In vitro scavenging activity for reactive oxygen species by N-substituted indole-2-carboxylic acid esters.

The hydroxyl radical (HO*)- and superoxide anion radical (O* (2))-scavenging activity, as well as the singlet oxygen ((1)O(2))-quenching property of N-substituted indole-2-carboxylic acid esters (INDs) were investigated by deoxyribose degradation assay, a chemiluminescence method and the electron spin resonance (ESR) spin-trapping technique. This novel group of compounds was developed as a search for cyclooxygenase-2 (COX-2)-selective enzyme inhibitors. The results obtained demonstrated that of the 16 compounds examined, five inhibited light emission from the superoxide anion radical (O* (2))-DMSO system by at least 60% at a concentration of 1 mmol/L, nine prevented the degradation of deoxyribose induced by the Fenton reaction system (range 3-78%) or scavenged hydroxyl radicals (HO*) directly (range 8-93%) and 14 showed the (1)O(2)-quenching effect (range 10-74%). These results indicate that majority of the indole esters tested possess the ability to scavenge O(-) (2) and HO radicals and to quench (1)O(2) directly, and consequently may be considered effective antioxidative agents.     

Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using l-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH) free radical scavenging activities of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-l-glycinate ethyl ester (c₅) had the strongest ability to scavenge free radicals with an IC₅₀ value of 18.6 µM. The acetylferuloyl amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides.     

Identification of chemical structure and free radical scavenging activity of diphlorethohydroxycarmalol isolated from a brown alga, Ishige okamurae

To obtain a natural antioxidant from a marine biomass, this study investigated the antioxidative activity of methanolic extracts from the marine brown alga, Ishige okamurae collected off Jeju Island. A potent free radical scavenging activity was detected in the ethyl acetate fraction containing polyphenolic compounds, and the potent antioxidant elucidated as a kind of phlorotannin, diphlorethohydroxycarmalol, by NMR and mass spectroscopic data. The free radical scavenging activities of the diphlorethohydroxycarmalol were investigated in relation to 1,1-diphenyl-2-picrylhydrazyl (DPPH), alkyl, and hydroxyl radicals using an electron spin resonance (ESR) system. The diphlorethohydroxycarmalol was found to scavenge DPPH (IC50=3.41 microM) and alkyl (IC50=4.92 microM) radicals more effectively than the commercial antioxidant, ascorbic acid. Therefore, these results present diphlorethohydroxycarmalol as a new phlorotannin with a potent antioxidative activity that could be useful in cosmetics, foods, and pharmaceuticals.     

Radical scavenging activity and oxidative modification of citrus dehydrin.

Dehydrins are ubiquitous proteins produced by plants in response to water stress. Their functions, however, are not fully understood. The overexpression of Citrus unshiu Marcov. dehydrin (CuCOR19) enhanced cold tolerance in transgenic plants by reducing lipid peroxidation promoted by cold stress, suggesting that the CuCOR19 protein directly scavenges radicals. In this paper, we report the radical scavenging activity and oxidative modification of CuCOR19. The hydroxyl radical generated by the Fe2+/H2O2 system and peroxyl radical generated from 2, 2'-azobis (2-amidinopropane) (AAPH) were scavenged by CuCOR19, but hydrogen peroxide and superoxide were not. The scavenging activity for the hydroxyl radical and peroxyl radical of CuCOR19 was more potent than that of mannitol, and approximately equal to that of serum albumin, which is known as an antioxidative protein in mammals. CuCOR19 was degraded by the hydroxyl radical and peroxyl radical in a time- and dose-dependent manner. Mannitol and thiourea inhibited the degradation. Analysis of the amino acid composition of CuCOR19 indicated that glycine, histidine, and lysine, which are major residues in many dehydrins, were targeted by the hydroxyl radical. These results suggest that CuCOR19 is a radical scavenging protein, and may reduce oxidative damage induced by water stress in plants.     

迎春花色素提取及抗氧化性研究

目的:提取迎春花黄色素研究其抗氧化性能。方法:利用对羟基自由基(OH-·)和超氧阴离子自由基(O2-·)的清除能力研究迎春花色素的抗氧化性能。结果:随着迎春花色素量的增加,其清除OH-·和O2-·的能力逐渐提高,最高分别可达31.5%和91.0%。结论:迎春花色素对超氧阴离子和羟基自由基均具有较强的清除能力。     

Synthesis and radical-scavenging activity of a dimethyl catechin analogue

Catechin analogue 1 with methyl substituents ortho to the catechol hydroxyl groups was synthesized to improve the antioxidant ability of (+)-catechin. The synthetic scheme involved a solid acid catalyzed Friedel–Crafts coupling of a cinnamyl alcohol derivative to 3,5-dibenzyloxyphenol followed by hydroxylation and then cyclization through an intermediate orthoester. The antioxidative radical scavenging activity of 1 against galvinoxyl radical, an oxyl radical, was found to be 28-fold more potent than (+)-catechin.     

