Systematic characterization of active components and therapeutic mechanisms of Chaihu Guizhi Ganjiang Decoction in metabolic dysfunction-associated steatohepatitis.

Chaihu Guizhi Ganjiang Decoction (CGGD) is a clinically proven prescription effective against metabolic dysfunction-associated steatohepatitis (MASH). However, its chemical constituents with the lipid-lowering and anti-inflammatory bioactivities and underlying mechanisms remain unclear. This study aimed to characterize the chemical profile of CGGD while elucidating their lipolytic and anti-inflammatory activities and mechanisms. Using UHPLC-Q-TOF-MS/MS, 209 compounds were identified in CGGD, categorized into 52 saponins, 103 flavonoids, 17 gingerols, 21 organic acids, and 16 others, with 13 putative unvalidated new compounds. Nineteen prototype components were detected in hepatic tissues, with saikosaponin A, baicalin, wogonoside, skullcapflavone II, and glycyrrhizin exhibiting strong binding affinities toward PPARα and TLR4 proteins. These components significantly reduced triglyceride (TG) accumulation in FFA-induced HepG2 steatosis cells, confirmed by oil red O staining and TG quantification. Mechanistically, saikosaponin A and baicalin upregulated PPARα and its downstream genes (CPT1A, FABP1, ACOX1) to promote fatty acid transport and oxidation. Meanwhile, saikosaponin A, baicalin, and wogonoside significantly suppressed mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in LPS-stimulated RAW264.7 macrophages, and saikosaponin A and wogonoside further inhibited the expression of TLR4, MyD88, and phosphorylated NFκB, thereby attenuating inflammation. In conclusion, this study established a comprehensive methodology for profiling the chemical constituents of CGGD and identified saikosaponin A, baicalin, wogonoside, skullcapflavone II, and glycyrrhizin as its key anti-MASH components. Their lipid-lowering and anti-inflammatory activities were mediated through the PPARα-regulated fatty acid metabolism pathway and the TLR4/MyD88/NFκB signaling pathway, respectively, offering a scientific basis for the clinical application of CGGD in MASH.