New C₂₁-steroidal glycosides with cytotoxic activities from Cynanchum otophyllum roots

Fourteen C₂₁-steroidal glycosides were isolated from the roots of Cynanchum otophyllum, including five previously undescribed compounds, namely cynotosides A-E (1-5). Their structures were determined by extensive NMR spectroscopic data, HR-ESI-MS and ECD calculation. All the compounds were tested for cytotoxic activities against four human cancer cell lines (MCF-7, HCT-116, HeLa, and HepG2). Among them, compounds 2, 4, 5, 7-9, 12, and 14 showed moderate cytotoxic activity with IC₅₀ values ranging from 11.19 to 38.83 μM. Compound 7 could induce both cell cycle arrest in the G0/G1 phase and apoptosis against HepG2 cells in a dose-dependent manner. Furthermore, a molecular docking analysis was performed to gain insights into the binding mode of compound 7 with cancer-related signal pathways.