Gallic acid oxidation by turnip peroxidase

Both ellagic and gallic acids non competitively inhibited guaiacol oxidation by turnip peroxidase. The K_i values were 3 and 26 μm for ellagic and gallic acid respectively. Enzymatic oxidation of gallic acid by the isolated major turnip peroxidase was characterized with respect to spectral behaviour, affinity constant and pH effect. The K_m for H₂O₂ and gallic acid are 2.5 and 8.0 mM for turnip peroxidase. The pH optimum for gallic acid oxidation is about 6.5 and the rate constant k₄ decreased with the increase of pH in presence of both guaiacol and Gallic acid. When the gallic acid oxidation products were subjected to chromatographic analysis, it was found to be converted mainly to ellagic and an unknown quinone.     

Generation of Probucol Radicals and Their Reduction by Ascorbate and Dihydrolipoic Acid in Human Low Density Lipoproteins

Probucol, 4.4'-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1.1-dimethyl)phenol], is a lipid regulating drug whose therapeutic potential depends on its antioxidant properties. Probucol and x-tocopherol were quantitatively compared in their ability to scavenge peroxyl radicals generatcd by the thermal decomposition of the lipid-soluble azo-initiator 2,2'-azo-bis(2,4-dimethyl-valeronitrile), AMVN, in dioleoylphos-phatidylcholine (DOPC) liposomes. Probucol showed 15-times lower peroxyl radical scavenging efficiency than x-tocopherol as measured by the effects on AMVN-induced luminol-dependent chemiluminescence. We suggest that probucol cannot protect x-tocopherol against its loss in the course of oxidation, although probucol is known to prevent lipid peroxidation in membranes and lipoproteins. In human low density lipoproteins (LDL) ESR signals of the probucol phenoxyl radical were detected upon incubation with lipoxygenase + linolenic acid or AMVN. Ascorbate was shown to reduce probucol radicals. Dihydro-lipoic acid alone was not able to reduce the probucol radical but in the presence of both ascorbate and dihydrolipoic acid a synergistic effect of a stepwise reduction was observed. This resulted from ascorbate-dependent reduction of probucol radicals and dihydrolipoic acid-dependent reduction of ascorbyl radicals. The oxidized form of dihydrolipoic acid, thioctic acid, did not affect probucol radicals either in the presence or in the absence of ascorbate.     

Evidence for an ascorbate shuttle for the transfer of reducing equivalents across chromaffin granule membranes

Adrenal chromaffin granules must shuttle reducing equivalents from the cytosol inward to reduce ascorbic acid oxidized during norepinephrine biosynthesis by intragranular dopamine-beta-hydroxylase. A transmembrane electron shuttle between the external (cytosolic) and intragranular ascorbate pools was demonstrated in vitro in intact bovine chromaffin granules undergoing tyramine- or dopamine-stimulated dopamine-beta-hydroxylase turnover. Incubation of intact chromaffin granules with tyramine results in a time-dependent decrease in reduced intragranular ascorbate and production of octopamine. The rate of ascorbate oxidation is a function of the extragranular concentrations of tyramine over the range 50 microM to 2 mM and is 95% inhibited by addition of the dopamine-beta-hydroxylase inhibitor disulfiram. The stoichiometry of octopamine synthesized/ascorbate oxidized closely approximates unity. The presence of extragranular dopamine also induces oxidation of intragranular ascorbate which is inhibited by blocking dopamine transport with reserpine. On the other hand, incubation with octopamine, which is also transported by the granules, causes no net decrease in reduced intragranular ascorbate. The presence of 400 microM extragranular ascorbate abolishes the observed tyramine-induced intragranular ascorbate oxidation. The addition of ascorbate extragranularly 30 min after addition of tyramine reverses the oxidation of intragranular ascorbate. The measurement of [14C]ascorbate distribution ratios in granule pellets and supernatants indicates that there is no transmembrane transport of ascorbate. Extravesicular NADH had no significant effect on matrix ascorbate levels during beta-hydroxylation. These data provide new in vitro evidence that chromaffin granules shuttle reducing equivalents inwardly from an extra- to an intravesicular ascorbate pool and that cytosolic ascorbate is the source of the intragranular reducing equivalents required during norepinephrine biosynthesis.     

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane,by ascorbate/dihydrolipoate in living HL-60 cells

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.     

Factors Affecting the Ascorbate- and Phenolic-dependent Generation of Hydrogen Peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H?0?), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H?0? production. H?0? gene ration from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% C0? was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% C0?. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H?0? generated by ascorbate and phenolic compounds, but there was still substantial H?0? generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H?0? generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H?0? generation is discussed.     

