Kinetic Study of Flavonoid Reactions with Stable Radicals

Article abstract of DOI:10.1021/jf049880h

The antiradical activities of some flavonols (kaempferol, quercetin, robinetin, quercetagetin, and myricetin), flavones (apigenin, baicalein, and luteolin), flavanones (naringenin and dihydroquercetin), and flavanols [(+)-catechin and (-)-epicatechin] wer

Full text of DOI:10.1021/jf049880h

2816 J. Agric. Food Chem. 2004, 52, 2816−2820
Kinetic Study of Flavonoid Reactions with Stable Radicals
VJERA BUTKOVICÄ ,† LEO KLASINC,† AND WOLF BORS*,§
The Rudjer Boskovic Institute, HR-10002 Zagreb, Croatia, and Institut Strahlenbiologie,
GSF Forschungszentrum fu¨r Umwelt und Gesundheit, D-85764 Neuherberg, Germany
The antiradical activities of some flavonols (kaempferol, quercetin, robinetin, quercetagetin, and
myricetin), flavones (apigenin, baicalein, and luteolin), flavanones (naringenin and dihydroquercetin),
and flavanols [(+)-catechin and (-)-epicatechin] were determined by measuring the reaction kinetics
with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and R,γ-bisdiphenylene-â-phenylallyl (BDPA) radicals. The
reactions, which follow the mixed second-order rate law, were investigated under pseudo-first-order
conditions by use of a large excess of flavonoids, and their stoichiometry was determined by
spectrophotometric titration. The results confirm stoichiometric factors of 1, 2, and 3 for flavonoids
with one, two, and three hydroxyl groups in the B-ring, respectively, excluding kaempferol, which,
despite a single OH group in the B-ring, has a factor of 2, which is explained by the 3-OH group
supporting the reaction with free radicals. Structure-activity considerations indicate for the present
series of flavonoids the importance of multiple OH substitutions and conjugation. The logarithms of
reaction rate constants with the OH, DPPH, and BDPA radicals correlate well with the reduction
potential of the flavonoids.
KEYWORDS: Flavonoids; antiradical activity; reaction rate constants; DPPH and BDPA radicals;
correlation with reduction potential; structure-activity relationship (SAR)
INTRODUCTION
Free radical scavenging capacity is primarily attributed to the
high reactivity of hydroxy substituents that participate in the
following reaction (F-OH ) flavonoid):
Flavonoids are compounds found in fruits, vegetables, and
certain beverages that have diverse biochemical (1) and anti-
oxidant effects (2-4). Flavonoids are benzo-γ-pyrone deriva-
tives consisting of phenolic and pyran rings, and most possess
high antioxidant and free radical scavenging activityies (5, 6).
The antioxidant activity of flavonoids and their metabolites in
vitro depends on the arrangements of functional groups about
the nuclear structure. Dietary flavonoids differ in the arrange-
ments of hydroxy, methoxy, and glycosidic groups and in the
conjugation between the C- and B-rings. The protective effects
of flavonoids in biological systems are ascribed to their capacity
to transfer electrons to free radicals, chelate metals, and activate
antioxidant enzymes. Here we are interested in the antiradical
activity of some flavonols, flavones, flavanones, and flavan-3-
ols (Scheme 1).
An imbalance between antioxidants and pro-oxidants results
in oxidative stress, leading to cellular damage. Oxidative stress
has been linked to cancer, atherosclerosis, ischemic injury,
inflammation, and neurodegenerative diseases, among others (7).
A well-known fact is that antioxidants are compounds that
protect cells against the damaging effects of reactive oxygen
species, for example, the superoxide anion radicals, hydroxy
radicals, and peroxyl radicals (8, 9).
F-OH + R• f F-O• + RH
(1)
Hydroxy groups donate hydrogen and an electron to radicals,
stabilizing them and giving rise to a relatively stable flavonoid
radical (10). Due mainly to its simplicity, the most popular assay
to determine radical-scavenging activities is the bleaching of
the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) (11,
12). Unfortunately, the method has never been standardized with
respect to absolute and relative concentrations of substrates and
the DPPH radical, the observation period, etc. Despite earlier
suggestions of defining the inverse of IC50 values as “antioxidant
reduction potential” (ARP) (13-15), most compilations list IC50
values (decrease of the initial DPPH concentration by 50%)
(16-19), data that, if obtained in different laboratories, cannot
be directly compared. In fact, the use of EC50/IC50 has been
severely criticized as it does not take into account the various
kinetic behaviors and the authors define the inverse value as
“antiradical efficiency” (AE) (20).
Although the generally accepted mechanism is the transfer
of hydrogen atoms to the DPPH radical, this has only recently
been verified by the identification of reaction products (21-
25). Interestingly enough, the earlier study (21) reported only
the formation of the B-ring o-quinones from (+)-catechin and
(-)-epicatechin, whereas the later studies also discovered
various dimers of the catechins (23, 24), formed via intermediary
quinone methides and phenolic coupling reactions (26).
* Author to whom correspondence should be addressed (e-mail
bors@gsf.de; fax +49-89 3187-2818).
† Rudjer Boskovic Institute.
§ GSF Forschungszentrum fu¨r Umwelt und Gesundheit.
10.1021/jf049880h CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/27/2004

