Innovative Food Science and Emerging Technologies 7 (2006) 140 – 146

www.elsevier.com/locate/ifset

The contribution of flavonoid C-ring on the DPPH free radical scavenging

efficiency. A kinetic approach for the 3V,4V-hydroxy substituted members
Dimitrios I. Tsimogiannis, Vassiliki Oreopoulou *
National Technical University of Athens, School of Chemical Engineering, 9 Iroon Polytehniou st, Zografou Campus 157 80, Athens, Greece

Received 14 July 2005; accepted 5 September 2005

Abstract

5,7,3V,4V-hydroxy-substituted flavonoids are considered as very efficient radical scavengers. This study examines the effect of the structural
elements of the C-ring, on the radical scavenging activity of these compounds. The impact of di-hydroxy substitution of A- or B-ring on the
activity of fully substituted C-ring flavonoids was also studied. Quercetin, luteolin, taxifolin, eriodictyol, rutin, (+)-catechin, ()-epicatechin,
fisetin and kaempferol were studied during the reaction with the DPPH radical; they revealed two distinctive steps of reaction, a first rapid and a
second slower. DPPH scavenging followed second order kinetics during the rapid step; stoichiometric factors and rate constants were determined.
Quercetin and fisetin scavenged four radicals by each molecule, while the other flavonoids scavenged two radicals with a consequent production
of B-ring diquinone. Also the rate constants were affected by C-ring structure. The kinetics of the slow step was much more complicated and the
contribution of C-ring was based on stoichiometry.
D 2005 Elsevier Ltd. All rights reserved.

Keywords: DPPH; Free radical scavenging; Flavonoids; Antioxidant

Industrial relevance: The oxidation of oils, fats and fat-containing foods is a major problem for the food industry, because it is directly related to economical,
nutritional, flavour, safety and storage problems. The use of antioxidant compounds can slow down the process of oxidation and thereby increase the shelf-life of the
products. Due to probable mutagenic and carcinogenic properties of several synthetic antioxidants, there is an increasing demand from consumers for their
replacement with natural compounds of similar action. Flavonoids are plant polyphenols, which display antioxidant activity as well as several beneficial
pharmacological and biochemical actions, thus could be used as natural antioxidant and upgrade the quality of a product. A better understanding of the reaction of
flavonoids with free radicals would elucidate their mode of action against peroxyl radicals. The relation of structure to antioxidant activity can enhance the recovery
of specific compounds from herbs or agro-industrial by-products and their exploitation as natural antioxidants.

1. Introduction
Flavonoids are a widespread group of naturally occurring
phenolic compounds in common edible fruits, leaves, seeds
and other parts of plants. The basic structure is the 2-phenyl-
chromane skeleton. The major subgroups are the flavones,
flavonols, flavanols, flavanones and anthocyanidins. The
classification is based on the substitution pattern of the C-ring,
i.e. the 2,3-double bond, the 3-OH, the 4-keto group and the
flavylium cation (Fig. 1).
It has been proven that flavonoids display a wide range of
pharmacological and biochemical actions such as antimicrobi-
al, antithrombotic, antimutagenic and anticarcinogenic activi-
* Corresponding author. Tel.: +30 2107723166; fax: +30 2107723163.
E-mail address: vasor@chemeng.ntua.gr (V. Oreopoulou).
1466-8564/$ - see front matter D 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2005.09.001
ties (Cook & Samman, 1996; Kandaswami & Middleton, 1997;
Rice-Evans, Miller, & Paganga, 1996; Sahu & Green, 1997). In
food systems flavonoids can act as free radical scavengers and
terminate the radical chain reactions that occur during the
oxidation of triglycerides. Therefore they present antioxidative
efficiency in oils, fats and emulsions (Das & Pereira, 1990;
Madhavi, Singhal, & Kulkarni, 1996; Milovanovic, Picuric-
Jovanovic, Vucelic-Radovic, & Vrbaski, 1996; Nieto et al.,
1993; Roedig-Penman & Gordon, 1998; Wanasundara &
Shahidi, 1994). A method widely used to predict the ability
of flavonoids to transfer H atoms to radicals is based on the free
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Brand-Wil-
liams, Cuvelier, & Berset, 1995; Sanchez-Moreno, Larrauri,
& Saura-Calixto, 1998).
Although it is confirmed that flavonoids with a polyhy-
droxylated substitution in the B-ring show high radical
 D.I. Tsimogiannis, V. Oreopoulou / Innovative Food Science and Emerging Technologies 7 (2006) 140 – 146 141

