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 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.
Eguchi
Ai
A
Division of Food Science and Biotechnology, Kyoto University, Graduate School of Agriculture, Kyoto, 606-8502, Japan.
Murakami
Akira
A
Ohigashi
Hajime
H
D000818:Animals; D000975:Antioxidants / Q000032:analysis*; D001681:Biological Assay / Q000379:methods*; D018938:Caco-2 Cells; D002454:Cell Differentiation; D002470:Cell Survival / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004553:Electric Conductivity; D005502:Food; D005504:Food Analysis; D018922:HL-60 Cells; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D013481:Superoxides / Q000378:metabolism; D019108:Tight Junctions
D000975:Antioxidants; D017077:Culture Media, Conditioned; D013481:Superoxides; D009569:Nitric Oxide