Antibacterial, Antioxidant, and Antiproliferative Activities of Corymbia citriodora and the Essential Oils of Eight Eucalyptus Species

Background: Essential oils (EOs) have shown antimicrobial, antioxidant, and antiproliferative activity, which may, alone or in combination with other substances, potentially be used for the development of new drugs. However, their chemical variability, depending on the species, varieties, or geographical origin (among other factors) determines different bioactivities that need to be evaluated. Methods: The antioxidant activity of Corymbia citriodora and eight Eucalyptus species EOs was determined using two different methods: the scavenging ability of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+•) and peroxyl free radicals or oxygen radical absorbance capacity (ORAC). Antibacterial activity was evaluated using the microorganisms Streptococcus pneumoniae (strains D39 and TIGR4), and Haemophilus influenza (strain DSM 9999). The essential oils’ minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was assessed using a microdilution method. The antiproliferative activity was determined using the THP-1 cell line (human acute monocytic leukaemia) with methylthiazolyldiphenyl-tetrazolium bromide assay (MTT). Results: Corymbia citriodora and Eucalyptus viminalis EOs showed the highest ABTS and peroxyl free radical scavenging capacity. Eucalyptus globulus EO showed a high potential to treat Streptococcus pneumoniae infections. Haemophilus influenzae was the respiratory pathogen that showed the highest resistance to all EOs, including tea tree EO. After 96 h of incubation, at 25 μg/mL, Eucalyptus radiata and Eucalyptus viminalis EOs showed highest cytotoxic activity against the THP-1 cell line. Conclusions: Despite their specific bioactivities, no single EO showed simultaneously good antioxidant, antimicrobial, and antiproliferative activity.

Gas chromatographic analyses were performed using a Perkin Elmer Autosystem XL gas chromatograph (Perkin Elmer, Shelton, CT, USA) equipped with two flame ionization detectors (FIDs), a data handling system, and a vaporizing injector port into which two columns of different polarities were installed: a DB-1 fused-silica column (100% dimethylpolysiloxane, 30 m × 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific Inc., Folsom, CA, USA) and a DB-17HT fused-silica column ((50%-phenyl)-methylpolysiloxane, 30 m × 0.25 mm i.d., film thickness 0.15 µm; J & W Scientific Inc.). The oven temperature was programmed to 45-175 • C, increasing at 3 • C/min, subsequently increased to 300 • C at to 15 • C/min, and then maintained isothermal for 10 min; injector and detector temperatures were 280 • C and 300 • C, respectively. The carrier gas, hydrogen, was adjusted to a linear velocity of 30 cm/s. The samples were injected using a split sampling technique, ratio 1:50. The volume of injection was 0.1 µL of a distilled n-pentane-oil solution (1:1). The percentage composition of the oils was computed by the normalization method from the GC peak areas, calculated as a mean value of two injections from each oil, without response factors.

Gas Chromatography-Mass Spectrometry (GC-MS)
The GC-MS unit consisted of a Perkin Elmer Autosystem XL gas chromatograph, equipped with DB-1 fused-silica column (30 m × 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific, Inc.), and interfaced with Perkin-Elmer Turbomass mass spectrometer (software version 5.4.2.1617, PerkinElmer, Shelton, CT, USA). Injector and oven temperatures were as above; transfer line temperature, 280 • C; ion source temperature, 220 • C; carrier gas, helium, adjusted to a linear velocity of 30 cm/s; split ratio, 1:40; ionization energy, 70 eV; scan range, 40-300 u; scan time, 1 s. The identity of the components was assigned by comparison of their retention indices, relative to C 9 -C 17 n-alkane indices, and GC-MS spectra from a laboratory-made library based upon the analyses of reference oils, laboratory-synthesized components, and commercial available standards.

