Compositional Studies and Bioactivity-Guided Fractionation of Acetylcholinesterase Inhibitors in Papaver nudicaule from Mongolia—The Role of Amurensinine

Papaver nudicaule L. is a medicinal plant traditionally used in Asian ethnomedicine, yet its phytochemical composition and biological activity remain insufficiently explored. This study bridges phytochemistry and neuroactive potential. It includes metabolite profiling with bioactivity-guided fractionation, performed to evaluate its potential as a source of acetylcholinesterase (AChE) inhibitors. The methanolic extract of the aerial parts was analysed using HPLC–ESI–QTOF-MS/MS, which enabled the tentative identification of 34 compounds, predominantly isoquinoline alkaloids and flavonoid derivatives. The extract was subsequently fractionated by centrifugal partition chromatography (CPC) using an optimised biphasic solvent system, yielding fractions enriched in alkaloid constituents. The obtained fractions were evaluated for AChE inhibitory activity, revealing significantly higher activity than that of the crude extract. The most active fractions exhibited marked inhibition based on the comparison with the reference compound berberine, indicating effective enrichment of bioactive metabolites. Further analysis demonstrated that the activity of the most potent fraction was associated with the presence of amurensinine, which was purified by preparative HPLC and subsequently identified by NMR and LC-MS. The Papaver nudicaule extract showed no significant cytotoxicity toward SH-SY5Y neuronal cells up to 200 µg/mL, whereas the amurensinine-containing fraction reduced cell viability only at higher concentrations (≥100 µg/mL). Notably, when expressed in the micromolar range, this effect corresponds to relatively weak cytotoxicity, suggesting a potential safety margin at lower, biologically relevant concentrations. These findings demonstrate that P. nudicaule possesses a highly diverse alkaloid profile and represents a promising natural source of compounds with potential relevance for the development of agents targeting neurodegenerative disorders.