Formation of nitric oxide, ethyl nitrite and an oxathiolone derivative of caffeic acid in a mixture of saliva and white wine

Article abstract of DOI:10.3109/10715760903486057

Reactions of salivary nitrite with components of wine were studied using an acidic mixture of saliva and wine. The formation of nitric oxide (NO) in the stomach after drinking wine was observed. The formation of NO was also observed in the mixture (pH 3.6) of saliva and wine, which was prepared by washing the oral cavity with wine. A part of the NO formation in the stomach and the oral cavity was due to the reduction of salivary nitrite by caffeic and ferulic acids present in wine. Ethyl nitrite produced by the reaction of salivary nitrite and ethyl alcohol in wine also contributed to the formation of NO. In addition to the above reactions, caffeic acid in wine could be transformed to the oxathiolone derivative, which might have pharmacological functions. The results obtained in this study may help in understanding the effects of drinking wine on human health.

Full text of DOI:10.3109/10715760903486057

Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: http://www.tandfonline.com/loi/ifra20
Formation of nitric oxide, ethyl nitrite and an
oxathiolone derivative of caffeic acid in a mixture
of saliva and white wine
Umeo Takahama, Mariko Tanaka & Sachiko Hirota
To cite this article: Umeo Takahama, Mariko Tanaka & Sachiko Hirota (2010) Formation of
nitric oxide, ethyl nitrite and an oxathiolone derivative of caffeic acid in a mixture of saliva and
white wine, Free Radical Research, 44:3, 293-303
To link to this article: http://dx.doi.org/10.3109/10715760903486057
Published online: 19 Feb 2010.
Submit your article to this journal
Article views: 16
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ifra20
Download by: [University of California, San Diego]
Date: 15 November 2015, At: 21:05

Free Radical Research, March 2010; 44(3): 293–303
Formation of nitric oxide, ethyl nitrite and an oxathiolone derivative
of caffeic acid in a mixture of saliva and white wine
UMEOTAKAHAMA1, MARIKOTANAKA1 & SACHIKO HIROTA2
1Department of Bioscience, Kyushu Dental College, Kitakyushu 803-8580, Japan, and 2Department of Nutritional Science,
KyushuWomen’s University, Kitakyushu 807-8586, Japan
(Received date: 27 June 2009; in revised form date: 5 November 2009)
Abstract
Reactions of salivary nitrite with components of wine were studied using an acidic mixture of saliva and wine. The forma-
tion of nitric oxide (NO) in the stomach after drinking wine was observed.The formation of NO was also observed in the
mixture (pH 3.6) of saliva and wine, which was prepared by washing the oral cavity with wine. A part of the NO formation
in the stomach and the oral cavity was due to the reduction of salivary nitrite by caffeic and ferulic acids present in wine.
Ethyl nitrite produced by the reaction of salivary nitrite and ethyl alcohol in wine also contributed to the formation of NO.
In addition to the above reactions, caffeic acid in wine could be transformed to the oxathiolone derivative, which might have
pharmacological functions. The results obtained in this study may help in understanding the effects of drinking wine on
human health.
Keywords: Ethyl nitrite, nitric oxide (NO), nitrite, oxathiolone derivative, saliva, stomach, wine
Abbreviations: AU, arbitrary unit; DTCS, N-(dithiocarboxy)sarcosine sodium salt.
Introduction
as a component of foods [8] and secreted into the oral
Wine has health-promoting properties and beneficial
effects on certain diseases, including heart disease
and cancer, which in turn are attributed to the anti-
oxidative function of phenolics in wine [1,2]. The
phenolics include a phenylacetic and hydroxycin-
namic acids (Figure 1A) and flavonoids. Recently, it
has been reported that the ingestion of red wine
results in an increase in the concentration of nitric
oxide (NO) in the stomach and this increase has been
proposed to be due to the reduction of salivary nitrite
by phenolics in wine [3] (Figure 1B). The possibility
of the formation of NO in the stomach by the reduc-
tion of salivary nitrite by dietary phenolics has also
been reported [4–7].The phenolics comprise querce-
tin, rutin, caffeic acid and chlorogenic acid [3–7].
Nitrite required for NO formation is produced in the
oral cavity by the reduction of nitrate,which is ingested
cavity as a component of saliva, by nitrate-reducing
bacteria [9–12].The nitrite produced in the oral cav-
ity is transformed into nitrous acid (pKaꢀ3.3) in the
stomach so that it can be reduced to NO by phenolics
[3–7]. NO formed in the stomach can increase the
gastric mucosal blood flow and the mucus thickness
[13,14]. When alcoholic beverages are ingested, in
addition to NO, ethyl nitrite, which can function as a
vasodilator [15], may also be produced in the stom-
ach by the reaction of ethyl alcohol with nitrous acid
(Figure 1C).The production of ethyl nitrite has been
reported in the acidic mixture of saliva and ethyl alco-
hol [15,16].
Thiocyanate is also a component of saliva and func-
tions as a substrate of salivary peroxidase producing
an antimicrobial component hypothiocyanate ion
(OSCN–) in the oral cavity [17]. In the stomach,
Correspondence: Umeo Takahama, Department of Bioscience, Kyushu Dental College, Kitakyushu 803-8580, Japan. Email: takahama@
kyu-dent.ac.jp
ISSN 1071-5762 print/ISSN 1029-2470 online © 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.3109/10715760903486057

