Biochimica et Biophysica Acta 1721 (2005) 174 – 184

http://www.elsevier.com/locate/bba

Regular paper

Evaluation of the antioxidant activity of flavonoids by bferric reducing

antioxidant powerQ assay and cyclic voltammetry
Omidreza Firuzia, Antonio Lacannaa, Rita Petruccib, Giancarlo Marrosub, Luciano Sasoa,*
a
Dipartimento di Farmacologia delle Sostanze Naturali e Fisiologia Generale, Universitá di Roma bLa SapienzaQ, P.le Aldo Moro 5, 00185 Rome, Italy
b
Dipartimento di Ingegneria C. M. M. P. M., Universitá di Roma bLa SapienzaQ, Via del Castro Laurenziano 7, 00161 Rome, Italy

Received 11 August 2004; received in revised form 16 October 2004; accepted 1 November 2004
Available online 22 December 2004

Abstract

Flavonoids, naturally occurring phenolic compounds, have recently been studied extensively for their antioxidant properties. The
structure–antioxidant activity relationships (SAR) of flavonoids have been evaluated against different free radicals, but bferric reducing
antioxidant powerQ (FRAP) assay, which determines directly the reducing capacity of a compound, has not been used for this purpose. In
this study, the antioxidant activities of 18 structurally different flavonoids were evaluated by FRAP assay modified to be used in 96-well
microplates. Furthermore, their oxidation potentials were also measured, which were in the range of +0.3 V (myricetin) to +1.2 V (5-
hydroxy flavone) and were in good agreement with FRAP assay results. Quercetin, fisetin and myricetin had the lowest oxidation potentials
and appeared the most active compounds in FRAP assay and were 3.02, 2.52 and 2.28 times more active than Trolox, respectively.
Indications were found that the o-dihydroxy structure in the B ring and the 3-hydroxy group and 2,3-double bond in the C ring give the
highest contribution to the antioxidant activity.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Antioxidant; Flavonoid; FRAP; Cyclic voltammetry

