The in-vitro evaluation of antibacterial, antifungal and cytotoxic properties of Marrubium vulgare L. essential oil grown in Tunisia.

Abstract

In order to validate its antiseptic and anticancer properties with respect to traditional uses, we have screened for the first time the antimicrobial activity of aerial parts of M. vulgare L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines.
The agar disk diffusion method was used to study the antibacterial activity of M. vulgare essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the antimicrobial activities of this essential oil. The in vitro cytotoxicity of M. vulgare essential oil was examined using a modified MTT assay; the viability and the IC50 were used to evaluate this test.
The antimicrobial activity of the essential oil was investigated in order to evaluate its efficacy against the different tested microorganisms. The present results results showed a significant activity against microorganisms especially Gram (+) bacteria with inhibition zones and minimal inhibitory concentration values in the range of 6.6-25.2 mm and 1120-2600 μg/ml, respectively, whereas Gram (-) bacteria exhibited a higher resistance. As far as the antifungal activity, among four strains tested, Botrytis cinerea exhibited the strongest activity with inhibition zones of 12.6 mm. However, Fusarium solani, Penicillium digitatum and Aspergillus niger were less sensitive to M. vulgare essential oil. About the citotoxicity assay, this finding indicate the capability of this essential oil to inhibited the proliferation of HeLa cell lines under some conditions with IC50 value of 0.258 μg/ml.
This investigation showed that the M. vulgare essential oil has a potent antimicrobial activity against some Gram (+) pathogenic bacteria and Botrytis cinerea fungi. The present studies confirm the use of this essential oil as anticancer agent. Further research is required to evaluate the practical values of therapeutic applications.

Full text

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RESEARCHOpen Access
The in-vitro evaluation of antibacterial, antifungal
and cytotoxic properties of Marrubium vulgare
L. essential oil grown in Tunisia
Zied Zarai1, Adel Kadri2*, Ines Ben Chobba3, Riadh Ben Mansour4, Ahmed Bekir5, Hafedh Mejdoub6and
Néji Gharsallah3
Abstract
Background: In order to validate its antiseptic and anticancer properties with respect to traditional uses, we have
screened for the first time the antimicrobial activity of aerial parts of M. vulgare L. essential oil against different
pathogenic microorganisms and the cytotoxic activity against HeLa cell lines.
Methods: The agar disk diffusion method was used to study the antibacterial activity of M. vulgare essential oil
against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory
concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the
antimicrobial activities of this essential oil. The in vitro cytotoxicity of M. vulgare essential oil was examined using a
modified MTT assay; the viability and the IC50were used to evaluate this test.
Results: The antimicrobial activity of the essential oil was investigated in order to evaluate its efficacy against the
different tested microorganisms. The present results results showed a significant activity against microorganisms
especially Gram (+) bacteria with inhibition zones and minimal inhibitory concentration values in the range of
6.6-25.2 mm and 1120-2600 μg/ml, respectively, whereas Gram (-) bacteria exhibited a higher resistance. As far as
the antifungal activity, among four strains tested, Botrytis cinerea exhibited the strongest activity with inhibition
zones of 12.6 mm. However, Fusarium solani, Penicillium digitatum and Aspergillus niger were less sensitive to M.
vulgare essential oil. About the citotoxicity assay, this finding indicate the capability of this essential oil to inhibited
the proliferation of HeLa cell lines under some conditions with IC50value of 0.258 μg/ml.
Conclusion: This investigation showed that the M. vulgare essential oil has a potent antimicrobial activity against
some Gram (+) pathogenic bacteria and Botrytis cinerea fungi. The present studies confirm the use of this essential
oil as anticancer agent. Further research is required to evaluate the practical values of therapeutic applications.