294 U. Takahama et al.
(A) Phenolic compounds found in this study
ethyl nitrite in the mixture of saliva and wine in the
oral cavity.
HO
R
HO
O
O
H3CO
OH
Materials and methods
OH
R = H: p-Coumaric acid
Reagents and wine
4-Hydroxy-3-methoxyphenylacetic acid
R = OH: Caffeic acid
R = H3CO: Ferulic acid
N-(Dithiocarboxy)sarcosine sodium salt (DTCS) was
obtained from Dojin (Kumamoto, Japan). 4-Hydroxy-
3-methoxyphenylacetic acid, 3,4-dihydroxybenzalde-
hyde, p-coumaric acid, caffeic acid, ferulic acid,
vanillin and ethyl nitrite were obtained from Wako
Pure Chem. Ind. (Osaka, Japan). California white
wine (table wine; pH ∼3.3) was obtained from a local
market. According to the information available from
the label on the bottle, the alcohol concentration of
this wine is 9.5% (v/v).
(B) Reduction of nitrous acid to NO by phenolics
.
HNO2 + PhOH
NO + PhO + H2O
.
(PhOH, Phenolics; PhO , phenoxy radicals)
(C) Reaction of ethanol with nitrous acid
CH3H2COH + HNO2
CH3CH2CONO + H2O
(D) Formation of an oxathiolone derivative of caffeic acid
HNO2
O
O
HO
HO
R
R
1
1
NO + H2O
NO in expelled air
(2)
(1)
It has been reported that wine induces the production
of NO in the stomach [3]. In this study, we, at first,
performed experiments to confirm the above-
mentioned result. Healthy volunteers (age range,
50–60 years) were fasted for 4 h after they had their
lunch. During the fasting, they could consume only
water whenever required.The voluntary regurgitation
of air was collected in a gas-tight bag after an intake
of 300 ml of carbonated water (Suntory Foods;Tokyo,
Japan) under fasting conditions. Fifteen-to-thirty
minutes after the collection of the expelled air, 150 ml
of white wine was ingested.Carbonated water (300 ml)
was given again 10–15 min after the ingestion of white
wine to collect the air expelled from the stomach in
the gas-tight bag.The protocol to collect the expelled
air was designed according to the methods described
in previous studies [3,24]. NO in the gas-tight bag was
quantified using a nitrogen oxide detector tube (11L,
Gastec;Ayase, Japan).The detector can detect (NO ꢁ
NO2) in the concentration range from 0.2–5 ppm.
HSCN
O
R
=
1
OH
H2O
HO
HO
R
O
1
HO
R
1
S
NH3
SCN
O
(4)
(3)
Figure 1. Chemical structures and reactions concerned in this
study. (1), (2), (3) and (4) in (D) are caffeic acid, its o-quinone
form, 2-thiocyanatecaffeic acid and oxathiolone derivative of caffeic
acid, respectively.
SCN– reacts with nitrous acid, producing nitrosyl
thiocyanate (ONSCN) that can be reduced rapidly
to NO by a component of gastric juice, ascorbic
acid[18].Inaddition,SCN– canreactwitho-quinones,
which are produced in the nitrous acid-dependent
oxidation of o-dihydroxyphenolics such as chloro-
genic acid and rutin, under acidic conditions [19,20].
The initial reaction products are 2-thiocyanate con-
jugates of chlorogenic acid and rutin that are trans-
formed to the oxathiolone derivatives by hydrolysis
[19,20]. Caffeic acid (Figure 1A), which has a cate-
chol group, is present in wine [21–23]. This suggests
that, in addition to NO, 2-thiocyanate conjugates of
caffeic acid, which could transform to an oxathiolone
derivative of caffeic acid, might be formed in the
stomach after drinking wine (Figure 1D).The acidity
of wine also led us postulate that NO, ethyl nitrite
and an oxathiolone derivative of caffeic acid might be
produced in the oral cavity after drinking wine.
Measurements of electron spin resonance spectra
Electron spin resonance (ESR) spectra were measured
using an ESR spectrometer JE1XG (JOEL, Tokyo,
Japan) at 25°C using a quartz flat cell (50 μl) under
the following conditions [4,5]: microwave power,
10 mW;scanning speed,5 mT/min;line width,0.5 mT;
and amplification, 250- or 1000-fold. NO produced
in the mixture of nitrite and wine was trapped by
Fe(DTCS)2. The Fe(DTCS)2 solution was prepared
by adding 0.03 ml of 100 mM FeCl3 to 1 ml of 10 mM
DTCS that was dissolved in 0.1 M sodium phosphate
(pH 7.6).
The objectives of this study were to investigate the
formation of ethyl nitrite and an oxathiolone deriva-
tive of caffeic acid in the stomach after the ingestion
of wine, confirming the formation of NO in the
stomach, and to investigate the formation of NO and
The formation of NO was studied in the following
reaction mixtures (Figure 2). (A) White wine itself
(pH 3.3; 0.25 ml) was incubated for 1 min after the

Formation of nitric oxide after drinking wine 295
mixture.The pH after the addition of the Fe(DTCS)2
was 6.6. As a control, the oral cavity was washed for
30 s with 5 ml of 50 mM citric acid/sodium citrate
buffer (pH 3.3). The volume of the mixture of saliva
and citrate buffer (in the following, the mixture of
saliva/citrate buffer) was ∼5.6 ml and the pH was
∼3.6. After keeping the mixture of saliva/citrate buffer
for a further 30 s, 0.25 ml of Fe(DTCS)2 was added
to 0.25 ml of the mixture. After the addition of
Fe(DTCS)2, the pH was 6.3. (D)The oral cavity was
washed with wine or citrate buffer as (C) and the pH
of the mixtures of saliva/wine and saliva/citrate buffer
was adjusted to 2 by adding a small volume of 1 M
HCl simulating the mixture of saliva and gastric juice.
The mixtures of pH 2 were incubated for 30 s and
then Fe(DTCS)2 (0.5 ml) was added to 0.25 ml of
the each mixture of saliva/wine and saliva/citrate buf-
fer (pH 2) to measure the ESR spectra. After the
addition of Fe(DTCS)2, the pH values were in the
range of 6.6–6.8.
(A)
(B)
Wine +
50 mM KCl-HCl (pH 1.3)
Wine (pH 3.3)
+ nitrite
Mixture (pH 1.9)
+ nitrite
Incubation
Incubation
+ Fe(DTCS)2
+ Fe(DTCS)2
Measurment of
ESR signal
Measurment of
ESR signal
(D)
(C)
Washing oralcavity
with wine or
citrate/citric acid
buffer (pH 3.3)
Washing oralcavity
with wine or
citrate/citric acid
buffer (pH 3.3)
HPLC
Formation of ethyl nitrite and changes in the concen-
tration of phenolics in wine were studied using a
Shim-pack CLC-ODS column (6 mm i.d. ꢂ 15 cm;
particle size, 5 μm) combined with a spectrophoto-
metric detector with a photodiode array (SPD M10A)
(Shimadzu; Kyoto, Japan).The reaction mixture con-
tained 1 mM NaNO2 in wine (pH 3.3) or in the mix-
ture of 0.5 ml of 50 mM KCl-HCl buffer (pH 1.3),
the pH of which was 1.94. After incubation for 1 min,
10 μl of each reaction mixture was applied to the
HPLC column described above. Ethyl nitrite was
separated using a mixture of methanol and 25 mM
KH2PO4 (3:2, v/v) as the mobile phase and detected
at 350 nm. Phenolics were separated using the mix-
ture of methanol and 25 mM KH2PO4 (1:2, v/v) as
the mobile phase and detected at 280 nm.
The flow rate of the mobile phases was 1 ml/min.
Products that were formed by the reaction of nitrite
with caffeic acid were also studied using the above-
mentioned HPLC system.The mobile phase used was
a mixture of methanol and 25 mM KH2PO4 (1:2, v/v),
and the flow rate was 1 ml/min.The reaction mixture
contained 0.1 mM caffeic acid and 0.1 mM NaNO2
in 50 mM KCl-HCl buffer (pH 2.0) or 0.1 mM caf-
feic acid and 0.2 mM NaNO2 in 50 mM sodium
citrate-HCl buffer (pH 3.3). After incubation for
defined periods, an aliquot (20 μl) of each reaction
mixture was applied to the HPLC column.
Mixture of saliva/wine
or saliva/citrate
(pH 3.6)
Mixture of saliva/wine
or saliva/citrate
(pH 3.6)
+ HCl
pH 2
Incubation
Incubation
+ Fe(DTCS)2
+ Fe(DTCS)2
Measurment of
ESR signal
Measurment of
ESR signal
Figure 2. Procedures to prepare the mixtures for measurement of
ESR spectra.
addition of various concentrations of NaNO2 and
then 0.25 ml of Fe(DTCS)2 was added. After the
addition of Fe(DTCS)2, the pH was 6.6. (B) the
white wine (0.125 ml) was mixed with 0.125 ml of
50 mM KCl-HCl buffer (pH 1.3). The pH of the
mixture was 1.94. After the addition of NaNO2, the
mixture was incubated for 1 min and then Fe(DTCS)2
(0.25ml)wasadded.Aftertheadditionof Fe(DTCS)2,
the pH was 6.6. (C) The oral cavity was washed for
30 s with 5 ml of white wine and the wine was spat
out into a beaker.The solution obtained by the wash-
ing should contain saliva and other components in
the oral cavity. In the following sections, the spit
obtained by the washing is referred to as the mixture
of saliva/wine.The volume and the pH of the mixture
of saliva/wine were ∼5 ml and ∼3.6, respectively. After
keeping the mixture of saliva/wine for a further 30 s,
0.25 ml of Fe(DTCS)2 was added to 0.25 ml of the
Preparation of an oxathiolone derivative of caffeic acid
The oxathiolone derivative of chlorogenic acid [(E)-
5’-(3-(7-hydroxy-2-oxobenzo[d][1,3]oxathiol-4-yl)
acryloyloxy)quinic acid] was prepared according to

