Rapid characterization of the chemical composition and metabolic transformation of flavonoids in Da-Cheng-Qi Decoction guided by feature-based molecular networking and sequential metabolism strategy

Abstract

Flavonoids in Da-Cheng-Qi Decoction (DCQD), especially polymethoxyflavones (PMFs) and hydroxylated polymethoxyflavones (OH-PMFs), remain to be further explored considering their various biological activities. This study proposed an integrated strategy combining feature-based molecular networking (FBMN) and sequential metabolism strategy for the flavonoids analysis in DCQD extracts and in vivo. Firstly, based on FBMN annotation, a total of 102 flavonoids were identified in DCQD, including 14 flavones, 18 flavanones, 3 flavonols, 8 PMFs, 16 OH-PMFs aglycones and 43 OH-PMFs glycosides, among which 35 OH-PMFs glycosides were first reported as potential new compounds and the isomers of PMFs and OH-PMFs were innovatively discriminated by RDA fragmentation ions ^1,3A⁺, ^1,4A⁺ and ^1,3B⁺. Then, 9 metabolites were identified in rat plasma and the dynamic metabolic transformation of flavonoids in vivo was characterized based on sequential metabolism strategy. Flavanones mainly undergo deglycosylation, sulfation and glucuronidation before entering the circulation system. The inter-transformation from PMFs to OH-PMFs by demethylation and hydroxylation, and the subsequent glucuronidation of OH-PMFs were observed in enterocytes and hepatocytes. The present study elucidated the flavonoids in DCQD and their metabolic transformation in vivo, which laid a material basis for the in-depth pharmacology and mechanism research of DCQD and provided guidance for the comprehensive chemical and metabolic characterization of traditional Chinese medicine formulas.