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Journal of Essential Oil Bearing Plants
ISSN: 0972-060X (Print) 0976-5026 (Online) Journal homepage: http://www.tandfonline.com/loi/teop20
Chemical Profile, Antioxidant and Antibacterial
Activity of Thyme and Oregano Essential Oils,
Thymol and Carvacrol and Their Possible
Synergism
Neda Gavaric, Sonja Smole Mozina, Nebojša Kladar & Biljana Bozin
To cite this article: Neda Gavaric, Sonja Smole Mozina, Nebojša Kladar & Biljana Bozin (2015)
Chemical Profile, Antioxidant and Antibacterial Activity of Thyme and Oregano Essential Oils,
Thymol and Carvacrol and Their Possible Synergism, Journal of Essential Oil Bearing Plants,
18:4, 1013-1021, DOI: 10.1080/0972060X.2014.971069
To link to this article: http://dx.doi.org/10.1080/0972060X.2014.971069
Published online: 04 Sep 2015.
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Chemical Profile, Antioxidant and Antibacterial Activity of Thyme and
Oregano Essential Oils, Thymol and Carvacrol and Their Possible Synergism
Neda Gavaric 1*, Sonja Smole Mozina 2, Nebojša Kladar 1, Biljana Bozin 1
1 Department of Pharmacy, Faculty of Medicine, University of Novi Sad,
Hajduk Veljkova 3, 21000 Novi Sad, Serbia
2 Department of Food Science and Technology, Biotechnical Faculty,
University of Ljubljana, Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia
Abstract: In this study, chemical composition, antioxidant and antibacterial activities of thyme and
oregano essential oils, thymol and carvacrol, as well as their possible synergism were investigated. For chemical
profiling of essential oils, GC-MS analysis was performed. Antioxidant capacity was assessed throughout
neutralisation of DPPH and OH radicals. For determination of antibacterial activity against selected gram-
positive (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Salmonella Infantis,
Escherichia coli O157:H7) broth micro-dilution method was used. Finally, the combination testing for potential
synergy of thyme and oregano essential oils, thymol and carvacrol was performed using checkerboard method
and evaluated with fractional inhibitory concentration. Thymol and carvacrol were found to be dominant
components of investigated essential oils. All studied samples expressed strong antioxidant and antibacterial
potential. In synergy testing, both combinations (thyme/oregano essential oils and thymol/carvacrol) were
found to have additive effect (FIC=0.75). These results confirm thyme and oregano essential oils, thymol and
carvacrol as well as their combinations for possible application as natural additives in different products
(food, cosmetics and nutriceuticals).
Key words: Thyme, oregano, essential oils, thymol, carvacrol, antioxidant activity, antibacterial
activity, antibacterial synergism.
Introduction
Many species of Thymus and Oreganum genera,
native to central and southern Europe, the Balkans
and Asia are widely used as medicinal and
culinary herbs 1. Among numerous species,
Thymus vulgaris L. and Origanum vulgare L.,
(Lamiaceae) are the most frequently used and
cultivated worldwide. Numerous studies
confirmed antioxidant, antibacterial, spasmolytic
and expectorant activity for thyme essential oil
(TEO) and antioxidant, antibacterial, spasmolityc
and carminative effect for oregano essential oil
(OEO )1,2,3,4. Both plants are widely used as spices
in food processing and in many pharmaceutical
preparations as herbal remedies 4.
In many representatives of Origanum and
Thymus oxygenated monoterpenes thymol and
carvacrol were found to be dominant components
4,5,6. They have been assessed for antioxidant and
antimicrobial activities since they are generally
recognised as safe (GRAS) by the United States
Food and Drug Administration (FDA). Also, they
have been registered by the European Com-
mission for use as flavouring agents in foodstuffs
*Corresponding author (Neda Gavaric)
E-mail: < nedalakic@gmail.com > © 2015, Har Krishan Bhalla & Sons
Received 03 July 2014; accepted in revised form 20 October 2014
TEOP 18 (4) 2015 pp 1013 - 1021 1013
ISSN Print: 0972-060X
ISSN Online: 0976-5026
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3,7. Both substances appear to influence the cell
membrane permeability causing the inhibition of
growth and death of vegetative bacterial cells, but
also influence the toxin production 8,9,10.
