Nitric Oxide (NO) Scavenging and NO Protecting Effects of Quercetin and Their Biological Significance in Vascular Smooth Muscle

Abstract

The flavonoid quercetin reduces blood pressure and endothelial dysfunction in animal models of hypertension. However, the results concerning the relationship between quercetin and NO present a complex picture. We have analyzed the mechanisms involved in the NO scavenging effects of quercetin and its repercussion on NO bioactivity in vascular smooth muscle. Quercetin scavenged NO with apparent zero-order kinetics with respect to NO. This effect was strongly dependent on the O(2) concentrations, so that NO decay at pH 7.4 could be fitted to the equation -d[NO]/dt = k x [O(2)] x [quercetin], where k was 0.15 M(-1) s(-1). The NO scavenger effects were prevented by superoxide dismutase (SOD), reduced by lowering pH, accompanied by O(2)(.) production and correlated with decreased NO bioactivity in rat aortic rings. However, under conditions of increased O(2)(.) concentrations, quercetin was a better scavenger of O(2)(.) than of NO. When NO scavenging by quercetin was prevented by addition of SOD, NO bioactivity was increased. Quercetin also prevented the inhibitory effects of the SOD inhibitor diethyldithiocarbamic acid (DETCA) on NO bioactivity. In the presence of DETCA, quercetin reduced tissue O(2)(.) as measured by nitro blue tetrazolium staining. In conclusion, quercetin exerts dual effects on O(2)(.) and NO. At physiological conditions of pH, O(2) concentrations and NO, quercetin effectively scavenged NO in the low micromolar range, and the rate-limiting step was the autooxidation of quercetin and the formation of O(2)(.). When the extracellular NO scavenging effect was prevented, quercetin increased the biological activity of NO, an effect related to its O(2)(.) scavenger properties and/or its inhibitory effect on tissue O(2)(.) generation.

Full text

Nitric Oxide (NO) Scavenging and NO Protecting Effects of
Quercetin and Their Biological Significance in Vascular Smooth
Muscle
Gustavo L
´
opez-L
´
opez, Laura Moreno, Angel Cogolludo, Milagros Galisteo, Manuel Ibarra,
Juan Duarte, Federica Lodi, Juan Tamargo, and Francisco Perez-Vizcaino
Department of Pharmacology, Institute of Pharmacology and Toxicology (CSIC), School of Medicine, University Complutense
of Madrid, Madrid, Spain (L.M., A.C., M.I., J.T., F.P-V); and Department of Pharmacology, School of Pharmacy, University of
Granada, Granada, Spain (M.G., J.D.)
Received July 7, 2003; accepted January 6, 2003 This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
The flavonoid quercetin reduces blood pressure and endothe-
lial dysfunction in animal models of hypertension. However, the
results concerning the relationship between quercetin and NO
present a complex picture. We have analyzed the mechanisms
involved in the NO scavenging effects of quercetin and its
repercussion on NO bioactivity in vascular smooth muscle.
Quercetin scavenged NO with apparent zero-order kinetics
with respect to NO. This effect was strongly dependent on the
O
2
concentrations, so that NO decay at pH 7.4 could be fitted
to the equation ⫺d[NO]/dt ⫽ k ⫻ [O
2
] ⫻ [quercetin], where k
was 0.15 M
⫺1
s
⫺1
. The NO scavenger effects were prevented
by superoxide dismutase (SOD), reduced by lowering pH, ac-
companied by O
2
.
production and correlated with decreased
NO bioactivity in rat aortic rings. However, under conditions of
increased O
2
.
concentrations, quercetin was a better scavenger
of O
2
.
than of NO. When NO scavenging by quercetin was
prevented by addition of SOD, NO bioactivity was increased.
Quercetin also prevented the inhibitory effects of the SOD
inhibitor diethyldithiocarbamic acid (DETCA) on NO bioactivity.
In the presence of DETCA, quercetin reduced tissue O
2
.
as
measured by nitro blue tetrazolium staining. In conclusion,
quercetin exerts dual effects on O
2
.
and NO. At physiological
conditions of pH, O
2
concentrations and NO, quercetin effec
-
tively scavenged NO in the low micromolar range, and the
rate-limiting step was the autooxidation of quercetin and the
formation of O
2
.
. When the extracellular NO scavenging effect
was prevented, quercetin increased the biological activity of
NO, an effect related to its O
2
.
scavenger properties and/or its
inhibitory effect on tissue O
2
.
generation.
Flavonoids are polyphenolic compounds widely distributed
in dietary fruits, vegetables, and wine. The average daily
intake in the occidental diet is ⬇23 mg, of which quercetin
represents 60 to 75% (Hertog et al., 1993; Sampson et al.,
2002). Epidemiological studies including more than 100,000
patients have shown an inverse association between dietary
flavonoid intake and mortality from coronary heart disease
and/or risk of stroke (Hertog et al., 1993; Rimm et al., 1996).
Although prospective randomized clinical trials are lacking,
several studies using animal models support these potential
protective effects of flavonoids in cardiovascular diseases
(Middleton et al., 2000). Interestingly, quercetin exerts sys-
temic and coronary vasodilator effects (Duarte et al., 1993;
Perez-Vizcaino et al., 2002); when given orally, it reduces
blood pressure, cardiac hypertrophy, and vascular remodel-
ing in spontaneously hypertensive (SHR) and nitric oxide
(NO)-deficient rats (Duarte et al., 2001, 2002). It also exerts
free radical-scavenging effects (Robak and Gryglewski, 1988;
Van Acker et al., 1996), inhibits low-density lipoprotein per-
oxidation, and reduces the progression of atherosclerosis in
vivo (Hayek et al., 1997).
The results concerning the relationship between quercetin
and NO yield a confusing picture. Regarding NO synthesis,
high concentrations of quercetin (⬎100
␮
M) inhibit the ac-
tivity of the endothelial (eNOS), neuronal, and inducible
isoforms of NO synthase (Chiesi and Schwaller, 1995). In
vivo, high doses of quercetin (above 200 mg/kg/d for 10 days)
Supported by a PR247/02-11710 Danone/UCM grant (to F.P.-V.) and by
grants from Comunidad Auto´noma de Madrid 08.04.36.2001 (to F.P.-V.) and
Comisio´n Interministerial de Ciencia y Tecnologica SAF2002-02304 (J.T.),
SAF2001-2953 (J.D.). A.C. and L.M. are supported by Red Cardiovascular and
Ministerio de Educacio´n, Cultura y Deporte, respectively.
ABBREVIATIONS: SHR, spontaneously hypertensive rat; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; NOS, nitric-oxide synthase;
XO, xanthine oxidase; DMSO, dimethyl sulfoxide; HX, hypoxanthine;; SOD, superoxide dismutase; cyt c, cytochrome c; NBT, nitro blue
tetrazolium; DETCA, diethyldithiocarbamic acid; DPI, diphenylene iodonium.
