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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, and
CATHERINE RICE-EVANS
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-ethyl-


benzothiazoline-6-sulfonic acid)] radical cation [18]
A number of assays have been introduced for the mea-
forms the basis of one of the spectrophotometric meth-
surement of the total antioxidant activity of body fluids
ods that have been applied to the measurement of the
[1– 6], food extracts [7–11], and pure compounds [7,12–
total antioxidant activity of solutions of pure sub-
16]. Each method relates to the generation of a different
stances [12,19,20], aqueous mixtures and beverages
radical, acting through a variety of mechanisms and the
[7,8]. The original ABTS•1 assay was based on the
measurement of a range of end points at a fixed time
activation of metmyoglobin with hydrogen peroxide in
point or over a range (reviewed in refs 13 and 17). Two
the presence of ABTS to produce the radical cation, in
types of approach have been taken, namely, the inhibi-
the presence or absence of antioxidants. This has been
tion assays in that the extent of the scavenging by hy-
criticized on the basis that the faster reacting antioxi-
drogen- or electron-donation of a pre-formed free radical
dants might also contribute to the reduction of the
is the marker of antioxidant activity, as well as assays
ferryl myoglobin radical. A more appropriate format
involving the presence of antioxidant system during the
for the assay is a decolorization technique in that the
generation of the radical.
radical is generated directly in a stable form prior to
reaction with putative antioxidants.
Address correspondence to: Professor Catherine Rice-Evans, Inter- The improved technique for the generation of
national Antioxidant Research Centre, Guy’s, King’s and St Thomas’ ABTS•1 described here involves the direct production of
School of Biomedical Sciences, Kings College–Guy’s Campus, St
Thomas’s Street, London SE1 9RT, UK; Tel: 144 0171-955-4240; the blue/green ABTS•1 chromophore through the reac-
Fax: 144 0171-955-4983. tion between ABTS and potassium persulfate. This has

1231
1232 R. RE et al.

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 concentra-
tion 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-tet-


ramethychroman-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-6-
sulfonic acid) diammonium salt, and potassium persul- Fig. 1. Absorption spectrum of the ABTS radical cation.

fate (di-potassium peroxdisulfate) were obtained from


Sigma-Aldrich (Poole, Dorset, UK) and HPLC grade Assay protocol— decolorization assay in ethanolic
ethanol from Rathburn Chemicals Ltd. (Walkerburn, solution
Peebleshire, Scotland).
ABTS was dissolved in water to a 7 mM concentra-
Hydroxycinnamates, anthocyanidins, and flavonoids
tion. ABTS radical cation (ABTS•1) was produced by
were obtained from Extrasynthese (Lyon-Nord, France),
reacting ABTS stock solution with 2.45 mM potassium
carotenoids, b-carotene and lycopene, from AOCS (Bit-
persulfate (final concentration) and allowing the mixture
terne, Hampshire), and ascorbic acid and a-tocopherol
to stand in the dark at room temperature for 12–16 h
from Sigma-Aldrich (95% pure). Stock solutions of the
before use (Fig. 1). Because ABTS and potassium per-
carotenoids were prepared in dichloromethane and con-
sulfate react stoichiometrically at a ratio of 1:0.5, this
centrations confirmed using the extinction coefficient. will result in incomplete oxidation of the ABTS. Oxida-
Stock solutions of flavonoids and hydroxycinnamates tion of the ABTS commenced immediately, but the ab-
were prepared by dissolution in ethanol and subsequently sorbance was not maximal and stable until more than 6 h
diluted in ethanol for introduction into the assay system had elapsed. The radical was stable in this form for more
at concentrations within the activity range of the assay than two days when stored in the dark at room temper-
(1.5 mM to 15 mM final concentration). Anthocyanidins ature. For the study of phenolic compounds and food
were diluted in acidic ethanol pH 1.3 to a concentration extracts, the ABTS•1 solution was diluted with ethanol
of 0.5 mM. Ascorbic acid and uric acid were prepared as and for plasma antioxidants with PBS, pH 7.4, to an
stock solutions in 18 MV water to a concentration of 5 absorbance of 0.70 (60.02) at 734 nm and equilibrated at
mM, and a-tocopherol in ethanol at 2 mM. None of the 30°C. Stock solutions of phenolics in ethanol, carote-
solvents interfere in the assay. noids in dichloromethane and plasma antioxidants in
The antioxidant activity was assessed as described water were diluted such that, after introduction of a 10-
below. Experiments were performed on the Hewlett- ml aliquot of each dilution into the assay, they produced
Packard spectrophotometer model HP 8453 (Cheadle between 20%– 80% inhibition of the blank absorbance.
Heath, Stockport Cheshire, UK) fitted with peltier tem- After addition of 1.0 ml of diluted ABTS•1 solution
perature control. (A734nm 5 0.700 6 0.020) to 10 ml of antioxidant com-
ABTS•1 decolorization assay 1233

