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-