0026-895X/04/6504-851–859$20.00
M
OLECULAR PHARMACOLOGY Vol. 65, No. 4
Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2796/1139064
Mol Pharmacol 65:851–859, 2004 Printed in U.S.A.
851

increased the activity, but not the expression, of vascular
eNOS (Benito et al., 2002), whereas at low doses (5 and 10
mg/kg/d for 5 weeks), no changes were observed in either
vascular eNOS or inducible NOS expression or total NOS
activity (Duarte et al., 2002). Thus, changes in NOS activity
seem to be unlikely at doses of quercetin equivalent to those
present in the diet. In addition, quercetin directly scavenges
the superoxide anion (O
2
.
) (Robak and Gryglewski, 1988) and
inhibits several O
2
.
-generating enzymes such as xanthine
oxidase (XO) (Hayashi et al., 1988; Chang et al., 1993) or the
neutrophil membrane NADPH oxidase complex (Tauber et
al., 1984). In SHR rats, quercetin reduced the oxidative sta-
tus, as indicated by lower concentrations of markers of such
oxidative stress as plasma and hepatic malondialdehyde and
urinary isoprostane-F
2
␣
(Duarte et al., 2001). By reducing
O
2
.
concentrations, quercetin is expected to protect NO
from O
2
.
-driven inactivation. In fact, it improved the endo
-
thelial function in SHR rats (Duarte et al., 2001), possibly
because of an enhanced NO bioavailability. It also in-
creased the levels of NO determined by electron paramag-
netic resonance spectroscopy in rat brain during global
ischemia and reperfusion (Shutenko et al., 1999). On the
other hand, quercetin and related flavonoids also scavenge
NO (Van Acker et al., 1995; Haenen and Bast, 1999).
However, the mechanisms of the NO-scavenging effects,
the potential NO protecting mechanisms, and their reper-
cussion on NO bioactivity are unclear.
Therefore, in the present study, we have analyzed the
mechanisms involved in the NO scavenging effect of querce-
tin. In addition, we have investigated the mechanisms of the
potential protective effects of quercetin on NO bioactivity in
vascular smooth muscle.
Materials and Methods
The studies have been carried out in accordance with the Decla-
ration of Helsinki.
Materials. All chemicals and drugs were from Sigma (St. Louis,
MO). Quercetin and diphenylene iodonium were dissolved in DMSO
daily, hypoxanthine (HX) was dissolved in 0.1% NaOH, and other
drugs were dissolved in distilled, deionized water to prepare 1, 10, or
100 mM stock solutions; further dilutions were prepared in the
working buffer. To prepare the NO solutions used for biological
activity, a vial containing 20 ml of Krebs’ solution at 37°C was
initially bubbled with N
2
for 15 min and then continuously bubbled
with NO (450 ppm from Air Liquide, Paris, France) resulting in a
concentration of 0.9 to 1
␮
M (as measured by the electrochemical
electrode described below; Lopez-Lopez et al., 2001).
NO Scavenging. NO concentrations were monitored with an
ISO-NO meter (World Precision Instruments Inc., Sarasota FL) cou-
pled to a data acquisition hardware (PowerLab, AD Instruments Pty
Ltd., Castle Hill, Australia) and data recording software (Chart
v4.1.2; AD Instruments) in a 20-ml thermostat-equipped, water-
jacketed chamber at 37° filled with HEPES-buffered solution (130
mM NaCl, 5 mM KCl, 1.2 mM MgCl
2
, 10 mM glucose, 1.5 mM CaCl
2
,
and 10 mM HEPES; pH 7.4 unless otherwise stated). The chamber
was bubbled with N
2
for ⬎15 min then slowly bubbled with NO at
450 ppm until the desired NO concentration (50–70 nM) was
achieved (within 3–5 min), and the O
2
concentration was adjusted by
addition of O
2
-saturated HEPES solution [bubbled with 100% O
2
(i.e., 1.05 mM)]. At the beginning of the experiment, the system was
closed with no headspace, under constant rapid stirring, NO concen-
tration was 50 ⫾ 10 nM, O
2
concentration varied from nominally 0 (⬍
0.1%) to 5, 10, or 20%, and pH was 7.0 or 7.4. Oxygen concentration
was simultaneously measured in the chamber with a Clark O
2
elec
-
trode (World Precision Instruments Inc., New Haven, CT) connected
to the PowerLab system. Quercetin (20
␮
l) was added to the chamber
with a microsyringe at a final concentration of 1 to 100
␮
M, and NO
concentration was followed in the presence of quercetin. In some
experiments, superoxide dismutase (SOD; 100 U/ml), XO (5 or 15
mU/ml) plus HX (100
␮
M), or pyrogallol (3 or 10
␮
M) were added 2
min before quercetin. Corrections were made for the spontaneous
degradation of NO under the different O
2
concentrations in the
presence of vehicle (20
␮
l of DMSO). The electrode was calibrated
daily according to the manufacturer by the conversion of known
concentrations of NaNO
2
into NO in the presence of H
2
SO
4
and KI.
Quercetin Auto-Oxidation and O
2
.
Released. UV-visible ab
-
sorption spectra of quercetin in HEPES-buffered solution was re-
corded in a spectrophotometer (6405; Jenway, Essex, UK) just after
dilution and after 48 h of exposure to room air O
2
at 37°C. The
oxidation of quercetin at 37°C, room air O
2
concentration, and under
constant stirring was followed at 380 nm for 10 h. To measure the
release of O
2
.
, quercetin (10, 50, or 100
␮
M) or vehicle (DMSO) was
added to a HEPES-buffered solution at pH 7.4 or 9.0 and incubated
at 37°C under room air for 5 min. O
2
.
release was determined by
measuring luminescence after automatic injection of lucigenin (5
␮
M) over 200 s in a scintillation counter (Lumat LB 9507; Berthold
Technologies, Bad Wildbad, Germany) at 5-s intervals and expressed
as relative luminescence units/min. In addition, O
2
.
release was also
determined by measuring SOD-inhibitable cytochrome c (cyt c) re-
duction, after changes in absorbance at 550 nm for 5 min, after
addition of cyt c (final concentration, 20
␮
M) in a 1-ml cuvette. The
experiments were performed with and without SOD (100 U/ml). O
2
.
production was calculated as the fraction of ferricytochrome c reduc-
tion inhibited by SOD and expressed as nanomolar per minute.
Computer Simulation. The solution technique employed to sim-
ulate NO consumption is based on time discretization followed by the
solution of the discrete equations using an iterative scheme and
updating the concentrations of reagents in each iterative step. The
simulation was reproducible when discrete time intervals were of 10
␮
s or shorter. Calculations were made using a Turbo Pascal program
created by the authors (available on request).
