Industrial Crops and Products 52 (2014) 1–7

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Industrial Crops and Products

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Antibacterial activity and synergistic effects between

Eucalyptus globulus leaf residues (essential oils and extracts)
and antibiotics against several isolates of respiratory tract
infections (Pseudomonas aeruginosa)
V. Pereira a , C. Dias b , M.C. Vasconcelos a,c , E. Rosa a,c , M.J. Saavedra a,b,∗
a
University of Trás-os-Montes e Alto Douro, Apartado 1013, 5000-801 Vila Real, Portugal
b
CECAV/UTAD – Veterinary and Animal Science Research Center, University of Trás-os-Montes e Alto Douro, Apartado 1013, 5000-801 Vila Real, Portugal
c
CITAB/UTAD – Centre for the Research and Technology for Agro-Environment and Biological Sciences, University of Trás-os-Montes e Alto Douro, Apartado
1013, 5000-801 Vila Real, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: This is a novel study of phenolic content and synergistic effects between extracts and Eucalyptus globulus
Received 13 March 2013 leaves essential oil and antibiotics against several isolates from respiratory tract infections (Pseudomonas
Received in revised form 28 August 2013 aeruginosa). The extraction yield obtained with polar solvents was substantially higher, with the maxi-
Accepted 14 September 2013
mum value found for methanol 70%, followed by methanol 100% and water. Of the 16 isolates tested, only
4 (MJH 4, MJH 15, MJH 21 and MJH 40) revealed susceptibly to the extracts tested. The MJH 4 revealed
Keywords:
the highest susceptibility of the 5 extracts tested (water, 70% methanol, 100% methanol, 100% acetone
Eucalyptus globulus residues
and 100% dichloromethane). The isolates tested showed the highest susceptibility to the extracts with
Leaf extracts
Essential oils
higher phenolic compounds content, i.e. the methanolics. The highest inhibition halo value obtained
Antibacterial activity was observed for the oil B against the isolate MJH 4 (19.3 mm), while the minimum value was obtained
Pseudomonas aeruginosa against the isolate MJH 207 (7.7 mm) with oil A. The MIC (minimum inhibitory concentration) value for
Synergistic effects the extracts ranged between 625.0 ␮g/mL and 2500.0 ␮g/mL, while it was also observed that the essen-
tial oil B was more efficient than oil A. The combination between antibiotic and extracts or essential oils
demonstrated an additive effect in 55% of the cases, meaning that the combined use of antibiotic and the
compound tested (extract or essential oil) revealed an increase of antibacterial activity. The use of these
compounds (extracts and oils) of E. globulus may represent an important source of bioactive compounds
and an alternative for the treatment of respiratory infectious diseases caused by P. aeruginosa.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction respiratory infections, rhinitis, and sinusitis (Sadlon and Lamson,

2010). These oils can be found in the leaves of more than 300
The Eucalyptus, a native genus from Australia belongs to Myr- species of this genus and less than 20 of these have ever been
taceae family and comprises about 900 species and subspecies exploited commercially for the production of essential oils rich
(Gilles et al., 2010; Hakki et al., 2010; Tyagi and Malik, 2011). Euca- in 1,8-cineole by pharmaceutical and cosmetic industries (Elaissi
lyptus has been used in folk medicine throughout the world and the et al., 2011). Essential oils are the odorous, volatile products of the
medicinal properties of these plants have been investigated. This secondary metabolism of an aromatic plant, which are often con-
family is an important source of essential oils with a wide range centrated in a particular organ of the plant such as leaves, stems,
of biological activities such as antibacterial, antifungal, analgesic bark or fruit and are stored in secretory cells, cavities, canals, epi-
and anti-inflammatory properties (Mulyaningsih et al., 2010) and dermic cells or glandular trichomes. Approximately 3000 essential
a long history of use against the effects of colds, influenza, other oils are known, 300 of which are commercially important espe-
cially for the pharmaceutical, agronomic, food, sanitary, cosmetic
and perfume industries (Bakkali et al., 2008; Gilles et al., 2010). A
number of studies have demonstrated the antimicrobial properties
∗ Corresponding author at: University of Trás-os-Montes e Alto Douro, Apartado
of Eucalyptus essential oils against a wide range of microorganisms
1013, 5000-801 Vila Real, Portugal.
E-mail addresses: vania.prp@gmail.com (V. Pereira), mcarmov@utad.pt (Gilles et al., 2010; Elaissi et al., 2011). These studies, however, are
(M.C. Vasconcelos), erosa@utad.pt (E. Rosa), saavedra@utad.pt (M.J. Saavedra). focused on a few Eucalyptus species, especially Eucalyptus globulus

