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

- Development of online assay for hyaluronidase inhibition by CE,
- Activity of a brown alga, Padina pavonica, against hyaluronidase was studied,
- Different extraction techniques were used and optimized to obtain active extracts,
- IC50 of water and ethyl acetate extracts toward hyaluronidase evaluated by CE,

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 60 cm total length and of 50 μm internal diameter, +20 kV voltage for separation in 50 mM ammonium acetate buffer (pH 9.0) and 200 nm wavelength for detection. The reaction mixture was incubated for 1 h and CE analysis time was about 11 min. A novel online CE-assay using transverse diffusion of laminar flow profiles for in-capillary reactant mixing allowed efficient monitoring of hyaluronidase kinetics with Km and Vmax equal to 0.46 ± 0.04 mg mL−1 and 137.1 ± 0.3 nM s−1 (r2 = 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; 1000 W; 2 min) presented the highest anti-hyaluronidase activity. The half maximal effective concentration (IC50) of water PLE extract was 0.04 ± 0.01 mg mL−1 (r2 = 0.99; n = 3). This value is comparable to the one obtained for Einsenia bicyclis phlorotannin fractions (IC50 = 0.03 mg mL−1), which makes Padina pavonica bioactivity very promising.