Determination of Antioxidant Activity
Antioxidant activity was determined with 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS +• ) free radical scavenging activity and oxygen radical absorbance capacity (ORAC) methods. For the ABTS method, the preformed radical monocation of ABTS +• was generated by dissolving ABTS in water (7 mM), adding potassium persulfate (2.45 mM, final concentration), and allowing the mixture to stand overnight in the dark at room temperature. Before the assay, the ABTS +• solution was diluted with ethanol to obtain a solution with absorbance of 0.70 at 734 nm [20]. For the assay, 10 µL of sample were added to 1000 µL of ABTS +• solution and left to stand for 6 min, after which the absorbance was read spectrophotometrically at 734 nm. For the control, the volume of sample was replaced by ethanol. The antioxidant activity of each sample was calculated as scavenging effect % (IA%, Inhibitory Activity Percentage) = [(A 0 − A 1 )/A 0 ] × 100, where A 0 is the absorbance of the control, and A 1 is the absorbance of the sample. The same procedure was repeated using diverse concentrations of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) [21]. The values obtained with the samples were compared with the curve for several Trolox concentrations and the final results given as µmol Trolox equivalent (TE)/g EOs. Tests were carried out in triplicate.
For the ORAC assay, fluorescein (FL) was used as fluorescent probe [22]. Briefly, in each well 150 µL of fluorescein working solution and 25 µL of the previously diluted sample, blank (75 mM phosphate buffer), or standard (Trolox) were added. The plate was covered with a lid and incubated in the preheated (37 • C) BioTek Synergy TM 4 Hybrid Microplate Reader (BioTek, Swindon, UK) for 10 min after shaking for 3 min. Then 2,2 -Azobis-2-methyl-propanimidamide dihydrochloride (AAPH) was added to each well of the plate, except for the control and blank wells. The final volume of the assay was 200 µL. The fluorescence was read every minute for 90 min at 485 nm excitation and 527 nm emissions. ORAC values were calculated according to [23]. Briefly, the net area under the curve (AUC) of the standard (Trolox) and samples was calculated. The standard curve was obtained by plotting Trolox concentrations against the average net AUC of the measurements for each concentration.
Final ORAC values were calculated using the regression equation between Trolox concentration and the net AUC and were expressed as µmol TE/g EO [24]. Tests were carried out in triplicate.

Determination of Antibacterial Activity
Antibacterial activity was determined against two strains of Streptococcus pneumoniae D39 and TIGR4, and Haemophilus influenza DSM 9999. Bacterial strains were kept at −70 • C in brain heart infusion (BHI) (Oxoid) supplemented with glycerol (final concentration 25%, v/v) until use. The cultures were recovered from freezing by growth in brain heart infusion supplemented with 5% (v/v) horse blood at 37 • C in microaerophilic conditions. Prior to the assay bacterial cultures were grown in Columbia agar (Oxoid) plates supplemented with 5% (v/v) horse blood at 37 • C in microaerophilic conditions for 24 h. From this plate a suspension with a turbidity value corresponding to 0.5 in the McFarland scale was prepared. From this suspension 0.1 mL was transferred to a new Columbia agar plate supplemented with 5% (v/v) horse blood. Sterile filter paper discs of 6 mm (Oxoid) were distributed on the agar surface and 3 µL of each essential oil was dispensed in the paper discs. Tea tree essential oil with 38% terpinen-4-ol (Optima Healthcare Ltd., Bradford, UK) was included as reference. Sterile n-propanol (essential oil solvent) and the antibiotic chloramphenicol (30 µg/disk) were used as control. Inhibition zones were evaluated after 24 h at 37 • C in microaerophilic conditions. The assays were done in triplicate.
The minimum inhibitory concentration (MIC) for the tested EOs was determined using a microdilution method [6,25]. Concentrations ranging from 0 to 0.4 mg/mL with increments of 0.05 mg/mL were tested. Three replicates for each bacterium were used. The recovery of the viability of each bacterium was evaluated, and the minimum concentration at which no viability was observed was considered as the minimum bactericidal concentration (MBC).
The growth inhibitory effect of EOs was measured using the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay [26]. THP-1 cells were seeded in 96-well plate at 5 × 10 3 cells/well and submitted to different EO concentrations (10-500 µg/mL). All samples were dissolved in dimethyl-sulfoxide (DMSO). The solvent concentration in the incubation medium never exceeded 0.5%. Control cultures had the equivalent concentration of DMSO. After treatment, cells were incubated (1 h) in the usual culture conditions after addition of the same volume of medium containing MTT (2 mg/mL). After this period, 150 µL HCl (0.1 M) in isopropanol was added to dissolve the blue formazan crystals formed owing to the reduction of MTT. Absorbance at 570 nm using a background reference wavelength of 630 nm was measured. Antiproliferative activity was expressed as the mean absorbance values for the negative control (DMSO-treated cells) standardized as 100% absorbance (i.e., no growth inhibition) and results were expressed as absorbance (% of control) versus essential oil concentration [24].

