Kinetic study of free-radical-scavenging action of flavonoids in homogeneous and aqueous triton X-100 micellar solutions

Article abstract of DOI:10.1021/jp9706745

Kinetic study of free-radical-scavenging action of six kinds of flavonoids (flavone, chrysin, flavonol, apigenin, rutin, and quercetin) has been performed. The second-order rate constants for the reaction of flavonoids with aroxyl (ArO^.) (k_s) and 5,7-diisopropyltocopheroxyl (Toc^.) radical (k_r) have been measured in ethanol, 2-propanol/water (5:1, v/v), and aqueous Triton X-100 micellar solution (5.0 wt %). The rate constants (k_s and k_r) observed for flavone, chrysin, and flavonol are very slow, indicating that the reactivities of 5- and 7-OH groups at A-ring and 3-OH group at C-ring are very weak and almost negligible. Rutin and quercetin with 3′- and 4′-OH groups at B-ring showed high reactivity, indicating that the o-dihydroxyl (catechol) structure in the B-ring is the obvious radical target site for flavonoids. The rate constants (k_s and k_r) obtained in micellar solution showed notable pH dependence. For instance, both the k_s and k_r values of rutin increased with increasing pH value from 4 to 11. Rutin is a tetrabasic acid and can exist in five different molecular forms, depending on the pH value. By comparing the k_s values with the mole fraction (f) of each molecular form of rutin, the reaction rate k_s1 for undissociated form (RuH₄), k_s2 for monoanion (RuH₃^-), k_s3 for dianion (RuH₂^2-), and k_s4 for trianion (RuH^3-) were determined; the values are 9.5 × 10 M^-1 s^-1,4.0 × 10² M^-1 s^-1, 3.8 × 10³ M^-1 s^-1, and 4.0 × 10³ M^-1 s^-1, respectively. The reaction rates (k_si) increase remarkably with increasing the anionic character of rutin, that is, the electron-donating capacity of rutin. It was found that quercetin and rutin have high activity in vitamin E regeneration.

Full text of DOI:10.1021/jp9706745

3746
J. Phys. Chem. A 1997, 101, 3746-3753
Kinetic Study of Free-Radical-Scavenging Action of Flavonoids in Homogeneous and
Aqueous Triton X-100 Micellar Solutions
Kazuo Mukai,* Wataru Oka, Keiko Watanabe, Yoshifumi Egawa, and Shin-ichi Nagaoka
Department of Chemistry, Faculty of Science, Ehime UniVersity, Matsuyama 790-77, Japan
Junji Terao
National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan
ReceiVed: August 25, 1996; In Final Form: February 26, 1997X
Kinetic study of free-radical-scavenging action of six kinds of flavonoids (flavone, chrysin, flavonol, apigenin,
rutin, and quercetin) has been performed. The second-order rate constants for the reaction of flavonoids with
aroxyl (ArO•) (ks) and 5,7-diisopropyltocopheroxyl (Toc•) radical (kr) have been measured in ethanol,
2-propanol/water (5:1, v/v), and aqueous Triton X-100 micellar solution (5.0 wt %). The rate constants (ks
and kr) observed for flavone, chrysin, and flavonol are very slow, indicating that the reactivities of 5- and
7-OH groups at A-ring and 3-OH group at C-ring are very weak and almost negligible. Rutin and quercetin
with 3′- and 4′-OH groups at B-ring showed high reactivity, indicating that the o-dihydroxyl (catechol) structure
in the B-ring is the obvious radical target site for flavonoids. The rate constants (ks and kr) obtained in
micellar solution showed notable pH dependence. For instance, both the ks and kr values of rutin increased
with increasing pH value from 4 to 11. Rutin is a tetrabasic acid and can exist in five different molecular
forms, depending on the pH value. By comparing the ks values with the mole fraction (f) of each molecular
-
form of rutin, the reaction rate ks1 for undissociated form (RuH4), ks2 for monoanion (RuH3 ), ks3 for dianion
(RuH22-), and ks4 for trianion (RuH3-) were determined; the values are 9.5 × 10 M-1 s-1, 4.0 × 102 M-1 s-1,
3.8 × 103 M-1 s-1, and 4.0 × 103 M-1 s-1, respectively. The reaction rates (ksi) increase remarkably with
increasing the anionic character of rutin, that is, the electron-donating capacity of rutin. It was found that
quercetin and rutin have high activity in vitamin E regeneration.
Introduction
methoxyphenyl)phenoxyl (ArO• (abbreviated to aroxyl), see
Figure 1) in ethanol solution (reaction 1), using stopped-flow
spectrophotometry:12,13
Flavonoids are phenol derivatives widely distributed in plants.
Flavonoids possess high antioxidant and free-radical-scavenging
activity in foods and plants.1-3 For instance, flavonoids have
been compared in a dose-response manner with vitamin C and
E and â-carotene and found to be powerful antioxidants using
an in vitro lipoprotein oxidation model.4,5 Several kinetic studies
have been performed for the reaction of flavonoids with active
ks
9
ArO• + TocH
8 ArOH + Toc•
(1)
kinh
LOO• + TocH 8 LOOH + Toc•
(2)
The second-order rate constants (ks) obtained are 5.12 × 103
(R-TocH), 2.24 × 103 (â-TocH), 2.42 × 103 (γ-TocH), and 1.00
× 103 (δ-TocH) M-1 s-1 in ethanol at 25.0 °C. The relative
rates (R:â:γ:δ ) 100:44:47:20) agree well with those obtained
from studies of the reactivities of tocopherols toward poly-
(peroxystyryl)peroxyl radicals (100:41:44:14) by the O2 con-
sumption method (reaction 2).14,15 The result suggests that the
relative reactivities of tocopherols in solution probably do not
depend on the kinds of oxyradicals (ArO• and LOO•) used.12,13
Recently, Foti et al.16 measured the reaction rates between
the phenoxyl radical and some natural phenolic antioxidants
including R-, γ-, and δ-tocopherol (reaction 3).
