Development and Application of an In-Capillary CE-DAD Method for the Inhibitory Screening of Natural Extracts Towards Acetylcholinesterase Enzyme

<b>Background:</b> The enzymatic activity of acetylcholinesterase (AChE) has been a focal point in neurodegenerative diseases research, particularly in relation to Alzheimer's disease. This is attributed to the significantly reduced levels of cholinergic neurons observed in Alzheimer's patients compared to healthy individuals. The strategy to mitigate the onset of these diseases in patients lies in the exploration of new potential AChE inhibitors with a focus also on natural extracts. A rapid and specific capillary electrophoresis method with direct ultraviolet detection (CZE-UV/Vis) was developed to screen natural extracts by assessing their potential to inhibit AChE. <b>Materials and Methods:</b> To enhance the specificity when analysing complex matrixes such as natural extracts, a sequential analysis approach based on the "sandwich model" was implemented using Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] (DTNB) as a colorimetric indicator. <b>Results:</b> A reference inhibitor, neostigmine, was used for system validation through IC₅₀ and K_i values determination by subsequent injections of acetylthiocholine substrate in the presence of neostigmine at increasing concentrations, and the enzyme combined with DTNB in borate-phosphate buffer (30 mM, pH 8.0). The enzymatic product was selectively detected at 412 nm. The validated system was applied to the analysis of seven natural extracts. <b>Conclusions:</b> Results demonstrated promising outcomes for identifying phytotherapeutic agents with potential applications in the prevention of neurodegenerative diseases. This method provides high selectivity and automation, offering a streamlined and effective approach for screening natural matrices containing potential AChE inhibitors.