Flavonoid Reactions with Stable Radicals
J. Agric. Food Chem., Vol. 52, No. 10, 2004 2817
Scheme 1. Backbone Structures of Flavonoids Investigated and of
the Stable Radicals
Figure 1. Spectrophotometric titration of DPPH with quercetin at 516 nm.
Table 1. Kinetic Data for the Reaction of the DPPH Radical with
Flavonoidsa
flavonoids
flavonols
systematic name
kF/L mol-1 s-1
kaempferol
3,5,7,4′-tetrahydroxyflavonol
2.38 × 103
7.0 × 102 (28)
4.76 × 102
6.2 × 102 b
1.89 × 103 (29)
1.14 × 102
1.4 × 102 b
4.4 × 103
quercetin
3,5,7,3′,4′-pentahydroxyflavonol
robinetin
3,7,3′,4′,5′-pentahydroxyflavonol
quercetagetin
myricetin
3,5,6,7,3′,4′-hexahydroxyflavonol
3,5,7,3′,4′,5′-hexahydroxyflavonol
7.55 × 101
1.3 × 102 b
flavones
apigenin
baicalein
luteolin
5,7,4′-trihydroxyflavone
5,6,7 -trihydroxyflavone
5,7,3′,4′-tetrahydroxyflavone
0.1
6.3 × 102
2.29 × 103
flavanone
naringenin
dihydroquercetin
flavanol
5,7,4′-trihydroxyflavanone
3,5,7,3′,4′-pentahydroxyflavanone
4.0
Yordanov (27) has thus far been the only one to attempt a
quantification of the DPPH procedure, whereas kinetic studies
to determine absolute rate constants of the hydrogen transfer to
the DPPH radical have been carried out with flavones by
Lindberg Madsen et al. (28) and Goupy et al. (29). In the present
work we have measured the second-order rate constants for the
reaction of some flavonoids with DPPH, comparing some of
the data with those obtained with R,γ-bisdiphenylene-â-phen-
ylallyl (BDPA) radicals.
2.2 × 102
(+)-catechin
3,5,7,3′,4′-flavanol
3,5,7,3′,4′-flavanol
5.3 × 102
1.09 × 103 (29)
4.89 × 102
(−)-epicatechin
1.12 × 103 (29)
a 25 °C, methanol solution. b In 1:1 H O/2-propanol (v/v).
2
margin of error in our experiments of ∼10%. Most of the reactions
were carried out in methanol as solvent, although in several experiments
a 1:1 mixture of 2-propanol/water was used.
MATERIALS AND METHODS
The flavonoids were of commercial origin (Fluka, Sigma, Extra-
synthese) and were used without further purification. The stable free
radicals DPPH and BDPA [complex with benzene (1:1)] were from
Aldrichsthe latter is no longer available. Methanol and 2-propanol
(Kemika) were distilled as needed. Doubly distilled water, additionally
purified by a passage through a Milli-Q system, was used.
Spectral and kinetic data were obtained using an HP Agilent 8453
diode array spectrophotometer and a Durrum D-110 stopped-flow
instrument. The kinetics were followed at the absorption maximum of
the DPPH (516 nm, log ꢀ ) 3.985) and BDPA (480 nm, log ꢀ ) 4.323)
radicals. The data were collected under pseudo-first-order conditions
by use of a large excess of flavonoids [(1.8-5.6) × 10-4 M] over the
concentration of DPPH radicals [(1.8-2.5) × 10-5 M], whereas in the
experiments with BDPA radicals the initial concentrations were of
flavonoids [(0.45-1.3) × 10-3 M] and BDPA [(3.8-8) × 10-5 M].
We chose BDPA as another stable radical reacting with flavonoids,
and its absorption in the visible region allowed us to measure the
kinetics. The data were evaluated with the program for the first-order
reaction on the UV-vis Chemstation.
RESULTS AND DISCUSSION
The DPPH radical scavenging method has been applied to
the evaluation of the antiradical activity of numerous substances
(11, 12). As mentioned earlier, most of these papers presented
the radical scavenging activity as EC50.
The stoichiometry for the reaction of several flavonoids was
determined by spectrophotometric titration at the wavelength
of the maximum absorption of the DPPH radicals (29; see
Figure 1). The ratios were 0.5 for (+)-catechin, quercetin,
luteolin, and kaempferol, 0.3 for myricetin and robinetin, and
1 for naringenin. The results confirm the overall 1:1 stoichi-
ometry for flavonoids with one hydroxy group in the B-ring,
1:2 stoichiometry for flavonoids with two hydroxy groups in
the B-ring (but also including kaempferol), that is, values of n
) 1, 2, and 3 for the stoichiometry factor, respectively.
The reaction of flavonoids and DPPH radicals followed the
mixed second-order rate law (eq 2), yielding the values of kF in
Table 1.
In the stoichiometric titrations, the titrant flavonoid was added slowly
to the DPPH solution until the equivalence point was reached with no
further change in absorbance. The quantity of the flavonoid that is
required to consume all of the DPPH radical is expressed as a ratio of
the concentrations of both reactants ([F]/[DPPH]). We assume an overall
-d[DPPH]/dt ) -nd[F]/dt ) kF[DPPH][F]
(2)