0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.09.032
2 V. Pereira et al. / Industrial Crops and Products 52 (2014) 1–7

oil, which has been shown to have a wide spectrum of antimi- (Petri dishes 90 mm of diameter) which were prepared with 20 mL
crobial activity (Mulyaningsih et al., 2010; Tohidpour et al., 2010; of Mueller-Hinton Agar (Oxoid), the paper disks were impregnated
Tyagi and Malik, 2011). Pseudomonas aeruginosa is a major noso- with 15 ␮L of the different extracts and essential oils and placed on
comial pathogen that can be isolated from environmental sources, the agar plate seeded with the respective bacteria. The plates were
particularly freshwater and soil, and is a rare skin commensal in incubated at 37 ◦ C for 24 h. Discs of gentamicin (commonly used
some individuals (Lavoie et al., 2011). One of its preferential niches for gram negative bacteria) were used as positive controls and discs
is the respiratory tract of Intensive Care Unit (ICU) patients with impregnated with DMSO were used as negative controls. After incu-
severe co-morbidities and receiving antibiotic treatments(s), espe- bation, the diameter in mm of the inhibitory or clear zones around
cially in patients with impaired host defenses. This bacterium is a the disk was recorded. All tests were performed in triplicate and
uniquely problematic combination of inherent resistance to many the antibacterial activity was expressed as the mean of inhibition
drug classes and its ability to acquire resistance to all relevant treat- diameters (mm) produced.
ments (El-Solh et al., 2009; Riou et al., 2010). In this study, we
aimed to investigate the antimicrobial properties and the synergis- 2.3.2. Determination of minimum inhibitory concentration
tic effects of essential oils and extracts from E. globulus combined The MIC was evaluated by a resazurin microdilution broth
with a commercial antibiotic against isolates of P. aeruginosa from assay for the microplates method. The bacteria to be tested
respiratory tract infections. To our knowledge so far there are no were picked from overnight cultures in Brain Heart agar (Oxoid).
studies on the synergistic effects of E. globulus residues (extracts A small portion of bacteria was transferred into a bottle with
or essential oils) with antibiotics. Thus, this study brings valuable 50 mL of Mueller-Hinton broth (Oxoid), capped and placed in an
new and valuable information to this area. incubator overnight at a 37 ◦ C. After 12–18 h of incubation, the
bacteria suspension was adjusted, using aseptic preparation, in
2. Materials and methods order to match the optical density in the range of 0.5–1.0 that
was measured at 500 nm. The resazurin solution was prepared
2.1. Plant material and strains by dissolving a 270 mg tablet in 40 mL of sterile distilled water.
A vortex mixer was used to ensure that it was a well-dissolved
The leaves of E. globulus were harvested in the Botanical Gar- and homogenous solution, followed by dilution until 50% with
den of the University of Trás-os-Montes e Alto Douro during the sterile distilled water. The plates used were prepared under
spring season. The essential oils (A and B) were acquired commer- aseptic conditions (96 well plate, Orange Scientific). A volume of
cially in food stores. Both oils were obtained by hydrodistillation. 100 ␮L of Mueller-Hinton broth was used in each well together
A through industrial processing while oil B is from a traditional with 100 ␮L of extract/essential oil in the first line or 50 ␮L of
source (smaller quantities production). According to the manufac- extract/oil in combination with 50 ␮L of antibiotic. From the first
turers, major component was cineol, with 79% in oil A and 83% in well (belonging to the first horizontal line) were taken 100 ␮L,
oil B. Almost all of the bacteria isolates were provided with the added to the next well and then this step is repeated to each of
collaboration of the medical microbiology laboratory of the Veteri- the following wells in the vertical line, allowing a serial dilution
nary Science Department of the University of Trás-os-Montes e Alto of decreasing concentration. For each extract, oil, antibiotic or
Douro and the Centro Hospitalar de Trás-os-Montes e Alto Douro. antibiotic in combination of extracts/oil, 15 different concen-
The isolates were obtained from patients (human and animal) with trations were tested: 10,000 ␮g/mL, 5000 ␮g/mL, 2500 ␮g/mL,
infections of the lower respiratory tract. One of the isolates used in 1250 ␮g/mL, 625 ␮g/mL, 313 ␮g/mL, 156 ␮g/mL, 78 ␮g/mL,
this study was obtained from the American Type Culture Collection 39 ␮g/mL, 19.5 ␮g/mL, 9.8 ␮g/mL, 4.88 ␮g/mL, 2.44 ␮g/mL,
(P. aeruginosa ATCC 10145). 1.22 ␮g/mL and 0.61 ␮g/mL. Note that for the essential oils were
considered the percentages of the dilutions performed, and there-
2.2. Preparation of plant extracts fore for the pure essential oil was considered 100% and for the
other concentrations 50%, 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.78%,
The leaves of E. globulus were freeze dried during 48 h, placed in 0.39%, 0.20%, 0.10%, 0.05%, 0.025%, 0.013% and 0.007%. For this
hermetic bags and then stored in a dark and dry place until extrac- reason instead of MIC we considered this as the antimicrobial
tion. The samples were subsequently crushed and then extracted potential (AP). In each of the wells were also added 20 ␮L of the
in triplicate with 10 mL of each solvent used (1:10), followed by bacteria suspension and 20 ␮L of resazurin solution (50%). The
water bath at 20 or 70 ◦ C. For the solvents water, acetone and plates were then placed in an incubator set at 37 ◦ C for 18–24 h. All
dichloromethane the extraction temperature was 20 ◦ C during tests were performed in triplicate and the MIC was then assessed
16 h; for the methanol 70% and 100% the extraction temperature visually by the color change of the resazurin in each well (blue to
was of 70 ◦ C during 1 h. This difference in the procedure is due to pink in the presence of bacteria growth). The synergistic effects
the polarity of the solvents and extraction efficiency. Samples were classification was made according to Timurkaynak et al. (2006)
filtered using filter paper (Whatman no. 1), followed by centrifu- and FICI (Fractional Inhibitory Concentration Index). Each of the
gation (1500 rpm) during 5 min and then evaporated in the rotary combinations was calculated according to the following formula:
evaporator at 40 ◦ C, with the exception of the samples extracted
MIC (or PA) of drug A in combination
with water that were again freeze dried. The obtained extracts were FICI =
MIC (or PA) of drug A alone
finally resuspended in dimethyl sulfoxide (DMSO) with the final
concentration of 10 mg/mL (10,000 ␮g/mL). The results were interpreted as follows: FICI ≤ 0.5 synergistic,
0.5 < FICI < 1 partially synergistic, FICI = 1 additive, 1 < FICI ≤ 4 indif-
2.3. Antibacterial testing ferent and FICI > 4 antagonistic.

