UHPLC-Q/TOF-MS Characterization of Danshen-Chuanxiong and Mechanistic Insights Into Its Protective Effects Against...

Danshen-Chuanxiong is a traditional herb pair used for cardiovascular and cerebrovascular disorders, but its chemical basis and potential effects in Alzheimer's disease (AD)-related endothelial injury remain incompletely defined. Here, an integrated analytical workflow combining reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) with quadrupole time-of-flight mass spectrometry (Q-TOF-MS), cellular assays, metabolomics, and network pharmacology was applied in an amyloid-β_1-42 (Aβ_1-42) oligomer-induced human brain microvascular endothelial cell (hBMEC) injury model. The complementary RPLC/HILIC and positive/negative electrospray-ionization strategy expanded coverage across a broad polarity range. UHPLC-Q/TOF-MS detected 90 compounds, mainly phenolic acids, phthalides, tanshinones, diterpenoids, and other aromatic or terpenoid constituents. The reductions in cell viability and zonula occludens-1 expression following Aβ_1-42 exposure confirmed the successful establishment of the cellular injury model, whereas pretreatment with Danshen, Chuanxiong, or their combination increased cell viability and alleviated Aβ_1-42-induced cellular injury. Untargeted metabolomics annotated 38 differential metabolites, of which 30 showed a recovery-associated shift toward the control state after Danshen-Chuanxiong treatment; the associated pathways included amino-acid, purine, glutathione, and glycerophospholipid metabolism. Integrative analysis prioritized seven candidate targets of Danshen-Chuanxiong, namely ACHE, VEGFA, NOS2, NOS3, AKR1B1, MDM2, and XDH. These findings provide a separation-MS-based chemical characterization and mechanistic framework for further validation of Danshen-Chuanxiong in AD-related endothelial injury models.