(+)-Catechin and Quercetin during Their Oxidation by Nitrite under the

May 2, 2014 - Department of Nutrition, Kyushu Women,s University, Kitakyushu 807-8586, ... Department of Bioscience, Kyushu Dental College, Kitakyushu...
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Interactions between (+)-Catechin and Quercetin during Their Oxidation by Nitrite under the Conditions Simulating the Stomach Sonja Veljovic-Jovanovic,† Filis Morina,† Ryo Yamauchi,‡ Sachiko Hirota,§ and Umeo Takahama*,∥ †

Institute for Multidisciplinary Research, University of Belgrade, Belgrade 11030, Republic of Serbia Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan § Department of Nutrition, Kyushu Women’s University, Kitakyushu 807-8586, Japan ∥ Department of Bioscience, Kyushu Dental College, Kitakyushu 803-8580, Japan ‡

ABSTRACT: When foods that contain catechins and quercetin glycosides are ingested, quercetin glycosides are hydrolyzed to quercetin during mastication by hydrolytic enzymes derived from oral bacteria and the generated quercetin aglycone is mixed with catechins in saliva. The present study deals with the interactions between (+)-catechin and quercetin during their reactions with nitrous acid under the conditions simulating the gastric lumen. Nitrous acid reacted with (+)-catechin producing 6,8dinitrosocatechin, and quercetin partially suppressed the dinitrosocatechin formation. Nitric oxide, which was produced by not only (+)-catechin/nitrous acid but also quercetin/nitrous acid systems, was used to produce 6,8-dinitrosocatechin. Furthermore, 6,8-dinitrosocatechin was oxidized by nitrous acid to the quinone form. The quinone formation was significantly suppressed by quercetin. Quercetin-dependent suppression of the above reactions accompanied the oxidation of quercetin, which was observed with the formation of 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone. Taking the above results into account, we proposed a possible mechanism of 6,8-dinitrosocatechin formation and discuss the importance of quercetin to prevent the quinone formation from 6,8-dinitrosocatechin in the gastric lumen, taking the interactions between quercetin and catechins into account. KEYWORDS: acidic conditions, (+)-catechin, nitrosation, nitrous acid, quercetin, redox reaction

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antimicrobial activity,18,19 inhibition of stress-induced gastric mucosal injury,20 increase in gastric blood flow, and mucus formation.21,22 The oxidation of catechins and quercetin by nitrous acid forms their semiquinone radicals as the intermediates.14,23 The reduction potential of the (+)-catechin semiquinone radical at pH 2 (Eo′) is 1.1 V,23 and this value is supposed to be higher than that of the quercetin semiquinone radical from the reduction potentials of the (+)-catechin semiquinone radical (Eo′ = 0.57 V) and the quercetin semiquinone radical (Eo′ = 0.33 V) at pH 7.0.23 These reduction potentials of (+)-catechin, quercetin, and their semiquinone radicals suggest that some interactions between (+)-catechin and quercetin are possible during their oxidation by nitrous acid, which may also occur in the gastric lumen. This paper deals with (i) the reactions of nitrous acid with (+)-catechin (1) and quercetin (2) (Figure 1) and (ii) the interactions between (+)-catechin and quercetin during their reactions with nitrous acid. The results are that nitrous acid transformed (+)-catechin into 6,8-dinitrosocatechin and dinitrosocatechin into its quinone form and that the above reactions were suppressed by quercetin accompanying the enhancement of quercetin oxidation. When the above results

INTRODUCTION Various polyphenols are contained in foods of plant origin. For example, onion contains both quercetin aglycone and its glucosides,1 and adzuki bean contains (+)-catechin in addition to quercetin aglycone and its glycosides.2,3 Furthermore, buckwheat seeds contain both catechins and a quercetin glycoside rutin,4 and apple contains quercetin glycosides with (+)-catechin and (−)-epicatetechin.5 When we take in foods prepared from the above plants, polyphenols are released into saliva during mastication and hydrolytic enzymes derived from microorganisms in the oral cavity can hydrolyze polyphenol glycosides into agylcones and sugars.6−8 Thus, catechins can be mixed with quercetin in the oral cavity. The mixtures of catechins and quercetin in saliva are swallowed into the gastric lumen, where the pH is around 2. Under such conditions, nitrite in saliva, the concentration of which is 0.05−1 mM,9 is transformed to an oxidizing agent, nitrous acid (pKa = 3.3) [Eo = 0.983 V; 0.865 V at pH 2.0 (calculated value)].10 The reduction potentials of (+)-catechin (Eo′ = 0.49 V at pH 2.0)11 and quercetin (Eo′ = 0.45 V at pH 2.0)12 suggest that these polyphenols can be oxidized by nitrous acid. In fact, nitrousacid-induced oxidation of (+)-catechin and quercetin has been reported.13,14 The oxidation products of catechins are dinitrosocatechins, in which 6,8-dinitrosocatechin is included,15,16 and a major oxidation product of quercetin is 2(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone.17 During the reactions of polyphenols with nitrous acid, nitric oxide (•NO) is produced.13,14 The functions of •NO produced from nitrous acid in the gastric lumen include © 2014 American Chemical Society

Received: Revised: Accepted: Published: 4951

February 18, 2014 April 30, 2014 May 2, 2014 May 2, 2014 dx.doi.org/10.1021/jf500860s | J. Agric. Food Chem. 2014, 62, 4951−4959