2.3.1. Kirby Bauer paper method 2.4. Total phenolics and individual phenolics (HPLC)
The bacteria tested (P. aeruginosa) were picked from overnight
cultures in Brain Heart agar (Oxoid, England). Antibacterial activity Essentially triplicate samples (3× 500 mg) of the eucalyptus
was tested using a modification of the disk diffusion method (Bauer samples were extracted in 10 mL screw-cap micro-tubes with the
et al., 1966). The suspensions were prepared in sterile saline solu- different solvents (see Section 2.2), with vortex mixing for 10 s
tion by adjusting the turbidity to match 0.5 McFarland standards (LABINCO L46 at maximum speed) every 5 min to optimize the
 V. Pereira et al. / Industrial Crops and Products 52 (2014) 1–7 3

Table 1
Antibacterial activitya of extracts and essential oils from E. globulus against several isolates of P. aeruginosa.

Gentamicin Water Methanol 70% Methanol 100% Acetone Dichloromethane Oil A Oil B DMSO

MJH 4 22.00 ± 0.00d 8.33 ± 0.33b 13.00 ± 0.00a 12.33 ± 0.33a 13.00 ± 0.58a 13.33 ± 0.33a 9.67 ± 0.33cde 19.33 ± 1.76a n.d.
MJH 15 16.00 ± 0.00h 9.67 ± 0.33a 11.67 ± 0.33b 10.67 ± 0.33bc 9.33 ± 0.33c 9.33 ± 0.33c 8.33 ± 0.33fg 12.67 ± 0.88b n.d.
MJH 21 20.50 ± 6.36c 7.00 ± 0.00c 11.00 ± 0.00c 11.00 ± 0.58b 11.00 ± 0.00b 11.00 ± 0.00b 9.00 ± 0.00ef 10.67 ± 0.67cd n.d.
MJH 24 12.00 ± 0.00k n.d. n.d. n.d. n.d. n.d. 8.00 ± 0.58g 9.33 ± 0.33cd n.d.
MJH 40 28.00 ± 0.00a 8.67 ± 0.67b 11.00 ± 0.58c 10.00 ± 0.33c 8.33 ± 0.33d 7.67 ± 0.33d 10.33 ± 0.33c 11.00 ± 0.57bc n.d.
MJH 67 18.00 ± 0.00f n.d. n.d. n.d. n.d. n.d. 9.33 ± 0.33de 9.33 ± 0.88cd n.d.
MJH 149 15.50 ± 0.71i n.d. n.d. n.d. n.d. n.d. 9.00 ± 0.00ef 10.67 ± 0.33cd n.d.
MJH 198 18.00 ± 0.00f n.d. n.d. n.d. n.d. n.d. 8.33 ± 0.33fg 10.00 ± 0.00cd n.d.
MJH 207 15.00 ± 0.00j n.d. n.d. n.d. n.d. n.d. 7.67 ± 0.33g 11.00 ± 0.58bc n.d.
MJH 215 19.00 ± 0.00e n.d. n.d. n.d. n.d. n.d. 8.33 ± 0.33efg 9.67 ± 0.33cd n.d.
MJH 216 26.00 ± 0.00b n.d. n.d. n.d. n.d. n.d. 13.33 ± 0.33a 10.33 ± 0.67cd n.d.
MJH 221 15.00 ± 0.00j n.d. n.d. n.d. n.d. n.d. 10.00 ± 0.00cd 9.00 ± 0.58d n.d.
MJH 260 16.00 ± 0.00h n.d. n.d. n.d. n.d. n.d. 9.00 ± 0.00ef 10.00 ± 0.00cd n.d.
MJH 279 25.00 ± 0.00c n.d. n.d. n.d. n.d. n.d. 9.00 ± 0.00ef 10.67 ± 0.33cd n.d.
MJH 287 18.00 ± 0.00f n.d. n.d. n.d. n.d. n.d. 12.00 ± 0.00b 9.33 ± 0.33cd n.d.
ATCC 10145 17.00 ± 0.00g n.d. n.d. n.d. n.d. n.d. 8.00 ± 0.57g 10.33 ± 0.00cd n.d.
a
Determined by the diameter of inhibition zones (mm); n.d. – non detect.
Data are expressed as means ± SE of triplicate experiments. Mean values in a row with different letters are significantly different at p < 0.05.

