Rapid Identification of Chemical Components and Screening of Beta–Klotho Agonists From Trichosanthes pericarpium Aqueous Extract Based on Affinity Ultrafiltration and Liquid Chromatography–Tandem Mass Spectrometry

ABSTRACT Trichosanthes pericarpium (TP), a natural product rich in pharmacologically active constituents, exhibits potential cardioprotective effects. This study employed a ligand fishing strategy combined with identification to efficiently characterize the chemical ingredients of TP and rapidly screen for Beta–Klotho (βKlotho) agonists, utilizing affinity ultrafiltration combined high–performance liquid chromatography–mass spectrometry (AUF–LC–MS). As a result, a total of 59 compounds were tentatively characterized, and 7 potential ligands were fished out when βKlotho acted as the targeted protein. Subsequently, three representative angling compounds, including diosmetin–7 ‐O –β –D –glucopyranoside, cucurbitacin B, and rutin, were further verified by molecular docking visualization analysis and in vitro activity validation assays. Cell viability assays demonstrated that all three aforementioned compounds significantly increase the viability of myocardial cells injured by ischemia and hypoxia. Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays demonstrated that these compounds significantly attenuated cardiomyocyte apoptosis. Collectively, this study establishes a novel strategy for the systematic characterization and screening of bioactive compounds in natural products, providing a solid foundation for the development of βKlotho agonists derived from TP.