Apoptosis Induction by CSEO, Compounds

The effects of CSEO, methyl isothiocyanate, hexadecanoic acid, and limonene on the induction of apoptosis were evaluated by the colorimetric protease (Sigma, Germany) method according to the manufacturer recommendations. In this way, the caspase-3-like activity level was measured based on the rate of color spectrophotometric produced through the release of a molecule (pNA attached to the substrate) under the enzyme caspase-3 activity. In brief, the promastigotes (1 × 10⁶) were incubated with CSEO, methyl isothiocyanate, hexadecanoic acid, and limonene at the concentrations of 6.25, 12.5, and 25 µg/mL for 24 h and were centrifuged at 700 rpm for 5 minutes at 4°C. Next, the cell residue was lysed, and the cell lysate was centrifuged again at 20,000 rpm for 10 minutes. Lastly, the supernatant of reaction (5 μl) was added to the 85 μl of buffer and 10 μl of caspase-3 (pNA-DEVD-Ac) solution and the mixture was incubated for 120 min at 37°C. The caspase-3-like activity was determined through the light absorption at 405 nm with the ELISA reader [27 (link)].