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Chemical Profile, Antioxidant and Antibacterial Activity of Thyme and Oregano Essential Oils, Thymol and Carvacrol and Their Possible Synergism

  • Faculty of Medicine, University of Novi Sad
  • Faculty of Medicine, University of Novi Sad

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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).
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Journal of Essential Oil Bearing Plants
ISSN: 0972-060X (Print) 0976-5026 (Online) Journal homepage:
Chemical Profile, Antioxidant and Antibacterial
Activity of Thyme and Oregano Essential Oils,
Thymol and Carvacrol and Their Possible
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
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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.
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: < > © 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
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
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
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-
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).
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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
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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
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
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
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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
% 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
μg/mL) (comb.2)(
μg/mL) (comb.2) index
μg/mL) (μμ
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
μg/mL) (comb.2)(
μg/mL) (comb.2) index
μg/mL) (μμ
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
Table 4. MIC of TEO, OEO, thymol, carvacrol and BHT
as positive control on investigated bacterial strains
Sample MIC (μμ
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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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.
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.
1. Bisset, N.G., Wichtl, M. (2001). Herbal Drugs and Phytopharmaceuticals, 2nd edition. CRC Press,
Boca Raton. London, New York, Washington D.C.
2. Dorman, H.J.D., Deans, S.G. (2000). Antimicrobial agents from plants: antibacterial activity of
plant volatile oils. J. Appl. Microbiol. 88: 308-316.
3. Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in foods-
a review. Int. J. Food Microbiol. 94: 223-253.
4. Tepe, B., Deferera, D., Sokmen, M., Polissiou, M., Sokmen, A. (2004). In vitro antimicrobial
and antioxidant activities of the essential oils and various extracts of Thymus eigii M. Zohary et
P.H. Davis. J. Agr. Food Chem. 52: 1132-1137.
5. Aligiannis, N., Kalpoutzakis, E., Mitaku, S., Chinou, I.B. (2001). Composition and antimicrobial
activity of the essential oil of two Origanum species. J. Agr. Food Chem. 49: 4168-4170.
6. Ozcan, M., Chalchat, J.C. (2004). Aroma profile of Thymus vulgaris L. growing wild in Turkey.
Bulg. J. Plant Physiol. 30 (3-4): 68-73.
7. Rivas, L., McDonnell, M.J., Burgess, C.M., O‘Brien, M., Navarro-Villa, A., Fanning, S.,
Duffy, G. (2010). Inhibition of verocytotoxigenic Escherichia coli in model broth and rumen
systems by carvacrol and thymol. Int. J. Food Microbiol. 139: 70-78.
8. Lambert, R.J.W., Skandamis, P.N., Coote, P.J., Nychas, G.J.E. (2001). A study of the minimum
inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. J.
Appl. Microbiol. 91: 453-462.
9. Ultee, A., Smid, E.J. (2001). Influence of carvacrol on growth and toxin production by Bacillus
cereus. Int. J. Food Microbiol. 64: 373-378.
10. Veldhuizen, E.J.A., Tjeerdsma-Van Bokhoven, J.L.M., Zweijtzer, C., Burt, S.A., Haagsman,
H.P. (2006). Structural requirements for the antimicrobial activity of carvacrol. J. Agr. Food
Chem. 54: 1874-1879.
11. Bozin, B., Mimica-Dukic, N., Samojlik, I., Jovin, E. (2007). Antimicrobial and antioxidant
properties of rosemary and sage (Rosmarinus officinalis L. and Salvia officinalis L., Lamiaceae)
essential oils. J. Agr. Food Chem. 55: 7879-7885.
12. Selmi, S., Sadok, S. (2008). The effect of natural antioxidant (Thymus vulgaris Linnaeus) on
flesh quality of tuna (Thunnus thynnus (Linnaeus)) during chilled storage. Pan Am. J. Aquatic.
Sci.3(1): 36-45.
13. Burt, S., Vlielanders, R., Haagsman, H.P., Veldhuizen, E.J.A. (2005). Increase in activity of
essential oil components carvacrol and thymol against Escherichia coli O157:H7 by addition of
food stabilizers. J. Food Protect. 68: 919-926.
14. Santiesteban-Lopez, A., Palou, E., Lopez-Malo, A. (2007). Susceptibility of food-borne bacteria
to binary combinations of antimicrobials at selected aw and pH. J. Appl. Microbiol. 102: 486-497.