extraction; replicates A and B had 1000 ␮L of 70% (v/v) methanol nonparametric Wilcoxon test was used. Statistical calculations
added, replicate C had 950 ␮L of 70% (v/v) methanol and 50 ␮L were based on confidence level equal or higher than 95% (p < 0.05
1 mg/mL naringin (internal standard) added (De Vasconcelos was considered statistically significant).
et al., 2007). Essentially the previously reported multi-purpose
reverse-phase method was used; a PhenomenexLuna C18 (2)
3. Results and discussion
(250 mm × 4.6 mm, 5 ␮m) main column, with a Security guard
pre-column (Phenomenex, United Kingdom) and a C18 cartridge
3.1. Extraction yield
was used in combination with a Thermo-Finnigan Surveyor HPLC
system (solvent de-gasser, quaternary pump, thermostatically
The extraction yield obtained from the samples of E. globu-
controlled auto-sampler set at 10 ◦ C, thermostatically controlled
lus leaves extracted with dichloromethane was the lowest (9%).
column oven set at 25 ◦ C, a photodiode array detector set to collect
The extraction yield of the compounds extracted with polar sol-
overall data from 200 to 600 nm, and selected wavelengths of 227,
vents is substantially higher, being the maximum value obtained
270, 370, and 520 nm). Peak identifications were confirmed from
with methanol 70%, followed by the methanol 100% and the
retention times, UV spectroscopic data, and direct comparison to
water, with yields of 33.0%, 27% and 21%, respectively. Previ-
pure standards. The total phenolics were determined using the
ous results (Vázquez et al., 2008; Pathamanathan et al., 2010)
Folin–Ciocalteau method previously described (Javanmardi et al.,
regarding the polar solvents presented lower values. The work
2003).
developed by Vázquez et al. (2008) in bark of E. globulus reported
the highest yield of extraction for the solvent water (7%) and the
2.5. Statistical analysis lowest for acetone (1%), while the work performed by another
author (Pathamanathan et al., 2010) showed that the higher yield
The data were analyzed using the statistical program SPSS ver- value was obtained with ethanol (4%), followed by water and
sion 14.0 (Statistical Package for the Social Sciences). The mean and methanol (4% and 3%). However in the present work, the methano-
standard deviation within samples were calculated for all cases. lic extracts showed a higher extraction yield when compared with
Because low sample numbers contribute to uneven variation, water.

Table 2
In vitro antimicrobial activitya for the combination of the extracts and essential oils with the antibiotic.

CN CN + 1 CN + 2 CN + 3 CN + 4 CN + 5 CN + OA CN + OB

MJH 4 22.00 ± 0.00d 25.00 ± 2.83a 23.50 ± 2.12b 22.00 ± 2.83b 26.00 ± 2.83a – 27.50 ± 0.71a 30.00 ± 1.41a
MJH 15 16.00 ± 0.00h 16.50 ± 2.12c 15.50 ± 0.71d 14.00 ± 1.41e 16.50 ± 2.12c – 19.00 ± 1.41ef 16.50 ± 0.71de
MJH 21 20.50 ± 6.36c 19.00 ± 2.83b 19.50 ± 0.71c 19.00 ± 1.41c 20.50 ± 0.71b – 26.50 ± 2.12bc 20.00 ± 5.66b
MJH 24 12.00 ± 0.00k 11.00 ± 1.41d 12.50 ± 0.71ef 13.50 ± 0.71ef 13.00 ± 1.41e – 20.00 ± 0.00e 20.00 ± 7.07cd
MJH 40 28.00 ± 0.00a 27.00 ± 1.41a 26.50 ± 2.12a 26.00 ± 0.00a 26.50 ± 0.71a – 31.50 ± 2.12a 23.00 ± 9.90a
MJH 67 18.00 ± 0.00f 17.00 ± 1.41bc 16.50 ± 2.12d 14.50 ± 0.71e 16.00 ± 1.41cd – 18.50 ± 0.71f 17.50 ± 2.12cde
MJH 149 15.50 ± 0.71i 16.50 ± 2.12c 16.00 ± 2.83d 14.00 ± 0.00e 14.50 ± 0.71cde – 18.50 ± 0.71f 16.50 ± 2.12de
MJH 198 18.00 ± 0.00f 15.00 ± 0.00c 15.00 ± 0.00de 13.50 ± 0.71ef 13.50 ± 0.71de – 16.50 ± 0.71f 17.00 ± 0.00cde
MJH 207 15.00 ± 0.00j 14.00 ± 1.41cd 12.00 ± 0.00f 11.50 ± 0.71f 13.00 ± 0.00e – 18.00 ± 0.00ef 17.50 ± 0.71de
MJH 215 19.00 ± 0.00e 17.00 ± 1.41bc 17.50 ± 0.71cd 17.50 ± 0.71cd 17.00 ± 1.41c – 20.00 ± 0.00e 19.50 ± 0.71c
MJH 216 26.00 ± 0.00b 24.50 ± 0.71a 24.00 ± 1.41ab 25.00 ± 1.41a 24.50 ± 0.71a – 23.00 ± 0.00d 22.50 ± 0.71b
MJH 221 15.00 ± 0.00j 16.50 ± 2.12c 16.50 ± 2.12d 15.50 ± 0.71de 15.50 ± 0.71cde – 17.50 ± 3.54ef 17.50 ± 2.12cde
MJH 260 16.00 ± 0.00h 16.00 ± 0.00c 15.00 ± 0.00de 14.50 ± 0.71e 14.50 ± 0.71cde – 17.00 ± 0.00f 15.50 ± 0.71e
MJH 279 25.00 ± 0.00c 26.50 ± 0.71a 25.00 ± 0.00ab 25.00 ± 0.00a 27.00 ± 0.00a – 24.50 ± 0.71cd 24.50 ± 0.71b
MJH 287 18.00 ± 0.00f 15.00 ± 0.00c 15.00 ± 0.00de 15.00 ± 0.00e 15.00 ± 0.00cdee – 16.50 ± 0.71f 15.50 ± 0.71e
ATCC 10145 17.00 ± 0.00g 16.50 ± 0.71c 15.00 ± 0.00de 14.50 ± 0.71e 14.50 ± 0.71cde – 17.50 ± 2.12ef 17.00 ± 1.41cde
a
Determined by the diameter of inhibition zones (mm).
Data are expressed as means ± SE of triplicate experiments. Mean values in a row with different letters are significantly different at p < 0.05.
CN – gentamicin; CN + 1 – gentamicin + water extract; CN + 2 – gentamicin + methanol 70% extract; CN + 3 – gentamicin + methanol 100%; CN + 4 – gentamicin + acetone
extract; CN + 5 – gentamicin + dichloromethane extract; CN + OA – gentamicin + oil A; CN + OB – gentamicin + Oil B.
4 V. Pereira et al. / Industrial Crops and Products 52 (2014) 1–7

