*4.1. Reagents*

Anhydrous sodium sulfate, ethanol, ethyl acetate, dimethyl sulfoxide (DMSO), eugenol, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), penicillin, streptomycin, N-benzyl-2-nitro-1H-imidazole-1-acetamide (Benznidazole), Brewer thioglycolate medium, RPMI 1640 medium, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT), sulfanilamide, H3PO4, N-(1-naphthyl)ethylenediamine and sodium nitrite were purchased from Sigma, St Louis, MO, USA. Giemsa's azur-eosinmethylene blue, Brain Heart Infusion broth, Mueller-Hinton agar and Mueller-Hinton broth were purchased from MERK, Darmstadt, Germany. The other bacteria culture medium were purchased from BD, Becton Dickinson, Franklin Lakes, NJ, USA. API® 20 E system was purchased from bioMérieux, Durham, NC, USA. Fetal bovine serum (FBS) was purchased from Gibco, Gaithersburg, MD, USA.

#### *4.2. Plant Material*

Authentic samples of the *A. rosaeodora* species were obtained from three trees cultivated at the Adolpho Ducke Forest Reserve, Highway AM-010, km 26 (latitude −2.908185, longitude −59.975457), Manaus, Brazil. Leaves and thin branches were harvested with a trimmer from the treetops in the dry season, March 2017. The taxonomic identification was undertaken by the Herbarium of the Department of Botany of the Universidade Federal do Amazonas, registry number 5982. The leaves were selected and dried in an oven at 37 ◦C for 48 h and sprayed in an electric knife mill at the Food and Water Quality Control Laboratory of the Federal University of Maranhão.

#### *4.3. Essential Oil Extraction*

The extraction of the essential oil of *A. rosaeodora* was carried out with 100 g of dried leaves from thin branches diluted in water in the proportion of 1:10 by hydrodistillation using the Clevenger system for 3 h at 100 ◦C. The essential oil collected were dried with anhydrous sodium sulfate (Na2SO4) and the final volume found was used to determine the yield through the mass/volume ratio by measuring the density. Mass/volume ratios were calculated from the mass (g) of the initial vegetal material and the volume (mL) of essential oil obtained after extraction. The essential oil samples were kept at 25 ◦C and then weighed. For the verification of biological activity in vitro, the essential oil and the reference drugs were diluted in DMSO and subsequently made serial dilutions in an appropriate culture medium until reaching a final concentration below 1% DMSO.

#### *4.4. Physical-Chemical Analysis of Essential Oil*

Physical-chemical analyzes performed on *A. rosaeodora* essential oil were: density, measured with a glass pycnometer; refractive index, calculated with an ABBE 2WAJ refractometer (PCE Instruments, Southampton, UK); the color and appearance, that were visually verified by three different people; and the determination of solubility, carried out through the ratio of 1:1 of oil and 70% ethanol until its complete solubilization.

#### *4.5. Gas Chromatography–Mass Spectrometry (GC–MS)*

The standard used in the development of the analytical methodology was linalool. Standard solutions of monoterpenes were prepared by dilution in absolute ethyl alcohol and chloroform at different concentrations. The essential oil of *A. rosaeodora* was solubilized in ethyl acetate and was analyzed by a gas chromatograph Shimadzu QP 5000 (Shimadzu Corp., Kyoto, Japan), a column used with a capillary ZB-5 ms (5% phenyl arylene 95% dimethylpolysiloxane) coupled to 70 eV (40–500 Da) HP 5MS electronic impact detector with a transfer temperature of 280 ◦C. In the analysis, 0.3 μL of ethyl acetate and helium

gas (99.99%) were injected at a temperature of 280 ◦C, using a split mode (1:10) with an initial temperature gradient of 40 to 300 ◦C.min−1, with a chromatographic run that lasted 30 min.

