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, Guys, Kings and St Thomas School of Biomedical Sciences, Kings CollegeGuys
Campus, London SE1 9RT, UK
(Received 4 August 1998; Revised 29 October 1998; Accepted 29 October 1998)
AbstractA method for the screening of antioxidant activity is reported as a decolorization assay applicable to both
lipophilic and hydrophilic antioxidants, including avonoids, hydroxycinnamates, carotenoids, and plasma antioxidants.
The pre-formed radical monocation of 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS

) is generated by
oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants.
The inuences 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.
KeywordsABTS 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 uids
[16], food extracts [711], 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 xed time
point or over a range (reviewed in refs 13 and 17). Two
types 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 radical
is the marker of antioxidant activity, as well as assays
involving the presence of antioxidant system during the
generation of the radical.
Generation of the ABTS [2,2-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 the
total antioxidant activity of solutions of pure sub-
stances [12,19,20], aqueous mixtures and beverages
[7,8]. The original ABTS

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 antioxi-
dants 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

described here involves the direct production of

the blue/green ABTS

chromophore through the reac-

tion between ABTS and potassium persulfate. This has
Address correspondence to: Professor Catherine Rice-Evans, Inter-
national Antioxidant Research Centre, Guys, Kings and St Thomas
School of Biomedical Sciences, Kings CollegeGuys Campus, St
Thomass Street, London SE1 9RT, UK; Tel: 44 0171-955-4240;
Fax: 44 0171-955-4983.
Free Radical Biology & Medicine, Vol. 26, Nos. 9/10, pp. 12311237, 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
1231
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

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, 2,2-azinobis(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salt, and potassium persul-
fate (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 avonoids
were obtained from Extrasynthese (Lyon-Nord, France),
carotenoids, -carotene and lycopene, from AOCS (Bit-
terne, Hampshire), and ascorbic acid and -tocopherol
from Sigma-Aldrich (95% pure). Stock solutions of the
carotenoids were prepared in dichloromethane and con-
centrations conrmed using the extinction coefcient.
Stock solutions of avonoids 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 M to 15 M nal 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 M water to a concentration of 5
mM, and -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 Hewlett-
Packard spectrophotometer model HP 8453 (Cheadle
Heath, Stockport Cheshire, UK) tted with peltier tem-
perature control.
Assay protocoldecolorization assay in ethanolic
solution
ABTS was dissolved in water to a 7 mM concentra-
tion. ABTS radical cation (ABTS

) was produced by
reacting ABTS stock solution with 2.45 mM potassium
persulfate (nal concentration) and allowing the mixture
to stand in the dark at room temperature for 1216 h
before use (Fig. 1). Because ABTS and potassium per-
sulfate react stoichiometrically at a ratio of 1:0.5, this
will 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 h
had elapsed. The radical was stable in this form for more
than two days when stored in the dark at room temper-
ature. For the study of phenolic compounds and food
extracts, the ABTS

solution was diluted with ethanol

and for plasma antioxidants with PBS, pH 7.4, to an
absorbance of 0.70 (0.02) at 734 nm and equilibrated at
30C. Stock solutions of phenolics in ethanol, carote-
noids in dichloromethane and plasma antioxidants in
water were diluted such that, after introduction of a 10-
l 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

solution
(A
734nm
0.700 0.020) to 10 l of antioxidant com-
Fig. 1. Absorption spectrum of the ABTS radical cation.
1232 R. RE et al.
pounds or Trolox standards (nal concentration 015
M) in ethanol or PBS the absorbance reading was taken
at 30C 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 percent-
age 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 concen-
tration-response curve for 5 sequentially and separately
prepared stock standards of Trolox is illustrated in Fig. 2.
Determination of the molar extinction coefcient () of
ABTS

at 734 nm
Dilutions of ABTS

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 coefcient of
ABTS

at 415 nm ( 3.6 10
4
mol
1
l cm
1
)
reported by Forni et al. [22], the extinction coefcient of
ABTS

at 734 has been calculated in water as 1.5 10

4
mol
1
l cm
1
549 (mean SD, n 9) and in ethanol
as 1.6 10
4
mol
1
l cm
1
606 (mean SD, n 8).
Under the conditions used here for the preparation of the
ABTS

, about 60% of the ABTS present was oxidized

to 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

, through measuring the reduction of

the radical cation as the percentage inhibition of absor-
bance at 734 nm. Figure 3 illustrates the effects of the
duration of interaction of specic antioxidants on the
suppression of the absorbance of the ABTS

radical
cation at 734 nm for Trolox, the standard reference
compound, compared with glutathione, uric acid, ascor-
bic acid, -tocopherol, and the avonoid aglycone anti-
oxidants, kaempferol, and cyanidin. The results demon-
strate that the reaction with ABTS

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

is plotted as a function of concentration in order

to 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, 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

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 specic time-point. To cal-
culate the TEAC, the gradient of the plot of the percent-
age 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 specic
time point and the calculated results for the avonoids,
carotenoids, some plasma antioxidants, and a represen-
tative 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
Fig. 2. Concentration-response curve for the absorbance at 734 nm for
ABTS

as a function of concentration of standard Trolox solution.