296 U. Takahama et al.
the method reported previously [19]. One milligram
of the compound was suspended in 2 ml of 2 M HCl
and incubated for 30 min at ∼92°C. After extraction
of the acidic solution with ethyl acetate, the ethyl
acetate fraction was analysed by HPLC using the
ODS column described above. The mobile phase
used was a mixture of methanol and 25 mM KH2PO4
(1:2, v/v). The results showed that the oxathiolone
derivative of chlorogenic acid, which had a retention
time of ∼10.3 min and absorption peaks at 230 and
309 nm in the above mobile phase, was transformed
to a new component. The new component had a
retention time of 16.5 min and absorption peaks at
227 and 293 nm. The component was considered to
be the oxathiolone derivative of caffeic acid because
the quinic acid moiety of the oxathiolone derivative
of chlorogenic acid would undergo hydrolysation dur-
ing the incubation under acidic conditions, producing
the oxathiolone derivative of caffeic acid.
might be mainly detected by the nitrogen oxide detec-
tor tube used in this study; the half-life of 1 ppm of
gaseous NO in the atmosphere was calculated to be
∼56 h using the rate constant of 1.4 ꢂ 104 M–2 s–1
[25]. Even if NO2 was produced by the reaction of
NO with O2, the concentration of NO2 would be
decreased by the reaction with NO leading to the
formation of N2O3.The concentration of NO detected
in the air expelled from the stomach was higher than
0.2 ppm and NO was not detected in the air exhaled
by breathing in this study,suggesting that NO detected
in this study was mainly from the stomach. It has been
reported that the concentration of NO in the air
exhaled by breathing is 4 ꢃ 1 ppb and the air expelled
from the stomach ranges from 0.6–2.6 ppm [24], sup-
porting the idea that NO detected in this study was
from the stomach.
Formation of NO in the mixture of saliva/wine
Figure 3 shows typical data for the formation of NO
in the mixtures of saliva/citrate buffer and saliva/wine
prepared by washing the oral cavity with citrate buffer
and wine, respectively, as shown in Figure 2C. No clear
ESR signal of NO-Fe(DTCS)2 was detected when
Fe(DTCS)2 was added to the mixture of saliva/citrate
Detection of 2-thiocyanatecaffeic acid
Mixed whole saliva was collected and filtered as
described previously [20].The mixture of whole saliva
filtrate (0.5 ml), wine (0.5 ml) and 50 mM KCl-HCl
buffer (pH 1.3, 1.0 ml) was prepared and the pH was
determined to be 1.9. Caffeic acid (10 μM), sodium
nitrite (0.1 mM) and NaSCN (0.5 mM) were added
when required. After the incubation for 10 min at
room temperature (∼ 25°C),the mixture was extracted
twice with 5 ml of ethyl acetate. The ethyl acetate
extracts were combined and dehydrated with sodium
sulphate. After the evaporation of ethyl acetate with a
rotary evaporator, the residue was dissolved in 0.2 ml
of the mixture of methanol and 25 mM KH2PO4
(1:2, v/v). An aliquot (20 μl) of the solution was
applied to the HPLC column described above to
separate 2-thiocyanate caffeic acid. The mixture of
methanol and 25 mM KH2PO4 (1:2, v/v) was used
as the mobile phase and the flow rate was 1 ml/min.
Results and discussion
Wine-induced increase in NO concentration in
expelled air
The concentration of (NO ꢁ NO2) in the air expelled
from the stomach increased from 0.70 ꢃ 0.51 ppm
to 2.04 ꢃ 1.48 ppm (nꢀ7: pꢀ0.039, paired t-test) by
the ingestion of white wine. This result is consistent
with a previous report that the ingestion of red wine
increases the concentration of NO in the air expelled
from the stomach [3]. In this study, NO might
be mainly detected by the (NO ꢁ NO2) detector tube
because it is a primary reaction product of nitrite
under the conditions of this study. The slow reaction
of gaseous NO with gaseous O2 may support that NO
Figure 3. Concentration of NO in spit prepared after washing the
oral cavity with acidic buffer or wine. (A)Washing with citrate/citric
acid buffer; (B) washing with wine; (C) after washing with citrate/
citric acid buffer, the pH of the spit was decreased to 2; (D) after
washing with wine, the pH of the spit was decreased to 2; (E) as
in (D) except for the addition of 0.1 mM NaNO2 just after the
decrease in pH. ESR spectra were started recording 2 min after the
addition of Fe(DTCS)2. Amplification, 1000-fold.

