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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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Τίτλος: Development of a thin-layer chromatography-angiotensin-converting enzyme bioassay coupled with digital image analysis for effect-directed screening of enzyme inhibitors.
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
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  – Url: https://www.doi.org/10.1016/j.chroma.2026.467324?
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  Data: 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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  Data: <searchLink fieldCode="AU" term="%22Obregón-Berg+C%22">Obregón-Berg C</searchLink>; Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile.<br /><searchLink fieldCode="AU" term="%22Galarce-Bustos+O%22">Galarce-Bustos O</searchLink>; Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile. Electronic address: oscargalarce@udec.cl.<br /><searchLink fieldCode="AU" term="%22Fernández+B%22">Fernández B</searchLink>; Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile.<br /><searchLink fieldCode="AU" term="%22Gutiérrez+A%22">Gutiérrez A</searchLink>; Laboratorio de Farmacognosia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile.<br /><searchLink fieldCode="AU" term="%22Pavón-Pérez+J%22">Pavón-Pérez J</searchLink>; 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.
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  Data: <searchLink fieldCode="JN" term="%229318488%22">Journal of chromatography. A</searchLink> [J Chromatogr A] 2026 Sep 27; Vol. 1785, pp. 467324. <i>Date of Electronic Publication: </i>2026 Aug 02.
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  Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22Elsevier%22">Elsevier </searchLink><i>Country of Publication: </i>Netherlands <i>NLM ID: </i>9318488 <i>Publication Model: </i>Print-Electronic <i>Cited Medium: </i>Internet <i>ISSN: </i>1873-3778 (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%2200219673%22">00219673 </searchLink><i>NLM ISO Abbreviation: </i>J Chromatogr A <i>Subsets: </i>MEDLINE
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  Data: <i>Original Publication</i>: Amsterdam ; New York : Elsevier, 1993-
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  Data: <searchLink fieldCode="MM" term="%22Angiotensin-Converting+Enzyme+Inhibitors%22">Angiotensin-Converting Enzyme Inhibitors*</searchLink>/<searchLink fieldCode="MM" term="%22Angiotensin-Converting+Enzyme+Inhibitors+analysis%22">analysis</searchLink> <br /><searchLink fieldCode="MM" term="%22Angiotensin-Converting+Enzyme+Inhibitors%22">Angiotensin-Converting Enzyme Inhibitors*</searchLink>/<searchLink fieldCode="MM" term="%22Angiotensin-Converting+Enzyme+Inhibitors+pharmacology%22">pharmacology</searchLink> <br /><searchLink fieldCode="MM" term="%22Angiotensin-Converting+Enzyme+Inhibitors%22">Angiotensin-Converting Enzyme Inhibitors*</searchLink>/<searchLink fieldCode="MM" term="%22Angiotensin-Converting+Enzyme+Inhibitors+chemistry%22">chemistry</searchLink> <br /><searchLink fieldCode="MM" term="%22Image+Processing%2C+Computer-Assisted%22">Image Processing, Computer-Assisted*</searchLink>/<searchLink fieldCode="MM" term="%22Image+Processing%2C+Computer-Assisted+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Biological+Assay%22">Biological Assay*</searchLink>/<searchLink fieldCode="MM" term="%22Biological+Assay+methods%22">methods</searchLink> <br /><searchLink fieldCode="MM" term="%22Peptidyl-Dipeptidase+A%22">Peptidyl-Dipeptidase A*</searchLink>/<searchLink fieldCode="MM" term="%22Peptidyl-Dipeptidase+A+metabolism%22">metabolism</searchLink><br /><searchLink fieldCode="MH" term="%22Chromatography%2C+Thin+Layer%22">Chromatography, Thin Layer</searchLink>/<searchLink fieldCode="MH" term="%22Chromatography%2C+Thin+Layer+methods%22">methods</searchLink> ; <searchLink fieldCode="MH" term="%22Aporphines%22">Aporphines</searchLink>/<searchLink fieldCode="MH" term="%22Aporphines+analysis%22">analysis</searchLink> ; <searchLink fieldCode="MH" term="%22Aporphines%22">Aporphines</searchLink>/<searchLink fieldCode="MH" term="%22Aporphines+chemistry%22">chemistry</searchLink> ; <searchLink fieldCode="MH" term="%22Hippurates%22">Hippurates</searchLink>/<searchLink fieldCode="MH" term="%22Hippurates+chemistry%22">chemistry</searchLink> ; <searchLink fieldCode="MH" term="%22Hippurates%22">Hippurates</searchLink>/<searchLink fieldCode="MH" term="%22Hippurates+metabolism%22">metabolism</searchLink> ; <searchLink fieldCode="MH" term="%22Reproducibility+of+Results%22">Reproducibility of Results</searchLink> ; <searchLink fieldCode="MH" term="%22Oligopeptides%22">Oligopeptides</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: 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 (R<superscript>2</superscript> = 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<superscript>-1</superscript> 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]<superscript>+</superscript>), boldine (m/z 328.10 [M+H]<superscript>+</superscript>), and laurotetanine (m/z 328.10 ([M+H]<superscript>+</superscript>). 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.<br /> (Copyright © 2026 Elsevier B.V. All rights reserved.)
– Name: Abstract
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  Data: 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.
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        StartPage: 467324
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      – TitleFull: 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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