OH OH OH
OH OH OH

HO O HO O HO O

O
OH O OH O OH O
OH O ERIODICTYOL
O LUTEOLIN
CH3 O
OH
OH OH
OH OH OH OH
3' OH
OH 2' 4'
B HO O
HO 8 O 1' 5'
RUTIN 7 1 2 6'
A C OH
6 3 OH
5 4 OH OH
OH
OH O
HO O (+)-CATECHIN
OH QUERCETIN OH
OH
HO O OH OH
OH OH O HO O
TAXIFOLIN
OH HO O
OH O OH
OH
KAEMPFEROL OH
(-)-EPICATECHIN
O

FISETIN

Fig. 1. The studied flavonoids: quercetin, kaempferol, fisetin (flavonols), rutin, luteolin (flavones), taxifolin (dihydroflavonol), eriodictyol (flavanone), (+)-catechin,
()-epicatechin (flavanols).

scavenging, the effect of the structure of the C-ring is not
clarified. Reports on the activity of flavonoids with different
C-ring structure in scavenging free radicals and retarding lipid
peroxidation are contradictory. The presence of the 2,3-double
bond increases the activity against free radicals (Rice-Evans
et al., 1996; Silva, Santos, Caroço, Rocha, Justino & Mira,
2002); the same effect was observed for canola oil by
Wanasundara and Shahidi (1994), but the results of Pratt and
Hudson (1990) on corn oil did not present any difference.
However, if the 2,3-double bond is not accompanied by a 3-
OH group its effect on increasing free radical scavenging
activity becomes lower according to Benavente-Garcia,
Castillo, Lorente, Ortuño, and Del Rio (2000) and Rice-
Evans et al. (1996), or even reversed according to Hotta,
Nagano, Ueda, Tsujino, Koyama, and Osakai (2002). Differ-
ences in experimental results concerning the substitution of
the position 3 are also reported. The results of Rice-Evans et
al. (1996) and Benavente-Garcia et al. (2000) indicate a
decrease in activity following the order 3-OH > 3-O-glyco-
syl > no substitution, while Silva et al. (2002) reported better
results with no substitute compared to O-glycosyl. The
aforementioned results concern flavonoids with a 2,3-double
bond, while no difference, between a 3-OH and no
substitution, was observed with compounds which lack the
double bond (Hotta et al., 2002). The presence of the 4-keto
group in compounds without a 2,3-double bond has been
shown to decrease the stoichiometry (Hotta et al., 2002) or
not to affect it (Silva et al., 2002). However, most researchers
(Benavente-Garcia et al., 2000; Rice-Evans et al., 1996; Silva
et al., 2002) agree that the presence of the 4-keto group with
a 2,3 double bond in 3-OH substituted compounds (quercetin)
offers maximun activity, although there are some contradic-
tory results (Hotta et al., 2002; Fukumoto & Mazza, 2000)
indicating that maximum activity is obtained with 3-OH
substitution alone (catechin).
Most of the references cited above concern studies that do
not include compounds representative of all the major
subgroups of flavonoids; therefore conclusions for the effect
of all the structural elements of C-ring are not always feasible.
Moreover, the scavenging activity was studied by using
different radicals (ABTS+ or DPPH) or different solvents.
The purpose of the present work was to study the free radical
scavenging activity of 5,7,3V,4V-hydroxy-substituted flavonoids
which are categorized among the most widespread in plants
and efficient members of the major subgroups. Taking into
consideration that all substances possess catecholic structure on
the B-ring, we tried to clarify how the substitution pattern of C-
ring affects the reactivity of the catecholic moiety. DPPH was
selected as the free radical as it reacts with the flavonoids
through hydrogen transfer and may predict reactivity against
lipid peroxidation.
2. Materials and methods
2.1. Materials
The flavonoids used were quercetin dihydrate (Fluka 99%),
fisetin hydrate (Aldrich, pure), (+)-catechin (Fluka, 98%), ()-
142 D.I. Tsimogiannis, V. Oreopoulou / Innovative Food Science and Emerging Technologies 7 (2006) 140 – 146
epicatechin (ItChem), eriodictyol (Extrasynthèse, pure), rutin
(Merck, Extra pure), luteolin (Sigma), taxifolin (Sigma),
kaempferol (ItChem). 2,2-diphenyl-1-picrylhydrazyl radical
(DPPH) was obtained from Aldrich (purity 95%).
2.2. DPPH free radical scavenging
All the DPPH scavenging measurements were held on a
digital spectrophotometer (Unicam Helios, Spectronic Unicam