Generation of Probucol Radicals and Their Reduction by Ascorbate and Dihydrolipoic Acid in Human Low Density Lipoproteins

Probucol, 4.4′-[(1-methylethylidene)bis(thio)]bis-[2,6-bis(1.1-dimethyl)phenol], is a lipid regulating drug whose therapeutic potential depends on its antioxidant properties. Probucol and x-tocopherol were quantitatively compared in their ability to scavenge peroxyl radicals generatcd by the thermal decomposition of the lipid-soluble azo-initiator 2,2′-azo-bis(2,4-dimethyl-valeronitrile), AMVN, in dioleoylphos-phatidylcholine (DOPC) liposomes. Probucol showed 15-times lower peroxyl radical scavenging efficiency than x-tocopherol as measured by the effects on AMVN-induced luminol-dependent chemiluminescence. We suggest that probucol cannot protect x-tocopherol against its loss in the course of oxidation, although probucol is known to prevent lipid peroxidation in membranes and lipoproteins. In human low density lipoproteins (LDL) ESR signals of the probucol phenoxyl radical were detected upon incubation with lipoxygenase + linolenic acid or AMVN. Ascorbate was shown to reduce probucol radicals. Dihydro-lipoic acid alone was not able to reduce the probucol radical but in the presence of both ascorbate and dihydrolipoic acid a synergistic effect of a stepwise reduction was observed. This resulted from ascorbate-dependent reduction of probucol radicals and dihydrolipoic acid-dependent reduction of ascorbyl radicals. The oxidized form of dihydrolipoic acid, thioctic acid, did not affect probucol radicals either in the presence or in the absence of ascorbate.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Lucigenin chemiluminescence in human seminal plasma

Seminal plasma protects spermatozoa from the detrimental effects of reactive oxygen species such as hydrogen peroxide. We investigated the lucigenin-dependent chemiluminescence in cell-free seminal plasma from andrological patients. The seminal plasma was separated from cells by centrifugation. In all seminal plasmas studied lucigenin-dependent chemiluminescence (LCL) was detected. The LCL showed a strong pH-dependence. The signal was stable if samples were stored at +4°C for up to 4 days or up to 8 days at -80°C. Filtration of the samples (0.45 and 0.22 μm pore size) did not lower their luminescence. The addition of superoxide dismutase (SOD) and ascorbic acid oxidase (AAO) lowered LCL nearly to baseline values while trolox and desferal showed moderate effect, whereas allopurinol had no effect. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radicals in seminal plasma. Physiological concentrations of ascorbic acid yielded SOD-inhibitable lucigenin-chemiluminescence. The nitroblue-tetrazolium assay showed that ascorbic acid in buffer solution produced formazan. Superoxide-anion radicals were not detected in seminal plasma by the spin-trap DEPMPO due to their low steady state concentration. It is concluded that in seminal plasma ascorbate reacts with molecular oxygen yielding ascorbyl radicals and superoxide anion. If lucigenin is added to seminal plasma, reducing substances present, such as ascorbate, reduce lucigenin to the corresponding radical; this radical reacts with molecular oxygen and also forms O₂^-2.. So LCL in human seminal plasma results from the autoxidation of ascorbate and the oxidation of the reduced lucigenin. While the physiological relevance of the former mechanism is unknown, the latter is an artifact.     

Factors affecting the ascorbate- and phenolic-dependent generation of hydrogen peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H

2

O

2

), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H

2

O

2

production. H

2

O

2

generation from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% CO

2

was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% CO

2

. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H

2

O

2

generated by ascorbate and phenolic compounds, but there was still substantial H

2

O

2

generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H

2

O

2

generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H

2

O

2

generation is discussed.     

The effect of ascorbic acid on the breakdown of arachidonate 15-hydroperoxide in the presence of iron salts and complexes

The decomposition of 15-hydroperoxide of arachidonic acid initiated by iron and some of its complexes in the presence of ascorbate was studied using UV-absorbing measurements of conjugated dienes. The kinetics of 15-HPAA decomposition by Fe++ and Fe+(+)-EDTA was very fast and could not be registered using conventional spectrophotometry. In the presence of ascorbate addition of Fe or its complexes with EDTA and resulted in 15-HPAA decomposition, which could be measured by the changes in the UV absorption. The decomposition rate was dependent on the amount of ascorbate oxidation products formed. Preincubation of ascorbate with iron prevented the 15-HPAA decomposition by Fe++ or its complexes.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Effects of Ascorbate on the Oxidation of Derivatives of Hydroxycinnamic Acid and the Mechanism of Oxidation of Sinapic Acid by Cell Wall-Bound Peroxidases

