Kinetic study of the antioxidant activity of pyrroloquinolinequinol (PQQH2, a reduced form of pyrroloquinolinequinone) in micellar solution

Article abstract of DOI:10.1021/jf802197d

Kinetic study of the aroxyl radical-scavenging action of pyrroloquinolinequinol [PQQH₂, a reduced form of pyrroloquinolinequinone (PQQ)] and water-soluble antioxidants (vitamin C, cysteine, glutathione, and uric acid) has been performed. The se

Full text of DOI:10.1021/jf802197d

450 J. Agric. Food Chem. 2009, 57, 450–456
Kinetic Study of the Antioxidant Activity of
Pyrroloquinolinequinol (PQQH2, a Reduced Form of
Pyrroloquinolinequinone) in Micellar Solution
AYA OUCHI,*,† MASAHIKO NAKANO,§ SHIN-ICHI NAGAOKA,† AND
KAZUO MUKAI*,†
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan, and
Niigata Research Laboratory, Mitsubishi Gas Chemical Company, Inc., Niigata 950-3112, Japan
Kinetic study of the aroxyl radical-scavenging action of pyrroloquinolinequinol [PQQH2, a reduced
form of pyrroloquinolinequinone (PQQ)] and water-soluble antioxidants (vitamin C, cysteine, glu-
tathione, and uric acid) has been performed. The second-order rate constants (ks) for the reaction of
aroxyl radical with PQQH2 and water-soluble antioxidants were measured in Triton X-100 micellar
solution (5.0 wt %) (pH 7.4), using stopped-flow and UV-visible spectrophotometers. The ks values
decreased in the order PQQH2 > vitamin C . cysteine > uric acid > glutathione. The aroxyl radical-
scavenging activity of PQQH2 was 7.4 times higher than that of vitamin C, which is well-known as
the most active water-soluble antioxidant. Furthermore, PQQNa2 (disodium salt of PQQ) was easily
reduced to PQQH2 by reaction of PQQNa2 with glutathione and cysteine in buffer solution (pH 7.4)
under nitrogen atmosphere. The result suggests that PQQ exists as a reduced form throughout the
cell and plays a role as antioxidant.
KEYWORDS: PQQ; pyrroloquinolinequinone; antioxidant activity; reaction rate; stopped-flow spectro-
photometer; free radicals; cofactor; glutathione; cysteine
In the present work, a kinetic study of the aroxyl (ArO•)
INTRODUCTION
radical scavenging activity of PQQH2 and water-soluble anti-
oxidants was performed in micellar solution, using stopped-
flow and UV-visible spectrophotometers. A stable ArO• radical
[2,6-di-tert-butyl-4-(4′-methoxyphenyl)phenoxyl] (Figure 1) was
used as a model of active free radicals such as LOO•, LO•, and
Toc•, as described in previous works (26-28). The preparation
of PQQH2 was performed by the reduction of PQQNa2 (diso-
dium salt of PQQ, see Figure 1) in buffer solution at pH 7.4
under nitrogen gas atmosphere, where not only NaBH4 but also
glutathione (GSH) and cysteine (Cys) were used as reducing
agents. The second-order rate constants (ks) for the reaction of
ArO• radical with PQQH2 and water-soluble antioxidants [GSH,
Cys, and uric acid (UA)] (see Figure 1) (reaction 1) have been
measured in 5.0 wt % Triton X-100 micellar solution (pH 7.4,
0.05 M phosphate buffer). The ks values obtained were compared
with that reported for vitamin C (Vit C) (29), which is well-
known as a typical water-soluble antioxidant. This is the first
report with the kinetic study of the free radical scavenging
activity of PQQH2.
Pyrroloquinolinequinone (PQQ), 4,5-dihydro-4,5-dioxo-1H-
pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid, has received
much attention in recent years owing to its several interesting
functions. PQQ is a cofactor of alcohol- and glucose-dehydro-
genases in bacteria (1-6). PQQ is known to be a nutritionally
important growth factor (7-9). PQQ nutritional status alters
lysine metabolism and modulates mitochondrial DNA content
in the mouse and rat (10). PQQ has also been reported to show
neuroprotective effects (11-13). PQQ was found in many kinds
of fruits and foods (14-16). Furthermore, the existence of small
amounts of free PQQ was found in eight human organs, plasma,
and urine and in three rat organs (17).