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injected into the column was 0.9 mL. The mobile phases were mixtures of methanol and 0.2% formic acid. The concentration of methanol was increased stepwise as follows: 14, 20, 25, and 33% (v/v), and each mobile phase was flowed for 5, 15, 15, and 25 min, respectively. Their flow rate was 9 mL/min. Components separated by the preparative HPLC were detected at 210, 280, and 320 nm using the spectrophotometric detector. A peak supposed to be 6,8dinitrosocatechin was eluted at about 7 min after changing the mobile phase to 25% methanol, and the peak component was collected. Methanol of the collected solution was removed in vacuo, and the residue was lyophilized. The yield was 27−35%. The above procedure was repeated 6 times, and about 50 mg of the lyophilizate was obtained. Reactions of Nitrite with Quercetin, (+)-Catechin, and 6,8Dinitrosocatechin. Interactions of quercetin with (+)-catechin or 6,8-dinitrosocatechin were studied in reaction mixtures (1 mL) that contained 0.1 mM (+)-catechin or 6,8-dinitrosocatechin and various concentrations of quercetin (0, 10, 30, or 100 μM) in 50 mM KCl− HCl (pH 2.0). When required, 2 mM ascorbic acid or 10 mM potassium thiocyanate was added to the above reaction mixtures. Reactions were initiated by the addition of 0.1 mM sodium nitrite. After incubation for defined periods, an aliquot (50 μL) of each reaction mixture was applied to a HPLC column to quantify (+)-catechin, dinitrosocatechin, quercetin, and their reaction products (see below). Interactions between quercetin and (+)-catechin or 6,8-dinitrosocatechin were also studied under anaerobic conditions. The reaction mixture contained 50 μM (+)-catechin or 6,8-dinitrosocatechin and various concentrations of quercetin. Air of the reaction mixtures was removed by bubbling argon gas for 2 min, and then reactions were initiated by the addition of 0.25 mM sodium nitrite. After incubation for 1 min, 50 μL of the reaction mixture was applied to the HPLC column to quantify (+)-catechin, quercetin, dinitrosocatechin, and their reaction products (see below). Analysis of Reaction Products by HPLC. Analytical HPLC was performed using a Shim-pack CLC-ODS column (15 cm × 6 mm inner diameter; particle size of 5 μm; Shimadzu). The mixture of methanol and 0.2% formic acid (1:2, v/v) was used to separate (+)-catechin (280 nm), 6,8-dinitrosocatechin (320 nm), and the oxidation product of quercetin (293 nm), when (+)-catechin and/or quercetin were reacted with nitrous acid. When 6,8-dinitrosocatechin was reacted with nitrous acid with and without quercetin, the mixture of methanol and 0.2% formic acid (2:5, v/v) was used as the mobile phase to detect the reaction products. The ratio of 2:1 (v/v) was used to detect quercetin (360 nm) in the above systems. The flow rate of the mobile phases was 1 mL/min. The concentration of each component was estimated from the area under the peak. Measurements of •NO Production. Nitrite-induced •NO production was studied using a Clark-type electrode at 30 °C with a polarization voltage of −0.7 V.24−26 The reaction mixture (2.0 mL) contained 50 μM (+)-catechin or 6,8-dinitrocatechin and various concentrations of quercetin in 50 mM KCl−HCl (pH 2.0). Reactions were initiated by the addition of 0.5 mM sodium nitrite after removing air from the reaction mixture by bubbling argon gas. The rate of •NO production was estimated from the slope. Amounts of •NO produced in the above systems were calibrated by measuring •NO production in ascorbic acid/nitrous acid systems. In the ascorbic acid/nitrous acid system, one molecule of ascorbic acid produces two molecules of •NO under acidic conditions by the following reaction if the concentration of nitrous acid is more than 2 times that of ascorbic acid:26,27

Figure 1. Compounds investigated in this study: (1) (+)-catechin, (2) quercetin, (3a) 6,8-dinitrosocatechin, (3b) 6,8-dinitrosoepicatechin, and (4) 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone.

are taken into account, possible interactions between (+)-catechin and quercetin are discussed.

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MATERIALS AND METHODS

Reagents. (+)-Catechin, (−)-epicatechin, and quercetin were obtained from Sigma-Aldrich Japan (Tokyo, Japan). 1-Hydroxy-2oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7) (purity > 90%) was from Dojindo (Kumamoto, Japan). Apparatus. High-performance liquid chromatography (HPLC) was carried out using a Shimadzu LC-10AS pump combined with a SPD M10Avp photodiode array detector (Shimadzu Co., Kyoto, Japan). Atmospheric pressure chemical ionization (APCI) mass spectra were obtained with a Shimadzu LCMS QP8000α quadrupole mass spectrometer equipped with an APCI ion source. The deflector voltage of the mass spectrometer was maintained at +50 V for positivemode measurement or at −50 V for negative-mode measurement. The sample was delivered into the ion source using an Ascentis express C18 column (15 cm × 2.1 mm inner diameter; particle size of 2 μm; Sigma-Aldrich Japan, Tokyo, Japan). The mobile phase was 35% methanol containing 0.2% formic acid, and the flow rate was 0.2 mL/ min. Proton nuclear magnetic resonance (1H NMR) spectra were recorded with an ECX-400P FT-NMR spectrometer (JEOL, Tokyo, Japan) with dimethyl sulfoxide-d6 (DMSO-d6) as the solvent and tetramethylsilane as the internal standard. 1H NMR was performed at 399.78 MHz, and the 1H−1H chemical shift correlated technique was employed to assign 1H shifts and couplings. Isolation of 6,8-Dinitrosocatechin. (+)-Catechin (29 mg) in 100 mL of 50 mM KCl−HCl (pH 2.0), which was equivalent to 1 mM (+)-catechin, was reacted with 5 mM sodium nitrite for 5 min under anaerobic conditions, and then 5 mM ascorbic acid was added to terminate the reaction. The reaction products were extracted with 50 mL of ethyl acetate 5 times. Ethyl acetate extracts were combined and washed with 50 mL of 0.1% HCl twice. After water was removed from the ethyl acetate extract with anhydrous sodium sulfate, the solvent was removed in vacuo with a rotary evaporator, the residue was dissolved in 1.5 mL of methanol, and then the methanol solution was diluted with 1.5 mL of 0.2% formic acid to apply to a preparative HPLC column. Preparative HPLC was performed using a Shim-pack PREPODS(H) kit (25 cm × 20 mm inner diameter; particle size of 5 μm; pore size of 10 nm; Shimadzu), and the volume of samples

ascorbic acid + 2HNO2 → dehydroascorbic acid + 2•NO + 2H 2O

(1)

