Identification of anti-NSCLC bioactive compounds from Euphorbia helioscopia L. through integrated pharmacological classification and metabolomics analysis.

Euphorbia helioscopia L. (commonly known as Zeqi) has been reported to possess anti-tumor potential, however, its bioactive constituents and underlying molecular mechanisms against non-small cell lung cancer (NSCLC) remain to be elucidated. This study aimed to systematically identify anti-NSCLC components and explore their associated mechanisms through integrated pharmacological classification and metabolomics analysis. Zeqi samples exhibiting distinct in vitro antiproliferative activities were first classified into relatively higher-activity (RHA) and relatively lower-activity (RLA) group, and then subjected to metabolomics profiling using ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) and subsequent univariate and multivariate statistical analysis. Four compounds (fraxetin, 2''-O-galloylhyperoside, isofraxidin and myricitrin 3-O-galactoside) were identified as candidate bioactive constituents associated with the antiproliferative activity of Zeqi. Among these, fraxetin exhibited the most potent inhibitory effect against NSCLC cells. Flow cytometric analysis revealed a dose-dependent increase in apoptotic rates. Transcriptomic analysis indicated that fraxetin regulates the expression of apoptosis-related genes and activates apoptotic pathways. Further reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses confirmed that fraxetin-induced apoptosis in NSCLC cells might be associated with its ability to upregulate Noxa and downregulate Mcl-1 at both transcriptional and translational levels. In conclusion, the successful application of integrated pharmacological classification and metabolomics analysis not only enabled the identification of anti-NSCLC active components in Zeqi but also provides a robust methodological framework for efficient discovery of bioactive lead compounds from complex traditional Chinese medicine (TCM) systems.