Study On Estrogenic Activities Of Icariin And Its Possible Metabolites In Vitro

Epimedii Herba is an important traditional Chinese herbal medicine widely used as a tonic and aphrodisiac in China, Japan and Korea, and has been proven to possess efficacy on sexual function disorder, menopause syndrome, osteoporosis and cardiovascular diseases et al. Recent studies have indicated that the extracts or drug preparations of Epimedii Herba possess classic phytoestrogen-like activities, therefore, we speculate that the in vivo pharmacological activity of Epimedii Herba is related to the estrogen-like activities of its component(s). Icariin (ICA) is considered to be the major active component of Epimedii Herba, therefore, the content of ICA is employed as the standard for quality control in Epimedii herba and its drug preparations. In vivo metabolism studies have shown that the estrogenic activities of metabolite usually markedly increase, such as biochanin A metabolized to genistein, formononetin to daidzein. It is most likely that the metabolism process of ICA in vivo is similar to that described above. To determine the material foundation of pharmacological efficacy in Epimedii herba and its drug preparations, and to provide scientific data for traditional Chinese herbal medicine entering international market, the present paper studied the estrogenic activity and its structure-relationship of ICA and its possible metabolites icaritin (ICT) and desmethylicaritin (DICT) through cell biology and molecular biology techniques.1. Synthesis and identification by MS of ICT and DICTICT was derived from ICA by hydrolysis using a removing-glycoside specific cellulase, the target product was extracted with ethyl acetate. El mass spectrum displayed a molecular ion peak at m/z 368 corresponding to that of icaritin. The lost atomic mass units are exactly equal to that of total molecular weight of a rhamnosyl and a glucosyl. DICT was derived from ICT demethylation by boron tribromide, the target product was extracted and dried as above described. El mass spectrum displayed a molecular ion peak at m/z 354 corresponding to that of DICT. The lost atomic mass units are exactly equal to that of total molecular weight of a methyl.2. The effects on cell proliferation of ICA ICT and DICTThe estrogenic activities were assessed by MCF-7 cells and T47D cells proliferation assay in vitro (E-SCREEN). MCF-7 cells and T47D cells were seeded into 24-well plate at appropriate density, and after 1 day the medium was changed to experimental medium (endogenous estrogen-free), cultured for 1 or 2 day(s) for MCF-7 cells and T47D cells, respectively, The medium was changed to fresh estrogen-free medium and the cells were exposed to test chemicals and positive control estradiol (?), and cultured for another 6 or 9 days for MCF-7 and T47D cells, respectively. Cell proliferation was assessed by MTT.ICT and DICT significantly enhanced the proliferation of MCF-7 cells in a concentration-dependent manner from 10to 10mol/L, and the maximal stimulation was observed at 10mol/L. Compared with control, relative preliferation (RP) for ICT and DICT were calculated as 4.15, 4.34 fold respectively (p<0.01, E2as 4.61 fold). Whereas, ICA did not present stimulation of cell growth. Also, ICT and DICT significantly enhanced the proliferation of T47D cells, and the maximal stimulation was observed at 10mol/L. Compared with control, RP for ICT and DICT were calculated asl.78, 1.36 fold respectively (p<0.01, E2 as 2.65 fold), and there were significantly different between ICT and DICT (p<0.01). These results that ICT and DICT possess estrogen-like activities, however, icariin appears to have no estrogenicity in vitro; the significantly different of stimulation on T47D cells betweenICT and DICT is probably relevant to the selectivity to ERp of DICT better than ICT.3. Inhibition of ICI 182, 780 on cell proliferationIn order to ensure that the cell proliferation of MCF-7 cells and T47D cells induced by test compounds were mediated by estrogen receptors, cells were simultaneously exposed to 10~7mol/L ICI in the presence of test chemicals, a...