(Five separately prepared stock standard solutions SD.)
Fig. 3. The effects of time on the suppression of the absorbance of the
ABTS

. Control ABTS

radical cation (}), Trolox 10 M (),

vitamin C 12 M (), -tocopherol 15 M (F), kaempferol 6 M (I),
cyanidin 5 M (), reduced glutathione 12 M (.), uric acid 6 M
().
1233 ABTS

decolorization assay
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 specic antioxidant and that
for 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 derived
from 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 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 avonoids [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 possi-
bility that some interaction occurred in the previous
assay of the polyphenols with ferryl myoglobin, prior to
the latters 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 val-
ues 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 struc-
ture 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
Fig. 4. The effects of concentration of the antioxidant on the inhibition of the ABTS

. (A) Kaempferol (r
2
0.966); (B) ascorbic acid (r
2
1); (C)
-tocopherol (r
2
0.995); (D) cyanidin (r
2
0.997); (E) glutathione (r
2
0.948); (F) uric acid (r
2
1); (G) Trolox (r
2
1); (H) orange juice (r
2
0.993).
1234 R. RE et al.
reaction and the inuence 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 demon-
strate 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 luteo-
lin 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 improve-
ment incorporating the radical cation and the antioxidant
both in the lipophilic phase. The reaction between the
carotenoids and ABTS

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 -carotene was signicantly 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 pro-
duce a two electron oxidation of ABTS to the radical
dication, that limits its denition and applicability.
The antioxidant activities of the plasma antioxidants,
ascorbic acid, -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 avonoids and hydroxycinnamates at 1 min, 4 min
and 6 min by the ABTS

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-
Table 1. Comparison Between the Antioxidant Activity as TEAC (mM) at Specic Time-Points
Compounds
AUC Persulfate
Decolorization Assay
TEAC Persulfate Decolorization Assay
TEAC Myoglobin/ABTS
Decolorization Assay
1 min 4 min 6 min 6 min
Hydroxycinnamates
Ferulic acid 1.75 0.04 1.69 0.04 1.84 0.06 1.90 0.05 1.90 0.02
p-Coumaric acid 1.56 0.04 1.51 0.03 1.82 0.05 2.00 0.07 2.22 0.06
Caffeic acid 0.99 0.05 0.99 0.05 0.98 0.06 NC 1.26 0.01
Flavon-3-ols
Quercetin 2.88 0.01 2.77 0.02 3.03 0.02 3.1 0.05 4.72 0.10
Kaempferol 1.02 0.06 1.02 0.07 1.02 0.06 NC 1.34 0.08
Flavones
Luteolin 1.49 0.03 1.29 0.04 1.76 0.03 2.06 0.03 2.10 0.05
Flavanones
Naringenin 0.72 0.07 0.58 0.09 0.89 0.05 1.14 0.08 1.53 0.05
Anthocyanidin
Delphinidin 4.8 0.18 4.64 0.18 5.01 0.19 4.44 0.11
Malvidin 1.80 0.06 1.76 0.12 1.85 0.09 NC 2.06 0.1
Cyanidin 2.38 0.20 2.30 0.19 2.48 0.22 NC 4.4 0.12
Plasma antioxidant
Ascorbic acid 1.05 0.02 1.05 0.02 1.05 0.02 NC 0.99 0.04
-Tocopherol 0.90 0.00 0.89 0.05 0.97 0.06 NC 0.97 0.01
Gluthatione 1.19 0.02 1.13 0.03 1.28 0.04 0.90 0.03
Uric acid 1.01 0.06 1.00 0.06 1.01 0.06 NC 1.02 0.06
Carotenoids
-Carotene 2.50 0.03 2.47 0.03 2.57 0.03 NC 1.9 0.01
Lycopene 3.04 0.13 3.01 0.13 3.08 0.10 NC 2.9 0.1
Food extracts
Orange juice
Blond (Ovale) 1.77 0.22 2.22 0.40 2.31 0.44
TAA mmol/kg dry wt TAA mmol/kg dry wt
Tomato
Aqueous/methanol 18.00 0.41 16.72 0.41 19.87 0.20
Lipophilic 6.70 0.21 6.50 0.21 7.02 0.21 NC
Applying the ABTS

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 SD 3, each performed in triplicate at 3 separate concentrations.
NC no change.
1235 ABTS

decolorization assay
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 ac-
count the varied rates of reaction of the antioxidants with
ABTS

. The calculation of AUC is derived from both

antioxidant concentration and reaction time and is there-
fore an overall measure of the abilities of the compounds
to scavenge free radicals compared to the standard
Trolox during the specic 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

at a dened time point, relative to Trolox. As a

screen for relative antioxidant activities of pure com-
pounds or food extracts, the antioxidant activity referred
to measurement at 4 min time point would seem to be
appropriate.
Acknowledgements We acknowledge nancial 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
AUCarea under curve
ABTS2,2-azinobis(3-ethylbenzothiazoline 6-sulfonic
acid)
TEACTrolox equivalent antioxidant activity
TAAtotal antioxidant activity
1237 ABTS

decolorization assay