Free Radical Biology & Medicine 52 (2012) 1242–1252

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Free Radical Biology & Medicine

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Original Contribution

Assessment of antioxidant capacity for scavenging free radicals in vitro: A rational

basis and practical application
Mizuki Takashima, Masanori Horie 1, Mototada Shichiri, Yoshihisa Hagihara, Yasukazu Yoshida, Etsuo Niki ⁎
Health Research Institute, National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka 563-8577, Japan

a r t i c l e i n f o a b s t r a c t

Article history: With increasing evidence showing the involvement of oxidative stress induced by free radicals in the devel-
Received 10 October 2011 opment of various diseases, the role of radical-scavenging antioxidants has received much attention. Al-
Revised 7 December 2011 though many randomized controlled clinical trials do not support the beneficial effects of indiscriminate
Accepted 14 January 2012
supplementation of antioxidants, more recent studies suggest that antioxidants such as vitamin E may be ef-
Available online 28 January 2012
fective for prevention and treatment of some diseases when given to the right subjects at the right time.
Keywords:
Many studies on the antioxidant capacity assessed by various available methods showed inconsistent results
Antioxidant capacity assessment and the assessment of antioxidant capacity has been the subject of extensive studies and arguments. This
Free radical study was performed to elucidate the basic chemistry required for the development of a reliable method
Oxidative stress for the assessment of antioxidant capacity for radical scavenging in vitro. In this study, the capacity of α-
Uric acid tocopherol and its related compounds, ascorbic acid, and uric acid for scavenging radicals was assessed
Vitamin C from their effects on the rate of decay of hydrophilic and lipophilic probes with various reactivities toward
Vitamin E free radicals induced by hydrophilic and lipophilic radicals in homogeneous solution and heterogeneous mi-
celle systems. Fluorescein, pyranine, and pyrogallol red were used as hydrophilic probes, and BODIPY and N,
N-diphenyl-p-phenylenediamine were used as lipophilic probes. We show that the rate and amount of rad-
ical scavenging by antioxidants, termed the antioxidant radical absorbance capacity, could be assessed by an
appropriate combination of radical initiator and probe. This method was applied to the assessment of radical-
scavenging capacity of human plasma, wine, and green tea powder.
© 2012 Elsevier Inc. All rights reserved.

Abundant biochemical, biological, and clinical evidence suggests
the involvement of oxidative stress induced by free radicals in the
pathogenesis of various diseases and accelerated aging [1]. This has
attracted much attention from scientists, physicians, and also the
general public to the role and beneficial effects of antioxidants in
the maintenance of human health and prevention and treatment of
diseases [2]. Many large-scale epidemiologic studies, such as the
WHO/MONICA study [3], Nurses’ Health Study [4], and Health Profes-
sionals’ follow-up study [5], have supported the beneficial effects of
vitamin E, the major radical-scavenging antioxidant in vivo, against
chronic diseases such as atherosclerosis and cardiovascular diseases.
However, in contrast to expectations, the results of large-scale
Abbreviations: AAPH, 2,2′-azobis(2-amidinopropane) dihydrochloride; AIPH,
2,2′-azobis[2-(2-imidazolin-2-yl)] dihydrochloride; BODIPY, 4,4-difluoro-5-(4-phe-
nyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indene-3-undecanoic acid; DPBQ, N,N′-
diphenyl-p-benzoquinone diimine; DPPD, N,N′-diphenyl-p-phenylenediamine;
MeO-AMVN, 2,2′-azobis(4-methoxy-2,2-dimethylvaleronitrile); ORAC, oxygen radical ab-
sorbance capacity; PGR, pyrogallol red; PMC, 2,2,5,7,8-pentamethyl-6-chromanol
⁎ Corresponding author. Fax: + 81 72 751 9964.
E-mail address: etsuo-niki@aist.go.jp (E. Niki).
1
Present address: Institute of Industrial Ecological Sciences, University of Occupa-
tional and Environmental Health of Japan, Kitakyushu, Fukuoka 807-8555, Japan.
randomized controlled clinical intervention studies of vitamin E
were disappointing and these results raised much criticism and argu-
ment [6-8]. It is now generally agreed that indiscriminate supplemen-
tation of antioxidants may not be always beneficial [9], but it may be
stated also that antioxidants should be effective if given to the right
subjects at the right time in the right amount. In fact, for example, vi-
tamin E was found to be effective in reducing cardiovascular events in
a subgroup with the haptoglobin 2-2 genotype, which results in insuf-
ficient sequestration of iron and increased oxidative stress [10].
To cope with oxidative stress, aerobic organisms have gained a po-
tent antioxidant network during evolution, in which many antioxi-
dants with diverse functions play their respective roles in vivo [11].
They include enzymes such as glutathione peroxidases and superox-
ide dismutase, proteins such as ferritin and ceruloplasmin, and
small molecules such as glutathione, vitamin C, and vitamin E. Radical
scavenging is one of the important functions in the antioxidant net-
work, because free radicals are produced inevitably in vivo from not
only exogenous sources such as radiation, smoking, and pollution
but also endogenous sources such as free metal ions, inflammation,
respiratory burst, and xenobiotic killing. These free radicals oxidize
randomly biologically essential molecules such as lipids, proteins,
sugars, and nucleic acids, which results in the loss of their physiolog-
ical functions and induction of deleterious effects.

