Industrial Crops & Products 158 (2020) 112964

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Industrial Crops & Products

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Variation in chemical profile of leaves essential oils from thirteen Tunisian

Eucalyptus species and evaluation of their antioxidant and
antibacterial properties
Hajer Limam a, b, Mariem Ben Jemaa b, Sonia Tammar b, Nour Ksibi a, b, Saber Khammassi b,
Selim Jallouli c, Giovanni Del Re d, Kamel Msaada b, *
a
College of Sciences of Tunis, Tunis El Manar University, 2092, Tunis, Tunisia
b
Laboratory of Aromatic and Medicinal Plants (LPAM), Biotechnology Center in Borj Cedria Technopole, BP. 901, Hammam-Lif, 2050, Tunisia
c
Laboratory of Bioactive Substances (LSBA), Biotechnology Center of Borj-Cédria, BP 901, 2050, Hammam-lif, Tunisia
d
Università dell’Aquila, Dipartimento di Ingegneria Industriale e dell’ Informazione e di Economia, Piazzale Ernesto Pontieri, Monteluco di Roio, 67100, L’Aquila, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: The aromatic medicinal plant Eucalyptus and its essential oils have been used since antiquity in folk medicine.
Eucalyptus leaves The interest in this genus has increased remarkably over the last decade mostly because of their various bio­
Essential oils logical activities. This study aimed to determine the chemical composition of essential oils of thirteen Eucalyptus
1.8-cineole
species leaves and their antioxidant and antibacterial activities. Obtained results showed that E. maidenii had the
Antioxidant activities
Antibacterial activities
highest essential oil yield (6.16 ± 1.55 %). Chemical composition (by GC/MS) was highly affected by the species
Interspecific variation effect factor (P < 0.001), with the abundance of the major compound 1,8-cineole (82.64 % in E. microcarpa) in most
Eucalyptus species. Essential oils exhibited a moderate antioxidant activity, but an interesting bacteriostatic effect
against all tested bacteria. Eucalyptus camaldulensis had the strongest bioactivities despite its major compound
was spathulenol with efficacy concentration of 50 % EC50 = 2.96 ± 0.11 mg/mL for the reducing power assay and
presented an important inhibition zone (25.33 ± 2.84) and lowest minimum inhibitory concentration
(MIC = 0.93 mg/mL) against Serratia marcescens bacteria. This result demonstrated that biological activities
could be explained by the synergistic effect between major and minor compounds.

1. Introduction
The Eucalyptus genus is indigenous to Australia belongs to the Myr­
taceae family that includes more than 800 species (Tyagi and Malik,
2011). As in worldwide, Eucalyptus is extensively cultivated in Tunisia
where 117 species had been introduced in 1957 (Boulekbache-Makhlouf
et al., 2013) and have been used in folk medicine. Recently, many
species of this aromatic medicinal plant were investigated for their
medicinal properties (Pereira et al., 2014). Eucalyptus leaves have a
crucial role against many diseases such as cancer, cardiovascular,
neurodegenerative diseases (Asgary et al., 2014; Msaada et al., 2015)
influenza, pulmonary tuberculosis, and other respiratory infections,
thanks to their richness on bioactive compounds including essential oils
and phenolic acids (Harkat-Madouria et al., 2015), that exhibit wide
range of biological effects and natural therapies like anti-inflammatory,
antifungal, analgesic, antimicrobial and antioxidative (Pereira et al.,
2014). In recent years, the natural compounds extract from eucalypts
leaves which is included in the list of Existing Food Additives [Notifi­
cation No. 120 (16 April 1996), Ministry of Health and Welfare, Japan]
(Amakura et al., 2009) were involved in food industry as natural food
additive by treatment and prevention of various ailments. (Costa et al.,
2015). Furthermore, Eucalyptus essential oils are having increasing in­
terest in various factories such as; pharmaceuticals, alternative medi­
cine, cosmetic, perfume and food industry (Goldbeck et al., 2014) thanks
do their potential multi-purpose functional use such as anesthetic,
antiseptic, astringent, deodorant, disinfectant etc (Bachir and Benali,
2012).
The different biological activities of these volatile oils can be referred
to the abundance of 1,8-cineole (ranged from 44 % to 84 %) (Goldbeck
et al., 2014). This chemical composition is related to environmental,
agronomic, (Topiar et al., 2015) and genetic (species) factors.
The eucalypts essential oils are within the 18 most commonly traded

* Corresponding author.
E-mail address: msaada.kamel@gmail.com (K. Msaada).

