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

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, 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 flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants.
The pre-formed radical monocation of 2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS1) 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.
KeywordsABTS 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 ABTS1 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
ABTS1 described here involves the direct production of
the blue/green ABTS1 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 [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 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, Guys, Kings and St Thomas
School of Biomedical Sciences, Kings CollegeGuys Campus, St
Thomass Street, London SE1 9RT, UK; Tel: 144 0171-955-4240;
Fax: 144 0171-955-4983.
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 ABTS1
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 (ABTS1) 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 1216 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 ABTS1 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
30C. 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 ABTS1 solution
(A734nm 5 0.700 6 0.020) to 10 ml of antioxidant com-

ABTS1 decolorization assay

Fig. 2. Concentration-response curve for the absorbance at 734 nm for

ABTS1 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 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 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
ABTS1 at 734 nm
Dilutions of ABTS1 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
ABTS1 at 415 nm (e 5 3.6 3 104 mol21l cm21)
reported by Forni et al. [22], the extinction coefficient of
ABTS1 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
ABTS1, 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

1233

Fig. 3. The effects of time on the suppression of the absorbance of the

ABTS1. Control ABTS1 radical cation (}), Trolox 10 mM (1),
vitamin C 12 mM (2), a-tocopherol 15 mM (F), kaempferol 6 mM (),
cyanidin 5 mM (), reduced glutathione 12 mM (), uric acid 6 mM
().

some plasma antioxidants, determined by the decolorization of the ABTS1, 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 ABTS1 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 ABTS1 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
ABTS1 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 ABTS1 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

1234

R. RE et al.

Fig. 4. The effects of concentration of the antioxidant on the inhibition of the ABTS1. (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 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 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

ABTS1 decolorization assay

1235

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 ABTS1 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 ABTS1 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 ABTS1 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-

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 ; naringenin 1; (B) delphinidin}; cyanidin ; malvidin ;
(C) ascorbic acid }; a-tocopherol ; (D) ferulic acid }; p-coumaric acid ; caffeic acid .

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
ABTS1. 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
ABTS1 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

AUCarea under curve

ABTS2,29-azinobis(3-ethylbenzothiazoline 6-sulfonic
acid)
TEACTrolox equivalent antioxidant activity
TAAtotal antioxidant activity