The amount of •NO produced by the addition of 50 μM ascorbic acid in the presence of 0.5 mM sodium nitrite in 50 mM KCl−HCl (pH 2.0) was essentially the same as that of •NO produced by 50 μM NOC 7 in the same buffer, one molecule of which produces two molecules of 4952

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•

NO,28 indicating the usefulness of an ascorbic acid/nitrous acid system to calibrate the amounts of •NO produced. Presentation of Data. Each experiment was repeated 3−8 times. Typical data or means with standard deviations (SDs) are presented in figures and tables. The statistical significance of the difference between groups was evaluated by Student’s t test.

nitrite under the conditions of simulating the gastric lumen, a component with a retention time of 5.2 min was detected by analytical HPLC (top panels of Figure 2). The component had

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RESULTS AND DISCUSSION Characterization of Isolated 6,8-Dinitrosocatechin. Liquid chromatography−mass spectrometry (LC−MS) analysis of the isolated dinitrosocatechin gave two peaks (3a and 3b) (Figure 1) with retention times of 2.1 and 3.4 min, respectively. Their APCI mass spectra were the same. Compound 3a at m/z (relative intensity, %): 123.1 (39), 270.1 (28), 285.1 (25, [315.1 − NO]+), 286.1 (38), 315.1 (100, [349.1 − (OH)2]+), 331.1 (30, [M − OH]+), and 349.1 (32, [M + H]+) with positive-ion mode or at m/z 153.0 (43), 180.1 (72), 196.0 (98), 311.1 (52), 313.1 (97, [347.1 − (OH)2]−), 331.1 (46, [M − OH]+), and 347.1 (100, [M − H]−) with negative-ion mode. Compound 3b at m/z (relative intensity, %): 123.1 (43), 270.0 (23), 285.1 (24, [315.1 − NO]+), 286.1 (35), 315.1 (100, [349.1 − (OH)2]+), 331.1 (23, [M − OH]+), and 349.1 (31, [M + H]+) with positive-ion mode or m/z 153.0 (45), 180.1 (81), 196.0 (87), 311.1 (62), 313.1 (90, [347.1 − (OH)2]−), 331.1 (33, [M − OH]−), and 347.1 (100, [M − H]−) with negative-ion mode, suggesting that compounds 3a and 3b are dinitrosocatechin isomers. This was supported by the result that compound 3b was produced when (−)-epicatechin was incubated with nitrite in 50 mM KCl−HCl (pH 2.0), the production of which was confirmed by the retention time and ultraviolet/visible (UV/vis) absorption spectrum. The mutual transformation of these dinitroso compounds has been reported.15 Furthermore, each peak showed a fragment ion at m/z 123.1 with positive mode, corresponding to a B-ring fragment of catechin. This supports that the B ring of each compound is not modified.15,29 The major fragment ion at m/z 196.0 with negative mode suggests that two NO groups could add to the A ring of catechins. The chemicals structures of compounds 3a and 3b were supported by NMR data. 1H NMR data indicated that isolated dinitrosocatechin was a mixture of 6,8-dinitrosocatechin and 6,8-dinitrosoepicatechin. 1H NMR (DMSO-d6) δ: 2.38 (dd, J = 7 and 18 Hz, 2/3H, Hc-4ax), 2.50 (dd, J = 4 and 18 Hz, 2/3H, Hc-4eq), 2.57 (m, 1/3H, He-4ax), 2.67 (dd, J = 4 and 18 Hz, 1/ 3H, He-4eq), 4.00 (m, 2/3H, Hc-3), 4.13 (m, 1/3H, He-3), 4.95 (d, J = 6 Hz, 2/3H, Hc-2), 5.07 (s, 1/3H, He-2), 5.34 (br s, 1H, 4-OH), 6.62 (dd, J = 1 and 8 Hz, 2/3H, Hc-6′), 6.63 (m, 1/3H, He-5′), 6.71 (s, 1/3H, He-6′), 6.72 (s, 2/3H, Hc-2′), 6.73 (d, J = 8 Hz, 2/3H, Hc-6′), 6.93 (s, 2/3H, He-2′), 8.92 (br s, 1H, OH), and 9.00 (br s, 1H, OH), where Hc is a proton signal from catechine and He is a proton signal from epicatechin. No signals assigned to H bound to C6 and C8 were detected, which were observed in (+)-catechin and (−)-epicatechin.15,30 This supported that nitrosation took place on the C6 and C8 positions of the A ring. Nitrosation on the A ring is consistent with the previous reports, which concerned the nitrosation of epicatechin and epigallocatechiin gallate by nitrous acid.15,16 The presence of 6,8-dinitrosocatechin (3a) and 6,8-dinitrosoepicatechin (3b) in the isolate was further supported by the order of elution, because catechin eluted earlier than epicatechin in reversed-phase columns.31 Reaction of (+)-Catechin with Nitrous Acid. When (+)-catechin (retention time of 5.4 min) was reacted with

Figure 2. Formation of dinitrosocatechin and an oxidation product of quercetin, Q293. The reaction mixture that contained 0.1 mM sodium nitrite and 0.1 mM (+)-catechin or quercetin in 50 mM KCl−HCl (pH 2.0) was incubated for 5 min. (Top) (+)-Catechin and dinitrosocatechin. (Middle) Q293. (Bottom) Absorption spectrum of each peak in the mobile phase (methanol and 0.2% formic acid = 1:2, v/v).

