Akt activation by Evodiae Fructus extract protects ovary against 4-vinylcyclohexene diepoxide-induced ovotoxicity

Evodiae Fructus (EF) is the dried, unripe fruit of Evodia rutaecarpa Benth., and one of the main components of traditional herbal prescriptions issued for the treatment of sterility caused by irregular menstruation in Korea. However, scientific evidence regarding the efficacy and action mechanism of EF is lacking.In this study, the authors established an in vitro screening tool to identify promising new drug candidates in herbal medicines for the prevention and treatment of premature ovarian failure. The protective effects of EF extracts against 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity were investigated and the molecular mechanism responsible was sought.EF extract was prepared by boiling EF in water and its quality was confirmed by high performance liquid chromatography. CHO-K1 (Chinese hamster ovary cells) and COV434 (human ovarian granulosa cells) cells were plated, pretreated with EF extract for 2h and then treated with 1.5mM or 0.5mM VCD for 24h, respectively. Cell viabilities were measured using an MTT assay, and protein levels were determined by western blotting.VCD significantly suppressed the viability of both CHO-K1 and COV434 cells in a dose-dependent manner and induced the apoptosis of CHO-K1 cells at 1.5mM. EF extract dose-dependently blocked the ovotoxicity induced by treatment with VCD. Furthermore, EF extract significantly activated Akt and downstream effectors such as mTOR and GSK-3β in CHO-K1 cells. The ability of EF extract to prevent cytotoxicity by VCD was antagonized by pretreatment of LY294002, a PI3K/Akt inhibitor.EF has the ability to protect ovary cells against VCD-induced ovotoxicity, probably via Akt activation. These results suggest that the beneficial effects of EF might be useful for preventing premature ovarian failure or unexplained infertility caused by environmental factors.