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Central European Journal of Biology
* E-mail: zstojanovicradic@yahoo.com
Research Article
1University of Niš, Faculty of Science and Mathematics,
Department of Biology and Ecology, 18 000 Niš, Serbia
2University of Novi Sad, Department of Biology and Ecology,
21 000 Novi Sad, Serbia
3University of Niš, Faculty of Medicine, Department of Pharmacy,
18 000 Niš, Serbia
4University of Niš, Faculty of Science and Mathematics,
Department of Chemistry, 18 000 Niš, Serbia
Tatjana Mihajilov-Krstev1, Dragan Radnović2, Dušanka Kitić3, Vesna Stankov Jovanović4,
Violeta Mitić4, Zorica Stojanović-Radić1*, Bojan Zlatković1
Chemical composition, antimicrobial, antioxidative
and anticholinesterase activity of
Satureja
montana
L. ssp
montana
essential oil
1. Introduction
Satureja montana L. (winter savory) is an aromatic plant,
traditionally used on the Balkan peninsula as a spice and
a natural food preservative [1]. Savory honey is a very
common ingredient in folk remedies for the treatment
of bronchitis. Also, it is used as choleretic, stimulant,
digestive, antiseptic for gastrointestinal tract [2] and for
the treatment of premature ejaculation [3]. A number of
investigations justied traditional utilization and have
conrmed various biological effects of this plant species
namely, antioxidant [4,5], antibacterial [4,6-10] and
antifungal [11]. Satureja montana essential oil showed
antiproliferative activity on human erythroleukemic
K562 cells [12]. Also, this essential oil showed the ability
to prevent in vitro peroxynitrite oxidation better than
antioxidants of reference such as ascorbic acid [13].
According to the avaliable literature, there are
chemical differences of this plants’ essential oils,
obtained from specimens collected at different
localities [4,14-16] and in different ontogenetic stages
[7,15,17]. It is important to note that in the mentioned
investigations, their antimicrobial activities were either
not tested or the authors didn’t investigated the relation
between chemical composition and the obtained activity
of the essential oil [8-11]. Some of these studies used
only disc diffusion method [4] or did not specify which
subspecies of the investigated plant [4,6,7] was used
for the essential oil isolation. On the Balkan peninsula,
genus Satureja is represented by nine species. Species
Cent. Eur. J. Biol. • 9(7) • 2014 • 668-677
DOI: 10.2478/s11535-014-0298-x
668
Received 27 January 2013; Accepted 04 December 2013
Keywords: Satureja montana L. ssp montana • Essential oil • Antimicrobial activity • Antioxidant activity • Cholinesterase inhibiton
Abstract: The present study investigates the chemical compositions of three Satureja montana L. ssp montana essential oils and correlates
chemical variability with biological activities. GC/MS analysis showed that with an increase in altitude (100-500-800 m), a higher
content of linalool, terpinen-4-ol and cis-sabinene hydrate was found, while the percentage of phenolic compounds, thymol and
carvacrol decreased. Antimicrobial activity of the essential oils was tested against 7 fungal and 23 bacterial strains. The essential
oil characterized by the highest content of phenols and alcohols exhibited the highest antimicrobial potential. The correlation
analysis showed that the major carriers of the obtained antioxidant activity are oxygenated monoterpenes. All essential oils
inhibited human serum cholinesterase activity. High antimicrobial potential, together with moderate antioxidant capacity and strong
inhibition of human serum cholinesterase, classies S. montana essential oil as a natural source of compounds that can be used
in the treatment of foodborne and neurological diseases, wound and other infections, as well as for general health improvement.
© Versita Sp. z o.o.
T. Mihajilov-Krstev
et al.
S. montana has three subspecies - ssp. variegata (Host)
P.W. Ball; ssp. montana L. and ssp. pisidica (Wettst.)
