Academic Journal
Development of a thin-layer chromatography-angiotensin-converting enzyme bioassay coupled with digital image analysis for effect-directed screening of enzyme inhibitors.
| Τίτλος: | Development of a thin-layer chromatography-angiotensin-converting enzyme bioassay coupled with digital image analysis for effect-directed screening of enzyme inhibitors. |
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| Συγγραφείς: | Obregón-Berg C; Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile., Galarce-Bustos O; Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile. Electronic address: oscargalarce@udec.cl., Fernández B; Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile., Gutiérrez A; Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile., Pavón-Pérez J; Departamento de Ciencia y Tecnología de los Alimentos, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile; Grupo Interdisciplinario de Biotecnología Marina (GIBMAR), Centro de Biotecnología, Universidad de Concepción, Concepción, Chile. |
| Πηγή: | Journal of chromatography. A [J Chromatogr A] 2026 Sep 27; Vol. 1785, pp. 467324. Date of Electronic Publication: 2026 Aug 02. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Elsevier Country of Publication: Netherlands NLM ID: 9318488 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-3778 (Electronic) Linking ISSN: 00219673 NLM ISO Abbreviation: J Chromatogr A Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Amsterdam ; New York : Elsevier, 1993- |
| Ιατρικοί όροι (MeSH): | Angiotensin-Converting Enzyme Inhibitors*/analysis , Angiotensin-Converting Enzyme Inhibitors*/pharmacology , Angiotensin-Converting Enzyme Inhibitors*/chemistry , Image Processing, Computer-Assisted*/methods , Biological Assay*/methods , Peptidyl-Dipeptidase A*/metabolism, Chromatography, Thin Layer/methods ; Aporphines/analysis ; Aporphines/chemistry ; Hippurates/chemistry ; Hippurates/metabolism ; Reproducibility of Results ; Oligopeptides |
| Περίληψη: | A novel thin-layer chromatography (TLC)-based bioassay coupled with digital image analysis (DIA) was developed for rapid and non-targeted screening of angiotensin-converting enzyme (ACE) inhibitors in complex natural matrices. Conventional ACE inhibitory assays lack spatial resolution and rely on indirect measurements, limiting their applicability for effect-directed analysis (EDA). In this study, an on-surface enzymatic assay was implemented using hippuryl-histidyl-leucine (HHL) as substrate, followed by in situ derivatization of the generated hippuric acid with p-dimethylaminobenzaldehyde (DMAB) in an acetone:acetic anhydride system, producing an orange chromophore directly on the TLC plate. To overcome the inherent subjectivity of visual evaluation, a MATLAB-based DIA workflow was introduced to quantify the colorimetric response as pseudo-absorbance, enabling objective optimization of assay parameters. The blue channel provided the highest analytical sensitivity (R2 = 0.9984), and no significant matrix effects were observed (p = 0.09), confirming the robustness of the approach. Optimal conditions were established at 180 mU mL-1 ACE, 3 mM HHL, 30 min incubation, and 10% (w/v) DMAB. The method was applied to P. boldus alkaloidal extracts, enabling clear detection and localization of inhibitory zones poorly resolved by visual inspection. Subsequent isolation by micro-preparative TLC followed by UHPLC-DAD-MS/MS analysis enabled tentative identification of three aporphine alkaloids: norboldine (m/z 314.20 [M+H]+), boldine (m/z 328.10 [M+H]+), and laurotetanine (m/z 328.10 ([M+H]+). This integrated TLC-ACE-DIA platform, complemented by GNPS-assisted MS/MS annotation, provides a rapid, cost-effective, high-throughput strategy for discovery and prioritization of ACE inhibitors in natural products, significantly enhancing analytical capabilities of effect-directed analysis workflows. (Copyright © 2026 Elsevier B.V. All rights reserved.) |
| Competing Interests: | Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. |
| Contributed Indexing: | Keywords: Aporphine alkaloids; Colorimetric signal processing; Effect-directed analysis (EDA); MS/MS-based annotation; Planar bioassay ACE; Pseudo-absorbance |
| Substance Nomenclature: | 0 (Angiotensin-Converting Enzyme Inhibitors) 31373-65-6 (hippuryl-histidyl-leucine) EC 3.4.15.1 (Peptidyl-Dipeptidase A) TE0865N2ET (hippuric acid) 0 (Aporphines) 0 (Hippurates) 0 (Oligopeptides) |
| Entry Date(s): | Date Created: 20260807 Date Completed: 20260812 Latest Revision: 20260812 |
| Update Code: | 20260813 |
| DOI: | 10.1016/j.chroma.2026.467324 |
| PMID: | 42567021 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1873-3778 |
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| DOI: | 10.1016/j.chroma.2026.467324 |