Neda Gavaric et al., / TEOP 18 (4) 2015 1013 - 1021 1020
Downloaded by [faculties of the University of Ljubljana] at 05:32 28 October 2015
15. Pei, R.S., Zhou, F., Ji, B.P., Xu, J. (2009). Evaluation of combined antibacterial effects of
eugenol, cinnamaldehyde, thymol, and carvacrol against E. coli with an improved method. J.
Food Sci. 74: 379-383.
16. Palaniappan, K., Holley, R.A. (2010). Use of natural antimicrobials to increase antibiotic suscep-
tibility of drug resistant bacteria. Int. J. Food Microbiol. 140: 164-168.
17. Garcia-Garcia, R., Lopez-Malo, A., Palou, E. (2011). Bactericidal Action of Binary and Ternary
Mixtures of Carvacrol, Thymol, and Eugenol against Listeria innocua. J. Food Sci. 76: 95-100.
18. Siavash Saei-Dehkordi, S., Fallah, A.A., Saeid Saei-Dehkordi, S., Kousha, S. (2012). Chemical
Composition and Antioxidative Activity of Echinophora platyloba DC. Essential Oil, and Its
Interaction with Natural Antimicrobials against Food-Borne Pathogens and Spoilage Organisms.
J. Food Sci. 77(11): M631-637.
19. Tutin, T.G., Heywood, H.V., Burges, N.A., Valentine, D.H., Walters, S.M., Webb, D.A. (2001).
Flora Euroapea Vol. 3., Diapensiaceae to Myoporaceae. Cambridge, U.K.: Cambridge University
20. European Pharmacopeia. (2002). 4th edition. Council of Europe . Strasbourg Cedex, France.
Vol. 2.8.12, p 183-184.
21. Adams, R.P. (2007). Identification of Essential Oil Components by Gas Chromato- graphy/
Mass Spectroscopy, 4th edition. Allured Publishing Co. Carol Stream, Illinois.
22. Mimica-Dukic, N., Bozin, B., Sokolovic, M., Simin, N. (2004). Antimicrobial and antioxidant
activities of Melissa officinalis L. (Lamiaceae) essential oil. J. Agr. Food Chem. 52: 2485-2489.
23. Klanènik, A., Piskernik, S., Jeršek, B., Smole-Mo•ina, S. (2010). Evaluation of diffusion and
dilution methods to determine the antibacterial activity of plant extracts. J. Microbiol. Meth.
81: 121-126.
24. Bozin, B., Mimica-Dukic, N., Simin, N., Anackov, G. (2006). Characterization of the volatile
composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant
activities of the entire oils. J. Agr. Food Chem. 54: 1822-1828.
25. Mockute, D., Bernotiene, G., Judzentiene, A. (2001). The essential oil of Origanum vulgare L.
ssp. vulgare growing wild in Vilnius district (Lithuania). Phytochemistry. 57: 65-69.
26. Sanchez-Moreno, C. (2002). Review: Methods used to evaluate the free radical scavenging
activity in foods and biological systems. Food Sci. Technol. Int. 8: 121-137.
27. Miladinovic, B., Kostic, M., Šavikin, K., Ðorðevic, B., Mihajlov-Krstev, T., •ivanovic, S.,
Kitic, D. (2014). Chemical Profile and Antioxidative and Antimicrobial Activity of Juices and
Extracts of 4 Black Currants Varieties (Ribes nigrum L.). J. Food Sci. 79(3): C301-309.
28. Jia, H.L., Ji, Q.L., Xing, S.L., Zhang, P.H., Zhu, G.L., Wang, X.H. (2010). Chemical com-
position and antioxidant, antimicrobial activities of the essential oils of Thymus marschallianus
Will. and Thymus proximus Serg. H.L. J. Food Sci. 75: 59-65.
29. Milos, M., Makota, D. (2012). Investigation of antioxidant synergisms and antagonisms among
thymol, carvacrol, thymoquinone and p-cymene in a model system using the Briggs-Rauscher
oscillating reaction. Food Chem. 131: 296-299.
30. Lahlou, M. (2004). Methods to study the phytochemistry and bioactivity of essential oils. Phy-
tother. Res. 18: 435-448.
31. Eteghad, S.S., Mirzaei, H., Pour, S.F. Kahnamui, S. (2009). Inhibitory effects of endemic
Thymus vulgaris and Mentha piperita essential oils on Escherichia coli O157:H7. Res. J. Biol.