Additionally, antioxidant effect of natural
compounds seems to play a very important role
since oxidation processes occur often in raw
material during processing but also in final
products of food and pharmaceutical industry.
Such oxidative deterioration together with the
growth of undesirable microorganisms results in
the development of spoilage, off-flavour, rancidity
and deterioration, rendering such products
unacceptable for human consumption 11,12.
However, the use of essential oils and their
components in food products is often limited
because effective antimicrobial concentrations
may exceed the acceptable sensory level in food
8. It has been well documented that a different
constituents of essential oils exhibit significant
antimicrobial action when tested separately. Yet,
there is some evidence of enhancement in anti-
microbial action when essential oils components
are used in combination with other antimicrobial
agents, both synthetic and natural and a variety
of treatments 13,14, 15,16,17,18.
In this study antimicrobial and antioxidant
activity of chemically well characterised thyme
and oregano essential oils, as well as their major
compounds thymol and carvacrol were evaluated.
Moreover, potential synergistic effects of
examined essential oils and isolated compounds
against some human pathogen strains of bacteria
have been reported.
Material and methods
Plant material
Leaves of cultivated plants of thyme (Thymus
vulgaris L., Lamiaceae) were collected in July
2012 in Padej (Vojvodina province, Republic of
Serbia). Oregano herb (Origanum vulgare L.,
Lamiaceae) was obtained from the Institute for
Studies on Medicinal Plants in Belgrade in 2012.
Voucher specimens of plants (thyme no. ThV-15/
12 and oregano no. Ov-16/12) 19 were confirmed
and deposited at the Herbarium of the Laboratory
of Pharmacognosy, Department of Pharmacy,
Faculty of Medicine, University of Novi Sad.
Plant material was air-dried and stored in double
paper bags in a dry place without direct sunlight
until hydro-distillation.
Isolation of essential oils
Essential oils were obtained by hydrodistillation
according to European Pharmacopoeia IV 20, with
n-hexane as a collecting solvent. The solvent was
removed under vacuum, essential oils were
dehydrated with anhydrous Na2SO4 and quantified
gravimetrically.
GC-MS analysis
Qualitative and quantitative analysis of the
investigated essential oils was performed on
Hewlett-Packard 5973-6890 GC-MS system,
operating in electron ionization (EI) mode at 70
eV, equipped with a split-splitless injector (200°C)
and a flame ionization detector (250°C). HP 5MS
(30 m × 0.25 mm; film thickness 0.25 μm)
capillary columns and helium as carrier gas (1
mL/min) were used. The temperature programs
were 60-280°C at a rate of 3°C/min and 60-260°C
at a rate of 3°C/min, respectively; the split ratio
was 1:10. Coelution and MS analysis identified
the separated compounds by comparing their
relative retention times with those of authentic
samples (Carl Roth GmbH, Karlsruhe, Germany).
For the components for which authentic
substances were not available the identification
was based on matching their retention indices and
mass spectra with NIST/NBS, Wiley libraries
spectra, and literature data 21.
Antioxidant activity
Antioxidant properties of the thyme and
oregano essential oils, as well as for thymol and
carvacrol were evaluated as free radical
scavenging capacity (RSC) on the DPPH and OH
radicals.
DPPH assay
The DPPH assay was carried out as described
before 11. The concentration of investigated
essential oils ranged from 0.0625 to 1 μg/mL and
for isolated compounds from 12.2 to 244.3 μg/
mL. Spectrophotometric analysis was performed
on Agilent Technologies 8453 UV-VIS spectro-
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photometer. As a positive control, terc-butylated
hydroxytoluene (BHT; Fluka, AG, Buchs,
Switzerland) was used.
