Micheloni Oscar Bernardo, Ramallo Ivana Ayelen, Farroni Abel Eduardo, Furlan Ricardo Luis Eugenio
Escuela de Ciencias Agrarias, Naturales y Ambientales, Universidad Nacional del Noroeste de la Provincia de Buenos Aires, Pergamino, Buenos Aires Argentina.
Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Suipacha 531, 2000 Rosario, Argentina.
J Food Sci Technol. 2024 Sep;61(9):1722-1732. doi: 10.1007/s13197-024-05946-w. Epub 2024 Mar 2.
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/cm, 0.239 µmoles/cm, and 84.04 U/cm) 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 Meyen extract and a peroxidase inhibitor in a 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.
The online version contains supplementary material available at 10.1007/s13197-024-05946-w.
薄层色谱生物自显影分析有助于获取活性图谱色谱，并通过观察复杂混合物中产生的抑制晕圈来协助确定其中的活性成分。过氧化物酶是一种与不同鲜切蔬菜褐变及多种疾病相关的酶。基于酶的琼脂糖固定化和2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)二铵盐/自由基阳离子(ABTS/ABTS)报告系统，开发了一种过氧化物酶生物自显影分析方法。从土豆中纯化过氧化物酶以检测特定抑制剂。为减少假阳性，还采用了非酶分析方法。当将含有琼脂糖、ABTS、过氧化氢和过氧化物酶的磷酸盐缓冲溶液倒在薄层色谱板上(终浓度：0.031毫摩尔/厘米、0.239微摩尔/厘米和84.04单位/厘米)并孵育70分钟时，可获得最佳结果。槲皮素的检测限和定量限分别为0.16微克和0.54微克。所开发的系统能够检测Meyen提取物中的槲皮素和L.提取物中的过氧化物酶抑制剂。因此，该分析方法能够在任何制备性分级分离之前检测复杂混合物中的抑制成分，并区分过氧化物酶抑制剂和ABTS自由基清除剂，有助于尽早做出可节省时间和资源的操作决策。
在线版本包含可在10.1007/s13197-024-05946-w获取的补充材料。
