Exploring the effective substances and mechanisms of action of ChaiHu ShuGan San in treating perimenopausal syndrome based on in vivo exposure profiles and steroid hormone metabolic networks.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Perimenopause is a transitional period in women marked by hormonal fluctuations, often resulting in symptoms such as hot flashes and mood disturbances. According to traditional Chinese medicine, these symptoms are linked to liver stagnation and kidney deficiency, and ChaiHu ShuGan San (CSS), a classical multi-herbal formulation, has been traditionally used to alleviate mood-related symptoms and modulate endocrine function in women during the perimenopausal transition, but the underlying mechanisms and active compounds remain unclear. AIM OF THE STUDY: This study aims to explore the active substances and potential mechanisms of CSS in treating PMS through steroid hormone metabolic pathways and in vivo exposure profiles. MATERIALS AND METHODS: A perimenopausal rat model was induced via ovariectomy. LC-MS/MS was employed to establish CSS metabolites profile and the TMAO (trimethylamine-N-oxide) level in rat plasma. High-exposure compounds underwent molecular docking and network pharmacology to explore interactions with steroid metabolism pathways. Biochemical markers HPA axis hormones (in plasma), IL-10 (in plasma) and FMO3 (flavin-containing monooxygenase 3, in rat liver and serum) levels were measured. The Cellular Thermal Shift Assay (CETSA) was used to provide in vitro validation of compounds binding to FMO3. RESULTS: CSS treatment was shown to regulate abnormal hormone levels, reduce triglycerides and cholesterol, and increase IL-10 levels, alleviating PMS symptoms in rats. LC-MS/MS identified liquiritigenin, isosakuranetin, and hesperetin as high-exposure components. Network pharmacology and molecular docking suggest that these three flavonoids interact with the FMO3 enzyme. CETSA results showed that these flavonoids directly bind to the FMO3 protein. CSS decreased the FMO3 level in rat liver and serum. CONCLUSIONS: CSS alleviates PMS symptoms in rats and ameliorates the abnormal changes in disease-related biochemical markers. Inhibition of FMO3 pathways is involved in the effects of CSS. This study provides further pharmacological and chemical justifications for the use of CSS in PMS management.