Ingenane Diterpenoids from Euphorbia peplus: Structure Elucidation and Autophagic Flux Activation Activity

Autophagy dysfunction is implicated in the pathogenesis of Alzheimer's disease (AD), and enhancing autophagic flux has been proposed as a potential strategy for addressing neurodegenerative diseases. To expand the structural diversity of ingenol esters and systematically evaluate their autophagic flux activation activity, a systematic phytochemical investigation of ingenane diterpenoids from Euphorbia peplus was conducted. A total of 13 ingenane-type compounds were isolated and identified, including two previously undescribed compounds, euphingenol A and B (1-2), together with 11 known analogs (3-13). Their structures were elucidated by extensive spectroscopic analyses (HRESIMS and NMR) and comparison with literature data. The compounds were evaluated for their bioactivity with flow cytometry in assays of autophagic flux in HM Cherry-GFP-LC3 (human microglia cells stably expressing the tandem monomeric mCherry-GFP-tagged LC3) cells. 17-O-benzoyl-20-deoxyingenol (3) significantly activated autophagic flux at concentrations of 10 μM and 40 μM, while euphingenol A (1) induced a dose-dependent increase, with structure-activity relationship analysis indicating that C-17 acylation enhances this bioactivity. These findings suggest that compound 3 warrants further investigation as a potential modulator of autophagic flux, possibly through binding to PKCδ (protein kinase C), with relevance to autophagy-related neurodegenerative conditions.