Liquid and Vapor Phase of Four Conifer-Derived Essential Oils: Comparison of Chemical Compositions and Antimicrobial and Antioxidant Properties

In this study, the chemical composition of the vapor and liquid phase of Pinus cembra L., Pinus mugo Turra, Picea abies L., and Abies Alba M. needles essential oils (EOs) was investigated by Headspace-Gas Chromatography/Mass Spectrometry (HS-GC/MS). In the examined EOs, a total of twenty-eight components were identified, most of which belong to the monoterpenes family. α-Pinene (16.6–44.0%), β-pinene (7.5–44.7%), limonene (9.5–32.5%), and γ-terpinene (0.3–19.7%) were the most abundant components of the liquid phase. Such major compounds were also detected in the vapor phase of all EOs, and α-pinene reached higher relative percentages than in the liquid phase. Then, both the liquid and vapor phases were evaluated in terms of antibacterial activity against three Gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, and Acinetobacter bohemicus) and two Gram-positive bacteria (Kocuria marina and Bacillus cereus) using a microwell dilution assay, disc diffusion assay, and vapor phase test. The lowest Minimum Inhibitory Concentration (MIC) (13.28 mg/mL) and Minimal Bactericidal Concentration (MBC) (26.56 mg/mL) values, which correspond to the highest antibacterial activities, were reported for P. abies EO against A. bohemicus and for A. alba EO against A. bohemicus and B. cereus. The vapor phase of all the tested EOs was more active than liquid phase, showing the inhibition halos from 41.00 ± 10.15 mm to 80.00 ± 0.00 mm for three bacterial strains (A. bohemicus, K. marina, and B. cereus). Furthermore, antioxidant activities were also investigated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays, and a concentration-dependent antioxidant capacity for all EOs was found. P. mugo EO showed the best antioxidant activity than the other Pinaceae EOs. The four Pinaceae EOs could be further investigated for their promising antibacterial and antioxidant properties, and, in particular, α-pinene seems to have interesting possibilities for use as a novel natural antibacterial agent.