1. Introduction have recently been studied extensively for their vast

Epidemiological studies have shown that a diet rich in
fruits and vegetables is associated with a decreased risk of
cardiovascular diseases [1] and certain cancers [2]. These
beneficial health effects have been attributed in part to the
presence of phenolic compounds in dietary plants, which
may exert their effects as a result of their antioxidant
properties [3,4].
Flavonoids are a large group of naturally occurring
phenolic compounds, which have been estimated to be
present in human diet with an average of 23 mg/day [5] and
Abbreviations: E ap, anodic potential; E cp, cathodic potential; FRAP,
ferric reducing antioxidant power; SAR, structure–activity relationship;
TEAC, Trolox equivalent antioxidant capacity; TPTZ, 2,4,6-Tris(2-pyr-
idyl)-s-triazine
* Corresponding author. Tel.: +39 06 499 12481; fax: +39 06 499
12480.
0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2004.11.001
antioxidant properties [6].
The antioxidant activities of flavonoids have been
evaluated against various reactive oxygen and nitrogen
species like 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
[7], hydroxyl radicals (HO!) [8], superoxide (O!2
) [9],
peroxyl radicals (ROO!) [10] and hypochlorite [11]. Since
the antioxidant activity of flavonoids varies considerably
according to their backbone structures and the variation
and kind of functional groups present in the chemical
structure, some reports have established structure–activity
relationships (SAR) for flavonoids [10–13]. However,
there are some discrepancies among these studies, which
is probably due to the different assay conditions. A few
authors have studied the SAR of flavonoids, applying the
methods which are commonly used for measuring the total
antioxidant capacity, like Trolox equivalent antioxidant
capacity (TEAC) method [14] and oxygen radical absorb-
ance capacity (ORAC) assay [15]. Another method
 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184
commonly used for measuring the total antioxidant
capacity is bferric reducing antioxidant powerQ (FRAP)
assay, a simple and reliable colorimetric method, proposed
by Benzie and Strain [16]. This method is based on the
ability of the antioxidants to reduce Fe3+ to Fe2+. While
the major part of the methods applied to evaluate the SAR
of flavonoids measure their capacity against an oxidant,
the FRAP assay measures directly the reducing capacity of
the substance, which is an important parameter for a
compound to be a good antioxidant [16]. Although this
method has proved to be simple and reliable without
important interferences, it has not been used to study the
SAR of flavonoids in a systematic evaluation of different
subclasses of flavonoids.
Moreover, the redox potentials of flavonoids determined
by cyclic voltammetry are considered good measures of their
antioxidant capacities. It is generally believed that in free
radical scavenging reactions, antioxidant phenolic com-
pounds function as hydrogen atom donators according to
Eq. (1) [12].
ROHYRO ! þ H ! ð1Þ
Although the anodic potential (E ap) is a measure of
oxidizability according to the Eq. (2), electrochemical data
provide useful information about the free radical scavenging
capacity of various compounds because of the similarities of
these two reactions.
ROHYRO ! þ Hþ þ e
ð2Þ
We evaluated the activity of flavonoids by the FRAP
assay, modifying the original method to be used in 96-well
microplates. The redox potentials of these compounds were
also measured to compare their electrochemical behavior
with data obtained by the FRAP assay.
2. Materials and methods
2.1. Reagents
Apigenin, ferrous sulfate, baicalein, (F)catechin,
chrysin, fisetin, hesperetin, 5-hydroxyflavone, 7-hydroxy-
flavones, ferric chloride hexahydrate, myricetin,
(F)naringenin, quercetin, rutin, taxifolin, 2,4,6-Tris(2-
pyridyl)-s-triazine (TPTZ) and Trolox ((F)-6-Hydroxy-
2,5,7,8-tetramethylchromane-2-carboxylic acid) were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
Daidzein, galangin, genistein and 3-hydroxyflavone were
obtained from Lancaster Synthesis (Lancashire, England).
Methanol was purchased from Carlo Erba (Milan, Italy).
Acetic acid glacial, sodium acetate trihydrate and kaemp-
ferol were obtained from Delchimica scientific glassware
s.r.l. (Naples, Italy), Merck (Darmstadt, Germany) and MP
Biomedicals (Irvine, USA), respectively.
175
2.2. Determination of the antioxidant activity by FRAP
assay
The FRAP assay proposed by Benzie and Strain [16]
was modified to be performed in 96-well microplates.
Briefly, to prepare the FRAP solution, 10 ml of acetate
buffer 300 mM, adjusted to pH 3.6 by the addition of
acetic acid, was mixed with 1 ml of ferric chloride
hexahydrate 20 mM dissolved in distilled water and 1 ml
of 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) 10 mM dis-
solved in HCl 40 mM. In 96-well microplates, 25 Al of
the substance under investigation dissolved at different
concentrations in the range 10–200 AM was placed in
quadruplicate. All substances were dissolved in methanol,
except for apigenin, which was dissolved in NaOH 0.02
M. FRAP solution (175 Al) freshly prepared and warmed
at 37 8C was added to three of these samples, while the
same volume of acetate buffer was added to the fourth
one (blank). The absorbance at 595 nm was monitored by
a microplate reader (Bio-Rad, model 3550) at different
time intervals for up to 150 min. The absorbances of the
blanks and that of the mixture of 175-Al FRAP solution
plus 25-Al methanol were subtracted from the absorbances
of the samples at each time interval to calculate the
absorbance change (DA). All substances were tested at
least in triplicate at 37 8C as described above.
The FRAP value at time interval t (FRAP valuet ) was
calculated according to the formula:


FRAP valuet ðMÞ ¼ Dat Fl=Dat Fe2þ  10
5
where Da tFl is the absorbance change after the time interval
t (4 and 60 min) relative to the tested flavonoid at the
concentration of 110
5 M and Da t Fe2+ is the absorbance
change at the same time interval relative to ferrous sulfate at
the same concentration.
Trolox was used to determine the intra- and interassay
coefficients of variation (CV) for quality control purposes. It
was dissolved in methanol at a concentration of 60 AM, kept
at
20 8C until use and analyzed as described above.
2.3. Cyclic voltammetry
A three-electrode multipolarograph (AMEL 472)
coupled with a digital x/y recorder (AMEL 863) was used
for the voltammetric measurements, carried out using a
static glassy-carbon electrode on nitrogen purged solution.
A platinum wire was used as a counter electrode, while a
saturated calomel electrode (SCE) (+0.242 V vs. NHE) was
employed as the reference. Before each measurement the
working electrode was polished with a Buehler polishing
cloth and different dimension alumina (1.0, 0.3 and 0.05 A)
solutions. The solution contained acetate buffer 300 mM pH
3.6 and HCl 20 mM, at a ratio of 10:2 to reproduce the same
analysis condition as in FRAP assay. Fe3+ was also analysed
176 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184

in the presence of TPTZ to obtain its cathodic potential (E cp) 2.4. Statistics
in the same condition as in the FRAP assay.
The substance under investigation was added to this Multiple comparisons were performed by one-way
solution at a final concentration of 0.1 mM. analysis of variance (ANOVA), followed by Fisher’s LSD