3.2. Antimicrobial activity

0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
3.2.1. Antibacterial screening

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
6.25
6.25
6.25
12.50
3.13
12.50
6.25
12.50
3.13
100.00
25.00
25.00
25.00
12.50
12.50
12.50
The results of the antibacterial screening are obtained by the

Oil B
measurement of the inhibition halo observed in mm (inhibi-
tion zone), for the each different compound and the strains of
P. aeruginosa tested. The results of the antibacterial screening of
extracts from E. globulus leaves are described in Table 1. Of the
16 isolates tested, only 4 (MJH 4, MJH 15, MJH 21 and MJH 40)

0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
revealed susceptibly to the extracts tested. From these 4 isolates,

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
the MJH 4 was the one that revealed higher susceptibility when

6.25
6.25
6.25
100.00
6.25
10.00
6.25
100.00
25.00
100.00
100.00
25.00
25.00
25.00
12.50
100.00
Oil A
compared with the other isolates, for all of the 5 extracts tested
(water, 70% methanol, 100% methanol, 100% acetone and 100%
dichloromethane). The lowest inhibition halo was obtained for the
isolate MJH 21 in the aqueous extract (7 mm). From the 4 iso-
lates that presented susceptibility to the extracts, 3 were obtained

0.025 ± 0.00

0.025 ± 0.00

0.025 ± 0.00

0.025 ± 0.00
Essential oils

0.78 ± 0.00
0.10 ± 0.00

0.10 ± 0.00

0.05 ± 0.00

0.05 ± 0.00
0.10 ± 0.00

0.01 ± 0.00
0.05 ± 0.00
0.01 ± 0.00

0.01 ± 0.00
from expectoration (MJH 4, MJH 15 and MJH 40) and only 1 iso-
late was provided from bronchial secretions (MJH 21). For all of

<0.007

<0.007
the isolates tested the antibiotic was the most efficient inhibiting

CN
the bacterial growth. In a previous study (Egwaikhide and Emua,
2010) with root extracts of E. globulus, the extract with the high-
est antibacterial activity was the methanolic extract, the same was
observed in the present study along with the solvents of acetone

Dicloromethane
and dichloromethane. These authors obtained an inhibition halo of

0.00
0.00
0.00

0.00
17.0 mm for P. aeruginosa, higher than the obtained in this study

1250 ±
1250 ±
625 ±

1250 ±
(13.0 mm) for the methanolic extract at 70%. Other reports (Khan

–
–
–
–
–
–
–
–
–
–
–
et al., 2009) revealed no activity against P. aeruginosa with the
ethanolic extract of leafs from E. globulus, which corroborates the
data obtained in this work for the majority of the isolates tested.

0.00
0.00
0.00

0.00
Although these authors used a higher concentration, the inhibi- Acetone
tion halos obtained ranged between 26.0 and 35.0 mm with the
Minimum inhibitory concentration (MIC) detected for the extractsa and antimicrobial potential (AP) for the essential oils.b

1250 ±
1250 ±
625 ±

1250 ±
–

–
–
–
–
–
–
–
–
–
–
–
same extract, but for different bacterial strains, namely Staphylo-
coccus mutans and Staphylococcus aureus. The inhibition halo values
obtained in the present study (for the 4 isolates) were similar to the
results obtained by Badrunnisa et al. (2011) with the leaf extracts
of Eucalyptus tereticornis against P. aeruginosa for the methanolic
Methanol 100%

extracts, which is also observed by other authors (Safaei-Ghomi

0.00
0.00
0.00

0.00

and Ahd, 2010) with aqueous extract of Eucalyptus intertexta and

1250 ±
2500 ±
625 ±

1250 ±

Eucalyptus lorgiflorens.
–

–
–
–
–
–
–
–
–
–
–
–
The essential oils used in this study presented activity against
the 16 isolates tested (Table 1). The highest inhibition halo value
obtained was observed for the oil B against the isolate MJH 4, with
19.3 mm of diameter, while the minimum value was of 7.7 mm for
Methanol 70%

the isolate MJH 207 with the oil A. For oil A the isolate that presented
0.00
0.00
0.00