•
free radicals, such as N3 , HO•, O2•-, t-BuO•, and LOO•, by the
pulse radiolysis technique.6-9 It has been reported that fla-
vonoids may act as efficient scavengers of these active free
radicals. However, the above kinetic studies have been gener-
ally performed at relatively high pH region (pH ) 11.5 and
10). Owing to the various dissociable phenolic hydroxyl groups
in flavonoids, it is expected that the reaction rates between
flavonoids and active free radical show notable pH dependence.9
Therefore, it is necessary to measure the free-radical-scavenging
rates of flavonoids at various pH in aqueous solution and in
organic solvents, in order to discuss the antioxidant activity of
the flavonoids in foods and biological systems. It was reported
that quercetin, epicatechin, and epicatechin gallate can act as a
chain-breaking antioxidant in the hydroperoxidation of methyl
linoleate and their peroxyl-radical-scavenging rates (kinh) are
5-20 times smaller than that of R-tocopherol in n-hexane/2-
propanol (1:1, v/v) solution.10,11 However, it is still obscure
how these flavonoids contribute to the inhibition of lipid
peroxidation.
The phenoxyl radical has been found to be roughly 100-
300 times more reactive than peroxyl radicals, i.e., k3
∼
[(1-3) × 102]kinh for the same phenolic antioxidants. The
relative reactivities obtained for tocopherols were R:â:γ )
100:29:6 in CH3CN at 20 °C. Such a high reactivity of
phenoxyl radical is interesting, because, for example, the
reactivity of the phenoxyl radical is expected to be similar to
that of the tyrosyl radical, as they described.
In previous works, we measured the reaction rates (ks) of R-,
â-, γ-, and δ-tocopherols (TocH’s) with 2,6-di-tert-butyl-4-(4-
k3
9
PhO• + TocH
8 PhOH + Toc•
(3)
In the present work, in order to clear the structure-activity
relationship in the scavenging reaction of free radical by
* To whom correspondence should be addressed.
X Abstract published in AdVance ACS Abstracts, May 1, 1997.
S1089-5639(97)00674-9 CCC: $14.00 © 1997 American Chemical Society

Free-Radical-Scavenging Action of Flavonoids
J. Phys. Chem. A, Vol. 101, No. 20, 1997 3747
Figure 2. Change in electronic absorption spectrum of aroxyl radical
(ArO•) during reaction of ArO• with rutin in ethanol solution at 25.0
°C. [ArO•]t)0 ) ∼0.049 mM, and [rutin]t)0 ) 0.388 mM. The spectra
were recorded at 7550 ms intervals. The arrow indicates a decrease in
absorbance with time.
Figure 1. Molecular structures of six kinds of flavonoids, aroxyl radical
(ArO•) and 5,7-diisopropyltocopheroxyl (Toc•).
flavonoids, we have measured the second-order rate constants
(ks and kr) for the reaction of six kinds of flavonoids (flavone,
chrysin, flavonol, apigenin, rutin, and quercetin) with aroxyl
radical (ArO•) and 5,7-diisopropyltocopheroxyl (Toc•) radicals
(see Figure 1) in ethanol, 2-propanol/water (5:1, v/v), and
aqueous Triton X-100 micellar solution (5.0 wt %; reactions 4
and 5).17,18 The rate constants obtained in micellar solution were
pH dependent because of the dissociation of various phenolic
hydroxyl groups in the flavonoids. The observed rates, ks and
kr, were compared to those of R-tocopherol, ubiquinol-10, and
vitamin C (L-ascorbic acid), which are well-known as most
popular biological antioxidants.
Figure 3. Dependence of pseudo-first-order rate constant (kobsd) on
concentration of rutin in ethanol.
micellar) solutions of flavonoids and ArO• (or Toc•) under
nitrogen atmosphere. If the reaction rates (ks and kr) were faster
than 1 M-1 s-1, the measurements of rate constant were
performed by using an Unisoku Model RS-450 stopped-flow
spectrophotometer. All measurements were performed at 25.0
( 0.5 °C.
Results
ks
9
Aroxyl-Radical-Scavenging Rate (ks) of Flavonoids in
Ethanol Solution. Measurements of the rate constant (ks) for
the reaction of aroxyl radical (ArO•) with flavonoids were
performed in ethanol solution at 25.0 °C. Figure 2 shows an
example of the interaction between ArO• (∼0.049 mM) and rutin
(0.388 mM) in ethanol solution. The pseudo-first-order rate
constant (kobsd) was obtained by following the decrease in
absorbance at 580 nm of the ArO• radical. The details of the
experiments were reported in a previous paper.12 ArO• radical
shows a slow natural decay in ethanol solution. Therefore, the
rate constant (kobsd) for ArO• bleaching is given by
ArO• + flavonoid
Toc• + flavonoid
8 ArOH + flavonoid•
(4)
(5)
kr
9
8 TocH + flavonoid•
Experimental Section
Flavone, chrysin, flavonol, apigenin, rutin, and quercetin are
commercially available. Aroxyl radical (ArO•) was prepared
according to the method of Rieker et al.19 The 5,7-diisopro-
pyltocopheroxyl (Toc•) radical is fairly stable and was prepared
by PbO2 oxidation of the corresponding 5,7-diisopropyltocol
in ethanol or in 2-propanol/water (5:1, v/v) solutions under a
nitrogen atmosphere.20,21 In the case of the reaction in micellar
solution, Toc• radical was prepared by the reaction between the
ArO• radical and 5,7-diisopropyltocol in aqueous Triton X-100
micellar solution (5.0 wt %) at 25 °C and was reacted
immediately with Triton X-100 micellar solution (5.0 wt %) of
flavonoids.17,18
kobsd ) k0 + ks[rutin]
(6)
where k0 is the rate constant for the natural decay of ArO• in
the medium, and ks is the second-order rate constant for the
reaction of ArO• with rutin. These parameters are obtained by
plotting kobsd against [rutin] (see Figure 3).22 The second-order
rate constant (ks) obtained for rutin is 1.42 × 10 M-1 s-1
.
