Flavonoids, which have hydroxyl substitutions in their structure of 2-phenyl-benzopyrone-4-one, are plant-derived components with low toxicity.1,2) Because flavonoids are excellent substrates for sulfatase and glucuronidase, flavonoids administered are predominantly metabolized by sulfo-and glucurono-conjugative routes in intestine and liver cells prior to excretion in vertebrates. [3][4][5] We have recently found that flavonoids have inhibitory effects on the phase II metabolic pathway of acetaminophen in cultured rat hepatocytes.6) This study has expanded our previous findings on the inhibitory effect of flavonoids on N-acetyl-conjugation of 5-aminosalicylic acid (5-ASA), which represents the therapeutically active moiety of the sulfasalazine molecule in the treatment of inflammatory bowel disease. [7][8][9][10] We report in this study for the first time that certain flavonoids also possess potency in inhibiting the formation of N-acetyl-5-ASA (5-AcASA) from 5-ASA, another phase II conjugation reaction, in rat cultured hepatocytes. The potency of such flavonoids in inhibiting the N-acetylation of 5-ASA in the cell-free enzymatic preparation was very similar to that in cultured hepatocytes. Our observations suggest the potential clinical application of flavonoids as the promoting agents in 5-ASA therapy for patients with inflammatory bowel diseases. MATERIALS AND METHODS MaterialsMaterials and chemical reagents were purchased from the following companies: flavonoids from Funakoshi Co. (Tokyo, Japan); 5-ASA, acetyl coenzyme A and other chemicals used for cell culture from Wako Pure Chemical Co. (Osaka, Japan); and the Develosil RPAQUEOUS C-30-UG-3 column (4.6 I.D.ϫ150 mm) from Nomura Chemical Co. (Aichi, Japan). 5-AcASA was synthesized by the reaction of 5-ASA with acetic anhydride under basic conditions, as described by other researchers. [11][12][13][14] N-Acetyl-Conjugation by Cultured Hepatocytes Hepatocytes were isolated by the collagenase perfusion method from male Wistar rats weighing 180-220 g provided with standard rat chow and water ad libitum.15) The isolated cells were diluted to make a concentration of 0.5ϫ10 6 cells/ml, in Williams medium containing dexamethasone (1 mM), insulin (0.1 mM), tri-iodothyronine (1 mg/ml), d-aminolevulinic acid hydrochloride (0.2 mM) and 10% fetal calf serum. The suspended cells were seeded on 35 mm plastic culture dishes at a density of 1ϫ10 6 cells/dish, then placed in an incubator in an atmosphere of 5% CO 2 95% air at 37°C. The monolayers of hepatocytes were cultured for 24 h in Eagle's MEM containing dexamethasone, insulin, tri-iodothyronine, daminolevulinic acid hydrochloride and 10% calf serum. Flavonoids and 5-ASA were dissolved in dimethyl sulfoxide and added to the cultures at definite concentrations, with the final concentration of dimethyl sulfoxide being about 1%. 5-ASA in a stock solution at 50 mM was added to the cultures at a final concentration of 200 mM at 10 min after the addition of flavonoids. After being incubated for 2 h at 37°C, 0.2 ml of the medi...