an absorption peak at 274 nm with a shoulder at 320 nm (bottom panel of Figure 2). The retention time and absorption spectrum were identical to those of isolated 6,8-dinitrosocatechin under the conditions of Figure 2, and its formation increased with the increase in the concentration of sodium nitrite from 0 to 1 mM. It has been reported that dinitrosocatechin and dinitrosoprocyanidin B2 are formed when the methanol extract of apple fruit is incubated with acidified saliva (pH 2.0) that contains 0.4 mM nitrite.32 Figure 3A shows a time course of nitrite-induced formation of 6,8-dinitrosocatechin from (+)-catechin. The concentration increased nearly linearly during the incubation for 10 min. Figure 3A also shows a time course of (+)-catechin consumption. Because (+)-catechin was not completely separated from 6,8-dinitrosocatechin, the concentration of (+)-catechin was estimated by subtracting the area of 6,8dinitrosocatechin at 280 nm, which was determined from the area of dinitrosocatechin at 320 nm, from the area of both components at 280 nm (Figure 2). Time courses of the formation of 6,8-dinitrosocatechin and the consumption of (+)-catechin indicate that the yield of 6,8-dinitrosocatechin was approximately 30% in molar ratio. The rate constant of (+)-catechin consumption was calculated from data of (+)-catechin consumed during 2 min of incubation, postulating that 1 mol of (+)-catechin reacted with 1 mol of nitrous acid, producing •NO and the (+)-catechin semiquinone radical as follows: 4953

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NO was produced by reactions 2 and 4 under the conditions in Figure 3, namely, aerobic conditions, •NO2 should be formed as follows (k = 8.8 × 106 M−2 s−1):35 2•NO + O2 → 2•NO2

(6)

•

NO2 may oxidize (+)-catechin and nitrosocatechins, producing their semiquinone radicals. Oxidation of Quercetin by Nitrous Acid. The oxidation product of quercetin, which was referred to as Q293 in this study, had a retention time of 8.3 min in the mobile phase, and the absorption spectrum had a peak at 293 nm, with a shoulder around 320 nm (middle and bottom panels of Figure 2). We have reported the formation of the component in the acidic mixture of quercetin and nitrite13 and determined its structure to be 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (4).17 Figure 3B shows time courses of nitrite-induced formation of Q293 from 0.1 mM quercetin in 50 mM KCl− HCl (pH 2.0). The concentration of Q293 increased nearly linearly during incubation at least for 3 min, and the rate increased with the increase in the concentration of nitrite from 0 to 1 mM. Reaction of Dinitrosocatechin with Nitrous Acid. Isolated 6,8-dinitrosocatechins (0.1 mM) were incubated with 0.1 mM nitrite for 5 min in 50 mM KCl−HCl (pH 2.0), and the reaction mixture was analyzed by HPLC (top panel of Figure 4). Two new components, P1 and P2, were detected, and their absorption peaks (270 nm) were shorter than those of compounds 3a and 3b (274 nm) (middle panel of Figure 4). The bottom panel of Figure 4 shows time courses of 6,8dinitrosocatechin (3a plus 3b) consumption and P1 and P2 formation. The formation of P1 slowed down more rapidly than that of P2, and the amount of P1 produced was smaller compared to that of P2, suggesting that P1 and P2 were formed from compounds 3b and 3a, respectively. The rate constant of the consumption of 6,8-dinitrosocatechin (3a plus 3b) was calculated from the decrease in the concentration during 2 min of incubation. The value was (6.5 ± 2.5) × 102 M−1 min−1 (n = 4) under the postulation of the following reaction:

Figure 3. Time courses of the formation of reaction products of (+)-catechin and quercetin under aerobic conditions. The reaction mixture (1 mL) contained 0.1 mM (+)-catechin or 0.1 mM quercetin and 0.1 mM sodium nitrite in 50 mM KCl−HCl (pH 2.0). (A) Formation of dinitrosocatechin: (●) (+)-catechin consumption and (○) dinitrosocatechin formation. (B) Formation of the oxidation product of quercetin, Q293. Each data point represents the mean with SD (n = 3).

( +)‐catechin + HNO2 → ( +)‐catechin radical + •NO + H 2O

(2)

The value was (6.7 ± 1.0) × 102 M−1 min−1 (n = 4). Nitriteinduced formation of the semiquinone radical of (+)-catechin has been reported under the conditions that simulate the gastric lumen,14 and the unpaired electron is localized in the A ring of the (+)-catechin semiquinone radical.23 The localization of the unpaired electron suggests that •NO produced by reaction 2 can bind to the A ring of the semiquinone radical, generating mononitrosocatechin, and then mononitrosocatechin is oxidized in the A ring to react with •NO, generating 6,8dinitrosocatechin as follows: ( +)‐catechin radical + •NO → NO−catechin

diNO−catechin + HNO2 → diNO−catechin radicals + •NO + H 2O

In addition to HNO2, •NO2 produced by reaction 6 can also oxidize dinitrosocatechins to the semiquinone radicals. Because dinitrosocatechins have a catechol structure in the B ring, it is expected that the semiquinone radicals of dinitrocatechins are transformed to o-quinones by disproportionation. If components of P1 and P2 were quinones, the peaks should disappear by ascorbic acid.36 In fact, when 2 mM ascorbic acid was added to the reaction mixture that had been incubated for 5 min under the conditions of Figure 4, peaks P1 and P2 disappeared, accompanying the increase in peak heights of compounds 3a and 3b to the original heights. This result supports the postulation that P1 and P2 are quinone forms of 6,8-dinitroscatechin and 6,8-dinitrosoepicatechin. Nitrous-acidinduced formation of quinone of dinitrosoepigallocatechin-3-Ogallate has been reported.16 o-Quinones of polyphenols, such as caffeic acid, chlorogenic acid, and rutin, react with thiocyanate at pH 2.0, producing thiocyanate conjugates, which are transformed to the oxathiolone derivatives by the addition of water and removal of ammonia.37−39 Then, the effects of sodium thiocyanate on the formation of P1 and P2 were studied at pH 2.0.