The kinetic data were obtained with a Shimadzu UV-2100S
spectrophotometer by mixing equal volumes of ethanol (or
2-propanol/water (5:1, v/v), or aqueous 5.0 wt % Triton X-100
Similar measurements were performed for the reaction of
ArO• with flavonoids in ethanol solution. The values of ks
obtained are listed in Table 1, together with those obtained for

3748 J. Phys. Chem. A, Vol. 101, No. 20, 1997
Mukai et al.
dant effect of R-tocopherol:21,31-33
TABLE 1: Second-Order Rate Constants (ks and kr) for the
Reaction of ArO• and Toc• with Flavonoids in Ethanol and
2-Propanol/Water (5:1, v/v) Solutions at 25.0 °C
kp
Toc• + LH
9
8 TocH + L•
(7)
e
e
ks (M-1 s-1
ArO•
)
kr (M-1 s-1
)
Toc•
The reaction rate constant (kp) between Toc• and methyl linoleate
is 1.26 × 10-2 M-1 s-1 in ethanol. The kr values of rutin and
quercetin are 1.03 × 10 and 2.98 × 102 M-1 s-1, respectively,
and are about from 3 to 4 orders of magnitude larger than that
(kp) of methyl linoleate. The result suggests that rutin and
quercetin may contribute to a tocopheroxyl-radical-scavenging
reaction in biological systems. In fact, suppression of the
R-tocopherol consumption by flavonoids was previously re-
ported in the case of oxidative modification of human low-
density lipoprotein treated with macrophage or metal ion.34,35
Further, the flavonoids in wine are hypothesized to act
synergistically with tocopherol to inhibit lipid peroxidation.36
pH Dependence of the Aroxyl- and Tocopheroxyl-Radical-
Scavenging Rates (ks and kr) of Flavonoids in Aqueous
Triton X-100 Micellar Solution. Measurements of the rate
constant (ks) for the reaction of ArO• with flavonoids were
performed at various pH values in aqueous Triton X-100
micellar solution (5.0 wt %). The problem with flavonoid
aglycones is their poor solubility in aqueous Triton X-100
micellar solution (see Table 2). Flavone is insoluble in Triton
X-100 micellar solution. Chrysin and flavonol are also insoluble
in Triton X-100 micellar solution at pH <10 and <11,
respectively. These compounds are soluble at higher pH region,
because of the dissociation of phenolic hydroxyl groups in the
molecule. The ks values of chrysin are <10-2 M-1 s-1 at pH
10 and 11, and the ks value of flavonol is 4.89 × 10-1 M-1 s-1
at pH 11. The aroxyl-radical-scavenging rates of chrysin and
flavonol are very slow in micellar solution, as observed for the
reaction in homogeneous solution. Quercetin having five OH
groups is also insoluble in micellar solution at pH ) 7, and
soluble at pH 8 increasing the ionic character of the molecule
at higher pH region. However, the absorption spectra of ArO•
(λmax ) 580 nm) and quercetin aroxyl radical (λmax ) 530 nm)
which is produced by the reaction between ArO• and quercetin
overlap each other,7 and we were unsuccessful in determining
the reaction rate.
On the other hand, rutin having sugar substituent at 3-position
is soluble in water, and we have succeeded in measuring the
rate constant (ks) at wide pH region between pH 3.5 and 11.
The ks values obtained are summarized in Tables 2 and 3,
together with those obtained for R-tocopherol, ubiquinol-10, and
BHT. Where pH is <3 or >11, ArO• radical and/or flavonoids
are unstable, and the measurement of the ks value was
unsuccessful. The pH dependence of the second-order rate
constants (ks) of rutin is shown in Figure 4. By increasing pH
values, the ks of rutin remains constant (ks ) 9.5 × 10 M-1
s-1) between pH 4.0 and 5.0, increases gradually from 1.17 ×
102 M-1 s-1 at pH 6.0 to 4.14 × 102 M-1 s-1 at pH 8.0, and
then increases rapidly from 5.32 × 102 M-1 s-1 at pH 8.25 to
3.89 × 103 M-1 s-1 at pH 11.0 (see Figure 4 and Table 3).
This pH dependence reflects a complex mechanism that will
be discussed later. Similarly, the pH effect on the reaction
between flavonoids and Toc• radical was studied. As listed in
Table 2, the tocopheroxyl-radical-scavenging rates (kr) of chrysin
and flavonol obtained at pH ) 11 are very slow. As shown in
Figure 5, the reaction rate (kr) of rutin also increases with
increasing pH value from 3.5 to 9.0, as observed for the reaction
between rutin and ArO• radical. The kr value at pH 9.0 (kr )
3.02 × 103 M-1 s-1) is about 3 orders of magnitude larger than
that at pH 3.5 (kr ) 3.60 M-1 s-1), as listed in Table 3. Visible
absorption of Toc• (λmax ) 417 nm) overlaps with that of
flavonoids
flavone
chrysin
flavonol
apigenin
rutin
quercetin
BHT
R-tocopherol
ubiquinol-10
Na+AsH-
methyl linoleate
ethanol
ethanol
iPrOH/H2O (5:1, v/v)
<10-2
<10-2
<10-2
∼10-2
6.25 × 10-1
a
∼10-2
∼10-1
5.60 × 10-2 6.83 × 10-1
4.73 × 10-1
1.42 × 10
b
a
1.03 × 10
7.40
2.98 × 102
2.93 × 102
3.5 × 10
5.12 × 103 c
5.19 × 103
insoluble
3.64 × 104 d
insoluble
5.33 × 104 d
6.32 × 104
1.26 × 10-2
a Absorption spectrum of Toc• radical overlaps with that of apigenin
aroxyl radical. b Absorption spectrum of ArO• radical overlaps with
that of quercetin aroxyl radical (see refs 7 and 8). c See ref 20. d See
ref 18. e Experimental errors <(7%.
R-tocopherol, ubiquinol-10, and BHT, which are well-known
as popular lipid-soluble antioxidants. However, in the case of
quercetin, the absorption spectra of ArO• (λmax ) 580 nm) and
quercetin aroxyl radical (λmax ) 530 nm) which is produced by
the reaction between ArO• and quercetin overlap each other,7
and we were unsuccessful in determining the reaction rate.
