Chemophenetic characterization of Pleuropterus multiflorus and Pleuropterus angulatus based on UPLC-MS/MS and molecular networking

Pleuropterus multiflorus and P. angulatus are two morphologically similar taxa widely used in traditional Chinese medicine but often confused in the herbal market. Their taxonomic relationship remains controversial, and chemical profiling provides complementary evidence for clarifying species boundaries. In this study, an untargeted UPLC–MS/MS approach combined with molecular networking (MN) and multivariate statistical analysis was employed to compare the leaf metabolomes of P . multiflorus and P. angulatus . A total of 46 flavonoid metabolites were structurally annotated, and 16 differential metabolites were identified as key contributors to their separation. MN analysis revealed that both taxa shared the same flavonol core structures, but differed markedly in modification diversity. P. angulatus exhibited a higher abundance of acylated flavonol glycosides and several biflavonoid dimers, whereas P . multiflorus was dominated by non-acylated glycosides. Eight previously unreported structural analogues were also annotated through MN-guided analysis. These compositional and structural differences, observed under common garden conditions, suggest a genetic basis for the metabolic variation. Although limited to the studied accessions, these findings provide preliminary chemophenetic evidence supporting the differentiation of P. angulatus from P. multiflorus . The integration of UPLC–MS/MS and MN demonstrates strong potential for resolving chemical variation among closely related taxa and contributes to improved species authentication, quality control, and conservation of medicinal plant resources.