Enhancing effect of a cysteinyl thiol on the antioxidant activity of flavonoids and identification of the antioxidative thiol adducts of myricetin.

The enhancing effect of a cysteinyl thiol N-benzoylcysteine methyl ester on the antioxidant activity of several flavonoids was investigated in a lipid oxidation system. Obvious enhancement was apparent for catechin, myricetin, quercetin, and taxifolin, the activity for myricetin being the most potent among them. An HPLC analysis of the products from the antioxidation reaction of myricetin in the presence of the thiol was carried out and the structures of the products were determined to clarify the enhancing effect chemically. The obtained data indicated that two thiol adducts on the B ring, and probably C-ring adducts, which were produced in the antioxidation process, exerted an enhancing effect on the antioxidant activity of myricetin.

Radical scavenging activity of spring mountain herbs in the Shikoku mountain area and identification of antiradical constituents by simple HPLC detection and LC-MS methods.

The functionality of spring mountain herbs, which were collected in the Kajigamori mountain area of Shikoku area in Japan, was investigated in the course of our studies for utilizing local plant resources. The radical scavenging activity of the extracts from seventeen herbs was measured. Among these herbs, two extracts from Polystichym ovato-paleaceum (Japanese name: Tsuyanashiinode) and Sambucus racemosa subsp. sieboldiana (Japanese name: Niwatoko) showed potent DPPH radical scavenging activity. The material evidence for the potent activity of the extracts was studied by a combination of our developed method for detecting antiradical compounds, LC-MS/MS, and enzymatic hydrolysis.

Reducing effects of polyphenols on metmyoglobin and the in vitro regeneration of bright meat color by polyphenols in the presence of cysteine.

The effect of polyphenols and related phenolic compounds on the reduction of metmyoglobin (MetMb) to oxymyoglobin (MbO2), in the presence of cysteine, was investigated. Caffeic acid, dihydrocaffeic acid, and hydroxtyrosol (600 µmol/L) did not show any reducing activity individually. However, their highly potent activity in the reduction of MetMb to MbO2 was observed in the presence of equimolar amounts of cysteine. On the basis of the analytical results for the redox reaction products generated during the MetMb-reducing reaction of caffeic acid, we proposed a mechanism for the polyphenol-mediated reduction of MetMb. As per the proposed mechanism, the antioxidant polyphenols having a catechol substructure can effectively reduce MetMb to MbO2 with chemical assistance from nucleophilic reactive thiol compounds such as cysteine. Moreover, cysteine-coupled polyphenols such as cysteinylcaffeic acids (which are coupling products of caffeic acid and cysteine) can be used as preserving agents for retaining the fresh meat color, because of their powerful reducing effect on MetMb. The reduction of MetMb to MbO2 changes the color of meat from brown to the more desirable bright red.

Metmyoglobin reduction by polyphenols and mechanism of the conversion of metmyoglobin to oxymyoglobin by quercetin.

The effect of antioxidant polyphenols and related phenolic compounds from plants on the reduction of metmyoglobin (MetMb) was investigated. Potent activity in the reduction of MetMb to oxymyoglobin (MbO2), a bright red protein in meat, was observed for three flavonols, kaempferol, myricetin, and quercetin, at 300 µmol/L against 60 µmol/L MetMb. Sinapic acid, catechin, nordihydroguaiaretic acid, taxifolin, morin, and ferulic acid promoted reduction at 600 µmol/L. A mechanism for the reduction by one of the active flavonols, quercetin, was proposed on the basis of analytical results for redox reaction products derived from quercetin. This suggested the importance of a high propensity toward reduction of the flavonol structure and rapid convertibility of the quinone form to the phenol form for the MbO2 reduction and the maintenance of the level of MbO2 produced.

Identification of a potent xanthine oxidase inhibitor from oxidation of caffeic acid.

Inhibitory activity of Fe-ion-catalyzed radical oxidation products from 22 types of phenolic compounds toward xanthine oxidase (XO) was investigated. Phenols are readily oxidizable compounds in nature and, thus, showed potent antioxidant activities. Among the phenols screened in this study, noticeable activity was observed in the oxidation product of caffeic acid, whereas almost no XO-inhibitory activity of caffeic acid was observed. Assay-guided purification of the oxidation product of caffeic acid afforded a highly potent XO inhibitor, with an IC50 value that was calculated to be 60 nmol L(-1), which indicated XO-inhibitory activity much stronger than that of allopurinol (IC50 = 1 µmol L(-1)), a potent XO inhibitor and excellent medicine for the treatment of gout. The chemical structure of this new XO inhibitor was investigated by one- and two-dimensional NMR and HR-ESI-MS analyses, and the unique tetracyclic structure was confirmed by synthesis starting from commercially available 1,2,4-trimethoxybenzene and 3,4-dimethoxylbenzoyl chloride.