2818 J. Agric. Food Chem., Vol. 52, No. 10, 2004
Butkovic´ et al.
The rate constants observed for (+)-dihydroquercetin (2.2 ×
102 L mol-1 s-1), quercetin (4.76 × 102 L mol-1 s-1), and
luteolin (2.29 × 103 L mol-1 s-1) indicate different activities,
which depend on their backbone structures. They all have 5,7-
di-OH substitution in the A-ring and 3′,4′-di-OH substitution
in the B-ring, but the former two have a 3-OH substitution at
the C-ring and the latter two a C2-C3 double bond. The
flavanone (+)-dihydroquercetin reacts 2 times more slowly than
quercetin and luteolin reacts 5 times more rapidly than quercetin
with DPPH radicals. These results reveal a strong (5-fold) effect
of removing a 3-OH group and smaller (2-fold) effect of adding
a C2-C3 double bond on reactivity increase.
Figure 2. Plot of kobs versus the concentrations of flavonols for the reaction
However, if we compare the structure and the rate constants
for the kaempferol, naringenin, and apigeninsall with a single
4′-OH substitution in the B-ring, which is considered to yield
low activityswe observe that kaempferol has considerable
affinity toward radicals, indicating that here the 3-OH group at
the C-ring supports the reaction with free radicals. The 3-OH
substitution is thought to increase the stability of the flavonoid
radical in this group of flavonoids because the torsion angle of
the B-ring with respect to the rest of the molecule strongly influ-
ences free radical scavenging ability. Flavonols and flavanols
with a 3-OH substitution are planar, whereas flavones and
flavanones, lacking this feature, are slightly twisted (30, 31).
The observed enhanced activity and stoichiometry factor of
2 for kaempferol can also be rationalized by the existence of a
p-quinone methide structure upon the hydrogen atom abstraction
by the DPPH radical
with DPPH radicals.
Under our experimental conditions the data show linear
dependence of the pseudo-first-order rate constants on flavonoids
concentration (Figure 2.)
The least-squares fits for the lines expressed by y ) ax + b
yield the second-order rate constants from the slope (dimension
L mol-1 s-1). The lack of intercept denotes the stability of the
DPPH radical. The rate constants observed for the reaction of
DPPH radicals with various flavonoids are presented in Table
1. The scavenging activity of flavonoids toward radicals was
found to vary depending on the group of flavonoids.
Although rate constants of only kaempferol and eriodictyol
with DPPH were measured by Lindberg Madsen et al. (28) under
similar pseudo-first-order conditions as we employed with (on
average) a 10-fold excess of the flavones, the values obtained
by Groupy et al. (29) are 2-3 times higher as consequence of
using the stoichiometry factor in eq 2. The latter authors used
a kinetic modeling approach assuming as initial reaction the
reduction of the DPPH radical and formation of a flavonoid
quinoneswhich in effect is a two-electron oxidation.
The rate constants for the reaction with BDPA radicals,
although much lower than for DPPH, decrease in the order
kaempferol (0.141 L mol-1 s-1), luteolin (0.131 L mol-1 s-1),
quercetin (0.091 L mol-1 s-1), and myricetin (0.026 L mol-1
s-1) and thus follow the same trend as observed for the reaction
of the flavonoids with the DPPH radical.