A cell wall fraction isolated from epicotyls of Vigna angularis,which contained both ionically and covalently bound peroxidases,rapidly oxidized p-coumaric, caffeic and ferulic acids and slowlyoxidized sinapic acid. The oxidation of sinapic acid was greatlyenhanced in the presence of p-coumaric, caffeic or ferulic acid.Ascorbate (20 µM) inhibited the oxidation of ferulic acidby about 70% and completely inhibited the oxidation of p-coumaricand ferulic acids. The cell wall fraction was capable of bindingferulic and sinapic acids but not caffeic acid. p-Coumaric acidbound only slightly to cell walls. The oxidation of p-coumaricand ferulic acids by KCl-washed cell walls was inhibited byabout 60% and 10%, respectively, by 20 µM ascorbate, butthe oxidation of caffeic acid was completely inhibited by ascorbateat less than 20 µM. The oxidation of derivatives of hydroxycinnamicacid by peroxidases released from cell walls by washing with1 M KCl was completely inhibited by ascorbate. These resultssuggest that the inhibition by ascorbate depends on the substituentgroup of the phenyl ring of the derivatives of hydroxycinnamicacid when the oxidation reaction is catalyzed by cell wall-boundperoxidases and that the oxidation of sinapic acid is mediatedby phenoxyl radicals of derivatives of hydroxycinnamic acidother than sinapic acid. (Received December 2, 1993; Accepted March 3, 1994)     

Enzymatic recycling of ascorbic acid from dehydroascorbic acid by glutathione‐like peptides in the extracellular loops of aminergic G‐protein coupled receptors

The intracellular recycling of ascorbic acid from dehydroascorbic acid by the glutathione–glutathione reductase system has been well‐characterized. We propose that extracellular recycling of ascorbic acid is performed in a similar manner by cysteine‐rich, glutathione‐like regions of the first and second extracellular loops of some aminergic receptors including adrenergic, histaminergic, and dopaminergic receptors. Previous research in our laboratory demonstrated that ascorbic acid binds to these receptors at a site on their first or second extracellular loops, significantly enhancing ligand activity, and apparently recycling hundreds of times their own concentration of ascorbate in an enzymatic fashion. In this study, we have synthesized 25 peptides from the first and second extracellular loops of aminergic and insulin receptors and compared them directly to glutathione for their ability to prevent the oxidation of ascorbate and to regenerate ascorbate from dehydroascorbic acid. Peptide sequences that mimic glutathione in containing a cysteine and a glutamic acid‐like amino acid also mimic glutathione activity in effects and in kinetics. Some (but not all) peptide sequences that contain one or more methionines instead of cysteine can significantly retard the oxidation of ascorbic acid but do not recycle it from dehydroascorbate into ascorbate. Peptides lacking both cysteines and methionines uniformly failed to alter significantly ascorbate or dehydroascorbate oxidation or reduction. We believe that this is the first proof that receptors may carry out both ligand binding and enzymatic activity extracellularly. Our results suggest the existence of a previously unknown extracellular system for recycling ascorbate. Copyright © 2016 John Wiley & Sons, Ltd.     

Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein.

Chain-breaking antioxidants such as butylated hydroxytoluene, alpha-tocopherol, and probucol have been shown to decrease markedly the oxidative modification of low density lipoprotein (LDL). Their mechanism of action appears to involve scavenging of LDL-lipid peroxyl radicals. The purpose of this study was to investigate the occurrence of radical reactions produced during oxidation of LDL and LDL-containing probucol initiated by lipoxygenase or copper. In addition, we have investigated the possibility of a synergistic interaction between ascorbate and probucol in inhibiting the oxidation of LDL. Incubation of LDL-containing probucol and lipoxygenase produced a composite electron spin resonance (ESR) spectrum due to the endogenous alpha-tocopheroxyl radical and probucol-derived phenoxyl radical. The spectral assignment was further verified by chemical oxidation of alpha-tocopherol and probucol. In the presence of ascorbic acid, these radicals in the LDL particle were reduced to their parent compounds with concomitant formation of the ascorbate radical. In both the peroxidation of linoleic acid and the copper-initiated peroxidation of LDL, the antioxidant activity of probucol was significantly increased by low (3-6 microM) concentrations of ascorbate. The probucol-dependent inhibition of LDL oxidation was enhanced in the presence of ascorbic acid. We conclude that the reaction between the phenoxyl radical of probucol and ascorbate results in a synergistic enhancement of the antioxidant capacity of these two compounds and speculate that such reactions could play a role in maintaining the antioxidant status of LDL during oxidative stress in vivo.     