https://doi.org/10.1016/j.indcrop.2020.112964
Received 9 May 2020; Received in revised form 17 September 2020; Accepted 21 September 2020
Available online 1 November 2020
0926-6690/© 2020 Elsevier B.V. All rights reserved.
H. Limam et al.
essential oils in the world (Goldbeck et al., 2014). Studies have proved
their antimicrobial properties against a wide range of microorganisms,
including a large spectrum of bacterial strains cause oral infectious
diseases and respiratory tract infections, such as Staphylococcus aureus,
Pseudomonas aeruginosa, Klebsiella pneumonia and Hemophilisinfluenza.
These lower cost and safer herbal antibiotics are increasingly replacing
the conventional chemical antibiotics that cost up to $29,069 per patient
(Ventola, 2018; Aslam et al., 2018; Andrade et al., 2014). The medicinal
plant Eucalyptus camaldulensis is a species of this genus which recognized
by its antioxidant and antimicrobial potentials (Harkat-Madouria et al.,
2015).
Even that we can found these essential oils in more than 300 species,
only fewer than 20 have been exploited commercially (Elaissi et al.,
2012). Therefore, the objective of this study is to prove the effectiveness
of the essential oils of thirteen species of Tunisian eucalypts as natural
antioxidants and antibiotics, by determining their chemical composition
and evaluating their antioxidant activity, and antibacterial capacity
against six strains with negative and positive gram based on disc diffu­
sion method and calculation minimum inhibitory and bactericidal
concentrations method (MIC and MBC). Some of these species are
investigated against these strains for the first time.
2. Material and methods
2.1. Plant materials and chemicals
The samples of leaves of thirteen species of the Eucalyptus genus
(globulus, maidenii, astringens, camaldulensis, lehmannii, melliodora,
erythrocorys, gomphocornuta, gomphocephala, oxidantes, microcarpa,
paniculata, angulosa) were harvested in September 2017, from Hammam
Jebli Al Haouaria (latitude 36◦ 57′ 19.2′′ (N), longitude 11◦ 05′ 39.5′′ (E),
altitude 28 m). In North Eastern of Tunisia, the average annual rainfall is
229 mm, and the annual means of temperature was 18.0 ◦ C. The botanic
identification was carried out by Pr. Abderrazek Smaoui (the botanist of
the biotechnology centre of Borj Cedria. Technopark, Tunisia). All sol­
vents and reagents were of analytical grade.
2.2. Essential oils extraction
The collected leaves were cleaned and dried in free air during 15
days. Three samples of 100 g of crushed leaves from each 13 species
were the base of hydrodistillation extraction by Clevenger apparatus for
3 h / 750 mL distilled water. The obtained essential oils were collected
and stored in airtight containers, in darkness and at temperature of
-20 ◦ C until further analysis and use. The yield of extracted oil was
calculated following the next equation
% yield of oil = [Weight of oil/Weight of dried leaves] × 100
2.3. Gas chromatography mass spectrometry (GC/MS) analysis
Analysis of essential oil volatile compounds by GC was carried out on
a Hewlett-Packard 6890 gas chromatograph (Palo Alto, CA, USA)
equipped with a flame ionization detector (FID) and an electronic
pressure control (EPC) injector. A polar polyethylene glycol (PEG) HP
Innowax capillary column (30 m × 0.25 mm, 0.25 mm film thickness;
Hewlett-Packard, CA, 127 USA) was used. The flow of the carrier gas
(N2) was 1.6 mL/min. The split ratio was 60:1. The analysis was per­
formed using the following temperature program: oven temperature
kept isothermally at 35 ◦ C for 10 min, increased from 35 ◦ C to 205 ◦ C at
the rate of 3 ◦ C/min and kept isothermally at 205 ◦ C for 10 min. Injector
and detector temperatures were held at 250 ◦ C and 300 ◦ C, respectively.
The individual peaks were identified by comparing their relative
retention indices to n-alkanes (C6-C22) with those of literature (Adams,
2
Industrial Crops & Products 158 (2020) 112964
2007) and/or with those authentic compounds available in our labora­
tory. Percentage compositions of samples were calculated according to
the area of the chromatographic peaks using the total ion current.
Volatile compounds analysis by GC/MS was performed on a gas
chromatograph HP 5890 (II) interfaced with a HP 5972 mass spec­
trometer (Palo Alto, CA, USA) with electron impact ionization (70 eV is
the ionization energy). A HP-5 MS capillary column (30 m × 0.25 mm,
coated with 5% phenyl methyl silicone, 95 % dimethylpolysiloxane,
0.25 mm film thickness; Hewlett-Packard, CA, USA) was used. The col­
umn temperature was programmed to rise from 50 ◦ C to 240 ◦ C at a rate
of 5 ◦ C/min. The ion source temperature was 230 ◦ C and the quadrupole
temperature was 150 ◦ C. The front inlet temperature was 250 ◦ C. The
carrier gas was helium with a flow rate of 1.2 mL/min; split ratio was
60:1. Scan time and mass range were 1 s and 40− 300 m/z at 3.62 amu/
scan, respectively. Identification of volatile compounds was made by
matching their recorded mass spectra with those stored in the Wiley/
NBS mass spectral library of the GC/MS data system and other published
mass spectra.
2.3.1. Free radical- scavenging activity
The scavenging 2.2-diphenyl-1-picrylhydrazyl (DPPH) free radical
assay was monitored according to Noumi et al. (2016). All samples of
different species were prepared at the same concentration which is
10 mg/mL in pure ethanol, an aliquot of 1 mL from each of them was
added to 0.25 mL of a 0.2 mmol/L DPPH methanolic solution (purple
coloration). After a vigorous vortex of obtained mixture, it was incu­
bated in the dark and at room temperature during 30 min. After this, a
bleaching of the purple colour of mixture was noted which reflate the
scavenging capacity of Eucalyptus leaves essential oils. The absorbance
was measured at 517 nm and this antioxidant capacity was calculated
according to the next formula;
IP % = [(A0 − At)/A0] × 100
Where; IP is the inhibition percentage of free radical DPPH, A0 is the
absorbance of control (DPPH + Ethanol) after 30 min and At is the
absorbance of the test sample (DPPH + plant extract) after 30 min.
2.3.2. The reducing power
The method of Singh et al. (2012) was followed with slight modifi­
cations, to evaluate the capacity of eucalypts ‘essential oils to reduce the
Fe3+ to Fe2+. This reaction of reductants traduced experimentally by the
changing of the colour of the test solution from yellow to green. 1 mL of
sample of essential oils at different concentrations diluted in pure
ethanol range from 1 to 40 mg/mL was mixed with 2.5 mL of a 0.2 M
sodium phosphate buffer (pH 6.6, prepared from 62.5 mL of a 0.2 M
Na2HPO4 and 37.5 mL of 0.2 M NaH2PO4H2O and 2.5 mL (1% w/v)
potassium ferricyanide K3 [K3Fe (CN)6]. The obtained mixtures were
incubated in a water bath at 50 ◦ C for 20 min. Then 2.5 mL of tri­
chloroacetic acid (TCA) (10 %, w/v) were added followed by a centri­
fugation of 10 min at 650 g, and the supernatant (2.5 mL) was mixed
with 2.5 mL of distilled water and 0.5 mL of ferric chloride solution (0.1
%, w/v). The absorbance was measured at 700 nm against blank sample
and ascorbic acid was used as a positive control. The EC50 value
(mg/mL) expressed the reducing power. This value calculated from the
absorbance graphic, where concentration corresponding to 0.5 absor­
bance. Increasing absorbance reflect increasing reducing power. Tests
were carried out in triplicate.
2.4. Antibacterial activity
The antibacterial activity was evaluated qualitatively and quantita­
tively using two different methods; discs diffusion agar by measuring the
inhibition zone diameter of growth bacteria and determination of min­
imum inhibitory concentrations (MIC) and minimum bactericidal con­
centrations (MBC). These methods are the most common in most
H. Limam et al.
laboratories because they don’t require sophisticated equipment, in fact
they are relatively inexpensive and rapid.
2.4.1. Origin and selection of microbial strains
Six human pathogenic bacteria were selected for the antimicrobial
studies. They were obtained from the stock culture of our Laboratory.
Both Gram-positive strains including; Enterococcus hiare (ATCC 10,541),
Bacillus licheniformis (ATCC 8480), Staphylococcus aureus (ATCC 6538)
and Gram-negative strains including; Pseudomonas aeruginosa (ATCC
9027), Serratia marcescens (ATCC 13,880), Escherichia coli (ATCC 8739)
were used in our tests.
2.4.2. Preparation of the inoculums
The bacteria strains were maintained onto nutrient agar favourable
for their growth (Mueller-Hinton broth) at 37◦C for 24 h, they were sub-
cultured before any antimicrobial test. To prepare inoculums, bacteria
were suspending in a sterile saline solution. The optical density of the
suspensions was adjusted from 0.4 to 0.6 at 405 nm, which corresponds
to a cell density close to that of 0.5 McFarland, matching to an inoculum
estimated at 106–108 colony-forming units per mL (CFU/ mL) (Benje­
maa et al., 2018)
2.4.3. Diffusion method on agar
In this method, the Mueller-Hinton agar plates were streaked by
previously prepared inoculums using a sterile swab. Essential oils of all
Eucalyptus species were impregnated onto sterilized paper discs (6 mm,
Whatman paper N◦ 5) with 10 μL of each plant extract. In the same
conditions the antibiotic streptomycin (0.5 mg/mL) and the same sol­
vent (10 % Dimethyl sulfoxide (DMSO), 1% tween 80, DW) employed in
the dilution of essential oils, were used as positive and negative control,
respectively. The plates were maintained at a room temperature and
then incubated for 24 h at 37 ◦ C. In the end, antibacterial activity was
evaluated by measuring in millimetres the diameter of resulting inhi­
bition zones (around and including discs diameter). Each experiment
was done in triplicate (Luís et al., 2014).
2.4.4. Determination of the minimum inhibitory concentration (MIC)
The antibacterial activity of extract essential oils was also evaluated
by determination of the minimal inhibitory concentration (MIC) ac­
cording to Duarte et al. (2012) with slight modifications, using the
method of microdilution broth in 96-well microplates. In each well of
these plates a volume of 170 μL of sterile Mueller Hinton broth was
dispersed, 10 μL of blue colored resazurin, 10 μL of the inoculum
Fig. 1. Logarithmic correlation of the essential oil yield of the 13 Eucalyptus species.
3
Industrial Crops & Products 158 (2020) 112964
previously prepared (0.5 McFarland) 106 CFU/mL of each bacterium,
and then various Eucalyptus essential oils at different concentrations
ranged from 0.23 mg/mL to 30 mg/mL (diluted in 10 % DMSO, 1%
tween 80, DW) were added. Plates were then incubated from 12 to 24 h
at 37 ◦ C, for the bacterial strain growth. The wells without bacteria and
those without essential oils were used as negative and positive growth