[18]. The studies investigating the chemical composition
of this species’ essential oils showed that there are
signicant differences at the subspecies level [19],
which affects their biological properties.
All mentioned facts prompted us to investigate the
chemical composition of this plant at the subspecies level
and to correlate differences in the chemical composition
with different biological activities: antimicrobial,
antioxidative and anticholinesterase.
2. Experimental Procedures
2.1 Plant Material
The aerial parts of wild growing S. montana ssp
montana were collected during the owering stage from
the three different localities in Montenegro: Sample 1
- near the city of Budva - 100 m; Sample 2 - Cetinje
road - 500 m and Sample 3 - Orjen Mt. - 800 m a.s.l.).
Voucher specimens No. 2-1930 were conrmed and
deposited at the Herbarium of the Department of Biology
and Ecology (BUNS Herbarium), Faculty of Natural
Sciences, University of Novi Sad.
2.1.1 Isolation of the essential oil
Air-drying of plant material was performed under shade
at room temperature for 10 days. Dried aerial parts
(100 g) were cut and subjected to hydro-distillation
for 3 h, using a Clevenger-type apparatus [20].
The resulting essential oil was dried over anhydrous
sodium sulfate and preserved in a sealed vial at 4°C
until further analysis.
2.1.2 Gas chromatography
GC (Gas Chromatography) analyses of the oils were
carried out on a GC HP-5890 II apparatus equipped
with the split-splitless injector, attached to HP-5 column
(25 m x 0.32 mm, 0.52 μm lm thickness) and tted
to FID (Flame Ionisation Detector). The carrier gas
(H2) ow rate was 1 ml min-1, split ratio 1:30, injector
temperature 250°C, and detector temperature 300°C,
while the column temperature was linearly programmed
from 40°C to 240°C at a rate of 4°C min-1.
2.1.3 Gas chromatography–mass spectrometry
The same analytical conditions were employed for
GC-MS analyses, where a HP G 1800C Series II GCD
system was used. The transfer line was heated to
260°C. Mass spectra were acquired in EI mode (70 eV),
in m/e range 40-400 (column HP-5MS 30 m x 0.25 mm,
0.25 μm lm thickness).
2.1.4 Identication of components
Oil components were identied by comparing their
mass spectra with those of Adams [21] Wiley 275
and NIST/NBS libraries [22]. The experimental values
of retention indices were determined by the use of
calibrated Automated Mass Spectral Deconvolution
and Identication System software (AMDIS ver.2.1.,
DTRA/NIST, 2002). The obtained results were correlated
with literature data [21] as well as by using other
sources (www.avornet.org; iowtv.pherobase.com).
For quantication, area percent data obtained by FID
were used.
2.2 Antimicrobial activity
2.2.1 Microbial strains
The antimicrobial activity of S. montana L. ssp montana
essential oil was evaluated using three groups of
microorganisms: (1) laboratory reference strains,
Bacillus subtilis ATCC 6633, Bacillus cereus ATCC
10876, Clostridium perfringens ATCC 19404, Listeria
monocytogenes ATCC 15313, Staphylococcus aureus
ATCC 6538, Staphylococcus aureus ATCC 25923
and Sarcina lutea ATCC 9341 (Gram (+) bacteria),
Escherichia coli ATCC 8739, Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 9027 and
Salmonella enteritidis ATCC 13076 (Gram (-) bacteria)
and Aspergillus niger ATCC 16404, Candida albicans
ATCC 10231 and Saccharomyces cerevisiae 112
Hefebank Weihenstephan (fungal microorganisms)
obtained from the American Type Culture Collection;
(2) clinical isolates from wounds: Klebsiella pneumoniae,
Klebsiella oxytoca, Escherichia coli, Proteus mirabilis,
Staphylococcus aureus, Staphylococcus sp.,
Streptococcus pyogenes, Pseudomonas aeruginosa,
Enterococcus sp., Enterobacter sp., Citrobacter
sp. and Acinetobacter sp. (Source: Center for
Microbiology, Institute for Public Health of Vojvodina,
Novi Sad, Serbia); and (3) fungal strains isolated
from matress dust: Aspergillus fumigatus, Aspergillus
restrictus, Penicillium chrysogenum and Acremonium
chrysogenum.