0891-5849/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2012.01.010
 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252
It is important to understand the action and capacity of antioxi-
dants. Numerous natural and synthetic antioxidants have been ex-
plored and their antioxidant capacity has been assessed by diverse
methods [11 and references cited therein]. These include 2-carboxy-
2,5,7,8-tetramethyl-6-chromanol (Trolox)-equivalent antioxidant ca-
pacity or 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) [12],
cupric-reducing antioxidant power [13], ferric-reducing antioxidant
power [14], and ORAC 2 (oxygen radical absorbance capacity) [15].
The assay method is selected often by ease of use and availability of
instrumentation. Many studies have reported inconsistent results
and very often there is a lack of correlation between activities deter-
mined on the same material by different assays and between activi-
ties determined by the same assay in different laboratories. Such
discrepancy is understandable because the methods employed mea-
sure different actions under different conditions; for example, some
assays measure hydrogen atom transfer activity, some measure elec-
tron transfer capability, and others measure other actions in homoge-
neous solutions and in heterogeneous suspensions. The assessment of
antioxidant capacity has been the subject of extensive study and ar-
gument and a need for the development and standardization of reli-
able procedures has been pointed out [11,16,17].
Among the many available methods, the ORAC method has been
used most widely, and the ORAC values measured for numerous
foods and fruits have been summarized by the U.S. Department of Ag-
riculture [18]. This method is based on the capacity of the antioxi-
dants to scavenge radicals in competition with the probe, resulting
in the suppression of the decay of the probe as measured by fluores-
cence or visible absorption. Although the ORAC method is simple and
convenient, this method has its inherent drawbacks: that is, it does
not distinguish between rate and amount of radical scavenging; the
ORAC value depends on the probe used; the most widely used
probe, fluorescein, overestimates the capacity of a weak antioxidant;
and the ORAC value does not correlate with the capacity for inhibition
of free radical-mediated oxidation [11,19,20].
In this study, in an attempt to develop a comprehensive method,
several kinds of hydrophilic and lipophilic probes having different re-
activities toward free radicals were used to assess the radical-
scavenging activity of water-soluble and lipid-soluble antioxidants
in heterogeneous as well as homogeneous systems. Fluorescein, pyr-
anine, and pyrogallol red (PGR) were used as water-soluble probes
and 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-
indene-3-undecanoic acid (BODIPY) and N,N′-diphenyl-p-phenylene-
diamine (DPPD) as lipid-soluble probes. Peroxyl radicals, which exert
similar reactivities independent of the alkyl groups, were generated
in either aqueous or lipid phase by using water-soluble or lipid-
soluble azo initiators. Peroxyl radicals were chosen as the target rad-
icals, because peroxyl radicals are the target radicals for antioxidants
to scavenge in vivo to inhibit oxidative damage of biologically essen-
tial molecules [21]. In contrast, hydroxyl and alkoxyl radicals are too
reactive for antioxidants to scavenge efficiently in competition with
lipids, proteins, and DNA, whereas superoxide and nitric oxide are
not reactive enough per se to attack biological molecules.
Furthermore, peroxyl radicals play a very important role in the
oxidative damage of biological molecules as a chain-carrying spe-
cies of lipid peroxidation. Free radical-mediated lipid peroxidation
proceeds by a chain mechanism to give diverse products, which
are cytotoxic and genotoxic and modify proteins and nucleic
acids. Thus, the inhibition of lipid peroxidation is an important
role of radical-scavenging antioxidants. α-Tocopherol, 2,2,5,7,8-
pentamethyl-6-chromanol (PMC), and Trolox were chosen as test
compounds having the same chemical reactivity toward radicals
but different solubilities, mobilities, and phase localizations. In ad-
dition, ascorbic acid and uric acid were tested as physiological hy-
drophilic antioxidants, and further, human plasma, wine, and green
tea powder were tested as examples containing complex mixtures
of antioxidants.
1243
Materials and methods
Materials
2R,4′R,8′R-α-Tocopherol and PMC were kindly provided by Eisai
Co., Ltd. (Tokyo, Japan). Trolox was purchased from Cayman Chemical
Co. (Ann Arbor, MI, USA). L-(+)-Ascorbic acid and uric acid were from
Wako Pure Chemical Industries (Osaka, Japan). Red and white wine
(Kikkoman Corp., Japan) and green tea powder (Kunitaro Co., Shizu-
oka, Japan) were used as commercial samples. As suggested by a sup-
plier, green tea powder (20 mg) was mixed vigorously with 10 ml
distilled water for 10 min at room temperature and the mixture was
centrifuged at 3500 rpm for 10 min at 4 °C. The supernatant was
used in the assessment of antioxidant capacity.
Azo compounds were used to generate free radicals at a constant
and controlled rate. Hydrophilic 2,2′-azobis(2-amidinopropane)
dihydrochloride (AAPH) and 2,2′-azobis[2-(2-imidazolin-2-yl)] dihy-
drochloride (AIPH) and lipophilic 2,2′-azobis-(2,4-dimethyllvaleroni-
trile) and 2,2′-azobis-(4-methoxy-2,4-dimethyllvaleronitrile)
(MeO-AMVN) were obtained from Wako Pure Chemical Industries. Pyr-
ogallolsulfonephthalein (pyrogallol red), fluorescein, and pyranine
were purchased from Sigma–Aldrich (Tokyo, Japan). The lipophilic
probes, BODIPY and DPPD, were purchased from Molecular Probes
(Invitrogen, Eugene, OR, USA) and Aldrich Chemical Co. (Milwaukee,
WI, USA), respectively.
Spectrophotometric measurement of probe decay
The rates of reaction of fluorescein, pyranine, PGR, and BODIPY
with free radicals were followed in the phosphate-buffered saline
(PBS) or micelle system by measuring the decay of the probe at 494,
454, 540, and 586 nm with a UV–visible light absorption spectropho-
tometer (Shimadzu UV-2450, Kyoto, Japan) equipped with a
thermostated cell maintained at 37 °C. The micelles were prepared
by vigorously mixing methyl palmitate and methyl linoleate
(0.5 vol% each of the total solution) in PBS, pH 7.4, containing 0.5 M
sodium dodecyl sulfate (SDS) with a vortex mixer for 2 min. The reac-
tion was started by the addition of the azo compound. The lag phase
was obtained graphically by extrapolating the slope of maximal probe
decay to the intersection with the slope of minimal probe decay at the
initial stage. The consumption rate of PGR was measured from the
slope of the decay curve against time at the initial stage.
Measurement of DPPD oxidation
DPPD is oxidized by free radicals to produce N,N′-diphenyl-p-ben-
zoquinone diimine (DPBQ), which has a strong absorption at 440 nm
[22,23]. The reaction of DPPD with free radicals and its inhibition by
antioxidant were followed in acetonitrile solution by measuring the
increase in DPBQ at 440 nm with a UV–visible light absorption spec-
trophotometer (Shimadzu UV-2450) at 37 °C.
Plasma preparation
Blood was collected in ethylenediaminetetraacetic acid-containing
tubes from healthy volunteers after overnight fasting. Plasma was
obtained by centrifugation at 3500 rpm for 10 min at 4 °C. The plasma
was dialyzed using a dialysis membrane for 18 h at 4 °C in PBS to re-
move ascorbic acid and other water-soluble low-molecular-weight an-
tioxidants. The study was conducted in accordance with the principles of
the Declaration of Helsinki and was approved by the ethics committee of
the National Institute of Advanced Industrial Science and Technology.
The volunteers gave written informed consent after a complete explana-
tion of the purpose of the study.
1244 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252

Table 1 These two factors should be assessed separately. In this study, they
Characteristics of the probes used in this study. were assessed by using probes with different reactivities toward per-
Probe log P ε (M− 1 cm− 1) λmax λex λem oxyl radicals. The characteristics of the five kinds of probes used in
(nm) (nm) (nm) this study, such as molar extinction coefficient and partition coeffi-
Fluorescein bb0 4.90 × 104 (4.17 × 104) 494 494 515 cient, are summarized in Table 1. Fluorescein, pyranine, and PGR are
Pyranine bb0 1.31 × 104 (1.32 × 104) 460 460 510 hydrophilic, whereas BODIPY and DPPD are lipophilic probes. The
Pyrogallol red bb0 3.22 × 104 (1.88 × 104) 540 consumption of the probe or the formation of the product from the
BODIPY 4.57 (1.15 × 105) 586 500 520
probe by the reaction with peroxyl radicals was measured from the
DPBQ — 6.52 × 103 440
change in absorbance at specific wavelengths in the visible region,
P, partition coefficient; ε, molar extinction coefficient; λmax, maximum absorption shown in Table 1, with a conventional UV–visible absorption spectro-
wavelength; λex and λem, fluorescence excitation and emission wavelength,
respectively. Numbers in parentheses are those in micelle system.
photometer, although they can be measured also with a fluorescence
spectrophotometer.
Accuracy and reproducibility
Assessment of radical-scavenging capacity in homogeneous solution
The experiments were satisfactorily reproducible for pure com-
pounds and also for the same biological and natural samples. The re- First, the capacity of hydrophilic antioxidants for peroxyl radical
sults of typical experiments are shown in the figures. scavenging was assessed by using pyranine, fluorescein, and PGR as
probes in PBS solution. Ascorbic acid, uric acid, and Trolox inhibited
Results the consumption of both fluorescein and pyranine completely and
produced a clear lag phase (Fig. 1). Substantially the same results
Characterization of probes were observed for both fluorescein and pyranine. The lag phase was
directly proportional to the concentrations of uric acid and Trolox
The capacity of antioxidants for radical scavenging should be for both fluorescein (Fig. 1D) and pyranine (Fig. 1H). The same lag
assessed from both the amount and the rate of radicals scavenged. phase was produced by uric acid and Trolox for both fluorescein

A B C
(Abs)t/(Abs)0, 494nm

(Abs)t/(Abs)0, 494nm
(Abs)t/(Abs)0, 494nm

Fluorescein Trolox, µM Fluorescein Ascorbic Acid, µM Fluorescein Uric Acid, µM

1 1 1
0 20 50

50 50
0.5 0.5 0.5 0 20
20
0

0 0 0
0 1000 2000 3000 0 1000 2000 3000 0 1000 2000 3000
Time, s Time, s Time, s

D E F
Pyranine Uric Acid,µM
(Abs)t/(Abs)0, 454nm

(Abs)t/(Abs)0, 454nm
(Abs)t/(Abs)0, 454nm

Pyranine Trolox, µM Pyranine Ascorbic Acid, µM

1 1 1

50 50
50
20 20
0 0 20
0.5 0.5 0.5
0

0 0 0
0 1000 2000 3000 0 1000 2000 3000 0 1000 2000 3000
Time, s Time, s Time, s

G H
2500 2500
Fluorescein Trolox Pyranine UA
y = 44.8x y = 43.7x
2000 R 2 = 0.995 2000 R 2 = 0.999
UA Trolox
Lag phase, s