EMEA, Cambridge, United Kingdom), according to the
method proposed by Brand-Williams et al. (1995), under
thermostated conditions at 25 -C. 3.9 mL of a daily-prepared
DPPH solution of 6.02  10 5 M in methanol was placed in a
cuvette and 0.1 ml of a methanolic solution of each antioxidant
was added. The absorbance of the mixture was being recorded
at 515 nm against a second cuvette with a blank solution of
DPPH so as to take into consideration the decomposition rate
of the radical and correct the decrease in absorbance during the
reaction with the flavonoids (Ancerewicz et al., 1998). The
same procedure was followed for different concentrations of
each antioxidant. The corrected absorbance values were plotted
against time.
3. Results and discussion
The structures of the studied flavonoids are presented in
Fig. 1. The arrows indicate the systematical changes in
structure, starting from quercetin, which contains all the
functional elements of the C-ring, i.e. 2,3-double bond, 3-
hydroxy and 4-keto substitution, to compounds in which
by turns only one of these groups has been missing. In
addition to catecholic flavonoids, kaempferol was examined
in this study to clarify the effect of the B-ring catecholic
structure on the reactivity of the fully substituted C-ring.
Also fisetin (7,3V,4V-flavonol) which lacks 5-OH was
studied in order to examine if the A-ring substituents
A B
6.00E-05
5.00E-05
4.00E-05
[DPPH] (M)
3.00E-05
2.00E-05
1.00E-05
0.00E+00
0 20 40
t (min)
Fig. 2. A. Kinetic curves of DPPH scavenging for taxifolin at various concentrations. B. The correlation of a with time of reaction (s) for taxifolin 10 5 M. The linear
part belongs to the rapid stage and for a Primary Stoichiometric Factor n = 1.99, R 2 is optimized.
conjugate with the structural elements of B- and C-ring as
far as catecholic flavonols are concerned.
The reaction of the flavonoids with DPPH was conducted
in methanol, as it is a solvent commonly used for this
reaction (Ancerewicz et al., 1998; Brand-Williams et al.,
1995; Sanchez-Moreno et al., 1998; Zhu, Wang, Wei, Lin,
Yang & Ho, 2001). Furthermore, the reactivity of flavonoids
against DPPH in methanol is more accurate for the
prediction of their reactivity in oils, compared to non-
alcoholic solvents, such as ethyl acetate (Tsimogiannis &
Oreopoulou, 2004). The DPPH radical has deep purple color
and absorbs strongly at 515 nm, whereas the yellowish
reduction product, DPPH2, does not. A reference curve of
absorbance (A) against DPPH concentration in methanol
([DPPH], M) was constructed and used for the calculation
of DPPH concentration at various reaction times. It is
expressed by the equation: A = 11490I[DPPH]  0.0024
(R 2 = 0.9999).
The scavenging of the free radical by each compound
resulted in similar pattern curves of remaining [DPPH] versus
time. Fig. 2A presents the curves obtained during the
scavenging of DPPH by various concentrations of taxifolin,
representatively. It was evidenced that DPPH scavenging could
be separated in two parts; a first rapid one and a second in
which the radical was being scavenged at a very slow rate. The
duration of the rapid scavenging of most flavonoids lasted less
than 90 s, while the respective slow reactions were taking place
for at least 30 min. In the first part the decrease of DPPH
concentration was 50 to 500 times more rapid than in the
second. The stoichiometries of the rapid and slow stage are
basic elements for the explanation of the antiradical activity,
because they could reveal the contribution of the different
functional groups to scavenging reactions. The rapid stage
could be possibly attributed to the very reactive o-hydroxyls of
B-ring. In such case a reaction between the flavonoid and
DPPH takes place to produce a far less active quinone which
1.6E+05
1.06E-06 M
2.12E-06 M
3.19E-06 M 1.4E+05
4.25E-06 M
5.31E-06 M 1.2E+05
6.37E-06 M
7.41E-06 M
8.49E-06 M 1.0E+05
1.06E-05 M
8.0E+04
α
6.0E+04
α = 1253.1t + 734.16
4.0E+04
R2 = 0.9993
n=1.99
2.0E+04
0.0E+00
60 80 0 20 40 60 80 100 120
t (s)
 D.I. Tsimogiannis, V. Oreopoulou / Innovative Food Science and Emerging Technologies 7 (2006) 140 – 146 143