As is clear from the ks values listed in Table 1, the rate of
the scavenging reaction of ArO• with the above flavonoids
increases in the order of flavone < chrysin < flavonol <
apigenin < rutin. Rutin has the highest scavenging activity
among these flavonoids. The rate constant of rutin (ks ) 1.42
× 10 M-1 s-1) is similar to that of BHT (ks ) 3.5 × 10 M-1
s-1). However, the ks value of rutin is approximately 2 orders
of magnitude smaller than that of R-tocopherol (ks ) 5.12 ×
103 M-1 s-1), which has the highest free-radical-scavenging
activity among natural phenolic antioxidants.15
Rates (kr) of Vitamin E Regeneration Reaction with
Flavonoids in Ethanol and 2-Propanol/Water (5:1, v/v)
Solutions. Measurements of the rate constant (kr) for the
reaction of Toc• with flavonoids were performed in ethanol and
iPrOH/H2O (5:1, v/v) solutions at 25 °C. The rate was measured
by following the decrease in absorbance at 420 nm of the Toc•
radical.17,18,21 In the case of quercetin, visible absorption of
Toc• at 420 nm overlaps with that of quercetin and quercetin
aroxyl radical,7 and thus the rate constant was determined by
analyzing the increase in absorbance at 560 nm of quercetin
aroxyl radical which is produced by the reaction of Toc• with
quercetin. The kr values obtained are summarized in Table 1,
together with those obtained for ubiquinol-10 and sodium
ascorbate anion (Na+AsH-). The reactions of R-tocopheroxyl
with vitamin C (ascorbate) and ubiquinol-10 are well-known
as the usual tocopherol regeneration reaction in biomembrane
systems.17,18,24-30
The kr values of flavonoids obtained in ethanol are similar
to corresponding those obtained in PrOH/H2O (5:1, v/v)
i
solution. As observed for the reaction between ArO• and
flavonoids, the kr values increase in the order of flavone <
chrysin < flavonol < apigenin < rutin < quercetin in both the
solutions. Quercetin has the highest scavenging activity among
these flavonoids. However, the rate constant (kr) of quercetin
is approximately 2 orders of magnitude smaller than those of
sodium ascorbate and ubiquinol-10.
It was reported that the hydrogen abstraction reaction (reaction
7) from unsaturated lipids, such as methyl linoleate and methyl
linolenate, by R-tocopheroxyl radical may relate to the prooxi-

Free-Radical-Scavenging Action of Flavonoids
J. Phys. Chem. A, Vol. 101, No. 20, 1997 3749
TABLE 2: Second-Order Rate Constants (ks and kr) for the Reaction of ArO• and Toc• with Flavonoids in Triton X-100
Micellar Solution (5.0 wt %) at 25.0 °C
ks (M-1 s-1)c (ArO•)
flavonoids
pH 7
pH 8
pH 9
pH 10
<10-2
insoluble
2.94 × 103
a
pH 11
chrysin
flavonol
rutin
quercetin
BHT
R-tocopherol
ubiquinol-10
insoluble
insoluble
2.28 × 102
insoluble
8.62 × 103
4.53 × 105
1.25× 105
insoluble
insoluble
4.14 × 102
a
insoluble
insoluble
2.65 × 103
a
<10-2
4.89 × 10-1
3.89 × 103
a
kr (M-1 s-1)c (Toc•)
chrysin
flavonol
rutin
quercetin
ubiquinol-10
ascorbic acid
methyl linoleate
insoluble
insoluble
insoluble
insoluble
3.02 × 103
1.14 × 105
2.47 × 103
∼10-2
∼10-2
∼3
insoluble
insoluble
insoluble
5.48 × 102
insoluble
1.64 × 103
b
b
3.73 × 104
3.38 × 105
9.24 × 105
2.49 × 103
2.40 × 10-2
2.56 × 103
2.30 × 103
a Absorption spectrum of ArO• radical overlaps with that of quercetin aroxyl radical (see refs 7 and 8). b Absorption spectrum of Toc• radical
overlaps with that of rutin aroxyl radical. c Experimental errors <(7%.
TABLE 3: pH Dependence of the Second-Order Rate
Constants (ks and kr) for the Reaction of Flavonoids with
Aroxyl (ArO•) and Tocopheroxyl (Toc•) Radicals in Triton
X-100 Micellar Solution at 25.0 °C
ks (M-1 s-1
ArO•
)
kr (M-1 s-1
Toc•
)
pH
rutin
rutin
3.60
4.33
5.04
quercetin
AsA
3.5
4.0
4.5
4.75
5.0
6.0
6.5
6.75
6.8
6.9
7.0
7.25
7.3
7.4
7.5
8.0
8.25
8.5
9.0
9.25
9.5
9.48 × 10
7.98 × 102
1.05 × 102
8.92 × 10
9.43 × 10
1.17 × 102
1.58 × 102
8.27
1.56 × 103
2.26 × 103
7.36 × 10
1.43 × 102
1.70 × 102
4.45 × 102
5.82 × 102
5.48 × 102
5.89 × 102
8.07 × 102
1.16 × 103
2.28 × 102
2.49 × 103
Figure 4. Plots of second-order rate constant (ks) for rutin (open circle)
-
versus pH and of mole fraction (f) of four rutin species (RuH4, RuH3
,
3.99 × 102
4.14 × 102
5.32 × 102
9.69 × 102
2.65 × 103
2.82 × 103
2.82 × 103
3.15 × 103
2.94 × 103
3.15 × 103
3.73 × 103
3.89 × 103
RuH22-, and RuH3-) versus pH (solid line).
1.64 × 103
3.73 × 104
2.56 × 103
2.47 × 103
2.04 × 103
1.01 × 105
Flavonol with 3-OH group at the C-ring shows weak reactivity.
These results clearly indicate that the 5- and 7-OH groups at
the A-ring and 3-OH group at the the C-ring do not contribute
to the free-radical-scavenging action by flavonoids. The reaction
rate of apigenin with 4′-OH group at B-ring is 4.73 × 10-1
M-1 s-1. This value is about 1 order of magnitude larger than
that of flavonol, suggesting that the 4′-OH group at the B-ring
contributes to the free-radical scavenging. On the other hand,
the ks value obtained for rutin which has 3′-OH and 4′-OH
groups at the B-ring is 30 times larger than that of apigenin.
