Simultaneous elastase-, hyaluronidase- and collagenase-capillary electrophoresis based assay. Application to evaluate the bioactivity of the red alga Jania rubens

Abstract

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.

Keywords: Algae; Capillary electrophoresis; Electroporation; Enzyme; Laser-induced fluorescence; Mass spectrometry.