Essential oils are well known as strong antimicrobial agents of plant origin. In spite of this, the antimicrobial synergism of essential oils isolated from different plant species is poorly investigated. The following study examines the synergism of the essential oils of Origanum vulgare L. and Thymus vulgaris L against pathogenic bacteria, Staphylococcus aureus and Salmonella typhimurium. First, the antibacterial effect of the oils was tested, and the minimal inhibitory concentrations (MIC) of both oils were determined using the microdilution method. To test whether the oils act synergistically, every possible combination of essential oil concentrations was used in a dynamic checkerboard method. The results indicated that the oils indeed acted synergistically with fractional inhibitory concentration indexes of 0.45 and 0.50.Projekat ministarstva br. 17303

Glamočlija, Jasmina

Nikolić, M.

Ristić, M.

Soković, Marina

Stojković, D.

Ćirić, Ana

Šiljegović, Jovana

English

 Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

Arch. Biol. Sci., Belgrade, 65 (2), 639-643, 2013 DOI:10.2298/ABS1302639S
639
INVESTIGATION ON ANTIBACTERIAL SYNERGISM OF ORIGANUM VULGARE AND  
THYMUS VULGARIS ESSENTIAL OILS
D. STOJKOVIĆ1, JASMINA GLAMOČLIJA1, ANA ĆIRIĆ1, M. NIKOLIĆ1, M. RISTIĆ2,  
JOVANA ŠILJEGOVIĆ1 and MARINA SOKOVIĆ1,*
1 University of Belgrade, Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”,  
11000 Belgrade, Serbia 
2 Institute for Medicinal Plant Research “Josif Pančić”, 11000 Belgrade, Serbia
Abstract - Essential oils are well known as strong antimicrobial agents of plant origin. In spite of this, the antimicrobial 
synergism of essential oils isolated from different plant species is poorly investigated. The following study examines the 
synergism of the essential oils of Origanum vulgare L. and Thymus vulgaris L against pathogenic bacteria, Staphylococcus 
aureus and Salmonella typhimurium. First, the antibacterial effect of the oils was tested, and the minimal inhibitory con-
centrations (MIC) of both oils were determined using the microdilution method. To test whether the oils act synergisti-
cally, every possible combination of essential oil concentrations was used in a dynamic checkerboard method. The results 
indicated that the oils indeed acted synergistically with fractional inhibitory concentration indexes of 0.45 and 0.50. 
Key words: Essential oil, synergism, Origanum vulgare, Thymus vulgaris, antimicrobial.
INTRODUCTION
Despite progress in medical care and treatment, in-
fectious diseases caused by pathogen microorganisms 
are still a major threat to public health (WHO, 2002). 
New drugs are introduced but they are expensive and 
relatively unavailable in developing countries (Okeke 
et al., 2005). Emerging resistance to synthetic prod-
ucts also poses a public concern. New strategies are 
needed, and in the last two decades a growing inter-
est in the use of essential oils as safe alternatives is 
increasing.
Essential oils (EO) are products of plant second-
ary metabolites. They are a mixture of compounds, 
mainly mono- and sesquiterpenes, carbohydrates, 
alcohols, aldehydes and ketones. The biological ac-
tivity is in direct dependence of the genetically in-
duced specific chemical composition of an EO. 
Origanum vulgare L. (oregano) is a spice herb 
from the family Lamiaceae, growing from 20-80 cm 
in height, with opposite leaves and purple flowers 
produced in erect spikes. It is native to western and 
southwestern Eurasia and the Mediterranean region. 
The main components of its EO are phenolic com-
pounds: carvacrol and thymol. Chemical composi-
tions vary depending on geographical region and 
session of collecting (Faleiro et al., 2002). Thymus 
vulgaris L. (thyme) is a low-growing herbaceous 
plant that hardly grows above 20 cm, forming a small 
shrub. A member of the Lamiaceae family, it is native 
to southern Europe. The main components of its EO 
are thymol, linalool or carvacrol depends on chemo-
type. The EOs of oregano and thyme are known to 
possess a wide range of biological activities (Adam 
et al., 1998; Sartoratto et al., 2004). Both plants are 
commonly used in foods, for flavor and aroma, in 
pharmaceutical and cosmetic industry.
640 D. STOJKOVIĆ ET AL.
There have been reports of that EOs are more 
strongly antimicrobial than their major components 
individually (Lachowicz et al., 1998) which indicates 
that the minor components play a important role and 
may have a synergistic effect. The synergistic effect of 
the major components of EOs has been reported in 
several studies (Ipek et al., 2005, Ettayebi et al., 2000, 
Hummelbrunner and Isman, 2001), however, exami-
nations of whole oil are rare.
The concept of synergisms appears to have a 
great potential and influence on the biological ac-
tivity of EO. For this reason, we have evaluated the 
synergistic potential of O. vulgare and T. vulgaris EO 
against pathogens.