0.00

the higher susceptibility was MJH 216 with an inhibition halo of

Extracts

1250 ±
625 ±
1250 ±

1250 ±

13.33 mm. Both the isolates (MJH 4 and MJH 216) were also the
–

–
–
–
–
–
–
–
–
–
–
–

more sensitive to the antibiotic, together with MJH 15, MJH 221 and
MJH 279. When compared the effectiveness of the oils against the
isolates, was observed that oil A was less efficient for 5 isolates (MJH
0.00
0.00
0.00

0.00

4, MJH 15, MJH 149, MJH 198 and MJH 207), while oil B revealed
lower efficiency for only 2 isolates (MJH 216 and MJH 287). Previous
1250 ±
2500 ±
1250 ±

2500 ±
Water

–
–
–
–
–
–
–
–
–
–
–

studies showed lower inhibition halo values for P. aeruginosa, in the

presence of other essential oils of Eucalyptus, when compared with
the ones obtained in the present work (Gilles et al., 2010; Elaissi
et al., 2011). Other reports with essential oils of E. globulus tested
Values expressed as % of dilution.

against several bacterial species revealed also lower inhibition halo

9.77 ± 0.00
2.44 ± 0.00
9.77 ± 0.00

4.88 ± 0.00

2.44 ± 0.00
4.88 ± 0.00
9.77 ± 0.00
2.44 ± 0.00

1.22 ± 0.00
4.88 ± 0.00
1.22 ± 0.00
2.44 ± 0.00
1.22 ± 0.00
78.00 ± 0.00

values (Fit et al., 2009; Tohidpour et al., 2010). However, a more

Values expressed as ␮g/mL.
<0.61

<0.61

recent work obtained similar inhibition halos for P. aeruginosa (15

CN

and 17 mm) (Damjanovic-Vratnica et al., 2011). On the other hand

a previous study reported that the essential oils of E. intertexta and
E. larogiflorens did not present any antimicrobial activity against P.
aeruginosa (Safaei-Ghomi and Ahd, 2010).
ATCC 10145

The results obtained for the evaluation of synergism in the initial

MJH 149
MJH 198

MJH 215
MJH 216
MJH 221

MJH 279
MJH 287
MJH 207

MJH 260
Isolates

MJH 15
MJH 21
MJH 24

MJH 67
MJH 40

antibacterial screening are described in Table 2. The combination

MJH 4
Table 3

between the antibiotic and the essential oil A revealed the highest
a

antibacterial values, with 8 isolates presenting synergism (MJH 4,

 V. Pereira et al. / Industrial Crops and Products 52 (2014) 1–7 5

Table 4
Minimum inhibitory concentration (MIC)a and antimicrobial potential (AP)b values for the combination of extracts/oils with the antibiotic.

Isolates CN CN + 1 Extracts Essential oils

CN + 2 CN + 3 CN + 4 CN + 5 CN CN + OA CN + OB

MJH 4 9.77 ± 0.00 39.00 ± 0.00 19.50 ± 0.00 9.77 ± 0.00 9.77 ± 0.00 – 0.10 ± 0.00 12.50 ± 0.00 0.78 ± 0.00
MJH 15 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 – 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00
MJH 21 9.77 ± 0.00 19.50 ± 0.00 19.50 ± 0.00 9.77 ± 0.00 9.77 ± 0.00 – 0.10 ± 0.00 0.78 ± 0.00 0.78 ± 0.00
MJH 24 78.00 ± 0.00 78.00 ± 0.00 39.00 ± 0.00 39.00 ± 0.00 19.5 ± 0.00 – 0.78 ± 0.00 3.13 ± 0.00 6.25 ± 0.00
MJH 40 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 – 0.05 ± 0.00 0.05 ± 0.00 0.05 ± 0.00
MJH 67 <0.61 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 – <0.007 0.02 ± 0.00 0.02 ± 0.00
MJH 149 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 <0.61 2.44 ± 0.00 – 0.02 ± 0.00 0.05 ± 0.00 0.02 ± 0.00
MJH 198 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 – 0.05 ± 0.00 0.05 ± 0.00 0.05 ± 0.00
MJH 207 9.77 ± 0.00 9.77 ± 0.00 9.77 ± 0.00 9.77 ± 0.00 9.77 ± 0.00 – 0.10 ± 0.00 0.10 ± 0.00 0.10 ± 0.00
MJH 215 2.44 ± 0.00 4.88 ± 0.00 2.44 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 – 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00
MJH 216 <0.61 <0.61 <0.61 1.22 ± 0.00 1.22 ± 0.00 – <0.007 <0.007 0.01 ± 0.00
MJH 221 1.22 ± 0.00 1.22 ± 0.00 1.22 ± 0.00 1.22 ± 0.00 1.22 ± 0.00 – 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
MJH 260 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 4.88 ± 0.00 – 0.05 ± 0.00 0.05 ± 0.00 0.05 ± 0.00
MJH 279 1.22 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 – 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.00
MJH 287 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 – 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00
ATCC 10145 1.22 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 2.44 ± 0.00 – 0.01 ± 0.00 0.02 ± 0.00 0.02 ± 0.00
a
Values expressed as ␮g/mL.
b
Values expressed as % of dilution.
CN – gentamicin; CN + 1 – gentamicin + water extract; CN + 2 – gentamicin + methanol 70% extract; CN + 3 – gentamicin + methanol 100%; CN + 4 – gentamicin + acetone
extract; CN + 5 – gentamicin + dichloromethane extract; CN + OA – gentamicin + oil A; CN + OB – gentamicin + oil B.

