Takayuki Oniki

Flavonoids and Some Other Phenolics as Substrates of Peroxidase: Physiological Significance of the Redox Reactions

2O2 without accumulating oxidation products of phenolics. Scavenging of H2O2 by the systems can proceed in vacuoles and the apoplast, because phenolics, AA and POX are normal components of the compartments. AA seems to control lignification because it reduces radicals of lignin monomers which are formed by POX-dependent reactions. On lignification, oxidation of sinapyl alcohol is enhanced by radicals of coniferyl alcohol and hydroxycinnamic acid esters when apoplastic POX rapidly oxidizes coniferyl alcohol and the esters but slowly oxidizes sinapyl alcohol. POX seems to participate in the browning of tobacco leaves and onion scales on aging. H2O2, which is required for the POX-dependent reactions, can be formed by autooxidation of the phenolics that are transformed to brown components. It is discussed that browning involves the formation of antimicrobial substances.

The presence of 4‐hydroxyphenylacetic acid in human saliva and the possibility of its nitration by salivary nitrite in the stomach

Human saliva contained 4‐hydroxyphenylacetic acid (HPA) (2–10 μM) and nitrite (60–300 μM). HPA was nitrated to 4‐hydroxy‐3‐nitrophenylacetic acid (NO2HPA) when HPA and sodium nitrite were mixed at pH 1.0. NO2HPA was also formed when saliva was incubated under acidic conditions. These results suggest that salivary HPA is nitrated to NO2HPA when saliva is swallowed into the stomach.

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.

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.

Free radicals produced by the oxidation of gallic acid and catechin derivatives

Gallic acid and catechin derivatives were oxidized in alkaline solution (pH 10.0–13.0) with K3[Fe(CN)6] under anaerobic conditions, and electron spin resonance (ESR) spectra of the radicals produced were measured. Gallic acid, epicatechin gallate, gallocatechin gallate, epigallocatechin, and epigallocatechin gallate showed hyperfine structures. Gallic acid was found to be oxidatively C—O coupled in alkaline solution (pH 10.5–12.0). It was found that an unpaired electron delocalized over gallocatechin gallate and epigallocatechin molecules, but was localized on the galloyl group and the A-ring of epicatechin gallate and epigallocatechin gallate. The galloyl group of gallo-catechin gallate was readily alkali-hydrolyzed but those of epicatechin gallate and epigallocatechin gallate were resistant to alkaline hydrolysis.

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.

Reactions of thiocyanate in the mixture of nitrite and hydrogen peroxide under acidic conditions : Investigation of the reactions simulating the mixture of saliva and gastric juice

Nitrite and SCN− in saliva can mixes with H2O2 in the stomach. The mixing can result in the formation of ONOOH. It is not yet known how salivary SCN− reacts with ONOOH. An objective of the present study was to elucidate the reaction between ONOOH and SCN− . In nitrite/H2O2 systems at pH 2, SCN− inhibited the consumption of nitrite and the formation of . SCN− enhanced the decomposition of ONOOH and H2O2 in HNO2/H2O2 systems. Accompanying the reactions, sulfate was formed, suggesting that ONOOH oxidized SCN− . SCN− inhibited the nitration of phenolics induced by HNO2/H2O2. The inhibition is discussed taking SCN− -dependent reduction of ONOOH to HNO2 into consideration. SCN− also inhibited H2O2-induced consumption of nitrite and nitration of phenolics in acidified saliva. The result obtained in this study suggests that salivary SCN− can reduce ONOOH to /HNO2 inhibiting nitrating reactions in the stomach.

3, 4-Dihydroxyphenylalanine Is Oxidized by Phenoxyl Radicals of Hydroxycinnamic Acid Esters in Leaves of Vicia faba L.

Mechanisms of oxidation of 3,4-dihydroxyphenylalanine (dopa) in leaves ofVicia faba have not yet been elucidated in details. The author hypothesized its oxidation by radicals of hydroxycinnamic acid esters that were generated by a peroxidase-dependent reaction in vacuoles. The results obtained in this study were followings. 1) Vacuolar peroxidase isolated from the leaves oxidized dopa more slowly than 4-coumaric and caffeic acid esters isolated from the leaves. 2) The hydroxycinnamic acid esters enhanced peroxidase-dependent oxidation of dopa and dopa suppressed their oxidation. 3) Degree of the enhancement was roughly correlated with rates of the oxidation of hydroxycinnamic acid esters. 4) The hydroxycinnamic acid esters increased levels of dopa radical in the presence of peroxidase. 5) In protoplasts of mesophyll cells ofV. faba, hydrogen peroxide-induced oxidation of dopa was faster than that of 4-coumaric acid and caffeic acid esters. These results support the above hypothesis that dopa in vacuoles is oxidized by phenoxyl radicals of hydroxycinnamic acid esters that are generated by vacuolar peroxidase.