Antioxidant Properties of 5,7-Dihydroxycoumarin Derivatives in in vitro Cell-free and Cell-containing Systems

Background: Coumarin is a well-known plant-derived natural product, which is extensively used as a biologically active compound. Coumarins that possess hydroxyl groups in their benzenoid ring were shown to have potent antioxidant and radical-scavenging properties in various experimental models. Purposes: During our search for new types of coumarin derivatives possessing antioxidant activity, we investigated the synthesis of 5,7-dihydroxycoumarin derivatives and attempted to find potential therapeutic candidates to treat oxidative stress. Methods: A series of different cell-free and cell-containing tests was conducted. In cell-free systems, these compounds were tested by means of three electron spin resonance assays [a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, a xanthine/xanthine oxidase system, and a hydrogen peroxide/sodium hydroxide/dimethyl sulfoxide]. In cell-containing systems, baicalein-induced hydroxyl radical formation by B16F10 cells was used to evaluate the antioxidant properties of the coumarins. Finally, we measured the protective capacity of these coumarins against oxidative damage caused by baicalein-induced cytotoxicity in B16F10 cells. Results: All of the tested compounds were potent DPPH and superoxide radical scavengers in the DPPH and xanthine/xanthine oxidase systems, respectively. In the hydrogen peroxide/sodium hydroxide/dimethyl sulfoxide system, Compound 4 was the best radical scavenger among the 11 compounds tested. Compounds 6 and 8 were also potent hydroxyl radical scavengers but increased the formation of methyl radicals. We further evaluated the effects of the test compounds on living cells, and only Compounds 1, 3, 5, and 6 inhibited baicalein-induced hydroxyl radical formation in B16F10 cells. However, none of them could reduce baicalein-induced cytotoxicity in B16F10 cells. Conclusions: The different methods used to assess the antioxidant activities of the tested compounds can give varying results depending on the specific free radical and solvent system being used. Certain 5,7-dihydroxy-coumarins possess high antioxidant activity in vitro. Baicalein-induced cytotoxicity in B16F10 cells did not seem to be completely related to ROS generation.