Introduction
Since ancient times, plants are a source of different kinds of compounds that humans used for their numerous biological activities and as a source for drug development [1]. Nowadays, the studies on antioxidant and antimicrobial activities of natural products are of considerable interest due to the importance of identifying and characterizing new bioactive molecules for applications in different fields as food preservation and packaging, antibiotically resistance phenomenon, and plant diseases.
Among the most abundant compounds, particular attention was paid to α-pinene, as it always reached higher percentages in the vapor phase than in the liquid phase of the investigated EOs. The compared values are as follows: (44.0% vs. 65.6%), (16.6% vs. 31.6%), (20.2% vs. 35.5%), and (30.8% vs. 51.3%) liquid and vapor phase in P. cembra, P. mugo, P. abies, and A. alba EOs, respectively ( Figure 1).  The antibacterial activities of the Pinaceae EOs were evaluated for three Gram-negative (Escherichia coli, Pseudomonas fluorescens, and Acinetobacter bohemicus) and two Grampositive bacteria (Kocuria marina and Bacillus cereus using micro dilution assay to determine Minimum Inhibitory Concentration (MIC) and the Minimal Bactericidal Concentration (MBC), and the MBC/MIC ratio defines an agent as bacteriostatic when the MBC/MIC ratio > 4 and as bactericidal when the MBC/MIC ratio ≤ 4 [31]. Furthermore, the disc diffusion assay by contact with the essential oil determined the diameter of bacterial growth inhibition zone (IZ), and the vapor phase test determined the antibacterial growth inhibition zone (Vapor IZ) by more volatile molecules of the EO in a preservative atmosphere.
The antibacterial results of the tested EOs are summarized in Tables 3-6 reporting the MIC, MBC, MBC/MIC ratio, IZ, and vapor IZ following the treatments for each bacterial strain. In Table 3   The antibacterial activities of the Pinaceae EOs were evaluated for three Gram-negative (Escherichia coli, Pseudomonas fluorescens, and Acinetobacter bohemicus) and two Gram-positive bacteria (Kocuria marina and Bacillus cereus using micro dilution assay to determine Minimum Inhibitory Concentration (MIC) and the Minimal Bactericidal Concentration (MBC), and the MBC/MIC ratio defines an agent as bacteriostatic when the MBC/MIC ratio > 4 and as bactericidal when the MBC/MIC ratio ≤ 4 [31]. Furthermore, the disc diffusion assay by contact with the essential oil determined the diameter of bacterial growth inhibition zone (IZ), and the vapor phase test determined the antibacterial growth inhibition zone (Vapor IZ) by more volatile molecules of the EO in a preservative atmosphere.
The antibacterial results of the tested EOs are summarized in Tables 3-6 reporting the MIC, MBC, MBC/MIC ratio, IZ, and vapor IZ following the treatments for each bacterial strain. In Table 3, the treatment with P. cembra EO showed MIC and MBC values of 53.12 mg/mL for E. coli, P. fluorescens, and K. marina, while MIC values were 26.56 mg/mL for A. bohemicus and B. cereus, and MBC values were 26.56 mg/mL and 53.12 mg/mL, respectively. MBC/MIC ratio defined the P. cembra EO as bactericidal against all bacterial strains. No effects were observed with the disc diffusion assay and with the vapor phase test for P. cembra EO against E. coli and P. fluorescens. The IZ and vapor IZ values were 17.67 ± 0.58 mm and 67.33 ± 2.52 mm for A. bohemicus, 9.33 ± 0.58 mm and 80.00 ± 0.00 mm for K. marina, and 11.67 ± 1.15 mm and 80.00 ± 0.00 mm for B. cereus, respectively.   52.16 mg/mL for B. cereus, respectively. The MBC/MIC ratio defined as bactericidal the P. mugo EO against all bacterial strains. No effects were observed by the disc diffusion assay and by the vapor phase test for P. cembra EO on P. fluorescens. P. mugo EO was not highly active against E. coli with an IZ value of 9.67 ± 0.58 mm, while no growth inhibition zone was observed by the vapor phase test. Higher antibacterial activity was observed for the other bacterial strains: IZ and vapor IZ values were 25.33 ± 4.51 mm and 41.00 ± 10.15 mm for A. bohemicus, 11.33 ± 1.15 mm and 80.00 ± 0.00 mm for K. marina, and 15.67 ± 1.15 mm and 76.67 ± 5.77 mm for B. cereus, respectively. The P. mugo EO vapor phase was more active than the liquid phase against A. bohemicus, K. marina, and B. cereus.

