LC-HRMS/MS-Guided Profiling and Biological Evaluation of Stachys duriaei Extracts: Anticancer and Vasorelaxant Mechanisms via Apoptosis and Endothelium-Dependent Pathways

<i>Stachys duriaei</i> (Lamiaceae) remains unexplored despite its pharmacological potential. In this study, for the first time, the antiproliferative, pro-apoptotic, cell cycle arrest, and vasorelaxant effects of the <i>n</i>-butanolic extract (BESD) and a VLC fraction (BF1SD) of <i>Stachys duriaei</i> were investigated. Antiproliferative activity was evaluated on PC3 and MDA-MB-231 cell lines via MTT assay (72 h). Apoptosis (Annexin V-FITC/PI) and cell cycle arrest (PI/RNase) were assessed by flow cytometry (24 h, 250-1000 µg/mL). Vasorelaxant effects were studied ex vivo on rat aortic rings. LC-HRMS/MS was used for phytochemical analysis. BESD showed higher antiproliferative activity (IC₅₀: 196 ± 6 µg/mL for PC3, 182 ± 8 µg/mL for MDA-MB-231) than BF1SD (IC₅₀: 281 ± 6 µg/mL and 273 ± 3 µg/mL, respectively). Apoptosis was dose-dependent, with BF1SD displaying a stronger effect at 1000 µg/mL (67.3 ± 0.5% vs. 49.9 ± 0.7% for BESD). BESD induced G2/M arrest, while BF1SD caused G0/G1 and G2/M arrest. Vasorelaxation was endothelium-dependent, likely mediated by NO. Identified compounds (hyperoside, luteolin-7-glucoside, and rutin) may contribute to these effects. BESD and BF1SD exhibit anticancer and vasorelaxant properties, indicating potential therapeutic use against cancer and cardiovascular diseases. Further studies are needed to isolate active compounds and confirm their effects in vivo.