NO Bioactivity in Rat Vascular Smooth Muscle. Preparation
of aortic rings from Wistar rats, force measurement under isometric
conditions, and endothelial cell removal were performed as described
previously (Perez-Vizcaino et al., 2002). After equilibration in Krebs’
solution, endothelium-denuded rings were stimulated with 30 mM
KCl until they reached a steady-state contractile response and NO
(70 nM) was added to the chamber, which induced a transient relax-
ant response (30–40% of the initial tone). Then, the tissues were
washed with Krebs’ solution containing 30 mM KCl, incubated for 20
min with the drugs to be tested or vehicle (except HX, which was
added in the last minute), and NO was again added. This second
relaxant response indicative of NO bioactivity in the presence of the
drugs or vehicle was expressed as a percentage of the initial response
to NO. In some aortic rings stimulated with 30 mM KCl and exposed
to vehicle or quercetin for 20 min, the responses to the vasodilator
sodium nitroprusside (3 nM) were recorded.
In Situ Detection of O
2
.
by Nitro Blue Tetrazolium Reduc
-
tion. Tissues were exposed to nitro blue tetrazolium (NBT) to allow
O
2
.
generated by the tissue to reduce the NBT to blue formazan
(Di Wang et al., 1998). Unfixed aortic rings were embedded in OCT
(R. A. Lamb Ltd., East Sussex, UK) and frozen in liquid nitrogen.
Frozen aortas were cryosectioned at 50
␮
m. The sections were then
incubated at 37°C in HEPES-buffered solution (composition as
above) for 1 h and then in HEPES solution containing NBT (100
␮
M)
plus DMSO or quercetin (10
␮
M) in the presence or absence of
DETCA (1 mM) for 90 min, dried, and coverslipped. High contrast
images of the sections were obtained in a DM IRB microscope (Leica,
Wetzlar, Germany) coupled to a Leica DC300F color digital camera.
Statistical Analysis. Data are expressed as means ⫾ S.E.M.; n
indicates the number of experiments. Statistical analysis was per-
formed using Student⬘s t test for paired observations or one-way
852 Lo´ pez-Lo´ pez et al.

analysis of variance followed by a Newman Keuls’ post hoc test.
Differences were considered statistically significant when P was less
than 0.05.
Results
NO Scavenging Effects of Quercetin. Under nominally
anoxic conditions ([O
2
] ⬍ 0.1%), NO, at physiological concen
-
trations (50 nM), decayed very slowly (Fig. 1). The NO decay
was accelerated by increasing concentrations of quercetin
with apparent zero-order kinetics as indicated by the linear
relationship of NO concentration and time. However, the
values of the observed rate constants (k
obs
), after correction
for the spontaneous degradation of NO in DMSO, were lin-
early related to the concentrations of quercetin (Fig. 1, inset).
Thus, the reaction could be fitted to a kinetic relationship
with first-order dependence in quercetin as follows
⫺d[NO]/dt ⫽ k
obs
⫽ k
1
⫻ [quercetin] (1)
The slope of the plot of k
obs
versus [quercetin] yields a
value of k
1
of 2.86 ⫻ 10
⫺7
s
⫺1
with an intercept near zero
(Fig. 1 inset). Because the concentration of quercetin was
almost constant as measured spectrophotometrically during
the 800-s period of study (but see below), a pseudo–zero-order
reaction can be expected.
Dependence on O
2
. Fig. 2
, A and B, shows the depen-
dence of NO decay on O
2
concentrations in the presence of
vehicle or 10
␮
M quercetin, respectively. These plots also
showed pseudo–zero-order kinetics. Because the concentra-
tion of O
2
was not apparently modified, as measured by the
O
2
electrode during the time of the experiment, the kinetics
of this reaction can be approximated to a first-order depen-
dence in O
2
as follows
⫺d[NO]/dt ⫽ k
obs
⫽ k
2
⫻ [O
2
] (2)
When k
obs
values in the presence of 10
␮
M quercetin, after
correction for the spontaneous degradation of NO in DMSO
at the different O
2
concentrations, were plotted against O
2
concentrations, the slope of this plot yielded a value of k
2
of
1.51⫻ 10
⫺6
s
⫺1
, with a near-zero intercept (Fig. 2B, inset).
Thus, a new equation explaining the kinetics of NO consump-
tion depending on both the concentration of quercetin and O
2
can be proposed
⫺d[NO]/dt ⫽ k
3
⫻ [O
2
] ⫻ [quercetin] (3)
where k
3
yields a value of 0.15 M
⫺1
s
⫺1
. The dashed lines in
Fig. 2B show that this equation predicts very accurately the
NO decay at different concentrations of O
2
.
Role of O
2
.
and pH on Quercetin-Induced NO Scav
-
enging Effects. The possible role of O
2
.
produced by querce
-
tin was analyzed by using SOD at 100 U/ml. Figure 3A shows
that SOD strongly reduced the rate of decay of NO induced by
10
␮
M quercetin (P ⬍ 0.01). However, albumin at a concen-
tration 10 times higher than SOD (2.5 mg/ml) or denaturated
SOD (boiled for 20 min) had no effect (not shown). Therefore,
it can be proposed that the mechanism of the NO scavenging
effect of quercetin is related to its oxidation and the genera-
tion of O
2
.
. In fact, quercetin can undergo auto-oxidation
when dissolved in aqueous buffer generating free radicals as
indicated by the generation of 5,5-dimethyl-1-pyrroline-N-
oxide-OH radicals detected by spin resonance spectroscopy
(Canada et al., 1990). Likewise, quercetin generated O
2
.
at
room air as measured by either the luminescence of lucigenin
(Fig. 3B) or by SOD-sensitive cyt c reduction (Fig. 3C). SOD
also strongly inhibited quercetin-induced lucigenin lumines-
cence (e.g., 100 U/ml SOD inhibited the effect of 10
␮
M
quercetin by 78% at pH 9). However, consistent with the
strong dependence on pH of quercetin auto-oxidation (Can-
Fig. 1. NO-scavenging effect of
quercetin in N
2
-saturated solution.
Initial concentration of NO was
50 ⫾ 10 nM and 20
␮
l of vehicle
(DMSO) or quercetin (final concen-
trations of 1–100
␮
M) were added
at time 0. The k
obs
calculated
(mean ⫾ S.E.M., n ⫽ 2, except
DMSO and 10
␮
M quercetin,
where n ⫽ 3), after correction for
the spontaneous degradation of
NO in DMSO, was plotted against
the concentration of quercetin (in-
set).
NO-Scavenging and NO-Protecting Effects of Quercetin 853

ada et al., 1990), generated O
2
.
was clearly observed at pH
9.0, whereas at pH 7.4, the potential O
2
.
generation could not
be detected by either technique, and only a nonsignificant
trend was observed with the cyt c reduction at the highest
concentration of quercetin. We also analyzed the pH depen-
dence of the NO-scavenging effects of quercetin. Figure 3A
shows that small changes in pH result in large changes in NO
consumption (e.g., lowering pH from 7.4 to 7.0 halved the
rate of NO consumption).
