Chemical composition, antioxidant, antibacterial and anti-quorum sensing activities of Eucalyptus globulus and Eucalyptus radiata essential oils

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Chemical composition, antioxidant, antibacterial and anti-quorum sensing activities of Eucalyptus globulus and Eucalyptus radiata essential oils Ângelo Luís a , Andreia Duarte a , Jorge Gominho b , Fernanda Domingues a , Ana Paula Duarte a,∗ a b

CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal CEF-ISA, Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal

a r t i c l e

i n f o

Article history: Received 25 June 2015 Received in revised form 26 October 2015 Accepted 30 October 2015 Available online xxx Keywords: Essential oils Eucalyptus globulus Eucalyptus radiata Antioxidant Antimicrobial Anti-quorum sensing

a b s t r a c t The interest in plant polyphenol antioxidants has increased remarkably over the last decade mostly because of their protective effects against different diseases, including cardiovascular, inflammatory and neurological diseases, cancer as well as for retarding aging. Many naturally occurring polyphenols found in plants and spices have also been shown to possess antimicrobial properties and could serve as a source of antimicrobial agents. Eucalyptus globulus and Eucalyptus radiata are well known species that provide essential oils. These oils are in great demand in the market, since they find a vast array of applications. The present study was performed to evaluate some bioactivities of the essential oils from E. globulus and E. radiata, namely their antioxidant, antibacterial and anti-quorum sensing properties. Moreover, its chemical composition was assessed and the potential synergistic activity with conventional antibiotics against Acinetobacter baumannii strains was also evaluated. The major component of the E. globulus oil was 1,8-cineole, also known as eucalyptol (63.81%), and in the E. radiata oil, the principal component was limonene (68.51%). It was possible to conclude that both eucalypt essential oils presented relevant radical scavenging properties and also had the capacity to inhibit the lipid peroxidation. The E. globulus oil antioxidant properties stand out when compared to the E. radiata oil. The E. radiata oil had a more pronounced antibacterial activity than E. globulus oil. The studied eucalypt essential oils can act as potential improving agents of antibiotics against A. baumannii, considering the synergic effect obtained between these oils and conventional antibiotics. Both eucalypt essential oils now studied can inhibit the quorum sensing phenomena, inhibiting quorum sensing-regulated violacein pigment production in bacteria without interfering with their growth. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The interest in antioxidants from plants, namely polyphenols, has increased extremely over the last 10 years, mostly because of their benefic properties in several diseases, including cardiovascular, inflammatory and neurological diseases, cancer, as well as for retarding aging (Asgary et al., 2014; Bastianetto and Quirion, 2002; Gomes de Melo et al., 2012; Lu and Foo, 1997; Scalbert et al., 2005; Wang et al., 2008). The generally accepted mechanism of action of these compounds is that free radical-scavenging activity of polyphenols contributes to reduce the oxidative stress and

∗ Corresponding author. Fax: +351 275 329 099. E-mail address: [email protected] (A.P. Duarte).

to prevent the development of diseases (Huang et al., 2001; Wang et al., 2008). Many plant and spices polyphenols, which naturally occurs, have also shown to have antimicrobial properties and could act as a source of antimicrobial agents (Kotzekidou et al., 2008; Luís et al., 2014a). The antimicrobial properties of plant extracts and essential oils (EOs) has been widely investigated against several human pathogenic microorganisms (Luís et al., 2014c; Andrade et al., 2014; Silva et al., 2011). Furthermore, the multidrug-resistant bacteria has coming out and it represents a challenge to treat the infections, which creates a true need to search for new substances with antimicrobial properties that can replace the conventional antibiotics to fight these microorganisms (Andrade et al., 2014). The emergence of resistance of Gram-negative strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii) has been broadly recognized (Mulyaningsih et al.,

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2011). A. baumannii is an opportunistic pathogen that is usually related with nosocomial infections and is associated with infections acquired mainly in intensive care units (Duarte et al., 2013). This species have the ability to adhere to surfaces and then to form biofilms, and for this reason it can survive for extensive periods in hospital environments (Duarte et al., 2013). Multidrug-resistant pathogens like A. baumannii, make it particularly urgent to search and discover new antimicrobial compounds, such as EOs, which when used in combination with conventional antibiotics could improve the overall efficacy of the treatment creating a synergistic effect (Duarte et al., 2012). Several Gram-negative bacterial strains use signal molecules, like N-acyl homoserine lactones (AHLs), to monitor their own population density (Singh et al., 2009). At a threshold population densities, AHLs interact with cellular receptors and trigger the expression of a set of target genes, including virulence, antibiotic production, biofilm formation, bioluminescence, mobility and swarming, in a process called quorum sensing (QS) (Singh et al., 2009). All these characteristics make the QS a novel approach for the development of new strategies to combat multidrug-resistant pathogens (Singh et al., 2009). There are many reports relating the chemical composition and the antioxidant, antimicrobial and anti-QS activities of EOs, with their use in several commercial preparations such as antimicrobials and antioxidants (Castilho et al., 2012). The mainly constituents of EOs are terpenoids, which are a low molecular weight compounds that can be easily transported across the cell membranes and then induce a range of biological activities, including antioxidant, and antibacterial (Loizzo et al., 2009). Among the EOs with antibacterial activity are the ones of Eucalyptus spp. (Goldbeck et al., 2014). These species are native from Australia, belong to the Myrtaceae family and are usually known as eucalypt, a name that represents more than 700 species worldwide (Goldbeck et al., 2014). The main component of the EOs from eucalypt is the terpene 1,8-cineole, also known as eucalyptol, being the amount of this compound dependent on the specific species (Goldbeck et al., 2014; Ishnava et al., 2013). The concentration of this compound varies between 44% and 84% and it is known to possess significant antimicrobial activity (Goldbeck et al., 2014). The EOs from eucalypt species are among the 18 most commonly traded EOs in the world (Goldbeck et al., 2014). Consequently, there is an increasing interest in their application as a natural additive for food, drugs and cosmetics, both in scientific research and industry (Brooker and Kleinig, 2006; Goldbeck et al., 2014; Ishnava et al., 2013). Eucalyptus globulus and Eucalyptus radiata are well known species that provide EOs which are in great demand by the consumers, since they can be used as anesthetic, antiseptic, astringent, deodorant, disinfectant, expectorant, febrifuge, fumigant, inhalant, insect repellant, and for a folk remedy for abscess, arthritis, asthma, boils, bronchitis, burns, flu, inflammation, rhinitis, worms, and wounds (Bachir and Benali, 2012; Elliot and Jones, 1986). Based on this information, the present study was performed to evaluate some bioactivities of the EOs from E. globulus and E. radiata, namely their antioxidant, antibacterial and anti-QS properties. Moreover, its chemical composition was assessed and the potential synergistic activity with conventional antibiotics against A. baumannii strains was also evaluated.

