Antileishmanial activity

Promastigote forms of L. amazonensis (WHOM/BR/75/Josefa), which were originally isolated by Cesar Augusto Cuba-Cuba (Universidade de Brasilia, Brazil) from a human case of diffuse cutaneous leishmaniasis. Parasites were cultured at 25 °C in Warren’s medium (brain-heart infusion plus haemin and folic acid) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco Invitrogen Corporation, New York, USA) in a tissue flask. Macrophages (J774-A1) were maintained in tissue flasks with RPMI-1640 (pH 7.2) (Gibco Invitrogen, Grand Island, NY, USA), added with sodium bicarbonate and L-glutamine (As annex), and supplemented with 10% FBS (Gibco Invitrogen, Grand Island, NY, USA) at 37 °C in a 5% CO₂ atmosphere²⁴.

For experiments, promastigote forms in the logarithmic phase (1 x 10⁶ cells/mL) were cultured on a 24-well plate in Warren’s medium supplemented with 10% of inactivated FBS in the presence or absence of different essential oils concentrations (1-100 µg/mL). Amphotericin B was used as positive control (0.01-10 µg/mL). After 72 h at 28 °C, cell growth was estimated using the colorimetric cell viability XTT assay (2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) (Sigma Chemical Co., St. Louis, MO, USA). All experiments were performed in duplicate, and the results expressed as log number cells/mL and as the percentage of growth inhibition concentration that inhibited cell growth in 50% (IC₅₀) was determined by nonlinear regression analysis²⁵.

For the antiproliferative activity on intracellular amastigotes, peritoneal macrophages from healthy BALB/c mice were harvested and plated (3 x 10⁵ cells/mL) in a 24-well plate with round coverslips using RPMI medium supplemented with 10% FBS to adhere for 2 h at 37 °C in 5% CO₂. Adhered macrophages were infected with promastigotes in the stationary growth phase using a ratio 1:7 at 34 °C for 4 h. Afterwards, non-interiorized parasites were removed and the infected culture was treated with different concentrations of essential oils (1 to 20 μg/mL) at 34 °C. Amphotericin B was used as a positive control (0.1-10 μg/mL). After 48 h, the coverslips were washed, fixed with methanol and stained with Giemsa. By counting 200 cells under a light microscope (Olympus CX 31) the percentage of infected cells and number of intracellular amastigotes were estimated. The survival index (percentage of infected cells x number of amastigotes per cell) was calculated and IC₅₀ values were then determined by nonlinear regression analysis²⁵.

The activity against intracellular amastigote forms was compared with toxicity in macrophages, yielding the selectivity index (SI; ratio of the CC₅₀ in J774-A1 and the IC₅₀ in protozoa).