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 (■),
Fig. 2. Concentration-response curve for the absorbance at 734 nm for cyanidin 5 mM (Œ), reduced glutathione 12 mM (✳), uric acid 6 mM
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 some plasma antioxidants, determined by the decoloriza-
mM) in ethanol or PBS the absorbance reading was taken tion of the ABTS•1, through measuring the reduction of
at 30°C exactly 1 min after initial mixing and up to 6 the radical cation as the percentage inhibition of absor-
min. Appropriate solvent blanks were run in each assay. bance at 734 nm. Figure 3 illustrates the effects of the
All determinations were carried out at least three times, duration of interaction of specific antioxidants on the
and in triplicate, on each occasion and at each separate suppression of the absorbance of the ABTS•1 radical
concentration of the standard and samples. The percent- cation at 734 nm for Trolox, the standard reference
age inhibition of absorbance at 734 nm is calculated and compound, compared with glutathione, uric acid, ascor-
plotted as a function of concentration of antioxidants and bic acid, a-tocopherol, and the flavonoid aglycone anti-
of Trolox for the standard reference data. The concen- oxidants, kaempferol, and cyanidin. The results demon-
tration-response curve for 5 sequentially and separately strate that the reaction with ABTS•1 is complete by 1
prepared stock standards of Trolox is illustrated in Fig. 2. min, except for cyanidin and glutathione that show a
further small inhibitory effect up to 4 min reaction.
Determination of the molar extinction coefficient (e) of The extent of inhibition of the absorbance of the
ABTS•1 at 734 nm ABTS•1 is plotted as a function of concentration in order
to determine the TEAC, that can be assessed as a func-
Dilutions of ABTS•1 solution, prepared as described tion of time. The dose-response curve obtained by anal-
above, were further diluted in ethanol and in ultra-pure ysis of a range of concentrations of antioxidant com-
water to give absorbance values of between 0.12 to 0.9 at pounds, Trolox standards and selected food extracts, at
415 nm (a dilution of between 1/50 and 1/400). The ratio selected time points in the reaction, 1, 4 and 6 min, in
between the absorbance at 415 nm and the absorbance at some cases, was plotted as the percentage inhibition of
734 nm was calculated at 5 different dilutions. From this the absorbance of the ABTS•1 solution as a function of
ratio and from the molar extinction coefficient of concentration of antioxidant (Fig. 4). The concentration
ABTS•1 at 415 nm (e 5 3.6 3 104 mol21l cm21) of antioxidant giving the same percentage inhibition of
reported by Forni et al. [22], the extinction coefficient of absorbance of the radical cation at 734 nm as 1 mM
ABTS•1 at 734 has been calculated in water as 1.5 3 104 Trolox was calculated in terms of the Trolox equivalent
mol21l cm21 6 549 (mean 6 SD, n 5 9) and in ethanol antioxidant activity at each specific time-point. To cal-
as 1.6 3 104 mol21l cm21 6 606 (mean 6 SD, n 5 8). culate the TEAC, the gradient of the plot of the percent-
Under the conditions used here for the preparation of the age inhibition of absorbance vs. concentration plot for
ABTS•1, about 60% of the ABTS present was oxidized the antioxidant in question is divided by the gradient of
to the radical cation form. the plot for Trolox. This gives the TEAC at the specific
time point and the calculated results for the flavonoids,
RESULTS AND DISCUSSION carotenoids, some plasma antioxidants, and a represen-
tative fruit and beverage sample are given in Table 1.
The method described gives a measure of the antiox- The antioxidant activity can also be expressed in
idant activity of the range of carotenoids, phenolics, and terms of the total contribution to the antioxidant activity
1234 R. RE et al.

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 reducing of the flavonoids [23], for which the values do
the curve, derived from plotting the gradient of the not attain the levels as in the myoglobin/ABTS assay
percentage inhibition / concentration plots as a function system. This is likely to be accounted for by the possi-
of time of reaction. The ratio between the area under the bility that some interaction occurred in the previous
curve for the reaction of the specific antioxidant and that assay of the polyphenols with ferryl myoglobin, prior to
for Trolox gives the relative antioxidant activity (AUC), the latter’s reaction with ABTS, and the complex nature
as in Fig. 5. of the procedure of the ferryl myoglobin assay in that the
The comparison between the antioxidant activity de- formation of the radical cation and its inhibition were
termined from the AUC, and the TEAC values derived occurring in the same time frame. Strube et al. [24]
from the decolorization assay at individual 1-min, 4-min, previously proposed this explanation for the higher val-
and 6-min time-points are tabulated relative to the orig- ues obtained for quercetin in the ferryl myoglobin/ABTS
inal TEAC value obtained from the ferryl myoglobin/ assay. It should be noted that quercetin has a lower half
TEAC assay. All the selected phenolics (except del- oxidation potential than luteolin, that is itself lower than
phindin) demonstrate lower TEAC values with the kaempferol, due to the importance of the catechol struc-
decolorization assay at the individual time-points of 1 ture in the B ring as well as the reducing 3-hydroxyl
and 4 min reaction than those obtained with the original group on the unsaturated C ring adjacent to a carbonyl
myoglobin/ABTS assay at 6 min. At 6 min the values are group [23].
close, excepting quercetin and cyanidin, among the most The results demonstrate the time-dependency of the
ABTS•1 decolorization assay 1235

