A simple thin-layer chromatography autography for the detection of peroxidase inhibitors

Autores
Micheloni, Oscar Bernardo; Ramallo, Ivana Ayelen; Farroni, Abel Eduardo; Furlan, Ricardo Luis Eugenio
Año de publicación
2024
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Thin layer chromatography bioautographic assays facilitate the acquisition of activity-profile chromatograms and assist in pinpointing active constituents within complex mixtures by observing the inhibition halos they produce. Peroxidase is an enzyme implicated in the browning of different fresh cut vegetables and in several diseases. A peroxidase bioautographic assay was developed, based on enzyme agarose immobilization and the 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/radical cation (ABTS/ABTS·+) reporter system. Peroxidase was purified from potatoes with the aim to detect specific inhibitors. To reduce false positives, a non-enzymatic assay was also employed. The best results are obtained when a solution containing agarose, ABTS, hydrogen peroxide, and peroxidase in phosphate buffer is poured over the TLC plate (final concentrations: 0.031 mmoles/cm2, 0.239 µmoles/cm2, and 84.04 U/cm2) and incubated for 70 min. Limit of detection and quantification for quercetin is 0.16 µg and 0.54 µg, respectively. The developed system is able to detect quercetin in a Solidago chilensis Meyen extract and a peroxidase inhibitor in a Cichorium intybus L. extract. Therefore, the assay can detect inhibitory constituents in complex mixtures and differentiate between peroxidase inhibitors and ABTS·+ radical scavengers before any preparative fractionation, helping to take early operational decisions that can save time and resources.
EEA Pergamino
Fil: Micheloni, O.B. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Escuela de Ciencias Agrarias, Naturales y Ambientales. Departamento de Ciencias Básicas; Argentina
Fil: Ramallo, I.A. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Farmacognosia; Argentina
Fil: Ramallo, I.A. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Farroni, Abel Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Laboratorio Calidad de Alimento, Suelo y Agua; Argentina
Fil: Farroni, Abel Eduardo. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Escuela de Ciencias Agrarias, Naturales y ambientales; Argentina
Fil: Furlan, Ricardo Luis Eugenio. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Farmacognosia; Argentina
Fil: Furlan, Ricardo Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fuente
Journal of Food Science and Technology : 1-11. (March 2024).
Materia
Peroxídasas
Inhibidores de Enzimas
Antioxidantes
Cromatografía
Peroxidases
Enzyme Inhibitors
Antioxidants
Chromatography
Bioautography
Browning
Nivel de accesibilidad
acceso restringido
Condiciones de uso
http://creativecommons.org/licenses/by-nc-sa/4.0/
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/16927
Acceder
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oai_identifier_str oai:localhost:20.500.12123/16927
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network_name_str INTA Digital (INTA)
spelling A simple thin-layer chromatography autography for the detection of peroxidase inhibitorsMicheloni, Oscar BernardoRamallo, Ivana AyelenFarroni, Abel EduardoFurlan, Ricardo Luis EugenioPeroxídasasInhibidores de EnzimasAntioxidantesCromatografíaPeroxidasesEnzyme InhibitorsAntioxidantsChromatographyBioautographyBrowningThin layer chromatography bioautographic assays facilitate the acquisition of activity-profile chromatograms and assist in pinpointing active constituents within complex mixtures by observing the inhibition halos they produce. Peroxidase is an enzyme implicated in the browning of different fresh cut vegetables and in several diseases. A peroxidase bioautographic assay was developed, based on enzyme agarose immobilization and the 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/radical cation (ABTS/ABTS·+) reporter system. Peroxidase was purified from potatoes with the aim to detect specific inhibitors. To reduce false positives, a non-enzymatic assay was also employed. The best results are obtained when a solution containing agarose, ABTS, hydrogen peroxide, and peroxidase in phosphate buffer is poured over the TLC plate (final concentrations: 0.031 mmoles/cm2, 0.239 µmoles/cm2, and 84.04 U/cm2) and incubated for 70 min. Limit of detection and quantification for quercetin is 0.16 µg and 0.54 µg, respectively. The developed system is able to detect quercetin in a Solidago chilensis Meyen extract and a peroxidase inhibitor in a Cichorium intybus L. extract. Therefore, the assay can detect inhibitory constituents in complex mixtures and differentiate between peroxidase inhibitors and ABTS·+ radical scavengers before any preparative fractionation, helping to take early operational decisions that can save time and resources.EEA PergaminoFil: Micheloni, O.B. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Escuela de Ciencias Agrarias, Naturales y Ambientales. Departamento de Ciencias Básicas; ArgentinaFil: Ramallo, I.A. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Farmacognosia; ArgentinaFil: Ramallo, I.A. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Farroni, Abel Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Laboratorio Calidad de Alimento, Suelo y Agua; ArgentinaFil: Farroni, Abel Eduardo. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Escuela de Ciencias Agrarias, Naturales y ambientales; ArgentinaFil: Furlan, Ricardo Luis Eugenio. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Farmacognosia; ArgentinaFil: Furlan, Ricardo Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaSpringer Nature2024-03-05T18:37:45Z2024-03-05T18:37:45Z2024-02info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/16927https://link.springer.com/article/10.1007/s13197-024-05946-w0022-11550975-8402 (online)https://doi.org/10.1007/s13197-024-05946-wJournal of Food Science and Technology : 1-11. (March 2024).reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repograntAgreement/INTA/2019-PD-E7-I152-001, Alimentos nutracéuticos, funcionales o para regímenes especialesinfo:eu-repo/semantics/restrictedAccesshttp://creativecommons.org/licenses/by-nc-sa/4.0/Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)2025-09-04T09:50:15Zoai:localhost:20.500.12123/16927instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-04 09:50:15.5INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
