著者

澤井 祐典

出版者

農業技術研究機構野菜茶業研究所

雑誌

野菜茶業研究所研究報告 (ISSN:13466984)

巻号頁・発行日

no.6, pp.23-58[含 英語文要旨], 2007-03

被引用文献数

1

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Tea (Camellia sinensis) leaves contain various antioxidants such as ascorbic acid, α-tocopherol and polyphe-nols. In this study, we have tried to clarify the molecular mechanisms underlying the antioxidative and radical-scavenging activities of these antioxidants and directly compared the reactivities of each antioxidant against the stable free-radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) using nuclear magnetic resonance (NMR) analysis. Tea catechins (flavan-3-ol derivatives) are a major constituent of tea polyphenols. In recent years, they have attracted much attention for their potential beneficial effects in disease prevention. In particular, the antioxidant activities of catechins seem to be most important in terms of their physiological function. First of all, (+) -catechin (0.015 mmoles) was reacted with DPPH (0.030 mmoles) in acetone-d_6 (0.7 ml) as a model reaction of tea catechins to elucidate the molecular mechanisms underlying the antioxidative and radical-scavenging activities of antioxidants. After the reaction, the purple color of DPPH faded gradually. The reac-tion mixture was subjected to 13^C NMR analysis. The spectra were compared with those of (+) -catechin. The characteristic signals of the B-ring disappeared, although the signals ascribable to the A and C-rings remained unchanged, and two carbonyl signals were clearly detected in the spectrum. These strongly suggest that two hydroxyl groups in the B-ring are more important as a radical scavenger among the four phenolic hydroxyl groups of (+) -catechin and the B-ring was changed to a quinone structure. We substantiate this by trapping the com-pound as an adduct of a 1, 2-phenylenediamine to an ο-quinone. (-) -Epicatechin was also confirmed to give a similar result. The appearance of two carbonyl signals indicated the same conclusion.Furthermore, (-) -epigallocatechin was reacted with DPPH. But no carbonyl signals appeared, although many original carbon signals decreased. The antioxidation mechanism of (-) -epigallocatechin (pyrogallol struc-ture) is likely to be different from those of (+) -catechin and (-) -epicatechin (catechol structures) . (-) -Epigallocatechin seems to give a rather stable radical and was not stabilized as an ο-quinone. We clarified that the differences in their antioxidative activities were due to the differences of the antioxidation mechanisms. A mixture of (-) -epicatechin (0.015 mmol) and (-) -epigallocatechin (0.015 mmol) was reacted with DPPH (0.030 mmol) and subjected to 13^C NMR analysis. Their relative radical-scavenging abilities were directly assessed by comparing the resulting spectrum with those of (-) -epicatechin and (-) -epigallocatechin. The intensities of most of the original carbon signals ascribable to (-) -epigallocatechin decreased. However, the signals of () -epicatechin, not only those of the A-ring but also those of the B-ring, remained. Therefore, this observation suggests that radicals of DPPH are scavenged more rapidly by (-) -epigallocatechin than by (-) -epicatechin. To further clarify the molecular mechanisms underlying the antioxidative and radical-scavenging activities of tea polyphenols, we investigated other model polyphenols with more simple structures. Catechol, (+) -taxifolin, eriodictyol and methyl hydrocaffeate were oxidized to ο-quinone by DPPH. However, quercetin, luteolin, me-thyl caffeate and ethyl protocatechuate were not stabilized as an ο-quinone, even though they possess a catechol structure. Thus, the difference in their antioxidation mechanisms may depend on the presence or absence of the conjugated olefinic or carbonyl double bond. The radical-scavenging ability of ο-dihydroxy phenolic compounds with a conjugated olefinic double bond (e.g. quercetin) was superior to that of compounds without this bond [e.g. (+) -taxifolin], while the ability of ο-dihydroxy phenolic compounds possessing a conjugated carbonyl bond (ethyl protocatechuate) was inferior to that of compounds lacking this bond (catechol). The molecular mechanisms underlying tl1e antioxidative and radical-scavenging activities of vicinal-trihydroxyl phenolic compounds were further investigated. All vicinal-trihydroxyl phenolic compounds (pyrogallol, myri-cetin and ethyl gallate) do not seem to be stabilized as ο-quinone and produce a rather stable radical. Vicinal-trihydroxyl phenolic compounds with a conjugated olefinic double bond (e.g. myricetin) had an inferior scaveng-ing ability as compared with compounds lacking this bond (e.g. pyrogallol) ,but myricetin was a better scaveng-er than compounds with a conjugated carbonyl double bond (e.g. ethyl gallate) . In addition, vicinal-trihydroxyl phenolic compounds (e.g. ethyl gallate) were superior to ο-dihydroxyl phenolic compounds (e.g. quercetin) ,and that the gallate moiety is more important than the ο-dihydroxyl group in the B-ring as a radical-scavenging active site in the (-) -epicatechin gallate structure. The order of radical-scavenging ability suggested by these results is summarized as follows : (-) -Epigallo-catechin, pyrogallol > myricetin > (-) -epicatechin gallate, ethyl gallate > quercetin, luteolin, methyl caffeate > () -epicatechin, (+) -catechin, (+) -taxifolin, eriodictyol, methyl hydrocaffeate, catechol > ethyl protocatechuate.Ascorbic acid is also known to possess potent antioxidative activity. When ascorbic acid was reacted with DPPH, it was oxidized to dehydroascorbic acid by DPPH Ascorbic acid scavenged DPPH radical faster than (+) -catechin. When ascorbic acid was added to the reaction mixture of (+) -catechin and DPPH, the two carbonyl signals ascribable to (+) -catechin in the oxidized form completely disappeared, suggesting that (+) -catechin in the oxidized form after radical-scavenging can be reduced to (+) -catechin by ascorbic acid. Ethyl gallate scavenged DPPH radical faster than (+) -catechin and ethyl gallate reduced the oxidized (+) -catechin. Ascor-bic acid also scavenged DPPH radical more rapidly than ethyl gallate. However, when ascorbic acid was added to the reaction mixture of ethyl gallate and DPPH, the signals of ascorbic acid did not disappear completely and ethyl gallate was not easily regenerated from the oxidized form generated after radical-scavenging. Ascorbic acid also scavenged radicals more rapidly than pyrogallol ; in other words, ascorbic acid possesses more potent antioxidative activity than all of the polyphenols described above. α -Tocopherol is stabilized as α -tocopheroxyl radicals when it is reacted with DPPH. Ascorbic acid scav enged radicals faster than α -tocopherol. However, α -tocopherol was not likely to be regenerated from α -tocopherol radical after radical-scavenging, even if it was present with ascorbic acid. α -Tocopherol scav-enged DPPH radicals faster than (+) -catechin and α -tocopherol reduced the oxidized (+) -catechin. The radical-scavenging ability of α -tocopherol was inferior to ascorbic acid but superior to (+) -catechin (and ethyl protocatechuate) . However, α -tocopherol and other polyphenols (e.g. ethyl gallate) except them had almost identical antioxidative activities. The results of our 13^C NMR study of the mechanisms of antioxidative effects of several antioxidants using DPPH can be summarized as follows. First, the structural changes of antioxidants after scavenging of radicals can be directly observed at the molecular level. This allowed us to determine the active site of each antioxidant in each radical-scavenging reaction. Second, the relative radical-scavenging activity (speed) can be compared be-tween antioxidants.