Abstract

Identifies CYP2C29 and CYP2D22 as dominant LDXYF-metabolizing enzymes. • UC suppresses hepatic microsomal metabolism of main LDXYF components. • UC reduces hepatic CYP2C29/CYP2D22 expression at mRNA and protein levels. • Altered FXR/PXR-related signaling accompanies metabolic suppression. Understanding the metabolic process of traditional Chinese prescription (TCP) during disease states and its underlying mechanisms is crucial for evaluating therapeutic efficacy and safety. Cytochrome P450 (CYP450)-mediated hepatic metabolism plays a key role in this process. Liandan Xiaoyan Formula (LDXYF), a Chinese herbal prescription used to treat enteritis, exhibits different pharmacokinetic characteristics and metabolic profiles in ulcerative colitis (UC). To elucidate its underlying metabolic mechanisms, UPLC-Q-Exactive Orbitrap MS/MS technology was employed to compare the metabolite profiles of its six main effective components in hepatic microsomes from control versus UC mice. Molecular docking was employed to predict the dominant CYP450 isoforms involved. Subsequently, a CYP450 enzyme selective inhibition assay was used to identify the metabolic enzyme phenotypes for each component. Finally, RT-qPCR and Western blot analyses were conducted to confirm the expression changes of key isoforms and their regulatory targets. Metabolite profiling revealed that the biotransformation of LDXYF components was markedly suppressed in UC mice, with hydrogenation, carboxylation, hydroxylation, and demethylation being the most attenuated. The CYP450 phenotypes were identified as: CYP2D22/2C29 for andrographolide; CYP2C29 for dehydroandrographolide; CYP2D22/2C29 for 14-deoxyandrographolide; CYP2C29 for 1-methoxycarbonyl-β-carboline; CYP2D22/2C29 for 4,5-dimethoxycanthin-6-one; and CYP2C29 for 5-hydroxy-4-methoxycanthin-6-one. Furthermore, hepatic CYP2D22 and CYP2C29 were confirmed to be down-regulated at both mRNA and protein levels in UC, which correlated with reduced expression of the b