Enhanced Estrogenic Activity in Water-Soluble Silybum marianum Complex via Microwave-Assisted Steviol Glycoside-Based Formulation

Background: Phytoestrogens are promising alternatives to menopausal hormone therapy, but their clinical application is limited by poor solubility and bioavailability. Silybum marianum (milk thistle) contains flavonolignans with reported estrogenic potential. However, its extract shows only modest estrogenic activity, and approaches to enhance its efficacy by improving solubility remain insufficiently explored. Methods: A water-soluble Silybum marianum complex (WS) was prepared using microwave-assisted processing with steviol glycoside (SG), and its phytochemical integrity was assessed by UPLC-QTOF-MS. Estrogenic activity was evaluated using cell-free ERα/ERβ competitive binding assays and, in MCF-7 cells, by assessing proliferation and ERα/ERK/AKT phosphorylation. In vivo efficacy was evaluated in a VCD-induced menopause model in female C57BL/6 mice by measuring plasma estradiol levels, uterine weight, and vaginal epithelial thickness. Results: WS retained major flavonolignans and incorporated SG-derived metabolites. WS bound both ERα and ERβ in cell-free assays. In MCF-7 cells, WS induced ERα, ERK, and AKT phosphorylation and cell proliferation; the proliferative response and ERK/AKT phosphorylation were abolished by the ER antagonist fulvestrant, and proliferation was comparable to that of genistein and daidzein. Neither Silybum marianum extract (SME) alone nor its simple physical combinations with SG reproduced the activity of WS, indicating that enhanced estrogenic activity was observed following microwave-assisted processing. In vivo, WS dose-dependently increased measured estradiol levels, increased uterine weight, and reversed vaginal atrophy. Conclusions: Microwave-assisted processing with SG significantly enhances the estrogenic activity of Silybum marianum. Our findings suggest that WS may serve as a promising phytoestrogenic candidate for further evaluation in the context of estrogen deficiency.