Antioxidant activity applying an improved ABTS radical cation decolorization assay

Free Radical Biology & Medicine, Vol. 26, Nos. 9/10, pp. 1231–1237, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(98)00315-3

Original Contribution ANTIOXIDANT ACTIVITY APPLYING AN IMPROVED ABTS RADICAL CATION DECOLORIZATION ASSAY ROBERTA RE, NICOLETTA PELLEGRINI, ANNA PROTEGGENTE, ANANTH PANNALA, MIN YANG, CATHERINE RICE-EVANS

and

International Antioxidant Research Centre, Guy’s, King’s and St Thomas’ School of Biomedical Sciences, Kings College–Guy’s Campus, London SE1 9RT, UK (Received 4 August 1998; Revised 29 October 1998; Accepted 29 October 1998)

Abstract—A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic 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 by oxidation 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 cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC 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 intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC 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

Generation of the ABTS [2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation [18] forms the basis of one of the spectrophotometric methods that have been applied to the measurement of the total antioxidant activity of solutions of pure substances [12,19,20], aqueous mixtures and beverages [7,8]. The original ABTS•1 assay was based on the activation of metmyoglobin with hydrogen peroxide in the presence of ABTS to produce the radical cation, in the presence or absence of antioxidants. This has been criticized on the basis that the faster reacting antioxidants might also contribute to the reduction of the ferryl myoglobin radical. A more appropriate format for the assay is a decolorization technique in that the radical is generated directly in a stable form prior to reaction with putative antioxidants. The improved technique for the generation of ABTS•1 described here involves the direct production of the blue/green ABTS•1 chromophore through the reaction between ABTS and potassium persulfate. This has

A number of assays have been introduced for the measurement 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 different radical, acting through a variety of mechanisms and the measurement of a range of end points at a fixed time point or over a range (reviewed in refs 13 and 17). Two types of approach have been taken, namely, the inhibition assays in that the extent of the scavenging by hydrogen- or electron-donation of a pre-formed free radical is the marker of antioxidant activity, as well as assays involving the presence of antioxidant system during the generation of the radical. Address correspondence to: Professor Catherine Rice-Evans, International Antioxidant Research Centre, Guy’s, King’s and St Thomas’ School of Biomedical Sciences, Kings College–Guy’s Campus, St Thomas’s Street, London SE1 9RT, UK; Tel: 144 0171-955-4240; Fax: 144 0171-955-4983. 1231

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absorption maxima at wavelengths 645 nm, 734 nm and 815 nm, as reported previously [1,13,17], as well as the more commonly used maximum at 415 nm. Addition of antioxidants to the pre-formed radical cation reduces it ABTS, to an extent and on a time-scale depending on the antioxidant activity, the concentration of the antioxidant and the duration of the reaction. Thus the extent of decolorization as percentage inhibition of the ABTS•1 radical cation is determined as a function of concentration and time and calculated relative to the reactivity of Trolox as a standard, under the same conditions. The method is applicable to the study of both water-soluble and lipid-soluble antioxidants, pure compounds, and food extracts.

MATERIALS AND METHODS

Trolox (Hoffman-La Roche) (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid; Aldrich Chemical Co., Gillingham, Dorset, UK) was used an antioxidant standard. Trolox (2.5 mM) was prepared in ethanol or 5 mM phosphate buffered saline, pH 7.4, (PBS), for use as a stock standard, as described previously [1]. Fresh working standards were prepared daily on dilution with ethanol. ABTS, 2,29-azinobis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt, and potassium persulfate (di-potassium peroxdisulfate) were obtained from Sigma-Aldrich (Poole, Dorset, UK) and HPLC grade ethanol from Rathburn Chemicals Ltd. (Walkerburn, Peebleshire, Scotland). Hydroxycinnamates, anthocyanidins, and flavonoids were obtained from Extrasynthese (Lyon-Nord, France), carotenoids, b-carotene and lycopene, from AOCS (Bitterne, Hampshire), and ascorbic acid and a-tocopherol from Sigma-Aldrich (95% pure). Stock solutions of the carotenoids were prepared in dichloromethane and concentrations confirmed using the extinction coefficient. Stock solutions of flavonoids and hydroxycinnamates were prepared by dissolution in ethanol and subsequently diluted in ethanol for introduction into the assay system at concentrations within the activity range of the assay (1.5 mM to 15 mM final concentration). Anthocyanidins were diluted in acidic ethanol pH 1.3 to a concentration of 0.5 mM. Ascorbic acid and uric acid were prepared as stock solutions in 18 MV water to a concentration of 5 mM, and a-tocopherol in ethanol at 2 mM. None of the solvents interfere in the assay. The antioxidant activity was assessed as described below. Experiments were performed on the HewlettPackard spectrophotometer model HP 8453 (Cheadle Heath, Stockport Cheshire, UK) fitted with peltier temperature control.