2. Material and methods 2.1. Eucalypt essential oils Both E. globulus and E. radiata EOs were acquired commercially from a local Pharmacy (Covilhã, Portugal). According to the accom-

panying leaflet, both these oils were obtained by hydrodistillation of leaves and small branches of the tree. The E. globulus EO has its origin in Spain, while the E. radiata EO in Australia. Both these EOs are marketed by the same company (Absolute Aromas Ltd., England) and are produced and certified as biological products to be used in humans (“Soil Association—Organic”), since this trademark belongs to “Aromatherapy Trade Council”. 2.2. Gas chromatography-mass spectrometry (GC–MS) analysis Both essential oils were analyzed in an Agilent 7890A gas chromatograph coupled with an ion trap spectrometer Agilent MS220. The compounds’ identification was assessed using a time database and confronted to the NIST12 mass database. An Agilent VF50 column was used (30 m length, 0.25 mm diameter and 0.25 ␮m thickness). The temperature was initiated at 50 ◦ C and maintained for 5 min; afterwards, the temperature was raised to 180 ◦ C at a rate of 2 ◦ C min−1 and this temperature was maintained for 30 min. The temperature of the injection port and transfer line was set at 230 ◦ C. The split injection mode (ratio 1:20) was adopted, and the carrier gas was helium at a constant flow rate of 1 mL min−1 . The mass spectrometer was operated in the electron ionization mode with an electron energy value of 10 ␮A. The identity of the components was ascertained based on their retention indices and their mass spectra which were compared with those obtained from available libraries. The analysis was repeated two times. 2.3. Antioxidant activity evaluation 2.3.1. DPPH scavenging assay The antioxidant activity of the eucalypt EOs and standards (gallic acid and quercetin (Sigma–Aldrich, USA)) was determined by the free radical scavenging activity method using the 2,2diphenyl-1-picrylhydrazyl (DPPH) radical (Sigma–Aldrich, USA), previously implemented for plant extracts and slightly modified here for EOs (Luís et al., 2014b; Scherer and Godoy, 2009). In brief, aliquots of several concentrations of the EOs or standards (diluted in methanol) (0.1 mL) were added to three DPPH methanolic solutions with different concentrations (3.9 mL): 0.2000, 0.1242 and 0.0800 mM, which were prepared by dissolving 39.4, 24.5 and 15.8 mg of the compound in 500 mL of methanol (Fluka, Milwaukee), respectively. These concentrations were selected due to the linearity range of DPPH solutions: above 0.2 mM the concentration is very high, and below 0.5 mM due to the low concentration, the color is very weak, having a limited range of absorbance reading. The control sample was a solution of 0.1 mL of methanol mixed with 3.9 mL of DPPH. After the incubation period (90 min) at room temperature in the dark, the absorbance was measured at 517 nm using a spectrophotometer (Helios–Omega, Thermo Scientific, USA). The radical scavenging activity was calculated using the following formula: Inhibition (%) =





(Abs0 − Abs1 ) × 100, Abs0

where Abs0 was the absorbance of the control and Abs1 was the absorbance in the presence of the test sample at different concentrations. The IC50 (%) (concentration providing 50% of inhibition) was determined using a calibration curve in the linear range of the graphic, by plotting the EO concentration vs. the corresponding scavenging effect. The antioxidant activity was expressed as the Antioxidant Activity Index (AAI), calculated as follows: AAI = (final concentration of DPPH in the control sample)/(IC50 ) (Scherer and Godoy, 2009). As a result, the AAI was determined considering the mass of DPPH and the mass of the EO in the reaction, resulting in a

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constant for each sample, independent of the concentration of DPPH and sample used. The AAI allowed the following classification for the antioxidant activity of the EOs: poor (AAI ≤ 0.5), moderate (0.5 < AAI ≤ 1.0), strong (1.0 < AAI < 2.0) or very strong (AAI ≥ 2.0) (Scherer and Godoy, 2009). Assays were carried out in duplicate and all DPPH solutions were prepared daily. 2.3.2. ˇ-Carotene bleaching test Subsequent to the preparation of a ␤-carotene (Sigma–Aldrich, USA) solution (20 mg mL−1 in chloroform), 20 ␮L was added to linoleic acid (Fluka, Milwaukee) (40 ␮L), Tween 40 (Sigma–Aldrich, USA) (400 mg) and chloroform (Scharlab, Spain) (1 mL). This mixture was then evaporated (45 ◦ C, 5 min) in a rotary vacuum evaporator to remove chloroform and immediately diluted with oxygenated distilled water (100 mL). The water was added slowly to the mixture, which was then vigorously agitated in order to obtain an emulsion. Afterwards, 5 mL of this emulsion were transferred to test tubes containing the methanolic dilutions of the EOs (300 ␮L). The control sample consisted in 5 mL of the emulsion and 300 ␮L of methanol. Standard butylated hydroxytoluene (BHT) (Fluka, Milwaukee) in methanol, at the same concentrations as the samples, was used as reference, since it is a synthetic antioxidant often used in food industry. The tubes were then softly shaken and placed in a water bath (50 ◦ C, 2 h). The absorbances of the reaction mixtures were finally measured at 470 nm, using a spectrophotometer (Helios–Omega, Thermo Scientific, USA), against a blank consisting of an emulsion without ␤-carotene. The measurements were carried out at initial time (t = 0 h) and at final time (t = 2 h). The antioxidant activity was measured in terms of percentage of inhibition of ␤-carotene’s oxidation, calculated as follows:



Inhibition (%) =

Abst=2 sample − Abst=2 control



Abst=0 control − Abst=2 control





,

where Abst = 2 was the absorbance at final time of incubation of the sample or control and Abst = 0 was the absorbance at initial time of incubation in the control (Luís et al., 2014b). The experiments were performed in triplicate. 2.4. Determination of antibacterial activity 2.4.1. Bacterial strains and culture media Ten different Gram-negative bacterial strains were used for the antimicrobial studies. Reference strains: P. aeruginosa ATCC 27853, E. coli ATCC 25922, K. pneumoniae ATCC 13883, Salmonella Typhimurium ATCC 13311, Acinetobacter baumannii LMG 1025 and A. baumannii LMG 1041. Clinical isolates: P. aeruginosa PA 08, P. aeruginosa PA 12/08, E. coli EC 08 and K. pneumoniae KP 08. The reference strains were acquired from either the American Type Culture Collection (ATCC) (USA) or the BCCM/LMG Bacteria Collection (Belgian Co-ordinated Collections of Micro-organisms, Belgium). Stock cultures were prepared and stored with 20% glycerol (Himedia, India) at −80 ◦ C. All the strains were sub-cultured on the corresponding appropriate agar Plate 24 h before any antimicrobial test. When cultured from stock, the strains were sub-cultured before use. All the bacterial strains used in the work were grown in Brain Heart Infusion Agar (BHI) (Liofilchem, Italy). 2.4.2. Disc diffusion assay and proline test to assess the mechanism of action Antimicrobial activity of both eucalypt EOs was evaluated by the disc diffusion assay, following the M2-A8 method as described by the Clinical Laboratory and Standards Institute (CLSI) for bacteria (M2-A8, 2003). Inoculums were prepared by suspending bacteria in a saline solution to a cell suspension of 0.5 McFarland (about 1 to 2 × 108 colony-forming units mL−1 (CFU mL−1 )) to

