Chem. Res. Toxicol.

1996, 9, 1305-1312 1305

A Quantum Chemical Explanation of the Antioxidant

Activity of Flavonoids
Saskia A. B. E. van Acker,*,†,‡ Marcel J. de Groot,† Dirk-Jan van den Berg,†,‡
Michèl N. J. L. Tromp,† Gabrielle Donné-Op den Kelder,†
Wim J. F. van der Vijgh,‡ and Aalt Bast†
Leiden/Amsterdam Center for Drug Research, Department of Pharmacochemistry, Faculty of
Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, and
Department of Medical Oncology, Free University Hospital, De Boelelaan 1117,
1081 HV Amsterdam, The Netherlands

Received June 13, 1996X

Flavonoids are a group of naturally occurring antioxidants, which over the past years have
gained tremendous interest because of their possible therapeutic applicability. The mechanism
of their antioxidant activity has been extensively studied over several decades. However, there
is still much confusion about the molecular mechanism of radical scavenging and the
relationship between structure and activity. Therefore, we have calculated the heat of formation
and the geometry of both the parent compound and the corresponding radical using the ab
initio program GAMESS. We have compared their differences in energy in order to gain insight
into the stability of the radical and the ease with which it is formed. We have also investigated
the spin density of the radical, to determine the delocalization possibilities. These calculated
data were compared with experimental data from ESR (hyperfine coupling constants) and
electrochemical oxidation (Ep/2) and were found to be in good agreement. By comparing the
geometries of several flavonoids, we were able to explain the structural dependency of the
antioxidant action of these compounds. The extremely good antioxidant activity of the flavonols
could be explained by the formation of an intramolecular hydrogen bond.

Introduction
Flavonoids are a group of polyphenolic compounds
ubiquitously found in fruits and vegetables. Because of
their broad pharmacological activity, they have recently
gained tremendous interest. In many traditional medi-
cines, part of the therapeutic effect may be ascribed to
the flavonoids. The pharmacological effect can be ex-
plained by their inhibition of certain enzymes and their
antioxidant activity (1). Many authors have attempted
to elucidate the structure-activity relationships (SAR)1
of this antioxidant activity (2-9). However, this is
hampered by the fact that antioxidant activity can be
considered to be determined by several factors, of which
lipophilicity (and thus uptake into the membranes, which
are often the site of action), iron chelation, and scaveng-
ing of free radicals are the most important. There
appears to be agreement now on the SAR of the free
radical scavenging activity: hydroxyl groups in ring B,
preferably a catechol moiety, are required for good
scavenging activity and a C2-C3 double bond in combi-
nation with a hydroxyl at C3 can further increase the
scavenging activity (Figure 1). There is, however, no
molecular explanation as to why these structural features
are important. Usually (10, 11), conjugation effects are
mentioned, but this does not explain why a double bond
without a hydroxyl at C3 does not have an increasing

