Affinity screening and cardioprotective evaluation of FRS2α-targeted components from the ethyl-acetate fraction of Gualou-Xiebai-Banxia decoction.

The renowned traditional Chinese medicine (TCM) classical prescription Gualou-Xiebai-Banxia decoction (GXBD) exhibits remarkable therapeutic effects on coronary artery disease (CAD). However, its material basis and molecular targets remain unclear. Our previous studies found that its ethyl acetate fraction (GXB-E) exhibited the most pronounced cardioprotective effect among its series extracts and could potentially activate the Fibroblast Growth Factor Receptor Substrate 2α (FRS2α), the crucial gatekeeper within FGF21/FGFR1/β-Klotho-FRS2α pathway. This study aimed to rapidly screen for FRS2α-targeted components from GXB-E and to verify their cardioprotective effects. An integrated strategy combining affinity ultrafiltration with UHPLC-Orbitrap-Exploris MS (AUF-LC-MS) was established to screen for affinity components, binding sites of which to FRS2α were inferred via molecular docking. Forty-nine compounds were identified in GXB-E, among which 10 showed specific binding affinity to FRS2α. These compounds interacted with the active site of FRS2α mainly through hydrogen bonds and other intermolecular forces. Among them, vanillic acid (VA) and cynaroside (Cyn) exhibited stronger binding energies. Both components significantly improved the growth state of myocardial cells (MCs) injured by ischemia-hypoxia, inhibited apoptosis and necrosis, and markedly increased cell viability. In zebrafish hearts injured by doxorubicin (DOX), they notably alleviated myocardial hypertrophy and pathological damage, reduced the SV-BA distance, pericardial edema area, and myocardial enzyme activity. Meanwhile, the decreased p-FRS2α level was dramatically upregulated by these two components in both the MCs and zebrafish models. This work validated the feasibility of using AUF-LC-MS for lead compound screening. Our findings help clarify the multi-component synergistic mechanism underlying the therapeutic effect of GXBD on CAD, lay a foundation for exploring novel CAD therapeutics derived from this TCM classic prescription.