Sachiko Hirota

Quercetin-Dependent Reduction of Salivary Nitrite to Nitric Oxide under Acidic Conditions and Interaction between Quercetin and Ascorbic Acid during the Reduction

A salivary component, nitrate, is reduced to nitrite in the oral cavity. Polyphenols in foods are mixed with nitrite in the saliva to be swallowed into the stomach. An objective of the present study is to elucidate reactions between a polyphenol quercetin and a nitrite under acidic conditions. Nitric oxide, which is formed by the reactions between nitrous acid and quercetin or ascorbic acid (AA), can be measured using an oxygen electrode in the saliva as well as a buffer solution. The initial oxidation of quercetin was inhibited by AA, and quercetin enhanced the oxidation of AA, suggesting AA-dependent reduction of quercetin radicals, which might be formed during the oxidation of quercetin by nitrous acid. On the basis of the above results, the usefulness of an oxygen electrode for the measurement of nitrite-dependent nitric oxide formation under acidic conditions is proposed and the possible mechanism of reduction of nitrous acid by quercetin and the interaction between quercetin and AA, which is a normal component in the gastric juice, for the reduction of nitrous acid is discussed.

Fatty Acids, Epicatechin-Dimethylgallate, and Rutin Interact with Buckwheat Starch Inhibiting Its Digestion by Amylase: Implications for the Decrease in Glycemic Index by Buckwheat Flour

Glycemic indexes of bread made from mixtures of wheat flour and buckwheat flour are lower than those made from wheat flour. To discuss the mechanism of the buckwheat flour-dependent decrease in glycemic indexes, the formation of a starch-iodine complex and amylase-catalyzed digestion of starch were studied using buckwheat flour itself and buckwheat flour from which fatty acids, rutin, and proanthocyanidins including flavan-3-ols had been extracted. Absorbance due to the formation of a starch-iodine complex was larger in extracted than control flour, and starch in extracted flour was more susceptible to pancreatin-induced digestion than starch in control flour. Fatty acids, which were found in the buckwheat flour extract, bound to amylose in the extracted flour, inhibiting its digestion by pancreatin. Rutin and epicatechin-dimethylgallate, which were also found in the extract, bound to both amylose and amylopectin in the extracted flour, inhibiting their digestion induced by pancreatin. We discussed from these results that the lower glycemic indexes of bread made from mixtures of wheat flour and buckwheat flour were due to binding of fatty acids, rutin, and epicatechin-dimethylgallate, which were contained in buckwheat flour, to wheat flour starch.

Inhibition of xanthine oxidase activity by an oxathiolanone derivative of quercetin

An oxathiolanone derivative of rutin could be produced in the stomach after the ingestion of rutin containing foods, and the oxathiolanone derivative could be hydrolysed to an oxathiolanone derivative of quercetin (quercetin-oxathiolanone) in the intestine. Quercetin-oxathiolanone as well as quercetin inhibited xanthine oxidase. Approximately 0.05μM quercetin-oxathiolanone inhibited the activity by 50%, whereas 50% inhibition by quercetin was observed at approximately 0.4μM. The results suggested that quercetin-oxathiolanone can be used as an effective inhibitor of xanthine oxidase and that the ingestion of rutin-rich foods may be useful to prevent the increase in the blood concentration of uric acid.

Proanthocyanidins in buckwheat flour can reduce salivary nitrite to nitric oxide in the stomach.

Buckwheat flour, which is used for various dishes in the world, is a good source of proanthocyanidins. Proanthocyanidins in the buckwheat flour reduced nitrous acid producing nitric oxide (NO) when the flour was suspended in acidified saliva or in acidic buffer solution in the presence of nitrite. The ingestion of dough prepared from buckwheat flour increased the concentration of NO in the air expelled from the stomach, suggesting that the proanthocyanidins also reduced nitrite to NO in the stomach. During the production of NO by the buckwheat flour/nitrous acid systems, oxidation, nitration, and nitrosation of proanthocyanidins proceeded. The increase in the concentration of NO could improve the activity of stomach helping the digestion of ingested foods and the nitration and nitrosation of the proanthocyanidins could contribute to the scavenging of reactive nitrogen oxide species generated from NO and nitrous acid.

Human salivary peroxidase-catalyzed oxidation of nitrite and nitration of salivary components 4-hydroxyphenylacetic acid and proteins

Human saliva contains high activities of peroxidase and high concentrations of nitrite (about 0.2 mM in average). If H2O2 is provided by bacteria and leukocytes in the oral cavity, peroxidase-dependent formation of reactive nitrogen species, which can nitrate phenolics like 4-hydroxyphenylacetic acid (HPA) and tyrosine residues in salivary proteins, is possible. H2O2-dependent oxidation of nitrite and H2O2-dependent nitration of HPA were observed in dialyzed saliva and by partially purified salivary peroxidase (SPX). The nitration was inhibited by a physiological electron donor to salivary peroxidase, SCN-. When concentrations of H2O2 and nitrite were increased, nitration of HPA was also observed in control (non-dialyzed) saliva. In addition, H2O2-dependent nitration of tyrosine residues in salivary proteins was observed in dialyzed saliva as an increase in absorbance around 420 nm at pH 7.2. Kinetic studies of the increase in absorbance indicated that sulfhydryl groups in salivary proteins as well as glutathione, ascorbate, urate and SCN- could inhibit the nitration. Since the nitration of proteins can lead to impairment of their functions, it is discussed how the oral cavity is protected from the damages caused by reactive nitrogen species under normal conditions and also discussed that reactive nitrogen species generated by the H2O2/nitrite/peroxidase system can participate in the host defence mechanism in the oral cavity.