Galloylglucoses of low molecular weight as mordant in electron microscopy. II. The moiety and functional groups possibly involved in the mordanting effect

Synthetic pentamonogalloylglucose applied to fixed tissues acts as a mordant, inducing high and diversified contrast similar to that obtained with natural gallotannins of low molecular weight (LMGG). By the separate use of each of the two moieties of the galloylglucose molecule, it was found that gallic acid is the mordanting agent. Glucose may contribute, however, to the effect by increasing the solubility and cross-linking potential of the compound, since the mordanting induced by gallic acid alone is weaker than that produced by its hexose esters. As suggested by results obtained with various phenolics and benzoic acid derivatives, the functional groups required for the mordanting effect of such agents are the carboxyl group, and at least one hydroxyl group concomitantly present on the benzene ring. In the case of galloylglucoses, it is assumed that the effect is due to hydrolysis products (gallic, digallic, or trigallic acids) or to the multiple hydroxyl groups of the intact molecule. Esters of gallic acid (propyl- and methylgallate), as well as pyrogallol, produce a "reversed staining" of all membranes, except for those of communicating (gap) junctions.     

Antioxidative activity of the blue pigment formed in a D-xylose-glycine reaction system

A blue compound was prepared from 1 M D-xylose and 0.1 M glycine, and designated Blue-M1, an intermediate color product of melanoidins. As melanoidins are well known to have antioxidative activity as well as high scavenging activity against active oxygen species, the antioxidative activity of Blue-M1 against the peroxidation of linoleic acid was investigated, in addition to the scavenging activity of Blue-M1 toward hydroxyl and DPPH radicals. Blue-M1 suppressed the peroxidation of linoleic acid as effectively as melanoidins did. The scavenging activity of Blue-M1 toward hydroxyl and DPPH radicals was also as strong as that of melanoidins. Blue-M1 showed higher activity with increasing concentration. The pyrrolopyrrole ring and a methine bridge between two pyrrolopyrrole rings in Blue-M1 could be related to the ability for radical scavenging activity, but not four carboxyl groups.     

Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems

Although salicylates have been used for centuries as treatment of inflammatory diseases, the mechanism of action of these drugs is still not clear. Aspirin (acetylsalicylic acid) and other nonsteroidal anti-inflammatory drugs (NSAID) inhibit prostaglandin biosynthesis, a property that appears to explain part of their anti-inflammatory activity. However, this mechanism does not appear to explain the anti-inflammatory properties of salicylic acid, which is a major metabolite of ASA in vivo. Results of prior studies in our laboratory have established that benzoic acid, the parent compound of the salicylate group of drugs, is decarboxylated and hydroxylated by the hydroxyl free radical (OH.) produced by stimulated granulocytes. These observations suggested that salicylates might be similarly metabolized by granulocytes. If so, the capacity of salicylates to rapidly react with OH. might relate directly to their known anti-inflammatory properties. Preliminary experiments established that salicylic acid and aspirin were decarboxylated by the hydroxyl free radical generated by the enzyme system xanthine-xanthine oxidase. We then studied the metabolism of salicylates by human granulocytes. Unstimulated granulocyte suspensions did not oxidize ASA or salicylic acid. However, suspensions stimulated by opsonized zymosan particles rapidly oxidized both substrates in pharmacological concentrations. The rate of oxidation of salicylic acid was 16-fold higher than benzoic acid, whereas the rate of oxidation of ASA was four-fold higher. The reaction was oxygen dependent and could be inhibited by known hydroxyl scavengers, particularly dimethylthiourea. The reaction could also be inhibited by superoxide dismutase and azide, indicating that O-2 and heme or an iron-dependent enzyme were required for the reaction. The reaction was not impaired by compounds known to react with the HOCL and the chloramines generated by stimulated PMN. Furthermore, salicylic acid in high concentrations did not impair the HMPS pathway, the production of O-2 or the production of H2O2 by granulocytes. These data provide evidence that salicylates are rapidly oxidized by the hydroxyl free radical produced by granulocytes and not O-2, H2O2, or HOCL. This capacity of salicylates to react rapidly and selectively react with OH. may directly relate to their anti-inflammatory properties. In addition, results of our experiments indicate that stimulated granulocytes acquire the capacity to metabolize these drugs. Therefore, several metabolites of salicylates may be produced at a site of inflammation, all of which may have altered biological activity compared with the parent compound.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

