A stepwise integrated strategy to explore quality markers of Qishen Yiqi dripping pills against myocardial ischemia

Background: Numerous experiments and clinical practices have demonstrated the effectiveness of Qishen Yiqi dripping pills (QSYQ) on myocardial ischemia (MI). However, the bioactive ingredients and mechanisms remain unclear, leading to huge gaps between quality control and biological effect of QSYQ. Discovering quality markers (Q-markers) based on effective components is crucial for ensuring stable quality and clinical effectiveness of QSYQ.

Purpose: To explore Q-markers of QSYQ against MI by a stepwise strategy integrating serum pharmacochemistry, network pharmacology, metabolomics, quantitative analysis, and cell experiments.

Methods: Firstly, liquid/gas chromatography-mass spectrometry was applied to characterize chemical profiles of QSYQ in vitro and in vivo. Based on the serum migrating constituents, a component-target-MI interaction network was constructed. Subsequently, pharmacodynamics and metabolomics were conducted to evaluate cardioprotective effect and potential mechanism of QSYQ. Next, conjoint analysis of network pharmacology and metabolomics was performed to screen candidate Q-markers. Finally, the measurability and bioactivity were validated to justify their usage as Q-markers.

Results: A total of 97 components were identified in QSYQ, 24 prototypes of which were detected in serum. The "component-target-disease" interaction network was constructed based on serum migrating constituents. Pharmacodynamic results showed that QSYQ effectively improved cardiac function, attenuated inflammatory cell infiltration, alleviated myocardial fibrosis, and reduced the levels of myocardial enzymes and oxidative stress in MI rats. Metabolomics study demonstrated that 59 metabolites were markedly altered in MI rats, 25 of which were significantly reversely regulated by QSYQ. After integrative analysis of network pharmacology and metabolomics, 12 components were selected as candidate Q-markers of QSYQ, and the contents were quantified. These candidate Q-markers displayed synergistic protective effects against H₂O₂-induced injury in H9c2 cells. Taken together, 12 components with properties of transitivity and traceability, effectiveness, measurability, and compatibility contribution were defined as representative Q-markers of QSYQ, including Astragaloside IV, Ononin, Calycosin, Formononetin, Rosmarinic acid, Cryptotanshinone, Salvianolic acid A, Tanshinol, Ginsenoside Rb1, Ginsenoside Rg1, Nerolidol, and Santalol.

Conclusion: In this study, a novel stepwise integrated strategy was presented for discovering Q-markers related to therapeutic effects of traditional Chinese medicine prescriptions. Twelve comprehensive and representative Q-markers of QSYQ were identified for the first time to improve its quality control.