Lag phase, s

y = 40.0x y = 41.4x
1500 R 2 = 1.00 1500 R 2 = 0.999

1000 AA
AA 1000 y = 23.6x
y = 21.2x
R 2 = 0.941
R 2 = 0.977
500 500

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
[IH],µM [IH],µM

Fig. 1. Effects of antioxidants on the consumption of fluorescein and pyranine induced by peroxyl radicals generated from AAPH. The consumption of (A–C) fluorescein and (E–G)
pyranine was followed by visible absorption at 494 and 454 nm, respectively, in the absence and presence of various concentrations of (A, E) Trolox, (B, F) ascorbic acid, and (C, G)
uric acid in PBS at 37 °C. The initial concentrations of fluorescein, pyranine, and AAPH were 10 μM, 50 μM, and 50 mM, respectively. Lag phase produced by antioxidants was plotted
against antioxidant concentration for (D) fluorescein and (H) pyranine.
 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252 1245

and pyranine. On the other hand, the lag phase produced by ascorbic
acid was the same at low concentration but became shorter than that
by Trolox or uric acid at higher concentrations and the plot of lag
phase against ascorbic acid concentration gave an upward curvature
line (Figs. 1D and H). This is due to the fact that the autoxidation of
ascorbic acid becomes more important with its increasing concentra-
tion and the apparent stoichiometric number becomes smaller with
increasing concentration [24].
When the reactivity of the antioxidant toward free radicals is
much higher than that of the probe, a clear lag phase is produced
and it is determined by the amount of radicals scavenged by the anti-
oxidant, that is, concentration and stoichiometric number of the anti-
oxidant, as expressed by Eq. (1),
lag phase ¼ n½IH =Ri ;
where n, [IH], and Ri are the stoichiometric number, that is, moles of
radicals scavenged by each antioxidant molecule; antioxidant con-
centration; and rate of radical flux from the azo initiator, respectively.
It is known that each Trolox molecule scavenges two peroxyl radical
molecules, that is, n = 2 [25]. The slope of the straight line for Trolox
in Figs. 1D and H, 2/Ri, is calculated as 41.4 and 44.8 s/μM, for fluores-
cein and pyranine, respectively, and the rate of free radical flux is cal-
culated as Ri = 4.83 × 10 − 8 and 4.46 × 10 − 8 M/s, respectively. The
rate of radical flux is expressed by Eq. (2),
Ri ¼ 2ekd½AAPH ;
where e and kd are the efficiency of free radical production and the
rate constant for the decomposition of AAPH [22]. The value ekd is cal-
culated from the average value of Ri = 4.65 × 10 − 8 M/s and the con-
centration of AAPH, 50 mM, as 4.65 × 10 7 s − 1 at 37 °C in PBS. The
total content of antioxidants in complex mixtures can be estimated
from the lag phase obtained experimentally and Eq. (1) by:
−8
n½IH ¼ lag phase  Ri ¼ 4:65  10 lag phaseðM Þ:
A different pattern was observed for PGR (Fig. 2). In contrast to
fluorescein and pyranine, PGR has much higher reactivity toward
peroxyl radicals and only ascorbic acid inhibited the consumption of
PGR efficiently and produced a lag phase, whereas Trolox suppressed
it partially and uric acid did not suppress it appreciably. The ratio of
the rates of consumption of PGR in the absence and presence of anti-
oxidant, R0 and RIH, respectively, is given by Eq. (4),
R0 =RIH ¼ 1 þ ðkIH ½IH =kPGR ½PGRÞ; ð4Þ
where kIH and kPGR are the rate constants for the reactions of peroxyl
radicals with antioxidant and PGR, respectively. The rate of consump-
tion of PGR was calculated from the decrease in the absorption at
540 nm at the initial stage. It was difficult to obtain the rate for ascor-
bic acid accurately (Fig. 2B). The plots of R0/RIH as a function of [IH]/
ð1Þ [PGR] in the presence of Trolox and uric acid are shown in Fig. 2E. A
satisfactorily straight line was obtained for each and from the slope
of these lines the ratio of the rate constants was obtained as kIH/
kPGR = 0.254 and 0.0173 for Trolox and uric acid, respectively.
Assessment of activity for scavenging radicals in micelle systems
Biological systems are heterogeneous and antioxidants are local-
ized either in the aqueous phase or in lipophilic compartments. The
localization of antioxidants and also free radicals affects the antioxi-
dant capacity for radical scavenging and it is necessary to assess the
antioxidant capacity in the heterogeneous systems for both hydro-
ð2Þ philic and lipophilic antioxidants. In this study, such effects were ex-
amined by using micelle systems composed of methyl linoleate and
methyl palmitate in PBS containing SDS. AAPH and fluorescein pro-
duced opaque suspensions and therefore AIPH was used as a hydro-
philic azo initiator and pyranine and PGR were used as hydrophilic
probes. MeO-AMVN and BODIPY were used also as lipophilic azo ini-
tiator and probe, respectively. Ascorbic acid, uric acid, and Trolox
were used as hydrophilic antioxidants and α-tocopherol and PMC as
lipophilic antioxidants.
ð3Þ As observed in homogeneous PBS solution, ascorbic acid, uric acid,
and Trolox inhibited the consumption of pyranine induced by AIPH in
the micelle system and produced a clear lag phase, which was propor-
tional to the antioxidant concentration (data not shown). Lipophilic

A B C
(Abs)t/(Abs)0, 540nm
(Abs)t/(Abs)0, 540nm
(Abs)t/(Abs)0, 540nm

PGR Trolox, µM PGR Ascorbic Acid, µM PGR Ascorbic Acid, µM

1 1 1

30 60 90 120 150
0
0.5 90 150 0.5
0 30

0 30 60 90 120 150
0 0 0.9
0 500 1000 1500 0 1000 2000 3000 4000 5000 0 1000 2000 3000 4000 5000
Time, s Time, s Time, s

D E
3
(Abs)t/(Abs)0, 540nm

PGR Uric Acid, µM

1

2 Trolox
y = 0.254x + 1
R0/RIH

R2 = 1.00
0.5 150
0 1
UA
y = 0.0173x + 1
R2 = 0.687
0 0
0 500 1000 1500 0 2 4 6
Time, s [IH]/[PGR], M/M

Fig. 2. Effects of antioxidant on the consumption of pyrogallol red (PGR) in PBS. The consumption of PGR induced by AAPH was followed by visible absorption at 540 nm in the ab-
sence and presence of various concentrations of (A) Trolox, (B, C) ascorbic acid, and (D) uric acid in PBS at 37 °C. The initial concentrations of PGR and AAPH were 30 μM and 50 mM,
respectively. (E) The ratio of the rate of consumption of PGR in the absence and presence of Trolox and uric acid, R0/RIH, was plotted against the molar ratio of antioxidant and PGR.
1246 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252

