Industrial Crops and Products 79 (2016) 274–282

Contents lists available at ScienceDirect

Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

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
CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal
b
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 a b s t r a c t

Article history: The interest in plant polyphenol antioxidants has increased remarkably over the last decade mostly
Received 25 June 2015 because of their protective effects against different diseases, including cardiovascular, inflammatory and
Received in revised form 26 October 2015 neurological diseases, cancer as well as for retarding aging. Many naturally occurring polyphenols found
Accepted 30 October 2015
in plants and spices have also been shown to possess antimicrobial properties and could serve as a source
Available online 21 November 2015
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.
Keywords:
The present study was performed to evaluate some bioactivities of the essential oils from E. globulus
Essential oils
Eucalyptus globulus
and E. radiata, namely their antioxidant, antibacterial and anti-quorum sensing properties. Moreover, its
Eucalyptus radiata chemical composition was assessed and the potential synergistic activity with conventional antibiotics
Antioxidant against Acinetobacter baumannii strains was also evaluated. The major component of the E. globulus oil
Antimicrobial was 1,8-cineole, also known as eucalyptol (63.81%), and in the E. radiata oil, the principal component
Anti-quorum sensing 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 pro-
nounced 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 quo-
rum 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 to prevent the development of diseases (Huang et al., 2001; Wang

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 cardiovas-
cular, 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 activ-
ity of polyphenols contributes to reduce the oxidative stress and
∗ Corresponding author. Fax: +351 275 329 099.
E-mail address: apcd@ubi.pt (A.P. Duarte).
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 Acineto-
bacter baumannii) has been broadly recognized (Mulyaningsih et al.,

http://dx.doi.org/10.1016/j.indcrop.2015.10.055
0926-6690/© 2015 Elsevier B.V. All rights reserved.
 Â. Luís et al. / Industrial Crops and Products 79 (2016) 274–282
2011). A. baumannii is an opportunistic pathogen that is usually
related with nosocomial infections and is associated with infec-
tions 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 popula-
tion densities, AHLs interact with cellular receptors and trigger
the expression of a set of target genes, including virulence, antibi-
otic 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 Euca-
lyptus 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 world-
wide (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 addi-
tive 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, fumi-
gant, inhalant, insect repellant, and for a folk remedy for abscess,
arthritis, asthma, boils, bronchitis, burns, flu, inflammation, rhini-
tis, 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 prop-
erties. 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-
275
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., Eng-
land) 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 chro-
matograph 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 2,2-
diphenyl-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 methano-
lic 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, Milwau-
kee), 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 tem-
perature 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 for-
mula:
 
(Abs0 − Abs1 )
Inhibition (%) = × 100,
Abs0
where Abs0 was the absorbance of the control and Abs1 was the
absorbance in the presence of the test sample at different concen-
trations. 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 correspond-
ing 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
276 Â. Luís et al. / Industrial Crops and Products 79 (2016) 274–282
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 mix-
ture 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 trans-
ferred 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 spectropho-
tometer (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:
 
Abst=2 sample − Abst=2 control
Inhibition (%) =  
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 Cul-
ture 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 (Hime-
dia, India) at −80 ◦ C. All the strains were sub-cultured on the
corresponding appropriate agar Plate 24 h before any antimicro-
bial 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 bacte-
ria 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
non-fastidious bacteria. Discs with a diameter of 6 mm were each
impregnated with 20 ␮L of EO. For the negative controls dimethyl-
sulfoxide (DMSO) (Scharlab, Spain) was used instead. Then, the
Müeller-Hinton Agar (MHA) (Liofilchem, Italy) plates were inocu-
lated, 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 dehydroge-
nase (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 Müeller-
Hinton 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 orienta-
tion, 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
 Â. Luís et al. / Industrial Crops and Products 79 (2016) 274–282
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 syn-
ergy 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 euca-
lypt EOs. The bacterial suspension of C. violaceum ATCC 12472 was
obtained by overnight aerobic growth (30 ◦ C, 250 rpm) in Luria-
Bertani (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 incu-
bation 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 subtract-
ing 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 diffu-
sion method, QSI caused by both eucalypt EOs was also quantified
by a broth assay. The inhibition of violacein production by C. vio-
laceum 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 con-
trol 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 measure-
ments was lower than 10% and p < 0.01 was assumed as statistical
difference between experimental points.
277
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 iso-
lates. 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 investi-
gated 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 iden-
tification 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 agree-
ment 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, anti-
tumor 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 fra-
grance 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 prod-
ucts 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 envi-
ronmental 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 abil-
ity to reduce free radical formation and to scavenge free radicals.
They were proposed as potential substitutes for synthetic antiox-
idants 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
278 Â. Luís et al. / Industrial Crops and Products 79 (2016) 274–282