Table 1
Chemical structures, FRAP values and oxidation potentials of the five main classes of the flavonoids tested in this study: flavones, flavonols, flavanones,
flavanols and isoflavones
Substances R3 R5 R6 R7 R3V R4V R5V FRAP FRAP E ap
value4 min value60 min (V)b
(AM)a (AM)a
(A) Flavones
Apigenin H OH H OH H OH H 0.9F0.3 i 1.4F0.5 l +0.90
Baicalein H OH OH OH H H H 22.5F2.3 de 38.9F2.5 f +0.34
Chrysin H OH H OH H H H 0j 0n NPc
5-OH Flavone H OH H H H H H 0j 0n +1.2
7-OH Flavone H H H OH H H H 0j 0n NPc

(B) Flavonols
Fisetin OH H H OH OH OH H 54.2F2.9 b 86.4F6.5 b +0.39
Galangin OH OH H OH H H H 15.4F0.6 f 33.9F3.7 g +0.59
3-OH Flavone OH H H H H H H 4.5F1.1 h 21.6F1.5 h +0.68
Kaempferol OH OH H OH H OH H 27.3F0.3 c 40.3F0.9 f +0.44
Myricetin OH OH H OH OH OH OH 49.0F1.9 b 74.1F2.5 c +0.30
Quercetin OH OH H OH OH OH H 65.0F4.8 a 95.9F5.4 a +0.39
Rutin ORd OH H OH OH OH H 21.9F1.1 de 57.1F1.2 d +0.46

(C) Flavanones
Hesperetin H OH H OH OH OMe H 13.3F0.6 g 21.1F0.4 i +0.71
Naringenin H OH H OH H OH H 0j 0.5F0.3 m +0.89
Taxifolin OH OH H OH OH OH H 24.1F1.4 d 33.9F2.6 g +0.46

(D) Flavanols
Catechin OH OH H OH OH OH H 20.7F1.5 e 45.1F1.7 e +0.45

(E) Isoflavones
Daidzein H H H OH H OH H 0.5F0.3 i 5.3F0.8 j +0.80
Genistein H OH H OH H OH H 0.5F0.1 i 3.5F0.3 k +0.79

(F) Known antioxidants

Trolox 21.5F1.7 de 21.6F1.8 i +0.30
Uric acid 23.2F2.1 de 29.7F1.8 h +0.53
Resveratrol 12.2F0.5 g 22.8F1.4 i +0.55
Values are the meansFS.D. of at least three experiments. Values within a column with different letters (a, b, c, etc.) are significantly different at Pb0.05.
a
FRAP value was calculated according to the formula reported in Materials and methods.
b
Anodic oxidation potential vs. SCE.
c
No peak was detected.
d
O-Rutinose.
e
O-Methyl.
 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184 177

formed using the software SigmaPlot 2002 for Windows

version 8.0. All experiments were repeated at last three
times.

3. Results

3.1. Determination of the antioxidant activity by FRAP

assay

A range of structurally different flavonoids and known

Fig. 1. Concentration–absorbance curve of quercetin assayed by the FRAP
method. Quercetin at different concentrations was incubated with FRAP
solution as described in Materials and methods and the absorbance was
monitored at 595 nm by a microplate reader. Absorbance change after 4 min
was plotted against quercetin concentration. The values are the means ±
standard deviations of triplicates.
test using the program SigmaStat version 3.00 for Windows
(SPSS Chicago, IL, USA). Data were considered statisti-
cally different at Pb0.05. Regression analyses were per-
antioxidants, resveratrol, Trolox and uric acid were analysed
by the FRAP assay (Table 1).
A linear correlation was observed between the absorb-
ance change at 595 nm and the substance concentration
up to a maximum of circa 1.7 absorbance change, for all
tested compounds except for chrysin, 5-hydroxyflavone
and 7-hydroxyflavone, which were inactive. Absorbance
change after 4 min at 37 8C in the presence of quercetin,
the most active flavonoid, was plotted against its
concentration and depicted in Fig. 1 as a representative
figure.
While ferrous sulfate, Trolox and uric acid caused a rapid
enhancement in the absorbance at 595 nm, which reached a

