A Fluorescence-Based Acetylcholinesterase Inhibition Assay for Bioactivity-Guided Identification of Neuroactive Protoberberine Alkaloids from Berberis julianae C.K. Schneid.

BACKGROUND/OBJECTIVES: The identification of acetylcholinesterase (AChE) inhibitors from complex natural matrices remains challenging due to the limitations of conventional colorimetric assays. This study aimed to develop a fluorescence-based AChE inhibition assay (FAIA) suitable for screening plant extracts, while simultaneously characterizing the metabolite profile of Berberis julianae fruits, identifying bioactive alkaloid-enriched fractions, and evaluating their cytotoxicity in neuronal cell models. METHODS: Methanolic extracts of B. julianae fruits were profiled by HPLC-ESI-QTOF-MS/MS and fractionated using centrifugal partition chromatography (CPC). The developed FAIA, based on 4-methylumbelliferyl acetate as a fluorogenic substrate, was optimized and validated using berberine as a reference inhibitor. CPC fractions and selected protoberberine alkaloids were screened for AChE-inhibitory activity, while cytotoxicity was assessed in differentiated and undifferentiated SH-SY5Y cells using the resazurin assay. RESULTS: Twenty-five metabolites, including eight isoquinoline alkaloids, were tentatively identified in the fruit extract. The optimized FAIA enabled reliable evaluation of AChE inhibition without the limitations associated with chromogenic assays. Among the CPC fractions, fraction 7 exhibited the strongest inhibitory activity. LC-MS analysis revealed that this fraction was enriched in protoberberine alkaloids, including berberine, palmatine, jatrorrhizine, magnocurarine, and demethyleneberberine. Cytotoxicity studies demonstrated concentration-dependent effects of both the isolated fractions and the individual alkaloids, with differentiated and undifferentiated SH-SY5Y cells exhibiting distinct sensitivity profiles. At 200 µg/mL, cell viability ranged from approximately 20-40% in undifferentiated SH-SY5Y cells and from approximately 25-45% in differentiated SH-SY5Y cells, depending on the CPC fraction. Among the tested protoberberine alkaloids, demethyleneberberine exhibited the lowest cytotoxic effect, maintaining the highest viability of differentiated SH-SY5Y cells, followed by jatrorrhizine and berberine. CONCLUSIONS: The proposed FAIA represents a sensitive and practical approach for screening AChE inhibitors in complex plant matrices. Combined with metabolomic profiling and CPC-based bioassay-guided fractionation, it enabled the identification of alkaloid-rich fractions of B. julianae with pronounced anti-AChE activity. These findings support the applicability of the developed workflow for natural-product-based drug discovery targeting neurodegenerative disorders.