* To whom correspondence and requests for reprints should be Figure 1. The structural subclasses of the flavonoids.
addressed at the Department of Pharmacochemistry, Faculty of
Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amster-
dam, The Netherlands. effect. Several other compounds which do not meet these
† Vrije Universiteit.
‡ Free University Hospital.
structural requirements are unexpectedly active, such as
X Abstract published in Advance ACS Abstracts, November 1, 1996. catechin (cyanidanol), which lacks the C2-C3 double
1 Abbreviations: SAR, structure-activity relationship; QSAR, quan-
bond and the 4-keto function. Thus until now, SAR has
titative structure-activity relationship; RHF, restricted Hartree Fock;
UHF, unrestricted Hartree Fock; DMA, distributed multipole analysis; only been descriptive, not explanatory by means of, for
LPO, lipid peroxidation. example, a quantitative relationship (QSAR).1
S0893-228x(96)00096-3 CCC: $12.00 © 1996 American Chemical Society
1306 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
The aim of the present investigation was to find a
possible explanation for the observations described above
and understand the effects of the structure on the
scavenging activity in a molecular way. Therefore, we
have used quantum chemical calculations to optimize the
geometry of both the molecule and the corresponding
radical and to compare the heat of formation. This can
give information about the ease with which the radical
is formed. The spin distribution gives insight into the
degree of delocalization and thus conjugation, which is
a measure for the stability of the radical.
Materials and Methods
Chemicals. Hesperidin (97%), diosmin (95%), fisetin, nar-
ingin, phloridzin (99%), phloretin (98%), and galangin were
obtained from Aldrich (Milwaukee, WI), and hesperetin, nar-
ingenin (95%), taxifolin, NADP, and horseradish peroxidase
were obtained from Sigma (St. Louis, MO). Rutin was obtained
from Merck (Darmstadt, Germany), and quercetin, myricetin
(>97%), pelargonidin chloride, apigenin (98%), and kaempferol
(96%) were purchased from Fluka (Buchs, Switzerland). Cya-
nidin chloride and luteolin (90%) were purchased from Roth
(Karlsruhe, Germany). The hydroxyethyl rutosides, trihydroxy-
ethyl quercetin, and (+)-catechin (cyanidanol) were a generous
gift from Zyma (Nyon, Switzerland). Hydrogen peroxide (30%)
was obtained from Baker (Deventer, The Netherlands). All
other chemicals were of the highest grade of purity available.
Half Peak Oxidation Potentials. The pH dependence of
the Ep/2 of monoHER and kaempferol was measured at several
pH values ranging from pH 2 to pH 13. The pH was changed
by adding either NaOH or HCl to a 50 mM phosphate buffer.
Flavonoids were dissolved in DMSO, diluted 1:1 with nano-
pure water, and further diluted in buffer, until a final concen-
tration of 100 µM. This buffer solution was gassed with oxygen-
free nitrogen for 5 min before each measurement, and nitrogen
was led over the solution while measuring. A platinum elec-
trode was used as a working electrode, with a platinum counter
electrode and a saturated calomel reference electrode. A scan
was made from -0.2 to 0.6 V with a scanning speed of 20 mV/s
on a PSTAT 10 potentiostat connected to an Autolab (Eco
Chemie, Utrecht, The Netherlands) and controlled by the
program General Purpose Electrochemical System 3.0 (Eco
Chemie, Utrecht, The Netherlands) run on an Olivetti PC M240.
Electron Spin Resonance. The ESR experiments were
performed on a Bruker ESP300 ESR spectrometer. Modulation
frequency was 100 kHz, the amplitude 1 G. Microwave fre-
quency was set at 9.79 GHz, with a power of 20 mW. Whenever
necessary, spectra were simulated using the program EPRcalc,
which was included in the operating software of this spectrom-
eter (see below).
The flavonoid solution in 100% DMSO (650 µL) was added
to a flat cell, and after placing it in the cavity, 250 µL of
saturated solution of KOH was added. This resulted in spon-
taneous oxidation by air as the oxidation potential was lowered
considerably.
Alternatively, flavonoids were dissolved in DMSO and diluted
with phosphate buffer, pH 7.4, rendering a solution with less
than 4% DMSO. To this solution H2O2 and horseradish per-
oxidase were added to initiate an oxidation reaction.
Both experiments were performed in the presence and
absence of Mg2+, which is known to stabilize catechol radicals.
This was done to increase the signal to noise ratio and to obtain
spectra of higher resolution. The resolution of ESR spectra at
pH 13 was considerably better than that of spectra measured