Statistical Analysis
Data were analysed by one-way analysis of variance (ANOVA) using IBM SPSS Statistics version 23 (IBM, Armonk, NY, USA). Tukey's test was used to determine the difference at 5% significance level.

Antioxidant Activity
The antioxidant activity of the EO was determined with an electron reaction-based method (the ABTS+ method) and a hydrogen reaction-based method (the ORAC method). With the TEAC method, Cc EO showed significantly higher antioxidant activity (5.08 µmol TE/g EO) than Eucalyptus EOs ( Table 2). The lowest antioxidant activity was observed with Eg, Ep, Er and Es EOs, without significant differences among them. The activities ranged from 0.10 in Es to 0.30 µmol TE/g in Eg EO. Cc EO showed the highest scavenging peroxyl radical activity (148.55 µmol TE/g EO) along with Ev EO (135.80 µmol TE/g EO) ( Table 2). Eu EO (37.14 µmol TE/g EO) showed the lowest peroxyl radical scavenging activity.
Results for the essential oils of Cc and Es are in agreement with those previously reported by Luis et al. [28] for these species. In Luis et al. [28] study, citronellal (78%) was the main component for Corymbia citriodora EO, and 1,8-cineole (82%) dominated in Eucalyptus smithii EO.
Citronellal shows moderate antioxidant activity due to its limited capacity to scavenge free radicals [29]. The antioxidant activity of citronellal may be related to one unpaired electron from the molecule allylic hydrogen of both CH 3 or CH 2 (Figure 1) that by resonance stabilize the radical formed after the reaction. Nevertheless, Wojtunik et al. [30] considered that only the presence of conjugation of π bonds would allow a resonance-stabilized structure. However, essential oils are complex mixtures and the antioxidant activity cannot be assigned to only one compound. The different constituents of EOs may act synergistically or antagonistically, leading to an overall activity that results from the interaction among several compounds. Ciesla et al. [29] studied the capacity of binary mixtures of some common EO terpenoids, for scavenging DPPH free radicals. They reported an antagonistic effect between citronellal and 1,8-cineole, both present in Cc EO. Nonetheless, the interaction of citronellal with other constituents could trigger a synergistic effect, responsible for the high level of activity observed in Cc EO. Antagonism was also observed by Ciesla et al. [29] for the binary mixture of 1,8-cineole and p-cymene. These constituents were the major Er EO compounds (Table 1). Eg, Ep, Er, and Es EOs, which showed the lowest free radical scavenger activities, had 1,8-cineole, α-pinene, and p-cymene as major constituents. These monoterpenes were previously reported as poor scavengers of DPPH free radicals [31]. γ-Terpinene, one of the main constituents of Ev EO, is considered a good free radical scavenger [29,32], possibly due to the delocalization of the unpaired electron along the molecule, owing to the two allylic hydrogens that can stabilize the resonance-structures ( Figure 1). Moreover, the binary mixture of γ-terpinene and 1,8-cineole (another major constituent of Ev EO) did not produce any significant effects. Despite the presence of an allylic hydrogen in p-cymene in conjugation with an aromatic ring, its abstraction from the molecule can be difficult due to steric hindrances, because hydrogen is hidden by the two CH 3 groups as well as by the aromatic ring ( Figure 1). unpaired electron along the molecule, owing to the two allylic hydrogens that can stabilize the resonance-structures ( Figure 1). Moreover, the binary mixture of γ-terpinene and 1,8-cineole (another major constituent of Ev EO) did not produce any significant effects. Despite the presence of an allylic hydrogen in p-cymene in conjugation with an aromatic ring, its abstraction from the molecule can be difficult due to steric hindrances, because hydrogen is hidden by the two CH3 groups as well as by the aromatic ring ( Figure 1).