α-tocopherol and PMC also inhibited pyranine consumption and pro- acid did not (Fig. 4). The lag phase was proportional to the antioxi-
duced a clear lag phase (data not shown). dant concentration (Fig. 4G). PMC suppressed the consumption of
The effects of antioxidants on PGR consumption induced by BODIPY more efficiently and produced a clearer and shorter lag
AIPH in the micelle system are shown in Fig. 3. In the presence phase than α-tocopherol (Fig. 5), probably because PMC is more mo-
of the same concentrations of antioxidant and PGR, ascorbic acid bile and capable of scavenging radicals more efficiently than α-
inhibited the consumption of PGR completely; Trolox, PMC, and tocopherol in micelle systems. The plot of lag phase against PMC con-
α-tocopherol suppressed partially; and uric acid did not exert a centration gave a straight line with a slope of 1.38 × 10 8 s/M, from
significant effect (Fig. 3A). The antioxidants suppressed PGR con- which ekd is calculated as 7.25 × 10 − 6 s − 1. This is in good agreement
sumption in a concentration-dependent manner. Ascorbic acid with the value of 7.10 × 10 − 6 s − 1 obtained previously [22]. This sys-
inhibited the consumption of PGR markedly and produced a clear tem is useful for the assessment of lipophilic antioxidant capacity.
lag phase (Fig. 3B). Distinct results were observed for the effects PMC may be a suitable reference compound.
of α-tocopherol, PMC, and Trolox on the consumption of PGR
(Figs. 3C, D, and E). Trolox suppressed the decay of PGR in a Measurement of ORAC value
concentration-dependent manner, whereas the effects of PMC and
α-tocopherol were less significant than Trolox and complex. They The ORAC method is the most widely used at present to assess the
exerted a considerable effect at 30 μM, above which, however, antioxidant capacity. It is calculated from the net increase produced
the effects were small, probably because those localized within by antioxidant in the integrated area under the curve of probe
the lipophilic compartment were unable to compete well with decay (AUC), that is, (AUC in the presence of antioxidant) − (AUC in
PGR against radicals in the aqueous phase. The effect of uric acid the absence of antioxidant) (Fig. 6A). Phycoerythrin and fluorescein
was small even at 420 μM, which is the upper level in human plas- have been used often as probes. In this study the ORAC values were
ma (Fig. 3F). The plot of R0/RIH against [IH]/[PGR] in this system is measured for several antioxidants in various systems using fluoresce-
shown in Fig. 3G. The slopes that correspond to kIH/kPGR for ascor- in, pyranine, and PGR as probes. As shown in Figs. 6B–D, the ORAC
bic acid, Trolox, and uric acid were 12, 1.0, and 0.02, respectively. value was proportional to the antioxidant concentration. As expected
BODIPY and MeO-AMVN were used as lipophilic probe and radical from Fig. 1, the ORAC values for Trolox and uric acid were equal when
source, respectively, in the micelle systems. α-Tocopherol, PMC, Tro- fluorescein or pyranine was used as a probe (Fig. 6B), whereas those
lox, and ascorbic acid suppressed the consumption of BODIPY induced for ascorbic acid became smaller at higher concentration because of
by MeO-AMVN effectively and produced a lag phase, whereas uric its autoxidation as described above. The apparent ORAC value

A B C
PGR PGR Trolox, µM
(Abs)t/(Abs)0, 540nm

Ascorbic Acid, µM
(Abs)t/(Abs)0, 540nm

(Abs)t/(Abs)0, 540nm
PGR [PGR]/[IH] = 1/1
1
1 1
AA
150
Trolox

0.5
UA 0 30 60 90 120
PMC
none a-T
0 30 60 90 120 150
0 0.4 0.4
0 500 1000 1500 0 1000 2000 3000 0 500 1000 1500
Time, s Time, s Time, s

D E F
(Abs)t/(Abs)0, 540nm
(Abs)t/(Abs)0, 540nm

(Abs)t/(Abs)0, 540nm

PGR PMC, µM PGR a-T, µM PGR Uric Acid, µM

1 1 1

420
150 0
0 150
0.4 0 0.4
0.4
0 500 1000 1500 0 500 1000 1500 0 500 1000 1500
Time, s Time, s Time, s

G
Trolox
6 y = 1.01x + 1
R0/RIH

4
PMC
y = 0.324x + 1
2 a -T
y = 0.232x + 1
UA
y = 0.0189x + 1
0
0 1 2 3 4 5 6 7
[IH]/[PGR], (M/M)

Fig. 3. Effects of antioxidants on the consumption of PGR in micelle systems. (A) The consumption of PGR induced by AIPH in the absence and presence of the same concentrations
of ascorbic acid, Trolox, PMC, α-tocopherol, and uric acid was followed. Consumption of PGR in the presence of various concentrations of (B) ascorbic acid, (C) Trolox, (D) PMC, (E)
α-tocopherol, and (F) uric acid is shown. (G) Plot of R0/RIH against [IH]/[PGR]. The initial concentrations of PGR and AIPH were 30 μM and 30 mM, respectively.
 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252 1247

A B C
(Abs)t/(Abs)0, 586nm BODIPY a-T, µM

(Abs)t/(Abs)0, 586nm
(Abs)t/(Abs)0, 586nm
BODIPY Trolox, µM BODIPY PMC, µM

1 1 1
15

15 9
9 15
0 9
0 0

0.5 0.5 0.5

0 1000 2000 3000 0 1000 2000 3000 0 1000 2000 3000
Time, s Time, s Time, s

D E F
BODIPY
(Abs)t/(Abs)0, 586nm

BODIPY [IH], 12µM

(Abs)t/(Abs)0, 586nm
Ascorbic Acid, µM

(Abs)t/(Abs)0, 586nm
BODIPY Uric Acid, µM

1 1 1

AA Trolox
15 PMC a-T
9 UA
0 15
none
0

0.5 0.5 0.5

0 1000 2000 3000 0 1000 2000 3000 0 1000 2000 3000
Time, s Time, s Time, s

G 4000
H 20
PMC
a-T y = 6.72x + 1
y = 199.7x R² = 0.979
R² = 0.999 15
3000 Trolox
Lag phase, s

PMC
y = 152.4x y = 4.30x + 1
R0/RIH

R² = 0.995 R² = 0.948

2000 Trolox
10 a-T
y = 123.6x y = 3.31x + 1
R² = 0.991 R² = 0.894
AA
y = 120.4x 5
1000 R² = 0.990 UA
y = 0.183x + 1
R² = 0.852
0
0 0 1 2 3
0 2 4 6 8 10 12 14 16 18
[IH], µM [IH]/[BODIPY], M/M

Fig. 4. Effects of antioxidants on the consumption of BODIPY in micelle systems. The consumption of BODIPY induced by MeO-AMVN was measured from the absorption at 586 nm
in the absence and presence of antioxidant: (A) Trolox, (B) PMC, (C) α-tocopherol, (D) ascorbic acid, (E) uric acid. (F) The results using 12 μM are compared. The lag phase against
[IH] and the ratio of R0/RIH against [IH]/[BODIPY] are shown in (G) and (H), respectively. The initial concentrations of BODIPY and MeO-AMVN were 6 μM and 1.0 mM, respectively.

A B C
(Abs)t/(Abs)0, 586nm

(Abs)t/(Abs)0, 586nm
(Abs)t/(Abs)0, 586nm

[IH] = 3µM BODIPY [IH] = 6µM BODIPY [IH] = 9µM

1 1 1
PMC
PMC PMC

a-T a-T
a-T
none none
none
0.9 0.9 0.9
0 1000 2000 3000 0 1000 2000 3000 0 1000 2000 3000
Time, s Time, s Time, s

D E 4000
BODIPY [IH] = 12µM
(Abs)t/(Abs)0, 586nm

1 a-T
Lag phase, s

3000 y = 182.8x
R 2 = 0.994
PMC
PMC
2000 y = 138.0x
R 2 = 0.991
a-T 1000
none
0.9 0
0 1000 2000 3000 0 5 10 15 20
Time, s [IH], µM

Fig. 5. Effects of PMC and α-tocopherol on the consumption of BODIPY in micelle systems. The conditions are the same as in Fig. 4.
1248 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252

A B
UA (Pyr)
1500 y = 31.0x Trolox (Pyr)
UA (Flu) y = 27.6x
y = 30.3x Trolox (Flu)

ORAC
1000 y = 25.5x
AA (Pyr)
y = 16.5x
500 AA (Flu)
y = 17.6x

0
0 10 20 30 40 50 60 70
[IH], µM
C D
4000 4000
PGR/AAPH AA PGR/AIPH AA
y = 22.1x 3000 y = 56.9x
3000
ORAC

ORAC
2000 2000 Trolox
y = 23.8x
Trolox
1000 y = 5.9x 1000 PMC
y = 8.8x
0 UA 0 a-T
UA
y = 0.4x y = 6.1x
y = -0.4x
-1000 -1000
0 60 120 180 0 10 20 30 40 50 60 70
[IH], µM [IH], µM