Some structure-activity relationship can been proposed from
this series of the flavonoids, pointing out the importance of the
structure of the B-ring and the influences from the rest of the
molecule. As observed, the kF values for the flavonols decrease
in the order quercetagetin > kaempferol > quercetin > robinetin
> myricetin. The flavonol quercetagetin, which contains a
pyrogallol-like substitution in the A-ring (5,6,7-tri-OH) and an
o-di-OH substitution in the B-ring, has the highest reactivity
with DPPH. It seems that besides the well-established impor-
tance of the latter, multiple substitution on the A-ring is playing
also an important role in hydrogen atom transfer reaction.
However, kaempferol with one hydroxy group in the B-ring
reacts more rapidly than quercetin with 3′,4′-di-OH substitution,
which is explained by interaction with its 3-OH substituent (vide
infra). Also, flavonols with a pyrogallol-like structure in the
B-ring show low reactivity.
analogous to recent suggestions by Rietjens and colleagues for
quercetin (32-34).
The abstraction of a hydrogen atom from flavonoids by
radicals proceeds probably through a polar transition state. The
reaction rates of flavonoids obtained in 2-propanol/water (1:1)
solutions were slightly higher than those obtained in methanol,
in agreement with the polarity of the solutions (35).
Skibstedt and co-workers were the first to correlate reduction
potentials of flavonoids with their antioxidant potential (28, 36).
As shown in Figure 3, we also note a linear correlation between
the reduction potentials of flavonoids and the logarithm of the
rate constants for the reaction with DPPH radicals. The flavone
luteolin and the flavanol (+)-catechin have reducing strengths
similar to that of flavonol kaempferol, which shows good
antiradical activity (37, 38). Similar correlations were found for
reactions with other radicals as different as OH (10) and BDPA,
which indicates the same mechanism. In fact, because all three
radicals are oxidative species, that is, they remove a hydrogen
atom (or H+/e-) from the flavonoid hydroxyl groups, a
correlation with the respective reduction potential should be
expected.
In kinetic investigations one often observes empirical cor-
relations of rate constants with other parameters, for example,
Hammett or Marcus values. These so-called linear free energy
relationships (LFER) are usually depicted as semilogarithmic
plots, log k ) m(E) + b. Both the slopes and the ordinate
intercepts are specific for each correlation. In our case the
reasonable similarity of the slopes (OH, log k ) 3.78x + 8.87;
DPPH, log k ) 5.84x + 1.21; BDPA, log k ) 3.28x - 1.84)
The rate constant of the flavone luteolin with 3′,4′-di-OH
substitution in the B-ring and 5,7-di-OH substitution in the
A-ring of 2.29 × 103 L mol-1 s-1 is almost as high as for
kaempferol. It is worth noting that apigenin (KF ) 0.1 L mol-1
s-1) with 4′-OH in the B-ring compared to luteolin reacts
significantly slower. The rate of reaction of baicalein, which
has no substitution in the B-ring, but a pyrogallol-like structure
on the A-ring (5,6,7-tri-OH), is between those found for luteolin
and apigenin.

Flavonoid Reactions with Stable Radicals
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