Research On Estrogenic Effect Of Flavonoids And Its Effect-structure Relationship In Vitro

Post-menopause Osteoporosis is induced by the sharp falling of estrogen level after menopause in women, and the consequent calcium lose. Estrogen Replacement Therapy (ERT) can release the menopausal symptoms, and prevent osteoporosis. Although the side effects of ERT of inducing endometrial cancer, breast cancer, colporrhagia etc. are inevitable. Phytoestrogen is an effective way to treat osteoporosis, it can both effect as estrogen and avoid the side effects of using E2, the classic ones of which are Daidzein and Genistein that belongs to isoflavoniods and are widely recognized as bearing the estrogenic like effects. In order to design better effective compounds to meet the needs of treating Post-menopause Osteoporosis and some other estrogen deficiency deseases. This paper employed E-SCREEN methods to evaluate the estrogenic like effects and the structure-effective relationship on some isoflavonoids. Thus, we deployed series of assays including:The establishment of E-SCREEN methods; Study on Estrogenic Activities of Genistein and Daidzein in vitro; Study on Estrogenic Activities of Icariin and Its Possible Metabolites in vitro; Study on Estrogenic Activities of Luteolin and Apigenin in vitro; Study on Estrogenic Activities of Naringenin and Apigenin in vitro.After exposing cells under each isoflavonoid with10-7,10-8,10-9,10-10mol·L-1concentration of each compound,evaluate the proliferation of MCF-7cells by MTT method, assess the expression by immunohistochemistry to detect the influence on cell cycle and proliferation; then assess the expression of ERa,ERβ,PR,PS2mRNA by Real-time RT-PCR. Protein was detected by Western-blot.Results of estrogenic effect assays:Genistein has a stronger activity than Apigenin; Genistein has stronger estrogen-like activities than Daidzein; Both Luteolin and Apigenin have a strong estrogen-like effect; Apigenin has stronger estrogen-like activities than Luteolin; icaritin is the strongest isoflavonoid among the four. Which is to say:①the location of ring B is of the essence, and it is stronger when located at position3;②the hydroxyl at the position5of the AC double ring contributes to the high estrogenic effect;③the hydroxyl at ring B position5'influence the estrogenic effect;④C-C enhances the effect at AC double ring between position2and3;⑤glycosyl affect the binding with ER, thus makes the estrogenic effect weak;⑥prenyl inhibites the estrogenic effect.This assay screened out several strong activity isoflavonoids:icaritin, genistein and so on. It basicly explained how the isoflavonoids works as estrogenic substance, and the relationship between structure and estrogenic activity. Besides, with some probe into the signaling of ERβ's special effect on adjusting, provide the basic researchment for further clinic research and research in vivio. As well, this paper provided the phytoestrogen with new evidence, revealed the possibility and beautiful future to substitute the ERT with phytoestrogen.