Neutralisation of OH radical
The scavenging capacity of essential oils,
thymol and carvacrol for hydroxyl radicals was
performed spectrophotometrically as desribed
before 22. Examined TEO and OEO concentration
ranged from 0.033 to 0.75 μg/mL and from 16.29
to 500 μg/mL for thymol and carvacrol.
The inhibition rate of deoxyribose degradation
or neutralisation of DPPH radical by the examined
essential oils and isolated compounds were
calculated by the following equation:
All tests were done in triplicate.
The IC50 values, which represented the concen-
trations of essential oils, thymol and carvacrol that
caused 50 % neutralisation of DPPH and OH
radicals were determined by regression analysis.
Antibacterial testing
Antibacterial activity against selected gram-
positive and gram-negative bacteria was assessed
using broth microdilution method. After the
determination of minimal inhibitory concentration
(MIC), investigated essential oils, as well as
thymol and carvacrol, as dominant compounds,
were tested for possible synergism.
Bacterial strains and growth conditions
Selected bacterial strains, namely B. cereus
WSBC 10530 (clinical isolate), S. aureus ATCC
25923 (clinical isolate), S. Infantis •MJ 106
(poultry meat isolate) and E. coli O157:H7 •M
370 (clinical isolate) were prepared as described
in Klacnik and others 23 and used for antibacterial
testing.
Broth microdilution method
Minimal inhibitory concentration was deter-
mined as presented before 23. Specifically, 50 μL
of investigated essential oils, thymol (32 mg/mL)
or carvacrol (16 mg/mL) was two-fold serially
diluted in 96-well microplate before bacterail
suspensions were added. All measurements of
MIC values were repeated in triplicate.
Synergy testing
Synergy testing with thyme and oregano
essential oils and thymol and carvacrol against
cultivated bacterial strains was carried out by
checkerboard assay. Two-fold serially diluted
concentrations of one component were dispensed
in the rows and two-fold serially diluted concen-
trations of the other component were dispensed
in the columns of a microplate. Inocula and color
indicators were added as in broth dilution method.
Thyme essential oil was tested against oregano
essential oil and carvacrol was tested against
thymol. MICs were determined for each compo-
nent in the presence of the second component,
and fractional inhibitory concentrations (FICs)
were calculated from these as follows:
The FIC index was obtained by adding the
individual FICs. The results were interpreted as
synergistic when the FIC index was <0.5, as
additive when the index was between 0.5 and 1.0,
as indifferent when the index was between 1.0
and 2.0, and antagonistic when the index was
>2.0 7,13.
Results and discussion
Chemical composition
The content of essential oils (w/w in dry matter)
was 1.029 % for TEO and 1.074 % for OEO.
Obtained results are in correlation with previously
published data 1. The detailed GC-MS analysis
results are presented in Table 1. The total number
of chemical constituents identified in essential oils
was 12 for thyme and 25 for oregano, representing
96.3 % and 95.2 % of the total oil content, res-
pectively.
The essential oil obtained from Thymus vulgaris
was found to be composed primarily of aromatic
oxygenated monoterpenes (57.8 %) followed by
the oxygenated monoterpenes (17.3 %). Aromatic
oxygenated monoterpene thymol (41.6 %),
carvacrol (12.0 %); oxygenated monoterpenes
borneol (8.9 %), linalool (6.8 %) and hydrocarbon
γ-terpinene (7.3 %) were identified as the main
constituents of thyme essential oil. Besides,
carvacrol methyl ether (3.9 %), sesquieterpenes
trans-caryophyllene (5.1 %) and caryophyllene
RSC (%) = 100 × (Ablank-Asample / Ablank)FIC(A) = MIC(A in the presence of B) /MIC(A alone) and
FIC(B) =MIC(B in the presence of A) /MIC(B alone).