A previous work demonstrated that PQQ prevents mitochon-
drial lipid peroxidation and the inactivation of the mitochondrial
respiratory chain from oxidative damage (18). PQQ protects
against cell injury associated with oxidative stress (19-21).
Recently, it was found that PQQ prevents cognitive deficit
caused by oxidative stress in rats (22). Therefore, PQQ is
thought to function as an antioxidant (23, 24). In fact, the
reduced form of PQQ [PQQH2 (pyrroloquinolinequinol), see
Figure 1] has high free radical scavenging properties (25).
ks
ArO• + PQQH2 f ArOH + PQQH•
(1)
* Authors to whom corresponding should be addressed [fax 81-89-
927-9590;e-mail(K.M.)mukai@chem.sci.ehime-u.ac.jp,(A.O.)oouchi@
chem.sci.ehime-u.ac.jp].
MATERIALS AND METHODS
Commercial glutathione (GSH) (Sigma-Aldrich), cysteine (Cys)
(Tokyo Kasei), uric acid (UA) (Sigma-Aldrich), and Triton X-100
(Nacalai Tesque) were used as received. Powder sample of PQQNa2
† Ehime University.
§ Mitsubishi Gas Chemical Co., Inc.
10.1021/jf802197d CCC: $40.75  2009 American Chemical Society
Published on Web 12/24/2008

Antioxidant Activity of Pyrroloquinolinequinol
J. Agric. Food Chem., Vol. 57, No. 2, 2009 451
Figure 2. (a) UV-visible absorption spectrum of PQQNa2 in 0.05 M
phosphate buffer solution (pH 7.4) at 25.0 °C. [PQQNa2] ) 7.16 × 10-5
M. (b) UV-visible absorption spectrum of PQQH2 obtained by the reaction
of PQQNa2 with cysteine in 0.05 M phosphate buffer solution (pH 7.4) at
Figure 1. Molecular structures of PQQNa2, PQQH2, vitamin C, cysteine,
glutathione, uric acid, and aroxyl radical (ArO•).
25.0 °C. [PQQNa2]t)0 ) 4.45 × 10-5 M and [cysteine]
) 5.78 ×
t)0
was kindly supplied from Mitsubishi Gas Chemical Co., Inc. The results
of the elemental analysis, the thermogravimetry, and the titration of
H2O due to Karl Fischer’s reagent indicated that PQQNa2 used is a
monohydrate of PQQNa2 (PQQNa2 H2O). ArO• radical was synthesized
according to the method reported in a previous paper (30), and the
corresponding ArO•-containing micellar dispersions were prepared
according to the method reported in a previous paper (28). The buffer
solution was prepared using distilled water treated with a Millipore Q
system, and its pH was adjusted to 7.4 using 0.05 M KH2PO4-Na2HPO4
buffer.
The kinetic data were obtained with a Unisoku model RSP-1000
stopped-flow spectrophotometer by mixing equal volumes of solutions
of antioxidants and ArO• under nitrogen atmosphere. The shortest time
for mixing two solutions and recording the first data point (that is, dead
time) was 10-20 ms. The reaction was monitored with either single-
wavelength detection or photodiode array detector attached to the
stopped-flow spectrophotometer. The reaction was studied under
pseudo-first-order conditions, and the observed rate constant (kobsd) was
evaluated in the usual way using a standard least-squares analysis. If
the reaction rates (ks) were slower than 1 M-1 s-1, the measurements
were performed by using a Shimadzu UV-2100S spectrophotometer.
All of the measurements were performed at 25.0 ( 0.5 °C. Experimental
errors in the rate constants (ks) were estimated to be about 10% in
micellar solution.
