Gas chromatography coupled to mass spectrometry (GC-MS) characterization and evaluation of antibacterial bioactivities of the essential oils from Piper arboreum Aubl., Piper aduncum L. e Piper gaudichaudianum Kunth

Collection and identification of plant material

The fresh leaves of the plants were collected (summer of 2017) in the Biological Reserve of Bom Jesus Biological Reserve, at Vale do Ribeira, Guaraqueçaba—PR, Brazil. A voucher specimen of each plant was deposited in the Botanical Museum of Curitiba and in the Herbarium of the Faculdades Integradas Espírita with numbers of 396412 (P. arboreum), 396411 (P. aduncum), and 396403 (P. gaudichaudianum). This research was registered with an SISGEN N. A216E5A and an SISBIO N. 49770-1.

Extraction and chemical characterization of the essential oils

The extraction of the essential oils was performed by the hydrodistillation method using the Clevenger-type apparatus. Fresh leaves of each plant (50 g) were crushed and placed in a glass flask with 1.0 L of distilled water, remaining in the boil for 4.5 h for extraction [19]. The leaves were dried with an electric dryer (FANEM—Mod. 320 SE) with air circulation at 40°C for 24 h. To determine the essential oil content in dry basis, the total mass of each essential oil obtained was considered in relation to the amount of the dry mass of the botanical material used in the extraction. After extraction, the samples were collected with a precision pipette and conditioned in a freezer until the analysis. Each oil was named as follow: essential oil of P. arboreum Aubl. (EOPar), essential oil of P. aduncum L. (EOPad), and essential oil of P. gaudichaudianum Kunth (EOPg).

The chemical constituents of the essential oils were identified by gas chromatography coupled to mass spectrometry (GC-MS). The essential oils were diluted to a concentration of 1% in dichloromethane and 1.0 μL of the solution was injected with a 1:20 flow split into a chromatograph (Agilent 6890—Palo Alto, CA) coupled to a mass selective detector (Agilent 5973N—Palo Alto, CA). The injector was maintained at 250°C, and the constituents were isolated in an HP-5MS capillary column (5%-phenyl–95%-dimethylpolysiloxane, 30 m × 0.25 mm × 0.25 μm) using helium as carrier gas (1.0 mL min^-1). The oven temperature was programmed from 60 to 240°C at a rate of 3°C min^-1. The mass detector was operated in electronic ionization mode (70 eV), at a rate of 3.15 s^-1 sweeps, and a mass band of 40 to 450 u. The transfer line was maintained at 260°C, with ion source at 230°C and analyzer (quadrupole) at 150°C.

For quantification, the diluted samples were injected into a chromatograph (Agilent 7890A—Palo Alto, CA) equipped with a flame ionization detector (FID), operated at 280°C. The same column and analytical conditions described above were employed, except for the carrier gas, which was hydrogen, at a flow rate of 1.5 mL min^-1. The percentage composition was obtained by the electronic integration of the FID signal by dividing the area of each component by the total area (area%).

The identification of the chemical constituents was performed by comparing their mass spectra with those of spectral libraries [20], [21] and by their linear retention indexes, calculated from the injection of a homologous series of hydrocarbons (C₇–C₂₆) and compared with data from the literature [22].

Antibiotics, culture media, and microorganisms

The liquid antibiotics gentamicin and amikacin were obtained from LaborClin, Brazil. Heart Infusion Agar (HIA) and Brain Heart Infusion (BHI) culture media were acquired from HIMEDIA. The microorganisms used in the tests were provided by the Laboratory of Microbiology and Molecular Biology of the Regional University of Cariri. The following standard and resistant bacterial strains were used throughout this study: Escherichia coli ATCC 25922, E. coli 06, Staphylococcus aureus ATCC 25923, and S. aureus 10.

Preparation of test solutions

The test solutions were prepared using 10 mg of each oil diluted in 0.5 mL of dimethyl sulfoxide. Each solution was diluted to a final concentration of 1024 μg/mL. The solutions of the oils at this concentration were used in the antibacterial and modulation tests. The antibiotics used in the tests were also prepared at an initial concentration of 1024 μg/mL.

Determination of the minimum inhibitory concentration (MIC) by direct contact

Bacterial samples were seeded in Petri dishes containing HIA and placed in an oven at 37°C for 24 h to grow. After this period, the samples were collected and diluted in test tubes in triplicate. Then, the turbidity of the solution was determined according to the McFarland scale.

To evaluate the antibacterial activity, 100 μL of the inoculum solution was added to each well of the microdilution plate. Then, the treatments were performed using 100 μL of each oil per column at final concentrations ranging from 512 to 0.5 μg/mL. Of note, all treatments were performed in triplicate. The plates were taken to an oven at 37°C for 24 h. Then, each well was added with 20 μL of resazurin (a colorimetric indicator) and 1 h later the MIC was determined by ocular observation [23], [24].

