[Establishment of an ovarian function-improving quality evaluation method of Liuwei Dihuang Wan based on...

To establish a bioassay method based on the determination of aromatase activity in vitro for evaluating the quality of Liuwei Dihuang Wan(LWDHW) in improving the ovarian function. An animal experiment was conducted to verify the protective effect of LWDHW on the ovaries in the mouse model of premature ovarian failure(POF) and its impact on aromatase activity. An aromatase activity assay, calibrated by the conversion rate of testosterone in vitro, was employed to investigate the effects of substrate(testosterone) concentration, aromatase concentration, cofactor(NADPH) concentration, and reaction time on the reaction system. The established in vitro reaction system was used to determine the effects of LWDHW from different commercially available manufacturers on aromatase activity, and the pharmacodynamic results were validated through cell experiments. The results showed that LWDHW significantly ameliorated the sex hormone imbalance, markedly alleviated ovarian pathological damage, and enhanced aromatase activity in the ovarian tissue of the mouse model of POF. The optimal conditions for the in vitro aromatase activity assay were established as follows: testosterone 1 mmol·L~(-1), aromatase 1 μg·mL~(-1), NADPH 1 mmol·L~(-1), and reaction time of 20 min. When the concentration of LWDHW was 5 mg·mL~(-1), the reaction system was stable. The promotion rates of aromatase activity varied among LWDHW samples from different manufacturers, and the results were consistent with the trend observed in the cell experiments of pharmacodynamics. Regarding the efficacy in improving the ovarian function, the in vitro aromatase activity assay established in this study is suitable for evaluating the role of LWDHW in regulating estrogen-related functions. The development of this bioassay method not only contributes to improving the quality control system for LWDHW but also provides a new approach for bioactivity assessment of TCM compound prescriptions.