Interaction between ascorbic acid and chlorogenic acid during the formation of nitric oxide in acidified saliva

When saliva and gastric juice are mixed, salivary nitrite is transformed to nitrous acid to produce nitric oxide (NO). The NO formation in acidified saliva was enhanced by ascorbic acid and chlorogenic acid. Thiocyanate ion (SCN(-)) also enhanced the transformation of nitrous acid to NO. During the NO formation in the presence of both ascorbic acid and chlorogenic acid, ascorbic acid was preferentially oxidized. Chlorogenic acid was oxidized after ascorbic acid had been oxidized. Ascorbyl radical was detected during the oxidation of ascorbic acid, and the radical intensity was decreased by chlorogenic acid. The decrease is discussed to be due to the reduction of the oxidation intermediate or product of chlorogenic acid by ascorbyl radical. The result obtained in this study suggests that ascorbic acid was preferentially oxidized and that not only ascorbic acid but also ascorbyl radical could interact with the oxidation intermediate or product of chlorogenic acid when chlorogenic acid was added to the mixture of saliva and gastric juice that contained ascorbic acid.     

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.     

Effects of the food additive sulfite on nitrite-dependent nitric oxide production under conditions simulating the mixture of saliva and gastric juice

The food additive sulfite is mixed with saliva, which contains nitrite, in the oral cavity, and the mixture is mixed with gastric juice in the stomach. In the stomach, salivary nitrite can be transformed to nitric oxide (NO). In this study, the effects of sulfite on nitrite-dependent NO production were investigated using acidified saliva (pH 2.6) and acidic buffer solutions (pH 2.0). Sulfite enhanced NO production in acidified saliva and acidic buffer solutions, and the enhancement increased with the increase in sulfite concentration from 0 to 0.1 mM, whereas suppressed NO production and the suppression increased as the concentration was increased over 0.2 mM. The enhancement was due to the increase in reaction rate between nitrous acid and nitrososulfonate (ONSO(3)(-)) that was formed by the reaction of nitrous acid with hydrogen sulfite, and the suppression was due to the increase in hydrogen sulfite-dependent consumption rate of ONSO(3)(-). A salivary component SCN(-) (1 mM) enhanced and suppressed NO production induced by 1 mM nitrite when sulfite concentrations were lower and higher than 1 mM, respectively. ONSO(3)(-) formed from hydrogen sulfite and nitrosyl thiocyanate (ONSCN), which was produced by the reaction of nitrous acid with SCN(-), seemed to contribute to the enhancement and suppression. NO production induced by nitrite/ascorbic acid systems was suppressed by sulfite, and the suppressive effects were decreased by SCN(-), whereas sulfite-induced suppression of NO production in nitrite/rutin systems was increased by SCN(-). During reactions of nitrite with sulfite in the presence and absence of SCN(-), oxygen was taken up. The oxygen uptake is discussed to be due to autoxidation of NO and radical chain reactions initiated by hydrogen sulfite radicals. The results of the present study suggest that sulfite can enhance and suppress nitrite-dependent NO production. It is discussed that radicals including hydrogen sulfite radicals can be formed through the reactions of nitrite and sulfite in the stomach.     

Inhibition of buckwheat starch digestion by the formation of starch/bile salt complexes: possibility of its occurrence in the intestine

During the digestion of starch in foods, starch is mixed with bile in the duodenum. Because fatty acids and some kinds of polyphenols could bind to starch, it was postulated that bile salts might also bind to starch. The purpose of this paper is to study the effects of bile and bile salts on starch/iodine complex formation and pancreatin-induced starch digestion. Bile suppressed starch/iodine complex formation and inhibited pancreatin-induced starch digestion slightly in control buckwheat starch, but did so significantly in buckwheat starch from which fatty acids and polyphenols had been extracted. Such significant suppression and inhibition by bile were also observed in a reagent soluble starch. The effects of cholate and taurocholate on the starch/iodine complex formation and the pancreatin-induced starch digestion were essentially the same as those of bile. Bile, cholate, and taurocholate suppressed amylose/iodine complex formation more significantly than amylopectin/iodine complex formation and inhibited pancreatin-induced amylose digestion more effectively than the digestion of amylopectin. It is concluded from the results that bile salts could bind to starch, especially amylose, the helical structures of which were not occupied by other molecules such as fatty acids and polyphenols, and that the binding resulted in the inhibition of starch digestion by pancreatin. The conclusion suggests that the function of bile salts can be discussed from the point of not only lipid digestion but also starch digestion.     

