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.

中文翻译：

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同时基于弹性蛋白酶、透明质酸酶和胶原酶毛细管电泳的测定。应用评价红藻 Jania rubens 的生物活性

为了从天然来源中寻找负担得起且高效的化妆品成分，并使用对生态负责的工具评估其生物活性，已经做出了许多努力。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，即透明质酸、弹性蛋白和胶原蛋白。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，而无需固定反应物或使用放射性。开发的方法用于评估微波或电化学辅助提取后红藻 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。CE具有双检测系统，LIF和HRMS串联，用于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 种酶活性。这些条件包括酶（用藻类提取物）在 37°C 下预孵育 10 分钟；在 37°C 下与底物孵育 10 分钟，在 90°C 下停止时间为 10 分钟。使用短端注射将 1.4 nL 的每种反应混合物共注射到 85 cm 总长的毛细管中。醋酸铵（50 mM，pH 9.0）用于电泳分离。在不到 10 分钟的时间内同时检测到所有底物和产物，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) 和出色的 LOQ (< 5 纳米）。使用这种基于 CE 的多重酶促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 具有显着影响。