The effect of poly(ethyleneglycol) esters on the partition of proteins and fragmented membranes in aqueous biphasic systems

The hydroxyl groups of poly(ethyleneglycol) have been esterified (partly) with a number of carboxylic acids. When these esters are included in dextranpoly(ethyleneglycol)-water biphasic systems the partitions of proteins and membranes between the two phases (and the interface) are in some cases strongly affected. The affinity of serum albumin for the poly(ethyleneglycol)-rich phase is strongly increased when the fatty acid group consists of more than 10 carbon atoms. The partition also depends on the number of double bonds in the fatty acid. A corresponding relationship is found for membranes from spinach chloroplasts. The partitions of ovalbumin, lysozyme (EC 3.2.1.17) and ribonuclease (EC 3.1.4.22) are not influenced by the fatty acid esters. Esters of dibasic carboxylic acids show a minute but marked effect on the partition of proteins in general while malate and tartrate esters affect strongly the partition of chloroplast membranes. The partitions of both proteins and membranes are influenced by poly(ethyleneglycol) deoxycholate. Experiments with malate dehydrogenase (EC 1.1.1.37), lactate dehydrogenase (EC 1.1.1.27), fumarase (EC 4.2.1.2), enolase (EC 4.2.1.11) and glutamate-oxaloacetate transaminase (EC 2.6.1.1) show that their partitions, measured on enzymic activity basis, is changed when esters of benzoic, linolenic, tartaric or deoxycholic acid are included in the biphasic system. The mechanism behind the effect of the esterified poly(ethyleneglycol) on the partition of biomaterial, in this type of aqueous biphasic systems, is discussed in terms of a direct binding of the esters to the partitioned material.     

Antioxidative properties of vanillic acid esters in multiple antioxidant assays

The antioxidative properties of vanillic acid esters were systematically evaluated by multiple assays to compare with the well-known antioxidants, vanillic acid and Trolox. We first performed assays with the model radicals, DPPH, galvinoxyl and ABTS cation (ABTS(?+)) types. Methyl vanillate, ethyl vanillate and butyl vanillate showed stronger activity than Trolox in the ABTS(?+)-scavenging assay, but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. In contrast, vanillic acid could quench the three radicals. We then evaluated their antioxidative activities by an ORAC assay and an oxidative hemolysis inhibition assay (OxHLIA), using physiologically relevant peroxyl radicals. Vanillic acid esters and vanillic acid exerted much stronger activity than Trolox in the ORAC assay and OxHLIA. The antioxidative activity by OxHLIA was strongly correlated to the lipophilicity of vanillic acid and its esters. These results indicate that the protective effect of vanillic acid esters against free radical-induced biomembrane damage increased with increasing lipophilicity.     

Detection of hydroxyl radical in the mitochondria of ischemic-reperfused myocardium by trapping with salicylate

Although the presence of free radicals has been indicated in ischemic-reperfused heart, the exact nature and source of these free radicals are not known. The present study utilized a chemical trap, salicylic acid, to trap hydroxyl radical which could be detected as hydroxylated benzoic acid using high pressure liquid chromatography. Since the hydroxylated product is extremely stable, heart was subjected to subcellular fractionation after ischemia and reperfusion, and each fraction was separately examined for the presence of hydroxyl radical. The results indicated for the first time the presence of hydroxyl radical in the mitochondrial fraction during early reperfusion, which decreased in intensity as the reperfusion progressed.     

Antioxidative activity of microbial metabolites of (-)-epigallocatechin gallate produced in rat intestines

The antioxidative activities of (-)-epigallocatechin gallate (EGCg) metabolites degraded by rat intestinal flora were investigated by a flow injection analysis coupled to an on-line antioxidant detection system with the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation. All of the metabolites were found to have antioxidative activity, suggesting that the EGCg metabolites may show antioxidative activity in the body.     

Antioxidative Activity of Microbial Metabolites of (−)-Epigallocatechin Gallate Produced in Rat Intestines

The antioxidative activities of (?)-epigallocatechin gallate (EGCg) metabolites degraded by rat intestinal flora were investigated by a flow injection analysis coupled to an on-line antioxidant detection system with the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation. All of the metabolites were found to have antioxidative activity, suggesting that the EGCg metabolites may show antioxidative activity in the body.     