MATERIALS AND METHODS
Essential oil isolation
Essential oils were isolated by hydrodistillation in a 
Clevenger type apparatus for 3 h. The essential oils 
obtained were stored at +4 °C until further tests.
Gas chromatography (GC) and gas  
chromatography/mass spectrometry (GC/MS)
Qualitative and quantitative analyses of the oils were 
performed using GC and GC/MS. The GC analysis of 
the oil was carried out on a GC HP-5890 II appara-
tus, equipped with a split/splitless injector, attached 
to an HP-5 column (25 m x 0.32 mm, 0.52 μm film 
thickness) and fitted to a flame ionization detector 
(FID). Carrier gas flow rate (H2) was 1 ml/min, split 
ratio 1:30, injector temperature was 250°C, detector 
temperature 300°C, while column temperature was 
linearly programmed from 40-240°C (at rate of 4°/
min). The same analytical conditions were employed 
for GC/MS analysis, where HP G 1800C Series II 
GCD system equipped with an HP-5MS column 
(30 m x 0.25 mm, 0.25 μm film thickness) was used. 
The transfer line was heated at 260°C. Mass spectra 
were acquired in EI mode (70 eV), in an m/z range 
of 40-400. Electron impact identification of indi-
vidual constituents was made by comparison of their 
retention times with those of analytical standards 
of available terpenoids, and by computer searching, 
matching mass spectra with those held in the Wiley 
275 library of mass spectra. Confirmation was done 
using a calibrated AMDIS program for the determi-
nation of experimental values for retention indices 
of recorded constituents and comparing them with 
those from the literature (Adams, 2001). For quanti-
fication purpose, area percent data obtained by FID 
were used.
Microorganisms and culture conditions
For the bioassays, Gram (-) bacterial species Salmo-
nella typhimurium (ATCC 13311), and Gram (+) 
bacterial species Staphylococcus aureus, were used. 
The organisms tested were from the Mycological 
Laboratory, Department of Plant Physiology, Insti-
tute for Biological Research “Siniša Stanković”, Bel-
grade, Serbia. Bacteria were maintained on Mueller-
Hinton agar (MHA).
Microdilution method
In order to investigate the antimicrobial activity of 
the isolated essential oils and the main constituents, 
the modified microdilution technique was used 
(Daouk et al., 1995). Bacterial species were cultured 
overnight at 37ºC in Tryptic Soy Broth (TSB) me-
dium The bacterial cell and fungal suspension was 
adjusted with sterile saline to a concentration of 1.0 x 
105 units in a final volume of 100 μl per well. The in-
ocula were stored at +4ºC for further use. Dilutions 
of the inocula were cultured on solid MHA for bac-
teria to verify the absence of contamination and to 
check the validity of the inoculum.
Minimum inhibitory concentration (MIC) de-
termination was performed by a serial dilution tech-
nique using 96-well microtiter plates. The investi-
gated essential oils (0.01-10.00 μl/ml) were added 
in TSB medium with inoculum. The microplates 
were incubated for 24 h at 37ºC for bacteria. Bacte-
rial growth was detected by the addition of 30 μl of a 
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetra-
zolium chloride (INT) water solution (0.5 mg/ml), 
(Sigma). The bacteria were again incubated at 37ºC 
INVESTIGATION ON ANTIBACTERIAL SYNERGISM OF ORIGANUM VULGARE AND THYMUS VULGARIS ESSENTIAL OILS 641
for 30 min, and in the wells where bacterial growth 
occurred, INT changed from yellow to purple. MIC 
values were defined as the lowest concentration of 
essential oil to completely inhibit microbial growth. 
Checkerboard method
This method was carried out using 96-well micro-
plates containing Tryptic Soy Broth (TSB) with es-
sential oil concentrations ranging from 1/16 - 4 × 
MIC and combined with each other on the plate in a 
checkerboard style. The bacterial inoculum was 1.0 
x 105 colony-forming unit (CFU) per well. The mi-
croplates were incubated for 24 h at 37ºC (Jacqueline 
et al., 2005). The fractional inhibitory concentration 
(FIC) index was calculated as follows: drug A (O. 
vulgare essential oil) FIC (drug A MIC combined/
drug A MIC alone) + drug B (T. vulgaris essential 
oil) FIC (drug B MIC combined/drug B MIC alone). 
Synergism was defined as FIC index < 0.5; additivity 
FIC index > 0.5-< 2; indifference FIC index > 2-< 4 
and antagonism FIC index > 4 (Lorian, 2005). The 
strains that had MIC > 32 μg/ml and MIC > 1024 μg/
ml were considered to be MICs of 64 μg/ml and 2048 
μg/ml, respectively.
RESULTS AND DISCUSSION
Results on the chemical composition of the essen-
tial oils of Origanum vulgare and Thymus vulgaris 
are presented in Table 1. The essential oil of O. vul-
gare was rich in carvacrol (64.50%), while p-cymene 
and γ-terpinene were also dominant constituents 
with slightly lower percentage (10.90% and 10.80%). 
T. vulgaris essential oil was abundant with thymol 
(48.92%), followed by p-cymene (18.99%). The other 