Keywords: Antimicrobial, cytotoxicity, essential oil, Marrubium vulgare L., pathogenic microorganisms, HeLa cell
lines
Background
The Lamiaceae plants was considered as one of the large
plant families used as a framework to evaluate the occur-
rence of typical secondary metabolites [1]. The genus
Marrubium comprises 10 species, which are found wild
in many regions of Tunisia. Among them, Marrubium
vulgare L. is a perennial herb of the Labiatae family
which is commonly known as “horehound” in Europe, or
“Marrubia” in Tunisia, is naturalized in North and South
America, the latter and Western Asia. It possesses tonic,
aromatic, stimulant, expectorant, diaphoretic and diuretic
properties. It is helpful for bronchial asthma and non-
productive cough. It was formerly much esteemed in
various uterine, visceral and hepatic affections and in
phthisis [2]. In the Mediterranean region, M. vulgare is
frequently used in folk medicine to cure a variety of dis-
eases. The plant is reported to possess hypoglycemic [3],
vasorelaxant [4], antihypertensive [5], analgesic [6,7],
* Correspondence: lukadel@yahoo.fr
2Laboratoire de Chimie des Substances Naturelles, Faculté des Sciences de
Sfax, B.P. 1171, 3000 Sfax, University of Sfax, Tunisia
Full list of author information is available at the end of the article
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© 2011 Zarai et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
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anti-inflammatory [8], antioxidant activity [9,10], antiede-
matogenic activity [11], and many other biological activ-
ities. In Tunisian folk medicine, it was used as
hypotensive, hypoglycemic and cardiotonic.
Recently, a large number of essential (volatile) oils and
their constituents have been investigated for their bio-
logical activity, notably antibacterial, antifungal, and anti-
oxidant properties [12-14]. Essential oils and their
components are gaining increasing interest as a natural
alternative to synthetic drugs [15], particularly against
microbial agents because of their relatively safe status wide
acceptance by consumers and their exploitation for poten-
tial multipurpose functional use. The chemical composi-
tions of M. vulgare essential oil from various origins have
been the subject of many studies. The literature reveals
the occurence of several chemotypes. From Lithuania, (Z)-
b-farnesene, b-caryophyllene, (E)-2-hexenal, a-humulene
and germacrene D were the main components of M. vul-
gare essential oil [16]. From Czech Republic, the main
constituents of M. vulgare essential oil were b-caryophyl-
lene and germacrene D [17]. From different region of Iran,
the main constituants of M. vulgare essential tricyclene,
b-pinene, bisabolol, b-elemone and isomenthon-8-thiol
[18], b-bisabolene, 8-cadinene and isocaryophyllene [19],
and bisabolene, b-caryophyllene, germacrene D and E-b-
farnesene [20], caryophyllene oxide, b-caryophyllene and
germacrene D [21].
The interest in plants with antimicrobial properties has
been revived because of current problems associated with
the use of antibiotics [22]. Therefore, essential oils and
other naturally occurring antimicrobials are attractive to
the food industry as well as imparting flavor [23]. More
recently, the essential oil of this plant was advocated for
their use as an antioxidant agent [10], but to the best of
our knowledge, there are no reports on the antimicrobial
properties and the cytotoxicity has been published.
Therefore, this paper was conducted to investigate for
the first time the antimicrobial properties against clinical
and pathogenic microorganisms and the cytotoxicity of
M. vulgare essential oil grown in Tunisia.
Methods
Chemicals, reagents and plant material
Chemicals and reagents were supported by Prolabo (Paris,
France) and Pharmacia (Uppsala, Swedeen). Plant materi-
als (aerial parts) of M. vulgare L. were grown in the vici-
nity of the village of Ouled Mnasser, with a latitude of
34.88 (34° 52’ 60 N) and a longitude of 9.13 (9° 7’ 60 E) in
Sidi Bouzid, Tunisia. The aerial parts of wild growing
plant have been collected during the period of June-July
2009. The plant materials were confirmed by a senior A.
bekir. Voucher specimens were deposited at ISET, Sfax
(Département de Génie des procédés) as Bekir 520.
Distillation of essential oil and GC/MS analysis conditions
The fresh aerial parts of M. vulgare (300 g) were hydro-
distilled using a Clevenger-type apparatus to recover the
essential oils for 4 h. The distilled essential oils were
dried over anhydrous sodium sulfate, filtered and stored
at +4°C.
The essential oil was analyzed using an Agilent-Tech-
nologies 6890 N Network GC system equipped with a
flame ionization detector and HP-5MS capillary column
(30 m × 0.25 mm, film thickness 0.25 μm; Agilent-Tech-
nologies, Little Falls, CA, USA). The injector and detector
temperatures were set at 250 and 280°C, respectively. The
column temperature was programmed from 35 to 250°C
at a rate of 5°C/min, with the lower and upper tempera-
tures being held for 3 and 10 min, respectively. The flow
rate of the carrier gas (helium) was 1.0 ml/min. A sample
of 1.0 μl was injected, using split mode (split ratio, 1:100).