Fig. 2. Absorbance changes in time in the presence of different compounds in the FRAP assay. Absorbance changes at 595 nm were monitored at different time
intervals in the presence of flavones (A), flavonols (B), flavanones (C), flavanols (D), isoflavones (E) and known antioxidants (F) as described in Materials and
methods. The values are the means ± standard deviations of triplicates.
178 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184
Fig. 3. Cyclic voltammograms of flavonoids. Three typical voltammograms
obtained by a glassy-carbon electrode as described in Materials and
methods are shown. These voltammograms represent typical well-defined
quasi-reversible (quercetin), well-defined irreversible (3-hydroxyflavone)
and broadened irreversible anodic peak (hesperetin).
plateau before 4 min (Fig. 2F), the absorbance in the
presence of flavonoids and resveratrol continued to increase
also after 4 min (Fig. 2A–F).
Fig. 4. Correlation between the results of the FRAP assay and electrochemical data. The antioxidant activities of different flavonoids were determined by the
FRAP assay and plotted against their anodic oxidation potentials. A good inverse correlation was found between these two methods (r: 0.907). The FRAP
values at 4 min are the means of at least three experiments.
FRAP values of the flavonoids and other antioxidants at
4 and 60 min were calculated according to the formula
described in Materials and methods and reported in Table 1.
There was a good linear correlation between FRAP values at
4 and 60 min (r=0.970).
FRAP values of Trolox at 4 min were used to
calculate the intra- and interassay coefficients of variation
(CV) which were 2.6% (n=16) and and 4.0% (n=38),
respectively.
3.2. Cyclic voltammetry
Cyclic voltammetry of flavonoids, Fe3+ and known
antioxidants resveratrol, Trolox and uric acid was carried
out using a glassy-carbon electrode as described in Materials
and methods (Table 1).
Cyclic voltammetry of Fe3+ showed a well-defined
cathodic peak (E cp=+0.33 V) to which corresponded a
broadened anodic peak at about +0.8 V related to the anodic
oxidation of Fe2+. A cathodic shift of E cp value to +0.18 V
was observed when measurement was carried out in the
FRAP solution, in which the reduction product Fe3+ was
evidenced by a thin intense blue-coloured layer on the
electrode.
No relevant anodic wave in the investigated range
(inversion potential 1.5 V) was found for chrysin and 7-
OH flavone. All other studied compounds showed an anodic
peak ranging between +0.30 and +1.20 V vs. SCE (Table 1)
and could be divided in three groups according to their
voltammograms:
(a) 5-Hydroxyflavone, apigenin, naringenin, daidzein,
genistein and hesperetin showed a broadened irrever-
sible anodic peak with E ap values ranging between
+0.71 and +1.20 V.
 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184 179

Fig. 5. Correlation of the total number of hydroxyl substitutions of flavonols with FRAP values (A) and with oxidation potentials (B). The FRAP values at 4
min (A) and the oxidation potentials (E ap) of the flavonoids belonging to flavonol subclass were plotted against their total number of hydroxyl groups. The
correlation with oxidation potentials (r: 960) was slightly better than that with FRAP values (r: 0.908).