2.3 Micro-well Dilution Assay
Minimum inhibitory (MIC) and bactericidal/fungicidal
concentrations (MBC/MFC) of essential oils were
determined by employing the broth micro-well dilution
method as described previously [22]. Bacterial
species were cultured at 37°C in Mueller Hinton agar
for bacteria and Sabouraud dextrose agar for fungi
(28 and 30°C). After 18 h of incubation, bacterial
suspensions were made in Mueller Hinton broth and
their turbidity was standardized to 0.5 McFarland.
669
Chemical composition, antimicrobial, antioxidative and anticholinesterase activity
of
Satureja montana
L. ssp
montana
essential oil
The nal density of bacterial and yeast’s inoculum
was 5 x 105. Suspensions of the molds were made
in sterile saline and their turbidity was conrmed
by viable counting in a Thoma chamber. Final size
of the mold’s inoculum in Sabouraud dextrose broth
was 1 x 104. Dimethylsulfoxide (DMSO) was used
to dissolve the essential oils. Then, serial dilutions
of the oils were made in a microtiter plate (dilution
factor 2). The inoculum was added to all wells and
the plates were cultivated at 37°C for 24 h (bacteria)
or at 30°C (C. albicans) and 28°C (molds) for 48 h.
All experiments were performed in triplicate, where
two growth controls consisting of corresponding
medium with DMSO (10%) were included. Antibiotics
Chloramphenicol, Streptomycin, Kanamycin and
Nystatin served as positive controls. One inoculated
well was included, to allow control of the adequacy
of the broth for organism growth. One non-inoculated
well, free of any antimicrobial agent, was included
to ensure medium sterility. The bacterial growth was
determined by adding 20 μl of 0.5% TTC (triphenyl
tetrazolium chloride) aqueous solution. Minimal
inhibitory concentration (MIC) was dened as the
lowest concentration of the oil that inhibited visible
growth (red colored pellet on the bottom of the wells
after the addition of TTC), while minimal bactericidal
concentration (MBC) was dened as the lowest oil
concentration that killed 99.9% of bacterial/fungal
cells. To determine MBC/MFC (minimal fungicidal
concentration), broth was taken from each well without
visible growth and inoculated in Mueller Hinton agar
(MHA) for 24 h at 37°C for bacteria or in Sabouraud
dextrose agar (SDA) for 48 h at 28°C (molds) and
30°C (yeast). The experiments were done in triplicate
and the mean values are presented.
2.4 Antioxidant assays
2.4.1 1,1-diphenyl-2-picrylhydrasyl (DPPH) radical
scavenging assay
Relatively stable organic radical DPPH has been widely
used in the determination of antioxidant activity of single
compounds as well as the different plant extracts [23].
The DPPH assay was performed as previously described
[24]. Radical scavenging activity (RSC) of the essential
oils was calculated applying following equation:
DPPH RSC(%)=100 (A0−A1/A0)
Where:
A0 - absorbance of the blank; A1 - absorbance of the
sample.
The EC50 values were determined by polynomial
regression analysis of the obtained DPPH-RSC values
(software MS Excel 07).