(3)

NO−catechin + HNO2 → NO−catechin radical + •NO + H 2O NO−catechin radical + •NO → diNO−catechin

(7)

(4) (5)

In the above reactions, NO−catechin and diNO−catechin are mononitrosocatechin and 6,8-dinitrosocatechin, respectively. The formation of mononitrosocatechin in catechins/nitrous acid systems15 and the reactions of •NO with phenoxyl radicals33 have been reported. Hypochlorous-acid-induced formation of 6,8-dichlorocatechins from catechins34 supports the localization of unpaired electrons in the A ring of catechins. Panzella et al.16 have reported the formation of 6,8dinitroepigallocatechin-3-O-gallate in (−)-epigallocatechin-3O-gallate/nitrous acid systems, and they proposed the reaction of epigallocatechin-gallate quinone with nitrous acid as the mechanism of 6,8-dinitrosoepicatechin-3-O-gallate formation. If 4954

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Figure 5. Interactions between quercetin and (+)-catechin or dinitrosocatechin. The reaction mixture (1 mL) that contained 0.1 mM sodium nitrite and 0.1 mM (+)-catechin or dinitrosocatechin in 50 mM KCl−HCl (pH 2.0) was incubated for 2 min. (Left vertical axis) Enhancement of Q293 production by catechin and dinitrosocatechin: (○) no addition, (□) (+)-catechin, (△) dinitrosocatechin. (Right vertical axis) Inhibition of the production of dinitrosocatechin and P1 + P2 by quercetin: (■) production of dinitrosocatechin from (+)-catechin and (▲) production of (P1 + P2) from dinitrosocatechin. Each data point represents the mean with SD (n = 3−4).

the presence and absence of quercetin, and 50 μM quercetin inhibited its production by about 60% (■). The concentration of (+)-catechin decreased during the formation of dinitrosocatechin, but the effects of quercetin on the decrease in the (+)-catechin concentration were unclear. The unclearness might be due to the small decrease in the (+)-catechin concentration during 2 min of incubation (see Figure 3) and/or quercetin-dependent preferential reduction of the mononitrosochatechin semiquinone radical rather than the (+)-catechin semiquinone radical for the inhibition of dinitrosocatechin formation. The effects of (+)-catechin on the nitrous-acid-induced decrease in the concentration of quercetin are shown in Table 1. From the data in the absence of (+)-catechin, the rate constant of the reaction between quercetin and nitrous acid was calculated, postulating the following reaction:

Figure 4. Reactions of dinitrosocatechin with nitrous acid. The reaction mixture (1 mL) contained 0.1 mM isolated dinitrosocatechin and 0.1 mM sodium nitrite in 50 mM KCl−HCl (pH 2.0). (Top) HPLC of reaction products of dinitrosocatechin at 5 min after initiation. Compound 3a, 6,8-dinitrosocatechin; compound 3b, 6,8dinitrosoepicatechin; and P1 and P2, products of compounds 3b and 3a, respectively. Mobile phase, methanol and 0.2% formic acid = 2:5, v/v. (Middle) Absorption spectra of compound 3a and P1 in the mobile phase. (Bottom) Time courses of (●) dinitrosocatechin (3a plus 3b) consumption and (○) P1 and (△) P2 formation. Each data point represents the mean with SD (n = 3−4).

Thiocyanate (10 mM) suppressed the formation of P1 and P2 in the mixture that contained 0.1 mM dinitrosocatechin and 0.1 mM sodium nitrite in 50 mM KCl−HCl (pH 2.0). In addition, 2−10 mM thiocyanate decreased the concentration of P1 and P2 when added to a reaction mixture, in which P1 and P2 had been produced. These results further support that P1 and P2 were quinones. Two new components (retention times of 3.9 and 5.8 min) were produced in the dinitrosocatechin/ thiocyanate/nitrous acid systems, and their molecular weights were estimated to be 405 by LC−MS, suggesting that the components were thiocyanate conjugates of 6,8-dinitrosocatechin and 6,8-dinitrosoepicatechin. Interactions between (+)-Catechin and Quercetin. Figure 5 shows effects of 0.1 mM (+)-catechin on the formation of Q293 in the presence of various concentrations of quercetin. The formation of Q293 increased with the increase in the concentration of quercetin in the absence of (+)-catechin, when the concentration of Q293 was determined 2 min after the initiation of reactions (○). (+)-Catechin enhanced the production of Q293 independent of the quercetin concentration, and the enhancement in the presence of 10 and 30 μM quercetin was about 1.8-fold (□). The above result indicates that (+)-catechin can enhance the oxidation of quercetin. 6,8-Dinitrosocatechin was produced irrespective of

quercetin + HNO2 → quercetin radical + •NO + H 2O (8)

Table 1. Enhancement of Nitrite-Induced Quercetin Oxidation by (+)-Catechin and 6,8-Dinitrosocatechina quercetin concentration after 2 min of incubation (μM) initial concentration of quercetin (μM)

10

30

no addition (+)-catechin 6,8-dinitrosocatechin

7.7 ± 0.7 (n = 6) 3.3 ± 0.1 (n = 3)b 2.9 ± 1.8 (n = 3)b

19.6 ± 1.3 (n = 6) 16.5 ± 1.8 (n = 3)b 7.2 ± 2.3 (n = 3)b

a

The reaction mixture (1 mL) that contained 0.1 mM sodium nitrite and 10 or 30 μM quercetin in 50 mM KCl−HCl (pH 2.0) in the presence and absence of 0.1 mM (+)-catechin or 6,8-dinitrosocatechin was incubated for 2 min. The quercetin concentration of the reaction mixture was determined by HPLC. Each data point represents the mean with SD (n = 3). bSignificant differences in quercetin oxidation with and without the addition of (+)-catechin or 6,8-dinitrosocatechin (p < 0.05). 4955

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Table 2. •NO Production in Nitrite/Quercetin Systems with and without (+)-Catechin or Dinitrosocatechina rate of •NO production (μM/min) quercetin (μM) 0 5 15 50

+50 μM catechin

no addition 0 8.3 ± 3.1 (n = 4) 27.6 ± 5.3 (n = 4) 77.0 ± 9.6 (n = 8)

16.7 16.5 42.5 95.5

± ± ± ±

+50 μM diNO−CAT

b

3.3 (n = 8) 3.7 (n = 4)b,c 9.1 (n = 4)c 14.9 (n = 3)c

28.0 39.0 63.3 132.7

± ± ± ±

3.3 (n = 6) 4.0 (n = 3)c 14.5 (n = 3)c 17.5 (n = 3)c

a The reaction mixture (2 mL) contained 0, 5, 15, or 50 μM quercetin in 50 mM KCl−HCl (pH 2.0) in the presence and absence of 50 μM (+)-catechin or dinitrosocatechin. Reactions were initiated by the addition of 0.5 mM sodium nitrite. Rates of •NO production were determined from the initial slopes. diNO−CAT = 6,8-dinitrosocatechin. Each data point represents the mean with SD. bNo significant difference. cSignificant differences in •NO production regarding the addition of the quercetin to catechin/nitrous acid or dinitrosocatechin/nitrous acid system (p < 0.05).