All quantifications were carried out using a built-in data-
handling program provided by the manufacturer of the
gas chromatograph. The composition was reported as a
relative percentage of the total peak area. The identifica-
tion of the essential oil constituents was based on a com-
parison of their retention times to n-alkanes, compared
to published data and spectra of authentic compounds.
Compounds were further identified and authenticated
using their mass spectra compared to the Wiley version
7.0 library.
Antimicrobial activity assay
Microbial strain
The essential oil of M. vulgare was individually tested
against a panel of microorganisms (Table 1). The antimi-
crobial activities of essential oil were determined against
sixteen of human-pathogenic microbial strains. The bac-
teria and fungi used were selected because they have
implicated with skin, oral and intestinal tract of man.
Twelve species of bacteria and four species of fungi as
shown in Table 1 were used in this study.
Table 1 Pathogenic bacteria and fungi used for the
antimicrobial assay
Bacteria
Staphylococcus aureus 1327
Micrococcus luteus
Enterobacter cloacae
Bacillus subtilis
Pseudomonas aeruginosa 27853
Escherchia coli 25922
Fungi
Botrytis cinerea
Penicillium digitatum
Staphylococcus epidermidis
Enterococcus faecalis
Staphylococcus aureus 25923
Bacillus cereus
Klebsielle pneumoniae WHO24
Fusarium solan
Aspergillus niger
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Agar diffusion method
The agar diffusion method was employed for the determi-
nation of antibacterial activities of M. vulgare essential oil
according to the method described by Berghe and Vlie-
tinck (1991) [24]. The essential oil extracts were dissolved
in 100% ethanol to a final concentration of 10 mg/ml and
sterilized by filtration trough 0.22 μm Nylon membrane
filter. The bacterial strains were cultured in a nutriment
broth for 24 hours. Then, 200 μl of each suspension
bacteria (106CFU estimated by absorbance at 600 nm)
was spread on Luria Broth agar. Bores were made by using
a sterile borer and were loaded with 10 μl of each sample
extract. Ethanol was used as negative control and ampicil-
lin (10 μg/puit) as positive reference standard. All the
plates were incubated at 37°C for 24 hours. Antibacterial
activity was evaluated by measuring the zone of inhibition
in millimetres. All experiments were done in triplicates.
Determination of the minimal inhibitory concentration
(MIC)
The minimal inhibitory concentration (MIC) values,
which represent the lowest essential oil concentration
that completely inhibits the growth of microorganisms,
were determined by a micro-well dilution method [25].
The inoculum of each bacterium was prepared and the
suspensions were adjusted to 106CFU/ml. All the
extracts were dissolved in 100% ethanol and then dilu-
tions series were prepared in a 96-well plate. Each well of
the microplate included 40 μl of the growth medium,
10 μl of inoculums and 50 μl of the diluted sample
extract. The ampicillin and ethanol are used as positive
and negative controls, respectively. The plates were then
covered with the sterile plate and incubated at 37°C for
24 h. After that, 40 μl of 3- (4, 5-dimethyl-thiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT) at a final con-
centration 0.5 mg/ml freshly prepared in water was
added to each well and incubated for 30 min. The change
to red colour indicated that the bacteria were biologically
active. The MIC was taken to the well, where no change
of colour of MTT was observed. The MIC values were
done in triplicate.
Antibacterial assay disc-diffusion method
An antibacterial activity of the essential oils was screened
against eight human pathogenic bacteria. The inhibitory
effect on bacterial growth was determined using agar-
disc diffusion assay [26,27]. The bacterial cultures were
first grown on Muller Hinton agar (MH) plates at 37°C
for 18 to 24 h prior to seeding onto the nutrient agar.
One or several colonies of the respective bacteria were
transferred into API suspension medium (bioMerieux)
and adjusted to 0.5 McFarland turbidity standards with a
Densimat (bioMerieux) [28,29]. The inocula of the
respective bacteria were streaked into MH agar plates
using a sterile swab and were then dried at 37°C during
15 min. A sterile filter disc having 6 mm of diameter was
placed at the surface of MH agar and 5 μl of the essential
oil was dropped onto each Whatman paper disc [30].