2.4.2 Oxygen radical absorbance capacity (ORAC) -
ABTS+ radical cation decolorisation assay
Antioxidant capacity of the oils was evaluated
using a Perkin Elmer Lambda 15 UV-VIS
spectrophotometer, using the improved ABTS+
method, as described by Re et al. [25]. ABTS+ radical
cation was generated by a reaction of 7 mM L-1 ABTS
(Sigma Aldrich, Germany) with 2.45 mM L-1 potassium
persulfate (Merck, Germany) until an absorbance of
0.700 ± 0.050 at 734 nm was reached. Generation of
radical before the antioxidants are added prevents
interference of compounds, which affect radical
formation. This modification makes the assay less
susceptible to artifacts and prevents overestimation
of antioxidant capacity [26]. When stable absorbance
is obtained, the antioxidant sample is added to the
reaction medium, and the antioxidant activity is
measured in terms of decolorization. Fifty microlitres
of diluted sample were mixed with 1.9 ml of diluted
ABTS+ solution. The mixture was allowed to stand
for 6 min at room temperature and the absorbance
was immediately recorded at 734 nm. Trolox (Sigma
Aldrich) solution (final concentration 0-15 µM L-1)
was used as a reference standard. The results
were expressed as Trolox equivalents (µM of trolox
equivalents per mg of essential oil).
2.4.3 Total reducing power assay Fe(III) to Fe(II)
Reducing power of the oils was determined as described
previously [24] and expressed in relation to the reducing
power of ascorbic acid as a positive control (Ascorbate
Equivalent Antioxidant Capacity).
2.5 Inhibition of cholinesterase
Modied Ellman’s method was applied for testing impact
of essential oils on pooled human serum cholinesterase
[27]. A total of 10 healthy volunteers (18-65 years old
from both sexes), from the Pirot General Hospital,
donated blood with written consent. According to the
questionnaire, none of them had serious medical
disorders, nor are or have been drug, cigarette,
or alcohol abusers. At least a month before the blood
donation, none of them had been taking any medication
[27].
2.6 Statistical analysis of data
The experimental results were expressed as
mean±standard deviation (SD) of three replicates.
Correlation coefcients to determine the relationship
between two variables (between different antioxidant
assays; tests and content of major groups of essential
oils constituents) were calculated using MS Excel
software (CORREL statistical function).
670
T. Mihajilov-Krstev
et al.
3. Results
Essential oils of wild growing S. montana ssp. montana,
(collected at three different locations) were isolated by
hydro distillation. The yields of the essential oils were
0.42, 0.40 and 0.38% (v/w) (S1 - Budva, S2 - Cetinje
and S3 – Orijen Mt., respectively). Qualitative and
quantitative data on the main constituents of essential
oils have been summarized in Table 1.
GC and GC/MS analyses revealed that the
essential oil from the S1 possess a high content of the
phenolic compounds thymol and carvacrol, followed by
linalool. Chemical composition of the S2 essential oil
is characterized by a high content of carvacrol, linalool
and terpinen-4-ol, followed by cis-sabinene hydrate.
Compared to the other components of the essential oils
isolated from S1 and S2, p-cymene and borneol had
signicantly higher content. Contrary to the previous
two samples, essential oil isolated from the S3 did not
contained any phenolic compounds, but had linalool,
cis-sabinene hydrate and terpinen-4-ol as the most abundant
components. All three samples had signicant amount of
the sesquiterpenes β-caryophyllene and caryophyllene-
oxide. Apart from the two mentioned compounds, the oil
from the S3 contained a signicant amount of nerolidol.
In the present paper, the essential oils were
tested using a broth microdilution assay against
a panel of 30 microorganisms including laboratory
control strains (ATCC) of bacteria and fungi, clinical
isolates from wounds and fungal isolates from
matress dust (Table 2). The antimicrobial assay
showed that the highest activity was exhibited by the
oil from the S1, with inhibitory/bactericidal activity
in the range 0.18-0.78 µL mL-1 (except against
P. aeruginosa, which was much more resistant)
while, the essential oils from the S2 and S3 samples
showed much higher efcacy against P. aeruginosa.
When considering Gram positive bacteria, all three
investigated oils showed signicant antimicrobial
effect in the range from 0.18-6.25 µL mL-1.
The obtained activity was in the range from
0.18-1.56 µL mL-1 against both tested S. aureus strains.