Antioxidant Activity
To determine the antioxidant activity of the four Pinaceae EOs, 2,2-Diphenyl-1picrylhydrazyl (DPPH) scavenging activity and 2,2 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assay, based on the reaction of the potential antioxidant with colored radicals, were carried out. The antioxidant activity results are reported in Table 7. In all EOs, a concentration-dependent antioxidant capacity was found. In both tests, P. mugo EO showed the highest antioxidant activity than the other Pinaceae EOs. This EO exhibits lower IC 50 values (3.08 µg/mL and 43.08 µg/mL for DPPH and ABTS assays, respectively) and higher TEAC values (7.65 mol/mg and 14.01 mol/mg for DPPH and ABTS assays, respectively). The second effective essential oil was the A. alba EO with IC 50 values of 7.84 µg/mL and 44.23 µg/mL and TEAC values of 1.63 mol/mg and 13.26 mol/mg in the DPPH method and ABTS method, respectively. The TEAC values of P. cembra and P. abies EOs were almost identical: 1.63 mol/mg and 1.68 mol/mg in the DPPH assay, respectively and 13.26 mol/mg for both the EOs in the ABTS assay. Taking into account the ABTS test, the IC 50 amount was 44.90 µg/mL in P. cembra EO and 45.00 µg/mL in A. alba EO. In the DPPH test, both EOs showed similar IC 50 values, too (13.01 µg/mL for P. cembra and 13.05 µg/mL for P. abies). A. alba EO remains the least effective in antioxidant capacity of the analyzed Pinaceae EOs.
The chemical composition of A. alba EO showed two components such as limonene and α-pinene with a similar percentage (32.5% and 30.8%) followed by camphene (11.2%) and β-pinene (7.5%). The same compounds were listed with an inverted trend in A. alba EO from Montenegro where β-pinene (32.8%) was the major component followed by α-pinene (17.3%) and camphene (16.7%) [41]. On the contrary, α-limonene (about 70%) and α-pinene (57%) were the major compounds in A. alba EO from seeds and cones respectively [42]. In A. alba EO from Poland, limonene was the component with the higher percentage (82.9%) detected in seed EO, whereas α-pinene (50.0%) was the main component in cone EO [43]. According to the literature [44] and on the basis of the reported data, it becomes evident that the chemical composition of the EOs from species belonging to the Pinaceae family can depend by multiple factors such as part of the plant examined, its geographic origin, and also, extraction methods and storage [45].
MIC and MBC values defined by the microwell dilution method were tested against E. coli, P. fluorescens and A. bohemicus Gram-negative bacteria strains and K. marina and B. cereus Gram-positive bacteria strains. The lowest MIC (13.28 mg/mL) and MBC (26.56 mg/mL) values, which correspond to the highest antibacterial activities, were reported for P. abies EO against A. bohemicus and for A. alba EO against A. bohemicus and B. cereus with 12.82 mg/mL (MIC) and 25.64 mg/mL (MBC).
The increase of antibiotically resistance phenomenon in human and animal pathologies has determined the intensification of research on new natural antimicrobial substances [19,46,47], and in this view, several studies were carried out to investigate the biological activities of Pinaceae EOs and the roles of their molecules. P. abies EO extracted by supercritical carbon dioxide was investigated for antimicrobial properties on E. coli using the isothermal calorimetry technique, and it inhibited the growth and interfered with the metabolic activity of the microorganism [48]. Kartnig et al. [49] determined the antibacterial activities of the essential oils of young pine shoots on different bacterial strains also from human patients, and significant activities were revealed against G+ bacteria strains and Candida species tested. Apetrei et al. [25] reported that needles and twigs The antibacterial activities of the four Pinaceae EOs were also confirmed by agar diffusion and disk volatilization methods by which the IZ and vapor IZ were measured in mm of inhibition halos. For all the tested EOs, the vapor phases were more active than the liquid phases, showing the inhibition halos from 41.00 ± 10.15 mm to 80.00 ± 0.00 mm for three bacterial strains (A. bohemicus, K. marina, and B. cereus). Concerning E. coli and P. fluorescens, a very low or null activity was reported. The results showed high activities of the EOs against A. bohemicus, K. marina, and B. cereus and a scarce or null activity against E. coli and P. fluorescens. The highest activities obtained by vapor phases of all EOs against A. bohemicus, K. marina, and B. cereus could be related with the presence of α-pinene. In the graph bar (Figure 1), the relative percentages of α-pinene were reported. It reached higher percentages in the vapor phase than in the liquid phase of all investigated EOs. In particular, liquid and vapor phase values were as follows: (44.0% vs. 65.6%), (16.6% vs. 31.6%), (20.2% vs. 35.5%), and (30.8% vs. 51.3%), in P. cembra, P. mugo, P. abies, and A. alba EOs respectively. These results suggest that α-pinene could play an important role for the detected antibacterial activity. Some papers reported α-pinene from Pinaceae EOs as the main compound showing good biological activity; it was the principal constituent (5.2-37.0%) in five Moroccan Pinus species EOs [50] and in Pinus peuce Griseb. EOs (12.89-27.34%) growing on three different locations in R. Macedonia [51].