under physiological conditions.
Electron Spin Resonance Simulations. ESR spectra were
also simulated by the program EPRcalc implemented on a
Bruker ESP300 ESR spectrometer. Hyperfine coupling con-
stants (a) were taken from the experimental spectra, from
Kuhnle et al. (12), and from the calculated spin densities (see
below).
van Acker et al.
Quantum Chemical Calculations. (A) Structural Op-
timization (Minimal Energy Conformation). Flavonoid
X-ray structures were taken from the Cambridge Structural
Database (13) at CAOS/CAMM Centre in Nijmegen. Quercetin
and (+)-catechin were used as found. Rutin was found without
hydrogen atoms, which were added using the molecular model-
ing program package ChemX (14). Taxifolin was constructed
in ChemX starting from the X-ray structure of naringenin.
An estimate for the global minimum structure was deter-
mined by conformational analysis with the software package
Macro Model (15-17) installed at the CAOS/CAMM Centre
(KUN, Nijmegen) by rotating around C2-C1′ and C3-sugar
axes and around axes of OH groups not involved in an H-bond
(Figure 1). Therefore, first the most suitable Macro Model
forcefield for these structures was determined. The AMBER
forcefield (18, 19) was found to have the least number of ill-
defined parameters, and was therefore used for the conforma-
tional analysis. For taxifolin and (+)-catechin the “ring-flip”
was included in the analysis by using the “closure bond” option.
In order to include this flexibility into the saturated C ring, the
O1-C2 bond was opened and rotation around C4-C3 and C3-
C2 was performed.
The lowest energy conformation was taken, except for (+)-
catechin, where the conformational analysis led to the wrong
stereoisomer around C2. In this case we therefore used both
the lowest energy conformation (the (-) isomer) and the lowest
energy conformation of the (+) isomer, which was the third
lowest energy conformation.
The aforementioned conformations were optimized using the
quantum chemical program package GAMESS-UK (20, 21) at
the Restricted Hartree Fock (RHF)1 level with the STO-3G
(Slater type orbitals comprised of 3 Gaussians) minimal basis
set (22) on a CRAY-98 supercomputer facility. On the resulting
geometry, a single point RHF calculation in a SV (split valence)
6-31G (23, 24) basis set was performed to derive the energy.
From these GAMESS-optimized structures, the structures of
apigenin, naringenin, galangin, kaempferol, diosmin, hesperetin,
and 3-OMe-quercetin were constructed by addition or removal
of OH or OMe groups. These structures were also optimized
using the procedure described above.
(B) Calculation of Spin Distribution and Differences
in Heat of Formation between Parent Molecule and
Corresponding Radical. A theoretical, quantum chemically
determined, suitable parameter for describing the abstraction
of a H• from an O-H bond is the difference in heat of formation
between the flavonoid and its corresponding radical (∆∆Hf). The
delocalization possibilities within the radical flavonoids will
largely contribute to the corresponding ∆∆Hf value.
In order to gain insight into the delocalization possibilities
of flavonoids (which are the reason for their antioxidant
activity), spin distributions were calculated for the radicals of
several main classes.
Ep/2 and ∆∆Hf both involve the breaking of an O-H bond
either hetero- or homolytically (see reaction 1 in discussion) and
are therefore expected to correlate well, but only in case the
proposed reaction mechanism is correct.
Geometry optimizations for the radicals were performed at
the unrestricted Hartree Fock (UHF) level with the STO-3G
basis set. To form the radical, an H• was removed from the 4′-
hydroxyl or, if this was not present, from the 3- or 3′-hydroxyl
which are also, but less, likely to be involved in a scavenging
reaction. The ∆Hf of the radical and the unpaired spin distribu-
tion were calculated using a single point (open shell RHF)
energy and distributed multipole analysis (DMA) calculation
(25) in a SV-631G basis set.
The use of stabilization energies (∆Hr, our ∆∆Hf) for the
prediction of the reaction of a flavonoid with a radical in
hydrogen abstraction reactions is illustrated by Korzekwa et al.
(26). They found that a linear (Brønsted) relationship exists
between stability of radicals (experimental bond dissociation
energy data) and activation energies (∆Hq) of hydrogen abstrac-
tion for similar reactions in a series of analogous substrates;
the relative order of hydrogen atom abstraction can be obtained
Antioxidant Activity of Flavonoids Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1307

