Amphotericin b

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.

Canine metastatic osteogenic osteosarcoma cells (D-17, BCRJ 0276, Lot 000573, Passage 239, Canis familiaris), originating from the American Type Culture Collection (Manassas, VA, USA), were purchased from the Rio de Janeiro Cell Bank (UFRJ – Rio de Janeiro, Brazil). Cells were grown in Dulbecco’s modified Eagle culture medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, and streptomycin (10,000 IU/mL, 10 mg/mL), and amphotericin B and glutamine L (all reagents from Cultilab, Campinas, Brazil) and kept in a humidified incubator at 37°C with an atmosphere of 5% CO₂.

Viruses were kindly provided by the Laboratório de Vírus at UFMG, Brazil. Zika virus (ZIKV) PE243 was obtained as described by Donald et al. [74 (link)]. The Vero (ATCC^® CCL-81™) cell line was used for the cytotoxicity assays and antiviral activity evaluation against ZIKV. Cells were cultured in Eagle’s minimum essential media (MEM) (Cultilab, Campinas, Brazil). The media was supplemented with 5% fetal bovine serum (FBS) (Cultilab, Brazil), in addition to 100 IU/mL penicillin (Cellofarm, Rio De Janeiro, Brazil), 100 μg/mL streptomycin (Merck, Darmstadt, Germany), and 0.25 μg/mL amphotericin B (Cultilab, Brazil). This work is registered in SisGen under number AA7854F.