Neda Gavaric et al., / TEOP 18 (4) 2015 1013 - 1021 1015
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Table 1. Chemical composition of thyme and oregano essential oils
Mass percentage (%)
No. Compounds R.I.aThyme Oregano
essential oil essential oil
Monoterpene Hydrocarbones 7.3 5.1
1β-Myrcene 991 - 0.9
2γ-Terpinene b1060 7.3 4.2
Oxygenated Monoterpenes 17.4 11.4
3trans-Sabinene hydrate 1098 1.6 -
4 Linalool b1097 6.8 4.8
5 Borneol b1169 8.9 6.1
6α-Terpineol b1189 - 0.5
Aromatic Oxygenated Monoterpenes 57.8 59.9
7 Thymol methyl eter 0.3 0.6
8 Carvacrol methyl eter 1245 3.9 0.9
9 Thymol b1290 41.6 4.5
10 p-Cymene-7-ol 1291 - 0.5
11 Carvacrol b1299 12.0 53.4
Sesquiterpene Hydrocarbons 6.4 8.7
12 α-Caryophyllene 1409 - 0.2
13 trans Caryophyllene b1419 5.1 1.6
14 Aromadendrene 1441 - 0.4
15 α-Himachalene 1451 - 0.2
16 α-Amorphene 1485 - 0.2
17 β-Himachalene 1505 - 1.6
18 β-Bisabolene 1506 - 4.5
19 δ-Cadinene 1523 1.3 -
Oxygenated Sesquiterpenes 4.6 8.3
20 Caryophyllene oxide 1583 4.6 4.5
21 α-Cadinol 1654 - 3.3
22 Farnesol b1725 - 0.1
23 cis-Lanceol 1761 - 0.3
24 Cedran-diol 1898 - 0.1
Diterpenes 0.5
25 Abietatriene 2057 - 0.5
Aromatic Hydrocarbons 1.1
26 Anisole 0918 -1.1
Aliphatic compounds 2.8 0.2
27 1-Octen-3-ol 979 2.8 -
28 Octadecane 1800 - 0.2
Identified compounds (%) 96.3 95.2
a Retention indices (R.I.) relative to C9-C24 n-alkanes on the HP 5MS column
b identification based on retention times of authentic compounds on HP 5MS column
Neda Gavaric et al., / TEOP 18 (4) 2015 1013 - 1021 1016
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oxide (4.6 %) were detected in considerable
amounts (Table 1). In the essential oil distillated
from Origanum vulgare, aromatic oxygenated
monoterpenes were elucidated as the major class
of compounds (59.9 %). Carvacrol was dominant
(53.4 %), followed by borneol (6.1 %), linalool
(4.8 %) and thymol (4.5 %). Considerable amount
of sesquiterpene hydrocarbons (8.8 %) and oxy-
genated sesquiterpenes (8.2 %) were determined,
with β-bisabolene (4.5 %) and caryophyllene
oxide (4.5 %) as the major substances. These
findings are in accordance with previously
published data on TEO 1,6,24 and OEO 24,25 with
slightly lower concentrations of thymol and
carvacrol and considerably higher amount of
sesquiterpenes.
Antioxidant activity
Oxidation processes are one of the leading
causes of deterioration of foods stuffs, therefore
additives that prevent oxidation are welcomed by
the food industry. Nowadays, the focus is on
additives of natural origin that are less toxic and
have smaller impact on the environment since the
global market is experiencing a trend of ‘green’
consumerism 3. The antioxidant potential of plant
products and pure compounds can be evaluated
with numerous assays. First, the screening of the
potential antioxidant activity is done by various
in vitro tests dealing with the different mechanism
of action such are lipid peroxidation of free radical
scavenging ability. In order to obtain relevant data,
it is advisable to use more than one method for
assessment of antioxidant activities of plant
products due to their complex composition 26,27.
Therefore, for determination of antioxidant poten-
tial of examined thyme and oregano essential oil,
as well as thymol and carvacrol as dominant
compounds, DPPH and OH radical scavenging
ability was measured.