MJH 15, MJH 24, MJH 40, MJH 149, MJH 207, MJH 215 and MJH 260). value of MIC ranged between 625 ␮g/mL and 2500 ␮g/mL. Simi-
On the other hand, the combination between the antibiotic and lar values were obtained in an earlier study with methanolic and
methanol 70%, methanol 100% and acetone extracts was observed dichloromethane extracts from Eucalyptus camaldulensis and Euca-
some antagonism in 7 of 16 isolates. For the isolates MJH 198 and lyptus torrelliano leaves against P. aeruginosa, being 313.0 ␮g/mL the
MJH 287 was visible an antagonistic effect for the all combinations minimum value of MIC observed (methanolic extract of E. calam-
between antibiotic and extracts/oils. Also for MJH 216 was observed dulensis) and 2500.0 ␮g/mL (methanolic extract of E. torrelliano)
antagonism with the exception of the combination between the the highest (Adeniyi and Ayepola, 2008). In other previous stud-
antibiotic and the methanol 100% extract, showing the same value ies ethanolic and hexane extracts of E. olida and E. staigerana leaves
of the one obtained for the antibiotic alone. were more effective against P. aeruginosa than the ones used in
the present study, with MIC values of 62.5 ␮g/mL–250.0 ␮g/mL for
3.2.2. Determination of minimum inhibitory concentration the methanolic extracts and of 250.0 ␮g/mL–313.0 ␮g/mL for the
According to Andrews (2001) the minimum inhibition con- ethanolic extracts (Dupont et al., 2006). However, in the study of
centration (MIC), can be defined as the minimum concentration Badrunnisa et al. (2011) with ethanolic and methanolic extracts
of antimicrobial agent that is able to inhibit the visible bacte- of E. tereticornis, tested against P. aeruginosa, the values obtained
rial growth after one incubation period of 18–24 h. This method were higher for the methanolic extract (10,000 ␮g/mL) and lower
has been used and is considered a fundamental instrument for for the ethanolic extract (2000 ␮g/mL). On the other hand, in other
the determination of susceptibility of microorganism to antimi- previous studies that evaluated several bacterial species, the value
crobial agents. In Table 3 are presented the values obtained for obtained was lower obtaining results of MICs for the aqueous
the MIC for the 4 isolates (MJH 4, MJH 15, MJH 21 and MJH 40) extract against P. aeruginosa of 32 ␮g/mL (Safaei-Ghomi and Ahd,
which in the initial screening presented some susceptibility to the 2010). In the study of Boulekbache-Makhlouf et al. (2013) that
extracts (water, 70% methanol, 100% methanol, 100% acetone and tested the activity of E. globulus leaves extracts (n-hexane) against
100% dichloromethane). The value of the MIC for the antibiotic two Gram-positive bacteria, were reported MIC values ranging
varied between 2.44 ␮g/mL and 9.77 ␮g/mL. For the extracts the between 30 and 80 ␮g/mL.

Table 5
FICI classification of the synergistic combinations tested.a

CN + 1 CN + 2 CN + 3 CN + 4 CN + OA CN + OB

MJH 4 3.99 1.10 1 1 127.55 7.96

MJH 15 1 1 1 1 1 1
MJH 21 1.10 1.10 1 1 7.96 7.96
MJH 24 1 0.5 0.5 0.25 4.01 8.01
MJH 40 1 1 1 1 1 1
MJH 67 n.d. n.d. n.d. n.d. n.d. n.d.
MJH 149 1 1 n.d. 1 2 1
MJH 198 1 1 1 1 1 1
MJH 207 1 1 1 1 1 1
MJH 215 2 1 2 2 1 1
MJH 216 n.d. n.d. n.d. n.d. n.d. n.d.
MJH 221 1 1 1 1.01 1 1
MJH 260 1 1 1 1 1 1
MJH 279 2 2 2 2 2 2
MJH 287 1 1 1 1 1 1
ATCC 10145 2 2 2 2 2 2
a
FICI = MIC (or AP) of drug in combination/MIC (or AP) of drug alone; FICI ≤ 0.5 – synergism; 0.5 < FICI < 1 – partially synergism; FICI = 1 – additive; 1 < FICI ≤ 4 – indifferent;
FICI > 4 antagonism.
6 V. Pereira et al. / Industrial Crops and Products 52 (2014) 1–7

Table 6
Total phenolic and individual phenolics content of E. globulus leave extracts.

Extract Total phenolicsa Quercetinb Luteolinb

Water 53.42 ± 2.25b 176.25 ± 21.25ab 156.97 ± 18.35a

Methanol 70% 62.10 ± 2.49a 206.51 ± 31.11a 161.57 ± 24.25a
Methanol 100% 47.16 ± 4.99c 144.48 ± 29.17b 93.70 ± 17.97b
Acetone 15.68 ± 0.69d 14.27 ± 3.39c 23.80 ± 6.16c
Dichloromethane 4.07 ± 0.33e – –

Data are presented as means ± SD mg gallic acid equivalents/gram dry weight.

a

Data are presented as means ± SD ␮g/g dry weight.

b

Data are expressed as means ± SE of triplicate experiments. Mean values in a column with different letters are significantly different at p < 0.05.

