Simultaneous Screening and Identification of Potential Bioactive Components Against MASLD in Guizhi Fuling Decoction by Integrating a Multitarget Affinity Ultrafiltration Strategy With UPLC‐Q‐TOF‐MS/MS

ABSTRACT Introduction As one of the most common chronic liver diseases worldwide, metabolic dysfunction‐associated steatotic liver disease (MASLD) imposes an escalating burden on global health systems. However, the efficacy of Guizhi Fuling decoction (GZFLD) against MASLD and its potential pharmacodynamic material basis remain elusive. Objectives This work aims to explore the potential bioactive constituents of GZFLD against MASLD by integrating a multitarget affinity ultrafiltration strategy with UPLC‐Q‐TOF‐MS/MS. Methods The efficacy of GZFLD against MASLD was initially evaluated using an in vitro HepG2 model of MASLD. Subsequently, potential active ingredients of GZFLD against MASLD were screened out via affinity ultrafiltration coupled with liquid chromatography–tandem mass spectrometry (AUF‐LC‐MS/MS) targeting multiple enzymes, including pancreatic lipase (PL), cyclooxygenase‐2 (COX‐2), proprotein convertase subtilisin/kexin type 9 (PCSK9), and interleukin‐6 (IL‐6). Finally, molecular docking simulations, enzyme inhibition assays, and cell experiments were performed to validate the activities of the candidate components. Results The results demonstrated that GZFLD significantly alleviated lipid accumulation and oxidative stress in the HepG2 model of MASLD, and a total of 18 potential bioactive ingredients were identified in GZFLD. Among them, benzoylpaeoniflorin, galloylpaeoniflorin, and ellagic acid are the main active compounds of GZFLD against MASLD. Furthermore, the results of molecular docking revealed that these components could strongly bind their corresponding target by various forces. The result of COX‐2 inhibition assay indicated that ellagic acid exhibited a strong COX‐2 inhibitory activity. Moreover, the results of cell verifications implied that ellagic acid and benzoylpaeoniflorin exhibited significant therapeutic potential on MASLD by modulating lipid metabolism and inflammatory responses. Conclusion GZFLD demonstrated noteworthy effects against MASLD by alleviating lipid accumulation and oxidative stress, and the potential bioactive components of GZFLD and their interaction network against MASLD were first explored and constructed by combining AUF‐LC‐MS/MS with multiple targets, which showcased a powerful new integrating strategy for the simultaneous screening and identification of bioactive compounds in complex Chinese medicines.