In the DPPH assay, the ability of TEO, OEO,
thymol and carvacrol to act as donors of hydrogen
atoms or electrons in transformation of DPPH• into
its reduced form DPPH-H was investigated. All
examined samples were able to reduce the stable,
purple-colored DPPH radical into yellow-colored
DPPH-H, reaching 50 % of reduction with IC50
values of 0.24 μg/mL for thyme, 0.2 μg/mL for
oregano, 70.06 μg/mL for thymol and 79.75 μg/
mL for carvacrol, respectively (Table 2).
Comparison of the DPPH scavenging activity
of the investigated essential oils and those
expressed by BHT (6.95 μg/mL) (Fig 1.) showed
that examined samples possessed stronger
antioxidant effects than BHT. On the other hand,
isolated compounds exhibited considerably lower
scavenging activity. These findings are in
correlation with the earlier published data on the
notable antiradical activity of both essential oils
4,24,28. The fact that investigated essential oils
Table 2. Neutralisation of DPPH radical (%) by Thymus vulgaris and Origanum vulgare
essential oils, thymol, carvacrol and BHT (as a positive control) in DPPH assay
DPPH assay
Source concentration (μμ
μμ
μg/mL)
0.0625 0.125 0.25 0.3125 0.375 0.5 0.625 0.75 1
TEO 20.45 32.6 48.65 56.24 62.41 66.72 - - -
OEO - 30.93 60.82 - - 78.27 89.64 91.38 94.33
12.5 25 37.5 50 75 100 125 -
Thymol 8.96 21.47 33.86 40.67 52.66 60.21 64.18 -
12.2 61.07 91.61 122.15 183.2 244.3 - -
Carvacrol 6.17 38.92 51.7 62.04 72.73 77.52 - -
0.0375 0.09375 0.1875 0.28125 0.375 0.469 0.562 -
BHT 8.02 17.92 26.75 33.87 42.74 47.45 46.02
Neda Gavaric et al., / TEOP 18 (4) 2015 1013 - 1021 1017
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exhibited higher antioxidant potential comparing
to isolated compounds may be an indication for
synergistic scavenging effect of different com-
pounds present in thyme and oregano 29.
Furthermore, RSC for hydroxyl radical of the
TEO, OEO, thymol and carvacrol are shown in
Table 3. All examined samples were able to
neutralise formed hydroxyl radicals reaching 50
% of reduction with IC50 values of 0.2 μg/mL for
thyme, 0.12 μg/mL for oregano, 28.82 μg/mL for
thymol and 63.6 μg/mL for carvacrol (Table 3).
Comparison of the OH radical scavenging activity
of the investigated essential oils and isolated
compounds with values reached for BHT in
previously presented paper 24 indicate stronger
antioxidant potential of thyme and oregano
essential oils as well as thymol and carvacrol.
Antibacterial testing
Antibacterial potential of aromatic plants have
been studied in details 3,30. Together with the
antioxidant activity it has been one of the most
examined features, important for food preser-
vation as well as for the control of human and
animal diseases of microbial origin. Although
numerous essential oils and isolated compounds
expressed notable antibacterial activity, their
influence on organoleptic qualities of foodstuffs
somewhat limit their full exploitation. Recently,
it has been suggested that interactions between
essential oils, their components and other food
ingredients and food additives need to be
Figure 1. Comparison of IC50 values of TEO, OEO, thymol, carvacrol
and BHT (as a positive control) in DPPH and deoxyribose assays
DPPH OH
% of neutralisation
Table 3. Neutralisation of OH radical (%) by Thymus vulgaris and Origanum vulgare
essential oils, thymol, carvacrol and BHT (as a positive control) in deoxyribose assay
OH radical neutralisation
Source concentration (μg/mL)
0.033 0.083 0.125 0.25 0.33 0.5 0.625 0.75
TEO - - 30.93 60.82 78.27 89.64 89.64 91.38
OEO 35.88 48.19 50.75 57.26 60.63 65.42 - -
33.3 83.3 166.67 250 333.3 500
Thymol 47.85 65.45 66.67 68.84 71.96 75.73
16.29 32.57 81.43 162.8 325.7
Carvacrol 23.27 37.88 59.56 68 43 75.01
0.05 0.5 5 50 100 500 1000
BHT - ---- --
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investigated. Synergistic effect could enable
maximum antibacterial activity of essential oil and
isolated compounds and minimise the concen-
trations required to achieve desirable effect.
