Integrated chemical profiling and lipidomics reveal that Mailuoning oral liquid alleviates diet-induced MASLD via PPARα-mediated lipid homeostasis and COX-2 inhibition, with identification of potential active constituents

Background Mailuoning oral liquid (MLN), a standardized five-herb formula, is used for ischemic vascular diseases and shows potential in ameliorating dyslipidemia. Our previous study showed MLN alleviates metabolic dysfunction-associated steatotic liver disease (MASLD) via peroxisome proliferator-activated receptor α (PPARα) activation, but its comprehensive chemical profile, systemic exposure, and bioactive constituents mediating both PPARα activation and cyclooxygenase-2 (COX-2) inhibition remain unclear. Methods High-fat, high-fructose diet-fed rats received MLN (5.4 or 10.8 mL/kg/day) for 8 weeks. Ultra-high-performance liquid chromatography coupled with Q-Exactive Orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS) profiled MLN chemicals and identified serum/liver prototypes. Biochemical profiles, glucose tolerance, histopathology, untargeted lipidomics, and targeted polyunsaturated fatty acid (PUFA) analysis were performed. PPARα and COX-2 signaling were examined by quantitative real-time polymerase chain reaction (qPCR), western blotting, and immunohistochemistry. Key constituents were screened for PPARα activation via dual luciferase reporter assay and molecular docking; COX-2 inhibition was validated by enzyme assays and molecular dynamics. Results A total of 143 compounds were characterized; 48 and 92 prototypes were enriched in serum and liver, respectively. MLN ameliorated dyslipidemia, glucose intolerance, steatosis, and inflammation. Lipidomics revealed bidirectional modulation of hepatic lipids, reversing triglyceride (TAG) accumulation and restoring phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) levels. Targeted analysis showed increased eicosapentaenoic acid (EPA) and normalized COX-2-derived prostaglandins (PGD 2 , PGE 2 ). MLN activated hepatic PPARα and downregulated COX-2. Seven serum-enriched compounds (chlorogenic acid, macranthoidin B, luteoloside, harpagide, angoroside C, oleanolic acid, betaine) were validated as direct PPARα agonists. Secologanoside and chikusetsusaponin IVa were identified as novel COX-2 inhibitors (half-maximal inhibitory concentration (IC 50 ) = 14.61 and 120.9 nM). Discussion MLN alleviates MASLD via dual PPARα activation and COX-2 inhibition, providing a mechanistic foundation for repurposing this herbal formula in early-stage MASLD.