Discovering VEGFR-targeting constituents from Chloranthus holostegius guided by affinity screening

Natural products remain an indispensable resource for pharmaceutical exploration. This investigation focused on discovering bioactive compounds that target vascular endothelial growth factor receptor 2 (VEGFR-2) from Chloranthus holostegius. Our phytochemical analysis led to the isolation and structural elucidation of six previously undescribed sesquiterpene derivatives (1‒6) and seven known analogs (7‒13). Comprehensive structural characterization was achieved through integrated high-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopic analysis, with the absolute configurations of the new compounds established via electronic circular dichroism calculations. Among the six undescribed compounds, 1 and 2 were identified as a rare lindenane-monoterpene heterodimer fused by a 1,2-dioxane moiety and an unprecedented lindenane sesquiterpenoid dimer featuring an oxaspiro[4.5]decane unit, respectively. Affinity ultrafiltration combined with ultra-performance liquid chromatography-mass spectrometry identified seven potential ligands for VEGFR-2 (2, 3, 7‒10, and 12). Molecular docking studies elucidated the interactions between these compounds and VEGFR-2. Subsequent in vivo evaluations in transgenic zebrafish models confirmed potent anti-angiogenic effects for multiple candidates, with compound 2 demonstrating the strongest anti-angiogenic effects in this study. This study validates C. holostegius as a promising source for anticancer drug development while identifying structurally novel VEGFR-2 inhibitors with demonstrated biological efficacy. The findings provide both a chemical foundation and pharmacological rationale for advancing natural product-based anti-angiogenic therapeutics.