Enrichment of steroidal saponins and glycoalkaloids enhances the antitumor activity of solanum nigrum berries by targeting glycolytic metabolism in NSCLC.

Solanum nigrum L. is a widely distributed ethnomedicinal plant with significant antitumor potential, primarily attributed to its abundant steroidal saponins and steroidal glycoalkaloids. However, compositional variability and safety concerns have historically hindered its clinical translation. In this study, we developed a standardized fraction of S. nigrum berries specifically enriched with steroidal saponins and glycoalkaloids (S-SNB), and systematically evaluated its chemical composition, therapeutic potential against non-small cell lung cancer (NSCLC), underly mechanisms, and safety profile. Compared with the conventional aqueous extract, S-SNB exhibited markedly enhanced tumor suppression in A549 and PC-9 xenograft models at substantially lower crude herb equivalent doses. Phytochemical characterization and bioactivity-guided analyses revealed solasonine, solamargine, macrostemonoside B, and solanigroside J as core bioactive constituents, with the latter reported here for their anti-NSCLC activity for the first time. From a mechanistic perspective, proteome-wide target profiling via limited proteolysis-mass spectrometry (LiP-MS) identified glycolytic pathway as a primary node of S-SNB, which was validated by functional metabolic assays showing suppressed glycolytic flux both in vitro and in vivo. Integrated biophysical, biochemical, and computational analyses further converged on PKM2 as a central target of the major active constituents, revealing a non-classical mode of regulation involving coordinated multi-component interactions rather than direct catalytic inhibition. Collectively, these findings demonstrate that enrichment of steroidal saponins and glycoalkaloids enhances both pharmacological potency and mechanistic clarity of S. nigrum against NSCLC, and establishes S-SNB as a chemically defined, safety-validated, and mechanism-informed botanical preparation, providing a robust scientific foundation for its development as an adjunctive oncology therapy.