**3. Discussion**

Essential oil may change depending on the chemical nature of its constituents and can be modified by air, light, heat, water and various impurities of natural origin or from falsifications. The changes can be recognized both by changes in their organoleptic characteristics (aroma, color, taste, transparency, fluidity), as well as the values of their chemical and physical parameters. Thus the density, refractive index, solubility, color and appearance were analyzed and the physical characteristics of the essential oil were similar to the pattern described in previous studies of *A. rosaeodora* [14].

Studies has identified and quantified chemical compounds of *A. rosaeodora* essential oil, revealing that this species has chemotypes similar to essential oil extracted in Belém, state of Pará, Brazil, with linalool (84.8%) as the major compound, followed by α-terpineol (2.9%), geraniol (1.0%), benzyl benzoate (0.6%) and minimal amounts of monoterpene hydrocarbons and oxygenated sesquiterpenes (9.2%) [15]. The same was observed in the study of *A. rosaeodora* essential oil extracted in São Paulo, Brazil, where the presence of linalool (81.45%), trans-linalool oxide (1.19%), R-terpineol (1.09%) were observed [16]. Almeida et al. (2013) also reported that linalool is the main compound in essential oil obtained from wood, leaves and branches of the Brazilian rosewood [17].

The disk diffusion test carried out against standard strain bacteria and against bacteria isolated from a marine environment evidenced antibacterial activity, preliminarily. The mixed sensibility observed to the several antibiotics revealed the resistance pattern of marine environment bacteria. The sensibility observed to *A. rosaeodora* essential oil and linalool showed that both compounds have activity against marine environment bacteria used in this study. It is known that the bacterial cell wall influences in an important way on the action of certain antibiotics. The difference between Gram-positive and Gramnegative bacterial walls would be one of the responses to antibiotic resistance between two bacteria [14]. Bacterial resistance is best evidenced in environmental bacteria in the last few years. With the advent of modernization, an increasing amount of antibiotics was released into the environment along with the residues from domestic, industrial,

agricultural and medical activities. This has ended up generating a selection of antibioticresistant bacteria or genes in the environment, which threatens the efficiency of antibiotics in fighting bacterial infections [18].

The MIC of *A. rosaeodora* essential oil resulted in concentrations lower than linalool. Holetz, et al. (2002) classifies samples that have MIC values below 100 μg/mL with good antibacterial activity; 100 to 500 μg/mL moderate; and 500 to 1000 μg/mL weak above 1000 μg/mL inactive [19]. Following this classification, *A. rosaeodora* essential oil showed moderate activity, while linalool displayed weak activity. The difference between the activity of both compounds may be related to the synergistic effect of the compounds present in the essential oil of *A. rosaeodora*. A synergistic interaction can be verified between the essential oils of *A. rosaeodora* and *Pelargonium graveolens* with gentamicin, and a very strong synergistic interaction against *Acinetobacter baumannii* ATCC 19606 (fractional inhibitory concentration/FIC index = 0.11) [20]. While research conducted with linalool showed low activity against Gram-positive and Gram-negative bacteria. Jabir et al. (2018) found that linalool loaded in gold nanoparticles modified with glutathione (LIN-GNPs) has effective antibacterial activity against Gram-positive bacteria, proving that LIN-GNPs acted on the bacterial cell membrane, in giving up and increasing cell wall permeability and stimulated reactive oxygen species (ROS) production that leads to bacterial nucleic acid damage [21].

Linalool is a compound widely used by the cosmetics industry [22] In the study by Herman et al. (2016) [10], a significant increase in antimicrobial efficacy was observed by the addition of linalool to essential oil, reducing its concentrations in products (cosmetics, medicine), making it possible to obtain its synergistic and additive effects. In addition, several studies have been reported on the commercial availability of oxidized linalool samples possibly causing allergic contact dermatitis [23–25]. Thus, essential oil from *A. rosaeodora* has potential applicability in edible and/or dermatological preparations.

Biological activity may be directly related to phenolic compounds as they are good electron donors and therefore have efficient antioxidant activity among secondary plant metabolites. These compounds are capable of control oxidative damage generated by reactive oxygen species or radicals [26]. We can also classify the antioxidant activity according to the excellent (IC50 < 15 μg/mL), good (15 μg/mL < IC50 < 50 μg/mL), medium (50 μg/mL < IC50 < 100 μg/mL), and weak activity (IC50 ≥ 100 μg/mL). *A. rosaeodora* essential oil antioxidant activity was considered good while the linalool was optimal, corroborating with previous studies that verified excellent antioxidant activity of *Aniba* species [26].

In traditional medicine, plant essential oils are known as a rich source of secondary metabolites with relevant biological activities, as an alternative in antiparasitic therapy [27,28]. The trypanocidal activity of essential oils of *Aniba* genus was described in the literature [14,29]. Currently, the drugs available for the treatment of Chagas disease are benznidazole and nifurtimox, which have limited efficacy, serious adverse effects and have been in use since the late 1960s [30]. Thus, in an attempt to search for new therapeutic alternatives for Chagas disease, we report the effect of *A. rosaeodora* essential oil and its main component linalool in the growth of epimastigote and intracellular amastigote forms of *T. cruzi*.

