Anti-ferroptotic lignan glycosides from Zanthoxylum avicennae : Isolation, structural characterization, and mechanistic evaluation

Introduction: Ferroptosis, a regulated cell death driven by iron-dependent lipid peroxidation, has been implicated in various liver diseases. Small molecules that counteract ferroptosis remain largely restricted to a few synthetic scaffolds, leaving natural products an underexplored source. Zanthoxylum avicennae (Lam.) DC. (Rutaceae) is used against inflammatory and hepatic conditions in Vietnamese traditional medicine. It contains diverse lignans and flavonoids capable of hydrogen-atom-transfer radical scavenging, the mechanism shared by established ferroptosis inhibitors. Its metabolites are therefore potential ferroptosis inhibitors. This study aimed to isolate, structurally characterize, and evaluate the anti-ferroptotic activity of its metabolites. Methods: Metabolites were isolated from the leaves of Z. avicennae by 80% ethanol reflux extraction, solvent partitioning and silica gel column chromatography. Structures were elucidated by ¹H and ¹³C NMR spectroscopy. Anti-ferroptotic activity was assessed in an optimized RSL3-triggered ferroptosis model in HepG2 cells, using the MTT assay. The most active compound was evaluated for anti-lipid peroxidation activity using BODIPY 581/591 C11, labile iron pool modulation by FerroOrange, radical scavenging and antioxidant capacity using the DPPH, the cell-free BODIPY 581/591 C11, and the FRAP assay. Results: (−)-isolariciresinol 3-α-O-β-D-glucopyranoside (compound 9), a lignan glycoside, showed anti-ferroptotic activity (EC50 = 5.89 μM) without detectable cytotoxicity at 30 μM. It suppressed lipid peroxidation but did not reduce the intracellular labile iron. Compound 9 quenched DPPH and AAPH-derived peroxyl radicals and displayed Fe3+-reducing capacity. Conclusion: Compound 9 from Z. avicennae is an initial hit possessing radical-scavenging-associated protection in an RSL3-induced ferroptosis model.