Evaluation of agonistic and antagonistic effects of unprenylated and prenylated flavonoids on estrogen receptor–α

Prenylation, which involves the addition of hydrophobic molecules, is considered to enhance the bioavailability and biological activity of flavonoids. However, the effect of prenylation on the estrogenic activity of flavonoids with different structures remains unclear. This study evaluated the estrogen receptor-α (ER-α) agonistic and antagonistic activities of estrogenic flavonoids in both unprenylated and prenylated forms using OECD standardized in vitro ER-α transactivation assay and in vivo uterine hypertrophy assay. A luciferase reporter assay using ER-α-HeLa-9903 cells revealed that twelve flavonoid compounds exhibited ER-α agonistic activity, and among them, only 6-prenylnaringenin (6-PN) exhibited ER-α antagonistic activity. Interestingly, except for 6-PN, prenylated flavonoids showed reduced or similar ER-α agonistic activity compared to their parent compounds. 6-PN, but not 8-prenylnaringenin, demonstrated both enhanced ER-α agonistic and antagonistic activity compared to its parent compound, naringenin. Among the tested compounds, coumestrol exhibited the most potent ER-α agonistic activity in both transactivation and uterotrophic assays. The uterotrophic effect of coumestrol at a dose of 25 mg/kg was stronger than that of 17β-estradiol at 200 μg/kg, as evidenced by changes in uterine weight and estrogen-responsive protein expression. These findings provide important insights into the relative estrogenic potency of flavonoids and the impact of prenylation on their activity.