Fig. 6. ORAC (oxygen radical absorbance capacity) values measured for several antioxidants in various systems. (A) ORAC is obtained from the net increase produced by antioxidant
in the integrated area under the curve of probe decay (AUC). (B) ORAC value as a function of antioxidant concentration. ORAC value was calculated from the AUC observed for the
decay of 10 μM fluorescein (open symbols) and 50 μM pyranine (solid symbols) in the absence and presence of Trolox (circle), ascorbic acid (square), or uric acid (triangle) induced
by 50 mM AAPH in PBS at 37 °C. (C) ORAC values measured using 30 μM pyrogallol red and 50 mM AAPH in PBS. Other conditions are the same as in (B). (D) ORAC values measured
using pyrogallol red and 50 mM AIPH in SDS micelles containing 0.5% methyl palmitate and 0.5% methyl linoleate. Trolox (open circle), ascorbic acid (open square), uric acid (open
triangle), α-tocopherol (solid circle), PMC (solid square).

decreased in the order of Trolox = uric acid > ascorbic acid when fluo- within the lipophilic domain are less effective than Trolox at scaveng-
rescein or pyranine was used as a probe in PBS, whereas the order ing radicals in the aqueous phase. These results clearly show that the
was different when PGR was used as a probe, that is, ascorbic acid > - ORAC value depends on the probe and conditions employed.
Trolox > uric acid. In homogeneous solution, the ORAC value is the
same for Trolox, PMC, and α-tocopherol. On the other hand, in the Application to a complex mixture for the assessment of
micelle systems, the order as assessed with PGR and AIPH as a radical-scavenging capacity
probe and initiator, respectively, was ascorbic acid > Trolox > α-to-
copherol, PMC > uric acid. The smaller ORAC values for α-tocopherol The above methods were applied for the assessment of antioxi-
and PMC compared to Trolox are ascribed to different localization of dant capacity of practical samples containing a complex mixture of
the antioxidants, that is, lipophilic α-tocopherol and PMC localized antioxidants. Human plasma, wine, and green tea powder were

A B C 3000
(Abs)t/(Abs)0, 454nm
(Abs)t/(Abs)0, 454nm

Intact Plasma, Volume% Pyranine Dialyzed Plasma, Volume%

1 1
Lag phase, s

2000 Intact Plasma

y = 267.4x
0.5 R 2 = 0.998
0.5 10
0 2 5 1000
0 2 4 6 8 10

0 0 0
0 2000 4000 6000 0 2000 4000 6000 0 5 10
Time, s Time, s [IH], Volume%

D E F 3.0
PGR Intact Plasma, Volume%
(Abs)t/(Abs)0, 540nm

PGR Dialyzed Plasma, Volume%

(Abs)t/(Abs)0, 540nm

1 1 Intact Plasma
y = 0.0885x + 1
2.0 R 2 = 0.755
R0/RIH

0.5 0.5
10 1.0 Dialyzed Plasma
4 10 y = 0.0198x + 1
0 R 2 = 0.916
0 2
0 0 0.0
0 500 1000 1500 0 500 1000 1500 0 5 10 15
Time, s Time, s [IH], Volume%

Fig. 7. Effects of human plasma on the consumption of pyranine and PGR. The consumption of (A, B) 50 μM pyranine and (D, E) 30 μM PGR induced by 50 mM AAPH was followed in
PBS at 37 °C as described under Materials and methods in the absence and presence of various concentrations of (A, D) intact plasma and (B, E) dialyzed plasma. Plots of the lag
phase and R0/RIH against plasma vol% are shown in (C) and (E), respectively.
 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252 1249

A tested as examples. The antioxidant capacity of plasma has been mea-

BODIPY Intact Plasma,Volume% sured often to assess antioxidant status in vivo, bioavailability of anti-

(Abs)t/(Abs)0,586nm
1 oxidants, or efficacy of antioxidant supplementation [19,25-30]. In
20
10 this study, intact and dialyzed plasma was tested. The dialysis
5 removes small hydrophilic molecules such as vitamin C and uric
2
0.5 0 acid, but vitamin E is not affected. Both intact and dialyzed plasma
delayed the decay of pyranine and PGR in a concentration-
dependent manner, the major difference being the production of a
lag phase by intact plasma, but not by dialyzed plasma (Fig. 7). As
0
0 2000 4000 6000 shown above, ascorbic acid and uric acid produced a clear lag phase
Time, s for pyranine. The lag phase produced by 100% intact plasma is esti-
B mated to be 3.4 × 10 4 s, which together with Ri = 4.65 × 10 − 8 M/s
BODIPY Dialyzed Plasma,Volume% gives the total antioxidant concentration as n[IH] = 1.58 mM, and if
(Abs)t/(Abs)0,586nm

1 the stoichiometric number is assumed as n = 2, the apparent antioxi-

20 dant concentration is obtained as 790 μM. Uric acid and proteins are
0 5 10
the major antioxidants in plasma. It may be noted that the rate of con-
0.5 sumption of pyranine in the presence of intact plasma after the lag
phase was similar to that in the presence of dialyzed plasma.
On the other hand, as shown above and in a previous report [30],
only ascorbic acid can produce a clear lag phase against the decay of
0
0 2000 4000 6000 PGR. The lag phase for PGR consumption increased in proportion to
Time, s the vol% of intact plasma (Fig. 7D), from which the lag phase for
C 100% plasma is estimated as 2.0 × 10 3 s. The value for n[IH] is calculat-
10 ed as 2.0 × 10 3 × 4.65 × 10 − 8 = 9.3 × 10 − 5 M, and if n is assumed as 2,
9 then the concentration of ascorbic acid in the plasma is obtained as
8
7 46 μM. The effect of dialyzed plasma on PGR consumption was quite
Intact Plasma
6 y = 0.380x + 1 small (Fig. 7E). The plot of R0/RIH as a function of [IH]/[PGR] gave a
R0/RIH

R² = 0.993
5 straight line, the slope being 0.0885 and 0.0198/vol% for intact and di-
4 alyzed plasma, respectively (Fig. 7F).
3
2 Dialyzed Plasma The effects of intact and dialyzed plasma on the consumption of
y = 0.106x + 1 BODIPY induced by MeO-AMVN in micelle systems are shown in
1 R² = 0.996
0 Fig. 8. The plot of R0/RIH against [IH]/[BODIPY] gave a straight line,
0 5 10 15 20 25
the slope being 0.380 (R 2 = 0.993) and 0.106 (R 2 = 0.996)/vol% for in-
[IH], Volume%
tact and dialyzed plasma, respectively.
The antioxidants contained in wine have received much atten-
Fig. 8. Effect of human plasma on the consumption of BODIPY induced by MeO-
AMVN in a micelle system. The consumption of 6 μM BODIPY induced by 1.0 mM tion for their direct antioxidant action and nonantioxidant function
MeO-AMVN in the absence and presence of (A) intact and (B) dialyzed plasma [31]. In this study, the antioxidant capacity of red and white wine
was followed in micelle systems at 37 °C. (C) The ratio R0/RIH was plotted against was assessed using fluorescein and PGR as probes (Fig. 9). Both
plasma vol%. red and white wine produced a clear lag phase against the con-
sumption of fluorescein, the lag phase by red wine being much

A B C 2000
(Abs)t/(Abs)0, 494nm

(Abs)t/(Abs)0, 494nm

Fluorescein Red Wine, Volume% Fluorescein White Wine, Volume%

1 1 10.0 Red Wine
1.0 1500
Lag phase, s

8.0 y = 1360x
0.7 2.0
R2 = 0.996
6.0
0.5
1000 White Wine
0.5 4.0 y = 157x
0.3
0.5
R2 = 0.950
500
0.1
0.0 0.0
0 0 0
0 1000 2000 3000 0 1000 2000 3000 0 2 4 6 8 10 12
Time, s Time, s [IH], Volume%
D E F
PGR PGR White Wine, Volume% 5
(Abs)t/(Abs)0, 540nm

Red Wine, Volume%

(Abs)t/(Abs)0, 540nm

Red Wine
1 1 4 y = 1.25x + 1
R 2 = 0.982
R0/RIH

2
White Wine
2.5 10 1 y = 0.0296x + 1
1.0 0 R 2 = 0.921
0.0
0.5 0.5 0
0 500 1000 0 500 1000 0 5 10 15
Time, s Time, s [IH], Volume%