The treated Petri dishes were incubated at 37°C for 18 to
24 h. The antibacterial activity was evaluated by measur-
ing the clear zone surrounding the Whatman paper.
Standard discs of the antibiotic ampicillin were applied as
a positive antibacterial controls.
Antibacterial assay dilution method
The minimal inhibitory concentration (MIC) of essential
oil was determined using the Mueller Hinton broth
(MHB) dilution method [31]. All tests were performed
in MHB supplemented with ethanol 5% [32]. Bacterial
strains were cultured overnight in MHB at 37°C. Tubes
of MHB containing various concentrations of oils were
inoculated with 10 μl bacterial inoculums adjusted to
106CFU/ml. They were incubated under shaking condi-
tions (100-120 rpm) at 37°C for 24 h [33,34]. Control
tubes without tested samples were essayed simulta-
neously. The essays were performed in triplicate. The
MIC was defined as the lowest concentration preventing
visible growth [35,36].
Antifungal assay disc-diffusion method
The biological activity against yeasts was determined by
employing disc agar diffusion method using Sabouraud
Dextrose agar [37]. An aliquot (5 μl) of the oil was depos-
ited on sterile paper discs (6 mm diameter) which were
subsequently placed in the centre of the inoculated Petri
dishes. After an incubation period of the 24 h at 30°C, the
inhibitory activity was compared to that of commercial
cycloheximide at a concentration of 10 mg/ml.
Cell lines and culture condition
HeLa cells (cervical cancer line, adherent) were used to
investigate the cytotoxicity effect of essential oil. This cell
line were grown in RPMI 1640 medium (Gibco) supple-
mented with 10% (v/v) foetal calf serum (FCS) and 2 mM
L-glutamin in tissue culture flasks (Nunc). They were
passed twice a week and kept at 37°C in a humidified
atmosphere of 95% air and 5% CO2.
MTT test
The proliferation rates of HeLa cells after treatment with
essential oils were determined by the colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay. The yellow compound MTT is reduced by
mitochondrial dehydrogenases to the water-insoluble
blue compound formazan, depending on the viability of
cells.
HeLa cells (4 ×104in each well) were incubated in 96-
well plates for 24 hours in the presence or absence of
essential oil. Twenty microlitres MTT solution (Sigma)
(5 mg mL-1in PBS) were added to each well. The plate
was incubated for 4 h at 37°C in a CO2-incubator. One
hundred and eighty microlitres of medium was removed
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from every well without disturbing the cell clusters. A
180 μl methanol/DMSO solution (50:50) was added to
each well, and the preparations were mixed thoroughly
on a plate shaker with the cell containing formazan crys-
tals. After all of the crystals were dissolved, the A570
values were determined with a microplate reader (ELx
800).
Results and Discussion
Antimicrobial assays
The antimicrobial activities of M. vulgare essential
oil against microorganisms examined in the present study
and their potency were qualitatively and quantitatively
assessed by the presence or absence of inhibition zones
and zone diameter (DD), the medium inhibitory concentra-
tion (IC50) and the minimal inhibitory concentration (MIC)
values. This essential oil displayed varied antibacterial and
antifungal activities across the studied pathogens. As can
be seen from Table 2, essential oil inhibited the growth of
bacterial strains produced a zone diameter of inhibition
from 6.6 to 25.2 mm for Gram (+) bacteria, along with
IC50and MIC values ranging from 560-1100 μg/ml and
1120-2600 μg/ml, respectively. Whereas, for Gram (-)
bacteria, no antimicrobial activities of essential oil tested
against all strains (Pseudomonas aeruginosa 27853,
Klebsielle pneumoniae WHO24, Escherichia coli 25922 and
Salmonella) has been revealed. Among Gram (+) bacteria,
the strongest activity of M. vulgare essential oil was
observed against Staphylococcus epidermidis (25.2 mm)
followed by Staphylococcus aureus 25923 (18 mm), Entero-
bacter cloacae (13.8 mm), Bacillus subtilis (13.2 mm),
Micrococcus luteus (12 mm) and Staphylococcus aureus
1327 (12 mm). However, Enterococcus faecalis (9.6 mm)
and Bacillus cereus (6.6 mm) exhibited moderate to weak
activities, respectively.