10-4 M.
is stable and shows absorption peaks at λmax ) 249 nm (ε )
26600 M-1 cm-1), 267 sh (20500), 331 (12700), and 477 (690)
(Figure 2A) in 0.05 M phosphate buffer solution (pH 7.4), as
listed in Table 1 (sh stands for shoulder). Catalytic oxidation
of thiols by PQQ was studied in previous works by Itoh et
al. (31, 32). The oxidation of benzenethiol and related thiol
derivatives by PQQ was performed in 0.1 M phosphate buffer
solution (containing 20% CH3CN, pH 6.2) under anaerobic
conditions, giving corresponding disulfide compounds in high
yield. PQQH2 is unstable in buffer solution (pH 7.4) under air
and easily oxidized to PQQ, as reported in previous works
(33-35). Consequently, in the present work, the reduction of
PQQNa2 to PQQH2 and the measurements of the reaction rate
constants were performed under strictly deaerated and nitrogen-
substituted conditions by using a Hamilton 1000 series gastight
syringe and sealing cap to avoid an oxidation of PQQH2.
The reduction of PQQNa2 was performed by using Cys and
GSH as reducing agent (reaction 2). For instance, by adding
the 0.05 M phosphate buffer solution of Cys (1.16 × 10-3 M)
to the solution of PQQNa2 (8.89 × 10-5 M) (1:1 in volume) at
room temperature, the absorption spectrum of PQQNa2 disap-
peared rapidly and changed to that of PQQH2 with absorption
peaks and shoulders at λmax ) 304, 340 sh, 405 sh, and 499
nm, as shown in Figure 2b. As shown in Figure 3, the
RESULTS AND DISCUSSION
PQQNa2 Is Reduced to PQQH2 by the Reaction with
Glutathione and Cysteine in Buffer Solution. The PQQNa2

452 J. Agric. Food Chem., Vol. 57, No. 2, 2009
Ouchi et al.
i
Table 1. Values of UV-Visible Absorption Maxima (λmax ) and Molar Extinction Coefficients (εi) of PQQNa2 and PQQH2 in Buffer Solution (pH 7.4) by Using
Several Reducing Agents and Experimental Methods
reducing agent (method)
λ
max1/nm (ε1/M-1 cm-1
)
λ
max2/nm (ε2/M-1 cm-1
)
λ
max3/nm (ε3/M-1 cm-1
)
λ
max4/nm (ε4/M-1 cm-1
)
PQQH2
PQQH2
PQQH2
PQQH2c
PQQNa2
PQQc
cysteine (UV-vis)a
NaBH4 (UV-vis)
glutathione (stopped-flow)b
H2 (PtO2) (UV-vis)
(UV-vis)
304.0 (40000)
304.0 (37400)
304.0 (42700)
302 (25050)
249 (26600)
249 (18400)
340 sh (11500)
340 sh (13800)
405 sh (2410)
405 sh (2610)
499 (1170)
499.0 (1170)
267 sh (20500)
331 (12700)
477 (692)
(UV-vis)
a UV-vis; the measurements were performed by a UV-visible spectrophotometer. b Stopped-flow; the measurements were performed by a stopped-flow spectrophotometer.
c The values in 0.05 M phosphate buffer solution (pH 7.0) were reported by Duine et al. (34).
Figure 3. Time dependence of the absorbance at 304 nm of PQQH2,
after the reduction of PQQNa2 with cysteine in 0.05 M phosphate buffer
(pH 7.4) at 25.0 °C: under nitrogen atmosphere (O); under air (b).
Figure 4. Change in absorption spectrum of PQQNa2 and PQQH2 during
[PQQNa2]t)0 ) 4.45 × 10-5 M and [cysteine]t)0 ) 5.78 × 10-4 M.
the reaction of PQQNa2 with glutathione in 0.05 M phosphate buffer
solution (pH 7.4) at 25.0 °C. [PQQNa2]t)0 ) 4.92 × 10-5 M. [Glutathione]
absorbance of PQQH2 at 304.0 nm increases rapidly, shows a
) 8.62 × 10-3 M. The spectra were recorded at 20 s intervals. The
arrow indicates a decrease (PQQNa2) and an increase (PQQH2) in
absorbance with time.
