.995); (D) cyanidin (r2 5 0.997); (E) glutathione (r2 5 0.948); (F) uric acid (r2 5 1); (G) Trolox (r2 5 1); (H) orange juice (r2

5 0.993).

1234 R. RE et al.

reaction and the influence of the selected time-point ofmeasurement on the reported antioxidant activity; thusthe determinants of the antioxidant activity are the extentof reduction and rate of reduction of the radical. Forexample, whereas caffeic acid and kaempferol demon-strate the lower extent of inhibition than ferulic acid andluteolin, respectively, the reactions of the former areessentially complete after 1 min reaction. Flavonoidsvaried in the range of times over which the reaction tookplace (Fig. 5). Whereas most phenolics had completedthe reaction at 4 min, some compounds especially luteo-lin and naringenin were still reacting. Expressing theresults as area under the curve can take these factors intoaccount.

The major improvement in the assay for lipophiliccompounds such as carotenoids is the design improve-ment incorporating the radical cation and the antioxidantboth in the lipophilic phase. The reaction between thecarotenoids and ABTS•1 is essentially complete after 1min, little further reaction taking place thereafter. The

antioxidant activity of lycopene was of the same order asobtained using previous methodology that produced theradical cation using manganese dioxide as oxidant [20].The value forb-carotene was significantly higher. Thismethod improves the assay also on the grounds thatapplication of manganese dioxide as oxidizing agent caninvolve molecular chemistry with the potential to pro-duce a two electron oxidation of ABTS to the radicaldication, that limits its definition and applicability.

The antioxidant activities of the plasma antioxidants,ascorbic acid,a-tocopherol, and uric acid, as well as thatof glutathione, are shown in Table 1. The TEAC valuesobtained are close to those obtained by myoglobin/ABTSassay [1,13], with the latter two being slightly higher.

There are differences between the TEAC values forthe flavonoids and hydroxycinnamates at 1 min, 4 minand 6 min by the ABTS•1 decolorization assay comparedwith the myoglobin/ABTS assay monitored at 6 min. Thelatter assay involved continuous formation of the ABTSradical cation from ferryl myoglobin, derived from met-

Table 1. Comparison Between the Antioxidant Activity as TEAC (mM) at Specific Time-Points

CompoundsAUC Persulfate

Decolorization Assay

TEAC Persulfate Decolorization AssayTEAC Myoglobin/ABTS

Decolorization Assay

1 min 4 min 6 min 6 min

HydroxycinnamatesFerulic acid 1.756 0.04 1.696 0.04 1.846 0.06 1.906 0.05 1.906 0.02p-Coumaric acid 1.566 0.04 1.516 0.03 1.826 0.05 2.006 0.07 2.226 0.06Caffeic acid 0.996 0.05 0.996 0.05 0.986 0.06 NC 1.266 0.01

Flavon-3-olsQuercetin 2.886 0.01 2.776 0.02 3.036 0.02 3.16 0.05 4.726 0.10Kaempferol 1.026 0.06 1.026 0.07 1.026 0.06 NC 1.346 0.08

FlavonesLuteolin 1.496 0.03 1.296 0.04 1.766 0.03 2.066 0.03 2.106 0.05

FlavanonesNaringenin 0.726 0.07 0.586 0.09 0.896 0.05 1.146 0.08 1.536 0.05

AnthocyanidinDelphinidin 4.86 0.18 4.646 0.18 5.016 0.19 4.446 0.11Malvidin 1.806 0.06 1.766 0.12 1.856 0.09 NC 2.066 0.1Cyanidin 2.386 0.20 2.306 0.19 2.486 0.22 NC 4.46 0.12

Plasma antioxidantAscorbic acid 1.056 0.02 1.056 0.02 1.056 0.02 NC 0.996 0.04a-Tocopherol 0.906 0.00 0.896 0.05 0.976 0.06 NC 0.976 0.01Gluthatione 1.196 0.02 1.136 0.03 1.286 0.04 0.906 0.03Uric acid 1.016 0.06 1.006 0.06 1.016 0.06 NC 1.026 0.06

Carotenoidsb-Carotene 2.506 0.03 2.476 0.03 2.576 0.03 NC 1.96 0.01Lycopene 3.046 0.13 3.016 0.13 3.086 0.10 NC 2.96 0.1

Food extractsOrange juice

Blond (Ovale) 1.776 0.22 2.226 0.40 2.316 0.44TAA mmol/kg dry wt TAA mmol/kg dry wt

TomatoAqueous/methanol 18.006 0.41 16.726 0.41 19.876 0.20Lipophilic 6.706 0.21 6.506 0.21 7.026 0.21 NC

Applying the ABTS•1 decolorization assay (based on potassium persulfate), the value derived from the area under the time-dependency curve andthe original TEAC assay based on ABTS/myoglobin assay [19].

n 1 SD 5 $ 3, each performed in triplicate at 3 separate concentrations.NC 5 no change.

1235ABTS•1 decolorization assay

myoglobin and hydrogen peroxide in the presence of thereductants. Preliminary fast kinetic studies (data notshown) indicate a biphasic reaction with a very rapidinitial phase, presumably indicative of the most reducinggroups followed by a slower phase.

The AUC method is an alternative way to describe theantioxidant activity of compounds when taking into ac-count the varied rates of reaction of the antioxidants withABTS•1. The calculation of AUC is derived from bothantioxidant concentration and reaction time and is there-fore an overall measure of the abilities of the compoundsto scavenge free radicals compared to the standardTrolox during the specific time range, taking into accountthe variation in value with time.

