From Activity Screening to Quality Control: UHPLC-MS/MS Analysis of Anti-Inflammatory Cyclodipeptides in Pinellia ternata.

This study investigated the anti-inflammatory material basis and quality control of Pinellia ternata (P. ternata) to provide a modern scientific interpretation for its therapeutic properties. First, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) analysis was used to analyze different polar fractions of P. ternata, resulting in the identification of 79 compounds. Next, an in vitro evaluation using an LPS-induced RAW264.7 cell inflammation model revealed that the ethyl acetate fraction exhibited the most significant inhibition of nitric oxide (NO) production. Three cyclodipeptides, cyclo (Pro-Leu), cyclo (Phe-Pro), and cyclo (Leu-Phe), which displayed notable differences from other fractions, were subsequently screened. Molecular docking studies showed binding free energies below -5 kcal/mol with inducible nitric oxide synthase (iNOS), indicating potential anti-inflammatory targeting properties. Cellular experiments further confirmed that the reduction in NO production induced by these cyclodipeptides ranged from 11.03% to 40.38%. To enable their simultaneous quantification, a method based on ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) in the multiple reaction monitoring (MRM) mode was established, meeting all analytical validation criteria. Application of this method to P. ternata samples from different origins and growth conditions demonstrated that the contents of these cyclodipeptides were significantly influenced by both the origin and cultivation method. In conclusion, this study preliminarily identifies cyclodipeptides as an important anti-inflammatory material basis of P. ternata, and the established quantitative method provides methodological support and data for constructing its quality evaluation system.