Formation of nitric oxide after drinking wine 297
buffer (trace A), whereas the signal was detected
when Fe(DTCS)2 was added to the mixture of saliva/
wine (trace B). The signal at 333.3 mT was from
Fe(DTCS)2 itself. When the pH of the mixture of
saliva/citrate buffer was decreased to 2, as in Figure
2D, a very weak signal of NO-Fe(DTCS)2 was
detected by the addition of Fe(DTCS)2 (trace C),
whereas when the pH of the mixture of saliva/wine
was decreased to 2, an intense signal was detected by
the addition of Fe(DTCS)2 (trace D). The signal
intensity of NO-Fe(DTCS)2 at pH 2 was stronger
than that at pH 3.6, indicating that the formation of
NO was faster at pH 2 than at 3.6. The addition of
0.1 mM nitrite to the mixture of saliva/wine (pH 2)
resulted in an enhanced formation of NO-Fe(DTCS)2
(trace E). The concentration of nitrite in the mixture
of saliva/wine was estimated to be 0.06 mM by com-
paring the signal intensity before and after the addi-
tion of 0.1 mM nitrite (Figure 3, traces D and E).
The result in Figure 3 indicates that NO was pro-
duced if wine was mixed with saliva in the oral cavity
and that the production of NO could continue after
swallowing the mixture into the stomach. Further-
more, the above-mentioned result suggests that sali-
vary nitrite contributed to the formation of NO.
The intensity of ESR signal of NO-Fe(DTCS)2 just
after the addition of Fe(DTCS)2 to the reaction mix-
ture would reflect the concentration of NO in the
mixture, and the signal intensity of NO-Fe(DTCS)2
would be kept constant if NO production ceased after
the addition of Fe(DTCS)2. However, a slow increase
in the intensity of the ESR signal was observed in
trace B in Figure 3 after the addition of Fe(DTCS)2,
and the rate was ∼0.1 arbitrary unit per min (AU/min).
Such a slow increase in the intensity of the signal was
also observed in trace E in Figure 3 and the rate was
∼0.6 AU/min. The slow increase in the ESR signal
will be discussed later.
nitrite with ethyl alcohol in wine. Possibility (iii) can
be excluded for the slow increase because the reaction
between nitrite and ethyl alcohol producing ethyl
nitrite does not proceed when the pH is higher
than 4 [15].
Nitrite (0.2 or 1 mM) was added to 25 mM citrate/
citric acid buffer (pH 3.3). After incubation for 1 min,
Fe(DTCS)2 was added. A small ESR signal of NO-
Fe(DTCS)2 was observed but the subsequent increase
in the signal of NO-Fe(DTCS)2 was not observed
(Figure 4, upper panel, closed circles). When nitrite
was added to 50 mM KCl-HCl buffer (pH 2.0) fol-
lowed by Fe(DTCS)2, a weak ESR signal of NO-
Fe(DTCS)2 was observed and the intensity of the
signal of NO-Fe(DTCS)2 did not increase subse-
quently (Figure 4, lower panel, closed circles).
Wine (pH 3.3)
1 mM NaNO
2
80
60
40
20
0.4 mM NaNO
2
0.2 mM NaNO
2
0.1 mM NaNO
0.2 mM NaNO
2
2
1 mM NaNO
2
0
6
0
2
4
8
10
140
Wine:50 mM KCl-HCl (pH 1.3) = 1:1 (v/v)
(final pH 1.94)
120
100
80
1 mM NaNO
2
Nitrite-induced formation of NO in wine
60
0.4 mM NaNO
2
Salivary nitrite seemed to contribute to the NO for-
mation in the mixture of saliva/wine. Next, we studied
the effects of nitrite on the formation of NO in wine
itself. No NO-Fe(DTCS)2 was detected in the mix-
ture of wine and Fe(DTCS)2, the pH of which was
6.6.After mixing Fe(DTCS)2 with wine, 1 mM nitrite
was added and then the ESR spectra were measured.
The ESR signal of NO-Fe(DTCS)2 was not detected
immediately after the addition of nitrite, but the sig-
nal intensity increased slowly during the incubation
at the rate of 0.2 AU/min. There were three possi-
bilities for the slow increase in the ESR signal inten-
sity of NO-Fe(DTCS)2: (i) self-decomposition of nitrite
producing NO, (ii) the reduction of nitrite to NO by
the reductants in wine, and (iii) the decomposition of
ethyl nitrite that was produced by the reaction of
40
0.2 mM NaNO
2
20
1 mM NaNO
2
0.2 mM NaNO
2
0
4
6
0
2
8
10
Time after the addition of Fe(DTCS) (min)
2
Figure 4. Time courses of nitrite-induced formation of NO-
Fe(DTCS)2 in wine. Upper panel. z, 25 mM citrate/citric acid
buffer (pH 3.4); {, wine itself (pH 3.3). Lower panel. z, 50 mM
KCl-HCl buffer (pH 2.0); {, mixture of wine and 50 mM KCl-
HCl buffer (pH 1.3) (1:1, v/v). Concentrations of nitrite shown in
the figure are the concentrations before the addition of Fe(DTCS)2.
Amplification, 250-fold.

