Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
Faculty of Science II, Department of Biology, Lebanese University, 90656, Jdeidet El Metn, Fanar, Lebanon.
Anal Chim Acta. 2018 Aug 22;1020:134-141. doi: 10.1016/j.aca.2018.03.004. Epub 2018 Mar 19.
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 °C; 10 min incubation with the substrate at 37 °C and 10 min stop-time at 90 °C. 1.4 nL of each reaction mixture were co-injected into a 85 cm total length capillary using short-end injection. Ammonium acetate (50 mM, 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.
已经有许多努力从天然来源中寻找经济实惠且高效的化妆品成分，并使用生态负责的工具来评估它们的生物活性。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成分透明质酸、弹性蛋白和胶原蛋白。本工作旨在开发一种单一的毛细管电泳方法，无需反应物固定化或放射性标记，即可同时监测这三种酶的活性。该方法用于监测微波或电化学辅助提取后的红色海藻 Jania rubens 的生物活性。为此，仔细选择了孵育时间、反应物浓度、分离缓冲液和检测系统。采用双检测系统的 CE、LIF 和串联的 HRMS 用于确保同时分析三种酶反应的底物和产物。优化的酶促条件允许使用相同的方案来评估三种酶的活性。这些条件包括在 37°C 下对酶（与海藻提取物）进行 10 分钟预孵育；在 37°C 下孵育 10 分钟，然后在 90°C 下停止 10 分钟。将每种反应混合物的 1.4nL 通过短端进样共注入 85cm 总长度的毛细管中。使用 50mM 乙酸铵（pH 9.0）进行电泳分离。所有底物和产物在不到 10 分钟内同时检测到，具有良好的峰对称性和效率，足够的日内和日间重现性（RSD<4.5%；n=3）和优异的 LOQ（<5nM）。使用这种基于多重 CE 的酶法测定获得的结果表明，Jania rubens 乙醇提取物对弹性蛋白酶、透明质酸酶和金属蛋白酶 MMP-1 具有显著影响。
