2.2.1. BNZ activity against extracellular epimastigote forms 
BNZ activity against epimastigote forms was determined using the method described by Rolón et al. (Rolón et al. , 2006) with some modifications. Briefly, 5 × 105 epimastigotes·mL–1 were treated by adding BNZ at a concentration range from 100 to 0.4 μM (200 μL·well–1 volumes) in 96-well plates at 28 ºC for 72 h. Untreated growth controls were also included. 20 μL of resazurin sodium salt (0.125 mg·mL–1) (Sigma-Aldrich) was added, and the plates were incubated for further 24 h. Finally, 5 μL of sodium dodecyl sulfate (10 % w/v) was added, and the absorbance was measured at 570 and 600 nm. The trypanocidal activity was expressed as the IC50 and IC90 (inhibitory concentrations 50 and 90) using GraphPad Prism 6 software. Each BNZ concentration was tested in triplicate in four separate determinations.