The results obtained for the determination of the antimicrobial Therefore, this species might be a good candidate for further devel-
potential (AP) of the essential oils tested in this work are described opment as a nutraceuticals. A previous study on the antibacterial
in Table 3. Similar to the results obtained with the extracts, also activity of methanolic extracts of E. camaldulensis reported the pres-
for the essential oils the antibiotic was the most effective. The val- ence of tannins and flavonoids (Babayi et al., 2004). Based in our
ues of the MICs ranged between 0.01–0.78% and 3.13–100% for the results it seems that for some isolates the extracts with higher
antibiotic and essential oils, respectively. Comparing the results of total, quercetin and luteolin have also higher antibacterial activ-
the two essential oils, we find that 8 isolates (MJH 4, MJH 15, MJH 21, ity. These results confirm the antibacterial potential of these two
MJH 149, MJH 215, MJH 221, MJH 260 and MJH 287) presented the phenolics. Thus, plants such as E. globulus, with higher content of
same value for the AP of both oils, however oil B was the most effec- quercetin and luteolin should be considered as important sources
tive for the other 8 isolates. In an earlier report was also observed of bioactivities.
a higher antimicrobial potential for the essential oil of E. globulus
when compared with the antibiotics used (ceftriaxone, amykacine 4. Conclusions
and tetracycline) (Damjanovic-Vratnica et al., 2011).
The results obtained for the determination of the MIC and AP The results obtained in this work revealed that the extraction
values relative to the synergism are described in the Table 4. The yield of the different extraction solvents used was not correlated
MIC/AP values for the antibiotic are in general lower than the ones with the antimicrobial activity observed. Of the isolates tested, only
found for the combination between the antibiotic and extracts/oils. 4 (MJH 4, MJH 15, MJH 21 and MJH 40) revealed susceptibly to
For isolate MJH 24 was observed that the MIC value for the combi- the extracts, with MJH 4 revealing the highest susceptibility. In
nation between the antibiotic with methanol 70%, methanol 100% general oil B showed higher values that oil A of inhibition halos
and acetone is lower than when tested with the antibiotic alone, against the isolates tested. For the majority of the isolates tested
in other words, the combination is more effective. When analyz- the antibiotic was more efficient than the extracts and essen-
ing the FICI classification (Table 5) we verified synergism effects for tial oils used. The combination of antibiotic with oil A was more
the isolate MJH 24 when combined the antibiotic with the methanol effective, showing synergism in 50% of the isolates. On the other
70%, methanol 100% and acetone extracts. On the other hand antag- hand, the methanol 70%, methanol 100% and acetone extracts
onism was observed for 3 isolates (MJH 4, MJH 21 and MJH 24) were the less effective, showing antagonism in 7 of the 16 isolates
when combining the antibiotic with the oils. Finally, 55% of the iso- tested. Regarding the MIC and AP values the antibiotic presented
lates presented additive effect when combining the antibiotic with most effective results, with lower values when compared with the
extracts/oils and for 20.8% the combination was indifferent. extracts and oils. For the synergism tests the MIC and AP values
revealed antagonism for only 3 isolates and for 55% of the cases
3.3. Total and individual phenolics content the combination of the antibiotic with the compounds (extracts
or oils) presented an additive effect. Antioxidant compounds such
Phenolic compounds are often associated with various posi- asphenolics and flavonoids are known for their antibacterial activ-
tive health effects including antioxidant effects, decreases in the ities which might explain the highest susceptibility of the isolates
risk of cardiovascular diseases, anticancer mechanisms and anti- tested to the methanolic extracts that presented the highest values
inflammatory properties. The total phenolics content found in of these compounds.
the extracts tested (Table 6) revealed that the highest values In light of the growing concern with antibiotic resistance these
were found in the 70% methanol extract (62.1 mg/g DW) while results show that the use of these residues (leaf extracts and oils)
the dichloromethane had the lowest values (4.1 mg/g DW). The of E. globulus may represent a potential valuable source of bioac-
total phenolic content values obtained for the several extracts tive compounds and an alternative for the treatment of respiratory
were all significantly different. On the other hand there were infectious diseases caused by P. aeruginosa. However, further stud-
no significant differences between the water and 70% methanol ies need to be performed in order to identify and quantify the
extracts regarding the content of the main individual pheno- constituents present in these extracts and oils that are involved
lics found, quercetin and luteolin. The highest values found of in the synergistic effects.
these compounds belonged to the 70% Methanol extract (206.5
and 161.6 mg/g DW, respectively), while the dichloromethane Acknowledgments
extracts showed values under the detection limits. The essential
oils tested (A and B) possess 79 and 83% of 1,8-cineole (eucalyptol), To the Centro Hospitalar de Trás-os-Montes e Alto Douro for
respectively. This is one of the major compounds found in euca- providing the strains. The Center of Veterinary and Animal Sci-
lyptus leaves essential oils. The content of phenolics found in the ence Research; Microbiology Laboratory of Veterinary Science
tested extracts might explain the antimicrobial activity differences Department and the Center for the Research and Technology for
obtained. Agro-Environment and Biological Sciences for financial support. To
Because of their antioxidant activity, leaf extracts of E. globu- FCT for the financial support of the scholarship of Maria do Car-
lus have been proposed as food additives (Amakura et al., 2009). moVasconcelos (SFRH/BPD/70310/2010).
 V. Pereira et al. / Industrial Crops and Products 52 (2014) 1–7 7

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