Auto-oxidation of quercetin for 48 h produced a change in
the UV-visible spectrum (Fig. 3D) with a reduction in the
absorbance of the bands at 270 and 380 nm. These bands are
supposed to be associated with the light absorption of the
benzoyl (A and B rings) and cinnamoyl systems (B and C
rings) in the flavonol structure, respectively (Wolfbeis et al.,
1984). The rate of quercetin consumption in HEPES-buffered
solution at 37°, pH 7.4, and room air O
2
concentration was
measured at 380 nm, and the process could be fitted to a
monoexponential decay (i.e., first order in quercetin) with a
k
obs
value of 0.0016 M
⫺1
s
⫺1
(Fig. 3E). In the simplest model,
the reaction of O
2
.
generation during quercetin auto-oxidation
can be assumed as a second-order reaction.
d[O
2
.
]/dt ⫽ k
4
⫻ [O
2
] ⫻ [quercetin] (4)
After this equation, the estimated value for k
4
from the plot
in Fig. 3E was 0.12 M
⫺1
s
⫺1
. The theoretical quercetin con
-
sumption under these conditions after eq. 4 is shown in Fig.
3F. The estimated values of k
3
and k
4
were very similar,
suggesting the relationship between eqs. 3 and 4. Therefore,
it can be proposed that O
2
.
generated in eq. 4 reacts rapidly
with NO after the equation.
⫺d[NO]/dt ⫽ k
5
⫻ [O
2
.
] ⫻ [NO] (5)
where k
5
is 5 ⫻ 10
9
M
⫺1
s
⫺1
(Koppenol, 1998). Therefore, the
lower rate of NO decay in the presence of SOD (Fig. 3, A and
B) can be explained by competition of SOD and NO for the
O
2
.
generated by quercetin because SOD dismutates O
2
.
with
a rate constant of 2 ⫻ 10
9
M
⫺1
s
⫺1
, which is in the same
range as k
5
(Koppenol, 1998). Thus, it can be proposed that
the NO-scavenging effects of quercetin occurs in two consec-
utive reactions:
Quercetin ⫹ O
2
O
¡
k
4
O
2
.
⫹ Q* (6)
O
2
.
⫹ NO
O
¡
k
5
ONOO
⫺
(7)
where Q* is the product of quercetin oxidation. However, it
should be noted that because we could not perform a reliable
quantitative measurement of O
2
.
production at pH 7.4, the
stoichiometry of the first reaction and eq. 4 is not guaranteed.
Because O
2
and quercetin concentrations remain fairly
constant during the time of the assay for NO consumption
(200–400 s) as measured with a Clark electrode and spectro-
Fig. 2. Kinetics of NO decay in DMSO (A) and in the presence of quercetin (B) as a function of O
2
concentration (0, 5, 10, and 20%). Initial concentration
of NO was 50 ⫾ 10 nM, and 20
␮
l of vehicle (DMSO) or quercetin (final concentration of 10
␮
M) was added at time 0. The dashed line shows the
predicted NO decay from eq. 1. The k
obs
calculated from three experiments (mean ⫾ S.E.M.), after correction for the spontaneous degradation of NO
in DMSO, was plotted against the concentration of O
2
(B, inset).
854 Lo´ pez-Lo´ pez et al.

photometrically, respectively, the rate of O
2
.
generation by
quercetin in eq. 4 can also be assumed to be constant. In
addition, because O
2
.
consumption in eq. 5 is several orders of
magnitude faster than O
2
.
generation in eq. 4, the latter is the
limiting step in NO consumption; i.e., the NO-scavenging
effects of quercetin are entirely dependent on the rate of its
auto-oxidation.
Computer Simulation. To simulate the kinetics of NO
consumption predicted by eqs. 4 and 5, we employed time
discretization and solved the discrete equations using an
iterative scheme. The discrete forms of the differential eqs. 4
and 5 are shown in eqs. 8 and 9, respectively.
⌬[O
2
.
] ⫽⫺⌬关O
2
兴 ⫽⫺⌬关quercetin兴 ⫽ k
4
⫻ 关O
2
兴 ⫻ 关quercetin兴 ⫻ ⌬t (8)
⫺⌬[NO] ⫽ k
5
⫻ [O
2
.
] ⫻ [NO] ⫻ ⌬t (9)
Then, the values of reagent concentrations after each iter-
ation step k are updated as follows:
[O
2
.
]
k⫹1
⫽ [O
2
.
]
k
⫹ ⌬[O
2
.
] ⫺ ⌬[NO] (10)
[NO]
k⫹1
⫽ [NO]
k
⫺ ⌬[NO] (11)
[O
2
]
k⫹1
⫽ [O
2
]
k
⫺ ⌬[O
2
] (12)
[quercetin]
k⫹1
⫽ [quercetin]
k
⫺ ⌬[quercetin] (13)
Equations 8 to 13 are solved in each iteration step for as
many iterations as required until NO is almost fully con-
sumed. Reproducible results were obtained using discrete
time intervals (⌬t)of10
␮
s or less. An example using 50 nM
NO, 20% O
2
, and 10
␮
M quercetin is shown in Fig. 4
. The
simulation accurately predicted the apparent zero-order re-
action of NO consumption. However, it should be noted that
this is not a true zero-order kinetics process; it just looks like
zero order because in eq. 5, as the concentration of one of the
substrates (NO) is reduced (Fig. 4C), the other (O
2
.
)isin
-
creased (Fig. 4D), so that the product of both, and hence the
Fig. 3. Role of O
2
.
and pH on the NO scavenging effects of quercetin. A, effects of SOD (100 U/ml) and lowering pH from 7.4 to 7.0 on the NO-scavenging
effect of 10
␮
M quercetin at 5% O
2
. The k
obs
calculated from three experiments (mean ⫾ S.E.M.) is shown in the inset. B and C, O
2
.
generation by
quercetin (mean ⫾ S.E.M.) at pH 7.4 and pH 9 measured by lucigenin luminescence (n ⫽ 3 and 6 for pH 7.4 and 9, respectively) (B) or by
SOD-inhibitable reduction of cyt c (n ⫽ 6) (C). D, UV spectra of quercetin (10
␮
M) just after dilution and after 48 h of exposure to room air O
2
at 37°C
(oxidized, dotted line). E, kinetics of quercetin (10
␮
M) oxidation at room air and 100% O
2
concentration measured by its absorbance at 380 nm. The
trace is the average of 2 experiments. F, quercetin (10
␮
M) consumption at room air predicted from eq. 4 where k
4
⫽ 0.15 M
⫺1
s
⫺1
.