Table 1. Flavonoids Used in This Study Divided into Table 2. Torsion Angles (deg) of Ring B in both Parent
Subclasses Compound and Radical Relative to O1
substituents flavonoid molecule radical
class compound 3 5 7 3′ 4′ 5′ quercetin -0.29 -0.19
luteolin 16.29 0.04
flavonols quercetin OH OH OH OH OH H
(+)-catechin 35.64 39.19
fisetin OH H OH OH OH H
apigenin 16.48
rutin ORua OH OH OH OH H
-0.05
diosmin 15.54 0.00
kaempferol OH OH OH H OH H
galangin 0.07
galangin OH OH OH H H H
-0.27
kaempferol 0.00
monoHER ORu OH OEtOH OH OH H
-0.14
taxifolin
flavanon(ol)s naringenin H OH OH H OH H
-27.64 -37.53
3-OMe-quercetin 0.04
hesperetin H OH OH OH OMe H
-23.58
hesperetin
taxifolin OH OH OH OH OH H
-42.28 -41.74
naringenin
flavones apigenin H OH OH H OH H
-42.73 -41.34
rutin 27.17 NDa
diosmin H OH ORu OH OMe H
luteolin H OH OH OH OH H a ND: not determined.
flavanols (+)-catechin OH OH OH OH OH H
anthocyanidins cyanidin OH OH OH OH OH H
a Ru: rutinose ()glucose-rhamnose).