Table 3. Enhancement of Quercetin Consumption by (+)-Catechin and 6,8-Dinitrosocatechin under Anaerobic Conditionsa concentration of quercetin (μM) 0 no addition 50 μM (+)-catechin 50 μM diNO−CAT

0 0 0

no addition 50 μM (+)-catechin 50 μM diNO−CAT

0 0 0

50 μM (+)-catechin

3.4 ±

50 μM diNO−CAT

14 ±

5 (Decrease in the Quercetin Concentration, μM) 2.5 ± 0.2 3.9 ± 0.4b 4.3 ± 1.1b (Formation of Q293, Arbitrary Units) 17 ± 3 86 ± 2b 230 ± 5b (Formation of diNO−CAT, μM) 0.8 3.2 ± 0.4 (Formation of P1 and P2, Arbitrary Units) 3 7 ± 2b

15

50

6.5 ± 0.4 8.4 ± 0.8b 10.0 ± 0.3b

21.1 ± 1.7 25.4 ± 1.6b 26.1 ± 0.6b

37 ± 6 109 ± 11b 264 ± 9b

83 ± 2 158 ± 9b 295 ± 9b

2.7 ± 0.4b

2.3 ± 0.5b

c

c

The reaction mixture (2 mL) contained 0, 5, 15, or 50 μM quercetin in 50 mM KCl−HCl (pH 2.0) in the presence and absence of 50 μM (+)-catechin or dinitrosocatechin. Reactions were initiated by the addition of 0.25 mM sodium nitrite, and the concentrations of reactants and products were determined at 1 min after the initiation of reactions. diNO−CAT = 6,8-dinitrosocatechin. Each data point represents the mean with SD (n = 3−6). bSignificant differences in the decrease and increase in concentrations of quercetin and Q293, respectively, in the absence and presence of catechin or dinitrosocatechin and significant differences in the formation of diNO−CAT and formation of P1 and P2 in relation to the absence of quercetin (p < 0.05). cBelow detection. a

The value was (15.0 ± 3.6) × 102 M−1 min−1 (n = 6), which was about 2.2-fold the rate constants of the reactions between nitrous acid and (+)-catechin and between nitrous acid and 6,8dinitrosocatechin (see above). The decrease in the concentration of quercetin was enhanced by 0.1 mM (+)-catechin by about 2.9- and 1.5-fold when the initial concentrations of quercetin were 10 and 30 μM, respectively. The degree of the enhancement was somewhat greater than that of the enhancement of Q293 formation by 0.1 mM (+)-catechin. (+)-Catechin-dependent enhancement of the quercetin consumption in nitrous acid/quercetin systems was observed in a concentration range of nitrite from 0.05 to 0.7 mM, suggesting that interactions between (+)-catechin and quercetin can occur under the conditions of the gastric lumen. The results in Table 1 and Figure 5 indicate that semiquinone radicals produced by reactions 2 and 4 can oxidize quercetin, enhancing the production of Q293. Interactions between Dinitrosocatechin and Quercetin. The effects of 0.1 mM 6,8-dinitrosocatechin on the formation of Q293 were also studied in the presence of various concentrations of quercetin (Figure 5). Dinitrosocatechin enhanced the formation of Q293, and the enhancement was about 4-fold in the presence of 10 and 30 μM quercetin (△). The production of P1 and P2 was effectively suppressed by quercetin, and 50% inhibition was observed at approximately 10 μM quercetin (▲). Table 1 shows the enhancement of nitrous-acid-induced oxidation of quercetin by 0.1 mM 6,8-

dinitrosocatechin. The degrees of the enhancement were about 3.1- and 2.3-fold in the presence of 10 and 30 μM quercetin, respectively. The results in Table 1 and Figure 5 suggest that the dinitrosocatechin semiquinone radical generated by the reaction with nitrous acid (reaction 7) can oxidize quercetin, suppressing the formation of dinitrosocatechin quinones and enhancing the formation of Q293. • NO Production under Anaerobic Conditions. Table 2 shows the rate of nitrous-acid-induced •NO production determined using a Clark-type electrode under anaerobic conditions. When 50 μM quercetin, (+)-catechin, or 6,8dinitroscatechin was present in the reaction mixture, the rate of • NO production was in the order (+)-catechin < 6,8dinitrosocatechin < quercetin. Such order of •NO production in a system simulating gastric lumen has been reported.40 The faster •NO production in the presence of quercetin compared to the presence of dinitrosocatechin could be explained by the difference in their reaction rate with nitrous acid (see above), and the much slower •NO production in the presence of (+)-catechin might be due to the reaction of semiquinone radicals derived from (+)-catechin with •NO by reactions 3 and 5. The rate of nitrous-acid-induced •NO production increased nearly linearly as a function of the quercetin concentration. In the presence of 50 μM (+)-catechin, however, quercetin did not increase the rate of •NO production as expected from the rate in the absence of (+)-catechin, when the concentration of 4956

