Phosphoramidon

To test for potential degradation of IL-6, 10 µl conditioned culture medium obtained from infection experiments was used either untreated, heat-treated (95°C/5 min), or mixed with 8 µl of different protease inhibitors (or respective controls) and then incubated with 2 µl (0.5 µg/µl) recombinant IL-6 (Peprotech, Rocky Hill) at 37°C. For inhibition of metalloproteases, phosphoramidon (Sigma-Aldrich) was used at a final concentration of 3.4 mM. Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK, Sigma-Aldrich), primarily inhibiting serine proteases, was used at a final concentration of 40 mM. Additionally, a proprietary, broad non-metalloprotease inhibitor was used (cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail, Roche, one tab dissolved in 500 µl water).

As previously described^{53 (link)}, 20 µL of plasma, 10 µL of substrate (5 mmol/L glutaryl-Ala-Ala-Phe-AMC; Peptides International, Louisville, KY, USA) and 50 µL of assay buffer (0.1 mol/L Tris–HCl, pH 7.6) were incubated at 37 °C for 30 min. The reaction was stopped by adding 10 µL of the NEP inhibitor phosphoramidon (0.1 mmol/L; Sigma, Oakville, ON, Canada) and incubating the samples on ice. Background controls were processed in the same manner except that phosphoramidon was added before the incubation at 37 °C. In the next step, the samples were incubated at 37 °C for 30 min with 10 µL of aminopeptidase M (500 mg/L, EMD Millipore, Etobicoke, ON, Canada) and 5 mmol/L EDTA. The reaction products were diluted in 3 mL of assay buffer, and the fluorescence was measured using a Tecan Infinite M1000 plate reader at an excitation wavelength of 360 nm and an emission wavelength of 440 nm. NEP activity was calculated from the difference between the sample (S) and control (C) values with the equation (S—C)/194^{53 (link)}.

For extraction, analytical grade solvents, Dichloromethane (DCM), Ethyl Acetate (EtOAc), and Ethanol (EtOH) were purchased from Carlo Erba Reactifs SDS (Val de Reuil, France). The aqueous solutions were prepared using deionized water. For TLC analysis, solvents were obtained from Fisher Scientific, while Sulphuric Acid (H₂SO₄) and Vanillin standard (98%) were purchased from Sigma-Aldrich (Steinheim, Germany). For HPLC analysis, MEthanol (MeOH), Acetonitrile (can), Water, and Acetic Acid were obtained from Fisher Scientific (Leicestershire, UK). ACN and Formic Acid used for LC–MS analysis were of LC–MS grade (Fisher Scientific, Loughborough, UK), while ultrapure water was obtained from a Milli-Q^® purification system (Merck Millipore, Darmstadt, Germany). Dimethyl sulfoxide (DMSO), DPPH (2,2-diphenyl-1-picrylhydrazyl), Gallic Acid used for DPPH Radical Scavenging assay, as well all reagents used for the three enzymatic assays, were purchased from Sigma-Aldrich. Kojic Acid (purity 99%), Elastatinal, and Phosphoramidon were used as the positive control for anti-tyrosinase, anti-elastase, and anti-collagenase assays, respectively, and were also purchased from Sigma-Aldrich.