Actions of melatonin in the reduction of oxidative stress

Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.     

A comparative study of antioxidative activities of cell-wall polysaccharides

Oxidative burst in plants is elicited by biotic and abiotic stressors. Analogously to some monosaccharides which act as intracellular antioxidants, cell-wall polysaccharides may be in charge of buffering free-radical production in the extracellular compartment under pronounced prooxidative settings. Although a wide range of plant polysaccharides have been examined for their antioxidative properties, this usually has not been done in a coherent and comparative manner and against biologically relevant reactive species. Here we show that different cell-wall polysaccharides, cellulose, pectin, d-galacto-d-mannan, arabinogalactan, and xylan, exhibit distinctive antioxidative activities against the hydroxyl radical (OH)-generating Fenton reaction and superoxide. We found, using an EPR spin-trapping method, that the main carriers of ‘anti-Fenton’ activity in the plant cell wall are pectin and xylan. They most likely act by binding metal ions in such a manner to allow the Fenton reaction, after which they scavenge OH. Such a mode of action is preferred by cells resulting in a safe degradation of H₂O₂. On the other hand, the polysaccharides examined showed similar superoxide scavenging capacities. We propose that plants may employ different antioxidative characteristics of polysaccharides to regulate their redox status by modifying the composition of the cell wall.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

Presence of Hydrogen Peroxide,a Source of Hydroxyl Radicals,in Acid Electrolyzed Water

Background

Acid electrolyzed water (AEW), which is produced through the electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution, is used as a disinfectant in various fields because of its potent antimicrobial activity. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity.

Methodology/Principal Findings

The aim of the present study is to detect hydroxyl radicals in AEW. The hydroxyl radicals in AEW prepared under different conditions were determined using an electron spin resonance (ESR) technique. A signal from 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH, an adduct of DMPO and the hydroxyl radical, was detected in AEW prepared by double or triple electrolyses of 1% NaCl but not of 0.1% NaCl solution. Then the presence of hydrogen peroxide as a proposed source of hydroxyl radicals was examined using a combination of ESR and a Fenton reaction. The DMPO-OH signal was clearly detected, even in AEW prepared by single electrolysis of 0.1% NaCl solution, when ferrous sulfate was added to induce a Fenton reaction, indicating the presence of hydrogen peroxide in the AEW. Since sodium formate, a hydroxyl radical scavenger, did not affect the bactericidal activity of AEW, it is concluded that the radical is unlikely to contribute to the antimicrobial activity of AEW, although a small amount of the radical is produced from hydrogen peroxide. Dimethyl sulfoxide, the other hydroxyl radical scavenger used in the present study, canceled the bactericidal activity of AEW, accompanied by complete depletion of free available chlorine, suggesting that hypochlorous acid is probably a major contributor to the antimicrobial activity.

Conclusions

It is strongly suggested that although hydrogen peroxide is present in AEW as a source of hydroxyl radicals, the antimicrobial activity of AEW does not depend on these radicals.     

Hydroxyl radical scavenging action of capsaicin

We recently reported that capsaicin (CAP) is capable of scavenging peroxyl radicals derived from 2,2'-azobis(2,4-dimethylvaleronitrile) as measured by electron spin resonance (ESR) spectroscopy. The present study describes the hydroxyl radical (HO*) scavenging ability of CAP as measured by DNA strand scission assay and by an ESR spin trapping technique with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The Fenton reaction [Fe(II)+ H(2)O(2) --> Fe(III) + HO* + HO(-)] was used as a source of HO*. The incubation of DNA with a mixture of FeSO(4) and H(2)O(2) caused DNA strand scission. The addition of CAP to the incubation mixture decreased the strand scission in a concentration-dependent manner. To understand the antioxidative mechanism of CAP, we used an ESR spin trapping technique. Kinetic competition studies using different concentrations of DMPO indicated that the decrease of the oxidative DNA damage was mainly due to the scavenging of HO* by CAP, not to the inhibition of the HO* generation system itself. We estimated the second order rate constants in the reaction of CAP and common HO* scavengers with HO* by kinetic competition studies. By comparison with the common HO* scavengers, CAP was found to scavenge HO* more effectively than mannitol, deoxyribose and ethanol, and to be equivalent to DMSO and benzoic acid, demonstrating that CAP is a potent HO* scavenger. The results suggest that CAP may act as an effective HO* scavenger as well as a peroxyl radical scavenger in biological systems.