Oxidation of quercetin by salivary components. Quercetin-dependent reduction of salivary nitrite under acidic conditions producing nitric oxide

Under acidic conditions, nitrite is protonated to nitrous acid (pK(a) = 3.2-3.4) that can be transformed into nitric oxide by self-decomposition and reduction. When sodium nitrite was mixed with quercetin at pH 1-2, quercetin was oxidized producing nitric oxide. In addition to quercetin, kaempferol and quercetin 4'-glucoside were also oxidized by nitrous acid, but oxidation of apigenin, luteolin, and rutin was slow compared to oxidation of the above flavonols. These results suggested that flavonols, which have a free hydroxyl group at carbon position 3, can readily reduce nitrous acid to nitric oxide. When the pH of saliva was decreased to 1-2, formation of nitric oxide was observed. The nitric oxide formation was enhanced by quercetin, and during this process quercetin was oxidized. These results indicate that there is a possibility of reactions between phenolics and nitrous acid derived from salivary nitrite in 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.     

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.     

Role of nitrite and nitric oxide in the processes of nitrification and denitrification in soil: Results from N tracer experiments

Recent research has proven soil nitrite to be a key element in understanding N-gas production (NO, N₂O, N₂) in soils. NO is widely accepted to be an obligatory intermediate of N₂O formation in the denitrification pathway. However, studies with native soils could not confirm NO as a N₂O precursor, and field experiments mainly revealed ammonium nitrification as the source of NO. The hypothesis was constructed, that the limited diffusion of NO in soil is the reason for this contradiction. To test this diffusion limitation hypothesis and to verify nitrite and NO as free intermediates in native soils we conducted through-flow (He/O₂ atmosphere) ¹⁵N tracer experiments using black earth soil in an experimental set up free of diffusion limitation. All of the three relevant inorganic N soil pools (ammonium, nitrite, nitrate) were ¹⁵N labelled in separate incubation experiments lasting 81 h based on the kinetic isotope method. During the experiments the partial pressure of O₂ was decreased in four steps from 20% to about 0%. The net NO emission increased up to 3.7 μg N kg⁻¹ h⁻¹ with decreasing O₂ partial pressure. Due to the special experimental set up with little to no obstructions of gas diffusion, only very low N₂O emission could be observed. As expected the content of the substrates ammonium, nitrate and nitrite remained almost constant over the incubation time. The ¹⁵N abundance of nitrite revealed high turnover rates. The contribution of nitrification of ammonium to the total nitrite production was approx. 88% under strong aerobic soil conditions but quickly decreased to zero with declining O₂ partial pressure. It is remarkable that already under the high partial pressure of 20% O₂ 12 % of nitrite is generated by nitrate denitrification, and under strict anaerobic conditions it increases to 100%. Nitrite is present in two separate endogenous pools at least, each one fed by the nitrification of ammonium or the denitrification of nitrate. The experiments clearly revealed that nitrite is almost 100% the direct precursor of NO formation under anaerobic as well as aerobic conditions. Emitted N₂O only originated to about 100% from NO under strict anaerobic conditions (0-0.2% O₂), providing evidence that NO is a free intermediate of N₂O formation by denitrification. To the best of our knowledge this is the first time that NO has been detected in a native soil as a free intermediate product of N₂O formation at denitrification. These results clearly verify the “diffusion limitation” hypothesis.     

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.     

Resistance of the caribou lichen Cladina stellaris (Opiz.) brodo to growth reduction by simulated acidic rain

Natural mats of C. stellaris growing in the subarctic lichen woodlands of northern Québec were treated in a randomized complete block design with solutions of simulated rain at pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 and 5.6. These solutions were acidified by addition of mixtures of sulfuric and nitric acids to give both 2 : 1 and 6 : 1 μequivalent ratios of SO _inf4 ^sup= : NO _inf3 ^sup? . After two years of acidification there was no significant effect of either pH or SO _inf4 ^sup= : NO _inf3 ^sup? ratio on the growth of C. stellaris, but thallus discoloration was evident below pH 3.5. After three years of acidification marginally significant (p = 0.05) but erratic depression of growth occurred under the 6 : 1 but not the 2 : 1 acidification regime, especially at pH 4.5 or less. Acid precipitation therefore only very gradually impairs the growth of C. stellaris, and the deleterious effects of acidification may be partially offset by nitrogen enrichment when precipitation is relatively rich in nitrate compared to sulfate ions.     

