Stevioside Modulates Protease Activity

Crude extracts of trophozoites previously treated with 9.53 mM stevioside for 24 h and untreated trophozoites were obtained for the determination of proteolytic activity by substrate degradation. One million trophozoites for control and STV-treated samples were harvested. For protein extraction, trophozoites were washed in 1× PBS and centrifuged for 5 min at 350× g 3 times. The pellet was resuspended in a lysis buffer and incubated on ice for 10 min. The supernatant was recovered after centrifugation for 2 min at 5600× g and quantified by the Bradford method. The electrophoretic development of 2 µg of total protein, for both controls and STV-treated samples, was carried out in 15% polyacrylamide gels copolymerized with 0.1% porcine skin gelatin. The gels were washed twice in a 2.5% Triton X-100 solution and incubated overnight into an activation buffer (100 mM Tris-HCl, 10 mM CaCl₂, pH 7) at 37 °C while stirring. Finally, the gels were washed with tridistilled water and stained with a Coomassie blue solution for 1 h at 37 °C. A densitometric analysis of the bands was performed by the ImageJ^® software 1.8.0.