298 U. Takahama et al.
The results indicate that the conversion of nitrite
to NO by self-decomposition could proceed at pH
values of 2 and 3.3 and that the self-decomposition
producing NO did not proceed after the addition
of Fe(DTCS)2, excluding possibility (i) and remain-
ing possibility (ii) for the slow formation of
NO-Fe(DTCS)2 that was observed by the addition of
1 mM nitrite to the mixture of Fe(DTCS)2 and wine,
the pH of which was pH 6.6.
in Figure 4 could be explained by possibility (iii),
namely, the decomposition of ethyl nitrite.
Taking the above-mentioned discussion into con-
sideration, the slow formation of NO-Fe(DTCS)2
after the addition of Fe(DTCS)2 to the acidic mixture
of saliva/wine (see description related to Figure 3) can
be explained as below. The concentrations of nitrite
in the mixtures of saliva/wine were estimated to be
0.06 and 0.16 mM in traces B and E, respectively
(Figure 3). If the slow formation of NO-Fe(DTCS)2
proceeded by possibility (ii), namely, by the reduction
of nitrite to NO by the reductants in wine after the
addition of Fe(DTCS)2 to the mixture of saliva/wine,
the rates of the slow formation of NO-Fe(DTCS)2
in traces B and E can be calculated to be 0.012
and 0.032 AU/min, respectively, using the rate of
NO-Fe(DTCS)2 formation in the presence of 1 mM
nitrite (0.2 AU/min, see above). The calculated rates
were much smaller than the rates of the slow forma-
tion of NO-Fe(DTCS)2 observed by the addition of
Fe(DTCS)2 to the mixture of saliva/wine (0.1 and
0.63 AU/min in traces B and E, respectively).
The result suggests that the slow formation of NO-
Fe(DTCS)2 observedaftertheadditionofFe(DTCS)2
to the mixture of saliva/wine might also be explained
by possibility (iii) (production of NO by the decom-
position of ethyl nitrite) but not by possibility (ii)
(reduction of nitrite to NO by the reductants in wine).
Since ethyl nitrite seemed to contribute to the slow
formation of NO-Fe(DTCS)2, we studied the nitrite-
induced formation of ethyl nitrite using wine.
Wine was incubated for 1 min after the addition of
nitrite, and then mixed with Fe(DTCS)2, as shown in
Figure 2A. The mixing of the wine with Fe(DTCS)2
resulted in the detection of the ESR signal of NO-
Fe(DTCS)2 (Figure 4, upper panel, open circles),
indicating the production of NO in the nitrite-
containing wine before the addition of Fe(DTCS)2.
Time courses of the increase in the ESR signal inten-
sity after the mixing of Fe(DTCS)2 with the nitrite-
containing wine showed slow increases in the intensity
of the ESR signal of NO-Fe(DTCS)2 following the
initial rapid increase. The initial intensity in the ESR
signal of NO-Fe(DTCS)2, which could be estimated
by the extrapolation of the slow increase to time zero
and would reflect the concentration of NO just before
the addition of Fe(DTCS)2, was dependent on the
concentration of nitrite added. The rate of the slow
increase was also dependent on the concentration of
nitrite added, and the rates were 1.1, 3.2, 6.7, and
14.2 AU/min in the presence of 0.1, 0.2, 0.4, and
1.0 mM nitrite, respectively. When nitrite was added
to the mixture of wine and 50 mM KCl-HCl buffer
(pH 1.3), followed by the addition of Fe(DTCS)2 as
in Figure 2B, the intensity of the ESR signal of NO-
Fe(DTCS)2 increased, as shown in the lower panel of
Figure 4. The result was essentially the same as the
result shown in the upper panel. The initial signal
intensity of NO-Fe(DTCS)2 was dependent on the
concentration of nitrite added and rates of the slow
increase were 1.7, 3.7 and 13.5 AU/min in the pres-
ence of 0.2, 0.4 and 1.0 mM nitrite, respectively.
The signal of NO-Fe(DTCS)2 detected just after
the mixing of Fe(DTCS)2 with the nitrite-containing
wine may reflect the concentration of NO in the reac-
tion mixture as described above, but the slow increase
in Figure 4 remains to be elucidated. The rate of the
slow increase of NO-Fe(DTCS)2 ,which was observed
after the mixing of Fe(DTCS)2 with wine that was
supplemented with 1 mM nitrite, was ∼14 AU/min
(Figure 4), whereas the rate of the formation of NO-
Fe(DTCS)2 after the addition of 1 mM nitrite to the
mixture of Fe(DTCS)2 and wine, which might pro-
ceed by possibility (ii), was 0.2 AU/min (see above).
Since ethyl nitrite could be produced under the for-
mer experimental conditions but not under the latter
experimental conditions and ethyl nitrite could
decompose producing NO [15,16], the slow increase
in the intensity of the ESR signal of NO-Fe(DTCS)2
Nitrite-induced formation of ethyl nitrite and oxidation
of hydroxycinnamic acids in wine
Figure 5 shows the nitrite-induced formation of ethyl
nitrite in the mixture of wine and 50 mM KCl-HCl
buffer (pH 1.3). During the incubation for 1 min, a
new peak was observed at a retention time of ∼8.2
min (compare Figures 5A and B). When wine itself
was incubated with 1 mM nitrite for 1 min the new
peak was also observed (data not shown). The reten-
tion time and the absorption spectrum (Figure 5C)
were identical to those of standard ethyl nitrite, sup-
porting the nitrite-induced formation of ethyl nitrite
in wine as in the acidic mixture of ethyl alcohol and
nitrite [15,16]. The concentration of ethyl nitrite in
wine decreased slowly with a half-life of ∼7 min. Since
it has been reported that ethyl alcohol can react with
nitrite producing ethyl nitrite when the pH is lower
than 4 [15], the above-mentioned result suggests that,
after the ingestion of wine, ethyl alcohol in wine can
be transformed to ethyl nitrite in the oral cavity and
in the stomach. The detection of ethyl nitrite in the
blood after the administration of ethyl alcohol has
been reported [26]. If ethyl nitrite is produced in the

Formation of nitric oxide after drinking wine 299
oral cavity and in the stomach after drinking wine, it
might also be detectable in the blood.
The components of peaks 2, 5 and 6 were identified
to be caffeic,p-coumaric and ferulic acids (Figure 1A),
respectively, by comparing the retention times and
absorption spectra with their standard compounds,
and their concentrations were estimated to be 10, 4.5
and 1.5 μM, respectively. The presence of these
hydroxycinnamic acids in wine has been reported
[20–23]. It is clear from Figure 6 that 1 mM nitrite
decreased the concentration of caffeic (peak 2) and
ferulic acid (peak 6) and the concentrations of caffeic
and ferulic acids after 1-min incubation were less than
10% of their initial concentrations. The component
of peak 1, the concentration of which was not
decreased by nitrite, was identified to be 4-hydroxy-
3-methoxyphenylacetic acid (Figure 1A) by compar-
ing the retention time and absorption spectrum with
the standard compound. The components of peaks 3
and 4 could not be identified, although nitrite
decreased the concentration of the component of
peak 4. When 1 mM nitrite was added to the wine
itself, the concentration of caffeic and ferulic acids
decreased slowly, and their concentrations after
20-min incubation were ∼10% of their initial concen-
trations (data not shown).
The formation of ethyl nitrite during the incuba-
tion for 1 min accompanied the disappearance of peak
X (compare Figures 5A and B) and the absorption
spectrum of peak X suggested that peak X might be
constituted of hydroxycinnamic acids.The concentra-
tion of the hydroxycinnamic acid-like components
was also decreased by the addition of 1 mM nitrite
to wine (pH 3.3), although the decrease in the con-
centration in wine (half-life, ∼7 min) was slower than
that in the mixture of wine and 50 mM KCl-HCl
(pH 1.3). One of the reasons for the difference in the
rate between the pH values might be the difference
in the concentration of nitrous acid that could con-
tribute to oxidation of hydroxycinnamic acids.
Hydroxycinnamic acids in wine were separated by
HPLC using the mixture of methanol and 25 mM
KH2PO4 (1:2, v/v) as the mobile phase. Six peaks
were separated when the mixture of wine and
50 mM KCl-HCl (pH 1.3) was analysed (Figure 6).
Figure 5. Nitrite-induced formation of ethyl nitrite in wine. Wine
(1 ml) was mixed with 1 ml of 50 mM KCl-HCl buffer (pH 1.3)
and then nitrite was added. (A) without nitrite; (B) 1 min after the
addition of 1 mM NaNO2; (C) absorption spectrum of ethyl nitrite
(EtO-NO in (B)). X in (A), a component disappeared by nitrite.
Figure 6. Nitrite-induced decrease in the concentrations of
components in wine.Wine (1 ml) was mixed with 1 ml of 50 mM
KCl-HCl buffer (pH 1.3). (A) Before the addition of nitrite; (B)
1 min after the addition of 1 mM nitrite.