maximum at t ∼ 100 min, and then decreases gradually. The
spectrum of the PQQH2 at tmax ) 100 min is shown in Figure
2b. The molar extinction coefficient (ε1) of PQQH2 was
calculated from the absorption spectra at t ) 100 min, by using
Lambert-Beer’s equation [absorbance (At) ) ε1[PQQH2]),
where the concentration of PQQH2 ([PQQH2]) was assumed to
be equal to that of PQQNa2 at t ) 0 s]. The εi values of PQQH2
obtained are listed in Table 1, together with that reported in a
previous work (34). The absorption spectrum of PQQH2 changed
to original PQQNa2 after 25 h; PQQH2 was comparatively stable
under strict nitrogen atmosphere. On the other hand, by
introducing air to the above buffer solution, the spectrum of
PQQH2 decreased rapidly and changed to that of the original
PQQNa2, as shown in Figure 3. Similar behavior was observed
for the reaction of PQQNa2 with GSH.
of the interaction between PQQNa2 (4.92 × 10-5 M) and GSH
(8.62 × 10-3 M) in 0.05 M phosphate buffer solution at 25 °C.
The spectra were recorded at 20 s intervals. The arrow shows
a decrease (V) in absorbance of PQQNa2 at 370 nm and an
increase (v) in absorbance of PQQH2 at 304 nm with time. Three
isosbestic points were clearly observed at 275, 348, and 410
nm, indicating that the reaction is simple and the contribution
of side reaction is negligible. It is clear that PQQH2 was
produced by the reaction of PQQNa2 with GSH. However, the
absorption spectrum of PQQNa2 [λmax ) 370 nm (ε3 ) 9440
M-1 cm-1) and 320 nm (ε3 ) 9690 M-1 cm-1)] at t ) 0 s (see
3
Figure 4) is very different from that of PQQNa2 [λmax ) 331
4
nm (ε3 ) 12700 M-1 cm-1) and λmax ) 477 nm (ε4 ) 692
PQQ + 2RSH f PQQH2 + RS-SR
M-1 cm-1)] without GSH (Figure 2a). Such an absorption
spectrum was also observed for the reaction of PQQNa2 with
Cys (data are not shown).
(RSH: cysteine and glutathione) (2)
Similarly, by adding 0.05 M phosphate buffer solution of
PQQNa2 (4.57 × 10-5 M) to the powder sample of 5.79 mg of
NaBH4 (3.83 × 10-4 M) under nitrogen atmosphere at room
temperature, the absorption spectrum of PQQNa2 disappeared
gradually and changed to that of PQQH2, as observed for the
reaction of PQQNa2 with Cys. The values of λmaxi and εi obtained
are also listed in Table 1. The values of ε1 (40000 and 37400
M-1 cm-1) obtained by the reduction due to Cys and NaBH4
As Itoh et al. (32) reported, a similar absorption spectrum
was obtained by reacting PQQ with benzenethiol (PhSH) in 0.05
M acetate buffer containing 20% CH3CN. The scheme of the
reduction of PQQ by PhSH was discussed by Itoh et al. As
they reported, one of the possible mechanisms is that involving
C-5 attack of the thiolate ion followed by breakdown to reduced
PQQ and the corresponding disulfide (see the scheme in ref
32).
are 1.63 and 1.49 times larger than that (25050 M-1 cm-1
)
reported (34), respectively. The values of molar extinction
coefficient (ε1) are very different from that reported.
Furthermore, the molar extinction coefficient (εi) of PQQH2
was also determined under nitrogen atmosphere by using a
stopped-flow spectrophotometer. Figure 4 shows an example
Figure 5 shows the time courses of the decrease in absorbance
at 370 nm of PQQNa2 and the increase in absorbance at 304
nm of PQQH2 observed when 0.05 M phosphate buffer solution
(pH 7.4) containing PQQNa2 (6.88 × 10-5 M) is mixed with a
0.05 M buffer solution of GSH (1.92 × 10-2 M) (1:1, v/v; final

Antioxidant Activity of Pyrroloquinolinequinol
J. Agric. Food Chem., Vol. 57, No. 2, 2009 453
Figure 5. Change in absorbance of PQQNa2 at 370 nm and PQQH2 at
304 nm during reaction of PQQNa2 with glutathione in 0.05 M phosphate
buffer solution (pH 7.4) at 25.0 °C. [PQQNa2]t)0 ) 3.44 × 10-5 M, and
[glutathione]t)0 ) 9.60 × 10-3 M. The decrease and increase in the
absorbance at 370 and 304 nm, respectively, are shown.