scavenges DPPH leading to a complex mixture of degradation The stoichiometries of the rapid step show that for all
products according to Eqs. (I) and (II). catecholic flavonoids, except quercetin and fisetin, the rapid
initial scavenging is accomplished by the two ortho-
Flavonoid þ nDPPH & †Quinone þ nDPPH2 ðIÞ hydroxyls of ring B, which donate their H atoms to reduce
DPPH and form the corresponding o-quinones. Alluis, Pérol,
Quinone þ mPPP H & YProducts þ mDPPH2 : ðIIÞ
El Hajji, and Dangles (2000) also distinguished the
According to Sang et al. (2003) and Brand-Williams et al. scavenging of DPPH in two steps and came to similar
(1995) the formation of quinones is governed by reversible conclusions as far as rutin and rutin derivatives are
reactions. On the contrary, the reactions that form the final concerned: they determined stoichiometries close to 2 during
products are irreversible since they involve fission of the initial the fast step, consistent with the formation of quinone
skeleton and production of radical termination compounds intermediates. Furthermore Goupy et al. (2003) reported that
(Goupy, Dufour, Loonis, & Dangles, 2003; Shahidi, Janitha, & polyphenols displaying a free 1,2-hydroxybenzene (catechol)
Wanasundara, 1992; Zhu, Huang, Yu, La Voie, Yang & Ho, gave stoichiometric values close to 2, per catechol group, in
2000; Zhu et al., 2001). agreement with the stepwise formation of quinones during
In order to avoid the reversibility of the primary reactions, the fast step.
DPPH was used in excess (even fifty-fold higher concentration Concerning quercetin, the experimental results showed that
than the antioxidant), a fact that rendered the first stage it scavenges four free radicals (Table 1). This is in complete
    o 
CDPPH 1 1 o 1 o  CAH
 ¼ kCDPPH ICAHn ` I ln I CAH  C  C DPPH  ln n
¼  kt ðIIIÞ
t o 1 o CDPPH n
n DPPH o
CDPPH
CAH  C
n
n DPPH
|fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl
ffl}
a