control, respectively. MIC was recorded as the lowest concentration of
essential oil that didn’t give a visible growth, at which the blue colour of
resazurin was maintained. Each assay was done on duplicates and on
independent times.
2.4.5. Determination of minimum bactericidal concentration (MBC)
The minimum bactericidal concentrations (MBC) were usually
higher than the minimum inhibitory concentration (MIC) (Karthikeyan
et al., 2011). To determine their values, a fresh nutrient broth was
inoculated with the content taken from the MIC well and from those that
precede it (higher concentrations). The lowest concentration of essential
oils at which no growth of the bacterial inoculum was observed, that
caused 99.9 % of the bacterial population death after incubation for
18–24 h at 37 ◦ C, was recorded as MBC (Duarte et al., 2013). Each
experiment was performed two independent times.
2.5. Statistical analyses
All analyses were conducted in triplicates and results were expressed
as mean values ± SD (standard deviations). The one-way and multivar­
iate analysis of variance (ANOVA) followed by Duncan multiple range
tests was used to compare means. At P< 0.05 the differences between
individual mean values were deemed to be significant. Statistical anal­
ysis was performed by using the statistical program package STATIS­
TICA (Statsoft, 1998). In addition, more than 50 compounds detected in
the oils samples were subjected to hierarchical cluster analysis (HCA) in
order to discriminate between the different studied species of Eucalyptus.
3. Results and discussion
3.1. Extraction yield of essential oils
The essential oils extracted by hydrodistillation from leaves of thir­
teen Eucalyptus species characterized by pleasant smell (odour), and
pale-yellow colour except E. camaldulensis species that had a specific
orange colour. The volatile oils’ yield of these studied species varied
significantly (P < 0.001) among species with percentage ranged from
H. Limam et al.
0.11 ± 0.006%–6.16 ± 1.55 % in E. gomphocornuta and E. maidenii,
respectively, related to interspecific variation (Fig. 1). Except
E. gomphocornuta and E. paniculata, all other species showed higher
values than that of E. globulus reported by Selvakumar et al. (2012) with
percentage of 0.70 %, and higher than that of eucalypts species
(E. deglupta, E. urophylla, E. citriodora and E. camaldulensis) investigated
by Chahomchuen et al. (2020) that showed the range 0.6− 0.8% (v/w) of
their oils yield from fresh leaf, this result of E. camaldulensis is compa­
rable of the result obtained in this current study. In addition to eucalypt,
the obtained essential oils yields are higher than other plant such as
Psidiumgua java with 0.45 % (Nisha et al., 2011), according to various
factors including; genotype, agronomic, environmental, age, and geo
climatic (Msaada et al., 2015). Many studies reported that extraction
techniques and condition may affect on the essential oil percentage re­
coveries and composition (Msaada et al., 2012; Bagheri et al., 2014).
The important economic potential of Eucalyptus was justified by the
traded of thousand tonnes of their oils every year on international
markets (Harkat-Madouria et al., 2015).
3.2. Chemical characterization of essential oils
The chromatographic analysis by gas chromatography coupled to
mass spectrometry (GC/MS) of thirteen Eucalyptus species essential oils
allowed the identification of 53 components. The details of GC/MS re­
sults were presented in Table 1, it showed a significant variation (P <
0.05) between various studied species in their identified components
that are highly affected by the species factor (P < 0.001). According to
Chahomchuen et al. (2020), the Eucalyptus oil chemical composition
depends upon the species. Indeed they reported that the oils of investi­
gated eucalyptus species are a complex mixture of 63 compounds, while
Boukhatem et al. (2014) identified only more than 20 derived from
terpenes and their oxygenated compounds. In this current study,
oxygenated monoterpenes constituted the major fraction of most Euca­
lyptus species oils where the lowest concentration was detected in
E. gomphocornuta (0.66 ± 0.03 %) and the highest one was presented by
E. microcarpa (91.38 ± 0.45 %). 1,8-cineole was the main component of
this terpene class with concentrations ranged from 48.44 ± 0.11%–
82.64 ± 0.14 % (in E. astringens and E. microcarpa, respectively) while
E. camaldulensis (Fig. 2) and E. gomphocornuta had oxygenated sesqui­
terpenes as a major class with concentration of 37.91 ± 0.48 % and
58.75 ± 0.56 %, presented by spathulenol (32.88 ± 0.40 %) and
o-cymene (12.03 ± 0.04 %), respectively as main compound. Cha­
homchuen et al. (2020) results showed that 1,8-cineole (eucalyptol) was
the main essential oil constituent of E. camaldulensis. The predominance
of 1,8-cineole obtained in our study was in agreement with the literature
but with a clear difference in its concentration. Ghaffar et al. (2015)
have reported a lower contents (56.5 %) in E. globulus leaves than those
found in our results (70.12 %), whereas the Eucalyptus species leucoxylon
obtained from Iran was significantly richer in 1,8-cineole (89.8 %) than
our eucalypt species (Sefidkon et al., 2009). Salehi et al. (2019) reported
based on various studies, that the main constituent of widely examined
Eucalyptus oils was 1,8-cineole. Indeed, the content of this particular
constituent (1,8-cineole or eucalyptol) is the determinant factor of the
Eucalyptus oil value for medicinal purposes. A study conducted in Korea
showed that the inhalation of 1,8-cineole could be useful for Pre and
postoperative management when compared with placebo (Kim et al.,
2014). While other researches monstered that other compounds such as
cryptone (17.8 %), spathulenol (17.0 %), and globulol (8.0 %) were the
main constituent of oils from leaves of young branches. In addition to
these genetic and geo climatic factors, the variation of the genotype
composition and contents could be explained by the organ factor too; In
the study of Bey-Ould Si Saida et al. (2016) the sesquiterpenic hydro­
carbon aromadendrene was the predominance volatile compound on of
the E. camaldulensis fruits essential oils (18.0 %) which is completely
different from our study and from Barra et al. (2010) results too, con­
ducted on E. camaldulensis leaves, these later have reported that the only
4
Industrial Crops & Products 158 (2020) 112964
sesquiterpinene present in these oils is spathulenol (2.1–15.5 %). The
high concentration of 1,8-cineole found in the majority of our eucalypt
species could be depend on the extraction technique (hydrodistillation),
as a lower percentage was obtained by Topiar et al. (2015) from
E. globulus (21.01 %) using the supercritical fluid extraction. The
hydrodistillation extraction is the best method to extract 1,8-cineole
from Eucalyptus (Harkat-Madouria et al., 2015). A cluster analysis was
carried out in order to determine the eventual relationship between the
13 Eucalyptus species on the basis of their essential oil composition.
Obtained results from the cluster analysis (Fig. 3) showed the existence
of three well defined groups represented by E. camaldulensis and
E. gomphocornuta as first cluster and E. paniculata and E. gomphocephala
as second cluster. The last group was represented by E. lehmannii and
E. melliodora suggesting similar essential oil composition. The remaining
Eucalyptus species were clearly distinguished from these three clusters.
In conclusion, the Eucalyptus species collected from the north-east of
Tunisa habitats biosynthesized essential oils with two chemotypes, the
first one 1,8-cineole and the second spathulenol. The reported essential
oil bioactive are characterized by pharmacological activities, economic
viability and low toxicity (Bey-Ould Si Saida et al., 2016) thanks to their
reaction with different environment’s organisms by inhibiting micro­
organisms or reducing oxidative stress.
3.3. Antioxidant activity
Secondary metabolites as essential oils of medicinal plants have been
qualified as natural antioxidants that could substitute synthetic antiox­
idants in many factories specifically in pharmacological and cosmetic
industry. The antioxidant capacity of the thirteen species eucalypt
essential oils has been evaluated using two different assays; DPPH and
reducing power. Table 2 showed the results of these both tests that are
highly depend on species factor (P < 0.001). The DPPH test evaluate the
capacity of essential oils to convert the free radical 2,2-diphenyl-1-pic­
rylhydrazyl to 2,2-diphenyl-1-picrylhydrazine which interpreted
experimentally by the discoloration of the deep violet colour of DPPH,
its degree differ from sample to another indicating the free radical
scavenging potential of each one (Sarikurkcu et al., 2009). As repre­
sented in Table 2, at concentration of 10 mg/mL the inhibition per­
centage (IP) of free radical DPPH ranged from 10.75 ± 1.05 % in
E. angulosa to 52.69 ± 4.59 % in E. camaldulensis. This latter can reveal
the IC50 value since it defined as the concentration of sample that
decrease the DPPH concentration to half of its initial value (Erkan et al.,
2008). Indeed, it can be compared with the IC50 reported in literature,
the essential oil obtained from aerial parts of E. camaldulensis collected
from Sardinia (Italy) showed a capacity of scavenging DPPH radical
ranged between 0.5 and 5.8 mmol/L (Barra et al., 2010).
Harkat-Madouri et al.(2015) have reported a similar radical scavenging
activity from E. globulus essential oil leaves to that obtained in present
study (IP = 23.66 ± 1.05 at C = 10 mg/mL), while Bey-Ould Si Said et al.
(2016) results showed a higher antioxidant activity from E. globulus
essential oil fruits (IP more than 35 % at C = 10 mg/mL). In addition to
E. camaldulensis, E. gomphocephala and E. gomphocornuta had the most
active radical scavenging activity with percentages of 32.8 ± 1.05 % and
48.92 ± 1.05 %, respectively.
The capacity of eucalypt essential oils leaves of reducing the metal
ion Fe 3+ was evaluated by determining the EC50 corresponding to the
concentration of the extract to 0.5 absorbance. As for DPPH antiradical
activity, statistical analysis of reducing power results indicated a high
effect species (P < 0.001). E. gomphocornuta showed the highest
reducing capacity (EC50 = 2.09 ± 0.05 mg/mL) followed by
E. camaldulensis (EC50 = 2.96 ± 0.11 mg/mL) and E. paniculata
(EC50 = 4 ± 0.18 mg/mL). Bey-Ould Si Said et al. (2016) have registered
a less important reducing capacity of the essential oil extracts from
E. globulus fruits (32.8 ± 1.8 mg/mL), than that found in our study ob­
tained from E. globulus leaves (18.79 ± 0.7 mg/mL). The both antioxi­
dant test results may prove that the antioxidant activity of the studied
 Table 1

