**2. Results**

#### *2.1. Physical and Chemical Characterization of A. rosaeodora Essential Oil*

The yield of *A. rosaeodora* essential oil obtained from dried leaves thin branches was 2.8%. The oil showed a yellow color and a clean appearance (Figure 1B), and presented a density of 0.89 g/mL at 25 ◦C, and a refractive index (ND 25) of 1.459. It was soluble in ethanol 70% in a ratio of 1:2. Chemical compounds identified and quantified in *A. rosaeodora* essential oil are presented in the chromatogram (Figure 2) and Table 1. Three compounds were identified and enumerated accordingly with elution order and retention time. The major constituent of *A. rosaeodora* essential oil was linalool with 93.60%. In addition, αterpinolene and linalool cis-oxide were identified and quantified at 3.37% and 3.03%, respectively.

**Figure 2.** Chromatogram of *Aniba rosaeodora* essential oil. \* TIC: total ion chromatogram.


**Table 1.** Chemical composition of *Aniba rosaeodora* essential oil.

1 Peak area percentage in relation to peak total area.

*2.2. Antimicrobial Activity of A. rosaeodora Essential Oil and Linalool*

Bacteria from the marine environment were evaluated by the disc-diffusion method against *A. rosaeodora*, linalool and several reference antibiotics (Table 2). The bacteria culture displayed inhibition halos ranging from 7 to 25 mm, besides the non-inhibition presented in some cultures. Comparing linalool with *A. rosaeodora* essential oil, we found that *A. rosaeodora* was more efficient against *Aeromonas caviae* and *Enterococcus faecalis* than the standard linalool. Linalool exhibited greater activity against *Klebsiella pneumonia* and *Providencia stuartii* than *A. rosaeodora* essential oil, while both compounds presented the same activity against *Aeromonas hydrophila*. The susceptibility test performed with antibiotics showed that *E. faecalis* was the strain that presented sensibility for all the antibiotics analyzed, while the other four strains displayed mixed sensibility to the antibiotics.

The preliminary antibacterial activity against standard strain bacteria evaluated by the disc-diffusion method showed a growth inhibitory halo on *A. rosaeodora* essential oil and linalool disks against Gram-positive (*Staphylococcus aureus*) and Gram-negative strains (*Escherichia coli*, *Pseudomonas aeruginosa* and *Salmonella* sp.) (Table 3). Gram-positive bacteria exhibited the highest inhibition halo from both essential oil and linalool. The minimum inhibitory concentration (MIC) of *A. rosaeodora* essential oil ranged from 250 to 450 μg/mL against the tested strains. As observed in the disc-diffusion method, *S. aureus* was the strain more sensible to *A. rosaeodora* essential oil and linalool activity by MIC analysis. Likewise, both methodologies showed that *A. rosaeodora* essential oil presented better antimicrobial activity than linalool to all strains analyzed.


**Table 2.** Inhibitory zone diameters of *Aniba rosaeodora* essential oil, linalool and antibiotics on different bacteria isolated from marine environment after 24 h of treatment.

Data represents mean ± standard deviation of experiment realized in triplicate.

**Table 3.** Inhibitory zone diameters and minimum inhibitory concentration of *Aniba rosaeodora* essential oil on different bacterial cultures after 24 h of treatment.


MIC: minimum inhibitory concentration; n.d.: not determined. Data represents mean ± standard deviation of experiment realized in triplicate.

#### *2.3. Antioxidant Activity of A. rosaeodora Essential Oil and Linalol*

*Aniba roseadora* essential oil and linalool presented antioxidant activity concentrationdependent, as observed in the graph that relates *A. rosaeodora* essential oil and linalool concentration versus the percentage of inhibition of the 2,2-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical (Figure 3). The calculated EC50 was 15.46 μg/mL for *A. rosaeodora* essential oil and 6.78 μg/mL for linalool.

**Figure 3.** Inhibition of the 2,2-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical by *Aniba rosaeodora* essential oil and linalool.