For the fungi strains, the disc diameter zones of inhibition
ranged from 6.4-12.6 mm, along with IC50and MIC values
ranging from 2190-3000 μg/ml and 1100-1180 μg/ml,
respectively. The maximal inhibition zones was obtained
for Botrytis cinerea, however Fusarium solani, Penicillium
digitatum and Aspergillus niger exhibited weak activity.
Our results suggest that Gram (+) bacteria are more sensi-
tive to the M. vulgare essential than Gram (-) bacteria. This
was consistent with the previous studies on other spices
and herbs [38,39]. This generally higher resistance among
Gram (-) bacteria could be ascribed to the presence of their
Table 2 Antibacterial and antifungal activity of the essential oil of M. vulgare using agar disc diffusion, IC50and
minimal inhibition concentration (MIC)
StrainsDDa
IC50b
MICc
DDd
Bacterial strains Gram (+)
Staphylococcus aureus
Staphylococcus epidermidis
Micrococcus luteus
Enterococcus faecalis
Enterobacter cloacae
Staphylococcus aureus 25923
Bacillus subtilis
Bacillus cereus
Bacterial strains Gram (-)
Botrytis cinerea
Pseudomonas aeruginosa 27853
Klebsielle pneumoniae WHO24
Escherchia coli 25922
Salmonella
Fungal strains
Botrytis cinerea
Fusarium solani
Penicillium digitatum
Aspergillus. niger
12.0 ± 0.5
25.2 ± 0.3
12.0 ± 0.5
9.6 ± 1.0
13.8 ± 0.6
18 ± 1.0
13.2 ± 1.0
6.6 ± 0.4
2500 ± 50
2200 ± 50
1120 ± 50
1140 ± 10
1500 ± 90
2600 ± 80
2130 ± 50
2113 ± 40
>1100.00
>590.00
>560.00
>680.00
>670.00
>1150.00
>890.00
>950.00
20 ± 0.5
26 ± 0.5
20 ± 1.5
25 ± 1.0
21 ± 1.4
24 ± 0.5
26 ± 0.6
21 ± 1.0
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
21 ± 0.5
20 ± 1.0
21 ± 0.9
22 ± 0.8
29 ± 1.0
12.6 ± 0.5
6.9 ± 0.5
6.6 ± 0.4
6.4 ± 0.0
2190 ± 12
3220 ± 20
3200 ± 15
3000 ± 50
>1100.00
>1190.00
>1120.00
>1180.00
29 ± 1.0
28 ± 0.6
21 ± 0.9
30 ± 0.5
Results are means of three different experiments,
aDD: Disc Diameter of inhibition (halo size) in (mm), E. oil 100 μg/disc,
bMIC: minimum inhibitory concentration (μg/ml),
cIC50: 50% inhibition concentration (μg/ml),
dDD: Disc Diameter of inhibition zone of ampicillin (10 μg/disc) and cycloheximide (10 μg/disc), were used as positive controls for bacteria and fungi,
respectively,
NS: not sensitive.
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outer membrane, surrounding the cell wall, which restricts
diffusion of hydrophobic compounds through its lipopoly-
saccharide covering. The absence of this barrier in Gram
(+) bacteria allows the direct contact of the essential oil’s
hydrophobic constituents with the phospholipids bilayer of
the cell membrane, causing either an increase of ion perme-
ability and leakage of vital intracellular constituents, or
impairment of the bacterial enzyme systems. Differences in
MIC values of bacteria may be related to the differential
susceptibility of bacterial cell wall, which is the functional
barrier to minor differences present in the outer membrane
in the cell wall composition [40]. The highest sensitivity of
Staphylococcus epidermidis and Staphylococcus aureus may
be due to its cell wall structure and outer membrane.
It is most likely that numerous components of the
essential oils play a crucial role in defining the features of
oils, lipophilic or hydrophilic attraction and fixation on
cell wall and membranes, and cellular distribution [41].