In this study broth microdilution method was
used to determine MIC of TEO, OEO, thymol and
carvacrol on selected gram-positive (Staphylo-
coccus aureus and Bacillus cereus) and gram-
negative (Salmonella Infantis, Escherichia coli)
bacterial strains. Results, shown in Table 4,
confirm all investigated samples as relatively
strong antibacterial agents. Obtained MIC values
are in accordance with previously published
literature 3,4,7,10,16,31.
Finally, the combination testing (thymol with
Table 5. Synergy testing of TEO/OEO and thymol/carvacrol
Bacterial MIC t1MIC t MIC c3MIC c FIC t FIC c FIC
strain (μμ
μμ
μg/mL) (comb.2)(
μμ
μμ
μg/mL) (comb.2) index
(μμ
μμ
μg/mL) (μμ
μμ
μg/mL)
S.aureus 250 62.5 125 62.5 0.25 0.5 0.75
Salmonella 250 125 125 31.25 0.5 0.25 0.75
E. coli 250 62.5 250 125 0.25 0.5 0.75
B. cereus 250 125 125 31.25 0.5 0.25 0.75
MIC teo4MIC teo MIC oeo 5MIC oeo FIC teo FICoeo FIC
(μμ
μμ
μg/mL) (comb.2)(
μμ
μμ
μg/mL) (comb.2) index
(μμ
μμ
μg/mL) (μμ
μμ
μg/mL)
S.aureus 0.6 0.3 0.5 0.25 0.5 0.5 1
Salmonella 0.8 0.2 1 0.5 0.25 0.5 0.75
E. coli 1.15 0.57 1.25 0.31 0.5 0.25 0.75
B. cereus 1 0.25 1 0.5 0.25 0.5 0.75
1 thymol; 2 in combination; 3 carvacrol
4 thyme essential oil; 5 oregano essential oil
carvacrol and TEO with OEO) was performed
using checkerboard method. Thymol and
carvacrol were found to have additive antibacterial
effect against all tested bacterial strains (Table 5).
This has been reported in several studies 8,14,15,32.
However there are very few data on possible
synergistic or additive effect of thyme and oregano
essential oils 33. In this research, additive anti-
bacterial action of TEO and OEO was confirmed
(Table 5). Their ability to inhibit microbial
growth was enhanced through combination in a
way that the lowest addition of one component
caused a significant increase of action. This may
help to achieve their acceptable sensory level in
food.
Table 4. MIC of TEO, OEO, thymol, carvacrol and BHT
as positive control on investigated bacterial strains
Sample MIC (μμ
μμ
μg/mL)
bacterial strain S.aureus B.cereus Salmonella E.coli
TEO 0.6 1 0.8 1.15
OEO 0.5 1 1 1.25
Thymol 250 250 250 250
Carvacrol 125 125 125 250
BHT 550 550 550 550
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Conclusion
Thyme and oregano essential oils and their
isolated constituents thymol and carvacrol have
noteworthy antioxidant and antibacterial action,
both considered to be important for their use as
food additives. Yet, their practical application
could be limited by their influence on organoleptic
properties of foodstuffs or antagonism with other
food ingredients. The synergistic effect of
essential oils or their components may be a
promising field that could lead to the optimization
of antioxidant and antibacterial activity.
Acknowledgements
The authors are thankful to ARRS, Slovenia and
Serbia for financing of the bilateral project BI-
034-SLO-SR-2010/11, The Provincial Secretariat
for Science and Technological Development of
Vojvodina (grant number 114-451-2056/2011-01)
and The Ministry of Science and Technological
Development, Republic of Serbia (grant number
OI 172058) for financial support.
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