practically a one-way reaction. Furthermore it could be
considered that during the rapid stage the reduction of DPPH
due to the action of the quinone is not significant, given that
reaction (II) is much slower than reaction (I). Practically the
kinetics of the two stages do not conflict.
Data of the rapid stage were analyzed for all concentrations
of each antioxidant to approach the kinetics of this stage. In all
cases a second order kinetics according to Eq. (III) performed
best fit to experimental data. The plot of the left term of Eq.
(III) (a) versus time is indicated in Fig. 2B for taxifolin,
representatively. The parameter n of Eq. (III) corresponds to
the number of scavenged radicals per molecule of antioxidant
as indicated in Eq (I), i.e. the stoichiometric factor of primary
stage (PSF). The PSF and rate constant k for each compound
were calculated as the average of the determined n i and k i from
the kinetic analysis of different flavonoid concentrations and
are presented in Table 1.
agreement with the finding of Dangles, Fargeix, and Dufour
(1999). The action of this flavonoid is due to the formation of
solvent adducts (addition of two methanol molecules on the 2,3
double bond of C-ring), thus prolonging the radical scavenging
activity by means of reducing the o-quinone in B-ring. The
regenerated o-diphenol still reacts with two additional mole-
cules of DPPH to form the corresponding diquinone, indicated
in Fig. 3. The other flavonoids that posses a 2,3-double bond
did not show similar behaviour, leading to the conclusion that
methanol addition to the 2,3-double bond depends on the
existence of the 3-OH hydrogen and the B-ring catecholic
structure. The first argument is confirmed by the reaction of
rutin and Luteolin, which lack the active hydrogen and the
whole 3-OH group, respectively, as indicated in Fig. 1, and
scavenged two radicals during the rapid step (Table 1), in
contrast to the four scavenged radicals by quercetin. Therefore
no methanol addition occurs. The second argument is

Table 1
Rate constants of rapid kinetics, moles DPPH scavenged per mole of each flavonoid during the rapid kinetics (PSF) and the total reaction (TSF) and the quantity of
flavonoid necessary to scavenge the 50% of the initial quantity of DPPH (EC50)
Compound tested PSF k TSF EC50
  (M 1s 1)      
molDPPH molDPPH molantiox gantiox
molantiox molantiox molDPPH KgPPPH