This feature is very important because, depending on
their component, the distribution of the oil in the cell
determines the different types of biological activities such
as antibacterial, antifungal and cytotoxicity. As reported
previously [10], the M. vulgare essential oils isolated by
hydrodistillation from the aerial parts was analyzed by
HP-5MS column. The general chemical composition of
the essential oil, the percentage contents, and retention
indices of the components are given in Table 3. Thirty
four components could be identified in the oil (100% of
the total oil). The essential oil is constituted mainly by
approximately equal amounts of oxygenated monoter-
penes (40.02%) and sesquiterpenes hydrocarbons
(42.70%). The major components were g-eudesmol
(11,93%), followed by b-citronellol (9,90%), Citronellyl
formate (9,50%) and germacrene D (9,37%). When com-
pared with previous studies [16-21], our study showed
that this essential oil possessed an original composition
with the main component of g-eudesmol (11.93%), which
is not observed elsewhere.
The antimicrobial properties of essential oils from aerial
part of M. vulgare are suspected to be associated, in part
with their high contents of oxygenated compounds
(46.21%). Several researchers also report mono- and sesqui-
terpenoids as the major components of essential oils which
are phenolic in nature [42,43]. It is therefore reasonable to
assume that their antimicrobial activity might be related to
the abundance of phenolic compounds. Referring to the
literature, the antimicrobial activity of the tested essential
oil can be related to the contribution of the mixture
between major (g-eudesmol, b-citronellol, citronellyl for-
mate and germacrene D) and minor (camphene, borneol)
[10] constituents, which known to have efficient antimicro-
bial properties [44,45]. In addition, germacrene-D is known
to have a strong effect on insect behavior [46] and has
significant antibacterial and antifungal activities [47].
Therefore, Essential oils always represent a complex mix-
ture of different chemical components, thus it is very diffi-
cult to reduce the antibacterial effect of the total oil to a
few active principles.
Compared to the positive control, ampicillin (belong-
ing to the penicillin group of beta-lactam antibiotics is
able to penetrate Gram positive and some Gram nega-
tive bacteria) and cycloheximide (generally used only in
in vitro research applications as a fungicide, and is not
suitable for human use as a therapeutic compound)
were found to possess lower activity than the tested
essential oil. We explain this by the fact, that pure com-
ponent, such as antibiotics give a more potent antimi-
crobial activity when compared to a complex mixture of
components such as essential oils.
Cytotoxicity assays
The effect of different concentrations (3.91-3000 μg/ml),
of M. vulgare essential oil on HeLa cell lines were studied.
As depicted in Table 4, they significantly decreased the
viability of HeLa in a dose dependent manner. For a
concentration up to 250 μg/ml, essential oil destructed
HeLa cells by 27%, however for a concentration higher
than 500 μg/ml, all HeLa cells were destructed. At lower
doses, the oil was tolerated by the cells and its IC50(the
concentration of the tested oil required to reduce the cell
survival fraction to 50% of the control) was 0.258 μg/ml.
The volatile oil displayed an excellent cytotoxic effect
towards the human tumor cell line. This makes the tested
oil certainly deserve some further investigation. Referring
to the literature, the cytotoxicity of the tested essential oils
may be due to the presence of some monoterpenes and
sesquiterpenes including a-Humulene [48] and isopre-
noids including geraniol [49]. These compounds were
reported to be active against the tumor cell lines, but in
our oil, they exist in small amount [10]. Although, the pre-
sence of germacrene D in a good amount can enhances
the cytoxicity of the tested oil. This results was confirmed
by Setzer et al., who reported that germacrene D has
exhibited a 7-fold stronger cytotoxic activity against the
Hs 578T cell line in comparison with a-pinene and limo-
nene and a 6-fold stronger action against Hs 578T and
Hep-G2 than 1,8-cineole, linalool, 4-terpineol and a-terpi-
neol [50]. Some reports support the relationship of cyto-
toxicity with antioxidant activity [51]. So the antioxidant
activity of M. vulgare essential oil might contribute to its
cytotoxic activity [45]. Finally, we noted that the synergis-
tic effects of these active chemicals with other constituents
of the essential oil should be taken into consideration.
This study also justifies and reinforces the use of this plant
on traditional medicine. Although all in vitro experiments
hold limitations with regards to possible in vivo efficacy.
The results of this study are very promising as it will serve
as a data base for researchers in these kinds of studies for
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indicating which essential oils and plant oils may be useful
for specific medical conditions. Furthermore, it is still
necessary to investigate in vivo bioactivity and cytoxicity
of the oil, to explore in more depth its potential beneficial
use in diseases and infections caused by microbes.