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non-fastidious bacteria. Discs with a diameter of 6 mm were each impregnated with 20 ␮L of EO. For the negative controls dimethylsulfoxide (DMSO) (Scharlab, Spain) was used instead. Then, the Müeller-Hinton Agar (MHA) (Liofilchem, Italy) plates were inoculated, allowed to dry and the discs previously prepared were placed over the agar. The plates that were inoculated with non-fastidious bacteria were incubated at 37 ◦ C for 24 h. Following incubation, all the plates were visually checked for inhibition zones and the diameters were measured in millimeters (Luís et al., 2014b). Each experiment was done three independent times. Other researchers (Kwon et al., 2007) have developed a model to study the mode of action of phenolic phytochemicals. This model is based on the rationale that small phenolics in phytochemical profiles could behave as proline analogs or proline analog mimics and could likely inhibit proline oxidation via proline dehydrogenase (Kwon et al., 2007). To perform the proline test, the previously optimized protocol was used (Luís et al., 2014c). Briefly, bacterial lawns were obtained in Petri dishes containing MHA as described above for the disc diffusion assay. Proline (Sigma–Aldrich, USA) was added into the medium to a final concentration of 0.5, 1.0, and 5.0 mM. Eucalypt EOs were added to paper discs with a diameter of 6 mm, and then those paper discs were placed on the surface of agar plates previously seeded. Those plates were incubated at 37 ◦ C for 24 h. The diameter of inhibition zone surrounding each disc was measured and the zone of inhibition was determined (Luís et al., 2014c). Each experiment was done three times. 2.4.3. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of eucalypt EOs were evaluated by the microdilution method described by Duarte et al. (2012). All the tests described in this section were performed in a MüellerHinton Broth (MHB) (Liofilchem, Italy) which was supplemented with DMSO (to maximum final concentration of 2% (v/v)) in order to increase oils solubility. Serial two-fold dilutions of both eucalypt oils (from 32 to 0.25 ␮L mL−1 ) were prepared in a 96-well plate (50 ␮L per well). Those wells to which no EOs had been added were used as a positive growth control. A diluted bacterial suspension in NaCl (Fisher Chemical, USA) 0.85% was added to each well, resulting in a final concentration of 5 × 105 CFU mL−1 , which was confirmed by the number of viable counts. Wells without bacteria were used as a negative growth control. The plates were incubated for 16–20 h at 37 ◦ C and the bacterial growth was assessed visually. The MIC was defined as the lowest EO concentration without visible growth (Duarte et al., 2012). Each assay was done at least three independent times. From the wells without visible growth, 10 ␮L were plated and after incubation the number of colonies was counted. The MBC was defined as the lowest EO concentration which caused the death of 99.9% of the bacterial inoculum (Duarte et al., 2013). At least three independent assays were performed. 2.4.4. Checkerboard titration The checkerboard assay described by Duarte et al. (2012) was employed. For that, two plates were prepared: the first one was used to serial two-fold dilutions of each EO in horizontal orientation, and the second one was used to make the antibiotic dilutions in the vertical orientation. Both dilutions were made in MHB (50 ␮L per well). Using a multichannel pipette (Eppendorf, Germany), 50 ␮L of the antibiotic were transferred to the first plate, and finally 50 ␮L of bacterial suspension (5 × 105 CFU mL−1 ) were added to each well and incubated for 16–20 h at 37 ◦ C. Wells with no EO were used as a positive control and without bacteria as negative control. The used concentrations of antibiotics and eucalypt EOs were selected on the basis of MIC values previously determined. The results of the combined effect of the antibiotics and eucalypt EOs

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were determined and expressed in terms of a fractional inhibitory concentration (FIC) index, which is equal to the sum of the FICs for each antimicrobial compound. The FIC is defined as the MIC of the drug when used in combination with the EO, divided by the MIC of the drug when used alone. The results were considered as a synergy if the FIC index (FICI) of the combination is ≤0.5, additive when it was 0.5 < FICI ≤ 1, subtractive when 1 < FICI < 4 or antagonism for FICI ≥ 4 (Duarte et al., 2012). Experiments were performed at least in three independent assays. 2.4.5. Evaluation of anti-quorum sensing activity The biomonitor strain Chromobacterium violaceum ATCC 12472 was employed to evaluate the anti-QS properties of both eucalypt EOs. The bacterial suspension of C. violaceum ATCC 12472 was obtained by overnight aerobic growth (30 ◦ C, 250 rpm) in LuriaBertani (LB) broth (Sigma–Aldrich, USA). 2.4.6. Disc diffusion test C. violaceum ATCC 12472 suspension was adjusted to an OD620 nm of 1 and the LB agar (Pronadisa, Sapain) plates were seeded. Sterile discs (6 mm diameter) impregnated with 20 ␮L of the EOs were placed over the plates and incubated (30 ◦ C, 24 h). Discs with DMSO were used as negative control. After the incubation period, the inhibition of the pigment production around the disc (a ring of colorless but viable cells) was checked. Antimicrobial activity is indicated by the lack of microbial growth. Therefore, the bacterial growth inhibition was measured as diameter 1 (d1 ) in mm while both bacterial growth and pigment inhibition were measured as total diameter 2 (d2 ) in mm. The QS inhibition (QSI), evaluated by the violacein pigment inhibition, was then determined by subtracting the diameter of bacterial growth inhibition (d1 ) from the total diameter (d2 ) (QSI = d2 –d1 ) (Borges et al., 2014). These experiments were performed in triplicate. 2.4.7. Violacein inhibition assay After the initial screening, using the qualitative agar disc diffusion method, QSI caused by both eucalypt EOs was also quantified by a broth assay. The inhibition of violacein production by C. violaceum ATCC 12472, when exposed to both eucalypt EOs, was quantified according to Borges et al. (2014). The C. violaceum ATCC 12472 suspension was adjusted to an OD620 nm of 0.1 and several EOs concentrations were added to the bacterial suspension. A control with the maximum percentage of DMSO used (0.06% (v/v)) was employed. After the incubation period (24 h, 30 ◦ C, 150 rpm), the violacein extraction was performed. Briefly, 1 mL of culture from each sample was centrifuged (11000 rpm, 10 min), in order to precipitate the insoluble violacein and bacterial cells. After that, the supernatant was discarded, and the pellet was solubilized in DMSO (1 mL), vortexed for 1 min to solubilize the violacein and centrifuged again (11000 rpm, 10 min) to remove the cells. Finally, 200 ␮L of the supernatant containing the violacein were transferred to a 96-well microtiter plate to measure the absorbance at 585 nm using a microplate reader (Bio-Rad 550, UK) (Borges et al., 2014). The assay was performed in three independent experiments. The results were expressed as percentage of violacein inhibition. 2.5. Statistical analysis Results were presented as mean values ± standard deviation or as modal values. In order to determine the measurements reproducibility, each assay was done in duplicate or triplicate, in independent times. Relative Standard Deviation of all measurements was lower than 10% and p < 0.01 was assumed as statistical difference between experimental points.

