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 p...
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2025-04-01
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| author | Francesca Rinaldi Sofia Salerno Elena Frigoli Giulia De Soricellis Gloria Brusotti Stefano Negri Matteo Radice Francesca Merlo Andrea Speltini Hellas Cena Enrica Calleri |
| author_facet | Francesca Rinaldi Sofia Salerno Elena Frigoli Giulia De Soricellis Gloria Brusotti Stefano Negri Matteo Radice Francesca Merlo Andrea Speltini Hellas Cena Enrica Calleri |
| author_sort | Francesca Rinaldi |
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| description | <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 IC<sub>50</sub> and K<sub>i</sub> 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. |
| format | Article |
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| institution | OA Journals |
| issn | 2218-1989 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | MDPI AG |
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| series | Metabolites |
| spelling | doaj-art-c44f40e203af44a18a744a6fc446471f2025-08-20T02:18:19ZengMDPI AGMetabolites2218-19892025-04-0115428310.3390/metabo15040283Development and Application of an In-Capillary CE-DAD Method for the Inhibitory Screening of Natural Extracts Towards Acetylcholinesterase EnzymeFrancesca Rinaldi0Sofia Salerno1Elena Frigoli2Giulia De Soricellis3Gloria Brusotti4Stefano Negri5Matteo Radice6Francesca Merlo7Andrea Speltini8Hellas Cena9Enrica Calleri10Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, ItalyDepartment of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, ItalyDepartment of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, ItalyDepartment of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, ItalyDepartment of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, ItalyNational Biodiversity Future Center (NBFC), 90133 Palermo, ItalyCampus Puyo, Universidad Estatal Amazónica, Km 2, Via Puyo-Tena, Puyo 160150, EcuadorDepartment of Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, ItalyDepartment of Chemistry, University of Pavia, Via Taramelli 12, 27100 Pavia, ItalyNational Biodiversity Future Center (NBFC), 90133 Palermo, ItalyDepartment of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy<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 IC<sub>50</sub> and K<sub>i</sub> 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.https://www.mdpi.com/2218-1989/15/4/283Alzheimer’s diseaseplant extractscapillary electrophoresis |
| spellingShingle | Francesca Rinaldi Sofia Salerno Elena Frigoli Giulia De Soricellis Gloria Brusotti Stefano Negri Matteo Radice Francesca Merlo Andrea Speltini Hellas Cena Enrica Calleri Development and Application of an In-Capillary CE-DAD Method for the Inhibitory Screening of Natural Extracts Towards Acetylcholinesterase Enzyme Metabolites Alzheimer’s disease plant extracts capillary electrophoresis |
| title | Development and Application of an In-Capillary CE-DAD Method for the Inhibitory Screening of Natural Extracts Towards Acetylcholinesterase Enzyme |
| title_full | Development and Application of an In-Capillary CE-DAD Method for the Inhibitory Screening of Natural Extracts Towards Acetylcholinesterase Enzyme |
| title_fullStr | Development and Application of an In-Capillary CE-DAD Method for the Inhibitory Screening of Natural Extracts Towards Acetylcholinesterase Enzyme |
| title_full_unstemmed | Development and Application of an In-Capillary CE-DAD Method for the Inhibitory Screening of Natural Extracts Towards Acetylcholinesterase Enzyme |
| title_short | Development and Application of an In-Capillary CE-DAD Method for the Inhibitory Screening of Natural Extracts Towards Acetylcholinesterase Enzyme |
| title_sort | development and application of an in capillary ce dad method for the inhibitory screening of natural extracts towards acetylcholinesterase enzyme |
| topic | Alzheimer’s disease plant extracts capillary electrophoresis |
| url | https://www.mdpi.com/2218-1989/15/4/283 |
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