Conclusion
In conclusion, our study can be considered as the first
report on the antimicrobial and cytotoxic properties of
M. vulgare volatile oil. The in vitro antimicrobial activity
of the obtained may well be due to the presence of
synergy, antagonism or additive effects of the tested
major components of the oils, which possess various
potency of activity. The results of the cytotoxic activity
against HeLa cell lines in this study are very promising
with regards to possible antineoplastic chemotherapy
and form a very sound basis for future research. Our
results are a contribution to a better valorization of this
Table 3 Chemical composition, retention indices and percentage composition of the M. vulgare essential oil
N° CompoundRI% Identification
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
N-trimethylsilyl trifluoroacetamide
N, N-bis trimethylsilyl trifluoroacetamide
a- pinene
Camphene
1,8-Cineole
a-thujone
1-Vinylcyclohexane
Camphor
Iso menthone
Borneol
b-citronellol
Geraniol
Citronellyl formate
Geranyl formate
a-copaene
b-Bourbonene
trans-caryophyllene
a-Muurolene
a-amorphene
a-Humulene
Neoalloocimene
neryl acetate
Germacrene-D
Ledene
b-bisabolene
δ-cadinene
a -agarofuran
Furan-2-one, 4-phenyltetrahydro
g-Eudesmol
b-Cubebene
Citronellyl butanoate
Geranyl tiglate
Cyclononasiloxane, octadecamethyl
Eicosamethylcyclodecasiloxane
764
857
932
948
1044
1131
1143
1174
1197
1199
1266
1295
1315
1344
1419
1429
1462
1484
1490
1495
1502
1512
1521
1534
1544
1559
1581
1616
1647
1674
1682
1712
2198
2264
2.35
0.97
1.16
0.49
3.72
2.29
0.75
1,03
0.57
0.61
9.90
2.74
9.50
6.25
1.37
1.96
2.15
0.63
0.81
0.68
0.91
3.41
9.37
5.35
0.86
3.30
0.42
1.44
11.93
1.52
0.66
5.53
3.08
2.29
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
MS, RI
Total identification
Yield (g/100 g dry weight)
Hydrocarbon monoterpenes
Oxygenated monoterpenes
Hydrocarbon sesquiterpenes
Oxygenated sesquiterpene
100
00.34
01.65
40.02
42.70
06.19
Zarai et al. Lipids in Health and Disease 2011, 10:161
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medicinal plant. Several other biological tests will be
worthwhile to search for more eventual activities of this
plant to characterize active principles, and assess toxicity
by laboratory assays.
Author details
1Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS, BPW,
1173 Sfax, University of Sfax, Tunisia.2Laboratoire de Chimie des Substances
Naturelles, Faculté des Sciences de Sfax, B.P. 1171, 3000 Sfax, University of
Sfax, Tunisia.3Laboratoire de Microbiologie Alimentaire, Faculté des sciences
de Sfax, B.P. 1171, 3000 Sfax, University of Sfax Tunisia.4Unité de recherche
Biotechnologie et pathologies, Institut Supérieur de Biotechnologie de Sfax,
University of Sfax, Tunisia.5Département de Génie des procédés, ISET Sfax,
Km 2,5 Rte de Mahdia, 3099 Sfax, University of Sfax, Tunisia.6Laboratoire de
Biochimie, Faculté des sciences de Sfax, B.P. 1171, 3000 Sfax, University of
Sfax, Tunisia.
Authors’ contributions
ZZ, AK, IBC and RBM carried out the experimental part such as extraction,
antibacterial, antifungal and cytotoxicity assays. AB contribute to the analysis
of the results. HM and NG supervised the work and corrected the
manuscript. Authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 23 August 2011 Accepted: 21 September 2011
Published: 21 September 2011
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14.
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Table 4 Cytotoxic activity of M. vulgare essential oil
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Oil (μg/ml) Mean OD570(nm)
0 0.971
3.910.836
7.810.815
15.630.798
31.250.780
62.50 0.756
125 0.717
250 0.707
500 0.015
10000.031
30000.023
% Viable HeLa cell line
100.00
86.10
83.93
82.18
80.94
77.85
73.84
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doi:10.1186/1476-511X-10-161
Cite this article as: Zarai et al.: The in-vitro evaluation of antibacterial,
antifungal and cytotoxic properties of Marrubium vulgare L. essential oil
grown in Tunisia. Lipids in Health and Disease 2011 10:161.
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