Macroalga Padina pavonica water extracts obtained by pressurized liquid extraction and microwave-assisted extraction inhibit hyaluronidase activity as shown by capillary electrophoresis

Abstract

Hyaluronidase degrades hyaluronic acid, the principal component of the extracellular matrix. Inhibition of this enzyme is thus expected to hinder skin aging. Brown alga Padina pavonica activity toward hyaluronidase was evaluated using capillary electrophoresis (CE)-based enzymatic assays. This green technique allows evaluation of the biological activity of the natural material in an economic manner. Pressurized liquid extraction (PLE), microwave assisted extraction (MAE), supercritical fluid extraction and electroporation extraction techniques were used. Extraction conditions were optimized to obtain cosmetically acceptable Padina pavonica extracts with the best inhibition activity. CE-based assays were conducted using only a few nanoliters of reactants, a capillary of 60cm total length and of 50μm internal diameter, +20kV voltage for separation in 50mM ammonium acetate buffer (pH 9.0) and 200nm wavelength for detection. The reaction mixture was incubated for 1h and CE analysis time was about 11min. A novel online CE-assay using transverse diffusion of laminar flow profiles for in-capillary reactant mixing allowed efficient monitoring of hyaluronidase kinetics with K and V equal to 0.46±0.04mgmL and 137.1±0.3nMs (r=0.99; n=3), respectively. These values compared well with literature, which validates the assay. Water extracts obtained by PLE (60°C; 2 cycles) and MAE (60°C; 1000W; 2min) presented the highest anti-hyaluronidase activity. The half maximal effective concentration (IC) of water PLE extract was 0.04±0.01mgmL (r=0.99; n=3). This value is comparable to the one obtained for Einsenia bicyclis phlorotannin fractions (IC=0.03mgmL), which makes Padina pavonica bioactivity very promising.

Advances in enzyme substrate analysis with capillary electrophoresis

“…Enzyme reactions can be monitored using capillary electrophoresis when the substrate has a different charge-to-size ratio from the product. Hydrolase, transferase, and lyase enzymes perform reactions that inherently change the size of the molecules [13–21], so the substrates can be separated from the products. This is depicted in the separation of deacetylated product shown in Fig.…”

“…Off-line analysis for enzymatic inhibition studies are performed by incubation of enzyme and substrate before the capillary electrophoresis analysis. The enzymatic reactions are then quenched and injected into the capillary for separation [14,15,20,21,28,29,38,40,55,65,71,72]. The substrates and products are separated and quantified for kinetic analysis of enzyme reaction and elucidation of the inhibitor mechanism and effectiveness.…”

“…On the other hand, the on-line approach initiates enzymatic reaction through mixing in-capillary. The on-line mixing of enzyme, substrate, and inhibitor has been accomplished through electrophoretically mediated microanalysis [58,59,62,63,66,73], transverse diffusion of laminar flow profiles [21,31,41,74,75], and pressure mediated microanalysis [45]. On-line analysis with the use of immobilized enzyme for inhibitor studies have also been developed and high throughput analysis was achieved [18,22,23,56,68,76–78].…”

“…This is achieved by determining the IC 50 value, which is defined as the inhibitor concentration necessary to obtain 50% of the original response in the absence of inhibitor for a given substrate concentration. The IC 50 is obtained by monitoring the product formation while varying the concentration of inhibitor [13,21–23,29,31,39,46,56,68,71,74,75,81,82]. The lower the IC 50 value is, the more effective the inhibitor.…”

“…The lower the IC 50 value is, the more effective the inhibitor. Alternatively, the percent differences of the product peak area in the absence and presence of inhibitor can also be used to estimate the inhibition efficiency of the inhibitor [13,21,56,68,73–77,82,83]. …”