Quercetin 3.98 T 0.12a 6433 T 381 6.74 T 0.08 0.078 T 0.004 60 T 3

()-Epicatechin 2.04 T 0.03 1608 T 129 – 0.087 64
(+)-Catechin 2.03 T 0.04 1542 T 152 – 0.077 57
Eriodictyol 2.02 T 0.03 1304 T 210 3.30 T 0.04 0.146 T 0.003 107 T 2
Rutin 1.98 T 0.04 4707 T 185 5.32 T 0.07 0.095 T 0.003 160 T 4
Luteolin 2.00 T 0.03 7737 T 1516 4.73 T 0.08 0.106 T 0.006 76 T 4
Taxifolin 1.99 T 0.05 1239 T 91 4.09 T 0.06 0.122 T 0.006 93 T 5
Kaempferol 1.87 T 0.09 11308 T 1590 1.87 T 0.09 0.249 T 0.019 181 T 7
Fisetin 4.13 T 0.15 6738 T 374 5.59 T 0.08 0.094 T 0.004 73 T 3
a
Mean value T SD.
144 D.I. Tsimogiannis, V. Oreopoulou / Innovative Food Science and Emerging Technologies 7 (2006) 140 – 146
O O
O O
MeO
HO O HO
OH
OH O OMe
quercetin
quinone
Fig. 3. The primary oxidation products of quercetin, fisetin and taxifolin, respectively.
confirmed by the reaction of kaempferol, with no B-ring
catecholic structure (Fig. 1) that scavenged two radicals (Table
1), whereas slow step was not observed. The slow step would
have been an indication that a weak scavenger was taking over.
Furthermore A-ring or its substituents do not influence the
primary reactions of flavonols; fisetin which lacks 5-OH not
only scavenged four radicals but also presented similar rate
constant (k = 6.7  103 M 1s 1) as quercetin (k = 6.4  103
M 1s 1). These results suggest that the methanol adducts,
which act further against free radicals, can be formed only by
catecholic flavonols.
Consequently the PSF of catecholic flavonoids is affected
by C-ring only when both 2,3-double bond and 3-OH exist
at the molecule. In all other cases of C-ring substitution the
molecules act identically; they scavenge two radicals.
Unlike, rate constants of the rapid kinetics are highly
affected by C-ring substitution (Table 1). The most valuable
characteristic appears to be the 2,3-double bond since
flavones and flavonols posses much higher rate constants
than the rest, saturated members.
Kaempferol exhibited the most rapid reaction with a rate
constant k = 1.1 104 M 1 s 1. This fact is reasonable
because at the 3V,4V-OH members, after the donation of one
hydrogen atom to DPPH, a hydrogen bond is settled between
the phenoxy radical and the hydrogen of the o-hydroxyl
(Zhang, Sun, & Wang, 2003). This H-bond suppresses the
delocalization of the unpaired electron through which the
donation of the second H can be achieved. Kaempferol is a
monohydroxy B-ring flavonoid and no intramolecular H-bond
occurs after scavenging one DPPH molecule. The unpaired
electron becomes highly delocalized and produces 10 reso-
nance structures, during the reaction of kaempferol with
DPPH which is presented in Fig. 4. All 3V,4V-OH members
possess lower rate constant values than kaempferol. Never-
theless these values are highly differentiated, a fact which
could be attributed to the number of resonance structures since
the higher the number of resonance structures, the lower the
demanded energy for the formation of the free radical. For
example, luteolin produces 9 resonance structures after the
donation of the first H to DPPH and the total rate constant for
the primary stage is 7.7  103 M 1 s 1 while eriodictyol,
which lacks 2,3-double bond, presents 8 resonance structures
and a respective rate constant 1.3  103 M 1 s 1. Rutin
reacted slower than luteolin due to steric effects of the 3-O-
rutinoside, while quercetin and fisetin had similar rate
constants.
O
O
MeO
O HO O
OH OH
O OMe OH O
fisetin taxifolin
quinone quinone
The effects of the C-ring structural characteristics are also
expressed through the secondary reactions, i.e. the reactions of
the produced quinones at the primary stage. However, the
secondary reactions are complicated; they lead to mixtures of
oxidation products and the second order kinetic model is not
applicable. The Total Stoichiometric Factor (TSF), representing
the scavenged molecules of DPPH per molecule of antioxidant
after the completion of the reaction was estimated through the
relevant curves, as the ones represented in Fig. 2A. The
scavenged DPPH of each curve at the end of the reaction was
plotted against the initial flavonoid concentration. The corre-
(scavenged)
lation CDPPH – C(initial)
AHn was linear for the majority of the
compounds (apart from the two flavanols) and the slope
revealed the scavenged DPPH molecules per molecule of
antioxidant, a value that represents the Total Stoichiometric
Factor (TSF) and is presented in Table 1. The total antioxidant
efficiency, expressed by EC50, which corresponds to the ratio
molantiox / molDPPH or gantiox / kgDPPH necessary to reduce 50%
of the initial concentration of DPPH is also presented in Table
1. Although TSFs of flavanols were not possible to be
(scavenged)
determined due to the lack of linearity between CDPPH -
(initial)
CAHn , the EC50 values were calculated graphically.
The stoichiometric factors concerning the slow scavenging
of the studied flavonoids vary and it is not safe to assume the
oxidative paths based on the observed stoichiometries,
especially since these factors are difficult to be approximated
to the nearest integer. To examine the effect of the different
structural groups of the C-ring on the scavenging efficiency
during the slow step, we tried to compare the observed
stoichiometric factors of compounds, which lack by turns only
one structural element, as indicated in Fig. 1. Rutin, luteolin
and eriodictyol reveal that the ether bonded 3-oxygen is
preferable than the non-substituted 3-C, while the loss of one
further substitution element of C-ring, i.e. the 2,3 double bond
leads to an additional decrease of the slow step radical
scavenging stoichiometry (rutin; 3.3 > luteolin; 2.7 > eriodic-
tyol; 1.3). In the case of quercetin a different primary
mechanism occurs, as already mentioned, which leads to
higher radical scavenging ability: not only the catecholic
hydroxyls participate during the reactions of the rapid part but
also the 2,3-double bond that becomes saturated because of the
concurrent methanol addition at the parent molecule. Therefore
the action of quercetin primary oxidation product, due to the
saturated 2,3 bond, is feasible to be compared with the
respective product of taxifolin due to structural similarities of
the two compounds that can be seen in Fig. 3. A slight decrease
 D.I. Tsimogiannis, V. Oreopoulou / Innovative Food Science and Emerging Technologies 7 (2006) 140 – 146 145