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quercetin was 5 μM. In the presence of 15 and 50 μM quercetin, quercetin increased the rate of •NO production significantly. These results support the postulation that semiquinone radicals of (+)-catechin and mononitrosocatechin can react with not only quercetin but also •NO produced by quercetin/nitrous acid systems and suggest that the rate of • NO consumption was limited by the rate of production of semiquinone radicals of (+)-catechin and mononitrosocatechin by reactions 3 and 5. 6,8-Dinitrosocatechin (50 μM) increased • NO production almost additively or more than additively at every concentration of quercetin examined. The result indicates that the reaction of •NO with the semiquinone radical of 6,8dinitrosocatechin will be slow or negligible, supporting the above conclusion that the radical will react rapidly with quercetin. Under the conditions of Table 2, N2O3 generated by reaction 941 appears not to contribute to the formation of dinitrosocatechin, because •NO production by reaction 9 was quite slow (less than 1 μM/min in the presence of 0.5 mM sodium nitrite). 2HNO2 ⇆ N2O3 + H 2O ⇆ •NO + •NO2 + H 2O

(9)

Nitrosonium cation (NO+) formed by reaction 1041 might not contribute significantly, either, to the formation of dinitrosocatechin. HNO2 + H+ ⇆ H 2NO2+ ⇆ NO+ + H 2O

Figure 6. Possible interactions among quercetin, (+)-catechin, and 6,8dinitrosocatechin. AA, ascorbic acid; DHA, dehydroascorbic acid; CAT and CAT•, (+)-catechin and its semiquinone radical; diNO−CAT and diNO−CAT•, 6,8-dinitrosocatechin and its semiquinone radical; diNO−CAT−quinone, quinone form of diNO−catechin; NO−CAT and NO−CAT•, mononitrosocatechin and its semiquinone radical, Q and Q•, quercetin and its semiquinone radical; Q293, an oxidation product of quercetin. Broken lines = slow reactions.

(10)

If NO contributed to the nitrosation, 5 μM quercetin should increase the rate of •NO production in the presence of (+)-catechin, effectively inhibiting the formation of dinitrosocatechin, because quercetin might reduce NO+ to •NO. Interactions between Quercetin and (+)-Catechin or Dinitrosocatechin under Anaerobic Conditions. The interactions between quercetin and (+)-catechin or 6,8dinitrosocatechin were also studied under anaerobic conditions (Table 3). (+)-Catechin (50 μM) and 6,8-dinitrosocatechin (50 μM) enhanced a nitrous-acid-induced decrease in the concentration of quercetin and the formation of Q293, irrespective of the concentration of quercetin. The results indicate that quercetin also interacted with (+)-catechin and dinitrosocatechin under anaerobic conditions. The interaction between quercetin and (+)-catechin is supported by the suppression of nitrous-acid-induced formation of 6,8-dinitrosocatechin by quercetin, and the interaction between quercetin and dinitrosocatechin is supported by the suppression of P1 and P2 formation by quercetin. Although the concentrations of (+)-catechin decreased by about 4 μM during the incubation for 1 min, effects of quercetin on the decrease in the concentration were unclear, similar to under aerobic conditions, supporting the slower reaction of the (+)-catechin semiquinone radical with quercetin than with NO. From the data in Tables 1 and 3 and Figure 5, we could conclude that the interactions between quercetin and (+)-catechin or 6,8-dinitrosocatechin were possible under semi-anaerobic conditions, such as the gastric lumen. Possibility of Interactions between Catechins and Quercetin in the Stomach. The reactions concerned in this study are summarized in Figure 6. The (+)-catechin semiquinone radical generated by the reaction of (+)-catechin with nitrous acid will react effectively with •NO, producing mononitrosocatechin. The reaction of the semiquinone radical with quercetin will not be rapid enough to suppress nitrousacid-induced consumption of (+)-catechin (represented as +

broken lines). Mononitrosocatechin also reacts with nitrous acid, producing the semiquinone radical, which can react with both •NO and quercetin, producing dinitrosocatechin and the quercetin semiquinone radical, respectively. The quercetin semiquinone radical generated by the above reactions is rapidly transformed to Q293 via the quercetin quinone form.12,42 Q293 has been reported to decompose to more stable 2,4,6trihydroxyphenylglyoxylic acid and 3,4-dihydroxybenzoic acid by oxidation.17 Dinitrosocatechin produced by the above reactions also reacts with nitrous acid producing •NO and the semiquinone radical that can be transformed to quinone by disproportionation. The molar ratios of (+)-catechin to quercetin glycosides were calculated to be about 0.7 and 0.5 in apple and buckwheat flour, respectively, and the ratios of (−)-epicatechin to quercetin glycosides were calculated to be about 3.2 and 3.5 in apple and buckwheat flour, respectively.3,4 Because parts of quercetin glycosides from foods are hydrolyzed in the oral cavity, as described in the Introduction,6−8 quercetin can coexist with (−)-epicatechin as well as (+)-catechin in gastric juice. There are no differences in the rate constants between (+)-catechin and (−)-epicatechin when these polyphenols react with superoxide anion radicals and singlet molecular oxygen,23 and the antioxidant activity of (+)-catechin is equivalent to that of (−)-epicatechin when measured using the 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid cation radical (ATBS• +) as an oxidant.43 Furthermore, 6,8-dinitorosoepicatechin produced in (−)-epicatechin/nitrous acid systems isomerizes to 6,8dinitrosocatechin.15 We can postulate from the results that (−)-epicatechin as well as (+)-catechin can interact with quercetin in the presence of nitrite under acidic conditions, as 4957