300 U. Takahama et al.
The nitrite-induced formation of NO accompany-
ing the decrease in concentration of the hydroxycin-
namic acids suggests that NO can be formed by the
hydroxycinnamic acid-dependent reduction of nitrous
acid when wine is mixed with saliva and when the pH
of the mixture is decreased to ∼2 in the stomach. In
addition to the hydroxycinnamic acids, catechins and
flavonols [22,23] might also contribute to the reduc-
tion of nitrous acid after the ingestion of wine. In this
study, nitrite-induced nitration and nitrosation of the
phenolics in wine were not investigated.
Reactions of nitrous acid with caffeic acid in the
presence of SCN–
It has been reported that caffeic acid reacts with
nitrite under acidic conditions and some of the prod-
ucts have been identified [27]. In this study, it was
shown that nitrite could react with caffeic acid in
wine. Since a salivary component SCN– can affect the
reactions between nitrous acid and o-diphenolics
[17,18], the effects of SCN– on the reaction between
nitrous acid and caffeic acid were investigated.When
caffeic acid was incubated with nitrite at pH 2 in the
absence of SCN–, peak p-1 was detected (Figure 7,
upper panel).The component of peak p-1 was identi-
fied to be 3,4-dihydroxybenzaldehyde by comparing
the retention time and absorption spectrum with the
standard compound, and the concentration increased
as a function of incubation time. The formation of
3,4-dihydroxybenzaldehyde in an acidic mixture of
nitrite and caffeic acid [27] and also in the mixture
of iron and a wine-like medium [28] has been
reported.
When caffeic acid was incubated with nitrite at
pH 2 in the presence of SCN–, in addition to peak
p-1, p-2 and p-3 were detected and their retention
times were ∼11.5 and 16 min, respectively (Figure 7,
upper panel). The absorption spectra of peak p-2
(maxima, 240 and 296 nm) and peak p-3 (maxima,
227 and 293 nm) (Figure 7, middle panel) resembled
2-thiocyanatechlorogenic acid (maxima, 240 and 311
nm) and the oxathiolone derivative of chlorogenic
acid (maxima, 230 and 311 nm) [19], respectively.
The concentration of the component of peak p-2
decreased accompanying the increase in the concen-
tration of the component of peak p-3 (Figure 7, lower
panel). Such kinetics has been observed for the for-
mation of the oxathiolone derivative of chlorogenic
acid from 2-thiocyanatechlorogenic acid [19]. The
retention time and absorption spectrum of the com-
ponent of peak p-3 were identical to those of the com-
ponent obtained by acid hydrolysis of the oxathiolone
derivative of chlorogenic acid, namely, (E)-5’-(3-(7-
hydroxy-2-oxobenzo[d][1,3]oxathiol-4-yl)acryloyloxy)
quinic acid. Therefore, we identified that the compo-
nent of peak p-3 was the oxathiolone derivative of
Figure 7. Effects of SCN– on nitrite-induced transformation of
caffeic acid.The reaction mixture (1 ml) contained 0.1 mM caffeic
acid and 0.1 mM NaNO2 in 50 mM KCl-HCl buffer (pH 2.0).
Upper panel: HPLC after incubation for 10 min. (A) without
SCN–; (B) 1 mM NaSCN. Middle panel: Absorption spectra. p-2,
spectrum of peak p-2; p-3, spectrum of peak p-3. Lower panel:
Change in the concentration of components as a function of
incubation time. c, caffeic acid; ꢀ, component of peak p-2;
U, component of peak p-3.
caffeic acid [(7-hydroxy-2-oxo-benz[1,3]oxathiol-4yl)
acrylic acid], which might be formed by hydrolysis of
2-thiocyanatecaffeic acid of peak p-2. The formation
of the components corresponding to peaks p-2 and
p-3 could not be detected when 0.1 mM caffeic acid
was incubated with 0.2 mM nitrite plus 1 mM NaSCN

