E3S Web of Conferences 319, 01032 (2021) https://doi.org/10.

1051/e3sconf/202131901032
VIGISAN 2021

Study of the sensitivity of certain bacteria –pathogenic human

health- to atlas cedar essential oils and antibiotics

Rabab Ez-Zriouli1*, Houda El Yacoubi1, And Atmane Rochdi1

1Laboratory of natural resources and sustainable development, Faculty of Sciences, University
Ibn Tofail, Kenitra 14000, Morocco.

Abstract. The spread of microbial resistance around the world is one of the most serious
threats to public health. Every year one to three antibiotics are released to the market, as
each antibiotic has a limited effective lifespan after which microorganisms develop
resistance. The need to identify new natural antimicrobial agents is an important research
goal at the global level. The use of volatile plant extracts as antimicrobial agents
represents a low risk for the development of resistance by microorganisms because these
products are made up of several phytocompounds from different groups which work in
synergy.The objective of this work is to test, in vitro, the antimicrobial activity of the
essential oil of cedrus atlantica against six bacterial strains; Salmonella sp, Staphylococcus
aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and
streptococcus sp. And three strains of yeast; Candida Tropicalis, Candida Albicans,
Saccharomyces Cerevisiae. With the aim of researching new effective antibacterial
substance with a broad spectrum of action.The results of the quantitative analysis showed
a richness in certain compounds identified by gas chromatography coupled with mass
spectrometry; β-Himachalene, α-Himachalene, himachalene oxide and Limonene. The
essential oil to show a significant antibacterial effect compared to the synthetic antibiotics
tested Piperacillin 30 µg (PRL30) and Ampicillin 10 µg (AMP 10). Key words: Cedrus
atlantica, essential oil, antimicrobial activity, GC / MS, antibiotics

1. Introduction
The fight against microbial attacks is based primarily on
the use of antibiotics. However, for several years, the This work is devoted to the study of the chemical
selection of multidrug-resistant strains has been observed composition of the essential oil of cedrus atlantica and
in human medicine, for which the abusive therapeutic its antimicrobial power on pathogenic microorganism.
use of antibiotics is largely responsible. In order to
preserve human health, the discovery of new molecules 2. Material and method
has become an absolute necessity[1]. The fields of
investigation are vast but the exploration of natural
resources and especially plants which have always been 2.1. Plant material and analysis by GC/MS
a source of inspiration for new drugs appears to be the
most promising because these constitute the largest Before extraction, the aerial parts of cedrus atlantica
reserve of active substances [2], [3]. were dried in the shade in a ventilated place. The EO is
Among the forest species of Morocco, the Atlas cedar extracted by hydrodistillation using a Clevenger type
has always been considered as the emblematic noble apparatus [8]. the extraction lasted 3 hours by placing
species of the Atlas Mountains (130,000 ha) and the Rif 250 g of the leaves of the plant in a flask with distilled
(15,000 ha), appreciated for its technological, ecological water. The oil obtained is then analyzed by gas
and biogeographical values[4]. chromatography coupled to a mass spectrum (GC / MS)
The cedar is a coniferous tree, which can reach a height of the (polarisQ) type, ion trap in electonic impact mode
of 30 to 40 m, of the Panacea family [5]. It is highly (EI) with ionization energy. from 70 ev. The column
sought after for its many uses (sawing, charcoal, etc.).[6] used is an apolar capillary column in silica of the type
The essential oils derived from C. atlantica are used in (Wcot Fused Silica), with a stationary phase (CP-
various products, such as medicines and perfumes[7]. SIL5CB), 50 m in length, the temperature of the column
*
Corresponding author: [email protected]