title A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
spellingShingle A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
Micheloni, Oscar Bernardo
Peroxídasas
Inhibidores de Enzimas
Antioxidantes
Cromatografía
Peroxidases
Enzyme Inhibitors
Antioxidants
Chromatography
Bioautography
Browning
title_short A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
title_full A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
title_fullStr A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
title_full_unstemmed A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
title_sort A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
dc.creator.none.fl_str_mv Micheloni, Oscar Bernardo
Ramallo, Ivana Ayelen
Farroni, Abel Eduardo
Furlan, Ricardo Luis Eugenio
author Micheloni, Oscar Bernardo
author_facet Micheloni, Oscar Bernardo
Ramallo, Ivana Ayelen
Farroni, Abel Eduardo
Furlan, Ricardo Luis Eugenio
author_role author
author2 Ramallo, Ivana Ayelen
Farroni, Abel Eduardo
Furlan, Ricardo Luis Eugenio
author2_role author
author
author
dc.subject.none.fl_str_mv Peroxídasas
Inhibidores de Enzimas
Antioxidantes
Cromatografía
Peroxidases
Enzyme Inhibitors
Antioxidants
Chromatography
Bioautography
Browning
topic Peroxídasas
Inhibidores de Enzimas
Antioxidantes
Cromatografía
Peroxidases
Enzyme Inhibitors
Antioxidants
Chromatography
Bioautography
Browning
dc.description.none.fl_txt_mv Thin layer chromatography bioautographic assays facilitate the acquisition of activity-profile chromatograms and assist in pinpointing active constituents within complex mixtures by observing the inhibition halos they produce. Peroxidase is an enzyme implicated in the browning of different fresh cut vegetables and in several diseases. A peroxidase bioautographic assay was developed, based on enzyme agarose immobilization and the 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/radical cation (ABTS/ABTS·+) reporter system. Peroxidase was purified from potatoes with the aim to detect specific inhibitors. To reduce false positives, a non-enzymatic assay was also employed. The best results are obtained when a solution containing agarose, ABTS, hydrogen peroxide, and peroxidase in phosphate buffer is poured over the TLC plate (final concentrations: 0.031 mmoles/cm2, 0.239 µmoles/cm2, and 84.04 U/cm2) and incubated for 70 min. Limit of detection and quantification for quercetin is 0.16 µg and 0.54 µg, respectively. The developed system is able to detect quercetin in a Solidago chilensis Meyen extract and a peroxidase inhibitor in a Cichorium intybus L. extract. Therefore, the assay can detect inhibitory constituents in complex mixtures and differentiate between peroxidase inhibitors and ABTS·+ radical scavengers before any preparative fractionation, helping to take early operational decisions that can save time and resources.
EEA Pergamino
Fil: Micheloni, O.B. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Escuela de Ciencias Agrarias, Naturales y Ambientales. Departamento de Ciencias Básicas; Argentina
Fil: Ramallo, I.A. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Farmacognosia; Argentina
Fil: Ramallo, I.A. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Farroni, Abel Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Laboratorio Calidad de Alimento, Suelo y Agua; Argentina
Fil: Farroni, Abel Eduardo. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Escuela de Ciencias Agrarias, Naturales y ambientales; Argentina
Fil: Furlan, Ricardo Luis Eugenio. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Farmacognosia; Argentina
Fil: Furlan, Ricardo Luis Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Thin layer chromatography bioautographic assays facilitate the acquisition of activity-profile chromatograms and assist in pinpointing active constituents within complex mixtures by observing the inhibition halos they produce. Peroxidase is an enzyme implicated in the browning of different fresh cut vegetables and in several diseases. A peroxidase bioautographic assay was developed, based on enzyme agarose immobilization and the 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/radical cation (ABTS/ABTS·+) reporter system. Peroxidase was purified from potatoes with the aim to detect specific inhibitors. To reduce false positives, a non-enzymatic assay was also employed. The best results are obtained when a solution containing agarose, ABTS, hydrogen peroxide, and peroxidase in phosphate buffer is poured over the TLC plate (final concentrations: 0.031 mmoles/cm2, 0.239 µmoles/cm2, and 84.04 U/cm2) and incubated for 70 min. Limit of detection and quantification for quercetin is 0.16 µg and 0.54 µg, respectively. The developed system is able to detect quercetin in a Solidago chilensis Meyen extract and a peroxidase inhibitor in a Cichorium intybus L. extract. Therefore, the assay can detect inhibitory constituents in complex mixtures and differentiate between peroxidase inhibitors and ABTS·+ radical scavengers before any preparative fractionation, helping to take early operational decisions that can save time and resources.
publishDate 2024
dc.date.none.fl_str_mv 2024-03-05T18:37:45Z
2024-03-05T18:37:45Z
2024-02
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/16927
https://link.springer.com/article/10.1007/s13197-024-05946-w
0022-1155
0975-8402 (online)
https://doi.org/10.1007/s13197-024-05946-w
url http://hdl.handle.net/20.500.12123/16927
https://link.springer.com/article/10.1007/s13197-024-05946-w
https://doi.org/10.1007/s13197-024-05946-w
identifier_str_mv 0022-1155
0975-8402 (online)
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repograntAgreement/INTA/2019-PD-E7-I152-001, Alimentos nutracéuticos, funcionales o para regímenes especiales
dc.rights.none.fl_str_mv info:eu-repo/semantics/restrictedAccess
http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
eu_rights_str_mv restrictedAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-sa/4.0/
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0)
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer Nature
publisher.none.fl_str_mv Springer Nature
dc.source.none.fl_str_mv Journal of Food Science and Technology : 1-11. (March 2024).
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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score 12.623145

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