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(5) Bhagwat, S.; Haytowitz, D. B.; Holden, J. M. USDA Database for the Flavonoid Content of Selected Foods, Release 3.1; United States Department of Agriculture (USDA): Washington, D.C., 2013; http:// www.ars.usda.gov/SP2UserFiles/Place/12354500/Data/Flav/Flav3-1. pdf. (6) Hirota, S.; Nishioka, T.; Shimoda, T.; Miura, K.; Ansai, T.; Takahama, U. Quercetin glucosides are hydrolyzed to quercetin in human oral cavity to participate in peroxidase-dependent scavenging of hydrogen peroxide. Food Sci. Technol. Res. 2001, 7, 239−245. (7) Browning, A. M.; Walle, U. K.; Walle, T. Flavonoid glycosides inhibit oral cancer proliferationRole of cellular uptake and hydrolysis to the aglycones. J. Pharm. Pharmacol. 2005, 57, 1037− 1041. (8) Walle, T.; Browning, A. M.; Steed, L. L.; Reed, S. G.; Walle, U. K. Flavonoid glucosides are hydrolyzed and thus activated in the oral cavity in humans. J. Nutr. 2005, 135, 48−52. (9) Pannala, A. S.; Mani, A. R.; Spencer, J. P. E.; Skinner, Y.; Bruckdorfer, K. R.; Moore, K. P.; Rice-Evans, C. A. The effect of dietary nitrate on salivary, plasma and urinary nitrate metabolism in humans. Free Radical Biol. Med. 2003, 34, 576−584. (10) Vanýsek, P. Electrochemical Series; Issuu, Inc.: Palo Alto, CA, 2010; http://issuu.com/time-to-wake-up/docs/electrochemical_ redox_potential. (11) Janeiro, P.; Brett, A. M. O. Catechin electrochemical oxidation mechanism. Anal. Chim. Acta 2004, 518, 109−115. (12) Brett, A. M. O.; Ghica, M.-E. Electrochemical oxidation of quercetin. Electroanalysis 2003, 15, 1745−1750. (13) Takahama, U.; Oniki, T.; Hirota, S. Oxidation of quercetin by salivary components. Quercetin-dependent reduction of salivary nitrite under acidic conditions producing nitric oxide. J. Agric. Food Chem. 2002, 50, 4317−4322. (14) 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 function for polyphenols with a catechol group? Free Radical Biol. Med. 2005, 39, 668−681. (15) Lee, S. Y. H.; Munerol, B.; Pollard, S.; Youdim, K. A.; Pannala, A. S.; Kuhnle, G. G. C.; Debnam, E. S.; Rice-Evans, C.; Spencer, J. P. E. The reaction of flavonols with nitrous acid protects against Nnitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth. Free Radical Biol. Med. 2006, 40, 323−334. (16) Panzella, L.; Manini, P.; Napolitano, A.; d’Ischia, M. The acidpromoted reaction of the green tea polyphenol epigallocatechin gallate with nitrite ions. Chem. Res. Toxicol. 2005, 18, 722−729. (17) Hirota, S.; Takahama, U.; Ly, T. N.; Yamauchi, R. Quercetindependent inhibition of nitration induced by peroxidase/H2O2/nitrite systems in human saliva and characterization of an oxidation product of quercetin formed during the inhibition. J. Agric. Food Chem. 2005, 53, 3265−3272. (18) Benjamin, N.; O’Driscoll, F.; Dougall, H.; Duncan, C.; Smith, L.; Golden, M.; McKenzie, H. Stomach NO. Nature 1994, 368, 502. (19) Xu, J.; Xu, X.; Verstraete, W. The bacteriacidal effect and chemical reactions of acidified nitrite under conditions simulating the stomach. J. Appl. Microbiol. 2001, 90, 523−529. (20) Miyoshi, M.; Kasahara, E.; Park, A. M.; Hiramoto, K.; Minamiyama, Y.; Takemura, S.; Sato, E. F.; Inoue, M. Dietary nitrate inhibits stress-induced gastric mucosal injury in the rat. Free Radical Res. 2003, 37, 85−90. (21) Björne, H. H.; Petersson, J.; Phillipson, M.; Weizberg, E.; Holm, L.; Lundberg, J. O. Nitrite in saliva increases gastric blood flow and mucuss thickness. J. Clin. Invest. 2004, 113, 106−114. (22) Petersson, J.; Phillipson, M.; Jansson, E. A.; Patzak, A.; Lundberg, J. O. Dietary nitrate increase gastic blood flow and mucus defense. Am. J. Physiol.: Gastrointest. Liver Physiol. 2007, 292, G718− G724. (23) Jovanovic, S. V.; Steenken, S.; Simic, M. G.; Hara, Y. Antioxidant properties of flavonoids: reduction potentials and electron transfer reactions of flavonoid radicals. In Flavonoids in Health and Disease;

shown in Figure 6. The postulation was supported by the result that 0.1 mM (−)-epicatechin enhanced nitrite-induced oxidation of 10, 30, and 100 μM quercetin, increasing the formation of Q293 in 50 mM KCl−HCl (pH 2.0) under aerobic and anaerobic conditions. It has been reported that (i) catechins can suppress nitrousacid-induced N-nitrosodimethylamine formation from dimethylamine producing dinitrosocatechins and (ii) dinitrosocatechins induce toxic effects in Caco-2 cells, which are cultured colon carcinoma cells.15 On the other hand, it is known that quinones are toxic because of their generation of reactive oxygen species and binding to DNA.44 Thus, quercetin has an important function to prevent the formation of reactive quinone from the dinitrosocatechin semiquinone radical. In addition to quercetin, ascorbic acid in gastric juice may also prevent the formation of quinones by reducing corresponding radicals (not shown in Figure 6). If dinitrosocatechin quinone is produced in the stomach, ascorbic acid in gastric juice can reduce quinone to the mother compound and salivary thiocyanate can transform quinone to a stable thiocyanate conjugate.37−39 Although there are some systems to scavenge the quinones of dinitrosocatechins in the gastric lumen, it is beneficial if the formation of the quinones is prevented. Quercetin and ascorbic acid are candidates to prevent the formation of quinones in the gastric lumen. Thus, it seems to be favorable to ingest foods rich in components, which reduce semiquinone radicals derived from mononitrosocatechins and dinitrosocatechins, to prevent the formation of toxic quinones. In this way, the clarification of chemical interactions among polyphenols under the conditions simulating the stomach may be important to prepare foods that are beneficial for human health.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +81-582-1131. Fax: +81-93-582-6000. E-mail: [email protected]. Funding

Part of this study was supported by Grants-in-Aid for Scientific Research (22500790 and 23500986) from the Ministry of Education and Science in Japan. Sonja Veljovic-Jovanovic and Filis Morina acknowledge the partial support from the Ministry of Education, Science, and Technological Development of the Republic of Serbia (III43010). Notes

The authors declare no competing financial interest.

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