2.2. Antileishmanial Activity of Leishmania amazonensis

Strains isolated from leishmaniasis (Leishmania amazonensis MHOM/BR/2009/{"type":"entrez-nucleotide","attrs":{"text":"M26361","term_id":"197967","term_text":"M26361"}}M26361) were obtained from the Evandro Chagas Institute, Ananindeua, Brazil.

The L. amazonensis promastigotes were cultivated at 26°C in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco®, Grand Island, NY, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL) [18].

Culture of promastigote forms in logarithm phase was adjusted to 5 × 10⁶ parasites/100 μL. The susceptibility testing was performed in 96-well plates. The extract, fraction, and lapachol were tested in triplicate in a concentration gradient (200 to 3.125 μg/mL). Negative control was performed with parasites and incubation medium. The positive control was made with amphotericin B (25–0.3906 μg/mL). After 24 h of incubation at 26°C in 5% de CO₂, 10 μL of tetrazolium salt (5 mg/mL) was added to each well, and the parasites were quantified in enzyme-linked immunosorbent-assay plate reader. The IC₅₀ was determined by linear regression (Graph Pad Prism version 5.04). The results were classified as follows: IC₅₀ ≤ 100 μg/mL were considered active, IC₅₀ between 101 and 200 μg/mL were considered moderate active, and IC₅₀ ≥ 200 μg/mL were considered to be inactive [18].

Modified THP-1 cell (4 × 10⁵ cells/0.1 mL) was cultured in RPMI-1640 (Roswell Park Memorial Institute 1640) medium (Sigma Aldrich®, USA), supplemented with 5% of fetal calf serum, kept in a 5% CO₂ atmosphere at 37°C with phorbol ester as inducing agent. The cells were added the circular coverslips (2 × 10⁵); then L. amazonensis promastigotes were added (5 × 10⁶). The samples treatment was performed with concentrations of 250, 125, and 62,5 μg/mL/24 h. The coverslips were removed and stained with Giemsa. After that, the infection rate of macrophages was determined.