The result indicates that the o-dihydroxyl (catechol) structure
in the B-ring is the obvious radical target site for rutin.
3.02 × 103
1.14 × 105
2.67 × 105
3.38 × 105
9.75
10.0
10.25
10.5
11.0
2.30 × 103
quercetin and quercetin aroxyl radical,7,8 and thus the kr value
for quercetin was determined by analyzing the increase in
absorbance at 560 nm of quercetin aroxyl radical. The second-
order rate constants (kr) of quercetin also increased with
increasing pH value from 8 to 10 (see Figure 6).
Similar behavior was observed for the reaction between Toc•
i
and flavonoids in ethanol and PrOH/H2O (5:1, v/v) solutions
(see Table 1), indicating that the existence of catechol structure
in B-ring is essential for the free-radical-scavenging of the
flavonoids.
Both quercetin and rutin, which is quercetin rutinoside at
3-position, have OH substituents at 3′-, 4′-, 5-, and 7-positions,
and we can expect similar rate constants (kr) for these flavonoids.
However, the kr values of rutin are 30-40 times smaller than
those of quercetin in homogeneous and micellar solutions as
listed in Tables 1 and 2. The π conjugation between the B-
Discussion
Structure-Activity Relationship of the Free-Radical-
Scavenging Reaction by Flavonoids in Homogeneous Solu-
tion. As listed in Table 1, the ks values obtained are less than
10-2 M-1 s-1 for flavone, about 10-2 M-1 s-1 for chrysin, and
5.60 × 10-2 M-1 s-1 for flavonol. Flavone having no OH
substituent shows no reactivity. The reactivity of chrysin with
two OH groups at A-ring is very weak and almost negligible.

3750 J. Phys. Chem. A, Vol. 101, No. 20, 1997
Mukai et al.
measurements in micellar solution increase in the order of
chrysin < flavonol , rutin < quercetin independent of pH
value, as listed in Table 2. On the other hand, in ethanol,
flavonoids will take undissociated forms, differing from those
in aqueous solution. However, the ks and kr values of flavonoids
observed in ethanol solution also increase in the order of flavone
< chrysin < flavonol < apigenin < rutin < quercetin. These
results indicate that the above determinants (i) and (ii) reported
by Bors et al. are important for free-radical scavenging in
flavonoids independent of both pH value and solvent system.
It seems that the existence of the 3-OH group at the C-ring and
the 5- and 7-OH groups at the A-ring is not necessary for radical-
scavenging, if the transition-metal ions such as Cu+ and Fe2+
ions are not included in the reaction system. The ability of
flavonoids to form complexes with metal ions relates to the
antioxidant action of flavonoids.39,40 Chelation of metal ions
renders them catalytically inactive. Recently, similar results
were obtained from the investigation of peroxyl-radical-
scavenging activities of quercetin and its monoglucosides at the
3-, 7-, and 4′-positions in n-hexane/2-propanol (1:1, v/v)
solution.11
Figure 5. Plots of second-order rate constant (kr) for the reaction of
5,7-diisopropyltocopheroxyl with rutin (open circle) and ascorbic acid
(closed circle) versus pH and of mole fraction (f) of four rutin species
versus pH (solid line).
Recently, Hendrickson et al.41 found that the effect of
flavonoids on microsomal phenol hydroxylase activity correlates
well with the oxidation potential (Ep) for flavonoid aglycones;
the flavonoids which have smaller Ep values show higher
inhibitions of phenol hydroxilase activity. Further, the correla-
tion between the Ep values of the flavonoids and their log IC50
of the doxorubicin induced lipid peroxidation has been reported
by Acker et al.37 As reported in previous works,13,20,42 the rate
constants of scavenging of ArO• (ks) by phenolic antioxidants
increased as the total electron donating capacity of the methyl
groups at the aromatic ring increased. A plot of log ks vs peak
oxidation potential (Ep) was found to be linear and the slope
was negative. As described above, the ks and kr values of
flavonoids increase in the order of chrysin < flavonol <
apigenin < rutin < quercetin independent of both pH value and
solvent system. On the other hand, the Ep values of the above
flavonoids reported by Hendrickson et al.41 and Acker et al.37
decrease in the order of chrysin > apigenin > flavonol > rutin
> quercetin. The result suggests that the flavonoids which have
smaller Ep values show higher free-radical-scavenging activity
and thus higher biological activity.
Figure 6. Plots of second-order rate constant (kr) for the reaction of
5,7-diisopropyltocopheroxyl with quercetin (open circle) and ascorbic
acid (closed circle) versus pH and of mole fraction (f) of four quercetin
species versus pH (solid line).
and C-rings in quercetin will decrease in rutin, because the
B-ring of rutin is considered to twist much more than that of
quercetin by the steric repulsion between the 6′- (or 2′-) ring
proton at the B-ring and rutinose group. In such a case, the
oxidation potential of rutin increases and thus the kr value will
decrease, as observed for the reaction of ArO• radical with many
phenolic antioxidants.13,20 In fact, the value of the peak
oxidation potential (Ep) of rutin (180 mV vs SCE) is higher
than that of quercetin (30 mV).37
Structure-Activity Relationship of the Free-Radical-
Scavenging Reaction by Flavonoids in Aqueous Micellar
Solution. In a previous work, a kinetic study of the reaction
between vitamin C (L-ascorbic acid, AsA) and a tocopheroxyl
radical (7-tert-butyl-5-isopropyltocopheroxyl) in aqueous Triton
X-100 micellar solution has been performed using stopped-flow
spectrophotometry.17 The second-order rate constants kr ob-
tained showed a notable pH dependence with a broad maximum
around pH 8. A good correlation between the rate constants
and the mole fraction of ascorbate monoanion was observed,
showing that ascorbate can regenerate the tocopherol from
tocopheroxyl in biological systems. Furthermore, the results
indicated that the undissociated form of ascorbic acid does not
have the ability to regenerate the tocopherol in aqueous solution.