Antibacterial Activity
Essential oils of thyme, tea tree, peppermint, anise, fennel, and eucalyptus may be used for the treatment of simple respiratory tract diseases [4]. Eucalyptus EOs have been used in the treatment of bronchitis, cold, cough, and in the symptomatic relief of catarrh of the upper respiratory tract [4]. Tea tree EO has been used for treating some respiratory infections, such as bronchitis, cold, and influenza [4].
S. pneumoniae, H. influenzae, and Staphylococcus aureus have been associated with community-acquired pneumonia, along with the less common Pseudomonas aeruginosa, Pneumocystis jirovecii, Moraxella catarrhalis, and other Gram-negative bacteria [33].
In the present work the capacity for inhibiting the growth of two strains of S. pneumoniae (D39 and TIGR4) and H. influenzae (DSM 9999) by Cc, Ed, Eg, Es and Ev EOs was evaluated and compared with that of tea tree EO and the antibiotic chloramphenicol (Table 3). All essential oils showed poor antibacterial activity in comparison with the antibiotic chloramphenicol, however differences could be observed among the EOs and bacterial strains (Table 3). For example, all EOs were less effective against S. pneumoniae TIGR4 in comparison with S. pneumoniae D39. For S. pneumoniae TIGR 4, tea tree EO showed better activity than Myrtaceae EOs, and the strain was even resistant to Ed EO. For H. influenzae, no significant differences (p > 0.05) were observed between the antibacterial activity-assayed Myrtaceae EOs (Table 3).
The MIC and MBC values were determined for Es, Eg, and tea tree EOs ( Table 4). The lowest MIC and MBC values were observed for Eg EO against S. pneumoniae TIGR 4, with values of 0.015 and 0.05 mg/mL, respectively. Both S. pneumoniae strains showed a similar MIC and MBC for the tea tree EO, namely 0.025 and 0.1 mg/mL, respectively. However, for the Es EO S. pneumoniae D39 showed a similar MIC value to S. pneumoniae TIGR 4, but the MBC value was the double of that of S. pneumoniae TIGR 4, showing an MBC of 0.3 mg/mL in contrast to TIGR 4 that showed an MBC of 0.15 mg/mL (Table 4). Such differences may be associated with serotype differences between the strains. Moreover, Eg EO, that in the diffusion agar test showed a very weak activity against S. pneumoniae TIGR 4, displayed an efficient activity in the microdilution assay, even in comparison with tea tree EO. These differences may be related to its low diffusion ability. Intriguing was the resistant behaviour of H. influenza that was able to growth at the highest EO concentration tested (0.4 mg/mL). Eg EO showed a better activity against S. pneumoniae, that can be related with its richness in 1,8-cineole and α-pinene. In addition, 1,8-cineole prevents the growth of other microorganisms, but when in association with other terpenes in an essential oil, such as camphene, α-pinene, globulol and limonene, it was more efficient against S. aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli and Candida albicans, and also biofilms of MRSA and P. aeruginosa [34].

