Tertiary Alcohol-Containing Polycyclic Polyprenylated Acylphloroglucinols from Hypericum scabrum Inhibit the Proliferation of Oral Squamous Cell Carcinoma CAL-27 cells
Background: Polycyclic polyprenylated acylphloroglucinols (PPAPs) represent a structurally diverse class of natural products with reported biological activities, yet their effects on oral squamous cell carcinoma (OSCC) remain insufficiently characterized. In this study, 14 tertiary alcohol-containing PPAPs, including 7 previously undescribed compounds, were isolated from Hypericum scabrum Linn and structurally elucidated by spectroscopic methods. Their antiproliferative activities were evaluated in CAL-27 human OSCC cells.Method: PPAPs were isolated and purified by means of column chromatography combined with semi-preparative techniques, then chemical structures were characterized using nuclear magnetic resonance (NMR) spectroscopy. The half-maximal inhibitory concentration (IC50) values of these phloroglucinol compounds against CAL-27 cells were determined via the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Furthermore, the in vitro mechanism underlying the action of these compounds on CAL-27 cells was elucidated through an integrated approach involving RNA sequencing (RNA-Seq), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis.Results: Fourteen tertiary alcohol-containing PPAPs were isolated and structurally characterized, including seven new analogs (1-7). Among these, compound 13 was found to be the potential cytotoxic active compound against CAL-27 during in vitro tests, with IC50 of 8.96 uM. Cell viability assays demonstrated that compound 13 exerted significant growth inhibitory effects in a dose-dependent manner. Flow cytometric analysis revealed cell cycle arrest at the G2/M phase and a marked increase in apoptotic cell population following treatment at the IC50 concentration. Further investigation showed that intracellular reactive oxygen species (ROS) levels were significantly elevated in treated cells, and co-treatment with the antioxidant cysteine partially attenuated ROS accumulation. These findings indicate that different cell death mechanisms may contribute to the cytotoxic effects of compound 13.Conclusion: These findings demonstrate that compound 13 suppresses proliferation and induces apoptosis in CAL-27 cells, with ROS accumulation potentially contributing to its cytotoxic effects.