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

Assay protocol— decolorization assay in ethanolic solution ABTS was dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS•1) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use (Fig. 1). Because ABTS and potassium persulfate react stoichiometrically at a ratio of 1:0.5, this will result in incomplete oxidation of the ABTS. Oxidation of the ABTS commenced immediately, but the absorbance was not maximal and stable until more than 6 h had elapsed. The radical was stable in this form for more than two days when stored in the dark at room temperature. For the study of phenolic compounds and food extracts, the ABTS•1 solution was diluted with ethanol and for plasma antioxidants with PBS, pH 7.4, to an absorbance of 0.70 (60.02) at 734 nm and equilibrated at 30°C. Stock solutions of phenolics in ethanol, carotenoids in dichloromethane and plasma antioxidants in water were diluted such that, after introduction of a 10ml aliquot of each dilution into the assay, they produced between 20%– 80% inhibition of the blank absorbance. After addition of 1.0 ml of diluted ABTS•1 solution (A734nm 5 0.700 6 0.020) to 10 ml of antioxidant com-

ABTS•1 decolorization assay

Fig. 2. Concentration-response curve for the absorbance at 734 nm for ABTS•1 as a function of concentration of standard Trolox solution. (Five separately prepared stock standard solutions 6 SD.)

pounds or Trolox standards (final concentration 0 –15 mM) in ethanol or PBS the absorbance reading was taken at 30°C exactly 1 min after initial mixing and up to 6 min. 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 separate concentration of the standard and samples. The percentage inhibition of absorbance at 734 nm is calculated and plotted as a function of concentration of antioxidants and of Trolox for the standard reference data. The concentration-response curve for 5 sequentially and separately prepared stock standards of Trolox is illustrated in Fig. 2. Determination of the molar extinction coefficient (e) of ABTS•1 at 734 nm Dilutions of ABTS•1 solution, prepared as described above, were further diluted in ethanol and in ultra-pure water to give absorbance values of between 0.12 to 0.9 at 415 nm (a dilution of between 1/50 and 1/400). The ratio between the absorbance at 415 nm and the absorbance at 734 nm was calculated at 5 different dilutions. From this ratio and from the molar extinction coefficient of ABTS•1 at 415 nm (e 5 3.6 3 104 mol21l cm21) reported by Forni et al. [22], the extinction coefficient of ABTS•1 at 734 has been calculated in water as 1.5 3 104 mol21l cm21 6 549 (mean 6 SD, n 5 9) and in ethanol as 1.6 3 104 mol21l cm21 6 606 (mean 6 SD, n 5 8). Under the conditions used here for the preparation of the ABTS•1, about 60% of the ABTS present was oxidized to the radical cation form. RESULTS AND DISCUSSION

The method described gives a measure of the antioxidant activity of the range of carotenoids, phenolics, and

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

some plasma antioxidants, determined by the decolorization of the ABTS•1, through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734 nm. Figure 3 illustrates the effects of the duration of interaction of specific antioxidants on the suppression of the absorbance of the ABTS•1 radical cation at 734 nm for Trolox, the standard reference compound, compared with glutathione, uric acid, ascorbic acid, a-tocopherol, and the flavonoid aglycone antioxidants, kaempferol, and cyanidin. The results demonstrate that the reaction with ABTS•1 is complete by 1 min, except for cyanidin and glutathione that show a further small inhibitory effect up to 4 min reaction. The extent of inhibition of the absorbance of the ABTS•1 is plotted as a function of concentration in order to determine the TEAC, that can be assessed as a function of time. The dose-response curve obtained by analysis of a range of concentrations of antioxidant compounds, Trolox standards and selected food extracts, at selected time points in the reaction, 1, 4 and 6 min, in some cases, was plotted as the percentage inhibition of the absorbance of the ABTS•1 solution as a function of concentration of antioxidant (Fig. 4). The concentration of antioxidant giving the same percentage inhibition of absorbance of the radical cation at 734 nm as 1 mM Trolox was calculated in terms of the Trolox equivalent antioxidant activity at each specific time-point. To calculate the TEAC, the gradient of the plot of the percentage inhibition of absorbance vs. concentration plot for the antioxidant in question is divided by the gradient of the plot for Trolox. This gives the TEAC at the specific time point and the calculated results for the flavonoids, carotenoids, some plasma antioxidants, and a representative fruit and beverage sample are given in Table 1. The antioxidant activity can also be expressed in terms of the total contribution to the antioxidant activity