Figure 6. Change in absorption spectrum of aroxyl (ArO•) during the
reaction of ArO• with PQQH2 at pH 7.4 in 5.0 wt % Triton X-100 micellar
solution at 25.0 °C. [PQQH2]t)0 ) 5.07 × 10-5 M. The spectra were
recorded at 2.4 s intervals. The arrow indicates decreases in absorbance
at 580 and 376 nm of ArO• and a decrease in absorbance at 304 nm of
PQQH2 with time. PQQH2 was prepared by the reaction of PQQNa2 with
GSH in phosphate buffer solution (pH 7.4).
concentration of GSH of 9.60 × 10-3 M). At t ∼ 20 min, the
absorbance of each peak approaches the minimum and the
maximum, respectively. The value of ε1 (42700 M-1 cm-1) for
PQQH2 was determined from the absorbance at t ) 20 min.
This value is similar to those obtained by the reduction due to
Cys and NaBH4 using UV-visible spectrophotometer (see
Table 1). The reduction of PQQ to PQQH2 is not a simple one-
electron reduction, but two-electron ones. Therefore, the increase
in absorbance at 304 nm of PQQH2 does not follow simple first-
order kinetics, showing a sigmoid curve (see Figure 5).
The λmax1 and ε1 values of PQQ and PQQH2 were reported
by Duine et al. (34), who performed the reduction of PQQ
by phenylhydrazine or H2 in the presence of PtO2 (see Table
Table 2. Rate Constants for the Reaction of PQQH2 and Water-Soluble
Antioxidants with ArO• Radical in 5.0 wt % Triton X-100 Micellar Solution
(pH 7.4) at 25.0 °C
antioxidant
PQQH2 (glutathione)a
[antioxidant]/M
kobsd/s-1
ks/M-1 s-1
1.81 × 103
5.34 × 10-4
9.35 × 10-4
18.7 × 10-4
0.297 × 10-4
5.93 × 10-4
11.8 × 10-4
0.852 × 10-3
2.56 × 10-3
3.41 × 10-3
2.95 × 10-3
8.86 × 10-3
1.25 × 10-3
2.49 × 10-3
4.98 × 10-3
1.25
1.74
3.62
0.121
1.04
2.34
PQQH2 (NaBH4)b
1.92 × 103
PQQNa2
glutathione
<10-1
1.22 × 10-1
1
0.574 × 10-3
0.816 × 10-3
0.878 × 10-3
2.52 × 10-3
8.17 × 10-3
1.05 × 10-3
1.87 × 10-3
3.34 × 10-3
1). The values of λmax for PQQ and PQQH2 are similar to
those obtained in the present work. On the other hand, both
of the values of ε1 reported for PQQ and PQQH2 are 1.44
and 1.49-1.70 times smaller than those obtained in the
present work. The sample of PQQNa2 H2O (formula weight
) 392.3) used in the present work was prepared by
evaporating water molecules from crude PQQNa2 sample at
120 °C under vacuum (5 Torr). The X-ray structure analysis
of a single crystal of PQQ compound was performed by
Ishida et al. (36), indicating that five H2O molecules are
included in the PQQ compound, that is, the chemical formula
of the PQQ compound is PQQNa2 · 5H2O (formula weight )
464.3). If Duine et al. used such a compound for the
measurement of UV spectrum, the value of ε will decrease
∼16%. However, the details of the difference in the value
of ε are not clear at present.
Free Radical Scavenging Activity of PQQH2 Is 7.4 Times
Higher than That of Vitamin C in Micellar Solution. ArO•
was stable in the absence of PQQH2 and showed absorption
peaks at λmax ) 580 and 376 nm in aqueous Triton X-100
micellar solution (5.0 wt %) (see Figure 6). PQQH2 was
prepared by the reduction of PQQNa2 (5.07 × 10-5 M) with
GSH (6.19 × 10-4 M) in 0.10 M phosphate buffer (pH 7.4)
under strict nitrogen atmosphere, to avoid an oxidation of
PQQH2, as described above. Upon addition of PQQH2 in 0.10
M phosphate buffer (pH 7.4) to 10.0 wt % Triton X-100 micellar
solution containing ArO• (1:1, v/v), the absorption spectrum of
ArO• disappeared immediately. Figure 6 shows an example of
the interaction between ArO• (∼7.5 × 10-5 M) and PQQH2
cysteine
uric acid
9.56 × 10-1
6.11 × 10-1
vitamin Cc
2.51 × 102
a PQQH2 was prepared by reducing PQQNa2 with glutathione. The concentrations
of glutathione used for the reduction of PQQNa2 were e2.40 × 10-2 M. b PQQH2
was prepared by reducing PQQNa2 with NaBH4. c See ref 29.