3. Results and discussion In this work, the antioxidant and antibacterial activities of EOs from E. globulus and E. radiata were studied. The antibacterial properties of these oils were evaluated against several strains of Gram-negative bacteria, both reference strains and clinical isolates. Moreover, the potential existence of synergisms between the oils and conventional antibiotics, against A. baumannii strains, was checked. Finally, the anti-QS properties of both EOs were investigated using the biomonitor strain C. violaceum ATCC 12472. The chemical composition of EOs was assessed by GC–MS analysis. 3.1. Essential oils chemical composition The GC–MS analysis of both eucalypt EOs resulted in a detection of 45 compounds in E. globulus oil, representing 90.32% of the oil (Table 1) and 72 compounds on E. radiata oil, resulting in the identification of 95.89% of the compounds in the oil (Table 1). Both oils were found to be rich in oxygenated monoterpenes, monoterpenes hydrocarbons and sesquiterpenoids. Major components of the E. globulus EO were 1,8-cineole, also known as eucalyptol (63.81%), ␣-pinene (16.06%), aromadendrene (3.68%) and o-cymene (2.35%). In the case of E. radiata EO, the principal components were limonene (68.51%), ␣-terpineol (8.60%), ␣-terpinyl acetate (6.07%) and ␣pinene (3.01%). It is known that Eucalyptus spp. EOs are rich in 1,8-cineole (Ben Hassine et al., 2012; Mulyaningsih et al., 2011), which is in agreement with the results obtained for the studied E. globulus essential oil. This monoterpene has been used for medicinal, flavor and fragrance purposes. 1,8-cineole exhibits mosquito repellency, antitumor properties, and anti-inflammatory activity (Mulyaningsih et al., 2011). Interestingly, the E. radiata oil is rich in limonene. This oil is sometimes preferred by aromatherapists, because its fragrance is more pleasant than the one from E. globulus oil and it appears to be useful for treating disorders of the respiratory tract (Mulyaningsih et al., 2011). High levels of limonene (59–88%) have been previously reported in individual citrus oils (Phillips et al., 2012). The use of this terpene as a platform chemical has been intensively investigated, and many new catalytic processes were reported affording valuable chemicals and polymers (Ciriminna et al., 2014). Indeed, limonene offers a wide range of potential products via chemical or biochemical catalytic conversion (Ciriminna et al., 2014). Other studies have found that the major compound of E. radiata oil is 1,8-cineole (Mulyaningsih et al., 2011). Some differences in EO chemical composition can occur from the same plant species probably due to genetic variation and different environmental factors (climate, harvesting seasons and geographical location) (Mulyaningsih et al., 2011). It was noteworthy that aromadendrene, o-cymene, ␣-terpineol and ␣-terpinyl acetate were commonly found in 1,8-cineole-rich oils, like eucalypt EOs, whereas the monoterpene ␣-pinene was present in all the EOs (Mulyaningsih et al., 2011). 3.2. Antioxidant activity EOs have been qualified as natural antioxidants due to their ability to reduce free radical formation and to scavenge free radicals. They were proposed as potential substitutes for synthetic antioxidants in specific sectors of food preservation (Horvathova et al., 2014). In this work, the antioxidant activity of the eucalypt EOs has been determined by two different test systems namely DPPH and ␤-carotene bleaching test (Table 2). In essence, the antioxidants react with the stable free radical 2,2-diphenyl-1-picrylhydrazyl (deep violet color) and convert it to 2,2-diphenyl-1-picrylhydrazine with discoloration (Sarikurkcu

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Table 1 Chemical composition (% of compound) of the eucalypt essential oils (T, trace, <0.05%). Eucalyptus globulus

Eucalyptus radiata

Compounds

Percentage (%)

Retention time (min)

Compounds

Percentage (%)

Retention time (min)

1,8-Cineole (Eucalyptol) ␣-Pinene Aromadendrene o-Cymene Alloaromandendrene Umbellulon Sabinene ␤-Caryophyllene p-Cymenene 3-Methoxy aceptophenone ␤-Gurjunene Camphene ␣-Phellandrene ␤-Pinene ␣-Caryophyllene Fenchol Thuja-2,4(10)-diene Terpinolene Tricyclene ␥-Muurolene ␣-Gurjunene ␣-Selinene ␣-Copaene Fenchene trans-Pinocamphone ␣-Campholenal Carvone Isoledene 10-epi-␥-Eudesmol ␥-Cadinene ␤-Bourbonene 9-epi-E-Caryophyllene -Cadinene 1,3,8-p-Menthatriene p-Cymene Camphor trans-Calamenene Isobornyl acetate Panasinsene ␣-Muurolene Verbenone E-Occimenone Carvotanacetone 3-Methyl-1-butanol acetate Tetradecene Total identified

63.81 16.06 3.68 2.35 0.74 0.67 0.48 0.26 0.25 0.23 0.23 0.19 0.18 0.11 0.11 0.10 0.09 0.08 0.06 0.06 0.06 0.06 0.05 0.05 0.05 T T T T T T T T T T T T T T T T T T T T 90.32 (45 compounds)

16.64 10.37 43.45 16.02 44.76 25.43 12.91 42.23 15.47 30.17 42.93 11.26 14.76 13.77 44.49 22.46 11.50 20.10 9.61 45.59 41.53 46.95 39.58 11.18 25.29 23.03 31.12 39.25 54.91 48.06 40.05 47.77 48.42 22.66 20.49 24.32 48.60 33.79 39.83 47.26 28.57 29.28 31.44 7.54 40.94

Limonene ␣-Terpineol ␣-Terpinyl acetate ␣-Pinene Terpinen-4-ol ␤-Pinene Sabinene o-Cymene Geranial Neral p-Cymene Linalool -Terpineol ␤-Caryophyllene (-)-Spathulenol Methyl-E-cinnamate ␣-Thujene Globulol Alloaromadendrene Cryptone Eremophyllene Caryophyllene oxide exo-2-Hydroxycineole acetate Viridiflorol Nerol ␤-Ocimene Ledol cis-Limonene oxide Linalyl isobutyrate p-Cymenol Longifolene Aromadendrene (-)-Carvone ␣-Epoxypinene Citronellal ␣-Humulene Camphene trans-Carveol cis-p-Mentha-2,8-dien-1-ol trans-Sabinenehydrate ␤-Elemene Isoborneol Citronellol acetate Cedrenol cis-p-Mentha-1(7),8-dien-2-ol cis-Jasmone ␣-Muurolene E,E-Farnesol cis-Piperitol Torreyol cis-Linalyl oxide ␥-Terpinene Terpinolene trans-p-Mentha-2,8-dien-1-ol Cuminaldehyde ␥-Gurjunene 6-Methyl-5-hepten-2-one cis-Verbenol trans-Pinocarveol ␣-Terpinen-7-al -2-Carene 6,7-Epoxymyrcene Methyl benzoate p-Cymenene cis-Linalyl oxide (pyranoid) Copaene Hexanol (Z)-3-Hexen-1-ol ␣-Phellandrene Pinocarvone Cyperene Sylvestrene Total identified