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) 63.81 16.64 Limonene 68.51 17.01

␣-Pinene 16.06 10.37 ␣-Terpineol 8.60 28.25
Aromadendrene 3.68 43.45 ␣-Terpinyl acetate 6.07 38.29
o-Cymene 2.35 16.02 ␣-Pinene 3.01 10.60
Alloaromandendrene 0.74 44.76 Terpinen-4-ol 1.61 27.06
Umbellulon 0.67 25.43 ␤-Pinene 1.13 14.06
Sabinene 0.48 12.91 Sabinene 0.97 12.94
␤-Caryophyllene 0.26 42.23 o-Cymene 0.69 16.42
p-Cymenene 0.25 15.47 Geranial 0.61 33.17
3-Methoxy aceptophenone 0.23 30.17 Neral 0.52 31.13
␤-Gurjunene 0.23 42.93 p-Cymene 0.51 16.34
Camphene 0.19 11.26 Linalool 0.40 21.55
␣-Phellandrene 0.18 14.76 -Terpineol 0.25 26.38
␤-Pinene 0.11 13.77 ␤-Caryophyllene 0.25 42.60
␣-Caryophyllene 0.11 44.49 (-)-Spathulenol 0.24 55.06
Fenchol 0.10 22.46 Methyl-E-cinnamate 0.19 40.68
Thuja-2,4(10)-diene 0.09 11.50 ␣-Thujene 0.18 10.19
Terpinolene 0.08 20.10 Globulol 0.15 55.55
Tricyclene 0.06 9.61 Alloaromadendrene 0.140 45.09
␥-Muurolene 0.06 45.59 Cryptone 0.13 27.51
␣-Gurjunene 0.06 41.53 Eremophyllene 0.10 47.03
␣-Selinene 0.06 46.95 Caryophyllene oxide 0.10 52.30
␣-Copaene 0.05 39.58 exo-2-Hydroxycineole acetate 0.10 37.74
Fenchene 0.05 11.18 Viridiflorol 0.10 53.04
trans-Pinocamphone 0.05 25.29 Nerol 0.08 30.17
␣-Campholenal T 23.03 ␤-Ocimene 0.07 17.86
Carvone T 31.12 Ledol 0.05 51.62
Isoledene T 39.25 cis-Limonene oxide T 23.73
10-epi-␥-Eudesmol T 54.91 Linalyl isobutyrate T 39.14
␥-Cadinene T 48.06 p-Cymenol T 27.63
␤-Bourbonene T 40.05 Longifolene T 41.86
9-epi-E-Caryophyllene T 47.77 Aromadendrene T 43.75
-Cadinene T 48.42 (-)-Carvone T 31.45
1,3,8-p-Menthatriene T 22.66 ␣-Epoxypinene T 21.32
p-Cymene T 20.49 Citronellal T 24.79
Camphor T 24.32 ␣-Humulene T 44.81
trans-Calamenene T 48.60 Camphene T 11.52
Isobornyl acetate T 33.79 trans-Carveol T 29.78
Panasinsene T 39.83 cis-p-Mentha-2,8-dien-1-ol T 24.03
␣-Muurolene T 47.26 trans-Sabinenehydrate T 23.14
Verbenone T 28.57 ␤-Elemene T 40.84
E-Occimenone T 29.28 Isoborneol T 24.42
Carvotanacetone T 31.44 Citronellol acetate T 38.55
3-Methyl-1-butanol acetate T 7.54 Cedrenol T 55.99
Tetradecene T 40.94 cis-p-Mentha-1(7),8-dien-2-ol T 30.68
Total identified 90.32 (45 compounds) cis-Jasmone T 41.18
␣-Muurolene T 47.52
E,E-Farnesol T 61.11
cis-Piperitol T 29.09
Torreyol T 56.52
cis-Linalyl oxide T 19.43
␥-Terpinene T 18.58
Terpinolene T 20.41
trans-p-Mentha-2,8-dien-1-ol T 22.98
Cuminaldehyde T 31.31
␥-Gurjunene T 46.84
6-Methyl-5-hepten-2-one T 13.84
cis-Verbenol T 24.64
trans-Pinocarveol T 24.22
␣-Terpinen-7-al T 34.25
-2-Carene T 14.90
6,7-Epoxymyrcene T 20.94
Methyl benzoate T 21.12
p-Cymenene T 20.80
cis-Linalyl oxide (pyranoid) T 26.77
Copaene T 39.58
Hexanol T 6.45
(Z)-3-Hexen-1-ol T 6.83
␣-Phellandrene T 15.06
Pinocarvone T 25.77
Cyperene T 42.08
Sylvestrene T 17.20
Total identified 95.89 (72 compounds)
 Â. Luís et al. / Industrial Crops and Products 79 (2016) 274–282 279