by simply calculating the energy difference between a compound

and its potential radical (∆E ) our ∆∆Hf).2 The relatively
tedious task of searching for and optimizing transition states
can thus be avoided (26). On the basis of these observations,
the molecule radical couple having the lowest ∆∆Hf value in
our calculations will most easily allow hydrogen atom abstrac-
tion by a radical, which is likely to be an important factor in
the scavenging reaction. The resulting flavonoid radical will
then be able to scavenge another radical; thus predictions will
depend at least on two phenomena: ease of hydrogen atom
abstraction (depending on the stabilization energies) and ease
of second radical scavenging (depending on the spin distribution
in the radical). The first process is probably rate limiting; the
second process is a critical one, because a very reactive flavonoid
radical (high amount of localized spin) will be able to start a
radical chain reaction and thus be a pro-oxidant.
Structures which were used for the calculations were quer-
cetin, luteolin, (+)-catechin, taxifolin, rutin, kaempferol, galan-
gin, naringenin, apigenin, diosmin, and hesperetin (Figure 1,
Table 1). Furthermore, a hypothetical structure of quercetin Figure 2. 3-D structures of quercetin (A) and luteolin (B) after
with a 3-OMe moiety was included. Structural variations are optimization using GAMESS.
C2-C3 double bond or saturated ring C, keto group or H at C4,
H, OH, or ORutinose at C3, and different hydroxylation patterns ture of the subclass, see Table 1) this torsion angle was
on ring B, which are the most common structural differences about 20° and thus ring B is not completely conjugated
between flavonoid subclasses. to the rest of the molecule, we investigated whether the
ESR Simulations with Calculated Spin Densities. Spin removal of the 3-OH would induce such a torsion angle.
densities as calculated by GAMESS were converted into a values The 3-OH was removed from Macro Model optimized
by O’Connell’s equation: quercetin, rendering luteolin. It appeared that the
torsion angle of ring B with the rest of the molecule was
a ) QFπ 17° and thus in agreement with flavone, which is a
similar structure, at least in ring C. Comparable results
where a is the hyperfine splitting constant, Q is O’Connell’s
were obtained for galangin, kaempferol, apigenin, and
constant which O’Connell determined to lie between 22.5 and
29 for aromatic compounds, and Fπ is the spin density in the
diosmin, of which compounds with a 3-OH are planar,
π-orbitals of the particular atom to which the hydrogen is and the ones lacking a 3-OH moiety are twisted, meaning
attached (27). The a values are entered into EPRcalc imple- that it can be stated in general that flavonols are planar
mented on a Bruker ESP300 ESR spectrometer for spectrum and that in flavones ring B is slightly ((20°) twisted
simulation. relative to the plane of ring A and C (Figure 2).
In (-)-catechin the 3-OH was found to have an H-bond
Results interaction with the hetero oxygen in ring C. H-bonds
were generally also found between the hydroxyl groups
Calculations. (A) Flavonoids. It was found that the on ring B (the catechol moiety) and between 5-OH and
optimized structure of quercetin was completely planar; 4-keto and 3-OH and 4-keto when appropriate (Figure
i.e., the torsion angle of ring B with the rest of the 3). These hydrogen bonds were indicated by the program
molecule was close to 0° (see Table 2 and Figure 2). This ChemX.
means that the molecule is completely conjugated. As (B) Flavonoid Radicals. The radicals of the flavones
there was one report in the literature (28) stating that were, in contrast to the parent compounds, planar and
in the very similar flavone (no substituents, basic struc- thus completely conjugated. Within a subclass, the same
behavior in geometry change can be observed when going
2 A Brønsted relationship is a linear correlation between ∆H and
r from molecule to radical.
∆Hq. These relationships are observed in a series of similar reactions
and suggest that a constant fraction of effects that stabilize or The ∆∆Hf per molecule radical couple is given in Table
destabilize the reactants or products is present in the transition state. 3 for the situation when there is an H-bond in the
1308 Chem. Res. Toxicol., Vol. 9, No. 8, 1996 van Acker et al.

Figure 3. Hydrogen bonds as indicated by the program

ChemX.