Antiproliferative Activity
The antiproliferative activities of Corymbia and Eucalyptus species EOs were studied with the THP-1 cell line (human acute monocytic leukaemia) using the MTT assay. Cells were incubated to increasing amounts of EOs for 24 h and 96 h (Figures 2 and 3).
EOs decreased viability of THP-1 cells in a dose-dependent manner. After one day, a great difference was observed between the antiproliferative activity of Eg and Es EOs and the other EOs, particularly at 50 µg/mL. Hence, Eg and Es EOs possessed the lowest antiproliferative activities. After 96 h incubation of THP1 cells with EOs, the differences were more evident for lower concentrations (10 and 25 µg/mL). At 25 µg/mL, it was possible to distinguish two EOs (Er and Ev) with significantly higher antiproliferative activity than the remaining EOs. At 50 µg/mL, these differences disappeared, because almost all EOs showed survival percentages <20%. The exceptions were Eg and Es EOs, that showed survival percentages above >70%, at 50 µg/mL. The main constituents of Eg EO were 1,8-cineole (64%) and α-pinene (20%), and in Es EO, 1,8-cineole represented 80% of the total EO ( Table 1). The major components of the Er and Ev EOs also included 1,8-cineole (48 and 46%, respectively), and other components such as p-cymene (13%) for Er and α-pinene (20%), and γ-terpinene (12%) for Ev EOs ( Table 1). The results suggested that 1,8-cineole has low antiproliferative activity on the THP-1 leukaemia cell line, although in the presence of p-cymene or γ-terpinene, the antiproliferative activity increased.

Conclusions
The highest scavenging of ABTS+ and peroxyl free radicals was observed in Cc and Ev EOs, and the lowest was detected in Eu EO. All essential oils were less effective against S. pneumoniae TIGR4 than against the D39 strain. MIC and MBC values showed that Eg EO is promising for the treatment of S. pneumoniae infections. EOs displayed low diffusion capacity and the antibacterial activity screening through microdilution test would be more appropriate. H. influenzae was the respiratory pathogen that showed the highest resistance to all EOs including that of tea tree. Eg and Es EOs showed the lowest antiproliferative activities on THP-1 cells. After 96 h incubation, the Er and Ev EOs showed the highest antiproliferative activity against THP-1 cells. Although further assays are required, the results suggest that the antiproliferative activity is challenged by the combination of 1,8-cineole with p-cymene or with γ-terpinene. Despite their specific bioactivities, none of the assayed

Conclusions
The highest scavenging of ABTS+ and peroxyl free radicals was observed in Cc and Ev EOs, and the lowest was detected in Eu EO. All essential oils were less effective against S. pneumoniae TIGR4 than against the D39 strain. MIC and MBC values showed that Eg EO is promising for the treatment of S. pneumoniae infections. EOs displayed low diffusion capacity and the antibacterial activity screening through microdilution test would be more appropriate. H. influenzae was the respiratory pathogen that showed the highest resistance to all EOs including that of tea tree. Eg and Es EOs showed the lowest antiproliferative activities on THP-1 cells. After 96 h incubation, the Er and Ev EOs showed the highest antiproliferative activity against THP-1 cells. Although further assays are required, the results suggest that the antiproliferative activity is challenged by the combination of 1,8-cineole with p-cymene or with γ-terpinene. Despite their specific bioactivities, none of the assayed

Conclusions
The highest scavenging of ABTS+ and peroxyl free radicals was observed in Cc and Ev EOs, and the lowest was detected in Eu EO. All essential oils were less effective against S. pneumoniae TIGR4 than against the D39 strain. MIC and MBC values showed that Eg EO is promising for the treatment of S. pneumoniae infections. EOs displayed low diffusion capacity and the antibacterial activity screening through microdilution test would be more appropriate. H. influenzae was the respiratory pathogen that showed the highest resistance to all EOs including that of tea tree. Eg and Es EOs showed the lowest antiproliferative activities on THP-1 cells. After 96 h incubation, the Er and Ev EOs showed the highest antiproliferative activity against THP-1 cells. Although further assays are required, the results suggest that the antiproliferative activity is challenged by the combination of 1,8-cineole with p-cymene or with γ-terpinene. Despite their specific bioactivities, none of the assayed EOs showed simultaneously good antibacterial, antioxidant, and antiproliferative capacity.
Supplementary Materials: The following are available online at www.mdpi.com/2305-6320/5/3/61/s1, Table S1: Percentage composition of the essential oil isolated from Eucalyptus delegatensis subsp. tasmaniensis Boland, previously included in the study of Sena et al. [27] under the former name of E. gigantea.