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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.

Antioxidants themselves are oxidized to prevent oxidation of other molecules. One may ask if the oxidized antioxidants are safe for humans. However, there is very little information on the toxicity of oxidized antioxidants. We previously identified cytotoxic compounds in the products from oxidation of sesamol, a potent antioxidant in sesame oil. In this study using a flow cytometer with fluorescent probes, we revealed cytotoxic characteristics of two isomeric dimers (dimer A and B) in rat thymocytes. Increase in cell lethality by dimer A was more profound than those by sesamol and dimer B. The incubation of cells with dimer A increased the populations of shrunken cells and the cells with phosphatidylserine exposed on outer surface of cell membranes. Since these phenomena are parameters for early stage of apoptosis, the results indicate that dimer A may promote the process of apoptosis. However, the population of the cells containing hypodiploid DNA, a parameter for late stage of apoptosis, was decreased by the incubation with dimer A. It was not the case for dimer B. Results indicate that dimer A may inhibit the degradation of DNA during apoptosis. Taken together, it is likely that dimer A exerts both proapoptotic action and inhibitory action on late stage of apoptosis in rat thymocytes.

The iron-ion catalyzed oxidation of the ethanol solution of rosmarinic acid, a potent antioxidant polyphenol of Lamiaceae (Labiatae) plants, afforded a highly tyrosinase-inhibitory active product. The structure of the active product in the oxidation product mixture was determined using extensive NMR spectroscopy to have a novel oxygen-containing seven-membered ring system. The formation mechanism of the unique ring structure from the catechol part of the rosmarinic acid was proposed.

Many effects of sesamol, an antioxidative constituent of sesame oil, have been reported for human health benefits due to its antioxidative action. However, we recently isolated two cytotoxic products, trimer and tetramer of sesamol, from oxidation of sesamol by an assay-guided purification. In this study, we have revealed some cytotoxic characteristics of these products in rat thymocytes and human leukemia K562 cells. Incubation of cells with trimer or tetramer at 10-30 microM for 24h significantly increased cell lethality and population of rat thymocytes containing hypodiploid DNA, suggesting cell death with DNA fragmentation, while it was not the case for 30 microM sesamol. The cytotoxic action of tetramer was more potent than that of trimer in rat thymocytes when their concentrations were 10-30 microM. The incubation of cells with 10 microM tetramer for 24h increased the population of cells with exposed phosphatidylserine, the activity of caspases, and the nick of DNA. These results indicate tetramer-induced apoptosis. In K562 cells, the incubation with tetramer at 10 microM for 72 h significantly inhibited the growth without affecting the lethality. However, tetramer at 30 microM significantly increased cell lethality. It is likely that tetramer exerts more cytotoxic action on normal non-proliferative cells (rat thymocytes) rather than proliferative cancer cells (human leukemia K562 cells). It may be necessary to consider the condition for preservation of sesamol and the safety of products from in vivo oxidation of sesamol for human health.

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.

Polyphenols have recently attracted much attention as potent antioxidants and related bioactive substances. These potent antioxidative polyphenols are very oxidizable due to their chemical properties, and their oxidation products must accumulate in the oxidizing foods when they are contained as the active ingredients. In this investigation, 30 polyphenols and related phenolics were oxidized with oxygen in the presence of a catalytic amount of Fe ions. Piceatannol, catechin, epicatechin, hydroxytyrosol, carnosol, and carnosic acid were oxidized very quickly. Sinapic acid, caffeic acid, chlorogenic acid, rosmarinic acid, gallic acid, propyl gallate, α-tocopherol, quercetin, and nordihydroguaiaretic acid were moderately oxidized. Protocatechuic acid, syringic acid, taxifolin, resveratrol, gentisic acid, secoisolariciresinol, and ellagic acid were oxidized for 19-20 days; however, their oxidation was very slow and did not complete. The other phenolics were not oxidized. The obtained oxidation products were next subjected to a lipoxygenase inhibition assay and the results compared to those of the corresponding phenols. Very interestingly, the oxidation product from resveratrol showed a high inhibitory activity, whereas resveratrol itself had no activity and its oxidation efficiency was low. To clarify the inhibition principle of the oxidation product, an LC-MS analysis was carried out on the oxidation product. The analytical results showed that they are the oligomeric and degraded compounds of resveratrol. Among them, the structures of three dimeric compounds were successfully identified, and their activity data clarified that the closed ring dimers were potent lipoxygenase inhibitors, whereas the opened ring dimer was not. It should be noted that resveratrol had almost no lipoxygenase inhibitory activity, contrary to some researchers' findings.

The antioxidative activity of secocyclolignanes was compared with that of the corresponding dibenzyl lignans for the first time. The radical scavenging activity of the secocyclolignanes was weaker than that of the corresponding dibenzyl lignans, the butane diol type showing the highest activity. The butane type of secocyclolignane exhibited the highest antioxidant activity of the unsaturated fatty acid.

Oxidation of rosmarinic acid (I) affords the highly tyrosinase inhibitory active product (II) (yield given in g).

Phytophenols of edible plants have recently attracted much attention as potent antioxidants and related bioactive substances. These antioxidative phytophenols are very oxidizable due to their chemical properties, and their oxidation products must accumulate in the oxidizing foods when they are contained as active ingredients. In this investigation, several phytophenols, which are well known as potent antioxidants in food science, were oxidized by oxygen in the presence of a catalytic amount of Ferric ion. Caffeic acid, catechin, chlorogenic acid, rosmarinic acid, and sesamol were quickly oxidized, whereas eugenol, resveratrol, rutin, and quercetin were not under the stated conditions. The oxidation product mixtures of the quickly oxidized phytophenols were next subjected to a cytotoxic assay using normal cells. Among the products, the oxidation product from sesamol showed a remarkably high cytotoxic activity. To clarify the cytotoxic principle of the oxidation products, an assay-guided fractionation and subsequent isolation of the oxidation compound of sesamol was carried out. Structure analysis of the isolated compounds revealed that they are new dimeric compounds (2-5) of sesamol. The cytotoxic activity of the dimers was evaluated from the population of dead cells by a flow cytometric analysis of rat thymocytes in the presence of 100 μM of each compound. Compound 2 showed the most potent cytotoxic activity among them. Compound 2 has a typical conjugated carbonyl moiety and the moiety possibly contributes to its high toxicity from a structure-activity point of view.