Different studies confirmed the antibacterial properties of α-pinene [52]. Freitas et al. [53] reported that α-pinene has antibacterial and antibiotic-modulating activities against S. aureus; it also increases the activity of norfloxacin against E. coli and norfloxacin and gentamicin against S. aureus. Furthermore, Hippeli et al. [54] described an antiinflammatory potential of P. mugo EOs and its main compound α-pinene, while Cole et al. [55] showed anti-proliferative activity on the MCF-7 cell line. On the other hand, Kurti et al. [37] attributed the antimicrobial activities of some Pinus species EOs from Kosovo to the hexane/diethyl ether fractions, which were mainly composed by oxygenated monoterpenes.
In the present study, the susceptibility of bacteria does not seem to be related with the features of the cell Gram-positive and Gram-negative bacteria wall structure, since the more sensitive bacteria strains, A. bohemicus, K. marina, and B. cereus do not belong to the same group. Generally, Gram-negative are more resistant than Gram-positive bacteria, because the cell wall does not allow the entrance into the cell of hydrophobic molecules present in the essential oils [56,57], although some exceptions have been shown [58,59]. In a comparative study of the essential oils from four Pinus species [30], it was found that the sensitivity of the tested bacterial pathogens cannot be related with the cell wall structure. Different mechanisms of action can explain the EOs antimicrobial activities, and their wide variety of molecular components can act at multiple levels [60].
The DPPH and ABTS assays demonstrated a significant antioxidant activity for all Pinaceae EOs. P. mugo EO was the more active with an IC 50 3.08 ± 0.65 and 43.08 ± 6.95 µg/mL for DPPH and ABTS assays, respectively. The values expressed in Trolox equivalent (TEAC) confirmed identical results. A comparative investigation has been carried out on P. halepensis EO chemical composition and antioxidant activities, with respect to the impact of geographic variation and environmental conditions [61].
The variety of compounds that are present in the investigated EOs confers them numerous biological properties, and their antioxidant activities could be related to the presence of monoterpenes. Wang et al. [62] studied the antioxidant activities of seven terpenoids found in wine, and among the tested compounds, α-pinene and limonene had the highest DPPH free radical scavenging and the highest reducing power. Wojtunik-Kulesza [63] reviewed the monoterpenes biological properties and antioxidant activities of α-pinene were also reported.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
To describe the chemical composition of the EOs, a gas chromatograph with a flame ionization detector (FID) directly coupled to a mass spectrometer (MS) Perkin Elmer Clarus 500 model (Waltham, MA, USA) was used. The GC was equipped with a Restek Stabilwax (fused-silica) polar capillary column. Helium was used as carrier gas at a flow rate of 1 mL/min. The injector was set to a 280 • C, and the oven temperature program was as follows: isothermal at 60 • C for 5 min, then ramped to 220 • C at a rate of 6 • C min −1 , and finally isothermal at 220 • C for 20 min. One uL of EO was diluted in 1 mL of methanol, and the injection volume was 1 µL. The Electron Impact-Mass Spectrometer (EI-MS) mass spectra were recorded at 70 eV (EI) and were scanned in the range of 40-500 m/z. The ion source and the connection parts temperature was 220 • C. The injector split ratio was 1:20. The GC-TIC mass spectra were obtained by the TurboMass data analysis software (Perkin Elmer). The identification of components was performed by matching their mass spectra with those stored in the Wiley and NIST 02 mass spectra libraries database. Furthermore, the linear retention indices (LRIs) (relative to C8-C30 aliphatic hydrocarbons, injected in the column at the same operating conditions described above) were calculated and compared with available retention data present in the literature. The relative percentages of all identified components were obtained by peak area normalization from GC-FID chromatograms without the use of an internal standard or correction factors and expressed in percentages. All analyses were repeated twice.