Bors et al.6-8,38 measured the second-order rate constants for
•
the reaction of flavonoids with HO•, N3 , and tBuO• radicals at
pH 11.5, using pulse radiolysis technique, and reported that three
structural groups in flavonoids are important determinants for
radical scavenging: (i) the 3′- and 4′-OH groups (catechol
structure) in the B-ring, which are the obvious radical target
site for all flavonoids; (ii) the 2,3-double bond in conjugation
with a 4-oxo function, which is responsible for electron
delocalization from the B-ring; (iii) the existence of both 3-
and 5-OH groups for maximal radical-scavenging activity.
Owing to the various dissociable phenolic hydroxyl groups
In this work, the rates of reaction (ks and kr) of rutin with
ArO• and Toc• in Triton X-100 micellar solutions have been
measured by varying pH value. The observed second-order rate
constants (ks and kr) increased with increasing pH value (see
Figures 4 and 5).
•
t
in flavonoids, the above active free radicals (HO•, N3 , and -
BuO•) react with phenolate anions at pH 11.5 rather than with
undissociated phenols. The ks and kr values obtained by the
Rutin is tetrabasic (see Figure 1) and can exist in five different
molecular forms, i.e., undissociated form (RuH4), monoanion

Free-Radical-Scavenging Action of Flavonoids
J. Phys. Chem. A, Vol. 101, No. 20, 1997 3751
(RuH3-), dianion (RuH22-), trianion (RuH3-), and tetraanion
(Ru4-), depending on the pH value. The equilibrium reactions
have the form:
Ka1
Ka2
Ka3
Ka4
RuH4 y z RuH3- y z RuH22- y z RuH3- y z Ru4- (8)
The pKa1, pKa2, and pKa3 values of rutin have been reported by
Jovanovic et al.9 The values are pKa1 ) 7.1, pKa2 ) 9.15, and
pKa3 ) 11.65. Therefore, the mole fractions (f) present as the
RuH4 molecule and the RuH3-, RuH22-, and RuH3- ions were
calculated as a function of pH.17 The analytical concentration
(Ca) is given in
-
Ca ) [RuH4] + [RuH3 ] + [RuH22-] + [RuH3-] (9)
The contribution from tetraanion (Ru4-) was neglected in eq 9,
because the pKa4 value is not reported. Mole fractions (f) present
as RuH4, RuH3-, RuH22-, and RuH3- are shown as functions
of pH in Figure 4. The result suggests that the reaction rates ks
increase with increasing the degree of dissociation in rutin.
If we assume that the ks1, ks2, ks3, and ks4 are the reaction
rates for undissociated (RuH4), monoanion (RuH3-), dianion
(RuH22-) and trianion (RuH3-) form of rutin, respectively, the
total rate ks will be expressed as
Figure 7. Plots of second-order rate constant (ks) for rutin (open circle)
versus pH and simulation curve (solid line).
tively. Therefore, as shown in Figure 8, the dissociation of rutin
is considered to proceed in the order of 7-OH, 3′-OH, 5-OH,
4′-OH, by increasing the pH value. The o-dihydroxyl (catechol)
structure in the B-ring is the obvious radical target site for
2-
rutin.6-9,11 It is reasonable that the ks3 value for RuH2 form
-
ks ) ks1f(RuH4) + ks2f(RuH3 ) + ks3f(RuH22-) +
-
is about 10 times larger than ks2 value for RuH3 form, as the
ks4f(RuH3-) (10)
dissociation in 3′-OH group at the B-ring proceeds at this stage.
Further, it is also reasonable that RuH22- and RuH3- forms show
rate constant similar to each other, because the B-ring takes
the same ionic structure in both the forms.
By comparing the observed pH dependence of ks with the pH
dependence of mole fraction, the values of ksi were deter-
mined: for instance, at pH 4.0 only the undissociated form of
rutin exists in solution, that is, f(RuH4) ) 1, and we can
immediately determine the ks1 value. At pH 6.5, both the
undissociated and mono anion form exist in solution, and the
mole fractions are f(RuH4) ) 0.799 and f(RuH3-) ) 0.201.
Consequently, we can determine the ks2 value, using eq 10.
Similarly, the ks3 value was determined. The ks1, ks2, and ks3
values obtained for three molecular forms of rutin are 9.5 ×
10, 4.0 × 102, and 3.8 × 103 M-1 s-1, respectively. The ks3
value is 40 times larger than the ks1 value. The result indicates
that the reaction rate ksi increases by increasing the anionic
character of rutin, that is, the electron-donating capacity of rutin.
By using these ks1, ks2, and ks3 values and by varying the ks4
value, we simulated the experimental data. As shown in Figure
7, good accordance between the observed rate constants ks and
theoretical curve was obtained for the ks4 value of 4.0 × 103
M-1 s-1, suggesting that each reaction rate estimated is
reasonable.
As is clear from the results shown in Figure 4 and listed in
Table 3, the ks of rutin increases rapidly from 5.32 × 102 M-1
s-1 at pH 8.25 to 3.89 × 103 M-1 s-1 at pH 11.0. A good
correlation between the rate constants (ks) and mole fraction (f)
of dianion form (RuH22-) was observed. The result shows that
the dianion (RuH22-) of rutin mainly contributes to the scaveng-
ing of free radical at this pH region (pH 8-11). On the other
hand, at lower pH region (7 < pH < 8) the undissociated form
(RuH4) and the monoanion (RuH3-) also contribute to the
scavenging of free radical.
A similar pH dependence of kr value was observed for the
reaction between tocopheroxyl and rutin, as shown in Figure 5.
At pH 3.5, only the undissociated form (RuH4) of rutin exists
in solution, and the kr1 value was immediately estimated to be
3.6 M-1 s-1. However, as is clear from the results shown in
Figure 5, the rapid increase of kr value does not correlate well
with the mole fraction (f) of the dianion (RuH22-) of rutin. If
each pKai value of rutin decreases by about 1 in this reaction,
a good accordance between the increase of kr and the increase
of mole fraction (f) of dianion form of rutin will be observed.
The reason for such a deviation is not clear at present.
