evaluation of the antioxidant activity of flavonoids by ferric reducing antioxidant power

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

Biochimica et Biophysica Ac

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,*

aDipartimento di Farmacologia delle Sostanze Naturali e Fisiologia Generale, Universita di Roma bLa SapienzaQ, P.le Aldo Moro 5, 00185 Rome, ItalybDipartimento di Ingegneria C. M. M. P. M., Universita 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

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

0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbagen.2004.11.001

Abbreviations: Eap, anodic potential; Ecp, 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.

have recently been studied extensively for their vast

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 (O2!�) [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

ta 1721 (2005) 174–184

O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184 175

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 (Eap) 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.

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 ofthe 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 Mð Þ ¼ DatFl=DatFe2þ� �

� 10�5

where DatFl is the absorbance change after the time interval

t (4 and 60 min) relative to the tested flavonoid at the

concentration of 1�10�5 M and DatFe2+ 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

O. Firuzi et al. / Biochimica et Biophysica Acta 1721 (2005) 174–184176

in the presence of TPTZ to obtain its cathodic potential (Ecp)

in the same condition as in the FRAP assay.

The substance under investigation was added to this

solution at a final concentration of 0.1 mM.

Table 1

Chemical structures, FRAP values and oxidation potentials of the five main clas

flavanols and isoflavones

Substances R3 R5 R6 R7 R3V R4V R5V

(A) Flavones

Apigenin H OH H OH H OH H

Baicalein H OH OH OH H H H

Chrysin H OH H OH H H H

5-OH Flavone H OH H H H H H

7-OH Flavone H H H OH H H H

(B) Flavonols

Fisetin OH H H OH OH OH H

Galangin OH OH H OH H H H

3-OH Flavone OH H H H H H H

Kaempferol OH OH H OH H OH H

Myricetin OH OH H OH OH OH OH

Quercetin OH OH H OH OH OH H

Rutin ORd OH H OH OH OH H

(C) Flavanones

Hesperetin H OH H OH OH OMe H

Naringenin H OH H OH H OH H

Taxifolin OH OH H OH OH OH H

(D) Flavanols

Catechin OH OH H OH OH OH H

(E) Isoflavones

Daidzein H H H OH H OH H

Genistein H OH H OH H OH H

(F) Known antioxidants

Trolox

Uric acid

Resveratrol

Values are the meansFS.D. of at least three experiments. Values within a columna FRAP value was calculated according to the formula reported in Materialsb Anodic oxidation potential vs. SCE.c No peak was detected.d O-Rutinose.e O-Methyl.

2.4. Statistics

Multiple comparisons were performed by one-way

analysis of variance (ANOVA), followed by Fisher’s LSD

ses of the flavonoids tested in this study: flavones, flavonols, flavanones,

FRAP

value4 min

(AM)a

FRAP

value60 min

(AM)a

Eap

(V)b

0.9F0.3 i 1.4F0.5 l +0.90

22.5F2.3 de 38.9F2.5 f +0.34

0 j 0 n NPc

0 j 0 n +1.2

0 j 0 n NPc

54.2F2.9 b 86.4F6.5 b +0.39

15.4F0.6 f 33.9F3.7 g +0.59

4.5F1.1 h 21.6F1.5 h +0.68

27.3F0.3 c 40.3F0.9 f +0.44

49.0F1.9 b 74.1F2.5 c +0.30

65.0F4.8 a 95.9F5.4 a +0.39

21.9F1.1 de 57.1F1.2 d +0.46

13.3F0.6 g 21.1F0.4 i +0.71

0 j 0.5F0.3 m +0.89

24.1F1.4 d 33.9F2.6 g +0.46

20.7F1.5 e 45.1F1.7 e +0.45

0.5F0.3 i 5.3F0.8 j +0.80

0.5F0.1 i 3.5F0.3 k +0.79

21.5F1.7 de 21.6F1.8 i +0.30

23.2F2.1 de 29.7F1.8 h +0.53

12.2F0.5 g 22.8F1.4 i +0.55

with different letters (a, b, c, etc.) are significantly different at Pb0.05.

and methods.

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-

Fig. 2. Absorbance changes in time in the presence of different compounds in the F

intervals in the presence of flavones (A), flavonols (B), flavanones (C), flavanols (D

methods. The values are the means ± standard deviations of triplicates.

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

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

RAP assay. Absorbance changes at 595 nm were monitored at different time

), isoflavones (E) and known antioxidants (F) as described in Materials and

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 dat

FRAP assay and plotted against their anodic oxidation potentials. A good invers

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.


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 (Ecp=+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 Ecp 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 Eap values ranging between

+0.71 and +1.20 V.

a. The antioxidant activities of different flavonoids were determined by the

e correlation was found between these two methods (r: 0.907). The FRAP

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 (Eap) 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 Eap 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 Eap 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 Eap (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


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

quercetinNfisetinzmyricetinNkaempferolNtaxifolinzbaicaleinzrutinzcatechinNgalanginNhesperetinN3-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.


We also measured the oxidation potentials (Eap) 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 Eap (Fig. 4).

The electron transfer between an oxidant and a

reductant, and the consequent redox reaction, occurs easily

when the cathodic potential (Ecp) of the oxidant is more

than the anodic potential (Eap) of the reductant. However,

a reaction can occur even when Ecp (oxidant) is less than

Eap (reductant) but the difference should not be bigger than

0.4 V. So the best conditions for an electron transfer

between Fe3+ (Ecp: +0.18) and studied compounds are for

flavonoids whose Eap 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 Eap ranging


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 Eap

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 Eap

values between +0.44 and +0.46 V. These were active

antioxidants in the FRAP method.

(d) quercetin, fisetin, baicalein and myricetin with Eap

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 Eap (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 struct

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 Eap 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 Eap (r: 0.676), while in the subclass of flavonols, FRAP

value4 min and Eap 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

ures after oxidation of flavonoids.


(FRAP value4 min: 15.4 AM, Eap: +0.59 V), apigenin (FRAP

value4 min: 0.9 AM, Eap: +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, Eap: +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 Eap 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 Eap 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, Eap:

+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, Eap: +0.39 V) and

galangin (FRAP value4 min: 15.4 AM, Eap: +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,

Eap: +0.89 V) is compared to hesperetin (FRAP value4 min:

13.3 AM, Eap: +0.71 V), which has and added methoxy

group in the B ring. Similarly, comparing apigenin (FRAP

value4 min: 0.9 AM, Eap: +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 Eap (+0.30 V), but its FRAP

value4 min was not as high as expected from its Eap and was

lower than the FRAP values4 min of quercetin and fisetin

(Table 1). Very low Eap 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 Eap and FRAP value was observed for baicalein,

another flavonoid containing pyrogallol that has a very low

Eap (+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 Eap 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


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 Eap (+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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evaluation of the antioxidant activity of flavonoids by ferric reducing antioxidant power

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