In the present study, *A. rosaeodora* essential oil showed activity against epimastigote forms. Literature data showed anti-*T. cruzi* activity in vitro in extracts and substances of different species of the *Aniba* genus of plants collected in the Amazon [29], with promising antileishmanial activity [14]. To understand whether linalool is responsible for the inhibitory activity, an analysis of linalool against epimastigote was performed. The results showed an inhibitory effect close to the values of *A. rosaeodora* essential oil. Therefore, it is worth inferring that the inhibitory effect of the essential oil occurs due to the high concentration of linalool, or due to a possible synergistic and/or additive effect of the constituents of the essential oil acting as trypanocidal agents [31].

Previous data demonstrated that the IC50/24 h for linalool was 162.5 μg/mL for epimastigotes and 264 μg/mL for *T. cruzi* trypomastigotes (Y strain) [32], corroborating with data presented in this study. However, linalool had a potent trypanocidal effect against the trypomastigote form of *T. cruzi* (clone Dm28c) derived from cells, with IC50/24 h of 306 ng/mL, indicating that different forms and/or origin and different strains may differ in their susceptibility to essential oil derivatives [33].

The search for new therapeutic drugs requires conditions that simulate the environment found by the parasite–cell interaction, therefore, the assay against intracellular amastigote forms of tripanosomatids may represent ideal conditions, with macrophages playing an important role in the evaluation of drug-mediated toxicity [34]. Thus, it was evaluated whether *A. rosaeodora* essential oil and linalool could inhibit *T. cruzi* intracellular amastigote. However, the inhibitory effect was observed only when infected cells were treated with linalool although in high concentration, while *A. rosaeodora* essential oil presented activity at an even higher concentration.

*Piper aduncum* essential oil (PaEO), with nerolidol (25.22%) and linalool (13.42%) as main constituents, effectively inhibits the intracellular survival/replication of T. cruzi amastigotes. PaEO at a concentration of 12.5 μg/mL decreased the rate of T. cruzi amastigote infection by 71.5%, with an IC50/24 h of 9 μg/mL. As linalool showed trypanocidal activity, with IC50/24 h of 306 ng/mL against trypomastigotes [33], it is possible to infer that activity against intracellular amastigote forms it is possibly due to linalool presence. In addition, previous data demonstrated that at low concentrations of purified linalool derived from the *Croton cajucara* essential oil, the number of parasites internalized in the macrophages decreased (treated before and after the interaction). On the other hand, no cytotoxic effects of essential oil and linalool were observed in peritoneal macrophages of Swiss mice and Vero cells [35]. As in our study, *A. rosaeodora* essential oil and linalool not exhibited cytotoxicity against peritoneal macrophages in the concentration range under analysis. As a result, linalool showed a select activity to the parasites when compared to mammalian cells [14].

Literature data with *L. infantum chagasi* determined that the post-interaction treatment with linalool has antiparasitic activity against intracellular amastigotes, inducing a decrease in the number of parasites within the macrophages [36]. In the same study, it was observed that linalool is capable of providing a drastic change in oxygen consumption, probably related to mitochondrial dysfunction. *P. aduncum* essential oil rich in linalool induced mitochondria dysfunction altering the mitochondrial membrane potential of the T. cruzi epimastigote [33]. Mitochondrial alterations as swelling and important changes in the organization of nuclear and kinetoplastic chromatins were observed by electron microscopy when *L. amazonensis* parasites were treated with *C. cajucara* essential oil [35]. Linalool may interfere with the integrity of protozoan mitochondria, however, further studies are needed to elucidate the mechanism involved in the trypanocidal activity observed in our study.

An indirect mechanism involved with antitrypanosomal activity is related with macrophage activation, particularly the nitric oxide (NO) induction. The NO-mediation directly kills *T. cruzi* in vitro [37]. Thus, we carried out an analysis of the nitrite quantification of *T. cruzi*-stimulated peritoneal macrophages treated with *A. rosaeodora* essential oil or linalool. However, a significant decrease in nitrite levels was observed in cells nonstimulated with *T. cruzi* and treated with *A. rosaeodora* essential oil or linalool. Reactive oxygen species decrease was also observed in cancer cells lines treated with *A. rosaeodora* essential oil, inhibiting apoptosis in these cells [13].

Otherwise, in *T. cruzi*-stimulated cells the treatment with *A. rosaeodora* essential oil or linalool did not significantly decrease nitrite levels. Linalool has a known anti-inflammatory activity [38] and inhibits NO formation in vitro [39], but interestingly, an in vitro experiment of macrophages treated with linalool (250 or 350 μg/mL) for 24 h before or after interactions with the *Leishmania infantum* was also not associated with any difference in NO production [36]. The inhibition of NO production observed in macrophages treated with *A.*

*rosaeodora* essential oil and linalool, although it is not associated with antitrypanosomal activity, is an interesting finding that should be better elucidated in further studies.

#### **4. Materials and Methods**