Table 3. Radical Stability Compared with Oxidation

Potential and Influence of a H-Bond in the Catechol
Moiety in Ring B
∆Hf ∆∆Hf mol ∆∆Hf
molecule to rad no H-bond Ep/2
molecule (au)a (kJ/mol) (kJ/mol) (mV)b
quercetin -1097.40 1667.3519 1684.2315 30
luteolin -1022.58 1672.8599 1697.8856 180
(+)-catechin -1024.89 1623.2839 1648.2500 160
apigenin -947.76 1676.9872 NDc >1000
diosmin -1061.59 1676.5389 ND >1000 Figure 5. Spin distribution of the quercetin and taxifolin
galangin -947.12 1683.5924 -c 320 radical.
kaempferol -1022.58 1671.5921 - 120
taxifolin -1098.56 1646.1754 1670.3985 150 radical scavengers because of their excellent delocaliza-
3-OMe-quercetin -1136.40 1675.9057 ND ND tion possibilities. However, it can also be seen that
hesperetin -1062.75 1671.5796 ND 400 delocalization is larger in compounds with a higher
naringenin 1650.3027 600
degree of conjugation, e.g., quercetin vs taxifolin. Ap-
-948.92 -
rutin -1704.41 ND ND 180
a 1 au (atomic unit) ) 2625.5 kJ/mol. b From (29). c Not present;
parently, this small difference in delocalization has a
large influence on antioxidant activity, as these highly
ND: not determined.
conjugated flavonoids have a higher in vitro activity than
their less conjugated counterparts (29).
Electron Spin Resonance. (A) Spectra Obtained
at pH 13. The luteolin spectrum consists of 6 peaks with
intensities 1:3:4:4:3:1 (Figure 6). An explanation is two
overlapping quartets, with the last two peaks of the first
quartet overlapping with the first two peaks of the last
quartet.
Spectra of quercetin are difficult to interpret. At
different scan speeds different spectra were found, indi-
cating that several consecutive reactions occur. Quer-
cetin is easily oxidized, even at physiological pH. Low-
ering the Ep/2 for other parts of the molecule (increasing
the oxidizability) by increasing the pH may give rise to
several different radicals, which are being formed at
different speeds. The spectrum of fisetin is very similar
to that of quercetin. Myricetin gives a triplet caused by
coupling of 2′-H and 6′-H.
The taxifolin spectrum shows two overlapping quartets
Figure 4. Correlation of ∆∆Hf with Ep/2 for quercetin, kaempfer- (Figure 6). It appears that also the 2-H is involved in
ol, luteolin, and galangin. coupling. From the quantum chemical calculations it
appears that this 2-H has a spin density close to that of
catechol moiety in ring B and for the situation when there
2′-H and 6′-H (Figure 5).3 This signal is split by 5′-H
is no H-bond. It can be seen that the H-bond interaction
which has a relatively high spin density. (+)-Catechin
has a large stabilizing effect on the catechol radical.
shows a similar spectrum, consisting of what appears to
When comparing ∆∆Hf and Ep/2, it can be seen that there
be two overlapping quartets, with the last peak of the
is a trend to correlate within each subclass, but only a
first quartet overlapping with the first peak of the last
quantitative correlation for the flavon(ol)s (Figure 4) and
quartet. Interpretation is the same as for taxifolin.
not for the whole group of flavonoids (Table 3). An
Kaempferol shows a spectrum which appears to be two
explanation may be that the Brønsted equation only
overlapping triplets (1:2:2:2:1) (Figure 6). Interpretation
holds within a subclass.
is difficult, as there are 2 × 2 equivalent protons in ring
From the spin distributions (Figure 5) it can be seen
B. Probably at this pH ring B is deprotonated and
that when oxidation takes place in ring B as is usually
oxidation occurs by a different mechanism than at
the case, almost all spin remains in ring B, even in case
physiological pH, as can be seen from the pH dependence
of a completely conjugated molecule such as quercetin.
Surprisingly, about 84% of all spin density remains on 3 Note that under the experimental conditions the catechol moiety
the O from where the H• is removed. This is quite in ring B is deprotonated, which gives rise to an increase in spin on
unexpected, as flavonoids are usually considered good H2′, C4′ and H6′.
Antioxidant Activity of Flavonoids
Figure 6. ESR spectra of luteolin (A), taxifolin (B), and
kaempferol at pH 13 (C) and 7.4 (D).
of the Ep/2 of kaempferol (Figure 7). It appears that at
pH >9, no protons are involved in the reaction, indicating
oxidation by removal of an electron only and not an H•.
The spectrum might be interpreted as 2 equivalent
protons, possibly 6-H and 8-H, and a proton on one of
the hydroxyl moieties in ring A, possibly 5-OH. Another
possibility is an H-bond between 2′-H or 6′-H and 3-OH,
which would also render 2 equivalent and one other
proton. This theory is further discussed below.
Rutin and monoHER show spectra consisting of two
triplets caused by the two equivalent protons 2′-H and
6′-H, and the proton at 5′, which has the highest spin
density.
No spectra could be obtained from all other flavonoids.
Probably their Ep/2 values were too high for them to be
spontaneously oxidized under these conditions. Ep/2
values for the flavonoids which could be oxidized were
<190 mV vs saturated calomel. The anthocyanidins
cyanidin and pelargonidin, which have very low oxidation
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1309
Figure 7. Relationship between Ep/2 and pH for kaempferol.
Figure 8. Influence of Mg2+ on the ESR spectrum of monoHER,
pH 7.4.
potentials (<10 mV), are probably oxidized in several
steps into nonradical products. This process appears to
be too fast to be detected using ESR.
(B) Spectra Obtained at Physiological pH (7.4).
Spectra obtained at physiological pH had usually more
noise than spectra obtained at high pH. In order to
obtain good spectra, sensitivity had to be increased 10-
100-fold. MonoHER shows a spectrum consisting of 2
triplets. Mg2+ causes a slight change in the spectrum,
but the form stays generally the same (Figure 8). Hy-
perfine coupling of protons in ring B is less ()smaller
spin density) due to complexation of Mg2+.
The spectrum of kaempferol shows a triplet, but with
a very poor signal to noise ratio. The triplet was also
obtained when simulating a spectrum with the spin
densities obtained from the quantum chemical calcula-
tions. At this pH, oxidation probably takes place at ring
B. Myricetin shows a singlet with hyperfine splitting.
The quercetin spectrum shows two triplets.
For all other flavonoids no spectra could be obtained
under these conditions, due to low oxidizability.
1310 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
(C) Simulated Spectra. Spectra were simulated
using the spin densities and an O’Connell’s constant of
25 (arbitrarily chosen) for kaempferol, rutin, fisetin, and
luteolin. They were in good agreement with the experi-
mental spectra. A complication, however, is that in
