Objective  To identify the material basis of Gualou Xiebai Banxia decoction (GXBD) in ameliorating myocardial hypoxia/reoxygenation (H/R) injury and to elucidate its cardioprotective molecular mechanism mediated through the promotion of mitophagy.

Methods   The standard decoction of GXBD was prepared by the traditional decoction method. Chemical fractions were obtained via ethanol precipitation and further separated and purified using D101 macroporous resin. An H/R injury model was established in H9c2 cardiomyocytes. The protective effects of the aqueouswashed fraction, the 40% ethanoleluted fraction and the 90% ethanoleluted fraction of GXBD against cardiomyocyte damage were evaluated by the CCK8 assay. UPLC-Q-TOFMS was employed to analyze and identify the chemical constituents of the 40% ethanoleluted fraction. Western Blot was performed to detect the protein expression levels of cysteinyl aspartate specific proteinase-3 (Caspase-3), Parkin, and FUN14 domain-containing protein 1 (FUNDC1) in each group, providing preliminary validation of the mechanism by which the active monomer chrysoeriol from GXBD attenuates H/Rinduced cardiomyocyte injury.

Results The aqueous-eluted, 40% ethanol-eluted, and 90% ethanol-eluted fractions all dose-dependently increased the survival rate of H/R-injured cardiomyocytes, with the 40% ethanol-eluted fraction showing superior efficacy. A total of 88 compounds, including chrysoeriol, were identified in the 40% ethanol-eluted fraction of the compound. Chrysoeriol significantly upregulated the expression of Parkin and FUNDC1, downregulated the expression of Caspase-3, and alleviated H/R-induced injury in cardiomyocytes.

Conclusion The active component chrysoeriol derived from the GXBD formula activates mitophagy and suppresses apoptosis, thereby ameliorating myocardial hypoxia/reoxygenation (H/R) injury.

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