A method for obtaining linear reciprocal plots with caeruloplasmin and its application in a study of the kinetic parameters of caeruloplasmin substrates

1. The curved plots of 1/v against 1/[S] obtained when caeruloplasmin oxidizes NN-dimethyl-p-phenylenediamine were investigated. The first free-radical oxidation product of caeruloplasmin oxidation of NN-dimethyl-p-phenylenediamine is required for curvature, as straight-line plots were obtained when activities were measured either before appreciable free-radical product had appeared or in the presence of ascorbate, which reduced it back to NN-dimethyl-p-phenylenediamine. 2. In the presence of ascorbate linear reciprocal-plots were obtained with all of the 37 substrates tested. V(max.) values varied over only an eightfold range and those for the 20 p-amino compounds over only a twofold range. K(m) values, however, varied over a 10(4)-fold range. The small range of V(max.) values indicates that the rate-limiting step in caeruloplasmin action is relatively independent of the nature of the substrate. K(m) values suggest that substrates bind primarily by ring electrons, although certain side-chain groups increased the K(m) in a manner unrelated to likely changes of ring-electron densities. A mechanism involving repulsion between negative charges on the substrate and the enzyme was supported by the variation of the K(m) of 5-hydroxyindol-3-ylacetic acid with pH.     

Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects.     

Nitric oxide-induced oxidant stress in endothelial cells: amelioration by ascorbic acid

Nitric oxide has multiple beneficial effects in the blood vessel wall. However, high concentrations of nitric oxide in the presence of hydroperoxides have been shown to damage cultured cells. In this work, the effect of relatively high concentrations of nitric oxide alone on the function and antioxidant status of a human endothelial cell line (EA.hy926) was tested. Nitric oxide generated from 0.1 to 0.5mM spermine NONOate generated reactive species in the cells detected by triazole formation from diaminofluorescein and by oxidation of dihydrofluorescein. Intracellular ascorbic acid decreased this oxidant stress. Spermine NONOate also decreased intracellular ascorbate concentrations, although reduced glutathione was not affected unless cells had also been caused to reduce dehydroascorbic acid to ascorbate. Nitric oxide predictably inhibited both endothelial nitric oxide synthase and glyceraldehyde 3-phosphate dehydrogenase, and ascorbate partially prevented inhibition of the latter enzyme. These results suggest that relatively high concentrations of nitric oxide can cause oxidant stress in endothelial cells that is ameliorated by ascorbic acid.     

Protein glycation inhibitory activities of Lawsonia inermis and its active principles

The protein glycation inhibitory activity of ethanolic extract of Lawsonia inermis (henna) plant tissues was evaluated in vitro using the model system of bovine serum albumin and glucose. Protein oxidation and glycation are posttranslational modifications that are implicated in the pathological development of many age-related disease processes. This study investigated the effects of Lawsonia inermis ethanolic extract and its components, on protein damage induced by a free radical generator in in vitro assay system. We found that alcoholic extract of Lawsonia inermis can effectively protect against protein damage and showed that its action is mainly due to Lawsone. In addition, the presence of gallic acid also plays an important role in the protective activity against protein oxidation and glycation. Two known compounds, namely, Lawsone and gallic acid previously isolated from this plant were subjected to glycation bioassay for the first time. It was found that the alcoholic extract, lawsone (1) and gallic acid (2) showed significant inhibition of Advanced Glycated End Products (AGEs) formation and exhibit 77.95%, 79.10% and 66.98% inhibition at a concentration of 1500 microg/mL, 1000 microg/mL and 1000 microM respectively. Lawsonia inermis, compounds 1 and 2 were found to be glycation inhibitors with IC(50) 82.06 +/- 0.13 microg/mL, 67.42 +/- 1.46 microM and 401.7 +/- 6. 23 microM respectively. This is the first report on the glycation activity of these compounds and alcoholic extract of Lawsonia inermis.     

Inhibition of lipoxygenase-dependent lipid peroxidation by quercetin: Mechanism of antioxidative function

Quercetin inhibited soybean lipoxygenase-1-dependent linoleic acid peroxidation. Two to three μM quercetin was required for 50% inhibition. During the inhibition, quercetin was oxidized. The oxidation was observed as an absorbance decrease at about 380 nm and an absorbance increase at about 335 nm. Inhibition of linoleic acid peroxidation by quercetin seems to be due to reduction by the reagent of the linoleic acid radical formed as an intermediate during lipoxygenation. Quercetin oxidation was suppressed by ascorbate under conditions when ascorbate did not affect lipoxygenase-dependent linoleic acid peroxidation. The results suggest that ascorbate can reduce the quercetin oxidized by the linoleic acid radical back to quercetin. Based on the results, the significance of a redox reaction between oxidized quercetin and ascorbate is discussed.     

Characteristics of H2O2-initiated oxidation of blood serum lipids by kinetic parameters of chemiluminescence

H2O2-initiated free radical oxidation of blood serum lipids was investigated by the chemiluminescence method. The first flash of the chemiluminescence was stimulated by H2O2 decomposition on the reaction, similar to Fenton reaction. The time of the chemiluminescence flash second maximum was correlated with the contents of antioxidants. This dependence had a linear kind and was characterized by the correlation coefficient--0.9898. With increases of concentration of such antioxidant as a hydroquinone, the time of chemiluminescence flash second maximum grew.