68.51 8.60 6.07 3.01 1.61 1.13 0.97 0.69 0.61 0.52 0.51 0.40 0.25 0.25 0.24 0.19 0.18 0.15 0.140 0.13 0.10 0.10 0.10 0.10 0.08 0.07 0.05 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 95.89 (72 compounds)

17.01 28.25 38.29 10.60 27.06 14.06 12.94 16.42 33.17 31.13 16.34 21.55 26.38 42.60 55.06 40.68 10.19 55.55 45.09 27.51 47.03 52.30 37.74 53.04 30.17 17.86 51.62 23.73 39.14 27.63 41.86 43.75 31.45 21.32 24.79 44.81 11.52 29.78 24.03 23.14 40.84 24.42 38.55 55.99 30.68 41.18 47.52 61.11 29.09 56.52 19.43 18.58 20.41 22.98 31.31 46.84 13.84 24.64 24.22 34.25 14.90 20.94 21.12 20.80 26.77 39.58 6.45 6.83 15.06 25.77 42.08 17.20

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Table 2 Antioxidant properties of the eucalypt essential oils measured by two different methods (Mean values ± standard deviation). Method

Parameters

Eucalyptus globulus (v/v)

Eucalyptus radiata (v/v)

Gallic acid (w/v)

Quercetin (w/v)

BHT (w/v)

DPPH scavenging assay

IC50 (%) AAI Antioxidant activity IC50 (%)

2.90 ± 0.35 1.74 ± 0.35 Strong 2.72 ± 0.01

4.56 ± 0.70 1.11 ± 0.21 Strong 6.54 ± 0.05

0.22 ± 0.01 22.77 ± 0.25 Very strong –

0.43 ± 0.04 12.17 ± 1.71 Very strong –

– – – 3.58 ± 0.02

␤-Carotene bleaching test

Table 3 Diameter of inhibition zones (mm) of the eucalypt essential oils and its effect on microbial growth inhibition in the presence of proline (Modal values). Strains/Diameters (mm)

P. aeruginosa ATCC 27853 P. aeruginosa PA 08 P. aeruginosa PA 12/08 E. coli ATCC 25922 E. coli EC 08 K. pneumoniae ATCC 13883 K. pneumoniae KP 08 S. ATCC 13311 A. baumannii LMG 1025 A. baumannii LMG 1041 DMSO

Eucalyptus globulus

Eucalyptus radiata

0 mM proline

0.5 mM proline

1 mM proline

5 mM proline

0 mM proline

0.5 mM proline

1 mM proline

5 mM proline

8 7 8 15 12 15 10 12 26 17 6

8 7 8 15 12 14 10 12 26 17 6

8 7 8 15 12 13 8 12 25 17 6

8 6 8 15 12 12 8 12 25 16 6

10 10 10 20 20 19 15 12 30 19 6

10 10 10 20 18 16 15 12 29 19 6

10 10 9 20 17 15 14 12 27 19 6

10 8 9 20 17 15 13 12 27 19 6

et al., 2009). The degree of discoloration indicates the free radical scavenging potential of the sample (Sarikurkcu et al., 2009). Regarding the IC50 values (Table 2), which are defined as the concentration of test material which is able to decrease the initial concentration of DPPH to half of its initial value (Erkan et al., 2008), it is possible to conclude that E. globulus oil has more pronounced antioxidant activity than E. radiata oil, since its IC50 value is lower than the one of E. radiata. Both eucalypt EOs presented strong antioxidant activity, when looking to its values of AAI. The antioxidant activity of Eucalyptus spp. EOs have also been reported by other researchers (Ben Hassine et al., 2012). These properties are related to their phytochemical profile, particularly with the polyphenols. Horvathova et al. (2014) reported previously that the 1,8-cineole showed various degrees of reducing power, radical scavenging, chelating, in addition to DNA-protective capacity (Horvathova et al., 2014). Limonene is also the principal constituent of other EOs with remarkable antioxidant activity (Amiri, 2012). Thus, it could be referred that the antioxidant activity of the eucalypt EOs here studied are mainly due to the presence of its major compounds, namely 1,8-cineole (E. globulus) and limonene (E. radiata). In the ␤-carotene bleaching test, ␤-carotene undergoes rapid discoloration in the absence of an antioxidant. This is because of the coupled oxidation of ␤-carotene and linoleic acid, which generates free radicals (Sarikurkcu et al., 2009). The linoleic acid free radical formed upon the abstraction of a hydrogen atom from one of its diallylic methylene group attacks the highly unsaturated ␤carotene molecules (Sarikurkcu et al., 2009). As a result, ␤-carotene is oxidized and broken down in part; subsequently the system loses its cromophore which give the characteristic orange color, which is monitored spectrophotometrically (Sarikurkcu et al., 2009). This test was selected to determine the antioxidant activity because it allows to assess the ability of EOs to inhibit lipid peroxidation and it is also useful because it is done in an emulsion, similarly to what is found in the food industry (Cruz et al., 2001). Table 2 lists the results obtained with this method and it is possible to observe that the E. globulus oil presented an IC50 lower than the one of BHT (synthetic antioxidant used as reference), which is an indicator of the great capacity of this oil to inhibit the lipid peroxidation, even better than BHT. This is a very promising result for the food preservation, since the unwanted side effects of synthetic antioxidants are widely known, namely liver damage or carcinogenesis (Xu et al., 2009). The antioxidant activity of E. radiata oil is also inferior to

the activity of E. globulus oil, similarly to what had already been found in the DPPH assay. These observations allow to conclude that 1,8-cineole has greater radical scavenging and lipid peroxidation inhibition properties than limonene, the major compounds of both oils. Nevertheless, it is important to mention, that the presence of synergistic effects could lead to the enhancement of the antioxidant properties of the isolated compounds. Considering the overall results in antioxidant activity, it was possible to conclude that both eucalypt EOs presented relevant radical scavenging properties and also had the capacity to inhibit the lipid peroxidation. The E. globulus oil antioxidant properties stand out when compared to the E. radiata oil. 3.3. Antibacterial properties, mechanism of action and synergistic activity In recent decades EOs and their components have attracted increased interest and consequently have been extensively investigated mainly in in vitro systems. Their effectiveness against a wide range of microorganisms is related to their hydrophobicity, which enables them to integrate into the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents (Horvathova et al., 2014). The antibacterial activity of both eucalypt EOs was studied in several strains of Gram-negative bacteria. The agar disc diffusion method was employed since it was found to be a simple, cheap and reproducible practical method (Bachir and Benali, 2012). Observing the results presented in Table 3 (0 mM proline), it is possible to conclude that E. radiata oil was more successful against the tested strains of microorganisms, since the diameter of inhibition zones are bigger than the ones obtained with the E. globulus oil. However, both oils had the ability to spread over the agar plates and to inhibit the growth of the microorganisms, particularly the strains of A. baumannii, which presented the greatest inhibition zones for both eucalypt oils. Kwon et al. (2007) had proposed that if proline dehydrogenase is inhibited by the phenolics, the energy metabolism of the bacterial cells is committed. Therefore, the rationale for the proline growth response assay was to evaluate whether small phenolics behave as proline analogs or proline mimics and, if so, if they could inhibit proline oxidation via proline dehydrogenase at the plasma membrane level in a prokaryotic cell, thus inhibiting bacterial growth

