Integrated LC-HRMS/MS-based Metabolomics and in vitro and in vivo Bioassays for Comparative Evaluation of Two Ethnomedicinal Paris Species and Their Official Counterparts

Paris species, known as “Chonglou” in traditional Chinese medicine, have been used clinically for millennia. Nevertheless, the variation patterns of their chemical constituents and biological activities among different species are still poorly understood and urgently need to be elucidated. Current research has largely focused on a few pharmacopoeial species, while many with ethnomedicinal value remain scientifically unexplored. This study presents the first integrated metabolomic and pharmacological comparison of two pharmacopoeial species, P. polyphylla Smith var. chinensis (PPC) and P. polyphylla Smith var. yunnanensis (PPY), with two ethnomedicinal species, P. thibetica France (PT) and P. fargesii France (PF). A comprehensive phytochemical profile was established using UPLC-Q-TOF-MS/MS, identifying 119 compounds, 85.7% of which were steroidal saponins. Significant inter-species variation in total saponin content was observed, with PF showing the highest average. Multivariate statistical analysis successfully identified species-specific markers and revealed unexpected chemotaxonomic affinities. Pharmacologically, all four species exhibited significant dual bioactivities. They demonstrated potent hemostatic effects, significantly reducing tail bleeding time and directly inducing platelet aggregation. Concurrently, the extracts showed considerable anti-hepatocarcinoma activity. Mechanistically, this anti-cancer effect involved the induction of S -phase cell cycle arrest and apoptosis, which were associated with down-regulation of Bcl-2 and suppression of c-Myc signaling. The above-mentioned findings confirmed PF and PT as promising complementary resources to the official pharmacopoeial species. This work provides a critical chemical and pharmacological foundation for their quality standardization and potential therapeutic application in hemostasis and liver cancer management.