Sci.4: 340-344.
32. Zheng, L., Bae, Y., Jung, K., Heu, S., Lee, S. (2013). Antimicrobial activity of natural anti-
microbial substances against spoilage bacteria isolated from fresh produce. Food Control. 32:
33. Gutierrez, J., Barry-Ryan, C., Bourke, P. (2008). The antimicrobial efficacy of plant essential
oil combinations and interactions with food ingredients. Int. J. Food Microbiol. 124:91-97.
Neda Gavaric et al., / TEOP 18 (4) 2015 1013 - 1021 1021
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... β-myrcene, followed by D-Limonene, were found to have a high Pa score (Pa = 0.582, 0.582, respectively), suggesting that the strong antifungal activity observed may be due to the presence of these two substances. Based on these findings, we projected that PVEO may have a good antifungal and poor antibacterial and antioxidant capabilities, due to the studied components, without overlooking the fact that synergies may be the origin of the powerful antibacterial and antioxidant activity found in the in vitro tests carried out, as described in several published reports [23][24][25][26]. ...
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Ptychotis verticillata Duby, referred to as Nûnkha in the local language, is a medicinal plant that is native to Morocco. This particular plant is a member of the Apiaceae family and has a longstanding history in traditional medicine and has been utilized for therapeutic purposes by practitioners for generations. The goal of this research is to uncover the phytochemical makeup of the essential oil extracted from P. verticillata, which is indigenous to the Touissite region in Eastern Morocco. The extraction of the essential oil of P. verticillata (PVEO) was accomplished through the use of hydro-distillation via a Clevenger apparatus. The chemical profile of the essential oil was then determined through analysis utilizing gas chromatography–mass spectrometry (GC/MS). The study findings indicated that the essential oil of P. verticillata is composed primarily of Carvacrol (37.05%), D-Limonene (22.97%), γ-Terpinene (15.97%), m-Cymene (12.14%) and Thymol (8.49%). The in vitro antioxidant potential of PVEO was evaluated using two methods: the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical trapping assay and the ferric reducing antioxidant power (FRAP) method. The data demonstrated considerable radical scavenging and relative antioxidative power. Escherichia coli, Staphylococcus aureus, Listeria innocua, and Pseudomonas aeruginosa were the most susceptible bacterial strains tested, while Geotrichum candidum, Candida albicans, and Rhodotorula glutinis were the most resilient fungi strains. PVEO had broad-spectrum antifungal and antibacterial properties. To elucidate the antioxidative and antibacterial characteristics of the identified molecules, we applied the methodology of molecular docking, a computational approach that forecasts the binding of a small molecule to a protein. Additionally, we utilized the Prediction of Activity Spectra for Substances (PASS) algorithm; Absorption, Distribution, Metabolism, and Excretion (ADME); and Pro-Tox II (to predict the toxicity in silico) tests to demonstrate PVEO’s identified compounds’ drug-likeness, pharmacokinetic properties, the anticipated safety features after ingestion, and the potential pharmacological activity. Finally, our findings scientifically confirm the ethnomedicinal usage and usefulness of this plant, which may be a promising source for future pharmaceutical development.
... hirtum (sin.: O. heracleoticum L.) is a perennial herbaceous plant widely distributed throughout Europe and North Africa both as a spontaneous and cultivated plant [5,6]. In addition to the use of fresh and dried leaves and flowers to flavor traditional Mediterranean dishes [6], oregano provides therapeutic benefits for human health due to its antioxidant, antimicrobial and antifungal properties [7][8][9]. ...
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Oregano, Origanum vulgare L., is a perennial herbaceous plant belonging to the Lamiaceae family. Oregano shows variations in biomass yield and essential oil (EO) content due to the influence of abiotic and biotic factors. The aim of this study was to assess the effect of different foliar applications based on calcium (Ca) and nitrogen (N) on morphological and productive parameters in oregano. Tests were carried out in Sicily (Italy) in 2020-2021. In each year, eight foliar applications were applied. Only flowers and leaves were used for the extraction of the EO. For all parameters in the study, except for plant height and inflorescence length, the highest values were found in treated plants with respect to the control. In plants treated with calcium and nitrogen, an increase of between 0.6 and 1.6 t ha −1 was observed for fresh yield, and an increase of between 0.5 and 0.9 t ha −1 was observed for dry yield. The increase in biomass yield led to an increase in EO yield of between 4 and 12 kg ha −1. The results highlight that foliar treatments with Ca in combination with N enable growth in crop production in environments that show poor water availability.