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Table 4 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of eucalypt essential oils (Modal values). Strains

P. aeruginosa ATCC 27853 P. aeruginosa PA 08 P. aeruginosa PA 12/08 E. coli ATCC 25922 E. coli EC 08 K. pneumoniae ATCC 13883 K. pneumoniae KP 08 S. ATCC 13311 A. baumannii LMG 1025 A. baumannii LMG 1041

Eucalyptus globulus

Eucalyptus radiata

MIC (␮L mL-1 )

MBC (␮L mL-1 )

MIC (␮L mL-1 )

MBC (␮L mL-1 )

32 32 32 32 32 16 32 32 4 8

32 32 32 32 32 16 32 32 4 8

32 16 32 16 16 16 16 32 8 8

32 16 32 16 16 16 16 32 8 8

(Kwon et al., 2007). If this is the case, then the addition of proline could overcome the inhibition of proline analog-type phenolics with an aromatic ring structure (Kwon et al., 2007). Analyzing the results obtained with the proline assay (Table 3), proline reversed the inhibitory effects of the eucalypt EOs for some microorganisms indicating that the site of action could be at the proline dehydrogenase level. Particularly, the action of E. radiata oil against the strain E. coli EC 08, is reversed with the increase of proline concentration in culture medium. The same effect is verified for K. pneumoniae ATCC 13883, for both oils. The growth of A. baumannii strains, in the presence of impregnated discs with both eucalypt EOs was also reversed in the presence of increasing proline concentrations, except for the strain of A. baumannii LMG 1041 with E. radiata oil, which value keeps constant. In other strains, namely P. aeruginosa PA 08, the effect of proline supplementation is only observed for the concentration of 5 mM. These results were similar to those obtained by Kwon et al. (2007) and Luís et al. (2014c); in which they observed the same proline reverse inhibitory effect, with extracts of clonal herbs of the family Lamiaceae and with some simple phenolic acids against strains of Staphylococcus aureus (Kwon et al., 2007; Luís et al., 2014c). The MIC and MBC values of both eucalypt oils are presented in Table 4 and vary from 4 to 32 ␮L mL−1 . As it was observed in the disc diffusion assay, the EO of E. radiata presented better antibacterial activity, since generally the MIC values for this oil were lower than the ones obtained for the E. globulus oil. Similar results, concerning MIC values were previously obtained for other EOs and microorganisms (Bachir and Benali, 2012; Silva et al., 2011). It was verified for all the strains, that the MBC values were equal to MIC values which is an indicator of the bactericidal activity of these EOs. The same conclusion was reported on a previous work (Duarte et al., 2012). The best results for the antibacterial activity were observed against both A. baumannii strains, considering their MIC values of eucalypt EOs, which varied from 4 to 8 ␮L mL−1 . These results led us to evaluate the existence of potential synergistic activity of these oils in the presence of some conventional antibiotics (cefoperazone, piperacillin, ciprofloxacin, tetracycline, chloramphenicol and gentamicin) in order to develop a new approach to potentiate the antimicrobial activity of these compounds by eucalypt EOs against A. baumannii. The MIC values of the selected antibiotics against the two reference strains of A. baumannii were previously published by Duarte et al. (2012) and ranged between 0.125 and 64 ␮g mL−1 (Duarte et al., 2012). The results of the checkerboard assay (Table 5) suggest a synergistic action between chloramphenicol, ciprofloxacin, tetracycline and both eucalypt EOs in A. baumannii strains. For gentamicin it was only observed synergism for the reference strain of A. baumannii LMG 1025. As can be observed in Table 5, the best FICI value (0.06), which indicates synergistic activity, was obtained by the combination of E. radiata oil and chloramphenicol against A. baumannii LMG 1041. The combinations of eucalypt EOs and cefoperazone or

Table 5 Fractional inhibitory concentration (FIC) and FIC indices (FICI) of eucalypt essential oils combined with conventional antibiotics against the strains of A. baumannii (Modal values).

E. globulus Cefoperazone E. globulus Piperacillin E. globulus Ciprofloxacin E. globulus Tetracycline E. globulus Chloramphenicol E. globulus Gentamicin E. radiata Cefoperazone E. radiata Piperacillin E. radiata Ciprofloxacin E. radiata Tetracycline E. radiata Chloramphenicol E. radiata Gentamicin

A. baumannii LMG 1025

A. baumannii LMG 1041

FIC index

FICI index

FIC index

FICI index

0.50 0.50 1.00 1.00 0.25 0.12 0.25 0.25 0.06 0.06 0.13 0.12 0.50 0.25 0.50 0.50 0.25 0.06 0.13 0.25 0.06 0.06 0.06 0.24

1.00

0.50 0.50 0.50 0.50 0.25 0.12 0.13 0.25 0.03 0.06 0.50 0.50 0.50 0.50 0.50 0.50 0.13 0.06 0.25 0.25 0.03 0.03 0.50 0.50

1.00

2.00 0.37 0.50 0.12 0.25 0.75 1.00 0.31 0.38 0.12 0.30

1.00 0.37 0.38 0.09 1.00 1.00 1.00 0.19 0.5 0.06 1.00

piperacillin had no synergistic effect against the studied strains. The results obtained in this work were consistent with previous observations in a study dealing with coriander seeds essential oil and the same strains of A. baumannii (Duarte et al., 2012). Notwithstanding these observations, further studies are necessary to clarify the mechanism of action of the synergistic combinations reported here, since the combination between EOs and antibiotics can affect several targets at the same time. Taking into account the evaluation of antibacterial properties of eucalypt EOs done in this work it is possible to conclude that E. radiata oil had a more pronounced antibacterial activity than E. globulus oil. Nevertheless, it seems that both oils can act as proline analogs, inhibiting proline oxidation via proline dehydrogenase. The studied eucalypt EOs can act as potential improving agents of antibiotics against A. baumannii, considering the synergic effect obtained between these oils and conventional antibiotics. 3.4. Anti-QS activity QS is a widespread prokaryotic intercellular communication system based on the signal molecules, known as autoinducers, relative to cell density (Abraham et al., 2011). QS plays a vital role in biofilm formation and virulence factor production in several bac-

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Table 6 Screening of eucalypt essential oils for anti-quorum sensing activity using Chromobacterium violaceum ATCC 12472 (Modal values). Diameters (mm)

d1

d2

QSI (d2 –d1 )

Eucalyptus globulus Eucalyptus radiata DMSO

25 45 6

35 65 6

10 20 0

100

4. Conclusions

90

% Violacein Inhibition

was verified that both oils were able to inhibit the violacein production in a concentration dependent manner. Same conclusions were reported in other studies regarding the inhibition of QS by natural products (Borges et al., 2014; Burt et al., 2014; Abraham et al., 2011; Singh et al., 2009). Overall, the eucalypt EOs now studied can inhibit the QS phenomena, inhibiting QS-regulated violacein pigment production in bacteria without interfering with their growth.

