Comprehensive exploration and comparison of chemical constituents of Paris polyphylla var. chinensis and Paris polyphylla var. yunnanensis through two-dimensional chromatography, metabolomic analysis and furostanosides quantitation.

Paris polyphylla var. chinensis (PPC) and Paris polyphylla var. yunnanensis (PPY) were recognized as two legal species of Paridis Rhizoma in the Chinese Pharmacopoeia, exhibiting distinct chemical profiles primarily in terms of spirostanosides content. However, the chemical differences of other types of steroidal saponins remained unclear, comprehensive component comparison is lacking. In this research, an offline two-dimensional separation system (comprising HILIC and C18) was established to thoroughly explore and compare the chemical components between PPC and PPY. Additionally, a nontargeted metabolomic approach utilizing high-resolution mass spectrometry was employed to identify differential metabolites. Consequently, a total of 444 metabolites were characterized, among which steroidal saponins were the most abundant components. Specifically, 394 steroidal saponins were identified based on their fragmentation patterns and chromatographic retention behaviors. The significant differences were observed in furostanosides and polyhydroxylated steroidal saponins between PPC and PPY. To enable quantitative analysis of furostanosides, the biosynthetic precursors of spirostanosides, a high-performance liquid chromatography method employing a HILIC column was developed for the first time. Similar to the profiles of spirostanosides, PPC contains a high abundance of proto-pennogenyl saponins, whereas PPY is rich in proto-diosgenyl saponins. These results facilitated a detailed understanding of chemical information of PPC and PPY, and improved quality control tactics of Paridis Rhizoma.