... Considering that in this study, ROS levels (i.e. quantification of H 2 O 2 levels) were similar between carvacroltreated groups and the control group, we believe that the greater antioxidant capacity of carvacrol detected through the ABTS and DPPH assays might have been due to its effect in reducing RNS (Jamali et al., 2021), such as nitric oxide (NO; Aristatile et al., 2015), and other types of ROS, for example, the hydroxyl radical ( • OH; Gavaric et al., 2015) and the superoxide anion (O 2 -; Aristatile et al., 2015). Although in the present study, blastocyst production was not affected by the addition of carvacrol during IVM, after carvacrol supplementation, the cleavage rate and the number of cells per blastocyst were reduced in a dose-dependent manner despite the increased antioxidant capacity. ...
Carvacrol (C 10 H 14 O), an efficient phenolic antioxidant substance for several cell types, may become a useful antioxidant for female germ cells and embryo culture. This study investigates the effects of carvacrol supplementation on bovine oocytes in in vitro maturation (IVM) and embryo production. In total, 1222 cumulus-oocyte complexes were cultured in TCM-199 þ alone (control treatment) or supplemented with carvacrol at the concentrations of 3 μM (Carv-3), 12.5 μM (Carv-12.5), or 25 μM (Carv-25). After IVM, the oocytes were subjected to in vitro fertilization and embryo production, and the spent medium post-IVM was used for evaluating the levels of reactive oxygen species and the antioxidant capacity (2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2 0-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid quantification). A greater (P < 0.05) antioxidant potential was observed in the spent medium of all carvacrol-treated groups compared with the control medium. Moreover, the addition of carvacrol to the maturation medium did not affect (P > 0.05) blastocyst production on days 7 and 10 of culture; however, the total number of cells per blastocyst was reduced (P < 0.05) in two carvacrol-treated groups (Carv-3 and Carv-25). In conclusion, carvacrol demonstrated a high antioxidant capacity in the spent medium after oocyte maturation; however, although embryo production was not affected, in general, carvacrol addition to IVM medium reduced the total number of cells per blastocyst. Therefore, due to the high antioxidant capacity of carvacrol, new experiments are warranted to investigate the beneficial effects of lower concentrations of carvacrol on embryo production in cattle and other species.
... These natural compounds have been extensively studied and may be responsible for the bioactivities of the essential oils containing them [10]. The modes of action of these natural bioactive compounds are under intense investigation, although some of them have been relatively well described. ...
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Plants have been used by humans since ancient times due their antimicrobial and medicinal properties. Essential oils (EOs) are complex mixtures of secondary plant metabolites, including terpenoids, phenylpropanoids, and other aromatic compounds. Cinnamomun verum and Thyme vulgaris EOs and their organic extracts exert numerous biological activities because of their major compounds, particularly thymol, carvacrol, eugenol, and benzoic and cinnamic acid. The structural motifs presented by these phytochemicals are responsible for their biological activities. Modification or hybridization of these structures could lead to new bio-based compounds with improved efficacy or multiple modes of action. In this work, we aimed to develop reliable methods of obtaining six hybrid molecules from the major constituents of C. verum and T. vulgaris EOs. For the first time, we tested their efficacy in the inhibition of the mycelium growth and spore germination of two of the most important phytopathogenic fungi, Fusarium oxysporum and Colletotrichum gloeosporioides, and one opportunistic human pathogen, Aspergillus niger. The cytotoxic activity of the obtained hybrids was assessed using the brine shrimp lethality assay. In addition, we report for the first time a biocatalytic process for the obtention of these bioactive hybrid molecules. The results of this work enable the possibility of using hybrid molecules based on the major constituents of EOs as active ingredients in strategic industries such as agriculture, aquaculture, and pharmaceuticals.