80 70 60 50

E. globulus

40

E. radiata

30 20 10 0 0.1

0.5 1 0.25 [essential oil] (µL mL-1)

5

Fig. 1. Percentage of violacein inhibition by the eucalypt essential oils (Mean values ± standard deviation).

terial species. Consequently, compounds that interfere with the QS system attenuating the bacterial pathogenicity are termed as antiQS compounds (Abraham et al., 2011). Such compounds neither kill the bacteria nor stop their growth and are less expected to develop resistance towards antibiotics (Abraham et al., 2011). In this work, the anti-QS activity of both eucalypt EOs was evaluated using the biomonitor strain Chromobacterium violaceum ATCC 12472. C. violaceum ATCC 12472 is a Gram-negative bacterium which synthesizes the purple pigment violacein, a QS-mediated trait regulated by C6-AHL (Tan et al., 2012). This wild type strain produces and responds to the cognate autoinducer molecules C6AHL and C4-AHL (Adonizio et al., 2006). Anti-QS compounds inhibit production of violacein making this strain excellent for screening the anti-QS activity (Adonizio et al., 2006). Regarding the results of the disc diffusion assay for screening of anti-QS activity of eucalypt EOs (Table 6), it was verified that both oils inhibited the violacein production by C. violaceum ATCC 12472, wherein E. radiata oil presented more anti-QS activity, since the diameter of violacein inhibition was twofold bigger than the one of E. globulus oil. Hence, loss of purple pigment by C. violaceum ATCC 12472 is indicative of QSI by the eucalypt EOs. The results now obtained for the disc diffusion assay were more promising than the ones obtained by other researchers (Borges et al., 2014; Singh et al., 2009), for example, some phytochemicals, studied by Borges et al., (2014); had not presented anti-QS activity with diameter of violacein inhibition equal to 0 mm (Borges et al., 2014). In order to evaluate the extent of QSI, the extraction and quantification of violacein from C. violaceum ATCC 12472 cultures in the absence and presence of eucalypt EOs at different concentrations were also performed. Fig. 1 shows the results of violacein inhibition by both oils. Violacein production is inhibited at all the EOs concentrations tested. The DMSO control presented 11.74 ± 1.10% of violacein production inhibition, which is lower than the results obtained for EOs (value not shown in the Fig. 1). Once again, E. radiata oil was more efficient in the inhibition of violacein production, with percentages of inhibition superiors to 50%, even at the lowest EO concentration. These results confirm the conclusions reached by the disc diffusion assay, namely, the anti-QS potential of these EOs and the best activity shown by the E. radiata oil. Moreover, it

In sum, the essential oils from E. globulus and E. radiata could be considered as potential antioxidant substitutes of synthetic ones, considering their radical scavenging properties as well as lipid peroxidation inhibition capacity. In addition, both oils have demonstrated great antibacterial activity against several Gram-negative bacteria and showed a synergistic effect with several conventional antibiotics against A. baumannii strains. These results, together with the verified anti-QS properties make these oils a possible alternative to the usual antibiotics or disinfectants. Further studies should be conducted in order to better understand the underlying mechanisms responsible for those bioactivities and also with the major compounds of these oils, 1,8-cineole and limonene. Acknowledgments Ângelo Luís acknowledges the research fellowship within the scope of the project titled “The below-ground biomass of Eucalyptus globulus: the forgotten component of forest sustainability” (PTDC/AGR-FOR/3872/2012) funded by Fundac¸ão para a Ciência e a Tecnologia (FCT). CICS-UBI and CEF-ISA are research units supported by the national funding of FCT through the program COMPETE (PEst-C/SAU/UI0709/2011 and PEst-OE/AGR/UI0239/2014, respectively). Authors would like to thank to Prof. Maria Stella Medina Martinez from CEBAS-CSIC (Murcia, Spain) for kindly provide the strain of Chromobacterium violaceum used in this work. References Abraham, S.V.P., Palani, A., Ramaswamy, B.R., Shunmugiah, K.P., Arumugam, V.R., 2011. Antiquorum sensing and antibiofilm potential of Capparis spinosa. Arch. Med. Res. 42, 658–668. Andrade, B.F., Nunes Barbosa, L., da Silva Probst, I., Fernandes Júnior, A., 2014. Antimicrobial activity of essential oils. J. Essent. Oil Res. 26, 34–40. Adonizio, A.L., Downum, K., Bennett, B.C., Mathee, K., 2006. Anti-quorum sensing activity of medicinal plants in southern Florida. J. Ethnopharmacol. 105, 427–435. Amiri, H., 2012. Volatile constituents and antioxidant activity of flowers, stems and leaves of Nasturtium officinale. R. Br. Nat. Prod. Res. 26, 109–115. Asgary, S., Sahebkar, A., Afshani, M.R., Keshvari, M., Haghjooyjavanmard, S., Rafieian-Kopaei, M., 2014. Clinical evaluation of blood pressure lowering, endothelial function improving, hypolipidemic and anti-inflammatory effects of pomegranate juice in hypertensive subjects. Phyther. Res. 28, 193–199. Bachir, R.G., Benali, M., 2012. Antibacterial activity of the essential oils from the leaves of Eucalyptus globulus against Escherichia coli and Staphylococcus aureus. Asian Pac. J. Trop. Biomed. 2, 739–742. Bastianetto, S., Quirion, R., 2002. Natural extracts as possible protective agents of brain aging. Neurobiol. Aging 23, 891–897. Ben Hassine, D., Abderrabba, M., Yvon, Y., Lebrihi, A., Mathieu, F., Couderc, F., Bouajila, J., 2012. Chemical composition and in vitro evaluation of the antioxidant and antimicrobial activities of Eucalyptus gillii essential oil and extracts. Molecules 17, 9540–9558. Borges, A., Serra, S., Cristina Abreu, A., Saavedra, M.J., Salgado, A., Simões, M., 2014. Evaluation of the effects of selected phytochemicals on quorum sensing inhibition and in vitro cytotoxicity. Biofouling 30, 183–195. Brooker, M.I.H., Kleinig, D.A., 2006. Field Guide to Eucalypts. Burt, S.A., Ojo-Fakunle, V.T.A., Woertman, J., Veldhuizen, E.J.A., 2014. The natural antimicrobial carvacrol inhibits quorum sensing in Chromobacterium violaceum and reduces bacterial biofilm formation at sub-lethal concentrations. PLoS One 9, e93414, http://dx.doi.org/10.1371/journal.pone.0093414. Castilho, P.C., Savluchinske-Feio, S., Weinhold, T.S., Gouveia, S.C., 2012. Evaluation of the antimicrobial and antioxidant activities of essential oils, extracts and their main components from oregano from Madeira Island, Portugal. Food Control 23, 552–558.