The TEAC values are obtained from the capacity ofan individual antioxidant or a mixture to inhibit theABTS•1 at a defined time point, relative to Trolox. As ascreen for relative antioxidant activities of pure com-pounds or food extracts, the antioxidant activity referredto measurement at 4 min time point would seem to beappropriate.

Acknowledgements— We acknowledge financial support from theMinistry of Agriculture, Fisheries and Food (Contract ANO448), theEuropean Union Fair program FAIRCT965077 for funding NicolettaPellegrini. We thank Dr. Nicholas J. Miller (Oxford Drug Trials Unit)for his participation in the initial development of the assay.

REFERENCES

[1] Miller, N. J.; Rice-Evans, C. A.; Davies, M. J.; Gopinathan, V.;Milner, A. A novel method for measuring antioxidant capacityand its application to monitoring the antioxidant status in prema-ture neonates.Clin. Sci.84:407–412; 1993.

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[3] Cao, G.; Verdon, C. P.; Wu, A. H. B.; Wang, H.; Prior, R. L.Automated assay of oxygen radical absorbance capacity with theCobas Fara II.Clin. Chem.41:1738–1744; 1995.

[4] Ghiselli, A.; Serafini, M.; Maiani, G.; Azzini, E.; Ferro-Luzzi, A.A fluorescence-based method for measuring total plasma antiox-idant capability.Free Radic. Biol. Med.18:29–36; 1995.

[5] Lonnrot, K.; Metsa-Ketela, T.; Molnar, G.; Ahonen, J.-P.;Latvala, M.; Peltola, J.; Pietila, T.; Alho, H. The effect of ascor-bate and ubiquinone supplementation on plasma and CSF totalantioxidant capacity.Free Radic. Biol. Med.21:211–217; 1996.

[6] Wayner, D. D. M.; Burton, G. W.; Ingold, K. U.; Locke, S.Quantitative measurement of the total peroxyl radical-trappingantioxidant capability of human blood plasma by controlled per-oxidation. The important contribution made by human plasmaproteins.FEBS Lett.187:33–37; 1985.

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[10] Wang, H.; Cao, G.; Prior, R. L. Total antioxidant capacity offruits. J. Agric. Food Chem.44:701–705; 1996.

Fig. 5. Profile of the variation of gradient of the percent inhibition vs. concentration plot of each antioxidant at 1 min and 4 min usedto measure the area under the curve (AUC) for the range of polyphenols, hydroxycinnamates, carotenoids, and antioxidant vitamins.The antioxidant activity derived from the AUC plot is calculated from the ratio of the area under the curve for the specific antioxidantin question to that for Trolox. (A) Quercetin}; luteolin ■; kaempferolŒ; naringenin1; (B) delphinidin}; cyanidin■; malvidinŒ;(C) ascorbic acid}; a-tocopherol■; (D) ferulic acid}; p-coumaric acid■; caffeic acidŒ.

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[11] Whitehead, T. P.; Robinson, D.; Allaway , S.; Syms, J.; Hale, A.Effect of red wine ingestion on the antioxidant capacity of serum.Clin. Chem.41:32–35; 1995.

[12] Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Structure-antioxi-dant activity relationships of flavonoids and phenolic acids.FreeRadic. Biol. Med.20:933–956; 1996.

[13] Miller, N. J.; Rice-Evans, C. A. Total antioxidant status in plasmaand body fluids.Methods Enzymol.234:279–293; 1994.

[14] Miller, N. J.; Castelluccio, C.; Tijburg, L.; Rice-Evans, C. A. Theantioxidant properties of thioflavines and their gallate esters—radical scavengers or metal chelator?FEBS Letts.392:40–44;1996.

[15] Kono, Y.; Shibata, H.; Kodama, Y.; Sawa, Y. The suppression ofthe N-nitrosating reaction by chlorogenic acid.Biochem. J.312:947–953; 1995.

[16] Arnao, M. B.; Casas, J. L.; del Rio, J. A.; Acosta, M.; Garcia-Canovas, F. An enzymatic colorimetric method for measuringnaringin using 2,29-azinobis (3 ethylbenzothiazoline-6-sulfonicacid).Anal. Biochem.185:335–338; 1990.

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[18] Wolfenden, B. S.; Willson, R. L. Radical-cations as referencechromogens in kinetic studies of one-electron transfer reactions:pulse radiolysis studies of 2,29-azinobis-(3-ethylbenzthiazoline-6-sulphonate).J. Chem. Soc. Perkin Trans.2:805–812; 1982.

[19] Rice-Evans, C. A.; Miller, N. J.; Bolwell, G. P.; Bramley, P. M.;Pridham, J. B. The relative antioxidant activities of plant-derivedpolyphenolic flavonoids.Free Radic. Res.22:375–383; 1995.

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Rice-Evans, C. A. Antioxidant activities of carotenes and xantho-phylls. FEBS Lett.384:240–242; 1996.

[21] Miller, N. J.; Rice Evans, C. A. Factors influencing the antioxi-dant activity determined by the ABTS•1 radical cation assay.FreeRadic. Res.26:195–199; 1997.

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ABBREVIATIONS

AUC—area under curveABTS—2,29-azinobis(3-ethylbenzothiazoline 6-sulfonic

acid)TEAC—Trolox equivalent antioxidant activityTAA—total antioxidant activity

1237ABTS•1 decolorization assay