Production of nitric oxide-derived reactive nitrogen species in human oral cavity and their scavenging by salivary redox components

Nitrite is reduced to nitric oxide (NO) in the oral cavity. The NO generated can react with molecular oxygen producing reactive nitrogen species. In this study, reduction of nitrite to NO was observed in bacterial fractions of saliva and whole saliva. Formation of reactive nitrogen species from NO was detected by measuring the transformation of 4,5-diaminofluorescein (DAF-2) to triazolfluorescein (DAF-2T). The transformation was fast in bacterial fractions but slow in whole saliva. Salivary components such as ascorbate, glutathione, uric acid and thiocyanate inhibited the transformation of DAF-2 to DAF-2T in bacterial fractions without affecting nitrite-dependent NO production. The inhibition was deduced to be due to scavenging of reactive nitrogen species, which were formed from NO, by the above reagents. The transformation of DAF-2 to DAF-2T was faster in bacterial fractions and whole saliva which were prepared 1–4 h after tooth brushing than those prepared immediately after toothbrushing. Increase in the rate as a function of time after toothbrushing seemed to be due to the increase in population of bacteria which could reduce nitrite to NO. The results obtained in this study suggest that reactive nitrogen species derived from NO are continuously formed in the oral cavity and that the reactive nitrogen species are effectively scavenged by salivary redox components in saliva but the scavenging is not complete.

Oxygen uptake during the mixing of saliva with ascorbic acid under acidic conditions: possibility of its occurrence in the stomach

Human saliva, which contains nitrite, is normally mixed with gastric juice, which contains ascorbic acid (AA). When saliva was mixed with an acidic buffer in the presence of 0.1 mM AA, rapid nitric oxide formation and oxygen uptake were observed. The oxygen uptake was due to the oxidation of nitric oxide, which was formed by AA‐dependent reduction of nitrite under acidic conditions, by molecular oxygen. A salivary component SCN− enhanced the nitric oxide formation and oxygen uptake by the AA/nitrite system. The oxygen uptake by the AA/nitrite/SCN− system was also observed in an acidic buffer solution. These results suggest that oxygen is normally taken up in the stomach when saliva and gastric juice are mixed.

Phenolic Components of Brown Scales of Onion Bulbs Produce Hydrogen Peroxide by Autooxidation

2O2 when the brown scales were suspended in water. Brown components isolated from the brown scales also transformed molecular oxygen into H2O2. During the autooxidation process, absorbance in the visible region was increased. On acid hydrolysis of the brown fraction, 2,4,6-trihydroxyphenylglyoxylic acid, 3,4-dihydroxybenzoic acid and the quinone form of benzoic acid were detected. In addition, glucose was detected as a sugar. 3,4-Dihydroxybenzoic acid was preferentially oxidized during autooxidation of the brown fraction. One of the oxidation products was the quinone form. Stable electron spin resonance (ESR) signals were detected in the brown fraction. New ESR signals appeared on oxidation of the brown fraction by hexacyanoferrate (III). One of the newly formed radicals seemed to have a 3,4-dihydroxyphenyl group. Based on these results, possible structures, mechanism of H2O2 formation and biological significance of the brown components are discussed.

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

Abstract 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.

Reduction of nitrous Acid to nitric oxide by coffee melanoidins and enhancement of the reduction by thiocyanate: possibility of its occurrence in the stomach.

Reactions of nitrous acid with freeze-dried instant coffee and its methanol-insoluble melanoidin fractions were studied at pH 2 in the presence and absence of thiocyanate (SCN (-)), simulating the mixture of coffee, saliva, and gastric juice. Coffee contained stable radicals, and the radical concentration increased by ferricyanide and decreased by ascorbic acid. This result indicates that the radical concentration was affected by the redox state of coffee and that the nature of the radical was due to quinhydrone structure that might be included in coffee melanoidins. Nitrite also increased the electron spin resonance (ESR) signal intensity at pH 2, suggesting that nitrite oxidized melanoidins producing nitric oxide (NO). The formation of NO could be detected by oxygen uptake due to the autoxidation of NO and using an NO-trapping agent. SCN (-) largely enhanced NO formation in coffee and methanol-insoluble melanoidin fractions but only slightly in a methanol-soluble fraction, and the enhancement accompanied the consumption of SCN (-) but did not accompany the formation of a stable ESR signal. The enhancement was explained by the reduction of NOSCN by melanoidins in methanol-insoluble fractions and that the consumption was due to binding of SCN (-) to melanoidins during their oxidation by nitrous acid. The result obtained in this study suggests that when coffee is ingested, in addition to chlorogenic acid and its isomers, melanoidins can also react with salivary nitrite and SCN (-) in the gastric lumen, producing NO.