All three tested oils exhibited activity against all tested
multiresistant strains in the range 3.13-50.0 µL mL-1,
with the exception of P. aeruginosa and Citrobacter
sp. (resistant to the highest tested concentrations
of the S2 and S3 oils), together with K. pneumoniae
(resistant to the highest tested concentrations
of the S3 oil). Essential oil isolated from the S1
exhibited the most prominent antimicrobial activity.
No. Constituents Sample 1
100 m*
Sample 2
500 m*
Sample 3
800 m*
1α-thujene 0.20 / /
2α-pinene 0.21 / /
3 camphene 0.25 / /
4 sabinene 1.04 1.05 0.26
5 1-octen-3-ol / 0.07 0.18
6β-myrcene 0.19 / /
7α-phellandrene / 0.08 /
8α-terpinene 0.62 0.18 0.65
9 p-cymene 6.61 5.38 1.39
10 β-phellandrene / 0.20 0.26
11 D-limonen 0.31 / /
12 1,8-cineole 0.13 0.13 /
13 cis-β-ocimene 0.24 0.09 0.47
14 γ-terpinene 2.11 0.06 2.39
15 cis-sabinene hydrate 3.50 14.61 23.05
16 cis-linalool oxide 0.48 0.33 /
17 terpinolene 0.60 0.35 0.27
18 trans-linalool oxide t 0.08 /
19 linalool 15.38 17.94 32.58
20 cis-p-menth-2-en-1-ol 0.33 0.76 1.38
21 terpine-1-ol / 0.44 0.33
22 trans-pinocarveol 0.29 / /
23 camphor 0.19 0.40 /
24 borneol 3.87 3.62 0.69
Table 1. Phytochemical composition (%) of Satureja montana ssp montana essential oils.
671
Chemical composition, antimicrobial, antioxidative and anticholinesterase activity
of
Satureja montana
L. ssp
montana
essential oil
No. Constituents Sample 1
100 m*
Sample 2
500 m*
Sample 3
800 m*
25 terpinen-4-ol 4.81 10.60 10.99
26 p-cymene-8-ol 0.30 0.69 /
27 α-terpineol 0.64 1.30 1.30
28 cis-piperitol / 0.39 /
29 trans-piperitol / 0.28 /
30 trans-carveol / 0.04 /
31 trans-dihydrocarvone 0.09 / /
32 thymol methyl ether 2.32 0.05 /
33 carvacrol methyl ether 2.30 0.69 /
34 carvone / 0.12 /
35 geraniol 0.19 0.12 /
36 geranial / 0.09 /
37 bornyl acetete / 0.06 /
38 thymol 24.69 2.46 /
39 carvacrol 15.19 24.46 /
40 myrtenyl acetate 0.15 / /
41 α-cubebene 0.08 / /
42 α-copaene / 0.04 /
43 β-bourbonene 0.15 0.32 0.98
44 β-elemene / / /
45 β-caryophyllene 4.38 3.37 2.57
46 β-copaene / 0.08 0.13
47 germacrene D / 0.38 2.34
48 aromadendrene / 0.14 /
49 cis-β-farnesene 0.12 / /
50 α-humulene 0.17 0.13 0.14
51 bicyclogermacrene / / 0.71
52 γ-muurolene 0.14 0.16 /
53 γ-crene D 0.24 / /
54 leden-viridiorene / 0.32 /
55 β-bisabolene 1.19 0.37 /
56 β-curcumene 0.14 0.14 /
57 δ-cadinene 0.27 0.25 0.27
58 elemol 0.24 0.47 2.72
69 nerolidol / / 9.36
60 spathulenol 0.39 1.32 /
61 caryophyllene oxide 3.96 3.30 1.27
62 maalinen / 0.14 0.37
63 β-eudesmol / 0.82 0.38
64 β-oplopenone 0.13 / /
65 τ-cadinol 0.40 / /
66 τ-muurolol 0.55 / /
67 β-bisabolol 0.26 / /
Monoterpene hydrocarbons 11.78 7.04 5.42
Oxygenated monoterpenes 75.53 80.08 70.77
Sesquiterpene hydrocarbons 11.39 10.93 20.86
Oxygenated sesquiterpenes 2.72 0.82 0.38
Identied (%) 47 compounds
(95.80%) 49 compounds (98.80%) 27 compounds (97.25%)
Oil yield (%) 0.42 0.40 0.38
SD - standard deviation 0.03 0.02 0.02
continuedTable 1. Phytochemical composition (%) of Satureja montana ssp montana essential oils.