NO-Scavenging and NO-Protecting Effects of Quercetin 855

rate of NO consumption, remains fairly constant. The model
is also consistent with apparently constant concentrations of
O
2
and quercetin (Fig. 4, A and B, respectively); e.g., using
the above conditions when NO is fully consumed after 160 s,
quercetin and O
2
were only decreased by 0.6% and 0.02%,
respectively. The model also predicts non–pseudo–zero-order
kinetics at concentrations of NO below 1 nM (Fig. 4E).
Effects of Quercetin on O
2
.
-Induced NO Scavenging.
Quercetin is a well known antioxidant and O
2
.
scavenger
(Robak and Gryglewski, 1988). Thus, we analyzed the effects
of quercetin on NO consumption induced by O
2
.
.O
2
.
was
generated by either XO (5 and 15 mU/ml) using HX as sub-
strate or nonenzymatically with pyrogallol (3 and 10
␮
M).
Figure 5 shows that XO and pyrogallol increased the rate of
NO consumption in a concentration-dependent manner and
that these effects were strongly and similarly reduced by 10
␮
M quercetin.
Effects on NO Bioactivity in Vascular Smooth Mus-
cle. In rat aortic rings, 30 mM KCl induced a sustained
contractile response (984 ⫾ 124 mg, in 11 control arteries).
Addition of NO (70 nM) induced a transient vasodilator effect
that reached a peak relaxant response of 41 ⫾ 4% of the
previous tone in control arteries. After washout with Krebs’
solution containing 30 mM KCl, quercetin (1 and 10
␮
M)
induced a weak vasodilator effect in precontracted aortas
(10 ⫾ 2% for 10
␮
M quercetin). After 20 min of exposure to
quercetin, the peak relaxant effects of NO were inhibited in a
concentration-dependent manner (Fig. 6). This inhibitory ef-
fect, consistent with the NO scavenging effect described
above, was independent of the presence of an intact endothe-
lium (data not shown).
When SOD was included in the bathing media, NO-in-
duced relaxation was only weakly but significantly increased
(Fig. 6). As described above, SOD prevented the inhibitory
effects of quercetin on NO consumption in the bathing media
(Fig. 3A). Furthermore, in the presence of SOD, quercetin not
only failed to reduce the biological activity of NO but also
increased it in a concentration-dependent manner. Further
addition of catalase (500 U/ml) did not modify the results
obtained in the presence of SOD (not shown). Because exog-
enously added SOD cannot be taken by the cells, we hypoth-
esized that quercetin scavenges NO in the bathing media, in
a SOD-inhibitable manner, but not intracellularly, where it
might exert opposite effects (i.e., protection of NO from
O
2
.
-induced inactivation).
The Cu
2⫹
chelator DETCA increases O
2
.
by inhibition of
Fig. 4. Computer simulation of the NO scavenging effects of quercetin.
Quercetin (A), O
2
(B), and NO (C) consumption, O
2
.
concentration (D) and
rate of NO consumption as a function of [NO] (E) predicted from a
computer simulation as described in the text. Initial concentrations of
NO, O
2
, quercetin, and O
2
.
were 50 nM, 20%, 10
␮
M, and 0, respectively.
Values of k
4
and k
5
were 0.15 M
⫺1
s
⫺1
and 5 ⫻ 10
9
M
⫺1
s
⫺1
, respectively.
Fig. 5. NO consumption by the O
2
.
generating systems XO using HX as
substrate (A) and pyrogallol (B) and its prevention by quercetin. Initial
concentration of NO was 50 ⫾ 10 nM, O
2
was 5%, and XO (15 mU/ml) plus
HX (100
␮
M) or pyrogallol (Pyr, 3
␮
M) were added at time 0 in the
presence of DMSO (control) or quercetin (final concentration of 10
␮
M). C,
k
obs
calculated (mean ⫾ S.E.M.) in the presence of XO with 5 (n ⫽ 4) or 15
mU/ml of XO plus HX (n ⫽ 3)or3or10
␮
M pyrogallol (n ⫽ 3) in the
presence of DMSO or quercetin, after correction for the spontaneous
degradation of NO. *, P ⬍ 0.05 quercetin versus DMSO.
Fig. 6. Effects of quercetin (QUER) on NO and sodium nitroprusside
bioactivity in endothelium-denuded aortic rings stimulated by 30 mM
KCl. Rings were challenged with 70 nM NO, then exposed to DMSO (0) or
quercetin (1 or 10
␮
M) in the absence (control) or presence of SOD (100
U/ml) or the SOD inhibitor DETCA (1 mM) for 20 min and the again
exposed to 70 nM NO. The vasodilator response to the second exposure to
NO was expressed as a percentage of the initial one (mean ⫾ S.E.M. of
8–11 experiments). The vasodilator response to nitroprusside (3 nM) was
recorded in KCl-stimulated rings exposed to DMSO (0) or quercetin (10
␮
M) for 20 min. The vasodilator responses to nitroprusside are expressed
(mean ⫾ S.E.M., n ⫽ 7 and 10 for 0 and 10
␮
M quercetin, respectively) as
a percentage of the response in DMSO. *, P ⬍ 0.05 versus DMSO.
856 Lo´ pez-Lo´ pez et al.

endogenous Cu/Zn-SOD activity (Cocco et al., 1981). This
drug (at 1 mM) strongly reduced the biological activity of NO
in rat aortic rings (Fig. 6) but quercetin (1 or 10
␮
M) fully
prevented these inhibitory effects of DETCA.
The vasodilator effects of sodium nitroprusside, as well as
those of NO, are caused by the activation of soluble guanylate
cyclase but, as opposed to those of NO, are unaffected by
exogenously or endogenously generated O
2
.
(Lopez-Lopez et
al., 2001). In contrast to the effects of NO, the vasodilator
effects of 3 nM nitroprusside (31 ⫾ 3% in control arteries)
were unaffected by quercetin (Fig. 6). This suggests that
quercetin is not influencing the pathway for NO/cyclic GMP-
induced vasodilatation beyond the activation of soluble guan-
ylate cyclase.
Simultaneous Measurements of NO Concentrations
and NO Bioactivity. Figure 7 shows the effects of several
drugs on NO-induced relaxation in aortic rings simulta-
neously with NO concentrations measured with the NO elec-
trode introduced in the bathing solution. Quercetin produced
a similar inhibition of both effects. SOD did not increase NO
concentrations, suggesting that O
2
.
concentrations were low
in the whole bath, but increased NO bioactivity, which can be
explained by the reduction of O
2
.
levels within the tissue. As
expected from the above results, in the presence of SOD,
quercetin-induced NO consumption was strongly reduced.
Furthermore, in SOD-treated arteries, not only did quercetin
fail to reduce NO-induced relaxation but also the relaxation
was even increased. All the above results pointed to different
effects of quercetin on NO in the bathing media and within
the tissue. To test this hypothesis, we incubated the aortas
with quercetin for 20 min and then washed the tissues in
quercetin-free media just before the addition of NO, so that
presumably only extracellular quercetin was removed. Under
these conditions (QUER⫹wash; Fig. 7), NO bioactivity in-
creased to an extent similar to that in the presence of SOD,
but NO concentrations were only weakly reduced. Therefore,
intracellular quercetin increased NO bioactivity even in the
absence of exogenously added SOD. The results of exposure
to quercetin followed by washing could be mimicked by ex-
posure to DPI, which inhibits flavin-containing enzymes, in-
cluding the main cellular source of O
2
.
