Simultaneous elastase-, hyaluronidase- and collagenase-capillary electrophoresis based assay. Application to evaluate the bioactivity of the red alga Jania rubens
Simultaneous elastase-, hyaluronidase- and collagenase-capillary electrophoresis based assay. Application to evaluate the bioactivity of the red alga Jania rubens
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DOI：
10.1016/j.aca.2018.03.004
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发表时间：
2018-08-22
影响因子：
6.2
通讯作者：
Nehme, Reine
中科院分区：
文献类型：
--
作者：
Fayad, Syntia;Tannoury, Mona;Nehme, Reine
人们已经做出了许多努力，从天然来源寻找负担得起且有效的化妆品成分，并使用对生态负责的工具评估其生物活性。透明质酸酶、弹性蛋白酶和胶原酶分别负责降解细胞外基质的主要组分，即透明质酸、弹性蛋白和胶原。这项工作的目的是开发一种单一的毛细管电泳方法来同时监测这三种酶的活性，而无需反应物固定或使用放射性。所开发的方法被用来评估后的微波或电化学辅助提取的红色金鱼藻的生物活性。为此，孵育时间，反应物浓度，分离缓冲液和检测系统进行了仔细选择。采用LIF和HRMS串联双检测系统的CE，确保同时分析三种酶促反应的底物和产物。优化的酶促条件允许使用相同的方案来评估3种酶的活性。这些条件包括在37 ° C下酶（与甘草提取物）预孵育10分钟;在37 ° C下与底物孵育10分钟和在90 ° C下停止10分钟。1.4使用短端注射将nL的每种反应混合物共注射到85 cm总长度的毛细管中。使用乙酸铵（50 mM，pH 9.0）进行电泳分离。所有底物和产物在不到10分钟内同时检测，具有良好的峰对称性和效率，足够的日内和日间重复性（RSD < 4.5%; n = 3）和优异的LOQ（< 5 nM）。使用这种基于多重CE的酶测定法获得的结果显示了红坚木乙醇提取物对弹性蛋白酶、透明质酸酶和金属蛋白酶MMP-1的显著影响。(C)2018爱思唯尔B. V.保留所有权利。
There have been many efforts to search for affordable and efficient cosmetic ingredients from natural sources and to evaluate their bioactivities using eco-responsible tools. Hyaluronidase, elastase and collagenase are responsible for the degradation of the main components of the extracellular matrix, namely the hyaluronic acid, elastin and collagen, respectively. The aim of this work was to develop a single capillary electrophoresis method to monitor simultaneously the activities of these three enzymes, without reactant immobilization or radioactivity use. The developed approach was used to evaluate the bioactivity of the red alga Jania rubens after microwave- or electrochemical-assisted extraction. For this purpose, the incubation time, the reactant concentrations, the separation buffer and the detection system were carefully chosen. CE with double detection system, LIF and HRMS connected in series, was used to ensure the simultaneous analysis of the substrates and products of the three enzymatic reactions. The optimized enzymatic conditions allowed the use of the same protocol to assess the 3 enzyme activities. These conditions consisted of 10 min pre-incubation of the enzyme (with alga extract) at 37 degrees C; 10 min incubation with the substrate at 37 degrees C and 10 min stop-time at 90 degrees C. 1.4 nL of each reaction mixture were co-injected into a 85 cm total length capillary using short-end injection. Ammonium acetate (50mM, pH 9.0) was used for electrophoretic separation. All substrates and products were simultaneously detected in less than 10 min with good peak symmetry and efficiency, sufficient intra-day and inter-day repeatabilities (RSD < 4.5%; n = 3) and excellent LOQ (< 5 nM). The results obtained using this multiple CE-based enzymatic assay showed the significant effect of Jania rubens ethanolic extracts on elastase, hyaluronidase and the metalloproteinase MMP-1. (C) 2018 Elsevier B.V. All rights reserved.