A simple thin-layer chromatography autography for the detection of peroxidase inhibitors
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...
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| Format: | info:ar-repo/semantics/artículo |
| Language: | Inglés |
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Springer Nature
2024
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| Online Access: | 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 |
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| author | Micheloni, Oscar Bernardo Ramallo, Ivana Ayelen Farroni, Abel Eduardo Furlan, Ricardo Luis Eugenio |
| author_browse | Farroni, Abel Eduardo Furlan, Ricardo Luis Eugenio Micheloni, Oscar Bernardo Ramallo, Ivana Ayelen |
| author_facet | Micheloni, Oscar Bernardo Ramallo, Ivana Ayelen Farroni, Abel Eduardo Furlan, Ricardo Luis Eugenio |
| author_sort | Micheloni, Oscar Bernardo |
| collection | INTA Digital |
| 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. |
| format | info:ar-repo/semantics/artículo |
| id | INTA16927 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2024 |
| publishDateRange | 2024 |
| publishDateSort | 2024 |
| publisher | Springer Nature |
| publisherStr | Springer Nature |
| record_format | dspace |
| spelling | INTA169272024-03-05T18:45:16Z A simple thin-layer chromatography autography for the detection of peroxidase inhibitors Micheloni, Oscar Bernardo Ramallo, Ivana Ayelen Farroni, Abel Eduardo Furlan, Ricardo Luis Eugenio Peroxídasas Inhibidores de Enzimas Antioxidantes Cromatografía Peroxidases Enzyme Inhibitors Antioxidants Chromatography Bioautography Browning 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 2024-03-05T18:37:45Z 2024-03-05T18:37:45Z 2024-02 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 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 eng info:eu-repograntAgreement/INTA/2019-PD-E7-I152-001, Alimentos nutracéuticos, funcionales o para regímenes especiales 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) application/pdf Springer Nature Journal of Food Science and Technology : 1-11. (March 2024). |
| spellingShingle | Peroxídasas Inhibidores de Enzimas Antioxidantes Cromatografía Peroxidases Enzyme Inhibitors Antioxidants Chromatography Bioautography Browning Micheloni, Oscar Bernardo Ramallo, Ivana Ayelen Farroni, Abel Eduardo Furlan, Ricardo Luis Eugenio 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 |
| 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_short | A simple thin-layer chromatography autography for the detection of peroxidase inhibitors |
| title_sort | simple thin layer chromatography autography for the detection of peroxidase inhibitors |
| topic | Peroxídasas Inhibidores de Enzimas Antioxidantes Cromatografía Peroxidases Enzyme Inhibitors Antioxidants Chromatography Bioautography Browning |
| 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 |
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