The antibiotic activity modulation assay was performed using amikacin and gentamicin according to the method described by [25]. Briefly, test tubes were added with 150 μL of the bacterial suspension in a solution containing 10% BHI medium and the essential oils at subinhibitory concentrations (MIC/8). Control tubes were prepared using 150 μL of the bacterial suspension in a solution containing 10% BHI medium. Then, 100 μL of these solutions were transferred to corresponding wells in the plate and 100 μL of the antibiotic were added to the first well and serially diluted. The treatments were performed in triplicate, and the MICs were determined as described above.

Statistical analysis

The results were expressed as mean ± standard deviation and differences were evaluated through analysis of variance followed by Bonferroni posttests using GraphPad Prism 6.0 software. Results with p < 0.05 were considered as statistically significant.

Chemical composition

The relative amounts of the individual components were calculated based on the GC peak area (FID response), which those having a percentage above 10% were considered as major components.

The essential oil of P. arboreum had a yield of 0.23%. A total of 15 components were identified, representing 83.58% of the composition (Table 1), of which, 5.57% are monoterpenes oxygenated, 44.57% are sesquiterpenes oxygenated, 33.43% are sesquiterpenes nonoxygenated, having as main constituents β-copaen-4-α-ol: 31.38%, muurola-4,10-(14)-diene-1-β-ol: 17.32%.

The volatile oil of P. aduncum L. was isolated in 0.35% and presented high concentration of sesquiterpenes nonoxygenated (45.45%) and sesquiterpenes oxygenated (44.52%; Table 1), with spathulenol as the major compound (25.92%). In addition, β-macrocarpene (9.52%), α-humulene (5.33%) aromadendrene (5.61), and β-copaen-4-α-ol (5.45%), were also found in considerable yield.

Distillation of the essential oil of P. gaudichaudianum presented a yield of 0.56%. Twenty-two components were identified, representing 87.04% of the composition (Table 1), of these 3.96% are monoterpenes hydrocarbon, 67.26% sesquiterpenes nonoxygenated and 15.82% sesquiterpenes oxygenated, having as main constituent germacrene B: 21.53%.

Determination of minimum inhibitory concentration

In microdilution tests, EOPar showed MICs = 512 μg/mL against standard (ATCC 6538) S. aureus, 128 μg/mL against multiresistant S. aureus, and ≥ 1024 μg/mL against standard (ATCC 25922) and resistant E. coli. The EOPad had MIC = 512 μg/mL against standard S. aureus, 16 μg/mL against multi-resistant S. aureus, ≥ 1024 μg/mL against standard E. coli and 813 μg/mL against resistant E. coli. The EOPg had MIC = 813 μg/mL against standard S. aureus and ≥ 1024 μg/mL against resistant S. aureus and standard and resistant E. coli (Table 2).

Modulation of antibiotic activity

In the evaluation of the modulating effect, the combination of the EOPar (Figure 1) with the antibiotics norfloxacin and gentamicin caused decrease of the MIC against S. aureus 10, indicating synergism in the association of these treatments, whereas for E. coli 06 only synergism was observed in the combination with gentamicin, norfloxacin did not show a significant effect (Figure 2).

Figure 1:

Modulatory effect of essential oil from Piper arboreum (EOPar) in the antibiotic activity of norfloxacin, gentamycin, and erytromycin against strains of Staphylococcus aureus 10.

Figure 2:

Modulatory effect of essential oil from Piper arboreum (EOPar) in the antibiotic activity of norfloxacin, gentamycin, and erytromycin against strains of Escherichia coli 06.

The combination of P. aduncum essential oil with norfloxacin resulted in increased MIC, gentamicin, and decreased MIC indicating respectively antagonism and synergism against S. aureus 10 and E. coli 06 (Figures 3 and 4).

Figure 3:

Modulatory effect of essential oil from Piper aduncum (EOPad) in the antibiotic activity of norfloxacin, gentamycin, and erytromycin against strains of Staphylococcus aureus 10.

Figure 4:

Modulatory effect of essential oil from Piper aduncum (EOPad) in the antibiotic activity of norfloxacin, gentamycin, and erytromycin against strains of Escherichia coli 06.

The association of the EOPg with the drugs norfloxacin and gentamicin presented a synergistic effect against S. aureus 10 causing a decrease in MIC (Figure 5) and an antagonistic effect against E. coli 06, provoking an increase in MIC (Figure 6). The antibiotic erythromycin did not present significant results against any of the strains used in this study.

Figure 5:

Modulatory effect of essential oil from Piper gaudichaudianum (EOPg) in the antibiotic activity of norfloxacin, gentamycin, and erytromycin against strains of Staphylococcus aureus 10.

Figure 6:

Modulatory effect of essential oil from Piper gaudichaudianum (EOPg) in the antibiotic activity of norfloxacin, gentamycin, and erytromycin against strains of Escherichia coli 06.