Comprehensive Metabolic Profiling and Enzyme Kinetics of Cis -2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-glucoside in Normal and Hyperlipidemic Rats

Cis-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (cis-THSG), a photoisomer of the major bioactive component in Polygoni Multiflori Radix Praeparata (He-Shou-Wu), has enhanced hypolipidemic activity but is associated with idiosyncratic hepatotoxicity. Its metabolic fate under pathological conditions and key clearance enzymes remain unclear.This study aimed to compare metabolite profiles of cis-THSG in normal and hyperlipidemic rats in vivo and in vitro, identify metabolites, and characterize enzyme kinetics in rat liver microsomes (RLM) and major CYP450 isoforms. Metabolites in plasma, urine, feces, bile, and intestinal bacteria incubation fluids were analyzed by UHPLC-Q-TOF-MS/MS. Enzymatic kinetics and CYP450 isoforms were investigated via chemical inhibition assays.A total of 27 metabolites were characterized; cis-THSG underwent rapid glucoside hydrolysis, and its aglycone underwent phase I and II reactions. Hyperlipidemia reduced metabolites to 20 and markedly increased plasma cis-THSG and aglycone abundance. Kinetic parameters were Km 32.86 μmol/L, Vmax 0.7285 μmol/(min·mg protein), CLint 0.02217 L/(min·mg protein). Rat liver microsomal CYP isoforms analogous to human CYP3A4 and CYP2C9 mediated cis-THSG oxidative metabolism.This study provides the first comprehensive metabolite map of cis-THSG, identifies its aglycone as a potential bioactive form, and reveals a disease-related metabolic shift. CYP phenotyping offers a mechanistic basis for predicting clinical drug-drug interactions.