Headspace GC-MS Analysis
The volatile chemical profile of essential oils was carried out with a Perkin Elmer Headspace Turbomatrix 40 (Waltham, MA, USA) autosampler connected to GC-MS [64,65]. One mL of the each EO was placed in 20 mL vials sealed with headspace PTFE-coated silicone rubber septa and caps. To optimize the headspace procedure for the determination of volatile organic compounds (VOCs), more operative parameters were optimized. The gas phase of the sealed vials was equilibrated for 20 min at 60 • C and was followed immediately by compound desorption into GC injector in splitless mode. Quantification of compounds was performed by GC-FID in the same conditions described in the previous paragraph.

Antibacterial Activities of the Pinaceae Essential Oils
The antibacterial activities were investigated by using different methods, the Minimal Inhibitory Concentration (MIC), the Minimal Bactericidal Concentration (MBC), the agar diffusion method, and Vapor Phase Test (VPT).

Bacterial Strains
Five bacterial strains from the culture collections of the Plant Cytology and Biotechnology Laboratory of Tuscia University were tested to evaluate the antibacterial activities of P. cembra L., P. mugo Turra, A. alba M., and P. abies L. essential oils: Escherichia coli ATCC 25922, Pseudomonas fluorescens ATCC 13525, and Acinetobacter bohemicus DSM 102855 among Gram-negative and Kocuria marina DSM 16420 and Bacillus cereus ATCC 10876 among Gram-positive. All tested bacterial strains were maintained on LB broth (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter, autoclaved at 121 • C for 20 min) with agar. Bacteria cultures were maintained at two different temperatures: 26 • C for P. fluorescens, A. bohemicus, and B. cereus and 37 • C for K. marina and E. coli. All inocula were prepared with fresh cultures plated the day before the test.

Minimum Inhibitory Concentration (MIC)
The MIC is defined as the lowest concentration of antimicrobial agent that completely inhibits the growth of the microorganism as detected by the unaided eye and was carried out according to the microwell dilution method. Briefly, 12 dilutions of the four essential oils in LB broth (for P. cembra from 53.12 to 0.01 mg/mL; for P. mugo from 52.16 to 0.01 mg/mL; for A. alba form 51.28 to 0.01 mg/mL and for P. abies from 53.12 to 0.01 mg/mL), a control with the same percentage of DMSO (from 6.25% to 0.003%) in Lysogeny broth, a growth control without treatments, a positive control with gentamicin diluted from 100 to 0.05 µg/mL, and a sterility control without bacteria were plated on 96 microwell plates. Then, 50 µL of bacterial inoculum, 10 6 CFU/mL, were added in each well, except for the sterility control, and the plates were incubated for 24 h at the corresponding temperature. The visualization of the inhibition activity was obtained by 20 µL of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (200 µg/mL, MTT) added to each well. The assay was carried out in triplicate. The MBC/MIC ratio was reported to interpret the activity of the essential oil, and an antimicrobial agent is considered bacteriostatic when the ratio MBC/MIC > 4 and bactericidal when the ratio MBC/MIC is ≤4 [31].

Minimum Bactericidal Concentration (MBC)
To verify the lowest concentration at which the tested essential oils kill the bacterial cells, which is defined the Minimum Bactericidal Concentration (MBC), 10 µL of the last four dilutions from microwell dilution method in which no bacteria growth was observed were plated on a Petri plate with LB agar and incubated for 24 h. The concentration at which no growth on agar was observed defined MBC values. The assay was carried out in triplicate.

Agar Diffusion Method
To determine the diameter of the halo inhibition of the bacteria growth induced by P. abies, A. alba, P. cembra, and P. mugo essential oils, the bacterial strains were suspended in LB broth to obtain a turbidity of 0.5 McFarland (approximately 10 8 Colony-Forming Unit/mL-CFU/mL) and then plated on LB broth with agar in a Petri plate. Sterile disks (6 mm diameter, Oxoid) were placed on the agar and impregnated with 10 µL of samples. Two µL of gentamicin from a stock solution (10 mg/mL) was used as a positive control After 24 h, the inhibitory activities of each essential oil were recorded as mm of halo diameter without growth [58] using a vernier caliper rule. The mean and the respective standard deviation (SD) of the measured halo in three independent experiments were recorded.

