*4.10. Parasites*

Parasite cultures employed in this study were *Trypanosoma cruzi* (SC2005 strain). Trypomastigote forms were obtained from Vero cells infected and used to infect the macrophages. Epimatigote forms were originated from the suspension of cell culture trypomastigotes in 3 mL of liver infusion tryptose (LIT) medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin and 100 μg/mL of streptomycin), and incubated in an oven at 28 ◦C until complete differentiation of parasites.

#### *4.11. Anti-Epimastigote Assay*

Epimastigote forms of *T. cruzi*, from a 2- to 4-day-old culture were incubated for 24 h in the absence or in the presence of different concentrations (1000–15.625 μg/mL) of *A. rosaeodora* essential oil or linalool, obtained by serial dilutions (1:2), at a final volume of 100 μL per well. The controls were identified as blank (wells without parasites), untreated control (parasites and DMSO 1%) and reference drug (benznidazole). Incubation took place in a 96-wells plate, in a BOD incubator at 28 ◦C in LIT medium using a parasite concentration of 10<sup>6</sup> promastigotes/mL. After 24 h, with the aid of the Neubauer chamber and light microscopy [46], viability was evaluated by counting parasites and the results

were used to calculate the IC50 (50% inhibition of parasite growth) following the formula: IC50 = (sample counting)/(control counting) ×100 [47].

#### *4.12. Animals and Ethical Statements*

BALB/c female mice from 4 to 6 weeks of age were purchased from the Institute of Science and Technology in Biomodels of the Institute of Science and Technology in Biomodels. All procedures were performed in accordance with the National Council for the Control of Animal Experimentation National Council for Animal Experimentation Control—CONCEA) and approved by the Ethics Committee on Animal Care and Utilization (CEUA/IOC—L018/2018).

#### *4.13. Peritoneal Macrophage Collection and Culture*

Peritoneal macrophages from BALB/c mice were collected after elicited with 3 mL 3% Brewer thioglycollate medium broth injection for 72 h, and maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 μg/mL of streptomycin, overnight at 37 ◦C and 5% CO2.

#### *4.14. Cytotoxicity Assay*

Peritoneal macrophages (5 × 10<sup>5</sup> cells/mL) were cultured in 96-well plates with different concentrations, obtained by serial dilutions (1:2), of *A. rosaeodora* essential oil or linalool (1000–7.8 μg/mL) or benznidazole (200–0.78 μg/mL) up to a final volume of 100 μL per well. The controls were categorized as blanks (wells with culture medium without cells), untreated control (cells and DMSO 1%) and reference drug (benznidazole). After 72 h, the cell viability was analyzed by the MTT colorimetric method [48]. Absorbance was measured in a spectrophotometer at 540 nm wavelength. The concentration inhibiting 50% of cell growth (CC50) was calculated following the formula: CC50 = (sample absorbanceblank absorbance)/(control absorbance-blank absorbance) × 100 [49].

#### *4.15. Activitiy Against Intracellular Amastigotes and Selectivity Index (SI)*

BALB/c peritoneal macrophages cultured in 24-well plates (5 × 10<sup>5</sup> cells/well), with coverslips, were infected with trypomastigote forms of *T. cruzi*, obtained from cultured Vero cells, using the ratio of parasite/cell 10:1, at 37 ◦C and 5% CO2 for 6 h. After incubation, well plates were washed with phosphate-buffered saline (PBS, pH 7.2) to remove the noninternalized parasites. The infected cells were treated with different concentrations of *A. rosaeodora* essential oil or linalool (1000–31.25 μg/mL), or benzonidazole (100–6.25 μg/mL) for 24 h. The amastigotes couting by analysis of light microscopy were carried out to determine the IC50 calculation. Selectivity index were obtained from the relationship of macrophage cytotoxicity and antiamastigote activity. Parameters of infection analysis were performed according to Teles et al. [50].

## *4.16. Nitrite Quantification*

BALB/c peritoneal macrophages (5 × 10<sup>6</sup> cells/mL) was treated with *A. rosaeodora* essential oil (500 μg/mL) or linalool (250 μg/mL), and either stimulated or not stimulated with *T. cruzi* trypomastigotes (5 × 10<sup>7</sup> parasites/mL) for 48 h. Nitrite quantification of the supernatant of the cells was performed with Griess reagent. Briefly, 50 μL of culture supernatant and 50 μL of Griess reagen<sup>t</sup> (25 μL of sulfanilamide 1% in 2.5% H3PO4 solution and 25 μL of N-(1-naphthyl)-ethylenediamine 0.1% solution) were added in 96-well plates. After incubation in a dark environment for 10 min, absorbance was obtained at 570 nm on the spectrophotometer. The nitrite values were obtained from the standard curve of sodium nitrite (100–1.5 μM) [51].

#### *4.17. Statistical Analysis*

The numerical results from at least two independent assays were expressed as mean ± standard deviation and the IC50 and CC50 determination were performed with the GraphPad Prism 7.00 software package (GraphPad Software, San Diego, CA, USA). Kruskal-Wallis and Dunn's multiple comparison test was used to analyze the data and the difference at *p* < 0.05 was considered significant.