(b) 3-Hydroxyflavone and galangin showed a well-
defined irreversible anodic peak with E ap values of
+0.68 and +0.59 V, respectively.
(c) Taxifolin, rutin, catechin, kaempferol, baicalein, quer-
cetin, fisetin and myricetin all showed a well-defined
quasi-reversible anodic peak with E ap values ranging
between +0.30 and +0.46 V.
Typical voltammograms are shown in Fig. 3.
The electrochemical data were in good agreement with
results obtained by the FRAP assay (Fig. 4).
While good correlations were not found between the total
number of hydroxyl groups of flavonoids and FRAP values
at 4 min (r: 0.656) nor E ap (r: 0.676), good correlations
were found in the group of flavonols between these
parameters (Fig. 5).
4. Discussion
The antioxidant activities of a range of structurally
different flavonoids were analysed by a modified FRAP
assay and their oxidation potentials were measured by
cyclic voltammetry (Table 1). The study involved five
flavonoid subclasses, flavones, flavonols, flavanones,
flavanols and isoflavones, to evaluate the SAR of these
compounds.
4.1. FRAP assay
The FRAP assay is based on the measurement of the
ability of the substance to reduce Fe3+ to Fe2+ and was
initially proposed to measure the total antioxidant capacity of
plasma [16] and then applied by the same authors to other
substrates [17]. Fe2+ is measured spectrophotometrically via
determination of its coloured complex with 2,4,6-Tris(2-
pyridyl)-s-triazine (TPTZ), which has a high absorbance at
595 nm. Since the antioxidant activity of a substance is
usually correlated directly to its reducing capacity, the FRAP
assay provides a reliable method to study the antioxidant
activity of various compounds [16]. This method has been
frequently used for a rapid evaluation of the total antioxidant
capacity of various food and beverages [18,19] and also
different plant extracts containing flavonoids [20]. Further-
more, it has been applied to measure the antioxidant activity
of dietary polyphenols and a limited number of flavonoids in
180 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184
vitro [21]. Based on our previous experience [11,22], we
modified the FRAP assay to be performed in 96-well
microplates, using a microplate reader for absorbance
measurements. Eighteen Structurally different flavonoids
and three compounds with known antioxidant properties,
resveratrol, Trolox and uric acid, were studied. This assay
showed to be highly reproducible; the intra- and interassay
coefficients of variation (CV) were 2.6% and 4.0%,
respectively. This represents a major advantage of the FRAP
assay compared to more complex methodologies.
It has been reported that the absorbance in the FRAP
assay in the presence of plasma remains unchanged after 4
min at 37 8C [16]. However, we observed that the
absorbance continued to increase also after 4 min in the
presence of flavonoids (Fig. 2A–E). This observation is also
supported by the study of Pulido et al. [21]. This
discrepancy may be explained by the fact that uric acid,
which is the major antioxidant responsible for absorbance
changes in plasma [16], unlike flavonoids, causes a rapid
enhancement of absorbance which reaches a plateau before
4 min (Fig. 2F). The same behavior was also shown by
Trolox, but resveratrol, which is a polyphenolic antioxidant,
caused an absorbance change similar to flavonoids (Fig.
2E). Due to this continuous change in absorbance, FRAP
values at two different time intervals, 4 and 60 min, were
defined as indices of antioxidant activity. Since the order of
antioxidant efficiency after 60 min remained unchanged
(data not shown), this time interval was chosen as the longer
time interval for reducing capacity determination. Absorb-
ance change induced by the substance under investigation at
the concentration of 10 AM, compared to the absorbance
change generated by the same concentration of ferrous
sulfate, established the FRAP values at 4 and 60 min (FRAP
value4 min and FRAP value60 min, respectively). Since it has
been proposed that the antioxidant activity of a substance
may change in different solvents [21,23], both ferrous
sulfate and all tested compounds (except for apigenin, which
was not soluble in methanol) were dissolved in methanol to
avoid variations caused by solvent.
The order of FRAP values4 min of flavonoids was
quercetinNfisetinzmyricetinNkaempferolNtaxifolinz
baicaleinzrutinzcatechinNgalanginNhesperetinN3-hyroxy-
flavoneNapigeninzdaidzeinzgenistein. Naringenin,
chrysin, 5-hyroxyflavone and 7-hyroxyflavone were inac-
tive after 4 min. The order of efficiency at 60 min appeared
to be slightly different and was quercetin NfisetinN
myricetinNrutinNcatechinNkaempferolzbaicaleinNtaxifolin-
NtaxifolinzgalanginN3-OH flavonezhesperetinNdaid-
zeinNgenisteinNapigeninNnaringenin. Chrysin, 5-
hyroxyflavone and 7-hyroxyflavone were inactive even
after 60 min. The absorbance enhancement showed different
