Biospecific isolation and characterization of angiogenesis-promoting ingredients in Buyang Huanwu decoction using affinity chromatography on rat brain microvascular endothelial cells combined with solid-phase extraction, and HPLC-MS/MS

Buyang Huanwu decoction (BHD) was reported to exert angiogenesis-promoting effects, but its active ingredients remain unknown. In this study, we developed a method to screen potential angiogenesis-promoting compounds in BHD, which involved biospecific isolation using live rat brain microvascular endothelial cells (rBMECs) and characterization using solid-phase extraction (SPE) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Six compounds showed binding affinity to rBMECs and were further identified as 6-hydroxykaempferol-di-O-glucoside, paeoniflorin, calycosin-7-O-β-D-glucoside, galloylpaeoniflorin, formononetin-7-O-β-D-glucoside, and (3R)-7,2′-hydroxy-3′,4′-dimethoxy-isoflavan. The results indicated that five of them except 6-hydroxykaempferol-di-O-glucoside showed a protective effect against oxygen glucose deprivation/reperfusion injury in rBMECs and upregulated the secretion of vascular endothelial growth factor and basic fibroblast growth factor, suggesting a mechanism underlying their angiogenic activity. Our findings suggest that biospecific live cell-based isolation combined with SPE and HPLC-MS/MS is an effective method for screening potential bioactive components in traditional Chinese medicines. [Display omitted] •We developed a method potential promoting angiogenesis compound screening in BHD.•Strata-X SPE column eluted by an optimized set of solvents was used.•Six compounds showed rBMECs binding activity and were identified using HPLC/MS.•Five of binding components were confirmed to have pharmacological activity.•Upregulation of VEGF and bFGF levels is one of the mechanisms to promote angiogenesis.