general the best ESR spectra were obtained at pH 13,
whereas the calculations were performed on the neutral
species. For flavonoids containing a catechol moiety in
ring B, it is known that at pH 13 the catechol moiety is
deprotonated, which will probably result in more spin in
positions 2′ and 6′. This would explain why these protons
have larger shifts in the ESR spectra at pH 13 than
would be expected from the calculations (see also under
“Spectra Obtained at pH 13”).
Discussion
Flavonoids are a group of polyphenolic compounds
ubiquitously found in fruits and vegetables. The daily
human intake in the Western countries was recently re-
estimated to be approximately 23 mg/day (30). In very
recent years, flavonoids have gained a tremendous inter-
est as possible therapeutics against a wide variety of
diseases, most of which involve radical damage. Until
now, the mechanism of action and structural require-
ments have not been fully understood.
Numerous authors have investigated the antioxidant
activity of flavonoids, and several attempts have been
made to elucidate the structure-activity relationships.
The investigation of the structure-activity relationships
is hampered by a low solubility in most assays, and thus
solvents such as DMSO and ethanol are needed, which
possess good radical scavenging activities themselves.
Furthermore, as flavonoids are known to be good transi-
tion metal chelators, most lipid peroxidation (LPO)1
inhibition assays measure a combination of transition
metal (usually iron) chelation and radical scavenging.
Although there is general agreement that flavonoids
possess both excellent iron chelating and radical scav-
enging properties (1, 3), there is much discussion and
contradiction regarding their relative contribution and
the SARs on the antioxidant activity.
The molecular properties which we used to investigate
a possible antioxidant mechanism of flavonoids were the
spin density distribution of the radical formed after
subtraction of a hydrogen atom (homolytic dissociation)
and the heat of formation of the radical relative to the
parent compound (∆∆Hf). The latter property is a
measure for the scavenging potential of the compound,
which is related to the measured oxidation potential for
the heterolytic dissociation of the O-H bond, a reaction
which appears to be very similar to the reaction of a
flavonoid with a radical:
flavo-OH f flavo-O• + e- + H+ (1)
(electrochemical oxidation, H+ involvement is demon-
strated by pH dependence of the reaction).
In an earlier study on vitamin E analogues (31), it was
demonstrated that the ∆∆Hf between parent molecule
and corresponding radical correlated well with experi-
mental scavenging parameters and led to an understand-
ing of the physiochemical properties necessary for anti-
oxidant activity. We also showed a good correlation
between Ep/2 and the scavenging activity for flavonoids
(29). In the present investigation, calculated parameters
van Acker et al.
are compared with experimental parameters, such as an
oxidation potential (Ep/2) and ESR data.
From the optimized geometries, it was concluded that
the torsion angle of ring B with the rest of the molecule
is correlated with scavenging activity, due to the in-
creased conjugation, which the planarity offers. For
example, it can be seen that, in rutin, the bulky sugar
moiety causes the loss of coplanarity of ring B with the
rest of the flavonoid. Therefore, rutin cannot use its full
delocalization potential and is less active as a scavenger
than quercetin. From luteolin it appears that even the
loss of the hydrogen bond with 3-OH causes a slight twist
of the ring. Apparently, the gain in delocalization is less
than the steric hindrance of both hydrogen’s. Our results
with luteolin are confirmed by the results of Cody et al.
(28), who found a torsion angle for ring B of 20° for a
flavonoid without a 3-OH (flavone). This might be an
explanation as to why the flavon-3-ols, such as quercetin,
kaempferol, and galangin, are such good antioxidants.
The extra H-bond probably overcomes the steric hin-
drance.
Our ESR data are in general agreement with Kuhnle
et al. (12), except for kaempferol. At high pH, they found
a typical p-benzosemiquinone anion radical (1:4:6:4:1)
which they cannot explain from the intact structure. Our
results merely indicate two overlapping triplets (1:2:2:
2:1; Figure 6). The Ep/2 vs pH dependence indicates that
at pH 13 the mechanism of oxidation is different from
oxidation at pH 7.4 (Figure 7). At high pH no protons
are involved in oxidation. The spectrum can be inter-
preted as 2 equivalent protons and one proton with
somewhat more spin density. This indicates that ring B
is probably not involved in oxidation at high pH as 2 sets
of 2 equivalent protons are present. Alternatives are the
protons in ring A, on C-6 and C-8. The third proton
might then be a proton on one of the hydroxyls in ring
A, which has not been deprotonated yet. A more plau-
sible explanation might be that the proton on 2′ or 6′ is
hydrogen bonded to the 3-OH, as appears to be the case
in quercetin and fisetin (see below). This then removes
so much spin density from this proton that coupling is
too small to be observed. In that case we would also
expect to observe 2 + 1 protons coupling.
When combining the assignment of hyperfine splitting
constants in Kuhnle et al. (12) with our calculations, it
can be concluded that, although the authors suggest that
a2′ and a5′ are equivalent, this must probably be a2′ and
a6′ which have comparable spin densities. Their as-
signed a6′ is more likely to belong to the proton on 5′,
which has a relatively high spin density. When taking
this into consideration, it can be seen that in quercetin
and fisetin (and 5- and 7-OMe-quercetin of Kuhnle) one
of the protons 2′ or 6′ has a hyperfine splitting constant
which is only 50% of that in luteolin and 3-OMe quercetin
(Table 4). This is an interesting observation, as our
calculations suggest an H-bond-like interaction between
the proton on 2′ or 6′ and the 3-OH moiety in flavon-3-
ols, such as quercetin, fisetin, and kaempferol. Com-
pounds which cannot form this H-bond, such as luteolin
and 3-OMe-quercetin, do not show this effect, which can
probably be explained by “leakage” of spin through this
H-bond to the 3-OH group. It also appears that it must
be a quite stable interaction, as it remains intact on the
ESR time scale, which is several minutes.
In saturated flavonoids, such as catechin and taxifolin,
a proton on ring C (on C2) is also involved in coupling.
This is a result of a hyperconjugation interaction between
Antioxidant Activity of Flavonoids Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1311