patterns in various flavonoids (Fig. 2A–E), but a good
correlation was observed between FRAP values at 4 and 60
min (r=0.970). Rutin and catechin showed the most
considerable changes in the order of activity and had much
higher values at 60 min. Pulido et al. [21] also found that the
reducing activities of catechin and rutin increased consid-
erably after 4 min. To rule out the involvement of a stable
rutin–Fe3+ or rutin–Fe2+ complex as well as an acidic
hydrolysis reaction of rutin to quercetin, a UV study was
carried out in the same medium. No absorbance shift was
observed for rutin in the presence of Fe3+ or Fe2+ at the
operative pH value. Furthermore, rutin UV spectrum did not
have any change after 2 h.
It is known that in addition to the stoichiometric
factor, kinetic factor is also important for a compound to
be a good antioxidant in vivo. Considering that FRAP
value4 min takes into account the kinetics of the reaction
with Fe3+ more than the FRAP value60 min, the former
was considered as the index of reducing capacity to study
the SAR.
4.2. Cyclic voltammetry
We also measured the oxidation potentials (E ap) of the
flavonoids in the same analysis conditions as the FRAP
assay (acetate buffer 300 mM pH 3.6) (Table 1).
Cyclic voltammetry has been applied to measure the
oxidizability of various compounds [24] and also flavonoids
in different buffers and pH [25,26], and good correlations
have been observed between redox potentials and antiox-
idant properties [27].
The antioxidant activity of a substance in the FRAP
assay depends mainly on the electron transfer from the
substance to Fe3+, which is determined by the redox
potentials of the involved compounds. Thus, the redox
potential of Fe3+ was also measured in the same medium to
evaluate the possibility of an electron transfer between Fe3+
and flavonoids.
4.3. Correlation between FRAP assay results and electro-
chemical data
Two applied methods were in good agreement and
an inverse correlation was observed between the FRAP
values4 min and the E ap (Fig. 4).
The electron transfer between an oxidant and a
reductant, and the consequent redox reaction, occurs easily
when the cathodic potential (E cp) of the oxidant is more
than the anodic potential (E ap) of the reductant. However,
a reaction can occur even when E cp (oxidant) is less than
E ap (reductant) but the difference should not be bigger than
0.4 V. So the best conditions for an electron transfer
between Fe3+ (E cp: +0.18) and studied compounds are for
flavonoids whose E ap are less than +0.7 V, while +0.6 to
+0.7 V values represent a limit beyond which electron
transfer may not be possible.
Thus, according to cyclic voltammetry results, the tested
compounds could be divided to four groups;
(a) 5-OH flavone, 7-OH flavone, chrysin, apigenin,
naringenin, daidzein and genistein with E ap ranging
 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184
from +0.79 to +1.2 V. These flavonoids showed no or
very low activity in the FRAP assay.
(b) 3-OH flavone, galangin and hesperetin with E ap
ranging between +0.59 and +0.71 V. This group of
flavonoids resulted to be weakly active in the FRAP
assay.
(c) catechin, taxifolin, rutin and kaempferol with E ap
values between +0.44 and +0.46 V. These were active
antioxidants in the FRAP method.
(d) quercetin, fisetin, baicalein and myricetin with E ap
ranging between +0.30 and +0.39 V. These flavonoids
were the most strong antioxidants in the FRAP assay.
4.4. SAR
Among the flavonoids tested in this study, quercetin,
fisetin and myricetin showed the highest FRAP values and
the lowest E ap (Table 1 and Fig. 2). All these three
flavonoids belong to the subclass of flavonols and have in
their chemical structures the combination of 3-hydroxy
group, 2,3-double bond and 4-oxo function in the C ring and
the catechol group in the B ring (Table 1). This may indicate
that these functional groups give a major contribution to the
reducing capability. The Fe3+ reducing activities of these
three compounds have been evaluated by other authors in
different analysis conditions and, in agreement with our
results, high reducing capacities have been reported for
them [28]. In another study, only myricetin and quercetin
were shown to be able to reduce effectively Fe3+ ions, while
other flavonoids appeared to be inactive [29].
The high activity of these compounds may be explained
by the fact that in the presence of catechol group in the B
ring and 3-OH and 2,3-double bond in the C ring, a bi-
Scheme 1. Generation of quinonic structures after oxidation of flavonoids.
181
electronic oxidation may occur which leads to the produc-
tion of two highly stable quinonic structures (Scheme 1A)
[30]. The presence of the third hydroxyl group on the B
ring, like in myricetin, shifts the E ap to even less positive
values due to the electron-donating effect of OH group.
These findings about the high antioxidant activity of
quercetin and flavonoids with similar structure are sup-
