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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.

We systematically evaluated the antioxidant activity of ethyl vanillin, a vanillin analog, as compared with the activities of vanillin and other vanillin analogs using multiple assay systems. Ethyl vanillin and vanillin exerted stronger antioxidant effects than did vanillyl alcohol or vanillic acid in the oxygen radical absorbance capacity (ORAC) assay, although the antioxidant activities of vanillyl alcohol and vanillic acid were clearly superior to those of ethyl vanillin and vanillin in the three model radical assays. The antioxidant activity of ethyl vanillin was much stronger than that of vanillin in the oxidative hemolysis inhibition assay, but was the same as that of vanillin in the ORAC assay. Oral administration of ethyl vanillin to mice increased the concentration of ethyl vanillic acid, and effectively raised antioxidant activity in the plasma as compared to the effect of vanillin. These data suggest that the antioxidant activity of ethyl vanillin might be more beneficial than has been thought in daily health practice.Key words: ethyl vanillin; ABTS radical cation; oxygen radical absorbance capacity (ORAC); oxidative hemolysis inhibition assay; plasma antioxidant activityThe association of reactive oxygen species and free radicals with many disease states is now well recognized, and antioxidants have attracted considerable attention.1,2) It has been reported that there are two types of antioxidants that scavenge radicals quickly and quench many radicals, and it has been proposed that reactivity should be assessed on the basis of both reaction rate and stoichiometry, 3) and that multiple methods should be used, since the activities of some antioxidants vary depending on the assay method.3-6) In addition, comparative studies using common antioxidants appear to be essential to clarify the biological significance of the activities of samples. We have assessed antioxidant activities with attention to the above-mentioned proposals and points of view. We found that 2-O--D-glucopyranosyl-L-ascorbic acid (AA-2G), a stable ascorbic acid derivative, exerted radical-scavenging activity toward unnatural model radicals, including DPPH radical [7][8][9][10] and ABTS radical cation (ABTS þ ). 9,11) The chemical properties of AA-2G as a radical scavenger were widely different from those of ascorbic acid, in that the reaction rate with these model radicals of AA-2G was much slower, but the long-term radical scavenging ability per molecule of AA-2G was superior to that of ascorbic acid. Recently, we reassessed the antioxidant activity of arbutin using five in vitro assay systems, though arbutin has been reported to possess weak antioxidant activity as compared to its precursor, hydroquinone.12) We found that arbutin exerted strong antioxidant activity, comparable or even superior to that of hydroquinone in the ABTS þ -scavenging assay, the ORAC assay, and two cell-based antioxidant assays.Vanillin is widely used in foods, beverages, cosmetics, and drugs. It has been reported to have multifunctional effects, including an...