Formation of nitric oxide after drinking wine 301
in 50 mM citrate-HCl buffer (pH 3.3). One of the
reasons for the failure of the detection at pH 3.3 may
be the much lower concentration of HSCN (pKa ≈
0.8) that can react with quinones producing a thio-
cyanate conjugate (Figure 1D).
Ferulic acid was readily oxidized and nitrosylated
by nitrite under acidic conditions and the products
including vanillin have been identified [29,30]. In this
study, the production of vanillin was confirmed in the
acidic mixture of ferulic acid and nitrite, but SCN–
had no significant effects on the formation of the reac-
tion products.
by increases in the concentrations of caffeic acid,
nitrite and SCN–.The concentration of caffeic acid in
wine can increase to ∼60 μM depending on the grapes
used to produce wine [21–23] and the salivary con-
centrations of nitrite and SCN– can increase to ∼1
and ∼2 mM, respectively [8,31,32]. Although 2-thio-
cyanatecaffeic acid was detected in the mixture of
saliva/wine/acidic buffer, the oxathiolone derivative of
caffeic acid could not be detected.The detection fail-
ure was due to the presence of a component in wine,
which absorbed UV light strongly and had a retention
time similar to that of the oxathiolone derivative
(Figures 7 and 8).
Thiocyanate conjugates, which transform to com-
ponents with an oxathiolone moiety, are formed in
the acidic mixtures of saliva and coffee [19] and saliva
and dough prepared from buckwheat flour [20].
In this study, caffeic acid in wine was suggested to be
transformed to the oxathiolone derivative of caffeic
acid in the stomach via 2-thiocyanatecaffeic acid.The
compounds with an oxathiolone moiety have been
reported to have some pharmacological functions
such as anti-fungal, bacteriostatic and cytostatic
activities [33,34]. In the compounds, 6-hydroxy-1,
3-benzoxathiol-2-one is used for the treatment of acne
[31] and some compounds with an oxathiolone moi-
ety have been indicated to be able to inhibit inhibitory
kappaB kinase beta [35] and carbonic anhydrase [36].
In addition, compounds with an oxathiolone moiety
are also reported to have alkyl radical scavenging
activity [37].
Formation of 2-thiocyanatecaffeic acid in the mixture
of saliva and wine
The components corresponding to peaks p-2 and p-3
were produced in the mixture of caffeic acid, nitrite
and SCN– in the buffer solution at pH 2. Then the
formation of 2-thiocyanatecaffeic acid (p-2) was
examined in the mixture of saliva/wine/acidic buffer
at pH 1.94 (Figure 8).The mixture was incubated for
10 min and extracted with ethyl acetate, but the com-
ponent corresponding to peak p-2 in could not be
detected in the extract.The addition of 10 μM caffeic
acid to the mixture seemed to result in the formation
of the component (an arrow in Figure 8A), which was
confirmed by the addition of 10 μM caffeic acid
together with 0.1 mM nitrite and 0.5 mM NaSCN
to the mixture (an arrow in Figure 8B).The retention
time (11.5 min) and absorption spectrum (240 and
296 nm) of the component were identical to those
of peak p-2 in Figure 7. If 2-thiocyanatecaffeic acid
was formed in the mixture of saliva/wine at pH 2, the
component would also be formed in the stomach after
drinking wine and its formation would be enhanced
Concluding remarks
In this study, it was shown that the mixture of saliva/
wine could produce NO under pH conditions of 2
and 3.6, indicating that the ingestion of wine resulted
in the formation of NO in the oral cavity as well as
in the stomach.In addition to NO,ethyl nitrite seemed
to be produced in the oral cavity as well as in the
stomach.Two pathways are possible for the formation
of NO after the ingestion of wine. One is the reduc-
tion of nitrite by phenolics that are contained in wine
and the other is the decomposition of ethyl nitrite
producing NO and ethyl radicals. It has been reported
that NO formed in the stomach can increase the
activities of the stomach [13,14,38]. Therefore, the
results obtained in this study indicate that the use of
wine as an aperitif can be beneficial. The increased
formation of NO after drinking wine may also result
in the inhibition of the growth of microorganisms
[39–41] and the increase in mucosal blood flow and
mucus thickness [13,14] in the stomach.These actions
of NO may aid in preventing stomach ulcers and gas-
tritis [42,43]. Ethyl nitrite formed in the stomach may
be transported from the stomach to the whole body
and may function as a vasodilator [15]. Oxathiolone
Figure 8. Formation of a thiocyanate conjugate of caffeic acid in
mixture of saliva and wine.The reaction mixture (2 ml) contained
0.5 ml of saliva, 0.5 ml of wine and 1 ml of 50 mM KCl-HCl buffer
(pH 1.3). (A) 10 μM caffeic acid; (B) A plus 0.1 mM NaNO2 and
0.5 mM NaSCN. Downward arrows, thiocyanate conjugate of
caffeic acid.

