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,
carotenoids, some plasma antioxidants, and a represen-
RESULTS AND DISCUSSION
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 ( r 2 5 0.966); (B) ascorbic acid ( r 2 5 1); (C) a-tocopherol ( r 2 5 0.995); (D) cyanidin ( r 2 5 0.997); (E) glutathione ( r 2 5 0.948); (F) uric acid ( r 2 5 1); (G) Trolox ( r 2 5 1); (H) orange juice ( r 2
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].