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/).
E3S Web of Conferences 319, 01032 (2021) https://doi.org/10.1051/e3sconf/202131901032
VIGISAN 2021

is programmed from 40 to 280 ° C at a rate of 3 ° C / 3. Results and discussion

min. The temperature of the injector is set at 240 ° C and
that of the detector (ionization source) is 200 ° C. The 3.1. Chemical composition of the volatil extract
flow rate of the carrier gas (helium) is set at 1 ml / min.
The volume of the sample injected is 1 μl of EO diluted
in methanol (1% v / v) the GC / MS analysis spectrum of the essential oil
isolated from the leaves of cedrus atlantica by
hydrodistillation is shown in figure 1. The essential
2.2. Microorganism
compound of this oil, the retention times and the
Bacterial Strains: Gram+ positif (Staphylococcus aureus, distribution air are summarized in Table 1. Eleven
streptococcus sp), Gram- negatif (Escherichia coli, compounds have been identified, the major constituents
salmonella sp, Klebsiella pneumoniae, Pseudomonas of which are; β-Himachalene (54.21%), γ –Himachalene)
Aeruginosa) 15.54%), Himachalene oxide (6.23%) and Limonene
Yeasts: Candida Tropicalis, Candida Albicans, (6.12%). Compared with the results reported in the
Saccharomyces Cerevisiae literature, the compounds found in the essential oil of
Antibiotics: The antibiotics used are: Piperacillin (PRL cedrus atlantica in high percentage in samples of plants
30), Ampicillin (AMP 10). from morocco are: α-pinene (14,85%), himachalene
(10.14%), β-himachalene (9,89%), σ-himachalene
(7,62%), and cis-α-atlantone (6,78%) [11].
others research al have shown that cedrus atlantica oil is
2.3. Testing for antibacterial activity by the rich in β-Himachalene with a content of about 31.24%,
diffusion method followed by α-himachalene (15.63%) and γ -
Himachalene (14.46%) [12].
From a microbial inoculum of 108 cfu / mL, petri dishes This difference in composition may be due to various
containing Muller-Hinton solid culture medium for factors such as geographic environment, harvest season,
bacteria and Sabouraud for yeasts were inoculated by genotype, temperature and location of drying, and
spreading. Then a sterile disc of Wattman paper 6 mm in method of extraction.
diameter soaked in 15 µl of the essential oil of cedrus
atlantica is placed in the center of each dish on the
surface of the agar medium. For the positive controls
antibiotic discs were used; Piperacillin 30µg and
Ampicillin 10µg. once the dishes are prepared, they are
incubated at 37 ° C. for 24 hours for the bacteria and 48
hours at 30 ° C. for the yeasts. The sensitivity of the
strains was assessed by measuring the diameters of the
zones of inhibition formed around the discs. Each test is
repeated three times under the same experimental
conditions to confirm the results [9].

2.4. Antibacterial activity test by the solid

medium dilution method Fig. 1. GC / MS analysis chromatographic profile of essential
oil of cedrus atlantica
The minimum inhibitory concentration (MIC) and the
minimum bactericidal or fungicidal concentration (MBC
/ MFC) of the volatile extract is determined by the
dilution method [10]. This technique consists of making Table 1. Chemical composition of Cerdus Atlantica essential
successive dilutions of the essential oil in the culture
medium. After solidification, the medium is inoculated oil by GC / MS
with the strain and incubated according to the ideal
conditions for each microorganism. The results give the Percentage Retention Time
Chemical Compounds
(%) (RT) /(min)
MIC as the lowest concentration for which we do not
observe growth with the naked eye.
2.5. Statistical analysis 3,72
β –sinensal 0,01
The data obtained were analyzed using the Genstat 8,66
Isoledene 0,04
software, a one-way variance (ANOVA) analysis was 12,67
Himachala-2,4-diene 0,1
performed in addition to the comparison of means 19,24
according to the Bonferroni test. Humulene 1,2
Himachalene 1.16 26.94
Isocaryophillene 0.91 27.16

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E3S Web of Conferences 319, 01032 (2021) https://doi.org/10.1051/e3sconf/202131901032
VIGISAN 2021

