Novel bioassay system for evaluating anti-oxidative activities of food items: Use of basolateral media from differentiated Caco-2 cells

Reactive oxygen and nitrogen species, including superoxide (O-2(-)) and nitric oxide (NO), are known to be mediators of oxidative stress and play pivotal roles in the onset of numerous life style-related diseases. While a number of studies have shown that naturally occurring anti-oxidants may be applicable for prevention and therapy for those diseases, most in vitro anti-oxidation tests reported have not provided significant insight into the absorption efficiency or metabolism of dietary anti-oxidants in the gastrointestinal tract. In the present study, we established a novel assay system by focusing on the bioconversion of food constituents using differentiated Caco-2 cells as a model of human intestinal epithelial cells. Various fresh food preparations [ginger, garlic, shimeji ( Hypsizigus marmoreus ), onion, carrot] were added to the apical side of differentiated Caco-2 monolayers. After incubation, the medium was recovered and tested for its inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced generation in differentiated HL-60 cells, and on combined lipopolysaccharide (LPS)- and interferon (IFN)-gamma -induced NO generation in RAW 264.7 macrophages. The garlic preparation (25% v/v) basolateral medium abolished generation without any cytotoxicity toward HL-60 cells, though it was cytotoxic to Caco-2 cells. In the NO generation tests, all of the food preparations showed notable inhibitory activity, while the garlic preparation (5% v/v) basolateral medium inhibited NO generation with substantial cytotoxicity toward RAW 264.7 cells. Interestingly, the carrot preparation (1% v/v) basolateral medium inhibited NO generation in both a concentration- and time-dependent manner without any cytotoxicity toward RAW 264.7 or Caco-2 cells, and its activities were higher than those of the carrot preparation alone (1% v/v). Our results indicate that the present assay system is appropriate and reliable for determination of the anti-oxidative efficacy of dietary phytochemicals in vivo .