Determination of rat urinary metabolites of icariin in vivo and estrogenic activities of its metabolites on MCF-7 cells.

To confirm that the estrogenic activity of icariin is based on the close relationship between the structures of its metabolites and the effects of their binding to target hormone receptors, the metabolism of icariin in rat urine was analyzed in vivo, and the estrogenic activity of its metabolites was measured in cultured MCF-7 human breast cancer cells, respectively. By CZE analysis, peaks corresponding to the relative positions of desmethylicaritin and icaritin were observed in the urine sample. Structural analysis following LC-ESI-MS revealed molecular ions [M-H]- of 512.8, 353.3, and 367.0 for metabolites consistent with those of icariside II, desmethylicaritin, and icaritin. Icariin, icaritin, and desmethylicaritin were analyzed for their estrogenicity using MCF7-cell proliferation (E-screen test). MCF-7 cells were cultured in an estradiol free medium and then exposed to 10(-8) to 10(-5) mol/L icariin and its metabolites, icaritin and desmethylicaritin, for 6 days. Icaritin and desmethylicaritin significantly increased cell proliferation, and the cell number increased from 1.61 to 4.14 fold compared with the untreated control, but the parent compound icariin failed to exhibit this effect. These results indicate that icariin is converted to icariside II, desmethylicaritin, and icaritin in vivo, and that the latter two act as a weak xenoestrogen on MCF-7 cells.