672
T. Mihajilov-Krstev
et al.
Essential oil (MIC/MBC in µL mL-1) Antibiotics (MIC/MBC in µg mL-1)
Microorganisms Sample 1
100 m*
Sample 2
500 m*
Sample 3
800 m* Strept. Chlor. Nyst. Kan.
Gram (-) sorce No.
Escherichia coli
ATCC 8739 0.78/0.78 0.78/3.13 3.13/6.25 8.0/8.0 nt nt nt
Escherichia coli
ATCC 25922 0.78/0.78 1.56/3.13 6.25/12.5 16.0/16.0 nt nt nt
Pseudomonas aeruginosa
ATCC 9027 11.25/11.25 0.20/0.78 3.13/6.25 8.0/8.0 nt nt nt
Salmonella enteritidis
ATCC 13076 0.78/0.78 0.78/1.56 6.25/6.25 4.0/4.0 nt nt nt
Gram (+) source No.
Bacillus subtillis
ATCC 6633 0.35/0.35 1.56/3.13 0.78/3.13 nt 8.0/8.0 nt nt
Bacillus cereus
ATCC 10876 0.35/6.25 1.56/6.25 0.78/6.25 4.0/16.0
Clostridium perfringens
ATCC 19404 0.18/0.18 0.39/0.39 0.39/0.39 nt 1.0/8.0 nt nt
Listeria monocytogenes
ATCC15313 0.35/0.78 0.78/1.56 0.78/1.56 nt 8.0/16.0 nt nt
Staphylococcus aureus
ATCC 25923 0.35/0.35 0.78/1.56 1.56/1.56 nt 1.0/8.0 nt nt
Staphylococcus aureus
ATCC 6538 0.18/0.18 0.39/0.78 1.56/1.56 nt 2.0/16.0 nt nt
Sarcina lutea
ATCC 9341 0.18/0.18 0.10/0.10 0.39/0.39 nt 0.5/2.0 nt nt
Clinical isolates of bacteria
from wounds
Klebsiella pneumoniae 25.0/25.0 6.25/25.0 >50.0/>50.0 nt nt nt >100/>100
Klebsiella oxytoca 12.5/12.5 12.5/12.5 25.0/25.0 nt nt nt 12.5/12.5
Escherichia coli 12.5/12.5 12.5/12.5 25.0/50.0 nt nt nt >100/>100
Proteus mirabilis 12.5/12.5 12.5/12.5 25.0/50.0 nt nt nt >100/>100
Staphylococcus aureus 6.25/12.5 12.5/12.5 12.5/12.5 nt nt nt >100/>100
Staphylococcus sp. 12.5/12.5 12.5/12.5 12.5/12.5 nt nt nt >100/>100
Streptococcus pyogenes 12.5/12.5 6.25/25.0 12.5/25.0 nt nt nt 25.0/25.0
Pseudomonas aeruginosa 50.0/50.0 >50.0/>50.0 50/>50.0 nt nt nt >100/>100
Enterococcus sp. 12.5/25.0 25.0/25.0 25.0/50.0 nt nt nt >100/>100
Enterobacter sp. 25.0/25.0 25.0/25.0 50.0/>50.0 nt nt nt 12.5/12.5
Citrobacter sp. 50.0/50.0 >50.0/>50.0 >50.0/>50.0 nt nt nt 25.0/25.0
Acinetobacter sp. 3.13/3.13 6.25/12.5 12.5/12.5 nt nt nt >100/>100
Fungi source No.