Fig. 9. Effects of red and white wine on the consumption of fluorescein and PGR. The consumption of (A, B) 6 μM fluorescein and (D, E) 30 μM PGR induced by 100 mM AAPH was
followed in the absence and presence of varying concentrations of wine in PBS at 37 °C. Lag phase and the ratio of the rate of PGR consumption in the absence and presence of wine
are plotted against wine vol% in (C) and (F).
1250 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252

longer than that by white wine. The lag phase was proportional to Table 2
the concentration of wine and obtained as lag phase = 1.36 × 10 3 Antioxidant capacity of human plasma, wine, and green tea as assessed by the methods
of this study.
and 1.57 × 10 2 V for red and white wine, respectively, where V is
the vol% of wine. As described above, the rate of radical formation Sample n[IH] (M) R0/RIH (/vol%) (relative)
with 100 mM AAPH is Ri = 2 × 4.65 × 10 − 7 × 0.1 = 9.30 × 10 − 8 M/s. Human plasma
The number of radicals scavenged by wine is calculated as Intact 2.48 × 10− 3 0.089 (1.0)
n[IH] = lag phase × Ri = 1.36 × 10 5 × 9.30 × 10 − 8 = 1.26 × 10 − 2 M and Dialyzed 0.020 (0.22)
−2
Red wine 1.26 × 10 1.25 (14)
1.57 × 10 4 × 9.30 × 10 − 8 = 1.46 × 10 − 3 M for red wine and white
White wine 1.46 × 10− 3 0.030 (0.34)
wine, respectively. The antioxidant capacity was reported to range Green tea 4.44 × 10− 3 1.57 (18)
from 0.08 to 1.2 and from 6.4 to 41.9 mM equivalents of Trolox for
n[IH], amount of radicals that can be scavenged; R0/RIH, relative reactivity toward
white wine and red wine, respectively [27]. Because wine contains peroxyl radicals.
polyphenolic compounds whose n is larger than 2, the molar con-
centration of antioxidants in wine should be smaller than the
above values. In any event, these results show that both red and As described above, the capacity for both the rate and the amount
white wine contain high concentrations of antioxidants. of scavenging radicals can be assessed for complex mixtures as well
Red wine suppressed the PGR decay concentration dependently, as for pure antioxidant compounds. The results for the three practical
but the effect of white wine was small (Figs. 9D and E). The ratio samples measured in this study are summarized in Table 2, which
R0/RIH was proportional to the wine concentration, the slope being shows that the amounts of radicals scavenged and the relative rate
1.25 (R 2 = 0.982) and 0.030 (R 2 = 0.921)/vol% for red wine and of scavenging radicals by antioxidants can be assessed by the
white wine, respectively (Fig. 9F). methods described above.
The antioxidant capacity of green tea powder was also assessed
using fluorescein and PGR as probes. As described under Materials Discussion
and methods, 20 mg green tea powder was dissolved in 10 ml water
and vortex-mixed, followed by centrifugation. The supernatant thus The free radical-scavenging antioxidants play important roles in
obtained suppressed the consumption of fluorescein and PGR induced the physiological defense network against oxidative stress. The as-
by AAPH in PBS and produced a lag phase against both fluorescein sessment of antioxidant capacity has been the subject of extensive
and PGR in a concentration-dependent manner (Fig. 10). The plot of studies and arguments in the past 2 decades. Numerous studies
lag phase against vol% of the green tea gave a straight line, lag using available methods resulted in inconsistent results, due in part
phase (s) = 955 × green tea (vol%). to improper specification of antioxidant capacity and inappropriate
The lag phase for 100% was 9.55 × 10 4 s and the amount of free radi- application of methods and interpretation of data. The term “antioxi-
cals scavenged was calculated as n[IH] = 9.55 × 10 4 × 4.65 × 10 − 8 = dant capacity” is used sometimes to mean capacity of antioxidants for
4.44 × 10 − 3 M. Because 20 mg green tea powder was dissolved in scavenging radicals, but in other cases it means the capacity to inhibit
10 ml water, the amount of radicals that could be scavenged was oxidation. These two “antioxidant capacities” are different issues. Fur-
estimated as 2.22 mol/g green tea powder. thermore, the capacity of scavenging radicals is determined by both
The supernatant suppressed the consumption of PGR. The initial the rate and the amount of scavenging radicals and these two factors
rate of PGR consumption was calculated and the plot of R0/RIH against should be assessed distinctly.
green tea vol% gave a straight line, the slope being 1.57 (R 2 = 0.977) This study was performed aiming at the development of a com-
(data not shown). prehensive method for the assessment of antioxidant radical-
scavenging capacity. As shown above, in the present method, the
rate of radical scavenging is assessed from the effect on the decay of
A a probe with high reactivity, such as pyrogallol red, and the amount
of radical scavenging is assessed from the effect on the decay of a
(Abs)t/(Abs)0, 494nm

Fluorescein Green Tea, Volume%

1 probe with low reactivity such as fluorescein and pyranine. Further-
more, the localization of the antioxidants and radicals is considered
in the design of experimental conditions and interpretation of data.
0.5 A number of methods have been developed and applied [11 and
1.0 2.0 references cited therein]. Of these, the ORAC method is used most
0.0 0.5
widely at present for pure compounds, foods, botanicals, nutraceuti-
cals, and commercial products. The ORAC assay is based upon the
0
0 2000 4000 6000 early work of Glazer [32] and was developed by Cao et al. [15]. The
Time, s ORAC value refers to the net protection area under the quenching
B curve of probe decay by antioxidant. Phycoerythrin [15] and fluores-
cein [33] have been used as probes, but other probes may be used
(Abs)t/(Abs)0, 540nm