H. Limam et al.
ANOVA analysis, effect of species and quantitative and qualitative essential oil composition of the 13 Eucalyptus species leaves.
Compound (%, w/w) 1 2 3 4 5 6 7 8 9 10 11 12 13 P

thujene nd nd nd 0.82(0.04) nd nd nd 0.23(0.02) nd nd nd nd nd 0.000***

α-pinene 17.15 10.04 28.24 0.52(0.01) 18.21 17.06 9.94(0.11) 0.43(0.01) 6.18(0.30) 0.79(0.02) 4.27(0.20) 8.21(0.40) 17.67 0.000***
(0.05) (0.11) (0.60) (0.60) (0.20) (0.60)
camphene nd nd nd nd 0.22(0.01) 0.21(0.00) 0.23(0.01) nd 0.85(0.03) nd 0.47(0.02) 0.26(0.01) 0.28(0.00) 0.000***
sabinene nd nd nd 0.19(0.01) nd nd nd nd nd nd nd nd nd 0.000***
β-pinene 0.52(0.02) 0.43(0.01) 0.38(0.02) nd 0.14(0.01) nd nd nd nd nd nd nd 0.19(0.01) 0.000***
α-phellandrene nd 0.58(0.04) nd nd nd nd nd 3.44(0.11) nd 0.85(0.02) nd 0.33(0.02) 0.14(0.01) 0.000***
α-terpinene nd nd nd 0.27(0.01) nd nd nd 0.23(0.02) nd nd nd nd nd 0.000***
o-cymene 0.79(0.04) 1.07(0.07) 1.02(0.02) 19.14 0.21(0.01) 0.26(0.01) 0.62(0.03) 12.03 16.72 1.51(0.03) 0.76(0.02) 13.53 1.38(0.08) 0.000***
(0.08) (0.04) (0.03) (0.14)
β-phellandrene nd nd nd nd nd nd nd 4.74(0.13) nd nd nd nd nd 0.000***
1.8-cineole 70.15 71.16 48.44 16.31 68.46 72.91 60.74 nd 64.19 64.4(0.11) 82.64 54.89 60.48 0.000***
(0.17) (0.14) (0.11) (0.14) (0.08) (0.16) (0.08) (0.12) (0.14) (0.07) (0.08)
υ-terpinene nd nd nd 1.30(0.02) 0.28(0.01) 0.30(0.01) 0.87(0.03) 0.27(0.02) nd nd nd 4.25(0.04) nd 0.000***
cyclopentene nd nd nd nd nd nd nd nd nd nd nd nd 0.13(0.03) 0.000***
butanoic acid nd nd nd nd nd nd nd 0.31(0.02) nd nd 0.22(0.01) 0.44(0.01) nd 0.000***
fenchol nd nd nd nd nd nd nd nd 0.68(0.03) nd 0.57(0.02) 0.27(0.01) 0.38(0.01) 0.000***
pinocarveol 1.45(0.03) nd nd nd nd 0.64(0.02) nd nd nd nd 3.19(0.11) 0.80(0.02) 8.04(0.03) 0.000***
trans-pinocarveol nd nd 1.64(0.11) nd nd nd 0.40(0.02) nd 4.31(0.04) nd nd nd 0.40(0.02) 0.000***
p-cymene nd 0.32(0.02) nd nd nd nd nd nd nd nd nd nd nd 0.000***
1-terpineol nd nd nd 0.54(0.03) nd nd nd nd nd nd nd nd nd 0.000***
pinocarvone 0.43(0.02) nd 0.29(0.01) nd nd nd nd nd 0.85(0.04) nd 0.48(0.02) 0.27(0.01) 1.58(0.04) 0.000***
borneol nd nd nd nd 0.29(0.01) nd 0.34(0.02) nd 2.21(0.06) nd 1.41(0.11) 0.52(0.02) 0.68(0.02) 0.000***
4-terpineol 0.26(0.01) 0.41(0.03) nd 4.22(0.14) 0.22(0.00) 0.27(0.01) 0.48(0.02) 0.66(0.03) 0.41(0.02) 0.27(0.01) 0.27(0.01) 1.65(0.11) 0.39(0.02) 0.000***
3-cyclohexene-1-methanol nd 5.63(0.14) nd nd 4.39(0.11) nd nd 5.25(0.08) nd nd nd nd 2.10(0.04) 0.000***
α-terpineol 1.22(0.05) nd nd 1.15(0.04) nd 4.28(0.02) 1.63(0.04) nd 1.44(0.01) 0.76(0.02) 2.82(0.04) 2.43(0.02) nd 0.000***
propanal nd nd nd 0.98(0.02) nd nd nd nd nd nd nd nd nd 0.000***
5