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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).

over the time range studied by calculating the area under the curve, derived from plotting the gradient of the percentage inhibition / concentration plots as a function of time of reaction. The ratio between the area under the curve for the reaction of the specific antioxidant and that for Trolox gives the relative antioxidant activity (AUC), as in Fig. 5. The comparison between the antioxidant activity determined from the AUC, and the TEAC values derived from the decolorization assay at individual 1-min, 4-min, and 6-min time-points are tabulated relative to the original TEAC value obtained from the ferryl myoglobin/ TEAC assay. All the selected phenolics (except delphindin) demonstrate lower TEAC values with the decolorization assay at the individual time-points of 1 and 4 min reaction than those obtained with the original myoglobin/ABTS assay at 6 min. At 6 min the values are close, excepting quercetin and cyanidin, among the most

reducing of the flavonoids [23], for which the values do not attain the levels as in the myoglobin/ABTS assay system. This is likely to be accounted for by the possibility that some interaction occurred in the previous assay of the polyphenols with ferryl myoglobin, prior to the latter’s reaction with ABTS, and the complex nature of the procedure of the ferryl myoglobin assay in that the formation of the radical cation and its inhibition were occurring in the same time frame. Strube et al. [24] previously proposed this explanation for the higher values obtained for quercetin in the ferryl myoglobin/ABTS assay. It should be noted that quercetin has a lower half oxidation potential than luteolin, that is itself lower than kaempferol, due to the importance of the catechol structure in the B ring as well as the reducing 3-hydroxyl group on the unsaturated C ring adjacent to a carbonyl group [23]. The results demonstrate the time-dependency of the

ABTS•1 decolorization assay

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Table 1. Comparison Between the Antioxidant Activity as TEAC (mM) at Specific Time-Points TEAC Myoglobin/ABTS Decolorization Assay

TEAC Persulfate Decolorization Assay Compounds Hydroxycinnamates Ferulic acid p-Coumaric acid Caffeic acid Flavon-3-ols Quercetin Kaempferol Flavones Luteolin Flavanones Naringenin Anthocyanidin Delphinidin Malvidin Cyanidin Plasma antioxidant Ascorbic acid a-Tocopherol Gluthatione Uric acid Carotenoids b-Carotene Lycopene Food extracts Orange juice Blond (Ovale) Tomato Aqueous/methanol Lipophilic

