Exploring the Cardioprotective Spectrum-Effect Relationship of Apocynum venetum L. Using a Zebrafish Model

Background/Objectives:Apocynum venetum L. leaves have long been used in traditional medicine and have shown cardioprotective potential, but the active constituents responsible for these effects remain unclear. This study aimed to identify cardioprotective constituents in the n-butanol fraction of A. venetum leaves and clarify their mechanisms through chemical profiling, zebrafish phenotyping, and cell-based validation. Methods: The n-butanol extract was fractionated into S1-S6 and characterized by HPLC and UPLC-QE-Orbitrap-MS/MS. Cardioprotective effects were assessed in zebrafish myocardial injury models. Spectrum-effect relationships were analyzed using grey relational analysis (GRA) and orthogonal partial least squares (OPLS), and key compounds were validated in zebrafish. Transcriptomic analysis, qRT-PCR, molecular docking, and isoprenaline (ISO)-induced H9c2 cell assays were used to explore mechanisms and synergistic effects. Results: Fingerprinting analysis of subfractions S1-S6 revealed 12 common peaks and ten compounds. S1-S4 showed stronger protection against zebrafish myocardial injury than S5-S6. Spectrum-effect analysis indicated that chlorogenic acid (CGA) and 4-caffeoylquinic acid (4-CQA) were the major activity-associated constituents. CGA produced the most pronounced cardioprotective effect in zebrafish and was associated with PPARα-related metabolic pathways. In H9c2 cells, CGA combined with myricetin 3-O-glucoside showed synergistic protection. Quantitative analysis showed that CGA and 4-CQA were predominantly distributed in S1-S3, consistent with the stronger activity of these fractions. Conclusions: This study applied a spectrum-effect relationship strategy to identify CGA and 4-CQA as major cardioprotective constituents in the n-butanol fraction of A. venetum leaves, supporting the potential synergistic contribution of CGA-dominated constituent combinations and providing a basis for further pharmacological investigation.