constituents were present in amounts less than 5%. 
Chemical analysis of O. vulgare by other authors 
(Tian and Lai, 2006; Masood et al., 2007) also showed 
that the main constituents of this oil are phenols: car-
vacrol and thymol. However, some reports (Bussata 
et al., 2007) revealed monoterpene hydrocarbons 
terpinen-4-ol and γ-terpinen as the main compo-
nents in chemical analysis. The results of chemical 
analysis of T. vulgaris are in accordance with previ-
ous studies (Rota et al., 2008, Hudaib et al., 2002) 
with major constituents being thymol and p-cymene. 
The high content of phenol compounds from this oil 
is responsible for its strong antimicrobial and anti-
oxidant activity.
Investigating the antimicrobial synergism of a 
combination of thyme and oregano essential oils we 
obtained the results presented in Table 1. These re-
sults indicated that oregano and thyme essential oils 
indeed expressed antimicrobial synergism. As for the 
S. aureus MIC results, it can be seen from Table 1 
that it was more sensitive than S. typhimurium to the 
effect of oregano essential oil. The MIC for oregano 
essential oil on S. aureus was 0.5 μl/ml, and for S. ty-
phimurium was 1.0 μl/ml. The essential oil of thyme 
expressed the same MICs on both bacteria tested (1.0 
μl/ml). The combined effect of thyme and oregano 
essential oils on S. aureus was synergistic in a com-
bination of MIC 0.2 μl/ml for oregano oil and 0.25 
μl/ml for thyme oil. The FIC index was calculated to 
be 0.45, which is <0.5 and according to the literature 
(Lorian, 2005) this value indicates compound syner-
gism. A synergistic effect of combined essential oils 
on S. typhimurium was also obtained with combined 
MICs of 0.5 μl/ml and 0.05 μl/ml, for oregano and 
thyme oils, respectively. The value for the FIC index 
was calculated to be 0.505, which is approximately 
<0.5 and the effect is also considered to be synergis-
tic. 
Plant essential oils are a potentially useful source 
of antimicrobial compounds. The results of different 
studies are difficult to compare, most probably be-
cause of the different test methods, bacterial strains 
and sources of antimicrobial samples used. Natu-
ral products demonstrate different modes of action 
(McAllister et al., 2000), and in order to achieve the 
desired inhibition effect, products belonging to dif-
ferent plants should be combined as shown in this 
study.
By mixing different essential oils, their concen-
tration can be significantly reduced, which possibly 
eliminates their undesired sensorial impact on anti-
microbial preparation. The antibacterial properties 
of terpenic compounds are due to the presence of de-
642 D. STOJKOVIĆ ET AL.
localized electrons in the molecule, which according 
to Ultee (2002) allows for easier deprotonation, play-
ing an important role in their antibacterial activity. It 
is reported that terpenoid compounds have a greater 
inhibiting effect on Gram-positive than on Gram-
negative bacteria (Nascimento et al., 2000) and our 
results confirm these previous findings.
In conclusion, O. vulgare and T. vulgaris are valu-
able sources of secondary metabolites with biologi-
cal activities. This study demonstrates the synergistic 
effect of a natural mixture of two popular plant es-
sential oils, O. vulgare and T. vulgaris. This potential 
should be further evaluated for application in mod-
ern medicine and industry against pathogen micro-
organisms.
Acknowledgments - The authors are grateful to the Ministry of 
Education and Science of Serbia for financial support (Grant 
№ 173032).
Table 1. Chemical composition of essential oils
Components O. vulgare % T. vulgaris % RI
α-thujene 1.9 1.17 931
α-pinene - 1.21 939
camphene - 0.83 948
sabinene 2.20 0.58 973
β-pinene - 0.41 980
β-myrcene - 1.06 991
δ-3-carene 2.20 - 1011
α-terpinene - 0.65 1018
p-cymene 10.90 18.99 1026
limonene - 0.46 1030
1,8-cineole - 0.76 1031
trans-ocimene - 1.30 1050
γ-terpinene 10.80 4.08 1068
linalool - 0.74 1098
camphor - 0.17 1143
borneol - 1.72 1165
terpin-4-ol - 1.78 1177
thymol-methyl-ether - 0.16 1235
carvacrol-methyl-ether - 1.73 1244
thymol 3.50 48.92 1290
carvacrol 64.50 3.45 1298
β-caryophyllene 2.50 3.45 1418
α-humulene - 0.30 1454
germacrene D - 0.33 1480
α-cadinene - 2.23 1538
Total 98.50 96.48
Table 2. Synergistic property of O. vulgare and T. vulgaris combined essential oils (μL/mL).
MIC oregano MIC thyme MIC (O1 + T2) FIC index
S. aureus 0.5 1.0 0.2 + 0.25 0.45
S. typhimurium 1.0 1.0 0.5 + 0.05 0.50
1Oregano, 2Thyme
INVESTIGATION ON ANTIBACTERIAL SYNERGISM OF ORIGANUM VULGARE AND THYMUS VULGARIS ESSENTIAL OILS 643
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Investigation on antibacterial synergism of Origanum vulgare and Thymus vulgaris essential oils

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Investigation on antibacterial synergism of Origanum vulgare and Thymus vulgaris essential oils

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