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 (%) 2.90 ± 0.35 4.56 ± 0.70 0.22 ± 0.01 0.43 ± 0.04 –
AAI 1.74 ± 0.35 1.11 ± 0.21 22.77 ± 0.25 12.17 ± 1.71 –
Antioxidant activity Strong Strong Very strong Very strong –
␤-Carotene bleaching test IC50 (%) 2.72 ± 0.01 6.54 ± 0.05 – – 3.58 ± 0.02

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) 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

P. aeruginosa ATCC 27853 8 8 8 8 10 10 10 10

P. aeruginosa PA 08 7 7 7 6 10 10 10 8
P. aeruginosa PA 12/08 8 8 8 8 10 10 9 9
E. coli ATCC 25922 15 15 15 15 20 20 20 20
E. coli EC 08 12 12 12 12 20 18 17 17
K. pneumoniae ATCC 13883 15 14 13 12 19 16 15 15
K. pneumoniae KP 08 10 10 8 8 15 15 14 13
S. ATCC 13311 12 12 12 12 12 12 12 12
A. baumannii LMG 1025 26 26 25 25 30 29 27 27
A. baumannii LMG 1041 17 17 17 16 19 19 19 19
DMSO 6 6 6 6 6 6 6 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 ini-
tial concentration of DPPH to half of its initial value (Erkan et al.,
2008), it is possible to conclude that E. globulus oil has more pro-
nounced 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 antioxi-
dant 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 stud-
ied 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 gen-
erates 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 preserva-
tion, 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 rad-
ical 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 investi-
gated 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). Observ-
ing 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. How-
ever, 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 mem-
brane level in a prokaryotic cell, thus inhibiting bacterial growth
280 Â. Luís et al. / Industrial Crops and Products 79 (2016) 274–282

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

Strains Eucalyptus globulus Eucalyptus radiata

MIC (␮L mL-1 ) MBC (␮L mL-1 ) MIC (␮L mL-1 ) MBC (␮L mL-1 )