2 methyl-3-phenylpropanal nd nd nd 0.34(0.01) nd nd nd nd nd nd nd nd nd 0.000***

phellandral nd nd nd 1.23(0.03) nd nd nd nd nd nd nd nd nd 0.000***
isoterpinolene nd nd nd nd nd nd nd nd nd nd 0.82(0.03) nd nd 0.000***
α-gurjunene nd nd nd nd nd nd nd 0.14(0.00) nd 0.30(0.01) nd nd nd 0.000***
caryophyllene nd nd 0.45(0.02) nd nd nd nd 1.76(0.01) nd 0.19(0.01) nd 0.97(0.02) nd 0.000***
aromadendrene 0.49(0.02) nd 2.37(0.14) nd nd nd 1.37(0.01) 1.43(0.01) nd 3.95(0.07) nd nd 0.38(0.01) 0.000***
allo-aromadendrene nd nd 0.33(0.01) 0.59(0.02) nd nd 0.15(0.01) 0.41(0.01) nd 0.39(0.02) nd 0.17(0.02) nd 0.000***
viridiflorene nd nd nd nd nd nd nd nd nd 0.52(0.01) nd nd nd 0.000***
bicyclogermacrene nd nd 0.5(0.01) nd nd nd 2.25(0.14) 0.74(0.02) nd nd nd 1.57(0.04) nd 0.000***
caryophyllene oxide nd nd nd nd nd nd nd 0.30(0.01) nd nd nd nd nd 0.000***
epiglobulol 0.31(0.02) nd 1.01(0.01) nd nd nd 0.36(0.01) 2.49(0.03) 0.24(0.01) 3.03(0.08) nd nd 0.25(0.01) 0.000***
naphthalene nd nd 0.67(0.03) nd nd nd 1.45(0.08) 1.52(0.01) nd 1.13(0.03) nd nd nd 0.000***
α-campholenal nd nd nd nd 6.22(0.14) 3.47(0.00) nd nd nd nd nd 0.58(0.02) nd 0.000***
spathulenol nd nd 1.8(0.11) 32.88 0.43(0.01) nd 2.32(0.07) 11.98 nd 0.52(0.0 nd 1.58(0.01) 0.35(0.02) 0.000***
(0.40) (0.09)

Industrial Crops & Products 158 (2020) 112964

globulol 2.05(0.14) 0.44(0.02) 8.24(0.11) nd 0.36(0.01) nd 6.29(0.11) 19.87 1.87(0.02) 14.84 0.41(0.01) 1.76(0.07) 2.63(0.04) 0.000***
(0.00) (0.07)
veridiflorol 0.51(0.01) nd 2.40(0.03) nd nd nd 5.46(0.30) 3.68(0.12) nd 2.75(0.01) nd 1.72(0.02) 0.66(0.04) 0.000***
pyrimidine nd nd 0.89(0. 06) nd nd nd nd nd nd nd nd nd nd 0.000***
ledol nd nd nd 0.89(0.01) nd nd nd nd nd nd nd nd nd 0.000***
allo-spathulenol nd nd nd 2.23(0.02) nd nd nd 0.79(0.02) nd nd nd nd nd 0.000***
isoaromadendrene epoxide nd nd nd nd nd nd nd nd nd 0.34(0.01) nd nd nd 0.000***
β-gurjunene nd nd nd nd nd nd nd 1.56(0.11) nd nd nd nd nd 0.000***
valencene nd nd nd nd nd nd nd nd nd nd nd 0.29(0.01) nd 0.000***
2-naphthalenemethanol 0.39(0.01) nd nd nd nd nd nd 2.43(0.02) nd nd nd nd 0.26(0.01) 0.000***
υ-eudesmol 0.49(0.01) 1.70(0.02) nd nd nd nd nd 2.55(0.11) nd nd nd nd nd 0.000***
isospathulenol nd nd nd 1.27(0.04) nd nd 0.51(0.02) 3.46(0.02) nd nd nd 0.42(0.03) nd 0.000***
β-eudesmol 2.22(0.04) 3.84(0.03) nd 0.64(0.01) nd nd nd 6.33(0.14) nd nd 1.05(0.02) 0.67(0.03) 0.73(0.03) 0.000***
(continued on next page)
 H. Limam et al. Industrial Crops & Products 158 (2020) 112964

eucalypt essential oils didn’t due only to the presence of the 1,8-cineole

1: E. globulus 2: E. maidenii, 3: E. astringens, 4: E. camaldulensis, 5: E. lehmannii, 6: E. melliodora, 7: E. erythrocorys, 8: E. gomphocornuta, 9: E. gomphocephala, 10: E. oxidantes, 11: E. microcarpa, 12: E. paniculata, 13: E. angulosa.
0.000***
0.000***
compound since the species, E. camaldulensis and E. gomphocornuta,
which had the strongest activity, had spathulenol (32.88 ± 0.40 %) and
o-cymene (12.03 ± 0.04 %) as major compounds, respectively (Table 1).
P

19.66(0.7) Previous studies showed that 1,8-cineole showed various degrees of

0.38(0.02)

4.62(0.14)

2.49(0.08)
different antioxidant capacities including reducing power and radical

(0.22)

(1.16)
71.95

99.10
scavenging (Horvathova et al., 2014). Whereas the results of study
nd
nd
13

carried out in previous time on the methanolic extracts of these 13 eu­

calypts species, showed higher antioxidant proprieties comparing to
0.71(0.02)

3.00(0.18)

6.86(0.18)

0.44(0.01)
these investigated essential oils, and to standard BHT for the DPPH test

Values of volatile essential oil percentages are the average three determinations (n = 3). Values in the brackets are the representative standard deviations. nd: not detected. P : probability.
(0.61)

(0.28)

(1.26)
with IC50 5.07 ± 0.54 for E. erythrocorys where the prevailing antioxi­
26.58

61.41

98.29
nd
12

dant in the extract was gallic acid (Limam et al., 2020).