[i.e., NAD(P)H oxidase]
(Di Wang et al., 1998). Opposite results (i.e., unaffected NO
concentration but reduced NO bioactivity) were obtained
with DETCA. Finally, when O
2
.
was increased in the whole
bath by XO plus HX, a parallel reduction in NO concentra-
tions and NO bioactivity was observed.
In Situ Detection of O
2
.
by Nitro Blue Tetrazolium
Reduction. Reduction of NBT by O
2
.
yields the insoluble blue
stain nitro blue formazan. The specificity of this reaction in
the rat aorta has been reported previously (Di Wang et al.,
1998). Incubation of unfixed control (DMSO-treated) aortic
ring sections with NBT (100
␮
M) resulted in light blue stain-
ing (Fig. 8), which was dramatically enhanced by coincuba-
tion with DETCA (1 mM). In the absence of DETCA, the
intensity of the blue staining was similar in sections incu-
bated with or without quercetin (10
␮
M), but a visible quer-
cetin-induced reduction of the staining was observed in DE-
TCA-treated sections.
Discussion
The lifetime of NO in biological systems, in part controlling
its steady-state concentration, reflects both the concentra-
tions and compartmentalization of reactive scavengers. In
this study, in the presence of O
2
and physiological NO con
-
centrations, NO scavenging by quercetin was faster than
previously estimated (Van Acker et al., 1995; Haenen and
Bast, 1999). Moreover, the reaction of NO and quercetin was
much faster than the reaction of NO with O
2
in aqueous
solutions (Ford et al., 1993). However, under physiological
conditions, the NO scavenging effect of quercetin occurs at a
Fig. 7. Effects of several drugs on NO bioactivity (f) in endothelium-
denuded aortic rings simultaneously measured with NO concentrations
(䡺) in the tissue bath: DMSO (n ⫽ 6), quercetin (QUER, 10
␮
M, n ⫽ 6),
SOD (100 U/ml, n ⫽ 5), SOD⫹QUER (n ⫽ 7), DPI (10
␮
M, n ⫽ 6), DETCA
(1 mM, n ⫽ 6), XO (5 U/ml) plus HX (100
␮
M) (XO⫹HX, n ⫽ 5).
QUER⫹wash (n ⫽ 6) indicate arteries exposed to 10
␮
M quercetin and
washed just before addition of NO. The responses to the second exposure
to NO were expressed as a percentage of the initial ones (mean ⫾ S.E.M.).
*, P ⬍ 0.05 versus DMSO. # P ⬍ 0.05 versus QUER.
Fig. 8. Effects of quercetin on O
2
.
production measured by the reduction
of NBT in intact rat aortic sections. Photomicrographs of sections (50
␮
m)
incubated with NBT (100
␮
M) plus DMSO or quercetin (10
␮
M) in the
presence or absence of DETCA (1 mM) for 90 min. Reduction of NBT by
O
2
.
yields the blue stain nitro blue formazan. These images are represen
-
tative of preparations from three rats. Magnification, 10⫻.
NO-Scavenging and NO-Protecting Effects of Quercetin 857

rate much slower than that of oxyhemoglobin or myoglobin
(Gow et al., 1999). The plasma concentrations of quercetin in
humans on a normal diet are in the low micromolar range
(0.3–2.2
␮
M; Scalbert and Williamson, 2000), but quercetin
rapidly penetrates the plasma membrane, reaching intracel-
lular concentrations about 10-fold higher than those in the
extracellular medium (Fiorani et al., 2002). However, these
concentrations are still lower than those of oxyhemoglobin,
which are in the high micromolar/low millimolar range. Nev-
ertheless, based on the different distribution of these scav-
engers (e.g., oxyhemoglobin is restricted to erythrocytes),
Haenen and Bast (1999) speculated that the NO-scavenging
effect of dietary flavonoids might be relevant. On the other
hand, the pseudo–zero-order kinetics on NO indicates that
the lower the NO concentration, the shorter its half-life.
Therefore, quercetin might contribute to limit NO diffusion
in vivo only at low NO concentrations at sites at which other
known NO scavengers are also in low concentrations. How-
ever, given the involvement of quercetin oxidation in its
NO-scavenging effects, this effect in vivo may be limited by
antioxidant enzymes and vitamins.
The kinetics of NO consumption by quercetin were best
fitted by a straight line, indicating apparent zero-order ki-
netics. In contrast, Van Acker et al. (1995), using higher NO
concentrations and single high (50
␮
M) concentrations of the
flavonoid, described their results as pseudo–first-order kinet-
ics, despite the fact that their plotting of ln [NO] versus time
did not appear to be a straight line. The present study dem-
onstrates that quercetin does not directly scavenge NO, but
the reaction involves the oxidation of quercetin and the pro-
duction of O
2
.
which rapidly reacts with NO at near diffusion
rate to produce peroxynitrite (Koppenol, 1998). The strong
inhibitory effect of SOD on quercetin-induced NO consump-
tion supports this conclusion even when it cannot be excluded
that other radicals, such as those derived from quercetin
oxidation, might also react with NO. The proposed reactions
could be simulated in a computer, predicting the pseudo–
zero-order rate at concentrations of NO above 1 nM (Fig. 4E).
The rate of auto-oxidation, and hence O
2
.
production, for
quercetin has been reported to be highly pH-dependent (Can-
ada et al., 1990). Quercetin and related flavonoids are weak
acids. Thus, the lower oxidation at acidic pH seems to reflect
the higher stability of the uncharged phenolic groups of quer-
cetin. Quercetin produced a characteristic 5,5-dimethyl-1-
pyrroline-N-oxide-OH radical, indicating O
2
-derived radical
production, that was detectable only at pH values ⱖ9 and
auto-oxidation, measured by O
2
consumption, at pH values
ⱖ8 (Canada et al., 1990). In the present experiments, quer-
cetin consistently and markedly increased O
2
.
production at
pH 9, although this increase did not reach statistical signif-
icance at pH 7.4. This may reflect the low concentrations of
O
2
.
produced at pH 7.4. The rates of O
2
.
generation (d[O
2
.
