A comparative study of the antioxidant/prooxidant activities of eugenol and isoeugenol with various concentrations and oxidation conditions

Eugenol (compound 1 in Fig. 1, 4-allyl-2-methyoxyphenol) and isoeugenol (compound 2 in Fig. 1, 4-propenyl-2-methoxyphenol), both used as a flavor agent in cosmetic and food products, have both prooxidant and antioxidant activities. Their adverse effects such as allergic and inflammatory reaction may be due to their prooxidant activity. To clarify the mechanisms of their cytotoxicity and the factors affecting their antioxidant/prooxidant activities, we investigated the cytotoxicity, ROS production, and cellular glutathione (GSH) levels induced by eugenol and isoeugenol in a human submandibular cell line. The cytotoxicity (MTT method) of eugenol was 1 order of magnitude lower than that of isoeugenol (CC50: eugenol, 0.395 mM; isoeugenol, 0.0523 mM); and ROS production (CDF staining) was induced significantly by isoeugenol, but not by eugenol. Under treatment with H2O2 (100 μM) plus horseradish peroxidase (1 μg/ml) for 30 min or with visible light irradiation for 5 min, eugenol caused biphasic ROS production characterized by enhanced at lower eugenol concentrations (5-10 μM) and decreased at higher concentrations (500 μM). In contrast, isoeugenol enhanced ROS production over a wide range of concentrations (5-500 μM). Isoeugenol at 1000 μM significantly reduced GSH levels compared with eugenol at the same concentration. The high cytotoxicity of isoeugenol may be attributed to its induction of high ROS production and low GSH levels, possibly as a result of benzyl radical formation. In contrast, the cytotoxicity of eugenol is likely to be mediated by ROS-independent mechanisms, possibly involving phenoxyl radicals and/or eugenol quinone methide.