Candida albicans
ATCC 10231 0.35/0.35 0.78/1.56 3.13/3.13 nt nt 16.0/16.0 nt
Saccharomyces cerevisiae
112. Hefeb. Weihenstephan 0.09/0.09 0.09/1.56 3.13/3.13 nt nt 16.0/16.0 nt
Aspergillus niger
ATCC 16404 0.18/0.18 1.56/1.56 3.13/3.13 nt nt 8.0/8.0 nt
Fungal isolates
Aspergillus fumigatus 0.04/0.38 0.09/0.75 0.09/0.38 nt nt 0.04/0.04 nt
Aspergillus restrictus 0.09/0.75 0.09/0.75 0.09/0.75 nt nt 0.08/0.08 nt
Penicillium chrysogenum 0.09/0.38 0.19/0.38 0.09/0.19 nt nt 0.04/0.04 nt
Acremonium chrysogenum 0.04/0.04 0.04/0.04 0.04/0.04 nt nt 0.04/0.04 nt
Table 2. Antimicrobial activity of Satureja montana ssp montana essential oils.
*altitude
Strept. – Streptomycin; Chlor. – Chloramphenicol; Nyst. – Nystatin; Kan. - Kanamycin, nt – not tested
673
Chemical composition, antimicrobial, antioxidative and anticholinesterase activity
of
Satureja montana
L. ssp
montana
essential oil
Monoterpens Oxigenated monoterpens Sesquiterpens Total monoterpens
TRP -0.8932*0.2043 0.2608 -0.3144
DPPH -0.3480 -0.9955* 0.9306*-0.9087*
ORAC 0.5690 0.9412*-0.9922* 0.9837*
Table 4. Relation between percentage of major groups of essential oil constituents and antioxidant activities.
AstericsmarksignicantcorrelatonsatP<0.05
It showed high activity against Acinetobacter sp.
(MIC=MBC=3.125 µL mL-1) and S. aureus
(MIC/MBC=6.25/12.5 µL mL-1), moderate activity
against K. oxytoca, P. mirabilis, Staphylococcus sp.,
S. pyogenes and E. coli (MIC=MBC=12.5 µL mL-1)
and the weakest activity against Citrobacter sp.
and P. aeruginosa (MIC=MBC=50.0 µL mL-1).
The same concentration exhibited both inhibitory
and bactericidal effect in most strains tested, except
in the cases of S. aureus and Enterococcus sp.
(MIC/MBC=12.5/25.0 µL mL-1) (Table 2). Comparison of
the obtained results with the antimicrobial activity of the
reference antibiotics (positive controls) showed that all
tested essential oils possess generaly high antimicrobial
activity. High activity against all tested fungal strains in
the range of 0.09-3.13 µL mL-1 (Table 2) was observed
with the tested essential oils. The special signicance of
this activity can be observed against pathogenic yeast
C. albicans and fungal lamentous strains from the genus
Aspergillus (A. niger, A. fumigatus and A. restrictus).
Antioxidant properties tested using three different
assays are presented in Table 3. Antioxidant (radical
scavenging) activity of all tree samples, obtained by
DPPH assay, was estimated as weak (approximativelly
10-fold weaker than BHT as reference standard).
Results obtained by ORAC assay exibited relatively poor
antioxidant capacity, which was in very good agreement
with DPPH assay (correlation coefcient of -0.9689,
p<0.05). Total reducing power estimated as Fe(III) to
Fe(II) for all samples can also be considered as weak.