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

TEAC Myoglobin/ABTS
TEAC Persulfate Decolorization Assay Decolorization Assay
AUC Persulfate
Compounds Decolorization Assay 1 min 4 min 6 min 6 min

Hydroxycinnamates
Ferulic acid 1.75 6 0.04 1.69 6 0.04 1.84 6 0.06 1.90 6 0.05 1.90 6 0.02
p-Coumaric acid 1.56 6 0.04 1.51 6 0.03 1.82 6 0.05 2.00 6 0.07 2.22 6 0.06
Caffeic acid 0.99 6 0.05 0.99 6 0.05 0.98 6 0.06 NC 1.26 6 0.01
Flavon-3-ols
Quercetin 2.88 6 0.01 2.77 6 0.02 3.03 6 0.02 3.1 6 0.05 4.72 6 0.10
Kaempferol 1.02 6 0.06 1.02 6 0.07 1.02 6 0.06 NC 1.34 6 0.08
Flavones
Luteolin 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
Flavanones
Naringenin 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
Anthocyanidin
Delphinidin 4.8 6 0.18 4.64 6 0.18 5.01 6 0.19 4.44 6 0.11
Malvidin 1.80 6 0.06 1.76 6 0.12 1.85 6 0.09 NC 2.06 6 0.1
Cyanidin 2.38 6 0.20 2.30 6 0.19 2.48 6 0.22 NC 4.4 6 0.12
Plasma antioxidant
Ascorbic acid 1.05 6 0.02 1.05 6 0.02 1.05 6 0.02 NC 0.99 6 0.04
a-Tocopherol 0.90 6 0.00 0.89 6 0.05 0.97 6 0.06 NC 0.97 6 0.01
Gluthatione 1.19 6 0.02 1.13 6 0.03 1.28 6 0.04 0.90 6 0.03
Uric acid 1.01 6 0.06 1.00 6 0.06 1.01 6 0.06 NC 1.02 6 0.06
Carotenoids
b-Carotene 2.50 6 0.03 2.47 6 0.03 2.57 6 0.03 NC 1.9 6 0.01
Lycopene 3.04 6 0.13 3.01 6 0.13 3.08 6 0.10 NC 2.9 6 0.1
Food extracts
Orange juice
Blond (Ovale) 1.77 6 0.22 2.22 6 0.40 2.31 6 0.44
TAA mmol/kg dry wt TAA mmol/kg dry wt

Tomato
Aqueous/methanol 18.00 6 0.41 16.72 6 0.41 19.87 6 0.20
Lipophilic 6.70 6 0.21 6.50 6 0.21 7.02 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 antioxidant activity of lycopene was of the same order as
measurement on the reported antioxidant activity; thus obtained using previous methodology that produced the
the determinants of the antioxidant activity are the extent radical cation using manganese dioxide as oxidant [20].
of reduction and rate of reduction of the radical. For The value for b-carotene was significantly higher. This
example, whereas caffeic acid and kaempferol demon- method improves the assay also on the grounds that
strate the lower extent of inhibition than ferulic acid and application of manganese dioxide as oxidizing agent can
luteolin, respectively, the reactions of the former are involve molecular chemistry with the potential to pro-
essentially complete after 1 min reaction. Flavonoids duce a two electron oxidation of ABTS to the radical
varied in the range of times over which the reaction took dication, that limits its definition and applicability.
place (Fig. 5). Whereas most phenolics had completed The antioxidant activities of the plasma antioxidants,
the reaction at 4 min, some compounds especially luteo- ascorbic acid, a-tocopherol, and uric acid, as well as that
lin and naringenin were still reacting. Expressing the of glutathione, are shown in Table 1. The TEAC values
results as area under the curve can take these factors into obtained are close to those obtained by myoglobin/ABTS
account. assay [1,13], with the latter two being slightly higher.
The major improvement in the assay for lipophilic There are differences between the TEAC values for
compounds such as carotenoids is the design improve- the flavonoids and hydroxycinnamates at 1 min, 4 min
ment incorporating the radical cation and the antioxidant and 6 min by the ABTS•1 decolorization assay compared
both in the lipophilic phase. The reaction between the with the myoglobin/ABTS assay monitored at 6 min. The
carotenoids and ABTS•1 is essentially complete after 1 latter assay involved continuous formation of the ABTS
min, little further reaction taking place thereafter. The radical cation from ferryl myoglobin, derived from met-
1236 R. RE et al.

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.

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