... As shown in Fig. 2, the chromatographic profiles obtained for the different spices and aromatic herbs studied are, in general terms, significantly different from each other, being able to identify some of the characteristic biomarkers of each studied spice and herb such as eugenol in clove, cinnamon and bay leaf; salicylaldehyde in cinnamon; or vanillin in vanilla. Nevertheless, it should be noted the considerable similarity existing between the chromatographic profiles of oregano and thyme, which is attributed to the fact that both herbs have a very similar composition (Gavaric et al., 2015;Parthasarathy et al., 2008). Among the compounds that are common in both herbs we can find, for example: γ-terpinene, linalool, borneol, thymol methyl ether, carvacrol methyl ether, trans-caryophyllene and caryophyllene oxide, all with a similar weight in the composition of both oregano and thyme, but not being the majority. ...
... To date, more than sixty flavonoids have been isolated and reported from Thymus genus plants (Li et al., 2019). There are several groups of flavonoids, such as flavones, flavanols, and dihydroflavonoids, shown in Table 4. Flavonoids function as UV filters, signaling molecules, allelopathic agents, frost, and drought protection agents, phytoalexins (Gavaric et al., 2015;Karaca and Ince, 2022a), detoxifiers, antimicrobials, and anti-herbivore factors (Lahlou et al., 2022). It is known that the C-3 position in ring C is linked with diverse groups, e.g., hydroxy, glucopyranosyl, rutinoside, and glucuronic acid (Table 4). ...
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The family Lamiaceae (Labiatae) has more than 236 genera and 7200 species. The genus Thymus is one of the most known genera of Lamiaceae, along with Salvia, Hyptis, Scutellaria, Stachys, Teucrium, and Plectranthus. The genus Thymus contains about 350 species, and the Mediterranean area (specifically Southern and Western Europe) is considered the origin of the genus Thymus. Today, many species are distributed throughout North Africa, Iberian Peninsula, Greece, and Türkiye. There are at least 45 Thymus species naturally grown in Türkiye, and the number of endemic species in Türkiye is increasing in comparison to previous records.
... The peculiar executive technique of Mali wooden sculpture, allows the combined use of T. vulgaris essential oil and hydro-alcoholic solutions, setting up a green strategy to counteract a spread microbial colonization on artwork surface, and the protection from re-colonization seems to be active after one year. Gas-chromatography analysis showed that carvacrol and thymol are the main compounds in both EO and HA solutions, and to these compounds is main attributable the antimicrobial activity [37,38]. ...
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In the last decades plants products are increasingly used also in defining innovative procedures for sustainable conservation of cultural heritage. Particularly, essential oils or hydro-alcoholic extracts have been fruitful applied to contrast microbial colonization on organic and inorganic artworks or insect infestation (Anobidae) having regard to the repellent action. In this paper, extracts from Asteraceae and Lamiaceae plant families are utilized to counteract widespread microbial colonization (bacteria, cyanobacteria, fungi) due to their antimicrobial activity. In order to define the adequate concentration correlating it to microbial species detected, the antimicrobial activity of natural products is preliminary in vitro assayed by Agar Disc and Well Plate Diffusion methods. Moreover, an integrated approach based on morphological analysis (optical, scanning and confocal electron microscopy), in vitro cultures (Nutrient or Sabouraud agar) and molecular investigation (genomic microbial DNA extraction, sequencing and sequence analysis) are routinely performed for the identification of largest number of microbial taxa. Besides, to enhance the antimicrobial activity different protocols have been performed, such as for Thymus vulgaris products directly applied on wooden sculpture surface as hydro-alcoholic extract, followed by exposure, in a dedicated chamber, to the volatile compound of the corresponding Essential Oil. In other case studies, the exposure to volatile compounds of Crithmum maritmum, Inula critmoides, Thymus vulgaris and Origanum vulgare essential oils was performed under controlled vacuum conditions, comparing the effects to environmental condition exposure. These studies confirm the possible use of plant extracts in replacing synthetic chemical biocides, in full respect of human health and environment sustainability.
Thymus species are well known as medicinal plants because of their biological and pharmacological properties. Thymus migricus Klokov & Desj. -Shos belongs to Lamiaceae. Young branches of this plant produce an essential oil that is abundant in potent and volatile compounds that have a variety of therapeutic uses. In this study, the natural composition of thymol in this plant’s essential oil was first detected with HPLC and Mass, and then the essential oil was extracted. The increase in the lipophilicity of the synthesized silylated derivatives was then examined by HPLC after numerous silylated derivatives of this phenolic compound were synthesized using various silylation reagents. These derivatives were then analyzed by FT-IR and 1HNMR analysis. The antibacterial activity of thymol with its silylated derivatives against gram-negative and gram-positive bacteria was next tested by diffusion method, after which the antioxidant activity of thymol with its silylated derivatives was assessed by two DPPH and FRAP methods.