]/dt)
by 10 and 100
␮
M quercetin predicted by eq. 4 at pH 7.4, 21%
O
2
, are as low as 18.8 and 188 nM/min, respectively, and the
lower limit of the cyt c reduction technique (as used herein) to
detect O
2
.
in a statistically significant manner is above those
values. Therefore, it seems unfeasible to detect such low O
2
.
concentrations at pH 7.4 using either the lucigenin or the cyt
c approach. However, it should be stressed that because of
the rapid reaction of NO with O
2
.
at low (physiological) NO
concentrations as used herein, a low rate of O
2
.
generation is
sufficient to inactivate NO. In addition, in the absence of NO,
quercetin itself may scavenge its own generated O
2
.
and avoid
the raise in O
2
.
concentrations at neutral pH. Consistent with
the pH dependence of O
2
.
generation, NO consumption by
quercetin was halved when pH was reduced from 7.4 to 7.0
(Fig. 3A).
Quercetin is a well known O
2
.
scavenger (Robak and Gry
-
glewski, 1988). Therefore, its expected protection of NO from
O
2
.
-driven inactivation is opposed to its NO-scavenging ef
-
fects. Our competition studies (Fig. 5) indicate that, under
conditions of increased O
2
.
, quercetin is a better scavenger of
O
2
.
than of NO.
Interestingly, quercetin was much less effective in scav-
enging NO at pH 7 (i.e., the intracellular pH), suggesting a
reduced NO consumption by quercetin intracellularly. On the
other hand, quercetin rapidly accumulates intracellularly
(Fiorani et al., 2002). In the cytosol, NO binds to the heme of
soluble guanylate cyclase with very rapid kinetics (⬎1.4 ⫻
10
8
M
⫺1
s
⫺1
), effectively competing with intracellular scav
-
engers (Zhao et al., 1999). In addition, the cells are endowed
with several mechanisms for both generating and metaboliz-
ing O
2
.
(Wolin et al., 2002), which may modify NO consump
-
tion by quercetin. Thus, it can be expected that the NO-
scavenging effect of quercetin in vivo would be different from
those in cell-free systems. For this reason, we analyzed its
effects on the biological activity of NO using rat aortic rings
as bioassay systems.
The NO consumption by quercetin correlated with a de-
creased NO bioactivity (i.e., vasorelaxation). However, when
this NO consumption in the bathing solution was prevented
by addition of SOD, NO bioactivity was increased. In addi-
tion, when NO consumption in the bath was minimized by
rapidly washing extracellular quercetin, the remaining intra-
cellular quercetin also increased NO bioactivity. Therefore,
under the conditions present in the intracellular medium, the
NO consumption by quercetin seems to be decreased, thus
favoring other mechanisms protecting NO bioactivity. The
lower intracellular NO consumption may exist because quer-
cetin cannot compete with the NO binding to soluble guany-
late cyclase or intracellular scavengers or because of the
presence of endogenous SOD isoforms or other antioxidant
mechanisms. In contrast, quercetin had no effects on the
vasodilator responses induced by sodium nitroprusside
(present study) or by forskolin (Duarte et al., 1993). Nitro-
prusside is not a classic spontaneous NO donor, and we are
unable to detect NO with the amperometric electrode upon
addition of nitroprusside to the bath at concentrations exert-
ing its maximal relaxant response (Lopez-Lopez et al., 2001).
The effects of nitroprusside, as well as those of NO, are
caused by the activation of soluble guanylate cyclase, indi-
cating that quercetin is not influencing the pathway for NO/
cyclic GMP-induced vasodilatation beyond the activation of
soluble guanylate cyclase. However, as opposed to NO, the
effects of nitroprusside are unaffected by exogenously or en-
dogenously generated O
2
.
; e.g., DETCA inhibits NO- but not
nitroprusside-induced relaxation (Lopez-Lopez et al., 2001),
suggesting that the differential effects of quercetin on NO-
and nitroprusside-induced relaxation are caused by changes
in tissue O
2
.
. NO bioactivity, but not extracellular NO con
-
centrations, could also be increased by DPI, which inhibits
flavin-containing enzymes, including the main cellular
source of O
2
.
(i.e., NAD(P)H oxidase). Conversely, NO bioac
-
tivity was reduced by DETCA, an inhibitor of the main de-
858 Lo´ pez-Lo´ pez et al.

grading enzyme of O
2
.
(i.e., Cu/Zn-SOD). Interestingly, quer
-
cetin prevented the reduction of NO bioactivity by DETCA.
Furthermore, in DETCA-treated arteries, quercetin lowered
intracellular O
2
.
as measured by the reduction of NBT in rat
aorta. Taken together, these results strongly suggest that
quercetin protects NO from endogenous O
2
.
-driven inactiva
-
tion and enhances its biological activity. Several mechanisms
may account for this effect, including its direct O
2
.
scavenger
effect (Robak and Gryglewski, 1988). In fact, we have dem-
onstrated that quercetin protects NO from O
2
.
generated by
XO or pyrogallol in a cell-free system. In addition, quercetin
may also inhibit several O
2
.
-generating enzymes such as XO
(Hayashi et al., 1988; Chang et al., 1993) or the neutrophil
membrane NADPH oxidase complex (Tauber et al., 1984).
This finding may help to explain the reversal by long-term
quercetin treatment of the impaired acetylcholine-induced
vasodilatation in spontaneously hypertensive rat (Duarte et
al., 2001), an animal model associated to increased oxidative
status (Suzuki et al., 1995).
In conclusion, at physiological conditions of pH, O
2
concen
-
trations, and NO, quercetin effectively scavenged NO in the
low micromolar range. Analysis of the kinetic data indicated
that the rate-limiting step was the autooxidation of quercetin
and the formation of O
2
.
, which rapidly reacts with NO at a
near diffusion rate. The pseudo–zero-order kinetics of the
reaction together with the concentrations and rate of NO
scavenging of quercetin relative to other physiological NO
scavengers suggests that quercetin might contribute to limit
NO diffusion in vivo only at low NO concentrations and at
sites where other known NO scavengers are in low concen-
trations. Quercetin was apparently a more effective scaven-
ger of O
2
.
than of NO under conditions of increased O
2
.
. When
the extracellular NO scavenging effect of quercetin was pre-
vented, it increased the biological activity of NO, an effect
apparently related to reduced tissue O
2
.
.
Acknowledgments
We are grateful to Dr. Ana Marco (Dept. of Mathematics, Univer-
sidad de Alcala´) for her help with the notations of the equations and
review of the computer simulation. We thank Cristina Rivas for
excellent technical assistance.
References
Benito S, Lopez D, Saiz MP, Buxaderas S, Sanchez J, Puig-Parellada P, and Mit-
javila MT (2002) A flavonoid-rich diet increases nitric oxide production in rat
aorta. Br J Pharmacol 135:910–916.
Canada AT, Giannella E, Nguyen TD, and Mason RP (1990) The production of
reactive oxygen species by dietary flavonols. Free Radic Biol Med 9:441– 449.
Chang WS, Lee YJ, Lu FJ, and Chiang HC (1993) Inhibitory effects of flavonoids on
xanthine oxidase. Anticancer Res 13:2165–2170.