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Ciriminna, R., Lomeli-Rodriguez, M., Demma Carà, P., Lopez-Sanchez, J.A., Pagliaro, M., 2014. Limonene: a versatile chemical of the bioeconomy. Chem. Commun. 50, 15288–15296. Cruz, J.M., Domínguez, J.M., Domínguez, H., Parajó, J.C., 2001. Antioxidant and antimicrobial effects of extracts from hydrolysates of lignocellulosic materials. J. Agric. Food Chem. 49, 2459–2464. Duarte, A., Ferreira, S., Oliveira, R., Domingues, F., 2013. Effect of coriander oil (Coriandrum sativum) on planktonic and biofilm cells of Acinetobacter baumannii. Nat. Prod. Commun. 8, 673–678. Duarte, A., Ferreira, S., Silva, F., Domingues, F.C., 2012. Synergistic activity of coriander oil and conventional antibiotics against Acinetobacter baumannii. Phytomedicine 19, 236–238. Elliot, W.R., Jones, D., 986. The Encyclopaedia of Australian plants. Erkan, N., Ayranci, G., Ayranci, E., 2008. Antioxidant activities of rosemary (Rosmarinus officinalis L.) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol. Food Chem. 110, 76–82. Goldbeck, J.C., Nascimento, J.E., Jacob, R.G., Fiorentini, Â.M., Silva, W.P., 2014. Bioactivity of essential oils from Eucalyptus globulus and Eucalyptus urograndis against planktonic cells and biofilms of Streptococcus mutans. Ind. Crops Prod. 60, 304–309. Gomes de Melo, J., de Sousa Araújo, T.A., Thijan Nobre de Almeida e Castro, V., Lyra de Vasconcelos Cabral, D., do Desterro Rodrigues, M., Carneiro do Nascimento, S., Hassine, D., Abderrabba, M., Yvon, Y., Lebrihi, A., Mathieu, F., Couderc, F., Bouajila, J., 2012. Chemical composition and in vitro evaluation of the antioxidant and antimicrobial activities of Eucalyptus gillii essential oil and extracts. Molecules 17, 9540–9558. Horvathova, E., Navarova, J., Galova, E., Sevcovicova, A., Chodakova, L., Snahnicanova, Z., Melusova, M., Kozics, K., Slamenova, D., 2014. Assessment of antioxidative, chelating, and DNA-protective effects of selected essential oil components (eugenol, carvacrol, thymol, borneol, eucalyptol) of plants and intact Rosmarinus officinalis oil. J. Agric. Food Chem. 62, 6632–6639. Huang, Y.L., Chen, C.C., Chen, Y., 2001. Identification and quantification of major polyphenols in grape seed. J. Nat. Prod. 64, 903–906. Ishnava, K.B., Chauhan, J.B., Barad, M.B., 2013. Anticariogenic and phytochemical evaluation of Eucalyptus glubules Labill. Saudi J. Biol. Sci. 20, 69–74. Kotzekidou, P., Giannakidis, P., Boulamatsis, A., 2008. Antimicrobial activity of some plant extracts and essential oils against foodborne pathogens in vitro and on the fate of inoculated pathogens in chocolate. LWT - Food Sci. Technol. 41, 119–127. Kwon, Y.-I., Apostolidis, E., Labbe, R.G., Shetty, K., 2007. Inhibition of Staphylococcus aureus by phenolic phytochemicals of selected clonal herbs species of Lamiaceae family and likely mode of action through proline oxidation. Food Biotechnol. 21, 71–89. Loizzo, M.R., Menichini, F., Conforti, F., Tundis, R., Bonesi, M., Saab, A.M., Statti, G.A., Cindio, B., De, H., oughton, P.J., Menichini, F., Frega, N.G., 2009. Chemical

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analysis, antioxidant, antiinflammatory and anticholinesterase activities of Origanum ehrenbergii Boiss and Origanum syriacum L. essential oils. Food Chem. 117, 174–180. Lu, Y., Foo, L.Y., 1997. Identification and quantification of major polyphenols in apple pomace. Food Chem. 59, 187–194. Luís, Â., Breitenfeld, L., Ferreira, S., Duarte, Â.P., Domingues, F., 2014a. Antimicrobial, antibiofilm and cytotoxic activities of Hakea sericea Schrader extracts. Pharmacogn. Mag. 10, S6–S13. Luís, Â., Neiva, D., Pereira, H., Gominho, J., Domingues, F., Duarte, Â.P., 2014b. Stumps of Eucalyptus globulus as a source of antioxidant and antimicrobial polyphenols. Molecules 19, 16428–16446. Luís, Â., Silva, F., Sousa, S., Duarte, Â.P., Domingues, F., 2014c. Antistaphylococcal and biofilm inhibitory activities of gallic, caffeic, and chlorogenic acids. Biofouling 30, 69–79. M2-A8, C., 2003. Padronizac¸ão dos Testes de Sensibilidade a Antimicrobianos por Disco-difusão: Norma Aprovada. p. Oitava Edic¸ão 23, 1. Mulyaningsih, S., Sporer, F., Reichling, J., Wink, M., 2011. Antibacterial activity of essential oils from Eucalyptus and of selected components against multidrug-resistant bacterial pathogens. Pharm. Biol. 49, 893–899. Phillips, C.A., Gkatzionis, K., Laird, K., Score, J., Kant, A., Fielder, M.D., Road, B.G., Gateway, T., Campus, S.B., Thames, K., 2012. Identification and quantification of the antimicrobial components of a citrus essential oil vapour. Nat. Prod. Commun. 7, 103–107. Sarikurkcu, C., Arisoy, K., Tepe, B., Cakir, A., Abali, G., Mete, E., 2009. Studies on the antioxidant activity of essential oil and different solvent extracts of Vitexagnuscastus L fruits from Turkey. Food Chem. Toxicol. 47, 2479–2483. Scalbert, A., Johnson, I.T., Saltmarsh, M., 2005. Polyphenols: antioxidants and beyond. Am. J. Clin. Nutr. 81, 215–217. Scherer, R., Godoy, H.T., 2009. Antioxidant activity index (AAI) by the 2,2-diphenyl-1-picrylhydrazyl method. Food Chem. 112, 654–658. Silva, F., Ferreira, S., Queiroz, J., Domingues, F., 2011. Coriander (Coriandrum sativum L.) essential oil: its antibacterial activity and mode of action evaluated by flow cytometry. J. Med. Microbiol. 60, 1479–1486. Singh, B.N., Singh, B.R., Singh, R.L., Prakash, D., Sarma, B.K., Singh, H.B., 2009. Antioxidant and anti-quorum sensing activities of green pod of Acacia nilotica L. Food Chem. Toxicol. 47, 778–786. Tan, L.Y., Yin, W.-F., Chan, K.-G., 2012. Silencing quorum sensing through extracts of Melicope lunuankenda. Sensors 12, 4339–4351. Wang, H., Zhao, M., Yang, B., Jiang, Y., Rao, G., 2008. Identification of polyphenols in tobacco leaf and their antioxidant and antimicrobial activities. Food Chem. 107, 1399–1406. Xu, W., Zhang, F., Luo, Y., Ma, L., Kou, X., Huang, K., 2009. Antioxidant activity of a water-soluble polysaccharide purified from Pteridium aquilinum. Carbohydr. Res. 344, 217–222.

Please cite this article in press as: Luís, Â., et al., Chemical composition, antioxidant, antibacterial and anti-quorum sensing activities of Eucalyptus globulus and Eucalyptus radiata essential oils. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.10.055