Formation of the thiocyanate conjugate of chlorogenic acid in coffee under acidic conditions in the presence of thiocyanate and nitrite: possible occurrence in the stomach

The objective of the present study was to elucidate how chlorogenic acid in coffee was transformed under acidic conditions simulating the mixture of saliva and gastric juice. When coffee was incubated in acidified saliva that contained nitrite and SCN-, in addition to nitric oxide (NO), four major components were detected. Two of the four components (components 3 and 4) were generated when chlorogenic acid was incubated in acidified saliva and when incubated in an acidic buffer solution in the presence of both nitrite and SCN-. By the incubation of chlorogenic acid in acidic nitrite in the absence of SCN-, components 3 and 4 were not formed but the quinone of chlorogenic acid and nitrated chlorogenic acid were formed. The result indicates that SCN- was indispensable for nitrous acid induced formation of components 3 and 4. Component 4 was isolated and its structure was determined to be (E)-5'-(3-(7-hydroxy-2-oxobenzo[d] [1,3]oxathiol-4-yl)acryloyloxy)quinic acid. Component 3, which was suggested to be 2-thiocyanatochlorogenic acid, seemed to be formed by the reaction between SCN- and the quinone of chlorogenic acid. As it has been reported that the quinone of chlorogenic acid can react with thiols and can decompose producing H2O2, the formation of component 4 can reduce the toxic effects of the quinone of chlorogenic acid.     

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.     

Chlorogenic acid in coffee can prevent the formation of dinitrogen trioxide by scavenging nitrogen dioxide generated in the human oral cavity

Coffee contains antioxidants like chlorogenic acid and its isomers. In this report, effects of coffee on the nitrite-induced N2O3 formation were studied using whole saliva and bacterial fraction prepared from the saliva. The formation of N2O3 was measured by fluorescence increase due to the transformation of 4,5-diaminofluorescein to triazolfluorescein. Coffee inhibited the nitrite-induced fluorescence increase, and 50% inhibition was observed at several microg of coffee/mL in bacterial fraction of saliva as well as whole saliva. During the inhibition of the fluorescence increase, concentration of chlorogenic acid and its isomers decreased. It is discussed that the reduction of NO2 by chlorogenic acid and its isomers contributed to the coffee-dependent inhibition of the fluorescence increase as N2O3 is formed from NO and NO2. When coffee was added to whole saliva, chlorogenic acid and its isomers bound to cells in the saliva. The rate of the fluorescence increase in bacterial fraction, which was prepared at defined periods after the ingestion of coffee, was increased to the rate before the ingestion of coffee with a half-time of about 1 h. This result suggests that chlorogenic acid and its isomers remained in the oral cavity for a few hours after ingestion of coffee. The significance of coffee drinking and rinsing of the mouth with coffee for the health of the oral cavity is proposed.     

Quercetin-dependent inhibition of nitration induced by peroxidase/H2O2/nitrite systems in human saliva and characterization of an oxidation product of quercetin formed during the inhibition

Local pH in the oral cavity can decrease to below 7 at the site where acid-producing bacteria are proliferating. Effects of pH on nitration of 4-hydroxyphenylacetic acid were studied using dialyzed human saliva. Dialyzed saliva nitrated 4-hydroxyphenylacetic acid to 4-hydroxy-3-nitrophenylacetic acid in the presence of nitrite and H(2)O(2). The rate of the nitration was dependent on pH, and the maximal rate was observed between pH 5.5 and 7.2. The optimum pH seemed to reflect rates of formation of nitrogen dioxide and 4-hydroxyphenylacetic acid radicals. Quercetin inhibited the nitration. The quercetin-dependent inhibition might be due to scavenging of nitrogen dioxide and 4-hydroxyphenylacetic acid radicals, which were formed by salivary peroxidase-dependent oxidation of nitrite and 4-hydroxyphenylacetic acid, respectively, and competition with nitrite and 4-hydroxyphenylacetic acid for peroxidase in saliva. An oxidation product of quercetin was formed during inhibition of the nitration by quercetin. The oxidation product was identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone. This component could also be oxidized by salivary peroxidase and nitrogen dioxide radicals. The oxidation products were 2,4,6-trihydroxyphenylglyoxylic and 3,4-dihydroxybenzoic acids. On the basis of the results, the significance of quercetin for inhibition of nitrogen dioxide formation and for scavenging of nitrogen dioxide radicals in the oral cavity is discussed.     

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.     

Regulation of CD8+ T cell development by thymus-specific proteasomes

Proteasomes are responsible for generating peptides presented by the class I major histocompatibility complex (MHC) molecules of the immune system. Here, we report the identification of a previously unrecognized catalytic subunit called beta5t. beta5t is expressed exclusively in cortical thymic epithelial cells, which are responsible for the positive selection of developing thymocytes. Although the chymotrypsin-like activity of proteasomes is considered to be important for the production of peptides with high affinities for MHC class I clefts, incorporation of beta5t into proteasomes in place of beta5 or beta5i selectively reduces this activity. We also found that beta5t-deficient mice displayed defective development of CD8(+) T cells in the thymus. Our results suggest a key role for beta5t in generating the MHC class I-restricted CD8(+) T cell repertoire during thymic selection.