Table 4. Measured and Calculated a Values 13 the catechol moiety of the flavonoid is deprotonated
molecule a2 a3 a5 a2′ a5′ a6′ and probably also other protons are lost.
This is supported by the observation that, with in-
quercetin 1.45a 2.70a 0.70a
1.05b 2.67b 1.05b creasing pH, the mechanism of electrochemical oxidation
luteolin 1.25a 1.60a 2.60a 1.40a does not change for monoHER, a catechol-containing
0.99b 1.84b 3.26b 1.84b flavonoid, whereas the current at the peak oxidation
15.2c 0.40c 108.2c 3.95c potential at pH >9 is only 50% of that at pH <7.4. It is
cyanidin 1.37a 3.16a 1.21a
(+)-catechin 1.90a 2.13c 1.23a 3.45a 1.08a
supposed that at physiological pH a two-step reaction
1.78b 1.13b 2.43b 1.13b occurs, resulting in sequential loss of two H•s. At high
1.05c 0.43c 111.8c 8.73c pH, when the catechol moiety is deprotonated, only one
fisetin 0.25a 1.40a 2.75a 0.75a H• is lost, resulting in a 50% lower current upon oxida-
1.22b 2.19b 1.22b tion.
apigenin 13.6c 0.73c 107.4c 0.58c
diosmin 0.88c 121.9c 0.35c 59.3c
In contrast to monoHER, kaempferol does show dif-
galangin 16.3c 0.48c 16.6c ferent mechanisms at high and physiological pH. At pH
kaempferol 2.13b 4.26b 2.13b >9 the Ep/2 does not decrease anymore with increasing
0.62c 112.4c 0.62c pH, indicating that at high pH the electrochemical
taxifolin 1.20a 1.15a 3.40a 1.08a oxidation does not involve the release of a proton in
1.30c 1.05b 3.32b 1.05b
0.65c 108.0c 6.80c addition to the electron.
3-OMe-quercetin 1.40a 2.80a 1.40a At physiological pH, oxidation probably occurs at the
hesperetin 0.42c 112.9c 0.30c 65.1c 4′-OH as is indicated by a triplet ESR spectrum, which
myricetin 0.95a 0.95a can be reproduced by simulation with calculated a values.
0.89b 0.89b Another possibility would be the 3-OH in combination
naringenin 1.33c 0.15c 0.18c 116.8c 0.15c
rutin 0.89b 2.35b 0.89b with the double bond, but in that case galangin would
a From Kuhnle et al. (12). a2′ and a5′ are indistinguishably
be expected to show the same behavior, which it does not.
Galangin has a much higher Ep/2 than kaempferol and
assigned for all compounds; a5′ was assigned as a6′ in the original
article. b From ESR measurement in present study. c Calculated does not oxidize to give an ESR spectrum. At high pH,
a values from spin density calculations. kaempferol gives a spectrum consisting of two triplets.
The hypothesis that it is the 4′-OH which is oxidized in
kaempferol and not the 3-OH (thus making the presence
the C2 proton and the π-orbital of the B ring. The of the H-bond very likely) is strengthened by the obser-
magnitude of splitting is, according to Kuhnle et al. (12), vations by Hendrickson et al. (33, 34). They determined
proportional to Fπ (the spin density in the π-orbital) at separate oxidation steps for rings A, B, and C for a
C1′ and the amount of orbital overlap with the proton at number of flavonoids. The 3-OH moiety in a flavonol has
C2. Variation of this coupling is attributed to the motion an oxidation potential around 400 mV vs Ag/AgCl, and
of ring B around the σ bond to the pyran ring. 7-hydroxyflavone has an oxidation potential of 900 mV
Relative values of a and calculated spin densities are vs Ag/AgCl. Therefore, it must be concluded from the
in good agreement with each other. The simulated Ep/2 of kaempferol (120 mV vs SCE) that the basic
luteolin spectrum, for instance, exactly reproduces our structure of the flavonol confides a unique oxidizability
experimental spectrum. to the 4′-OH moiety, probably via the formation of an
From both hyperfine splitting constants and spin H-bond between 2′-H or 6′-H and 3-OH.
densities it can be seen that in the saturated flavans Interpretation of spectra at physiological pH can be
more spin is present in ring B than in the highly aided by the calculated spin densities, although calcula-
conjugated quercetin and fisetin (Table 4 and Figure 5). tions are performed on neutral species. This largely
Other flavonols also have less spin in ring B than the limits the use of calculating spin densities for interpreta-
flavans, but the difference is not as large as for quercetin tion of spectra measured at high pH. Deprotonation
and fisetin. Flavans have a relatively higher a5′ com- leaves extra negative charge on the molecule, which
pared to flavones. This is also in agreement with the largely influences the electron delocalization of the
calculations. radical. This negative charge appears to increase spin
Another interesting observation is the fact that the on C-2′ and C-6′ as coupling of these protons is more
radical is largely localized on the oxygen on 4′ and further evident at high pH.
spin remains mainly in ring B. This is quite unexpected In conclusion, it can be stated that there is a correlation
as it is generally believed that extensive delocalization between Ep/2 and ∆∆Hf, but only for the flavon(ol)s. No
of spin results in high antioxidant activity due to correlation was found for the flavonoids from all sub-
stabilization of the antioxidant radical. It does, however, classes together. This indicates that flavon(ol)s and
agree with our earlier suggested theory that it is mainly flavanes do not have the same fraction of effects that
ring B that determines the antioxidant activity and the stabilize or destabilize the transition state and that the
basic flavonoid structure has only a small influence (29). Brønsted equation does not hold between subclasses, but
The influence of the basic structure increases when only within a subclass. Furthermore, oxidation of the
antioxidant activity of ring B decreases, as is the case in flavonoid takes place in ring B for catechol-containing
kaempferol and galangin, where it can compensate the flavonoids. For other flavonoids, both oxidation site as
decreasing activity, and apigenin, where this is not the well as mechanism depend on the pH at which the
case. oxidation takes place. Finally, from both experimental
It is expected that the ESR assays at pH 13 and 7.4 and calculated data, there are strong indications that the
may result in different species; at pH 7.4 the catechol 3-OH moiety plays an important role in the antioxidant
moiety of a flavonoid is probably protonated, due to loss activity of flavones, via its interaction with ring B
of a more acidic proton in ring A (32). For comparison, through a hydrogen bond. In this way, it “fixates” the
catechol itself is partially deprotonated at pH 7.4. At pH position of ring B in the same plane as rings A and C.
1312 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
This might well be the explanation for the excellent
antioxidant activity of the flavon-3-ols, which are among
the most active flavonoid antioxidants.
Acknowledgment. The authors thank the Depart-
ment of Pharmaceutical Analysis, University of Utrecht,
The Netherlands, for their hospitality and the stimulat-
ing discussions on the subject of electrochemistry. Dr.
G. Golden of Zyma, Nyon, Switzerland, is gratefully
acknowledged for the generous gift of the hydroxyethyl
rutosides. The Netherlands Computer Facilities (NCF)
is gratefully acknowledged for financial support for
computer facilities. The project was further supported
by IKA 92-127 grant awarded by the Dutch Cancer
Society.
Supporting Information Available: Bond lengths of the
functional groups in parent compound and radical (1 page).
Ordering information is given on any current masthead page.
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