#### *2.4. Cytotoxicity, Antitrypanosomal Activivy and Selectivity Index of A. rosaeodora Essential Oil and Linalool*

The activity of *A. rosaeodora* essential oil and linalool was evaluated against the epimastigote and intracellular amastigote forms of *Trypanosoma cruzi*, as well as its cytotoxic effect against mammal cells. Both compounds presented concentration-dependent inhibitory activity against epimastigote and intracellular amastigote forms of *T. cruzi* (Figure 4). The inhibitory concentration for 50% of parasites (IC50) values for epimastigote forms was lower for *A. rosaeodora* essential oil than linalool. Analyzing the activity against different forms of the *T. cruzi*, *A. rosaeodora* essential oil exhibited IC50 value against epimastigote 6.0-fold higher in comparison to the IC50 against intracellular amastigotes forms. In contrast, linalool was 3.65-fold more effective against *T. cruzi* intracellular amastigote when compared to *A. rosaeodora* essential oil. Both compounds presented higher IC50 values when compared to benznidazole. Cytotoxicity assay revealed that *A. roseadora* essential oil and linalool not showed toxicity for BALB/c peritoneal macrophages even at the highest concentration analyzed (1000 μg/mL). Thus, linalool exhibited higher SI value than *A. roseadora* essential oil (Table 4).

**Figure 4.** Activity of *Aniba rosaeodora* essential oil and linalool against *Trypanosoma cruzi* epimastigote (**A**) and intracellular amastigote forms (**B**) after 24 h of treatment. Data represent media ± standard deviation of three independent experiment realized in triplicate.


**Table 4.** BALB/c peritoneal macrophage cytotoxicity, trypanocidal activity and selectivity index of *Aniba rosaeodora* essential oil and linalool.

IC50: inhibitory concentration for 50% of parasites; CC50: cytotoxic concentration for 50% of cells; SI: selectivity index, obtained from the ratio CC50/IC50 intracellular amastigote. Data represents mean ± standard deviation of at least two independent experiments carried out in triplicate.

The parameters of infection analysis (Figure 5) showed that *A. rosaeodora* essential oil treatment displayed significant low number of amastigotes per 100 cells at 1000 μg/mL (*p* = 0.0001) and 500 μg/mL (*p* = 0.0011) (Figure 5A). Linalool showed a low number of amastigotes per 100 cells at 500 μg/mL (*p* = 0.0001), 250 μg/mL (*p* = 0.0014) and 125 μg/mL (*p* = 0.0290) (Figure 5B). On the other hand, the treatment with *A. rosaeodora* essential oil and linalool only presented a significant low mean number of amastigotes per infected cells at 1000 μg/mL (*p* = 0.0398, Figure 5C) and 500 μg/mL (*p* = 0.0229, Figure 5D), respectively. The alterations in intracellular amastigotes of *T. cruzi* after treatment with *A. rosaeodora* essential oil and linalool are represented in photomicrography images of Figure 5E.

**Figure 5.** BALB/c peritoneal macrophages infected with *Trypanosoma cruzi* and treated for 24 h with *Aniba rosaeodora* essential oil or linalool. (**A**–**D**) Parameters of infection and (**E**) light microscopy after *A. rosaeodora* or linalool treatment at 1000 or 500 μg/mL respectively. Intracellular amastigotes inside macrophages (black arrows) and non-internalized parasite (red arrows). The images and data (mean ± standard deviation) represent two independent experiments performed in quadruplicate. \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 when compared with untreated infected cells by Kruskal–Wallis and Dunn's multiple comparison test. Giemsa, 40× objective.

#### *2.5. Nitrite Quantification in T. Cruzi-Infected Peritoneal Macrophages Treated with A. rosaeodora Essential Oil and Linalool*

The nitrite quantification in the supernatant of BALB/c peritoneal macrophages showed low nitrite levels in cells treated with *A. rosaeodora* essential oil (0.150 ± 0.220 μM NaNO2, *p* = 0.0259) and linalool (0.175 ± 0.146 μM NaNO2, *p* = 0.0490) when compared to untreated-unstimulated cells (1.129 ± 0.501 μM NaNO2). In *T. cruzi*-stimulated cells, although nitrite levels after treatment with *A. rosaeodora* essential oil (0.952 ± 0.779 μM NaNO2) and linalool (1.047 ± 0.702 μM NaNO2) were lower than stimulated-untreated cells (1.347 ± 0.416 μM NaNO2), the difference was not statistically significant for both compounds (*p* = 0.945 and *p* > 0.999, respectively) (Figure 6).

**Figure 6.** Nitrite quantification in the supernatant of the BALB/c peritoneal macrophage treated with *Aniba rosaeodora* essential oil (500 μg/mL) or linalool (125 μg /mL), and stimulated or not with *Trypanosoma cruzi*. Data represents mean ± standard deviation of experiment realized in sextuplicate; \* *p* < 0.05, \*\*\* *p* < 0.001 when compared with untreated and unstimulated macrophages by Kruskal–Wallis and Dunn's multiple comparison test.