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Plant based natural products have been widely used as antibacterial and insect repellent agents globally. Because of growing resistance in bacterial plant pathogens and urban pests to current methods of control, combined with the long-and short-term negative impact of certain chemical controls in humans, non-target organisms, and the environment, finding alternative methods is necessary to prevent and/or mitigate losses caused by these pathogens and pests. The antibacterial and insect repellent activities of essential oils of novel cultivars of catnip (Nepeta cataria L. cv. CR9) and oregano (Origanum vulgare L. cv. Pierre) rich in the terpenes nepetalactone and carvacrol, respectively, were evaluated using the agar well diffusion assay and petri dish repellency assay. The essential oils exhibit moderate to high antibacterial activity against three plant pathogens, Pseudomonas cichorii, Pseudomonas syringae and Xanthomonas perforans of economic interest and the individual essential oils, their mixtures and carvacrol possess strong insect repellent activity against the common bed bug (Cimex lectularius L.), an urban pest of major significance to public health. In this study, the essential oils of catnip and oregano were determined to be promising candidates for further evaluation and development as antibacterial agents and plant-based insect repellents with applications in agriculture and urban pest management. (2023) Volatile metabolites from new cultivars of catnip and oregano as potential antibacterial and insect repellent agents.
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Adams, R. P. 2007. Identification of essential oil components by gas chromatography/ mass spectrometry, 4th Edition. Allured Publ., Carol Stream, IL Is out of print, but you can obtain a free pdf of it at
Unlabelled: The aim of this article was to determine the chemical composition (anthocyanin and ascorbic acid contents, total phenols, and tannins), and the antioxidative, and antimicrobial activities of the juices and methanol extracts of 4 varieties-Tenah, Triton, Ben Sarek, and Ometa. The juices were made during 2008, 2009, and 2010 from the berries grown on the same location. Statistically significant differences were found in all the tested parameters of each variety for every year. Ometa variety exhibited the best antioxidative activity. The highest ascorbic acid value had Tenah juice from 2010 (211 mg/100 g) and Ben Sarek methanol extract (656 mg/100 g). Delphinidin-3-ruthenoside was the dominant anthocyanin in Triton, Tenah, and Ben Sarek variety during all 3 y and Ometa in 2010 where Ometa in 2008 and 2009 had slightly higher content of cyanidin-3-ruthenoside. The minimum inhibitory and microbicidal concentrations ranged from MIC/MBC(MFC) = 62.5 to 500 mg/mL for juices and 0.4 to 500 mg/mL for extracts. There was no significant difference in the effect of the juices against Gram(+) and Gram(-) bacteria. All the extracts acted in lower concentrations than juices. Practical application: Data contained in this article provide an insight into the effects the weather condition has on the content of the bioactive compounds in genetically different types of black currants, during the 3-y observation period. This article is relevant to the commercial application of black currants-based dietary supplements.
The aim of this study was to investigate the efficacy of natural antimicrobial substances for inhibiting vegetable spoilage bacteria. Natural antimicrobial compounds (carvacrol, thymol, eugenol, cinnamic acid, nisin, and chitosan), organic acids (acetic acid and lactic acid), and chemical sanitizers (sodium hypochlorite and chlorine dioxide) were evaluated for their antibacterial activities, as single and combination treatments, against 15 spoilage bacteria isolated from vegetables, using the agar disc diffusion and broth dilution methods. Carvacrol, thymol, and eugenol showed strong inhibitory effects compared to those of the other antimicrobial substances, and their average minimum inhibitory concentration (MIC) values against 15 spoilage bacteria were 167, 648, and 168 μg/ml, respectively. When they were combined, four kinds (carvacrol + thymol, carvacrol + eugenol, thymol + eugenol, and carvarol + thymol + eugenol) of the combination formulas showed higher antibacterial effect than others against spoilage bacteria, with average MIC values of 47, 43, 59, and 42 μg/ml, respectively. However, two combinations (carvacrol + thymol, and carvacrol + thymol + eugenol) showed the strongest inhibitory effects against bacteria in fresh vegetables among all treatments. These results could be used for the development of new sanitation or preservation methods to improve freshness and to extend the shelf-life of fresh produce.