Identification of novel bioactive metabolites of 5-demethylnobiletin in mice

SCOPE: Biotransformation of dietary components is important for their in vivo biological activities after oral ingestion. Herein, we investigated biotransformation of 5-demethylnobiletin (a polymethoxyflavone found in citrus fruits) in mice, and its implication in the inhibition of human colon cancer cells. METHODS AND RESULTS: Urine samples were collected from mice fed with 5-demethylnobiletin (1) and analyzed by LC-ESI-MS and HPLC coupled with an electrochemical detector. Three major metabolites were identified as 5,3'-didemethylnobiletin (2), 5,4'-didemethylnobiletin (3), and 5,3',4'-tridemethylnobiletin (4) by comparing their ESI-MS and HPLC profiles with those of authentic standards synthesized by a multistep route. Cell viability assay in human colon cancer cells demonstrated that all three metabolites of 5-demethylnobiletin, especially 5,3'-didemethylnobiletin (2), showed much stronger inhibitory effects on cancer cell growth than 5-demethylnobiletin. For example, metabolites (2-4) showed IC50 of 0.12, 5.5, and 4.2 μM in SW620 cells, respectively, while 5-demethylnobiletin at 10 μM only caused 37% inhibition after 72 h of treatment. CONCLUSION: Three novel metabolites were identified in mice after oral administration of 5-demethylniobiletin. These metabolites exhibited strong inhibitory effects against human colon cancer cells. Our results provide a first report on these bioactive metabolites and warrant further investigation on their molecular mechanism of actions.