As shown in Figure 6, the rate constant (kr) of quercetin also
showed notable pH dependence. Using the values of pKa1
)
6.74, pKa2 ) 9.02, and pKa3 ) 11.55 for quercetin,9,44 the mole
3-
fractions (f) of QuH5, QuH4-, QuH32-, and QuH2 forms of
quercetin were calculated as a function of pH. A good
correlation between the rate constants and the mole fraction (f)
of dianion (QuH32-) of quercetin was observed, as shown in
Figure 6. The result shows that dianion form of quercetin can
regenerate the tocopherol from tocopheroxyl, as found for the
reaction between ArO• and rutin. The reaction rate (kr3) for
dianion form (QuH32-) of quercetin was estimated to be about
4.0 × 105 M-1 s-1, assuming that the kr1 and kr2 values are
small compared to kr3 and negligible, and kr3 equals kr4 for
trianion form (QuH23-).
Comparison between Rates of Vitamin E Regeneration
Reaction with Flavonoids and Vitamin C in Aqueous Triton
X-100 Micellar Solution. To compare the reaction rates (kr)
obtained for rutin and quercetin with those for vitamin C (L-
ascorbic acid), pH dependence of tocopheroxyl-radical-scaveng-
ing rate (kr) of vitamin C has been measured in aqueous Triton
X-100 micellar solution (5.0 wt %). The second-order rate
constants (kr) obtained showed notable pH dependence with a
broad maximum around pH 8, as shown in Figure 5.17 For
instance, the kr values obtained are 7.98 × 102 M-1 s-1 at pH
The pKa values of several flavonoids and a series of
substituted catechols were reported by Slabbert.43 By comparing
the pKa values of flavonoids with those of substituted catechols,
A
the pKa values of 7- and 5-OH groups at the A-ring in
flavonoids are estimated to be 6.74-7.07 and 11.55, respec-
tively. Further, the pKaB values of 3′- and 4′-OH groups at the
B-ring are reported to be 8.77-9.02 and 13.20-13.25, respec-

3752 J. Phys. Chem. A, Vol. 101, No. 20, 1997
Mukai et al.
Figure 8. Five different molecular forms (RuH4, RuH3-, RuH22-, RuH3-, and Ru4-) of rutin in aqueous solution and their reaction rates, ksi.
4.0, 2.49 × 103 M-1 s-1 at pH 7.0, and 2.45 × 103 M-1 s-1 at
pH 9.0. As shown in Figure 5 and as listed in Table 3, the
rates of vitamin E regeneration reaction with rutin are much
slower than corresponding those of ascorbic acid at the low-
pH region (pH 4-6). However, the rate is similar to each other
at around pH 7-9. Further, the rate constants (kr) obtained for
quercetin are 15 and 150 times larger than those of ascorbic
acid at pH 8.0 and 10.0, respectively, as shown in Figure 6 and
as listed in Table 3. The reaction rate (kp) between Toc• and
methyl linoleate (reaction 6) is 2.4 × 10-1 M-1 s-1 in micellar
solution. The kr values of rutin and quercetin are much faster
than that (kp) of methyl linoleate. Especially, quercetin has high
activity in vitamin E regeneration. However, the tocopheroxyl-
radical-scavenging rates (kr) of rutin and quercetin are smaller
than that (kr ) 9.24 × 105 M-1 s-1 at pH ) 7.0) of ubiquinol-
10, which is also well-known as vitamin E regeneration
compounds in biomembrane systems.18,27-30
The structure-activity relationship in the scavenging reaction
of free radical by flavonoids in solution has been discussed. It
has been found that quercetin and rutin have high activity in
vitamin E regeneration. The results of the present kinetic study
should provide a foundation for the interpretation of reactions
of flavonoids with active free radicals, such as lipid peroxyl
and R-tocopheroxyl radicals in more complex biological sys-
tems.
References and Notes
(1) Larson, R. A. Phytochemistry 1988, 27, 969.
(2) Shahidi, F.; Wanasundara, P. K. Crit. ReV. Food Sci. Nutr. 1992,
32, 67.
(3) Pratt, D. E. 'Natural Antioxidants from Plant Material. In Phenolic
Compounds in Food and Their Effects on Health II; Huang, M.-T., Ho,
C.-T., Eds.; American Chemical Society: Washington, DC, 1992.
(4) Vinson, J. A.; Jang, J.; Dabbagh, Y. A.; Serry, M. M.; Cai, S. J.
Agric. Food Chem. 1995, 43, 2798.
(5) Vinson, J. A.; Dabbagh, Y. A.; Serry, M. M.; Jang, J. J. Agric.
Flavonoids are found in high concentration in fruits, fruit
juices, and wines. There exists a significant inverse association
between flavonoid intake (especially wine consumption in
various countries) and heart disease mortality.45,46 Of special
importance to heart disease is low-density lipoprotein (LDL)
oxidation. It has been reported that rutin and quercetin exhibit
lipoprotein-bound antioxidant activity using an in vitro oxidation
model for heart disease.4,5 The results of the present kinetic
study suggest that quercetin and rutin may function as vitamin
E regeneration compounds in biological systems. In fact,
suppression of the R-tocopherol consumption by flavonoids was
previously reported in the case of oxidative modification of
human LDL treated with macrophage or metal ion.34,35 Further,
the flavonoids in wine are hypothesized to act synergistically
with tocopherol to inhibit lipid peroxidation.36
Food Chem. 1995, 43, 2800.
(6) Erben-Russ, M.; Michel, C.; Bors, W.; Saran, M. J. Phys. Chem.
1987, 91, 2362.
(7) Bors, W.; Saran, M. Free Radicals Res. Commun. 1987, 2, 289.
(8) Bors, W.; Heller, W.; Michel, C.; Saran, M. Methods Enzymol.
1990, 186, 343.
(9) Jovanovic, S. V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic,
M. G. J. Am. Chem. Soc. 1994, 116, 4846.
(10) Terao, J.; Piskula, M.; Yao, Q. Arch. Biochem. Biophys. 1994, 308,
278.
(11) Ioku, K.; Tsushida, T.; Takei, Y.; Nakatani, N.; Terao, J. Biochim.
Biophys. Acta 1995, 1234, 99.