γ –Himachalene 15.54 28.4 them more permeable. This promotes the escape of ions
54.21
and the loss of cell content and ultimately cell death.
β-Himachalene 29.79
[18], [19], [20].
α-Pinene 1.08 30.52 In addition, the antimicrobial activities of extracts of
Cymene 2.68 30.67 aromatic and medicinal plants depend on various factors;
3-Carene 1.42 31.49 solvent used for the extraction, conditions under which
the plant grew, strains chosen and the method for
Himachalene oxide 6.23 32.47 determining antimicrobial activity ...[21].
δ -Cadinene 3.24 32.65
Longifolene 3.17 33.61
4. Conclusion
Limonene 6.12 34.55
The essential oil analyzed is characterized by a very
Total 97,11
diverse chemical composition. As the results obtained in
microbiology during this research relate to the
concentrations used, the results of our work constitute a
first step in the search for alternative control treatments
3.2. Antibacterial activity
against bacterial and fungal diseases. This study will be
The results of the antimicrobial activity are shown in complemented by further confirmations on other plant
Tables 2 and 3 the analysis of variance to show that the extracts and other strains.
differences between the strains are not significant (P>
0.05) for the essential oil and very highly significant for
Table 2. Analysis of variance
the Ampicillin (P <0.001). The comparison of the means,
the Bonferroni test to show that there is a variability EO : Essential oil, AMP : Ampicillin 10µg, DL : Degree of
between the classes i.e. the essential oil and the liberty, SS : Sum of the squares of the deviations, MS : Mean
Ampicillin did not react in the same way vis-à-vis the
of the squares of the deviations, P : probability
strains tested. Regarding the antibiotic Piperacillin, it
has not shown any effect on all microorganisms. Usually source of variation DL SS MS P
the yeasts appeared very sensitive in the presence of the
essential oil followed by Staphylococcus aureus,
streptococcus sp and salmonella sp EO
Strains 8 937,33 117,17 0,065
According to the method of dilution on a solid medium, Residual 16 781,11 48,82
the values of MIC and MBC / MFC of EO with respect
Strains 5 444,4444 88,8889 <,001
to the microorganisms tested are grouped together in AMP
table 4. For yeasts, the strain most sensitive to the effect Residual 10 8,8889 0,3889
of the essential oil is S. Cerevisiae for which the MIC
value of 0.5 μl / ml is the minimum EO concentration
capable of inhibiting the growth of this strain, therefore
Table 3. Comparison of means ( Bonferroni Test)
the extract to exert a fungistatic effect on the yeast. In
the case of bacteria, S. aureus to show a significant AMP: Ampicillin 10µg, PRL : Piperacillin 30 µg, NT : not
sensitivity to EO compared to other bacteria, in fact the tested , a and b represent the classes generated by the
10 μl / ml concentration of the extract in the culture
comparison of Bonferroni means
medium has a bactericidal power with respect to this
strain. For the other strains, it is estimated that at higher means of the diameters of the inhibition zones (mm)
concentrations, EO may give better results than those
obtained in this study. Strains EO AMP 10 PRL 30
Some authors have advanced the resistance of C.Albicans 20,33 a NT NT
Staphylococcus aureus compared to other essential oil S. Cerevisiae 20,33 a NT NT
strains of cedrus atlantica [13]. Others have proven that
strains react differently depending on the composition of C.Tropicalis 15,33 a NT NT
cedarwood oil, and the components of essential oils are S.Aureus 14,67 a 13,333 a 0,000 a
classified according to their antimicrobial potency in the Strypto sp 12,67 a 0,000 b 0,000 a
following descending order: Alcohol> ketones>
Salmonella sp 10,33 a 0,000 b 0,000 a
Hydrocarbons [14], [15]. Usually the antimicrobial
activity is due to the different chemical compounds E.Coli 9,67 a 0,000 b 0,000 a
present in this extract, including the major compounds K.Pneumoniae 9,67 a 0,000 b 0,000 a
and the synergistic effect of the minor compounds [16],
Ps. Aeruginosa 0,00 a 0,000 b 0,000 a
[17]. These molecules have an important characteristic,
namely hydrophobicity, which allows them to react with
the membrane lipids of the bacterial cell and the
mitochondria, disrupting their structures and making

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