302 U. Takahama et al.
nitrite is formed upon reaction of nitrite and ethanol under
gastric conditions. Free Radic Biol Med 2008;45:404–412.
[16] Takahama U, Tanaka M, Hirota S. Transformation of ethyl
alcohol to ethyl nitrite in acidified saliva: possibility of its
occurrence in the stomach. Arch Biochem Biophys 2008;
475:135–139.
derivatives are produced in the stomach but not in
the oral cavity after the ingestion of phenolics and,
at present, their functions have not been fully
elucidated.
[17] Tenovuo J. Nonimmunoglobulin defense factors in human
saliva. In: Tenovuo JO, editor. Human saliva: Clinical chem-
istry and microbilology. Vol II. Boca Raton, FL: CRC Press;
1989. p. 55–91.
Declaration of interest:The authors report no con-
flicts of interest.The authors alone are responsible for
the content and writing of the paper.
[18] Licht WR, Tannenbaum SR, Deen WM. Use of ascorbic aicd
to inhibit nitrosation: kinetic and mass transfer consideration
for an in vitro system. Carcinogenesis 1988;9:365–372.
[19] Takahama U,Tanaka M, Oniki T, Hirota S,Yamauchi R. For-
mation of thiocyanate conjugate of chlorogenic acid in coffee
under acidic conditions in the presence of thiocyanate and
nitrite: possible occurrence in the stomach. J Agric Food
Chem 2007;55:4169–4176.
References
[1] Teissedre PL, Landrault T. Wine phenolics: contribution to
dietary intake and bioavailability. Food Res Int 2000;33:
461–467.
[2] Serafini M, Laranjinha JA, Almeida LM, Maiani G. Inhibition
of human LDL lipid peroxidation by phenol-rich beverages
and their impact on plasma total antioxidant capacity in
humans. J Nutr Biochem 2000;11:585–590.
[20] Takahama U,Tanaka M, Hirota S,Yamauchi R. Formation of
an oxathiolone compound from rutin in acidic mixture of
saliva and buckwheat dough: possibility of its occurrence in
the stomach. Food Chem 2009;116:214–219.
[3] Gago B, Lundberg JO, Barbosa RM, Laranjinha J. Red wine-
dependent reduction of nitrite to nitric oxide in the stomach.
Free Radic Biol Med 2007;43:1233–1242.
[4] Takahama U, Oniki T, Hirota S. Oxidation of quercetin by
salivary components. Quercetin-dependent reduction of sali-
vary nitrite under acidic conditions producing nitric oxide.
J Agric Food Chem 2002;50:4317–4322.
[5] Takahama U, Yamamoto A, Hirota S, Oniki T. Quercetin-
dependent reduction of nitrite to nitric oxide under acidic
conditions and interaction between quercetin and ascorbic
acid during the reduction. J Agric Food Chem 2003;51:
6014–6020.
[21] Caccetta RA-A, Croft KD, Beilin LJ, Puddey IB. Ingestion of
red wine significantly increases plasma phenolic acid concen-
trations but does not acutely affect ex vivo lipoprotein oxidiz-
ability. Am J Clin Nutr 2000;71:67–74.
[22] Soleas GJ, Dam J, Carey M, Goldberg DM. Toward the
fingerprinting of wines: cultivar-related patterns of poly-
phenolic constituents in Ontario wines. J Agric Food Chem
1997;45:3871–3880.
[23] Kallithraka S, Salacha MI, Tzourou I. Changes in phenolic
composition and antioxidant activity of white wine during
bottle storage: accelerated browning test versus bottle storage.
Food Chem 2008;113:500–505.
[6] Takahama U,Tanaka M, Hirota S. Interaction between ascor-
bic acid and chlorogenic acid during the formation of nitric
oxide in acidified saliva. J Agric Food Chem 2008;56:
10406–10413.
[7] Peri L, Pietraforte D, Scorza G, Napolitano A, Fogliano V,
Minetti M. Apples increase nitric oxide production by human
saliva at the acidic pH of the stomach: a new biological func-
tion for polyphenols with a catechol group? Free Radic Biol
Med 2005;3:668–681.
[8] Pannala AS, Mani AR, Spencer JPE, Skinner Y, Bruckdorfer
KR, Moore KP, Rice-Evans CA. The effect of dietary nitrate
on salivary, plasma and urinary nitrate metabolism in humans.
Free Radic Biol Med 2003;34:576–584.
[24] Lundberg JO,Weitzberg E, Lundberg JM, Alving K. Intragas-
tric nitric oxide production in humans: measurement in
expelled air. Gut 1994;35:1543–1546.
[25] Hisatsne IC, Zafonte L. Kinetic study of some third-order
reactions of nitric oxide. J Phys Chem 1969;73:2980–2989.
[26] Deng XB, Bludeau P, Deitrich RA. Formation of ethyl nitrite
in vivo after ethanol administration. Alcohol 2004;34:
217–223.
[27] Napolitano A, d’Ischia M. New insights into the acid-
promoted reaction of caffeic acid and its esters with nitrite:
decarboxylation drives chain nitrosation pathways toward
novel oxime derivatives and oxidation/fragmentation products
thereof. J Org Chem 2002;67:803–810.
[9] Palmerini CA, Palombari R, Perito S, Arienti G. NO synthe-
sis in human saliva. Free Radic Res 2003;37:29–31.
[10] Zetterquist W, Pedroletti C, Lundberg JO, Alving K. Salivary
contribution to exhaled nitric oxide. Eur Respir J 1999;13:
327–333.
[28] Lutter M, Clark AC, Prenzler PD, Scollary GR. Oxidation of
caffeic acid in a wine-like medium: production of dihydroben-
zaldehyde and its subsequent reactions with (ꢁ)-catechin.
Food Chem 2007;105:968–975.
[29] Rousseau B, Rosazza JPN. Reaction of ferulic acid with
[11] Doel JJ, Hector MP, Amirtham CV, Al-Anzan LA, Benjamin N,
Allaker RP. Protective effect of salivary nitrite and microbial
nitrate reductase activity against caries. Eur J Oral Sci
2004;112:424–428.
[12] Benjamin N, Hector MP, Rogers M, Allaker RP. Evaluation
of bacterial nitrate reduction in the human oral cavity. Eur J
Oral Sci 2005;113:14–19.
nitrite: formation
of
7-hydroxy-6-methoxy-1,2-(4H)-
benzoxazin-4-one. J Agric Food Chem 1998;46:3314–3317.
[30] Kikugawa K, Hakamada T, Hasunuma M, Kurechi T. Reac-
tion of p-hydroxycinnamic acid derivatives with nitrite and its
relevance to nitrosoamin formation. J Agric Food Chem
1983;31:780–785.
[31] Galanti LM. Specificity of salivary thiocyanate as marker of
cigarette smoking is not affected by alimentary sources. Clin
Chem 1997;43:184–185.
[13] Björne H, Petersson J, Phillipson M, Weitzberg E, Holm L,
Lundberg JO. Nitrite in saliva increases gastric mucosal blood
flow and mucus thickness.
106–114.
[14] Whittle BJ, Lopez-Belmonte J, Moncada S. Regulation of gas-
tric mucosal integrity by endogenous nitric oxide: interactions
with prostanoids and sensory neuropeptides in the rat. Br J
Pharmacol 1990;99:607–611.
J
Clinic Invest 2004;113:
[32] Tsuge K, Kataoka M, SetoY. Cyanide and thiocyanaide levels
in blood and saliva of healthy adult volunteers. J Health Envi-
ron 2000;46:343–350.
[33] Lius V, Sennerfeldt P. Local treatment of acne with thiox-
olone. Lakartidningen 1979;76:39–41.
[34] Konieczny MT, Konieczny W, Sabisz M, Skladanowski A,
Wakiec R, Augustynowicz-Kopec E, Zwolska Z. Synthesis of
isomeric, oxathiolone fused to chalcones, and comparison of
[15] Gago B, Nyström T, Cavaleiro C, Rocha BS, Barbosa RM,
Laranjinha J, Lundberg JO. The potent vasodilator ethyl

Formation of nitric oxide after drinking wine 303
their activity toward various microorganisms and human can-
cer cells line. Chem Pharm Bull 2007;55:817–820.
[35] Kim BH, Roh E, Lee HY, Lee IJ, Ahn B, Jung SH, Lee H,
Han SB, KimY. Benzoxathiole derivative blocks lipopolysac-
charide-induced nuclear factor-kappaB activation and nuclear
factor-kappaB-regulated gene transcription through inactivat-
ing inhibitory kappaB kinase beta. Mol Pharmacol 2008;73:
1309–1318.
[36] Innocenti A, Maresca A, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors; thioxolone versus sulfonamides for
obtaining isozyme-selective inhibitors? Bioorg Med Chem
Lett 2008;18:3938–3941.
[37] Povalishev VN, Polozov GI, Shadyro OI. Effects of α-tocopherol
and related compounds on reactions involving various organic
radicals. Bioorg Med Chem Lett 2006;16:1236–1239.
[38] Desai KM, SessaWC,Vane JR. Involvement of nitric oxide in
the reflex relaxation of the stomach to accommodate food and
fluid. Nature 1991;351:477–479.
[39] Benjamin N, O’Driscoll F, Dougall H, Duncan C, McKenzie H.
Stomach NO sysnthesis. Nature 1994;368:502.
[40] Yu H, Sato EF, Nagata K, Nishikawa M, Kashiba M, Arakawa
T, Kobayashi K, Tamura T, Inoue M. Oxygen-dependent
regulation of the respiration and growth of Escherichia coli by
nitric oxide. FEBS Lett 1997;409:161–165.
[41] Xu J, Xu X,VerstraeteW.The bactericidal effect and chemical
reactions of acidified nitrite under conditions simulating the
stomach. J Appl Microbiol 2001;90:523–529.
[42] Larauche M, Anton PM, Gracia-Villar R,TheodorouV, Frex-
inos J, Bueno L, Fioramonti J. Protective effect of dietary
nitrate on experimental gastritis in rates. Br J Nutr 2003;
89:777–786.
[43] Jansson EA, Petersson J, Reinders C, Sobko T, Bjorne H,
Phillipson M, Weitberg E, Holm L, Lundberg JO. Protection
from nonsteroidal anti-inflammatory drug (NSAID)-induced
gastric ulcers by dietary nitrate. Free Radic Biol Med
2007;42:510–518.
This paper was first published online on Early Online on 25
January 2010.