L glutamine

Promastigotes of L. braziliensis (MHOM/Br/75/M2903) were cultivated in the BHI medium (Himedia, Mumbai, India) supplemented with 10% inactivated fetal bovine serum (FBS) (Cultilab, So Paulo, Brazil), L-glutamine, penicillin at 100 UI/mL, and streptomycin at 100 μg/mL (Cultilab, So Paulo, Brazil) and kept in a BOD incubator at 25°C.

The influence of TPG on the proliferation capacity of tumor cells was investigated through the colony formation assay. Initially, 5 × 10⁵ MB49 cells were cultured in 6-well microplates with 5 mL of high-glucose DMEM medium complemented with 2 mmol/L of L-glutamine (Cultilab), 10% FCS (Cultilab), and 1% penicillin/streptomycin (Vitrocell Embriolife).The microplate was incubated for 48 h at 37 °C in a 5% carbon dioxide atmosphere. When the cell monolayer reached 90% confluence, the supernatant was discarded, and the cells were exposed for another 24 h to 5 mL of a solution of complete DMEM and TPG at different concentrations (25 and 50%). After that, adherent cells were collected from the surface of the wells using trypsin (2.5%), washed/neutralized with DMEM + 10% FBS, centrifuged at 200 g, and resuspended in 1 mL of DMEM. The cell quantification of the control group (without exposure to TPG) was performed with 0.4% Trypan blue (Life Technologies) using a Neubauer chamber.
The cells from the control group (0 percent TPG) were diluted in a volume of complete DMEM determined to be able to culture 1 × 10³ viable MB49 cells/well. Using 48-well microplates, the same volume was utilized to plate cells from the other TPG groups in quintuplicate. The microplates were kept in an incubator for 48 h until the colony-forming units were observed.

Human umbilical vein endothelial cells were purchased from the Rio de Janeiro Cell Bank (UFRJ, Brazil) originated from the ATCC (American Type Culture Collection, USA). Cultivation was performed in a humidified incubator at 37°C and 5% CO₂ in bottles using modified Eagle's Dulbecco's medium (DMEM) plus 10% fetal bovine serum, amphotericin B, and L glutamine (Cultilab, Brazil).
After the cultivation step, the cells were quantified in a Neubauer chamber and seeded onto 96-well plates containing 200 μL DMEM medium at 1×10⁴ concentrations. Then, the medium was discarded, and the wells containing cells were treated with PV3 at concentrations of 10, 20, 30, and 250 µg/mL for 24 h. The negative control group was treated with extract diluent, 0.9% (w/v) sodium chloride solution. The positive control group was treated with diluent and, after 24 h, was exposed to 3% (v/v) hydrogen peroxide (H₂O₂) for one hour. All assays were performed in three independent and triplicated experiments.