Studies on Separation and Their Anti-Inflammatory Activity Evaluation of Lupane-Triterpenoid Aglycones from Leaves of <i>Acanthopanax Gracilistylus</i> W.W.Smith by HSCCC

Objective Lupane-type triterpenoid aglycones from Acanthopanax gracilistylus leaves (AGS) are underexplored compared to roots. This study aimed to establish an efficient isolation method and evaluate their anti-inflammatory potential. Methods Methanol extracts of AGS leaves were separated using high-speed counter-current chromatography (HSCCC) coupled with preparative HPLC. Six compounds were purified and structurally confirmed by NMR spectroscopy. Anti-inflammatory activity was assessed in LPS-stimulated RAW264.7 macrophages via nitric oxide (NO) production, with cytotoxicity measured by MTT assay. Results one lupane-triterpenoid and five Lupane-triterpenoid aglycones were obtained at purities of 94–97%. Cpds 6 and 7 sig-nificantly inhibited NO production by 57–64% at 20 µg/mL without affecting cell viability, whereas Cpds 1 and 2 were inactive, and Cpds 4 and 5 displayed a moderate impact. Additionally, Cpd 4 had moderate cytotoxicity. The integrated HSCCC-prep-HPLC strategy provided high-purity compounds efficiently with minimal solvent use. Conclusion This study presents an effective approach for isolating bioactive lupane-triterpenoid aglycones from AGS leaves. Cpds 6 and 7 demonstrate promising anti-inflammatory activity, supporting further pharmacological exploration.