P. aeruginosa ATCC 27853 32 32 32 32

P. aeruginosa PA 08 32 32 16 16
P. aeruginosa PA 12/08 32 32 32 32
E. coli ATCC 25922 32 32 16 16
E. coli EC 08 32 32 16 16
K. pneumoniae ATCC 13883 16 16 16 16
K. pneumoniae KP 08 32 32 16 16
S. ATCC 13311 32 32 32 32
A. baumannii LMG 1025 4 4 8 8
A. baumannii LMG 1041 8 8 8 8

(Kwon et al., 2007). If this is the case, then the addition of pro- Table 5
Fractional inhibitory concentration (FIC) and FIC indices (FICI) of eucalypt essen-
line could overcome the inhibition of proline analog-type phenolics
tial oils combined with conventional antibiotics against the strains of A. baumannii
with an aromatic ring structure (Kwon et al., 2007). Analyzing the (Modal values).
results obtained with the proline assay (Table 3), proline reversed
A. baumannii LMG 1025 A. baumannii LMG 1041
the inhibitory effects of the eucalypt EOs for some microorganisms
indicating that the site of action could be at the proline dehydroge- FIC index FICI index FIC index FICI index
nase level. Particularly, the action of E. radiata oil against the strain E. globulus 0.50 1.00 0.50 1.00
E. coli EC 08, is reversed with the increase of proline concentration in Cefoperazone 0.50 0.50
culture medium. The same effect is verified for K. pneumoniae ATCC E. globulus 1.00 2.00 0.50 1.00
13883, for both oils. The growth of A. baumannii strains, in the pres- Piperacillin 1.00 0.50
E. globulus 0.25 0.37 0.25 0.37
ence of impregnated discs with both eucalypt EOs was also reversed Ciprofloxacin 0.12 0.12
in the presence of increasing proline concentrations, except for the E. globulus 0.25 0.50 0.13 0.38
strain of A. baumannii LMG 1041 with E. radiata oil, which value Tetracycline 0.25 0.25
keeps constant. In other strains, namely P. aeruginosa PA 08, the E. globulus 0.06 0.12 0.03 0.09
Chloramphenicol 0.06 0.06
effect of proline supplementation is only observed for the concen-
E. globulus 0.13 0.25 0.50 1.00
tration of 5 mM. These results were similar to those obtained by Gentamicin 0.12 0.50
Kwon et al. (2007) and Luís et al. (2014c); in which they observed E. radiata 0.50 0.75 0.50 1.00
the same proline reverse inhibitory effect, with extracts of clonal Cefoperazone 0.25 0.50
herbs of the family Lamiaceae and with some simple phenolic acids E. radiata 0.50 1.00 0.50 1.00
Piperacillin 0.50 0.50
against strains of Staphylococcus aureus (Kwon et al., 2007; Luís
E. radiata 0.25 0.31 0.13 0.19
et al., 2014c). Ciprofloxacin 0.06 0.06
The MIC and MBC values of both eucalypt oils are presented in E. radiata 0.13 0.38 0.25 0.5
Table 4 and vary from 4 to 32 ␮L mL−1 . As it was observed in the disc Tetracycline 0.25 0.25
E. radiata 0.06 0.12 0.03 0.06
diffusion assay, the EO of E. radiata presented better antibacterial
Chloramphenicol 0.06 0.03
activity, since generally the MIC values for this oil were lower than E. radiata 0.06 0.30 0.50 1.00
the ones obtained for the E. globulus oil. Similar results, concerning Gentamicin 0.24 0.50
MIC values were previously obtained for other EOs and microor-
ganisms (Bachir and Benali, 2012; Silva et al., 2011). It was verified
for all the strains, that the MBC values were equal to MIC values piperacillin had no synergistic effect against the studied strains.
which is an indicator of the bactericidal activity of these EOs. The The results obtained in this work were consistent with previous
same conclusion was reported on a previous work (Duarte et al., observations in a study dealing with coriander seeds essential oil
2012). The best results for the antibacterial activity were observed and the same strains of A. baumannii (Duarte et al., 2012). Notwith-
against both A. baumannii strains, considering their MIC values of standing these observations, further studies are necessary to clarify
eucalypt EOs, which varied from 4 to 8 ␮L mL−1 . These results led the mechanism of action of the synergistic combinations reported
us to evaluate the existence of potential synergistic activity of these here, since the combination between EOs and antibiotics can affect
oils in the presence of some conventional antibiotics (cefopera- several targets at the same time.
