While flavonoids have been studied extensively for estrogen receptor activity, they have not been well studied for their ability to modify progesterone receptor (PR) and glucocorticoid receptor (GR) signaling. Three flavonoid compounds, tangeretin, wogonin, and baicalein, were selected for testing for PR and GR activity based on their structural similarity to known phytoprogesterone-like compounds. Each compound was docked in the binding pocket of PR and GR. Of these compounds, baicalein was predicted to be most likely to bind to both receptors. A fluorescence polarization competitive binding assay for PR and GR confirmed that baicalein binds to both the PR and GR with IC 50 values of 15.30 μM and 19.26 μM, respectively. In Ishikawa PR-B and T47D cells, baicalein acted as a PR antagonist in a hormone response element (HRE) luciferase (Luc) assay. In OVCAR5 cells, which only express GR, baicalein was a GR agonist via an HRE/Luc assay and induced GR target genes, FKBP5 and GILZ. RU486, a PR and GR antagonist, abrogated baicalein's activity in OVCAR5 cells, confirming baicalein's activity is mediated through the GR. In vivo, baicalein administered intraperitoneally to female mice twice a week for 4 weeks at a dose of 25 mg/kg induced the GR target gene GILZ in the reproductive tract, which was blocked by RU486. In summary, baicalein has PR antagonist and GR agonist activity in vitro and demonstrates GR agonist activity in the uterus in vivo.

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Trifolium pratense L. (red clover) is a popular botanical supplement used for women’s health. Irilone isolated from red clover previously demonstrated progestogenic potentiation activity. In this study, irilone enhanced progesterone signaling was determined to not occur due to post-translational phosphorylation or by reducing progesterone receptor (PR) protein levels but instead increased PR protein levels in T47D breast cancer cells, which could be blocked by estrogen receptor (ER) antagonists, suggesting an ER dependent effect. Further, irilone increased luciferase activity from a hormone responsive element in a cell line that lacked ER and PR but expressed the glucocorticoid receptor (GR). A siRNA knockdown of GR in Ishikawa PR-B endometrial cancer cells reduced irilone’s ability to enhance progesterone signaling. In an ovariectomized CD-1 mouse model, irilone did not induce uterine epithelial cell proliferation. The mechanism of action of irilone gives insight into PR crosstalk with other steroid hormone receptors, which can be important for understanding botanicals that are used for women’s health.