ported also by the annotations of other authors, which tested
these compounds by different antioxidant methods
[12,14,15].
It is generally believed that an increase in the number of
hydroxyl groups enhances the antioxidant activity of the
flavonoids [15]. In a previous study, applying a microplate-
based method developed in our laboratory [31], we found
that the hypochlorite scavenging activity of flavonoids was
correlated with their number of hydroxyl substitutions [11].
In this study, when all subclasses of flavonoids were taken
into account, poor correlations were found between the total
number of OH groups and the FRAP value4 min (r: 0.656)
and E ap (r: 0.676), while in the subclass of flavonols, FRAP
value4 min and E ap correlated well with the number of OH
groups (Fig. 5). These observations indicate that among the
compounds having the same basic structure, the number of
OH groups is the determinant factor for the antioxidant
activity, but when compared compounds belong to different
subclasses, other factors like the structure of the C ring
should also be concerned.
To evaluate the importance of each individual OH group
in the A and C rings, 3-, 5- and 7-monohydroxyflavones
were tested. Only 3-hydroxyflavone was moderately active
in the FRAP assay (FRAP value4 min: 4.5 AM) (Table 1),
showing the important role of OH group in position 3 in the
C ring. The same notion can be proved comparing galangin
182 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184
(FRAP value4 min: 15.4 AM, E ap: +0.59 V), apigenin (FRAP
value4 min: 0.9 AM, E ap: +0.90 V) and chrysin (inactive),
which have similar structures in the C ring and the same OH
groups in the A ring. Galangin with 3-OH is much more
active than other two flavonoids which lack 3-OH and the
presence of 4V-OH in apigenin does not compensate for the
absence of 3-OH.
The importance of 3-hydroxy group to the reducing
capacity can be seen also in kaempferol (FRAP value4 min:
27.3 AM, E ap: +0.44 V), which compared to the corre-
sponding compound lacking 3-OH group, which is apige-
nin, is much more active. The high reactivity of kaempferol
may also be explained by the fact that its oxidation may
follow the bi-electronic oxidation reaction depicted in
Scheme 1B, which involves 3-OH and 2,3-double bond in
the C ring and 4V-OH in the B ring and leads to the
formation of a stable quinone [32].
In agreement with our results, other studies have shown
the importance of 3-hydroxy group to the scavenging
activity against hypochlorite [11], peroxyl radical [33],
peroxynitrite [13] and superoxide radical [34].
While the 3-OH group in the C ring seemed to be very
important to the antioxidant activity, OH groups in the A
ring in 5- and 7-hydroxyflavone did not seem to give any
contribution to the activity; the FRAP values of these
compounds at 4 and 60 min were 0. The electrochemical
data also supported this notion (Table 1). It is known that
anodic oxidation of monohydric phenols occurs at quite
high positive potentials through different anodic reactions
according to medium, electrode material and substrate, to
yield the corresponding phenoxy radical or the phenoxo-
nium ion that can successively undergo different secondary
reactions [35]. In fact, the cyclic voltammetry of 5-OH
flavone showed a broadened and poorly reproducible anodic
wave with the highest E ap among all studied compounds
(+1.2 V), while 7-OH flavone was inactive in applied
voltage range. In flavonoids with a higher number of
hydroxyl groups, similar phenomena were observed; remov-
ing 5-OH from quercetin as in fisetin decreases only slightly
the FRAP activity and E ap remains unchanged (Table 1).
This also confirms again that in the oxidation of quercetin,
the A ring OH groups are not probably involved (Scheme
1A). It has also been reported in other studies that OH
groups on the A ring did not have significant contribution to
antioxidant activity [12].
However, when three hydroxyl groups are present in the
A ring like in baicalein (FRAP value4 min: 22.5 AM, E ap:
+0.34 V), pyrogallol group seems to become an important
factor to the activity. Indeed, baicalein was the only
compound of which its antioxidant capacity is due to its
A ring OH groups.
The importance of hydroxyl groups in the B ring to the
antioxidant activity can be shown, comparing two flavonols,
quercetin (FRAP value4 min: 65.0 AM, E ap: +0.39 V) and
galangin (FRAP value4 min: 15.4 AM, E ap: +0.59 V).
Quercetin with 3V,4V-dihydroxy substitution in the B ring
is much more active than galangin, which lacks both OH
groups in the B ring. This phenomenon is best explained by
the formation of stable oxidation products in quercetin
(Scheme 1A). However, in the absence of 2,3-double bond
or 3-OH in the C ring like in taxifolin, rutin and catechin,
the oxidation may follow another pattern, leading to the
formation of o-benzoquinone in the B ring (Scheme 1C).