The difference in chemical composition of essential oils assigned to
6.32(0.27)

1.46(0.03)

0.22(0.01)

various factors previously mentioned (geo climatic, genetic and epige­

netic) could be the reason of this difference in various studies, since the
(0.45)

(0.76)
91.38

99.38

biological and therapeutic activities of Eucalyptus oils depend upon their

nd
nd

nd
11

chemical composition content (Chahomchuen et al., 2020; Aleksic Sabo

4.28(0.10)

5.35(0.23)

and Knezevic, 2019). All eucalypt studied species exhibited a moderate

antioxidant activity since their essential oils showed lower activity than
(0.14)

(0.18)

(0.10)
65.43

21.48

96.54

that of the standards BHT and ascorbic acid with

nd
nd
10

IC50 = 32.80 ± 1.63 μg/mL and EC50 = 59 ± 1.13 μg/mL, for the DPPH
and reducing power tests, respectively. Chahomchuen et al. (2020) re­
2.11(0.03)

sults suggest Eucalyptus essential oil as a potent inhibitor of oxidative

(0.36)

(0.32)

(0.36)
23.75

74.09

99.95

stress. Aleksic Sabo and Knezevic. (2019) reported that E. camaldulensis

nd
nd

nd

nd
9

flower essential oil has the potential to be developed into a skin care
product since it inhibited melanogenesis by down-regulating both
7.30(0.02)

0.66(0.03)

6.04(0.21)

7.99(0.12)

mitogen-activated protein kinases (MAPK) and protein kinase A (PKA)

(0.36)

(0.56)

(1.28)
22.89

58.75

96.33

signaling pathways through its antioxidant properties. Except its anti­

nd
8

oxidant property, several studies have also assessed the efficacy of

Eucalyptus extract chewing gum in oral malodor and periodontal health.
0.46(0.02)

3.77(0.32)
(0.26)

(0.18)

(0.53)

(1.29)
13.11

63.59

15.40

95.87

3.4. Antibacterial activity

nd

nd
7

3.4.1. Agar diffusion test

Our results showed that the solvent (10 % DMSO, 1% tween 80, DW)
(0.22)

(0.21)

(0.43)
17.83

81.57

99.40

employed in the dilution of essential oils didn’t have any antibacterial

nd
nd

nd

nd

nd
6

capacity against all tested bacterial strains. According to our results

presented in Table 3, a substantial antimicrobial activity was exhibited
0.79(0.02)

4.39(0.11)

by all eucalypt essential oils against the six tested bacteria, since the
(0.64)

(0.23)

(1.00)
19.06

75.19

99.43

lowest inhibition zone registered was 9 ± 0.01 mm against Baccilus

nd
nd

nd
5

licheniformis from E. gomphocornuta essential oil. Indeed, E. astringens

presented the best activity against Pseudomonas aeruginosa
0.59(0.04)

0.98(0.02)

(33.33 ± 3.26 mm, IZ) while Elaissi et al. (2012) have obtained a much
(0.17)

(0.39)

(0.48)

(1.10)
22.24

23.79

37.91

85.51

weaker activity (6.00 ± 0.00 mm, IZ) from the same Eucalyptus species
nd
nd
4

(E. astringens) against the same bacterial strain (P. aeruginosa). E. globulus
have the second strongest activity followed by E. camaldulensis against
0.89(0. 06)
3.65(0.36)

Baccilus licheniformis and Serratia marcescens with 25.33 ± 0.65 mm and

(0.67)

(0.23)

(0.26)

(1.52)
30.31

50.37

13.45

98.67

25.33 ± 2.84 as inhibition zone, respectively. Various researches re­

nd
nd

ported the capacity of E. camaldulensis essential oil to affect biofilm

3

formation (Aleksic Sabo and Knezevic, 2019).

4.32(0.01)

5.63(0.14)

In addition, the antibiotic streptomycin used as positive control

(0.25)

(0.17)

(0.08)

(0.64)
12.44

71.57

10.30

99.94

possessed a less efficient antibacterial capacity than most of the Euca­

nd

nd

lyptus studied species essential oils. This effectiveness against wide range
2

of bacterial strains could be explained by their hydrophobicity and

1.29(0.02)

0.49(0.04)

6.87(0.24)

0.39(0.01)

permeability to the lipids of cell membrane and mitochondria, which

(0.11)

(0.28)

(0.68)

causing leakage of cell contents. (Horvathova et al., 2014). This anti­

18.46

73.51

99.72
nd

bacterial activity of eucalypt essential oils could be due to their richness

1

in 1,8-cineole, which known by its most potent biological activity and

Monoterpene Hydrocarbons

Oxygenated Sesquiterpenes

good antimicrobial activity (Salehi, et al., 2019), and the presence of

Oxygenated Monoterpenes

other compound as α-pinene, that are known by their antibacterial ac­

Compound (%, w/w)

Grouped compounds
Table 1 (continued )

tivity (Chikhoune et al., 2013; Elaissi et al., 2012). Indeed, the syner­
Hydrocarbons

gistic effect between these both major compounds, for the most of
Total identified
Sesquiterpenic

Eucalyptus studied specie, could be also behind the antibacterial activity.

α-eudesmol
t-muurolol

Same synergistic effect has been reported between aromadendrene and

Others

1,8-cineole against B. subtilis and S. aureus in the study of Mulyaningsih

et al. (2010). However, E. camaldulensis indicated one of the strongest

6
H. Limam et al.
Fig. 2. Example of a chromatographic profile of E. camaldulensis essential oil with its major compounds obtained by GC–MS analysis.
antibacterial activity, although it had spathulenol ant not 1,8-cineole as
major compound. The statistical analysis indicated a high significant
(P < 0.001) difference among different Eucalyptus species oils in their
antibacterial activity against all bacterial strains. According to obtained
results, all tested strains showed sensitivity to the studied eucalypts oils,
with different degrees ranged from intermediate (13 mm > D > 6 mm) to
sensitive (D > 13 mm). Based on their sensitivity, the tested strains were
classified as follow; Pseudomonas aeruginosa > Staphyllococcus aureus >
Baccilus licheniformis > Serratia marcescens > Escherchia coli > Entero­
coccus hiare. However, a different classification has been observed for
the strain sensitivity to the used antibiotic Streptomycin. Elaissi et al.
(2012) and Bey-Ould Si Said et al. (2016) have reported that Staphylo­
coccus aureus, gram-positive bacteria, was the most sensitive strain to
E. houseana and E. odorata leaves essential oils and E. globulus fruits
essential oil. In addition, higher inhibition was detected against Pseu­
domonas aeruginosa which is a major nosocomial pathogen that has the
respiratory tract as one of its preferential niches (Riou et al., 2010).
Different result was obtained by Gilles et al. (2010) for the P. aeruginosa
as it was the most resistant to the essential oils tested. Boulekbache-­
Makhlouf et al. (2013) have founded a weak antibacterial activity of
E. globulus fruit extract against B. subtilis which is similar to that of tannic
acid with inhibition zones of 5.33 ± 1.15 mm and 5.5 ± 0.5 mm,
respectively.
3.4.2. Determination of minimum inhibitory (MIC) and bactericidal (MBC)
concentrations
The result of MIC and MBC values of the thirteen tested eucalypt oils
are listed in Table 4, as it was indicated in the first antibacterial test (disc
diffusion assay), this second test confirmed the sensitivity of the tested
Gram− and Gram+ bacteria to essential oils and a clear variation on the
MIC values among eucalypt species were observed too, these values
7
Industrial Crops & Products 158 (2020) 112964
were ranged from 0.93 mg/mL to 30 mg/mL. Luís et al. (2014) have
reported a range of MIC values of eucalypt essential oils varied from 4 to
32 μL/mL. Eucalyptus camaldulensis essential oil showed the best anti­
bacterial activity against all tested bacterial strains since its MIC values
were lower than those obtained for the other species, and its lowest MIC
was against Serratia marcescens (0.93 mg/mL). This result reflected that
the antibacterial capacity could not due only to the abundance of the
major compound 1,8-cineole, since the major compound for
E. camaldulensis was spathulenol followed by o-cymene than 1,8-cineole
(Fig. 2). In that case spathulenol and its synergistic effect with the both
other compounds could justify this antibacterial property. Indeed, spa­
thulenol could has also a better antimicrobial activity than 1,8-cineole,
the same finding was reported by other researchers for the compound
aromadendrene (Bey-Ould Si Saida et al., 2016) followed by citronellol
and citronellal that were found to be the most active (Salehi, et al.,
2019). Aleksic Sabo and Knezevic. (2019) have mentioned that of
E. camaldulensis extracts have been examined for its antibacterial effects
in a wider extent than its essential oils, and the inhibitory concentrations
being in broad range from 0.08 μg/mL to 200 mg/mL. Tests done on
animal models proved that extracts leaves of E. camaldulensis and its
constituents possess numerous other beneficial effects except antimi­
crobial one. Its gastro intestinal effect reported to decrease gastric acid
production and thus appear useful for the treatment of gastric ulcers.
The both Eucalyptus species E. erythrycorys and E. gomphocornuta were
characterized by the second strongest antibacterial capacity of their
leave essential oils against Escherchia coli with MIC of 1.87 mg/mL for
the both species. The highest MIC values were shown against the Gram-
negative Pseudomonas aeruginosa, which reflects the resistance of this
bacterial strain. Same conclusion was reported by Tyagi and Malik
(2011) about the resistance of the same strain (P. aeruginosa) to the
essential oil of E. globulus (MIC = 9 mg/mL). The published data for
H. Limam et al. Industrial Crops & Products 158 (2020) 112964