#### *4.6. Bacteria from Marine Enviroment*

Bacteria strains isolated from the marine environment *Aeromonas caviae, Aeromonas hydrophila, Enterococcus faecalis, Klebsiella pneumoniae* and *Providencia stuartii* were gently provided by the Laboratory of Microbiology of the Water Quality Control Program at the Federal University of Maranhão. Water samples were aseptically collected from approximately 30 cm below the water surface of the Jansen lagoon, Maranhão Brazil (latitude −2.499629, longitude −44.301211). Then, the samples were transported to the Microbiology Laboratory of the Federal University of Maranhão in isothermal boxes containing ice to perform the identification. To *Aeromonas* isolation, successive decimal dilutions of water samples (10−<sup>1</sup> to <sup>10</sup>−7) were prepared in alkaline peptone water (APA), with subsequent distribution of 1 mL aliquots in five series of five tubes containing tryptic soy broth (TSB Broth) and 0.1 mL in plates containing the selective medium, agar gelatin phosphate salt (GSP Agar) (duplicates), both with 20 μg/mL of ampicillin, an antibiotic used as an inhibitor of the accompanying microbiota of *Aeromonas*. Colonies suspected of being *Aeromonas* were seeded in tilted BD trypticase soy agar (TSA agar) tubes, followed by incubation at 28 ◦C for 24 h. After, the cultures on TSA agar were subjected to biochemical tests of oxidase, catalase, gas production from glucose for species identification, indole production, O/129 resistance, amino acid decarboxylation (test on triple sugar agar and iron—TSI agar), motility: nitrate reduction, esculine hydrolysis, Voges–Proskauer (VP) assay, carbohydrate fermentation and growth at 3% and 6% sodium chloride. To Enterobacteriaceae isolation and identification of *Klebsiella pneumoniae* and *Providencia stuartii* in the water samples, initially, 25 mL of each sample were homogenized in 225 mL of brain and heart infusion broth (BHI broth) and incubated in a bacteriological oven at 37 ◦C for three hours. After the incubation period, the entire inoculum was transferred to 250 mL of broth for *Escherichia coli* and incubated at 37 ◦C for 24 h. Isolation was performed using selective and differential media, methylene blue eosin agar (EMB agar) and MacConkey sorbitol agar (MCS agar). For the identification of the species, initially five colonies were selected from the selective culture media, small colonies with metallic green or black without gloss in EMB agar and those of intense pink color (positive sorbitol) and yellow (negative sorbitol) in MCS agar. Then, the colonies were isolated in tubes containing TSA agar inclined with subsequent incubation at 37 ◦C for 24 h. Biochemical identification was performed using conventional tests: indole, simmons citrate, methyl red, VP, malonate, carbohydrate fermentation—sorbitol, rhamnose, mannitol, arabinose, inositol and raffinose, decarboxylation of amino acids lysine and ornithine, motility and H2S production in sulfide indole motility (SIM) agar [40] and by the API® 20 E system. For *Enterococcus* research, 9 mL of each sample were diluted in 90 mL of buffered peptide water and incubated for 24 h/35 ◦C. Subsequently dilutions (10−<sup>1</sup> to <sup>10</sup>−7) and the highest dilution were plated on M-*Enterococcus* agar and incubated at 35 ◦C for 48 h. Brick red colonies were inoculated on TSI agar. Tubes that showed suggestive characteristics were analyzed by acid ramp, acid-base, H2S (-), catalase, oxidase, 6% NaCl, glucose and esculin tests, and by the API® 20 E system.

#### *4.7. Bacterial Strains and Culture Conditions*

To perform the preliminary antimicrobial tests, the standard strains *Escherichia coli* (Migula) Castellani and Chalmers (ATCC® 25922™), *Staphylococcus aureus* subsp. *aureus* Rosenbach (ATCC® 12600™), *Pseudomonas aeruginosa* (Schroeter) Migula (ATCC® 27853™) and *Salmonella enterica* subsp. *enterica* (ex Kauffmann and Edwards) Le Minor and *Popoff serovar* Choleraesuis (ATCC® 12011™) were used. The tests were carried out at the Microbiology Laboratory of the Federal University of Maranhão. The strains were grown in BHI broth for 24 h at 37 ◦C and the inoculum was adjusted to a cell concentration of 10<sup>8</sup> colony

forming unit (CFU)/mL following the MacFarland scale, recommended by the Clinical and Laboratory Standards Institute [41].

#### *4.8. Antimicrobial Assays*

During the preliminary test of diffusion in solid medium, 100 μL of inoculum of each bacterium sown on Mueller–Hinton agar plates were used, and on the agar surface, a paper disc impregnated with 50 μL of essential oil of *A. rosaeodora*, standard linalool or reference drugs were added; then the plates were incubated at 35 ◦C and after 24 h the inhibition zone was measured with a millimeter rule [42]. The MIC was also performed according to the broth dilution methodology performed in triplicate with the same bacteria used in the diffusion tests in solid medium [41]. Initially, serial dilutions were performed resulting in concentrations of 5–1000 μg/mL of *A. rosaeodora* essential oil, linalool or reference drugs and transferred to a test tube containing Mueller-Hinton broth. To each concentration, 100 μL of the microbial suspension containing 1.5 × 10<sup>8</sup> CFU/mL were added and subsequently incubated at 35 ◦C for 24 h. It was also reserved control of broth sterility and bacterial growth. After the incubation period, the MIC was determined, being defined as the lowest concentration that visibly inhibited bacterial growth (absence of visible turbidity). To confirm growth inhibition, the broth was subjected to the microbial seeding test of the inoculum on the surface of the plate-count agar.

#### *4.9. Antioxidant Assay*

Antioxidant activity was assessed using a reaction mixture of 2,2-azinobis- (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 3840 μg/mL with 88 μL of 37,840 μg/mL potassium persulfate solution left in the dark at room temperature for 16 h giving rise to the ABTS radical which was diluted in ethanol to obtain an absorbance of 0.7 to 734 nm. The results were obtained in a dark environment, in which 30 μL of each concentration of essential oil (200 to 15 μg/mL) and eugenol (90 to 5 μg/mL) was transferred in test tubes containing 3.0 mL of the cation radical ABTS and homogenized on a tube shaker, and after 6 min the absorbance of the reaction mixture was read on a spectrophotometer at a length of 734 nm [43]. The analyses were carried out in triplicates and the determination of the activity was demonstrated as percentage of inhibition (% I) of the ABTS radical cation according to the equation: % inhibition = (absorbance of the solution of the radical ABTS— absorbance of the sample)/(solution of ABTS absorbance radical) × 100 [44]. We also verified the efficient concentration or EC50% that represents the concentration necessary to sequester 50% of the ABTS root. The essential oil will be considered active when it has an EC50 < 500 μg/mL [45].