OH

HO O

kaempferol
OH
OH O
DPPH

DPPH 2
O OH

HO O HO O

or
OH O
OH O OH O

OH OH

HO O H HO O H

OH OH
OH O DPPH OH O

DPPH 2

HO O

kaempferol quinone
O
OH O

Fig. 4. Proposed mechanism of DPPH radical scavenging by kaempferol.

of the slow step stoichiometry is observed in the case of
taxifolin, indicating that the 2,3-dimethoxy substitution of
quercetin primary oxidation product might be contributing to
the scavenging ability. The product of primary oxidation of
eriodictyol, which lacks one more structural element is again
the least active (quercetin; 2.8  taxifolin; 2.1 > Eriodictyol;
1.3). It is obvious from the above comparisons that the gradual
loss of structural elements of C-ring decreases the radical
scavenging action of the 5,7,3V,4V-flavonoids. On the other
hand the comparison of the slow step stoichiometric factors of
luteolin and taxifolin (2.7 and 2.1, respectively) indicates that
the 2,3-double bond offers a slightly higher scavenging ability
compared to the 3-OH group.
(+)-Catechin and ()-epicatechin, the only flavonoids that
lack the 4-carbonyl group, shown identical action to other
flavonoids, as far as the stoichiometric factors of the rapid step
are concerned; each antioxidant scavenged two radicals. The
stoichiometries of the slow step were not feasible to be
determined since the reactive concentration of the radical was
not linearly correlated with the initial concentration of each
antioxidant. Increasing the molar ratio flavanol/DPPH resulted
in a decrease of the observed stoichiometric factor. However, at
relatively low molar ratios (< 0.1) the two catechins appear to
possess the highest stoichiometric factors in slow kinetics, as
(scavenged) (initial)
indicated by the major slopes presented by CDPPH – CAH n
curves. The products of catechins oxidation are complex and
quinone formation is probably followed by dimerization
(Goupy et al., 2003). Flavanols lacking two structural elements
at C-ring, i.e. the 2,3-doudle bond and the 4-carbonyl could be
compared with quercetin and taxifolin. The fact that flavanols
show increased slow step stoichiometry compared to quercetin
and taxifolin is a paradox since it does not obey the rule of
decrease of antioxidant action with the loss of the C-ring
structural elements.
According to the total stoichiometric factors or the EC50 the
classification of the flavonoids is quercetin > fisetin > rutin > -
luteolin > taxifolin > eriodictyol > kaempferol. Catechin and epi-
catechin are classified close to quercetin according to the EC50
parameters.
Conclusively flavonoids are highly dependent on the C-ring
substitution that affects the radical scavenging activity differ-
ently as far as the stoichiometry and rate in both primary
(attributed to B-ring OH) and secondary reactions are
concerned. Full substitution of C-ring provides to the catecholic
146 D.I. Tsimogiannis, V. Oreopoulou / Innovative Food Science and Emerging Technologies 7 (2006) 140 – 146
flavonoids the maximal scavenging capacity and significant
reaction rates, while the overall activity of fully substituted
compounds is determined mainly by the rapid reactions. The
lack of one or more C-ring structural elements results in major
reduction of the stoichiometry during the rapid stage (PSF). The
primary reaction rates, on the other hand, are highly associated
with the 2,3-double bond. The total stoichiometric factor
presents gradual reduction with the lack of C-ring substituents
in chromanone-type flavonoids, while the chromane-type
catechins do not obey this rule. Nevertheless the contribution
of the slow reactions on the overall activity of flavonoids
increases when C-ring is not fully substituted.
As far as A-ring is concerned, monohydroxy-substitution
does not affect the stoichiometric factor and rate constant of the
primary reactions but results in reduced stoichiomeric factors
of secondary reactions, compared to the dihydroxy-substitu-
tion. On the contrary o-dihydroxy-substitution of B-ring affects
markedly the stoichiometry of primary reactions.
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