Chemical composition of essential oils from different parts of Protium heptaphyllum (Aubl.) Marchand and their in vitro antibacterial activity

Instituto Federal de Educação, Ciência e Tecnologia Goiano, Rio Verde, GO, Brazil; Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil; Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Franca, SP, Brazil; d Instituto Federal de Educação, Ciência e Tecnologia do Triângulo Mineiro, Uberlândia, MG, Brazil. *Corresponding author: maykermiranda@iftm.edu.br


Experimental
The plant material was collected at "Instituto Federal Goiano -Campus Iporá" in the city of Iporá (16°24'14.9''S and 51°06'40.0''W), State of Goiás, Brazil, in July 2017, at 9 a.m. The plant was identified by the botanist Luzia Francisca de Souza and a voucher specimen of Protium heptaphyllum (HJ1036) was deposited at the Herbarium Jataiense Professor Germano Guarim Neto. The essential oils of P. heptaphyllum were extracted from leaves and unripe and ripe fruits by hydrodistillation in a Clevenger-type apparatus for 2 h.
Hydrodistillation was performed in triplicate. The plant material was divided into three samples of 500 g each, and 500 mL of distilled water was added to each sample. After manual collection of the essential oils (EOs), traces of water remaining in the oil were removed with anhydrous sodium sulfate, which was followed by filtration. The EOs were stored in an amber bottle and kept in a refrigerator at 4°C until analysis. The EO yield was calculated from the weight of the fresh leaves, unripe and ripe fruits and expressed as the average of the triplicate analyses.
EOs were dissolved in ethyl ether and analyzed by Gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) using the Shimadzu QP5000 Plus and GCMS2010 Plus (Shimadzu Corporation, Kyoto, Japan) systems. The temperature of the column in the GC-FID was programmed to rise from 60 to 240°C at 3°C/min and was held at 240°C for 5 min; the carrier gas was H 2 at a flow rate of 1.0 mL/min. The equipment was set to operate in the injection mode; the injection volume was 0.1 µL (split ratio of 1:10), and the injector and detector temperatures were 240 and 280°C, respectively. The relative concentrations of the components were obtained by normalizing the peak areas (%). The relative areas consisted of the average of triplicate GC-FID analyses. The GC-MS conditions and the identification of the essential oils have been previously reported (Melo et al., 2015). Identification of the volatile components of essential oils of P. heptaphyllum (Table S1) was based on their retention indices on an Rtx-5MS (30 m X 0.25 mm; 0.250 µm) capillary column under the same operating conditions used for GC relative to a homologous series of n-alkanes (C 8 -C 20 ). The structures were computer-matched with Wiley 7, NIST 08, and FFNSC 1.2, and their fragmentation patterns were compared with literature data (Adams, 2007). . Standardization of each microorganism suspension was carried out as previously described (Ferreira et al., 2010). The EOs samples were dissolved in DMSO (Merck, Darmstadt, Germany) at 1 mg/mL and diluted in tryptic soy broth (Difco) so that concentrations in the range from 400 to 3.9 µg/mL would be achieved. The final DMSO concentration was 5% (v/v) and this solution was used as negative control. One inoculated well was included to control the adequacy of the broth for organism growth. One noninoculated well free of antimicrobial agent was also included to ensure medium sterility.
Chlorhexidine dihydrochloride (CHD) (C8527 Sigma) was dissolved in tryptic soy broth (Difco) and used as positive control at concentrations ranging from 59.0 to 0.115 µg/mL. The microplates (96 well) were sealed with plastic film and incubated at 37 o C for 24h as described above. After incubation, 30 µL of 0.02% resazurin (199303 Sigma, Stl Louis, MO, USA) aqueous solution were poured into each microplate well to indicate microorganism viability.
The MIC values were determined as the lowest concentration of the EOs capable of inhibiting microorganism growth. Three replicate assays were carried out for each microorganism.