OBJECTIVE  To explore the active components and mechanisms of flavonoids from new "Zhe Eight Flavors" Quzhou Fructus Aurantii(fruit of Citrus aurantium 'Changshan-huyou') against non-small cell lung cancer(NSCLC) through UPLC-Q-TOF-MS/MS and network pharmacology, and experiment verification.

METHODS  The main chemical substances of pure total flavonoids from fruit of Citrus aurantium 'Changshan-huyou'(PTFC) were detected using UPLC-Q-TOF-MS/MS. The potential active components and target proteins of PTFC were collected through TCMSP, ETCM, BATMAN-TCM, and Swiss Target Prediction databases. NSCLC disease target sets were constructed using GeneCards, CTD, Disgenet, and OMIM databases. The intersection of potential targets of PTFC and NSCLC targets was obtained to identify key target proteins, and a protein-protein interaction network(PPT) was constructed. GO function and KEGG pathway enrichment analysis were performed on core targets, and components-targets-pathways–diseases network was established. The molecular docking was conducted to verify the binding activity between the active ingredients of PTFC and the core target SMAD3. The effects of PTFC on the vitality of H1299 NSCLC cells were analyzed through proliferation, cloning, wound healing, and migration assays. The changes in mitochondrial membrane potential in H1299 cells were observed by JC-1 staining. RT-qPCR and western blotting were used to verify the regulatory effect of PTFC on the key target SMAD3 predicted by network pharmacology.

RESULTS  A total of 148 compounds were identified from PTFC, with 12 compounds considered potential active ingredients, primarily acting on core targets such as SRC, ESR1, AKT1, and SMAD3. GO enrichment analysis results suggested that PTFC might be involved in biological processes including apoptosis, protein phosphorylation, and epidermal growth factor receptor signaling pathway. KEGG enrichment analysis revealed that PTFC might exert anti-NSCLC effects through the PI3K-Akt signaling pathway and FoxO signaling pathway. The molecular docking experiments showed that 10 active ingredients exhibited good binding affinity with the SMAD3 protein target. Experimental results demonstrated that PTFC could inhibit proliferation, clonogenicity, wound healing, and migration capabilities of NSCLC, leading to a decrease in mitochondrial membrane potential. RT-qPCR results showed significant downregulation of SMAD3 mRNA expression, and western blotting analysis confirmed reduced p-SMAD2/3 protein expression, thereby validating the reliability of network pharmacology predictions.

CONCLUSION This study elucidates the potential molecular mechanism by which PTFC target SMAD3 to treat NSCLC, laying a foundation for its clinical application in NSCLC.