Integrative network pharmacology and experimental validation of anti-inflammatory triterpenoids from hawthorn leaves.

A network pharmacology approach was employed to identify key bioactive compounds and core targets in hawthorn leaves with potential anti-inflammatory properties. The predicted biological effects and underlying mechanisms were systematically validated through target enzyme activity evaluations, molecular docking simulations, LPS-induced inflammatory models in RAW264.7 macrophages, western blot and quantitative real-time PCR (qRT-PCR) analyses. Molecular docking studies revealed strong binding affinities of triterpenoids 99, 102, and 116 to SRC (Proto-Oncogene Tyrosine-Protein Kinase Src), a critical regulator of inflammatory signaling pathways. These interactions were further substantiated by enzymatic activity assays and macrophage-based inflammatory models. Notably, the triterpenoids showed strong anti-inflammatory properties by significantly reducing nitric oxide (NO) release and altering the expression of inflammatory genes in LPS-stimulated RAW264.7 macrophages. Among them, triterpenoid 99 demonstrated the most pronounced activity, primarily by downregulating SRC mRNA and protein expression. These findings provide compelling scientific evidence supporting the use of hawthorn leaves as a natural reservoir of anti-inflammatory agents, offering a strong foundation for future pharmacological and therapeutic developments.