This may explain the lower activity of these three
compounds compared to quercetin. However, they remain
still more active than other flavonoids, which do not possess
the catechol group in the B ring (Table 1).
The presence of an electron-donating moiety like
methoxy group in the B ring seems to confer a higher
reducing capability, when naringenin (FRAP value4 min: 0,
E ap: +0.89 V) is compared to hesperetin (FRAP value4 min:
13.3 AM, E ap: +0.71 V), which has and added methoxy
group in the B ring. Similarly, comparing apigenin (FRAP
value4 min: 0.9 AM, E ap: +0.90 V) with chrysin (not active),
two flavones which differ only in the presence of 4V-OH in
the B ring, the effect of OH group in the B ring can be
proved (Table 1).
The importance of o-dihydroxy substitution in the B ring
to the antioxidant activity of flavonoids found in this study
is similar to findings of other authors, which studied the
peroxyl radical (ROO!) scavenging [15] and peroxynitrite
scavenging activities of flavonoids [13].
Myricetin, which is a flavonol with a pyrogallol group in
the B ring, showed the lowest E ap (+0.30 V), but its FRAP
value4 min was not as high as expected from its E ap and was
lower than the FRAP values4 min of quercetin and fisetin
(Table 1). Very low E ap has also been reported for myricetin
by others [36], but its antioxidant activity seems to vary
according to the test system. In TEAC assay [14] and
against DPPH radical [37], it resulted to be less active than
quercetin, while in ORAC assay it showed to be more
effective than quercetin [15]. Similarly, the discrepancy
between E ap and FRAP value was observed for baicalein,
another flavonoid containing pyrogallol that has a very low
E ap (+0.34 V) but intermediate FRAP value4 min (22.5 AM)
(Table 1 and Fig. 4). Thus, it can be deduced that pyrogallol
confers a low E ap but not necessarily a high FRAP value.
This may be explained by the fact that in catechol-
containing molecules a nucleophilic attack on the first
oxidation product may regenerate the catechol moiety and
render it available for further oxidation, while this phenom-
enon is less probable in the presence of pyrogallol, which
hinders this reaction due to both steric and electronic effects.
The influence of the 2,3-double bond on the antioxidant
activity of quercetin was shown comparing it to taxifolin,
which lacks this double bond. However, in the absence of
important structural features like catechol and 3-hydroxy,
the presence of 2,3-double bond does not seem to have a
major impact on the activity. For example, in apigenin it
does not change considerably the activity compared to
naringenin. Likewise, chrysin, which is a flavone with 2,3-
double bond, without catechol and 3-hydroxy groups was
 O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184
inactive in the FRAP assay even after 60 min. Similarly, in
cyclic voltammetry, chrysin did not show any oxidation
peak. The resorcinol moiety in chrysin behaves like a
monohydric phenol, with also probable electrode absorption
phenomena [35]. The low antioxidant activity of chrysin has
also been observed by other authors that reported a
negligible activity in iron and AAPH-induced lipid perox-
idation inhibition [12] and a low Fe3+ reducing activity for
this compound [28].
Isoflavonoids genistein and daidzein showed low activi-
ties compared to major part of the other subclasses of fla-
vonoids (Table 1), probably due to their low total number of
OH groups. Genistein (FRAP value4 min: 0.5 AM) has similar
FRAP value with its isomer apigenin (FRAP value4 min: 0.9
AM), but its E ap (+0.79 V) is lower than apigenin (+0.90 V),
which might be due to a better stabilization of the phenoxy
radical in genistein.
5. Conclusions
In conclusion, the antioxidant activities of different
flavonoids from various subclasses were measured by the
FRAP assay and their oxidation potentials were determined
by cyclic voltammetry. A good correlation was observed
between the FRAP assay and electrochemical results,
which confirms the reliability of FRAP assay as a method
for the evaluation of the antioxidant activity of various
compounds. Most of the flavonoids tested in this study
showed to be active compared to known antioxidants
resveratrol, Trolox and uric acid. Hydroxyl groups and
especially catechol moiety, 3-OH and 2,3-double bond
showed to be the most important factors in determining
high antioxidant activity. However, the possibility of
hydrogen bond interactions between hydroxy groups as
donators and 4-oxo moiety as acceptor [38] or kinetic
factors, which may influence the antioxidant activity,
should also be considered. These compounds, which can
be absorbed in the gastrointestinal tract [39] and seem to
be directly involved in the in vivo antioxidant defences
[40], may have potential therapeutic effects in diseases in
which oxidative stress is involved.
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