Integrated LC-HRMS and in silico prediction discovery a hydroxylated protopanaxatriol metabolite in vivo with enhanced anti-inflammatory activity.

BACKGROUND: Protopanaxatriol (PPT), a key aglycone of ginsenosides, exhibits significant anti-inflammatory potential but suffers from poor oral bioavailability. After oral administration, PPT is transformed by gastric acid, gut microbiota, and liver enzymes into metabolites with enhanced bioactivity. METHODS: We employed an integrated metabolomics strategy combining LC-HRMS, in silico prediction, and molecular networking to systematically characterize PPT metabolism in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. Metabolites were identified across serum, urine, and feces samples. RESULTS: A total of 20 metabolites were identified, including the novel 25-hydroxy-PPT (25OH-PPT), discovered for the first time. Mechanistic in vitro assays demonstrated that 25OH-PPT was exclusively generated under simulated gastric conditions via an acid-catalyzed, non-enzymatic hydroxylation pathway. Functional evaluation showed that 25OH-PPT exhibited significantly stronger anti-inflammatory activity than PPT (IC50 = 1.52 μM vs. 40.34 μM in RAW 264.7 cells). CONCLUSIONS: These findings uncover a new metabolic activation pathway of PPT, providing mechanistic insights into its pharmacological effects and offering new perspectives for designing PPT-based therapeutics.