PGR Green Tea, Volume%

1 and their decay may be followed by fluorescence or visible absorption.
It is claimed that the ORAC method is useful because it shows lag
phase, initial rate, and total extent of inhibition in a single value. How-
0.5 10.0 ever, several inherent drawbacks may be pointed out. First, the ORAC
5.0 value does not distinguish between quality and quantity, that is, rate
2.0 and amount of radical scavenging by the antioxidant. For example,
0.0
0 the higher ORAC value of mixture A compared to B may be due to
0 1000 2000 3000 higher reactivity of A toward the radical compared to B, or higher con-
Time, s tent of antioxidant in A than in B, or both. Further, the ORAC value de-
pends on the probe used. Fluorescein is used often as a probe, but as
Fig. 10. Effects of green tea on the consumption of fluorescein and PGR. The consump-
tion of (A) 6 μM fluorescein and (B) 30 μM PGR induced by 50 mM AAPH was followed
shown in Fig. 1, Trolox and uric acid, and ascorbic acid at low concen-
in the absence and presence of varying concentrations of green tea in PBS at 37 °C as trations, gave the same ORAC values. On the other hand, quite a differ-
described under Materials and methods. ent capacity was assessed from the ORAC values obtained by using
 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252
PGR as a probe. In general, the ORAC assay measured by a probe with
low reactivity toward radicals such as fluorescein and pyranine over-
estimates the capacity of antioxidants with low reactivity. This prob-
lem also applies to the total antioxidant capacity in plasma or other
biological fluids. Care should be taken in the interpretation of ORAC
values. It may be pointed out that it is difficult to measure rate and
amount of radical scavenging separately by using only one probe.
It was reported that the relative contributions of peroxyl radical
trapping by antioxidants in human plasma were urate 35–65%, pro-
teins 10–50%, ascorbate 0–24%, and vitamin E 5–10% [34]. Similarly,
it was reported that uric acid accounted for approximately two-
thirds of total antioxidant capacity of human plasma [35]. The high
relative contribution of uric acid does not mean uric acid is the
major antioxidant in human plasma. The results of this study clearly
show that the reactivity of uric acid toward peroxyl radical is much
lower than that of ascorbate. In agreement with this notion, when
plasma or whole blood is oxidized by free radicals, ascorbic acid is
consumed first and uric acid is spared [36,37]. Both uric acid and
ascorbic acid are important physiological antioxidants, but they are
essentially different in that ascorbic acid is more reactive toward
radicals than uric acid and that, more importantly, ascorbic acid in-
hibits lipid peroxidation synergistically with vitamin E, whereas
uric acid cannot. This is because ascorbic acid reduces α-
tocopheroxyl radical derived from α-tocopherol to regenerate α-
tocopherol and inhibit pro-oxidant action of α-tocopherol, whereas
uric acid cannot do so [38,39].
Another important issue is the localization of antioxidants and ox-
idizing free radicals. The effects of phase separation on the action of
antioxidants were first studied in the reduction of α-tocopheroxyl
radical by ascorbic acid by using water-soluble and lipid-soluble azo
initiators in liposomal membranes [40,41]. The antioxidant capacity
has been measured for both hydrophilic and lipophilic antioxidants
in heterogeneous systems such as micelles, microemulsions, isolated
low-density lipoprotein suspensions, plasma, erythrocytes, and cul-
tured cells [11,42-46]. Cyclodextrin was used as a solubility enhancer
for lipophilic antioxidants [47]. The capacity of antioxidants to scav-
enge free radicals localized in different domains becomes smaller
than that in the same domain. It was also found that the capacity of
ascorbic acid to scavenge radicals becomes smaller as the radicals
go deeper into the membrane or lipoprotein [48]. These physical ef-
fects should be considered in the assessment and interpretation of
antioxidant capacity. The micelle systems using lipids and surfactant
are convenient for the assessment of antioxidant capacity in hetero-
geneous systems. It may be noteworthy that the electric charge of
the micelle, which is determined by the surfactant, may affect appar-
ent antioxidant capacity [22].
It should be added that the capacity for scavenging radicals does
not always correlate with the capacity to inhibit oxidation. The inhibi-
tion of lipid peroxidation is one of the important roles of antioxidants
and the capacity for inhibition of lipid peroxidation should be
assessed separately under controlled conditions [11].
Oxidative stress and damage are mediated by multiple species,
some by free radicals and some by nonradicals. These oxidant species
have different characteristics such as reactivity, selectivity, phase lo-
calization, and solubility. The antioxidant capacity depends on the ac-
tive species. For example, uric acid is as potent as ascorbic acid against
hypochlorite, and uric acid is more potent than Trolox against hypo-
chlorite and peroxynitrite [49]. This makes it difficult to develop a
universal method by which antioxidant capacity can be measured ac-
curately and quantitatively. A good method should meet several re-
quirements: simple, sensitive, reproducible, repeatable, available
instrumentation, high-throughput, adaptable for both hydrophilic
and lipophilic antioxidants, and little artificial interference. The loss
of unstable antioxidants such as ascorbic acid and ubiquinol by artifi-
cial oxidation during preparation and storage of the samples should
be carefully avoided. Obviously, the antioxidant capacity measured
1251
in vitro cannot be extrapolated simply to the in vivo situation, be-
cause bioavailability, metabolism, and biotransformation as well as
chemical reactivity are important determinant in vivo. Plasma may
serve as a useful sample to monitor and assess the capacity of antiox-
idants contained in the diet and in supplements for radical scaveng-
ing and inhibition of oxidation in vivo.
In conclusion, the antioxidant radical absorbance capacity may be
assessed as follows:
1. The amount of radicals that can be scavenged by antioxidants or
the concentration of antioxidants in a complex mixture is mea-
sured from the lag phase for the consumption of fluorescein, pyra-
nine, or BODIPY induced by an azo initiator in solution or a micelle
system. The probe and azo compound should be selected consider-
ing the reaction medium.
2. The reactivity of antioxidants or mixtures is measured from their
effect on the consumption of PGR or BODIPY in solution or a mi-
celle system induced by an azo initiator.
3. The relative antioxidant capacity may be assessed from the pattern
of the decay of the probe or more quantitatively by the kinetic
analysis as shown in this article.
References
[1] Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine, 4th edition.
Clarendon, Oxford; 2007.
[2] Aldini, G., Yeum, K.-J., Niki, E., Russell, R.M. (Eds.), 2010]. Biomarkers for Antioxi-
dant Defense and Oxidative Damage: Principles and Practical Applications. Wiley–
Blackwell, Ames; 2010.
[3] Gey, K. F.; Puska, P. Plasma vitamin E and A inversely correlated to mortality from
ischemic heart disease in cross-cultural epidemiology. Ann. N. Y. Acad. Sci. 570:
268–282; 1989.
[4] Stampfer, M. J.; Hennekens, C. H.; Manson, J. E.; Colditz, G. A.; Rosner, B.; Willett,
W. C. Vitamin E consumption and the risk of coronary heart disease in women. N.
Engl. J. Med. 328:1444–1449; 1993.
[5] Rimm, E. B.; Stampfer, M. J.; Ascheio, A.; Giovannucci, E.; Colditz, G. A.; Willett,
W. C. Vitamin E consumption and the risk of coronary heart disease in men. N.
Engl. J. Med. 328:1450–1456; 1993.
[6] Cordero, Z.; Drogan, D.; Weikert, C.; Boeing, H. Vitamin E and risk of cardiovascu-
lar disease: a review of epidemiologic and clinical trial studies. Crit. Rev. Food Sci.
Nutr. 50:420–440; 2010.
[7] Morris, M. C.; Tangney, C. C. A potential design flaw of randomized trials of vita-
min supplements. JAMA 305:1348–1349; 2011.
[8] Niki, E. Do free radicals play causal role in atherosclerosis? Low density lipopro-
tein oxidation and vitamin E revisited. J. Clin. Biochem. Nutr. 48:3–7; 2011.
[9] Dotan, Y.; Pinchuk, I.; Lichtenberg, D.; Leshno, M. Decision analysis supports the
paradigm that indiscriminate supplementation of vitamin E does more harm
than good. Arterioscler. Thromb. Vasc. Biol. 29:1304–1309; 2009.