(12) Mukai, K.; Watanabe, Y.; Uemoto, U.; Ishizu, K. Bull. Chem. Soc.
Jpn. 1986, 59, 3113.
(13) Mukai, K.; Fukuda, K.; Tajima, K.; Ishizu, K. J. Org. Chem. 1988,
53, 430.
(14) Burton, G. W.; Ingold, K. U. J. Am. Chem. Soc. 1981, 103, 6472
and references therein.
(15) Burton, G. W.; Doba, T.; Gabe, R. J.; Hughes, L.; Lee, F. L.; Prasad,
U.; Ingold, K. U. J. Am. Chem. Soc. 1985, 107, 7053.
(16) Foti, M.; Ingold, K. U.; Lusztyk, J. J. Am. Chem. Soc. 1994, 116,
9440.
Conclusion
(17) Mukai, K.; Nishimura, M.; Kikuchi, S. J. Biol. Chem. 1991, 266,
Detailed kinetic studies have been performed for the reaction
of aroxyl (ArO•) and 5,7-diisopropyltocopheroxyl (Toc•) radicals
with several flavonoids in homogeneous and micellar solutions.
274.
(18) Mukai, K.; Itoh, S.; Morimoto, H. J. Biol. Chem. 1992, 267, 22277.
(19) Rieker, A.; Scheffler, K. Liebigs Ann. Chem. 1965, 689, 78.

Free-Radical-Scavenging Action of Flavonoids
J. Phys. Chem. A, Vol. 101, No. 20, 1997 3753
(20) Mukai, K.; Kageyama, Y.; Ishida, T.; Fukuda, K. J. Org. Chem.
1989, 54, 552.
(33) Nagaoka, S.; Okauchi, Y.; Urano, S.; Nagashima, U.; Mukai, K. J.
Am. Chem. Soc. 1990, 112, 8921.
(34) De Whalley, C. V.; Rankin, S. M.; Hoult, R. S.; Jessup, W.; Leake,
D. S. Biochem. Pharmacol. 1990, 39, 1743.
(35) Jessup, W.; Rankin, S. M.; De Walley, C. V.; Hoult, J. R. S.; Scott,
J.; Leake, D. S. Biochem. J. 1990, 265, 399.
(36) Negre-Salvayre, A.; Alfany, A.; Hariton, C.; Salvayre, R. Phar-
macology 1991, 42, 262.
(37) Acker, S. A. B. E.; Berg, D.-J.; Tromp, M. N. J. L.; Griffioen, D.
H.; Bennekom, W. P.; Vijgh, W. J. F.; Bast, A. Free Radical Biol. Med.
1996, 20, 331.
(38) Bors, W.; Michel, C.; Schikora, S. Free Radical Biol. Med. 1995,
19, 45.
(39) Hudson, B. J. F.; Lewis, J. I. Food Chem. 1983, 10, 47.
(40) Afanas’ev, I. B.; Dorozhko, A. I.; Brodskii, A. V.; Kostyuk, V.
A.; Potapovitch, A. I. Biochem. Pharmacol. 1989, 38, 1763.
(41) Hendrickson, H. P.; Sahafayen, M.; Bell, M. A.; Kaufman, A. D.;
Hadwiger, M. E.; Lunte, C. E. J. Pharm. Biomed. Anal. 1994, 12, 335.
(42) Howard, J. A.; Ingold, K. U. Can. J. Chem. 1963, 41, 2800.
(43) Slabbert, N. P. Tetrahedron 1977, 33, 821.
(44) Kennedy, J. A.; Munro, M. H. G.; Powell, H. K. J.; Porter, L. J.;
Yeap, F. L. Aust. J. Chem. 1984, 38, 885.
(45) Hertog, M. G. L.; Feskens, E. G. M.; Lollman, P. C. H.; Katan, M.
B.; Kromhout, D. The Lancet 1993, 342, 1007.
(46) Hertog, M. G. L.; Kromhout, D.; Aravans, C.; Blackburn, H.;
Buzina, R.; Fidanza, F.; Giampaoli, S.; Jansen, A.; Menotti, A.; Nedeljkovic,
S.; Pekkarinen, M.; Simic, B. S.; Toshima, H.; Feskens, E. J. M.; Hollman,
P. C. H.; Katan, M. Arch. Int. Med. 1995, 155, 381.
(21) Mukai, K.; Okauchi, Y. Lipids 1989, 24, 936.
(22) There is an equilibrium in the present reaction, and thus the decay
rate of the aroxyl radical (-d[ArO•]/dt ) kobsd[ArO•]) will be affected by
the reverse reaction, as the reaction proceeds. The reverse reaction was
judged to be unimportant under our conditions (for example, in the case of
rutin, [ArO•] 0.049 mM and [rutin] ) 0.388-0.933 mM), since good
pseudo-first-order plots were obtained for at least the first 70% of reaction
and kobsd vs [rutin] plot showed a good linear relationship.23
(23) Valgimigli, L.; Banks, J. T.; Ingold, K. U.; Lusztyk, J. J. Am. Chem.
Soc. 1995, 117, 1966.
(24) Packer, J. E.; Slater, T. F.; Willson, R. L. Nature 1979, 278, 737.
(25) Scarpa, M.; Rigo, A.; Maiorino, M.; Ursini, F.; Gregolin, C.
Biochim. Biophys. Acta 1984, 801, 215.
(26) Mukai, K.; Kikuchi, S.; Urano, S. Biochim. Biophys. Acta 1990,
1035, 77.
(27) Kagan, V.; Serbinova, E.; Packer, L. Biochem. Biophys. Res.
Commun. 1990, 169, 851.
(28) Frei, B.; Kim, M. C.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 4879.
(29) Yamamoto, Y.; Komuro, E.; Niki, E. J. Nutr. Sci. Vitaminol. 1990,
36, 505.
(30) Stocker, R.; Bowry, V. W.; Frei, B. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 1646 and references therein.
(31) Loury, M.; Bloch, C.; Francois, R. ReV. Fr. Corps Gras 1966, 13,
747.
(32) Terao, J.; Matsushita, S. Lipids 1986, 21, 255 and references therein.