Chemical evidence for the synergistic effect of a cysteinyl thiol on the antioxidant activity of caffeic and dihydrocaffeic esters.

Antioxidant activity of methyl caffeate and methyl dihydrocaffeate in the presence of a cysteinyl thiol was measured in an azo-initiator-induced lipid oxidation system. The coexistence of the thiol was observed to display a synergistic effect on the antioxidant activity of both caffeates. The synergism was observed mainly with respect to the elongation of the induction period, rather than the inhibition rate for lipid oxidation. For methyl caffeate, the maximum elongation of the induction period was observed in the presence of more than two equivalents of the thiol, whereas the maximum effect on the activity of methyl dihydrocaffeate was observed in the presence of more than three equivalents of the thiol. These synergistic effects were analysed by high-performance liquid chromatography and liquid chromatography-mass spectrometry analyses of the intermediates produced during the antioxidation period. The analytical results clarified that the mono-thiol adduct of methyl caffeate and the mono- and di-thiol adducts of methyl dihydrocaffeate contributed to the synergism in the antioxidant activity of both caffeates.

Effect of polyphenols on oxymyoglobin oxidation: prooxidant activity of polyphenols in vitro and inhibition by amino acids.

Effects of various plant phenolics, including polyphenols, on the oxidation of oxymyoglobin were investigated. Most phenolics promoted the oxidation of oxymyoglobin at both pH 5.4 and 7.4. Potent oxidation-promoting activity was observed by several efficient antioxidant polyphenols with a catechol moiety. Therefore, effects of the catechol structure were investigated using dihydrocaffeic acid analogues. The results clarified that ortho- or para-substituted diphenol structures were important for promoting the oxidation of oxymyoglobin. Inhibition of such prooxidant activity for oxymyoglobin by dihydrocaffeic acid was also investigated. Although the required concentration was relatively higher than that of dihydrocaffeic acid, several amino acids inhibited the oxidation. Among these, cysteine was the most potent. Although cysteine alone completely inhibited oxidation at a concentration above 1 mmol/L, 0.1 mmol/L cysteine showed oxidation-promoting activity. In the presence of 0.1 mmol/L dihydrocaffeic acid, in the range of 0.01 mmol/L to 1 mmol/L cysteine, 0.1 mmol/L cysteine showed the most efficient inhibition. These results suggest the possibility of the formation of some equimolar complexes of dihydrocaffeic acid and cysteine such as 5'-cysteinyl dihydrocaffeic acid, which may be produced during the prooxidation of dihydrocaffeic acid, contributing to the inhibition of the oxidation of oxymyoglobin.

Chemical evidence for potent xanthine oxidase inhibitory activity of Glechoma hederacea var. grandis leaves (kakidoushi-cha).

In this study, chemical evidence for the potent xanthine oxidase inhibitory activity of "kakidoushi-cha" (dry leaves of Glechoma hederacea var. grandis), a traditional folk tea consumed in Japan, was clarified on the basis of structure identification of the active constituents. Assay-guided fractionation and purification afforded 15 compounds from the most active chromatographic fraction of an extract of the tea. Two flavonoids, apigenin and luteolin, showed remarkable inhibitory activity against xanthine oxidase (XO). The contribution of these flavonoid constituents to the observed XO inhibitory activity of the methanol and boiling-water extracts of the tea was estimated to be ca. 35% and ca. 18%, respectively.

Synergistic increase in cell lethality by dieldrin and H2O2 in rat thymocytes: effect of dieldrin on the cells exposed to oxidative stress.

Dieldrin, one of persistent pesticides, is highly resistant to biotic and abiotic degradation. It is accumulated in organisms. Recent studies suggest that dieldrin exerts a potent cytotoxic action on cells exposed to oxidative stress. In this study, the effect of dieldrin on rat thymocytes exposed to hydrogen peroxide (H2O2)-induced oxidative stress was examined. Dieldrin at 5µM and H2O2 at 300µM slightly increased cell lethality from a control value of 5.4±0.5% (mean±standard deviation of four experiments) to 7.8±1.3% and 9.0±0.3%, respectively. Simultaneous application of dieldrin and H2O2 significantly increased cell lethality to 46.2±1.8%. The synergistic increase in cell lethality was dependent on dieldrin concentration (0.3-5µM) but not on H2O2 concentration (30-300µM). Dieldrin accelerated H2O2-induced cell death, which was estimated with the help of annexin V-FITC and propidium iodide. Presence of either dieldrin or H2O2 decreased the cellular content of nonprotein thiol and increased intracellular Zn(2+) concentration. The combination of dieldrin and H2O2 further pronounced these effects. TPEN, a chelator of intracellular Zn(2+), significantly attenuated the synergistic increase in cell lethality induced by dieldrin and H2O2. It is, therefore, suggested that dieldrin augments the cytotoxicity of H2O2 in a Zn(2+)-dependent manner.