Fig. 3. Cluster analysis of the 13 Eucalyptus species based on their essential oil composition (Table 1).

were reported by Pankey and Sabath (2004) for the E. camaldulensis

Table 2 essential oils where the most sensitive bacteria are Gram negative
Antioxidant properties against; reducing power and DPPH radical of Eucalyptus
A. baumannii and V. parahaemolyticus. The variation in inhibitory con­
essential oils.
centrations could be depending on extraction method, plant properties,
Species Reducing power (EC50 in mg/ DPPH (IP % for C = 10 mg/ and model organism (Aleksic Sabo and Knezevic, 2019). The latest
mL) mL)
classification was different from that obtained by the disc diffusion test,
E. globulus 18.79 ± 0.70d 23.66 ± 1.05f where the most sensitive (S. marcescens) and the most resistant
E. maidenii 17.61 ± 1.12e 24.73 ± 1.05e
(P. aeruginosa) were both Gram-negative strains. These results suggest
E. astringens 7.46 ± 0.26 h 19.89 ± 1.05 g
E. camaldulensis 2.96 ± 0.11 L 52.69 ± 4.59a Eucalyptus essential oils as a potent inhibitor of pathogenic microor­
E. lehmannii 20.14 ± 0.49c 15.05 ± 1.05 j ganisms as mentioned by Chahomchuen et al. (2020).
E. melliodora 43.1 ± 1.74a 19.35 ± 1.83 h The antibacterial action of essential oils is bellowed to their mech­
E. erythrocorys 8.45 ± 0.75 g 29.57 ± 1.05d anisms of action. Indeed oil hydrophobicity increases cell permeability
E. gomphocornuta 2.09 ± 0.05m 32.8 ± 1.05c
E. gomphocephala 4.99 ± 0.04i 48.92 ± 1.05b
and consequent leakage of cell constituents, this effect on the bacterial
E. oxidantes 4.57 ± 0.07 j 16.13 ± 1.83i envelopes may affect stability of other cellular structures in a cascade
E. microcarpa 41.6 ± 1.73b 12.37 ± 1.05k type of action. Essential oils may cause cell wall and membrane
E. paniculata 4 ± 0.18k 23.66 ± 1.05f disturbance which further can lead to the significant loss of intracellular
E. angulosa 12.59 ± 0.34f 10.75 ± 1.05 L
ATP, induction the synthesis of heat shock proteins, pH disturbance, and
d.l 12 12
F 1073.56 216.40 intracytoplasmic changes (e.g. coagulation, periplasmic space enlarge­
P 0.000*** 0.000*** ment) (Aleksic Sabo and Knezevic, 2019).
Ascorbic acid EC50 = 59 ± 1.13 μg/mL Although that MIC values founded in our study were comparable to
BHT IC50 = 32.80 ± 1.63 μg/mL those reported in the literature, even that some of them were lower, MBC
d.l: degree of freedom, F: Fisher value, P: Probability, ***: significant effect at values of al tested strains were much higher (MBC > 30 mg/mL). Ac­
P < 0.001.The means of three determinations in the same column followed by cording to the study of Biyiti et al. (2004) about founding a better
the letters (a–l) are significantly different at P < 0.05. evaluation of antibacterial effect of bioactive by calculating the MBC /
DPPH: 2.2-diphenyl-1-picrylhydrazyl (free radical), IP %: inhibition percentage, MIC ratio, all studied eucalypt essential oils exert bacteriostatic and not
C: concentration, EC50: efficacy concentration of 50 %. bactericidal effects against all bacteria since the ratio MBC / MIC > 2.
Salehi et al. (2019) have reported that due to differences on cell wall
P. aeruginosa are in the same range which conform its highly resistant to sub-structures, investigated oils and their components were almost
antimicrobial agents (Aleksic Sabo and Knezevic, 2019). Thereafter, this inactive against multi-drug resistant Gram negative bacteria. Obtained
quantitative evaluation of the eucalypt essential oils antibacterial ca­ result could be explained by the fact that this bacterium is able to
pacity gave us a sensitivity classification of the tested bacterial strains as convert to the endospore form under unfavorable conditions (Ahmad
follow: Serratia marcescens > Escherchia coli > Enterococcus hiare > and Beg, 2001; Boulekbache-Makhlouf et al., 2013) and they resume
Staphyllococcus aureus > Baccilus licheniformis > Pseudomonas aeruginosa. their normal forms with the return of favourable vital conditions. Other
In comparison to Gram negative bacteria, Gram positive are consider mechanisms such as motility, antibiotic production, virulence, compe­
more sensitive to essential oils, but it cannot be applied for essential oils tition amongst populations, conjugation, and biofilm formation allow
tested of Eucalyptus species since the most sensitive bacteria are both bacteria to regulate some physiological activities that improve them. In
Gram negative (Serratia marcescens and Escherichia coli). Similar results fact bacteria adopt an intercellular communication system Quorum

8
H. Limam et al. Industrial Crops & Products 158 (2020) 112964

Table 3
Effect of species, ANOVA analysis and antibacterial activity of essential oils of different Eucalyptus species; Inhibition zone (IZ) in diameter.
Strain

Species Positive Gram (+) Negative Gram (-)

Enterococcus hiare Baccilus licheniformis Staphyllococcus aureus Pseudomonas aeruginosa Serratia marcescens Escherchia coli
(ATCC 10,541) (ATCC 8480) (ATCC 6538) (ATCC 9027) (ATCC 13,880) (ATCC 8739)
IZ mm

