2.3.1. Cytotoxicity in Non-Tumor Liver Cell Primary Culture

Hepatotoxic activity was evaluated following the method described by Abreu et al. [26], using a primary cell culture (PLP2) prepared from a porcine liver. Extracts were tested at a final concentration range from 400 to 1.56 μg/mL. Briefly, a cell culture was prepared from a freshly harvested porcine liver (obtained from a local slaughter house) and designated as PLP2 [26]. Briefly, the liver tissue was rinsed in Hank's balanced salt solution containing 100 U/mL penicillin + 100 μg/mL streptomycin, and was divided into 1 <sup>×</sup> 1 mm3 explants. Some of them were placed into 25 cm2 tissue flasks

containing Dulbecco's Modified Eagle's medium (DMEM, supplemented with 10% fetal bovine serum, 2 mM non-essential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin) and incubated at 37 ◦C under a humidified atmosphere with 5% CO2. A phase contrast microscope was used for direct monitoring of the cell cultivation every 2 to 3 days. Before reaching the confluence, cells were subcultured and plated in 96-well plates at a density of 1.0 <sup>×</sup> 104 cells/well, and cultivated in commercial DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The results were measured through the Sulforhodamine B method, where the amount of pigmented cells is directly proportional to the total protein mass and therefore to the number of bounded cells. Results were expressed as GI50 values (concentration that inhibits 50% of cell growth) and Ellipticine was used as a positive control.