zone, piperacillin, ciprofloxacin, tetracycline, chloramphenicol and Taking into account the evaluation of antibacterial properties
gentamicin) in order to develop a new approach to potentiate the of eucalypt EOs done in this work it is possible to conclude that
antimicrobial activity of these compounds by eucalypt EOs against E. radiata oil had a more pronounced antibacterial activity than E.
A. baumannii. The MIC values of the selected antibiotics against the globulus oil. Nevertheless, it seems that both oils can act as proline
two reference strains of A. baumannii were previously published analogs, inhibiting proline oxidation via proline dehydrogenase.
by Duarte et al. (2012) and ranged between 0.125 and 64 ␮g mL−1 The studied eucalypt EOs can act as potential improving agents
(Duarte et al., 2012). of antibiotics against A. baumannii, considering the synergic effect
The results of the checkerboard assay (Table 5) suggest a syner- obtained between these oils and conventional antibiotics.
gistic action between chloramphenicol, ciprofloxacin, tetracycline
and both eucalypt EOs in A. baumannii strains. For gentamicin it was
3.4. Anti-QS activity
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),
QS is a widespread prokaryotic intercellular communication
which indicates synergistic activity, was obtained by the combi-
system based on the signal molecules, known as autoinducers, rel-
nation of E. radiata oil and chloramphenicol against A. baumannii
ative to cell density (Abraham et al., 2011). QS plays a vital role in
LMG 1041. The combinations of eucalypt EOs and cefoperazone or
biofilm formation and virulence factor production in several bac-
 Â. Luís et al. / Industrial Crops and Products 79 (2016) 274–282
Table 6
Screening of eucalypt essential oils for anti-quorum sensing activity using Chro-
mobacterium violaceum ATCC 12472 (Modal values).
Diameters (mm) d1 d2
Eucalyptus globulus 25 35
Eucalyptus radiata 45 65
DMSO 6 6
100
90
80
% Violacein Inhibition
70
60
50
40
30
20
10
0 be conducted in order to better understand the underlying mech-
0.1 0.25 0.5 1 5
[essential oil] (µL mL-1)
Fig. 1. Percentage of violacein inhibition by the eucalypt essential oils (Mean val-
ues ± standard deviation).
terial species. Consequently, compounds that interfere with the QS
system attenuating the bacterial pathogenicity are termed as anti-
QS 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 evalu-
ated 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 C6-
AHL 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 quan-
tification 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 inhibi-
tion 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. radi-
ata 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
281
was verified that both oils were able to inhibit the violacein pro-
duction in a concentration dependent manner. Same conclusions
were reported in other studies regarding the inhibition of QS by
QSI (d2 –d1 ) natural products (Borges et al., 2014; Burt et al., 2014; Abraham
10 et al., 2011; Singh et al., 2009).
20 Overall, the eucalypt EOs now studied can inhibit the QS phe-
0 nomena, inhibiting QS-regulated violacein pigment production in
bacteria without interfering with their growth.
4. Conclusions
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 demon-
E. globulus strated great antibacterial activity against several Gram-negative
E. radiata 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 alterna-
tive to the usual antibiotics or disinfectants. Further studies should
anisms 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 Euca-
lyptus globulus: the forgotten component of forest sustainability”
(PTDC/AGR-FOR/3872/2012) funded by Fundação 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, respec-
tively). 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.
282 Â. Luís et al. / Industrial Crops and Products 79 (2016) 274–282
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
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. Padronização dos Testes de Sensibilidade a Antimicrobianos por
Disco-difusão: Norma Aprovada. p. Oitava Edição 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.