E. globulus 11.00 ± 1.13d 25.33 ± 0.65a 20.66 ± 1.30b 20.66 ± 0.65b 11.33 ± 0.65d 15.66 ± 3.26c
E. maidenii 11.33 ± 0.65f 20.33 ± 0.65a 17.00 ± 1.13c 19.00 ± 1.13b 11.66 ± 0.65e 12.66 ± 0.65d
E. astringens 14.66 ± 1.30e 18.66 ± 1.72c 21.66 ± 0.65b 33.33 ± 3.26a 13.66 ± 0.65f 15.33 ± 0.65d
E. camaldulensis 12.33 ± 0.65f 17.66 ± 0.65c 20.66 ± 0.65b 16.00 ± 1.13d 25.33 ± 2.84a 15.33 ± 1.72e
E. lehmannii 17.66 ± 1.30c 12.66 ± 2.35e 19.66 ± 1.30b 22.33 ± 0.65a 13.33 ± 0.65d 11.66 ± 0.65f
E. melliodora 13.66 ± 0.65c 12.33 ± 0.65de 20.00 ± 1.95a 11.33 ± 0.65e 18.66 ± 0.65b 12.66 ± 0.65d
E. erythrocorys 15.00 ± 1.13d 15.00 ± 0.01d 1966 ± 0.65b 24.00 ± 1.07a 12.00 ± 1.13e 15.33 ± 0.65c
E. gomphocornuta 15.33 ± 0.65b 9.00 ± 0.01f 11.00 ± 0.00e 11.33 ± 1.30d 23.33 ± 0.65a 12.33 ± 1.72c
E. gomphocephala 14.00 ± 1.13e 17.66 ± 1.30b 17.66 ± 0.65b 16.66 ± 0.65c 19.33 ± 1.30a 16.00 ± 1.13d
E. oxidantes 17.33 ± 0.65b 13.33 ± 0.65d 16.33 ± 0.65c 17.33 ± 2.35b 21.00 ± 1.13a 13.33 ± 1.72d
E. microcarpa 13.66 ± 0.65c 20.33 ± 0.65b 20.33 ± 1.72b 21.33 ± 0.65a 13.33 ± 0.65 cd 13.00 ± 1.13d
E. paniculata 11.00 ± 0.00e 16.00 ± 1.13b 16.33 ± 0.65ab 16.66 ± 4.57a 13.66 ± 1.72c 11.66 ± 0.65d
E. angulosa 13.33 ± 0.65f 17.33 ± 0.65d 20.66 ± 0.65c 23.00 ± 1.13a 16.66 ± 0.65e 22 ± 1.99b
d.f 12 12 12 12 12 12
F 20.26 62.01 28.94 27.30 58.50 11.46
P 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000***
Streptomycin 12.33 ± 0.65 13.66 ± 0.65 13 ± 0.01 14 ± 0.01 11.66 ± 0.65 15 ± 1.95
(0.5 mg/mL)

Means of inhibition zone (IZ) are the average three determinations (n = 3). These values with different letters (a–f) are significantly different at P < 0.05.
***P < 0.001.df: degree of freedom, F: Fisher value, and P: probability.

sensing (QS) allows it to share information about cell density and adjust
gene expression accordingly. There are reports of anti-QS activity of
essential oils Eucalyptus genus. However, future detailed studies of
anti-QS and anti-biofilm Eucalyptus activity are needed in order to
confirm this assumption (Aleksic Sabo and Knezevic, 2019). To Thus,
based on this current report, the antibacterial activity of Eucalyptus
essential oils may indicate their potential usefulness as a microbiostatic,
disinfectant agent or antiseptic. Eucalypts have the potential to be used
as antibacterial and antifungal agents in cosmetic and pharmaceutical
products, due to its low toxicity and natural origin; for most Eucalyptus 1,
8-cineole-rich essential oils, the recommended human oral and dermal
dose limit is 10 %. The hytomedicine 1,8-cineole capsules and Myrtol®
standardized (GeloMyrtol®, GeloMyrtol forte®) have gained a huge
attention due to its potential benefits in various respiratory conditions
including chronic sinusitis. chronic bronchitis, chronic obstructive pul­
monary disease, and asthma (Salehi et al., 2019).
4. Conclusion
In general, this study of the essential oils of thirteen eucalypt species
provides a potential application for different industries such as phar­
maceutical. The bioactive compounds of this plant can be considered as
a source of natural antibiotics and antioxidants. Indeed, they showed a
respectable antioxidant activity compared to literature, especially their
reducing power capacity. The current work reports the interesting
antibacterial activity, especially a great bacteriostatic effect of the
Eucalyptus leaves essential oil against both positive and negative Gram
pathogenic bacteria. In fact, this antimicrobial activity was related to the
presence of the indispensable moderate and minor compounds in addi­
tion to the major compound 1,8-cineole. In order to better understand
the mechanisms and the role of those bioactive compounds in the
studied bioactivities, further studies should be conducted.

Table 4
Antibacterial activity of essential oils of different species of Eucalyptus; determination of minimum inhibitory concentration (MIC) and minimum bactericidal con­
centration (MBC).
Strain

Positive Gram (+) Negative Gram (-)

Species
Enterococcus hiare Baccilus licheniformis Staphyllococcus Aureus Pseudomonas aeruginosa Serratia marcescens Escherchia coli
(ATCC 10,541 (ATCC 8480) (ATCC 6538) (ATCC 9027) (ATCC 13,880) (ATCC 8739)
MIC mg/mL

E. globulus 7.5 7.5 7.5 7.5 7.5 7.5

E. maidenii 7.5 15 15 15 7.5 7.5
E. astingens 7.5 3.75 7.5 3.75 3.75 3.75
E. camaldulensis 1.87 1.87 1.78 1.87 0.93 1.87
E. lehmannii 15 30 30 30 3.75 7.5
E. melliodora 7.5 15 3.75 30 7.5 7.5
E. erythrycorys 7.5 7.5 7.5 7.5 3.75 1.87
E. gomphocornuta 3.75 15 15 7.5 1.87 1.87
E. gomphocephala 7.5 7.5 7.5 7.5 3.75 3.75
E. oxidantes 3.75 7.5 3.75 3.75 3.75 3.75
E. microcarpa 7.5 7.5 3.75 3.75 3.75 7.5
E. paniculata 15 30 30 30 3.75 3.75
E. angulosa 7.5 7.5 3.75 7.5 7.5 3.75
30 mg/mL < MBC

9
H. Limam et al.
Authors credit statement
Hajer Limam: Contributed to all the experimental process, data
analysis and results interpretation as well as paper preparation.
Mariem Ben Jemaa: was involved in the experimental process, sta­
tistical analysis and contributed to the preparation of the final manu­
script version.
Sonia Tammar and Nour Ksibi: were responsible for the technical
support.
Saber Khammassi: performed all the extraction especially essential
oil.
Selim Jallouli is responsible on the essential oil analysis by GC and
GC/MS.
Giovanni Del Re and Kamel Msaada: supervised the project and co­
ordinated experimental process.
All authors discussed the results and implications and commented on
the manuscript at all stages. All the authors take full responsibility for
the content of the paper.
Declaration of Competing Interest
The authors declare that there is no conflict of interests regarding the
publication of this paper.
Acknowledgments
This work was supported by the Tunisian Ministry of Higher Edu­
cation and Scientific Research (LR 15 CBBC06).
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