(5.07 × 10-5 M) in phosphate buffer (pH 7.4). Higher
concentrations of PQQNa2 (that is, PQQH2) ([PQQH2] g 5.34
× 10-4 M) (see Table 2) were used for the measurement of
the reaction rate, because the condition [PQQH2] > [ArO•] is
necessary to determine the pseudo-first-order rate constant (kobsd).
By analyzing the decay curve of ArO• radical at 580 nm, the
kobsd value was determined. ArO• showed a slow natural decay
in Triton X-100 micellar solution. Therefore, the kobsd value for
ArO• bleaching is given by eq 3
kobsd ) ko+ks[PQQH2]
(3)
where ko 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 PQQH2. These parameters are obtained
by plotting kobsd against [PQQH2], as shown in Figure 7. The
ks value obtained for PQQH2 at pH 7.4 is 1.81 × 103 M-1

454 J. Agric. Food Chem., Vol. 57, No. 2, 2009
Ouchi et al.
Figure 8. Dependence of pseudo-first-order rate constants (kobsd) on
concentration of water-soluble antioxidants (cysteine, glutathione, and uric
acid) at pH 7.4 in 5.0 wt % Triton X-100 micellar solution.
Figure 7. Dependence of pseudo-first-order rate constants (kobsd) on
concentration of PQQH2 at pH 7.4 in 5.0 wt % Triton X-100 micellar
solution. PQQH2 was prepared by the reaction of PQQNa2 with glutathione
(O) and NaBH4 (b).
C, Cys, GSH, and UA with stable 5,7-di-isopropyl-toco-
pheroxyl radical were performed in 5.0 wt % aqueous Triton
X-100 micellar solution, and the second-order rate constants
(kr) for these reactions were determined (39). The kr value
of Vit C was found to be 3-4 orders of magnitude larger
than those of Cys, GSH, and UA in micellar solution. These
reactions are regarded as a model for regeneration of R-TocH
by the water-soluble antioxidants in human blood, and the
relative contributions of each antioxidant to the total regen-
eration in blood were discussed.
As described in the Introduction, the reduced form of PQQ
(PQQH2) functions as a radical scavenger. In fact, it has been
reported that PQQH2 shows higher reactivity than R-tocopherol
toward galvinoxyl radical and peroxyl radical in acetonitrile-
DMSO (93:2, v/v) solution (25). PQQH2 reduced the R-toco-
pheroxyl radical and spared R-tocopherol in the oxidation of
methyl linoleate in the same solution. These results suggest that
PQQH2 may act as a potent antioxidant, particularly in
combination with R-tocopherol. However, the second-order rate
constants for the above reactions have not been reported, because
PQQH2 is unstable and it is not easy to determine the
concentration of PQQH2 in the reaction mixture, as described
above.
In the present work, first, we tried to measure the reaction
rates between R-Toc• and PQQH2 in micellar solution. However,
we were unsuccessful in determining the rate constant (kr),
because R-Toc• is unstable and absorption of R-Toc• at 430 nm
overlaps those of PQQH2 and PQQ, as shown in Figure 2.
Therefore, the stable ArO• radical having an absorption maxi-
mum at λmax ) 580 nm was used for the measurements of the
free radical scavenging activity of PQQH2. As described above,
it has been found that the ks value of PQQH2 is 7.4 times larger
than that of Vit C and 3-4 orders of magnitude larger than
those of Cys, GSH, and UA.
