Objective To systematically characterize the metabolites of polygala oligosaccharide esters (PROEs) in rats, thereby providing evidence for elucidating the pharmacodynamic material basis of Polygala tenuifolia. Method Plasma samples from rats after oral administration of PROEs were analyzed using UPLC-QTOF-MS/MS. Feature-based molecular networking (FBMN) was employed to visually cluster and screen for potential metabolites. The identities of predicted metabolites were further confirmed by comparison with reference standards prepared via in vitro enzymatic incubation and chemical isomerization synthesis. Results FBMN effectively clustered structurally similar metabolites, such as glucuronidated and sulfated products, from the mass spectrometry data. A total of 59 metabolites were identified in rat plasma, including 14 free phenolic acids, 21 sulfated organic acids, 17 glucuronidated organic acids, and seven other compounds. No intact PROEs were detected. The metabolic pathway analysis revealed that PROEs were rapidly hydrolyzed in vivo to generate various phenolic acids (e.g., sinapic acid, ferulic acid, syringic acid), which subsequently underwent extensive phase I metabolism (reduction, demethylation, dehydroxylation) and predominantly phase II metabolism (sulfation and glucuronidation). Conclusion Following oral administration, PROEs enter the systemic circulation primarily as hydrolyzed organic acids and their extensive phase II conjugates. This study demonstrates that the combination of UPLC-Qtof-MS/MS and FBMN provides an efficient and comprehensive strategy for identifying the in vivo metabolites of PROEs. This approach offers a basis for revealing the active constituents of P. tenuifolia and serves as an effective method for elucidating the metabolic profiles of traditional Chinese medicines.

Polygala tenuifolia Willd.  /  oligosaccharide ester  /  Feature-based molecular networking  /  UPLC-QTOF-MS/MS  /  metabolite  /  metabolic pathway  /  sinapic acid  /  ferulic acid