Vapor Phase Test (VPT)
The antibacterial activity of the Pinaceae essential oils in the vapor phase was evaluated by the modified disk volatilization method [66,67]. LB agar were poured into an 80 mm plastic Petri dish and a lower amount into its cover. Each bacterial suspension containing 10 8 CFU/mL was plated on the LB agar medium. Then, 10 µL of tested essential oils were added to a 6 mm sterile disk and placed on agar in the covered Petri plate. Liquid LB agar was put in the space between the cover and the base of the Petri dishes to facilitate the sealing and to prevent any vapor leakage. The Petri plates were incubated for 24 h in an inverted position, and afterwards, the inhibition halos were measured. Negative controls were carried out without the essential. All VPTs were carried out in triplicate.

Antioxidant Activity
To assess the antioxidant activity of the four Pinaceae essential oils, DPPH radical scavenging activity and ABTS radical scavenging assay, which are based on the reaction of the potential antioxidant with colored radicals, were carried out.

DPPH Scavenging Activity Assay
In DPPH radical scavenging assay, the Pinaceae essential oils antioxidant activities were calculated against the 1,1-diphenyl-2-picrilidrazil radical (DPPH•) using the method described by Sanchez-Moreno et al. [68]. First, 100 µL of fresh solution of a solid crystalline DPPH• (0.2 mM) in methanol were added to 100 µL of 12 geometric dilutions in methanol of each essential oil inside a 96-well plate. Geometric dilutions of the samples in methanol were used as sample blanks. In blank DPPH samples, essential oils were omitted. As a positive control, dilutions were prepared starting from Trolox solution (1 mM) in methanol. The samples were incubated for 30 min in the dark at room temperature, and the absorbances decreases were measured at 517 nm using a Tecan Sunrise TM UV-vis spectrophotometer. The assay was repeated three times.

ABTS Radical Scavenging Assay
The radical scavenging activities of the Pinaceae essential oils were also calculated using the ABTS (2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assay described by Re et al. [69] with some modifications. The radical cation ABTS+• was produced by reacting ABTS aqueous solution (7 mM) with K 2 S 2 O 8 (140 mM) following an incubation for 16 h in the dark at room temperature before use. The ABTS+• solution was diluted with ethanol to reach an absorbance of 0.70 ± 0.02 at 734 nm, and 1980 µL was mixed with 20 µL of the essential oil dilutions in ethanol. The resulting solutions were incubated for 5 min at room temperature. Afterwards, the absorbances were measured at 734 nm using a Jasco V-630 UV-Visible spectrophotometer and using Spectra Manager TM software. Furthermore, the absorbance of the ABTS+• blank, consisting of 20 µL of ethanol dissolved in 1980 µL of ABTS+• solution, was measured. The assay was repeated three times.

IC 50 and TEAC Calculation
Trolox and samples calibration curves were obtained by plotting the inhibition ratio against sample concentrations. The inhibition ratio was calculated using the following formula: The IC50 parameter was calculated using the sample calibration curve. A lower value of the IC50 parameter correspond to a lower concentration of the EO that can scavenge 50% of DPPH· molecules; therefore, it indicates a higher antioxidant activity.
The Trolox equivalent antioxidant capacity (TEAC) index was obtained from the ratio between the Trolox IC50 (µM) and the sample IC50 (mg/L):

Statistical Analysis
The results were expressed as means ± standard deviation (SD). The one-way analysis of variance test (ANOVA) using GraphPad Prism software (GraphPad Prism 5.0, GraphPad Software, Inc., San Diego, CA, USA) was used to evaluate statistical discrepancies between the groups (p values < 0.05).

Conclusions
In this study, for the first time, the chemical composition of the liquid and vapor phase of four Pinaceae EOs was investigated by the HS-GC/MS technique. The results of analyses showed that these EOs are rich in monoterpenoids and highlight that α-pinene, one of the main compounds, is more abundant in the vapor phase of each oil than in the liquid phase. The antimicrobial and antioxidant activities were also reported and compared. The vapor phase of each EO resulted more active against the investigated bacterial strains.
The biological effects of the Pinaceae EOs combined with their bioavailability makes them promising sources for possible application in different fields such as pharmacology, pharmacognosy, and phytochemistry.