Ubiquinone-10 (and -9) and PQQ are p- and o-benzoquinone
derivatives, respectively. Ubiquinol-10 and -9 (the reduced forms
of ubiquinone-10 and -9) are well-known as representative lipid-
soluble antioxidants. Ubiquinone-10 and -9 are reduced by
NADPH (and enzyme) in tissues and exist as the reduced forms
in human and animal tissues (40). PQQ is also reduced by
NADPH (41). On the other hand, ubiquinone-10 and -9 are not
reduced by Cys and GSH in contrast to PQQ. This is because
ubiquinone-9 and -10 have smaller π-electron systems than PQQ
s-1 and ko ) 0.182 s-1. GSH molecules that were not
consumed for the reduction of PQQNa2 will remain in the
reaction mixture. This GSH ([GSH] < 2.40 × 10-2 M, see
Table 2) will also react with ArO•. However, the contribution
of this reaction will be negligible, because the rate constant
(ks) is 4 orders of magnitude smaller than that for PQQH2,
as described below.
Similarly, PQQH2 was prepared by the reduction due to
NaBH4 in 0.10 M buffer solution and reacted with 10.0 wt %
Triton X-100 micellar solution containing ArO• (1:1, v/v). As
shown in Figure 7, the ks value (1.92 × 103 M-1 s-1) obtained
is similar to that by the reduction due to GSH.
The measurement of the rate constant for the reaction of
PQQNa2 with ArO• was also performed in Triton X-100 micellar
solution at pH 7.4, showing that the reaction between ArO• and
PQQNa2 is negligible.
Similar measurements were performed for the reaction of
ArO• with water-soluble antioxidants (Cys, GSH, and UA) at
pH 7.4 in Triton X-100 micellar solution. The rate constants
(ks) for the reaction of Cys, GSH, and UA with ArO• are
expected to be much less than that for PQQH2, and thus the
higher concentrations of antioxidants were used for the mea-
surements (see Table 2). By analyzing the decay curve of ArO•
radical at 580 nm, the kobsd values were determined. kobsd versus
[antioxidant] plots are shown in Figure 8. The ks values obtained
are listed in Table 2, together with that (2.51 × 102 M-1 s-1
)
reported for vitamin C (29).
As listed in Table 2, the rate constants (ks) of PQQH2 and
water-soluble antioxidants decrease in the order
PQQH2 > Vit C . Cys > UA > GSH > PQQNa2 (4)
The ks value of PQQH2 at pH 7.4 is 7.4 ( 0.2 times larger than
that of Vit C. The rate constants of Cys, GSH, and UA are
(1.22-9.56) × 10-1 M-1 s-1 and are about 3-4 orders of
magnitude smaller than that of PQQH2 in micellar solution.
Oxidative damage of biomembrane and tissues by active
oxygen free radicals and its protection by biological anti-
oxidants have attracted much attention. Water-soluble anti-
oxidants (Vit C, Cys, GSH, and UA) suppress free radical
mediated chain oxidation of lipids of cell membrane (37, 38).
In a previous work, the kinetic studies of the reaction of Vit

Antioxidant Activity of Pyrroloquinolinequinol
J. Agric. Food Chem., Vol. 57, No. 2, 2009 455
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In the present work, it has been found that PQQNa2 is easily
reduced by GSH and Cys in buffer solution (pH 7.4), and results
in PQQH2. Cys is a proteinaceous thiol, and GSH is a major
nonproteinaceous thiol. These thiols exist not only in plasma
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plays a role as antioxidant. As described in the Introduction,
PQQ was found in many kinds of fruits and foods and in several
tissues, plasma, and urine of humans and rats (15-17). The
ArO• radical scavenging rate constant (ks) of PQQH2 was found
to be 3-4 orders of magnitude larger than those of Cys, GSH,
and UA and 7.4 times larger than that of Vit C at pH 7.4 in
micellar solution. The results of the present kinetic study suggest
that PQQH2 has high activity for the free radical cavenging (and/
or the tocopherol regeneration) and contributes to the prevention
of oxidative damage in tissues.
ACKNOWLEDGMENT
We are very grateful to Professor Fernado Antunes of the
University of Lisbon for his kind guidance with the experiments
under strictly deaerated and nitrogen-substituted conditions
during his stay in Ehime University. We are also grateful to
Akiko Nitta of Ehime University for her kind help in the early
stage of the measurement of the reaction rates.
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Received for review July 21, 2008. Revised manuscript received October
26, 2008. Accepted November 18, 2008.
JF802197D