Rinaldi Francesca, Salerno Sofia, Frigoli Elena, De Soricellis Giulia, Brusotti Gloria, Negri Stefano, Radice Matteo, Merlo Francesca, Speltini Andrea, Cena Hellas, Calleri Enrica
Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy.
National Biodiversity Future Center (NBFC), 90133 Palermo, Italy.
Metabolites. 2025 Apr 18;15(4):283. doi: 10.3390/metabo15040283.
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. 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. A reference inhibitor, neostigmine, was used for system validation through IC and K 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. 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.
乙酰胆碱酯酶（AChE）的酶活性一直是神经退行性疾病研究的焦点，特别是与阿尔茨海默病相关的研究。这归因于与健康个体相比，阿尔茨海默病患者中观察到的胆碱能神经元水平显著降低。减轻患者这些疾病发作的策略在于探索新的潜在AChE抑制剂，同时也关注天然提取物。开发了一种具有直接紫外检测功能的快速、特异性毛细管电泳方法（CZE-UV/Vis），通过评估天然提取物抑制AChE的潜力来筛选它们。为了在分析天然提取物等复杂基质时提高特异性，采用基于“三明治模型”的顺序分析方法，使用Ellman试剂[5,5'-二硫代双-(2-硝基苯甲酸)]（DTNB）作为比色指示剂。通过在存在浓度不断增加的新斯的明的情况下，随后注射乙酰硫代胆碱底物，并将酶与DTNB在硼酸盐-磷酸盐缓冲液（30 mM，pH 8.0）中结合，测定IC和K值，使用参考抑制剂新斯的明对系统进行验证。在412 nm处选择性检测酶促产物。经验证的系统应用于七种天然提取物的分析。结果表明，在鉴定具有预防神经退行性疾病潜在应用的植物治疗剂方面取得了有希望的成果。该方法具有高选择性和自动化，为筛选含有潜在AChE抑制剂的天然基质提供了一种简化且有效的方法。
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