Chiesi M and Schwaller R (1995) Inhibition of constitutive endothelial NO-synthase
activity by tannin and quercetin. Biochem Pharmacol 14:495–501.
Cocco D, Calabresse L, Rigo A, Argese E, and Rotillo G (1981) Re-examination of the
reaction of diethyldithiocarbamate with the copper of superoxide dismutase. J Biol
Chem 256:8983– 8996.
Di Wang H, Pagano PJ, Du Y, Cayatte AJ, Quinn MT, Brecher P, and Cohen RA
(1998) Superoxide anion from the adventitia of the rat thoracic aorta inactivates
nitric oxide. Circ Res 82:810– 818.
Duarte J, Perez-Vizcaino F, Zarzuelo A, Jimenez J, and Tamargo J (1993) Vasodi-
lator effects of quercetin on isolated rat vascular smooth muscle. Eur J Pharmacol
239:1–7.
Duarte J, Perez-Palencia R, Vargas F, Ocete MA, Perez-Vizcaino F, Zarzuelo A, and
Tamargo J (2001) Antihypertensive effects of the flavonoid quercetin in spontane-
ously hypertensive rats. Br J Pharmacol 133:117–124.
Duarte J, Jimenez R, O’Valle F, Galisteo M, Perez-Palencia R, Vargas F, Perez-
Vizcaino F, Zarzuelo A, and Tamargo J (2002) Protective effects of the flavonoid
quercetin in chronic nitric oxide deficient rats. J Hypertension 20:1843–1854.
Fiorani M, De Sanctis R, De Bellis R, and Dacha M (2002) Intracellular flavonoids as
electron donors for extracellular ferricyanide reduction in human erythrocytes.
Free Radic Biol Med 32:64–72.
Ford PC, Wink DA, and Stanbury DM (1993) Autoxidation kinetics of aqueous nitric
oxide. FEBS Lett 326:1–3.
Gow AJ, Luchsinger BP, Pawloski JR, Singel DJ, and Stamler JS (1999) The
oxyhemoglobin reaction of nitric oxide. Proc Natl Acad Sci USA 96:9027–9032.
Haenen GRMM and Bast A (1999) Nitric oxide radical scavenging of flavonoids.
Methods Enzymol 301:490 –503.
Hayashi T, Sawa K, Kawasaki M, Arisawa M, Shimizu M, and Morita N (1988)
Inhibition of cow’s milk xanthine oxidase by flavonoids. J Nat Prod 51:345–348.
Hayek T, Fuhrman B, Vaya J, Rosenblat M, Belinky P, Coleman R, Elis A, Aviram
M (1997) Reduced progression of atherosclerosis in apolipoprotein E-deficient mice
after consumption of red wine, or its polyphenols quercetin or catechin, is associ-
ated with reduced susceptibility of LDL to oxidation and aggregation. Arterioscler
Thromb Vasc Biol 17:2744 –2752.
Hertog MGL, Feskens EJM, Hollman PCH, Katan MB, and Kromhout D (1993)
Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen
Elderly Study. Lancet 342:1007–1111.
Koppenol WH (1998) The basic chemistry of nitrogen monoxide and peroxynitrite.
Free Rad Biol Med 25:385–391.
Lopez-Lopez JG, Perez-Vizcaino F, Cogolludo AL, Ibarra M, Zaragoza-Arnaez F, and
Tamargo J (2001) Nitric oxide- and nitric oxide donors-induced relaxation and its
modulation by oxidative stress in piglet pulmonary arteries. Br J Pharmacol
133:615–624.
Middleton E, Kandaswami C, and Theoharides CT (2000) The effects of plant
flavonoids on mammalian cells: implications for inflammation, heart disease and
cancer. Pharmacol Rev 52:673–751.
Perez-Vizcaino F, Ibarra M, Cogolludo AL, Duarte J, Zaragoza-Arnaez F, Moreno L,
Lopez-Lopez JG, and Tamargo J (2002) Endothelium-independent vasodilator
effects of the flavonoid quercetin and its methylated metabolites in rat conduc-
tance and resistance arteries. J Pharmacol Exp Ther 302:66 –72.
Rimm ER, Katan MB, Ascherio A, Stampfer M, and Willet W (1996) Relation
between intake of flavonoids and risk for coronary heart disease in male health
professionals. Ann Intern Med 125:384–389.
Robak J and Gryglewski RJ (1988) Flavonoids are scavengers of superoxide anions.
Biochem Pharmacol 37:837– 841.
Sampson L, Rimm E, Hollman PC, de Vries JH, and Katan MB (2002) Flavonol and
flavone intakes in US health professionals. J Am Diet Assoc 102:1414 –1420.
Scalbert A and Williamson G (2000) Dietary intake and bioavailability of polyphe-
nols. J Nutr 130:2073S–2085S.
Shutenko Z, Henry Y, Pinard E, Seylaz J, Potier P, Berthet F, Girard P, and
Sercombe R (1999) Influence of the antioxidant quercetin in vivo on the level of
nitric oxide determined by electron paramagnetic resonance in rat brain during
global ischemia and reperfusion. Biochem Pharmacol 57:199 –208.
Suzuki H, Swei A, Zweifach BW, and Schmid-Schoenbein GW (1995) In vivo evidence
for microvascular oxidative stress in spontaneously hypertensive rats: hydroethi-
dine microfluorography. Hypertension 25:1083–1089.
Tauber AI, Fay JR, and Marletta MA (1984) Flavonoid inhibition of the human
neutrophil NADPH-oxidase. Biochem Pharmacol 33:1367–1369.
Van Acker SA, Tromp MN, Haenen GR, van der Vijgh WJ, and Bast A (1995)
Flavonoids as scavengers of nitric oxide radical. Biochem Biophys Res Commun
214:755–759.
Van Acker SA, Van-den Berg DJ, Tromp MNJL, Griffioen DH, van Bennekom WP,
van der Vijgh WJf, and Bast A (1996) Structural aspects of antioxidant activity of
flavonoids. Free Radic Biol Med 20:331–342.
Wolfbeis OB, Leiner M, and Hochmuth P (1984) Absorption and fluorescence spectra,
pKa values and fluorescence lifetimes of monohydroxyflavones and monomethoxy-
flavones. Ber Bunsenges Phys Chem 88:759 –767.
Wolin MS, Gupte SA, and Oeckler RA (2002) Superoxide in the vascular system.
J Vasc Res 39:191–207.
Zhao Y, Brandish PE, Ballou DP, and Marletta MA (1999) A molecular basis for
nitric oxide sensing by soluble guanylate cyclase. Proc Natl Acad Sci USA 96:
14753–14758.
Address correspondence to: Francisco Pe´rez-Vizcaı´no, Department of Phar-
macology, School of Medicine, University Complutense of Madrid, 28040 Ma-
drid, Spain. E-mail: fperez@med.ucm.es
NO-Scavenging and NO-Protecting Effects of Quercetin 859