AUC Persulfate Decolorization Assay

1 min

4 min

6 min

6 min

1.75 6 0.04 1.56 6 0.04 0.99 6 0.05

1.69 6 0.04 1.51 6 0.03 0.99 6 0.05

1.84 6 0.06 1.82 6 0.05 0.98 6 0.06

1.90 6 0.05 2.00 6 0.07 NC

1.90 6 0.02 2.22 6 0.06 1.26 6 0.01

2.88 6 0.01 1.02 6 0.06

2.77 6 0.02 1.02 6 0.07

3.03 6 0.02 1.02 6 0.06

3.1 6 0.05 NC

4.72 6 0.10 1.34 6 0.08

1.49 6 0.03

1.29 6 0.04

1.76 6 0.03

2.06 6 0.03

2.10 6 0.05

0.72 6 0.07

0.58 6 0.09

0.89 6 0.05

1.14 6 0.08

1.53 6 0.05

4.8 6 0.18 1.80 6 0.06 2.38 6 0.20

4.64 6 0.18 1.76 6 0.12 2.30 6 0.19

5.01 6 0.19 1.85 6 0.09 2.48 6 0.22

NC NC

4.44 6 0.11 2.06 6 0.1 4.4 6 0.12

1.05 6 0.02 0.90 6 0.00 1.19 6 0.02 1.01 6 0.06

1.05 6 0.02 0.89 6 0.05 1.13 6 0.03 1.00 6 0.06

1.05 6 0.02 0.97 6 0.06 1.28 6 0.04 1.01 6 0.06

NC NC NC

0.99 6 0.04 0.97 6 0.01 0.90 6 0.03 1.02 6 0.06

2.50 6 0.03 3.04 6 0.13

2.47 6 0.03 3.01 6 0.13

2.57 6 0.03 3.08 6 0.10

NC NC

1.9 6 0.01 2.9 6 0.1

1.77 6 0.22 TAA mmol/kg dry wt

2.22 6 0.40 TAA mmol/kg dry wt

2.31 6 0.44

16.72 6 0.41 6.50 6 0.21

19.87 6 0.20 7.02 6 0.21

18.00 6 0.41 6.70 6 0.21

NC

Applying the ABTS•1 decolorization assay (based on potassium persulfate), the value derived from the area under the time-dependency curve and the 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.

reaction and the influence of the selected time-point of measurement on the reported antioxidant activity; thus the determinants of the antioxidant activity are the extent of reduction and rate of reduction of the radical. For example, whereas caffeic acid and kaempferol demonstrate the lower extent of inhibition than ferulic acid and luteolin, respectively, the reactions of the former are essentially complete after 1 min reaction. Flavonoids varied in the range of times over which the reaction took place (Fig. 5). Whereas most phenolics had completed the reaction at 4 min, some compounds especially luteolin and naringenin were still reacting. Expressing the results as area under the curve can take these factors into account. The major improvement in the assay for lipophilic compounds such as carotenoids is the design improvement incorporating the radical cation and the antioxidant both in the lipophilic phase. The reaction between the carotenoids and ABTS•1 is essentially complete after 1 min, little further reaction taking place thereafter. The

antioxidant activity of lycopene was of the same order as obtained using previous methodology that produced the radical cation using manganese dioxide as oxidant [20]. The value for b-carotene was significantly higher. This method improves the assay also on the grounds that application of manganese dioxide as oxidizing agent can involve molecular chemistry with the potential to produce a two electron oxidation of ABTS to the radical dication, that limits its definition and applicability. The antioxidant activities of the plasma antioxidants, ascorbic acid, a-tocopherol, and uric acid, as well as that of glutathione, are shown in Table 1. The TEAC values obtained are close to those obtained by myoglobin/ABTS assay [1,13], with the latter two being slightly higher. There are differences between the TEAC values for the flavonoids and hydroxycinnamates at 1 min, 4 min and 6 min by the ABTS•1 decolorization assay compared with the myoglobin/ABTS assay monitored at 6 min. The latter assay involved continuous formation of the ABTS radical cation from ferryl myoglobin, derived from met-

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Fig. 5. Profile of the variation of gradient of the percent inhibition vs. concentration plot of each antioxidant at 1 min and 4 min used to 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 antioxidant in question to that for Trolox. (A) Quercetin }; luteolin ■; kaempferol Œ; naringenin 1; (B) delphinidin}; cyanidin ■; malvidin Œ; (C) ascorbic acid }; a-tocopherol ■; (D) ferulic acid }; p-coumaric acid ■; caffeic acid Œ.

myoglobin and hydrogen peroxide in the presence of the reductants. Preliminary fast kinetic studies (data not shown) indicate a biphasic reaction with a very rapid initial phase, presumably indicative of the most reducing groups followed by a slower phase. The AUC method is an alternative way to describe the antioxidant activity of compounds when taking into account the varied rates of reaction of the antioxidants with ABTS•1. The calculation of AUC is derived from both antioxidant concentration and reaction time and is therefore an overall measure of the abilities of the compounds to scavenge free radicals compared to the standard Trolox during the specific time range, taking into account the variation in value with time. The TEAC values are obtained from the capacity of an individual antioxidant or a mixture to inhibit the ABTS•1 at a defined time point, relative to Trolox. As a screen for relative antioxidant activities of pure compounds or food extracts, the antioxidant activity referred to measurement at 4 min time point would seem to be appropriate. Acknowledgements — We acknowledge financial support from the Ministry of Agriculture, Fisheries and Food (Contract ANO448), the European Union Fair program FAIRCT965077 for funding Nicoletta Pellegrini. We thank Dr. Nicholas J. Miller (Oxford Drug Trials Unit) for his participation in the initial development of the assay.

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ABBREVIATIONS

AUC—area under curve ABTS—2,29-azinobis(3-ethylbenzothiazoline 6-sulfonic acid) TEAC—Trolox equivalent antioxidant activity TAA—total antioxidant activity