Effects of Ruscus aculeatus Rhizome Extract on Human Cerebral Microvascular Endothelial Cells Under Normoxic and...

Ruscus aculeatus L. (butcher's broom) is traditionally used in the management of chronic venous disorders; however, the molecular mechanisms underlying its vascular activity remain insufficiently characterized. This study investigated the effects of an 80% ethanolic rhizome extract on human cerebral microvascular endothelial hCMEC/D3 cells under normoxic and hypoxic conditions. The extract was phytochemically characterized using ultra-high-performance liquid chromatography-diode array detection-high-resolution mass spectrometry (UHPLC-DAD-HRMS), while its biological effects were evaluated by assessing cell viability and the secretion of selected cytokines, including transforming growth factor beta (TGF-β) isoforms, using Bio-Plex multiplex assays. Phytochemical analysis confirmed steroidal saponins as the predominant constituents and also revealed minor phenolic compounds, including hydroxycinnamic acid amides. The extract (10 and 50 μg/mL) was well tolerated by hCMEC/D3 cells and did not significantly reduce cell viability under either normoxic or hypoxic conditions. Its effects on cytokine secretion depended on oxygen availability and the mediator examined. Under hypoxia, the extract partially shifted interleukin-6 (IL-6) and interleukin-8 (IL-8) secretion toward normoxic levels and attenuated interferon alpha (IFN-α)-induced tumor necrosis factor alpha (TNF-α) production, whereas a significant increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) relative to the untreated control was observed only with the combination of 50 μg/mL extract and IFN-α. Hypoxia also differentially affected TGF-β isoforms, increasing TGF-β1 and decreasing TGF-β2, while treatment with the extract shifted both responses toward the profile observed under normoxia; TGF-β3 remained largely unchanged. Overall, these findings indicate that R. aculeatus rhizome extract does not exert a uniform anti-inflammatory effect but rather modulates endothelial secretory responses in an oxygen- and mediator-dependent manner. These observations provide new insights into the endothelial effects of R. aculeatus that may contribute to its vascular activity.