Investigation of the changes in low molecular weight organic acids and other physiological parameters by nitric oxide application in two wheat cultivars exposed to boron stress

Boron (B) is one of the essential nutrients for the growth of plants, but its high concentrations are toxic for plants. Thus, B toxicity is a big challenge in crop cultivation. Nitric oxide (NO) is a small signaling molecule that has cytoprotective roles in plants. We investigated whether exogenous sodium nitroprusside (SNP), which is a NO donor, may succeed to alleviate B-induced toxicity in wheat cultivars. Seedlings were grown for 10 days in a growth chamber at 25°C with 16 hr light–8 hr dark photo cycle. After high B application, the effects of SNP on growth parameters; electrolyte leakage (EL); changes in reactive oxygen species [contents of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and proline]; the activities of antioxidant enzymes [glutathione peroxidase (GSHPx), glutathione reductase (GR), and glutathione S-transferase (GST)] and nitrate reductase (NR); and low molecular weight organic acid (LMWOAs) contents and also chlorophyll and total carotenoid contents were investigated in both shoots and roots of two different wheat cultivars. All experiments were carried out in triplicate. 0.2 mM SNP application ameliorated the chlorophyll and total carotenoid contents, and growth parameters such as shoot length, root length, and fresh weight in both wheat cultivars exposed to B stress. SNP reduced the B-induced lipid peroxidation, EL, and proline and H₂O₂ content in wheat cultivars. SNP application also increased the activities of NR and antioxidant enzymes, including GSHPx, GR, and GST in wheat cultivars exposed to B toxicity. All of the tested LMWOAs including succinic, propionic, butyric, oxalic, formic, malic, malonic, and benzoic acids were increased by SNP treatment in the shoots and roots of both wheat cultivars exposed to B toxicity. In conclusion, results obtained from this study have demonstrated that interactive effects of SNP with B considerably reduced the toxic effects of B in wheat cultivars.     

Total nitrogen in plant material determinated by means of dry combustion: A possible alternative to determination by Kjeldahl digestion

Abstract

The use of dry combustion‐based analyzer (CHN‐600, LECO Corporation, St. Joseph, MI) for nitrogen (N) determination in plant material is described. The method was evalueted in inter‐laboratory proficiency test (International Plant‐Analytical Exchange) and compared with results obtained by the Kjeldahl method. Differences between methods were small and results were in very close agreement. However when differences did occur, correction for nitrates did not fully explain differences between the two methods. A high content of nitrates can lead to serious differences between results obtained by the Dumas and Kjeldahl methods. Accuracy for the dry combustion method was tested with certified reference materials. Long‐term evaluation of repeatability of a determination varied from 1% to 2% with respect to material analyzed or content of nitrogen.     

Ingestion of sand and soil by phytophagous earthworm Eudrilus eugeniae: a finding of relevance to earthworm ecology as well as vermitechnology

Epigeic earthworms are phytophagous in habit and are believed to prefer organic matter, principally phytomass, in various degrees of decay. To check whether epigeics will ingest soil/sand even when there is luxury availability of phytomass, the present work was carried out. It was seen that not only soil but even sand particles were ingested by epigeic (Eudrilus eugeniae) tested by us, even as there was availability of phytomass and cow manure in abundance. But the consumption of sand and soil decreased as the days progressed. Moreover, the ingestion of sand and soil with phytomass and cow dung did not show any significant influence on growth and survival of earthworms. It did increase the vermicast mass due to the presence of soil/sand as was confirmed by microphotography using polarizing microscope. The findings have important implications in the design of vermireactors for maximizing their efficiency.     

Identification of 9(E),11(E)-18:2 fatty acid methyl ester at trace level in thermal stressed olive oils by GC coupled to acetonitrile CI-MS and CI-MS/MS, a possible marker for adulteration by addition of deodorized olive oil

The olive oil market is suffering from sophisticated illegal treatments. One common adulteration process consists of the addition to virgin olive oil of lower quality oils, such as "lampante" oil, an inexpensive oil and with some organoleptic defects, which is then submitted to thermal deodorization under vacuum processes for removal of the undesired flavor components. Such a blending may not have a huge influence on the chemical composition and may not significantly affect the parameters usually checked as quality indicators, although the organoleptic properties may change. As a consequence, a major effort is being devoted to find reliable markers able to unmask such adulterations. We report here the complete characterization of a compound, detected at trace levels exclusively in thermal stressed oils, which could be a candidate marker for adulteration. The investigation, carried out by GC-MS and GC-MS/MS, provided its complete structure, including the stereochemistry, shown to be a 9(E),11(E)-18:2 fatty acid methyl ester. Experimental data also confirmed the influence of both temperature and heating time on formation and concentration of this compound.