[10] Milman, U.; Blum, S.; Shapira, C.; Aronson, D.; Miller-Rotan, R.; Anbinder, Y.;
Alshiek, J.; Bennett, L.; Kostenko, M.; Landau, M.; Keider, S.; Levy, Y.; Khemlin,
A.; Radan, A.; Levy, A. P. Vitamin E supplementation reduces cardiovascular
events in a subgroup of middle-aged individuals with both type 2 diabetes melli-
tus and the haptoglobin 2-2 genotype: a prospective double-blinded clinical trial.
Arterioscler. Thromb. Vasc. Biol. 28:341–347; 2008.
[11] Niki, E. Assessment of antioxidant capacity in vitro and in vivo. Free Radic. Biol.
Med. 49:503–515; 2010.
[12] Miller, N. J.; Diplock, A. T.; Rice-Evans, C.; Davies, M. J.; Gopinathan, V.; Milner, A.
A novel method for measuring antioxidant capacity and its application to moni-
toring the antioxidant status in premature neonates. Clin. Sci. 84:407–412; 1993.
[13] Apak, R.; Guclu, K. G.; Ozulrek, M.; Karademir, S. E. Novel total antioxidant capac-
ity index for dietary polyphenols and vitamins C and E, using their cupric ion re-
ducing capability in the presence of neocuproine: CUPRAC method. J. Agric. Food
Chem. 52:7970–7981; 2004.
[14] Benzie, I. E. F.; Strain, J. J. The ferric reducing ability of plasma (FRAP) as a measure
of “antioxidant power”: the FRAP assay. Anal. Biochem. 239:70–76; 1996.
[15] Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-radical absorbance capacity assay for
antioxidants. Free Radic. Biol. Med. 14:303–311; 1993.
[16] Schaich, K. M. Developing a rational basis for selection of antioxidant screening
and testing methods. Acta Hortic. 709:79–94; 2006.
[17] Frankel, E. N.; Finley, J. W. How to standardize the multiplicity of methods to eval-
uate natural antioxidants. J. Agric. Food Chem. 56:4901–4908; 2008.
[18] U.S. Department of Agriculture; Agricultural Research Service Oxygen radical ab-
sorbance capacity (ORAC) of selected foods, release 2. Nutrient Data Laboratory
home page. http://www.ars.usda.gov/nutrientdata/orac (Date accessed Novem-
ber 11, 2011); 2010.
[19] Niki, E.; Omata, Y.; Fukuhara, A.; Saito, Y.; Yoshida, Y. Assessment of radical scav-
enging capacity and lipid peroxidation inhibiting capacity of antioxidant. J. Agric.
Food Chem. 56:8255–8260; 2008.
[20] Litescu, S. C.; Eremia, S.; Radu, G. L. Methods for the determination of antioxidant
capacity in food and raw materials. Adv. Exp. Med. Biol. 698:241–249; 2011.
1252 M. Takashima et al. / Free Radical Biology & Medicine 52 (2012) 1242–1252
[21] Niki, E.; Noguchi, N.; Tsuchihashi, H.; Gotoh, N. Interaction among vitamin C, vi-
tamin E, and β-carotene. Am. J. Clin. Nutr. 62:1322S–1326S; 1995.
[22] Noguchi, N.; Yamashita, H.; Gotoh, N.; Yamamoto, Y.; Numano, R.; Niki, E. 2,2′-
Azobis(4-methoxy-2,4-dimethylvaleronitrile), a new lipid soluble azo initiator:
application to oxidation of lipids and low-density lipoprotein in solution and in
aqueous dispersions. Free Radic. Biol. Med. 24:259–268; 1998.
[23] Shi, H.; Noguchi, N.; Niki, E. Comparative study on dynamics of antioxidative ac-
tion of α-tocopheryl hydroquinone, ubiquinol, and α-tocopherol against lipid
peroxidation. Free Radic. Biol. Med. 27:334–346; 1999.
[24] Wayner, D. D.; Burton, G. W.; Ingold, K. U. The antioxidant efficiency of vitamin C
is concentration-dependent. Biochim. Biophys. Acta 884:119–123; 1986.
[25] Wayner, D. D. M.; Burton, G. W.; Ingold, K. U.; Locke, S. Quantitative measurement
of the total, peroxyl radical-trapping antioxidant capacity of human blood plasma
by controlled peroxidation. FEBS Lett. 187:33–37; 1985.
[26] Ghiselli, A.; Searfini, M. Measurements of the antioxidant capacity in human plas-
ma. Biofactors 6:451–453; 1997.
[27] Tubaro, F.; Rapuzzi, P.; Ursini, F. Kinetic analysis of antioxidant capacity of wine.
Biofactors 9:37–47; 1999.
[28] Ghiselli, A.; Serafini, M.; Natella, F.; Scaccini, C. Total antioxidant capacity as a tool
to assess redox status: critical view and experimental data. Free Radic. Biol. Med.
29:1106–1114; 2000.
[29] Prior, R. L.; Gu, L.; Wu, X.; Jacob, R. A.; Sotoudeh, G.; Kader, A. A.; Cook, R. A. Plas-
ma antioxidant capacity changes following a meal as a measure of the ability of a
food to alter in vivo antioxidant status. J. Am. Coll. Nutr. 26:170–181; 2007.
[30] Torres, P.; Galleguillos, P.; Lissi, E.; Lopez-Alarcon, C. Antioxidant capacity of
human plasma and human urine: simultaneous evaluation of the ORAC index
and ascorbic acid concentration employing pyrogallol red as probe. Bioorg. Med.
Chem. 16:9171–9175; 2008.
[31] Gresele, P.; Cerletti, C.; Guglielmini, G.; Pignatelli, P.; de Gaetano, G.; Violi, F. Ef-
fects of resveratrol and other wine polyphenols on vascular function: an update.
J. Nutr. Biochem. 22:201–211; 2011.
[32] Glazer, A. N. Phycoerythrin fluorescence based assay for reactive oxygen species.
Methods Enzymol. 186:161–168; 1990.
[33] Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and validation of an im-
proved oxygen radical absorbance capacity assay using fluorescein as the fluores-
cent probe. J. Agric. Food Chem. 49:4619–4626; 2001.
[34] Wayner, D. D. M.; Burton, G. W.; Ingold, K. U.; Barclay, L. R. C.; Locke, S. J. The rel-
ative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl
radical-trapping antioxidant activity of human blood plasma. Biochim. Biophys.
Acta 924:408–419; 1987.
[35] Lussibnoli, S.; Fraccaroli, M.; Andrioli, G.; Brocco, G.; Bellavite, P. A microplate-
based colorimetric assay of the total peroxyl radical trapping capability of
human plasma. Anal. Biochem. 269:38–44; 1999.
[36] Niki, E.; Yamamoto, Y.; Takahashi, M.; Yamamoto, K.; Yamamoto, Y.; Komuro,
E.; Miki, M.; Yasuda, H.; Mino, M. Free radical-mediated damage of blood and
its inhibition by antioxidants. J. Nutr. Sci. Vitaminol. 34:507–512 (Tokyo);
1988.
[37] Frei, B.; England, L.; Ames, B. N. Ascorbate is an outstanding antioxidant in human
blood plasma. Proc. Natl. Acad. Sci. U. S. A. 86:6377–6381; 1989.
[38] Sato, K.; Niki, E.; Shimasaki, H. Free radical-mediated chain oxidation of low den-
sity lipoprotein and its synergistic inhibition by vitamin E and vitamin C. Arch.
Biochem. Biophys. 279:402–405; 1990.
[39] Thomas, S. R.; Stocker, R. Molecular action of vitamin E in lipoprotein oxidation:
implications for atherosclerosis. Free Radic. Biol. Med. 28:1795–1805; 2000.
[40] Niki, E.; Kawakami, A.; Yamamoto, Y.; Kamiya, Y. Synergistic inhibition of oxida-
tion of phosphatidylcholine liposome in aqueous dispersion by vitamin E and vi-
tamin C. Bull. Chem. Soc. Jpn. 58:1971–1975; 1985.
[41] Doba, T.; Burton, G. W.; Ingold, K. U. Antioxidant and co-antioxidant activity of vi-
tamin C: the effect of vitamin C, either alone or in the presence of vitamin E or a
water-soluble vitamin E analogue, upon the peroxidation of aqueous multilamel-
lar phospholipid liposomes. Biochim. Biophys. Acta 835:298–303; 1985.
[42] Aldini, G.; Yeum, K. J.; Russell, R. M.; Krinsky, N. I. A method to measure the oxi-
dizability of both the aqueous and lipid compartments of plasma. Free Radic. Biol.
Med. 31:1043–1050; 2001.
[43] Yeum, K. J.; Russell, R. M.; Krinsky, N. I.; Aldini, G. Biomarkers of antioxidant ca-
pacity in the hydrophilic and lipophilic compartments of human plasma. Arch.
Biochem. Biophys. 430:97–103; 2004.
[44] Omata, Y.; Saito, Y.; Yoshida, Y.; Niki, E. Simple assessment of radical scavenging
capacity of beverages. J. Agric. Food Chem. 56:3386–3390; 2008.
[45] Sim, W. L. S.; Han, M. Y.; Huang, D. Quantitation of antioxidant capacity in a
microemulsion system: synergistic effects of chlorogenic acid with α-
tocopherol. J. Agric. Food Chem. 57:3409–3414; 2009.
[46] Romero, M.; Rojano, B.; Mella-Raipan, J.; Pessoa-Mahana, C. D.; Lissi, E.; Lopez-
Alarcon, C. Antioxidant capacity of pure compounds and complex mixtures eval-
uated by the ORAC–pyrogallol red assay in the presence of Triton X-100 micelles.
Molecules 15:6152–6157; 2010.
[47] Huang, D.; Ou, B.; Hampsch-Woodill, M. Development and validation of oxygen
radical absorbance capacity assay for lipophilic antioxidants using randomly
methylated β-cyclodextrin as the solubility enhancer. J. Agric. Food Chem. 50:
1815–1821; 2002.
[48] Gotoh, N.; Noguchi, N.; Tsuchiya, J.; Morita, K.; Sakai, H.; Shimasaki, H.; Niki, E. In-
hibition of oxidation of low density lipoprotein by vitamin E and related com-
pounds. Free Radic. Res. 24:123–134; 1996.
[49] Robaszkiewicz, A.; Bartosz, G. Estimation of antioxidant capacity against patho-
physiologically relevant oxidants using pyrogallol red. Biochem. Biophys. Res.
Commun. 390:659–661; 2009.