The best antioxidant properties were exibited by S2,
followed by S1, while S3 showed the lowest antioxidant
capacity among the tested oil samples. With an aim to
elucidate which group of essential oil constituents is
responsible for specic antioxidant activity, correlation
analysis was performed and the results are presented
in Table 4. The correlation analysis showed that the
major carriers of the antioxidant activity are oxygenated
monoterpenes, which showed the highest correlation
with DPPH method.
In addition to antimicrobial activity, determination and
estimation of the antioxidant activity, anticholinesterase
activities of the essential oils were measured.
All of the tested oils displayed anticholinesterase
activity, where S1 exhibited the strongest inhibition of
human serum cholinesterase (-85.71±0.24%), while S2
and S3 had similar inhibition percents (-51.16±0.13 and
49.17±0.11%, respectively).
4. Discussion
Literature data concerning S. montana showed
differences in the chemical composition of essential oils
with change in altitude of the plant (growth) collection
[4,12,16]. Beside this, it was found that the specimens
collected during the owering season had the highest
content of the carvacrol and thymol and their precursors
p-cymene and γ-terpinene [7,15,17]. Finally, differences
in the chemical compositions not only reects the genetic
variability of the genus Satureja [19], but also the variability
within the same species, as in the case of the S. hortensis
[28]. Unfortunately, only two studies using plant material
have identied the plant to its subspecies level [14,29],
which makes it difcult to compare data from literature.
All mentioned facts prompted us to collect plant material
Table 3. Antioxidant properties of Satureja montana essential oils estimated by DPPH, ORAC and total reducing power Fe (III) to Fe (II).
*EC50 (BHT)=41.6±0.4 µg/mL
DPPH(EC50)*
(µg/mL)
ORAC
(TE, µg/mL per mg of essential oil)
Total reducing power
(ascorbate equivalents - µg/mL of ascorbic acid per mg of essential oil)
S1 494.1±2.5 101.1±0.9 120.7±0.4
S2 426.3±1.4 106.3±0.9 211.4±1.2
S3 593.2±2.9 76.8±0.8 166.2±0.8
674
T. Mihajilov-Krstev
et al.
from the same Satureja subspecies (S. montana ssp.
montana), in the same developmental stage (owering
period), from three localities at different altitudes and to
investigate the relation between chemical composition
and biological activity of essential oils from S. montana
ssp. montana essential. Yields of essential oils isolated
from plant material were between 0.38 - 0.42% (v/w),
which is relatively lower compared to other reports in
literature where the yield of this oil was reported to range
between 0.22% and 2.8% [16,29]. It can be noted that
the essential oils collected from all three localities had
similar yields, but number of constituents showed evident
decrease, which was related to the increase in altitude.
Monoterpene hydrocarbons (oxygenated monoterpenes)
were the dominant class of compounds in the oils of
all three samples. Although the oil from the sample S1
has the lowest content of sesquiterpenes, this class
of compounds is more diverse than in the other two
samples. Obtained data showed the correlation between
altitude increment and higher content of alcohols
linalool (15.38%; 17.94% and 32.58%, respectively) and
terpinen-4-ol (0%, 10.60% and 10.99%, respectively).
Also, a similar correlation was found in the case of
the aldehide cis-sabinene hydrate (0%, 14.61% and
23.05%, respectively), but the content of the major
phenol compounds (thymol and carvacrol) signicantly
decreased with the increase of the altitude. Similar trends
were also present in the study of Slavkovska [14], where
increasing the altitude (200-600 m) caused a reduction
of the phenol compounds by two fold, as well as the
increase in the content of alcohols (linalool 8.1-2.8% and
borneol 7.1-10.6%) in the composition of the S. montana
ssp. montana essential oil. Decrease of the carvacrol
content in the essential oil of S. montana collected in
Albania (55.95%-39.53%-40.51%-21.07%-2.21%) was
also corelated with increase in altitude (4 m-15 m-207
m-750 m-800 m a.s.l.) [16]. Essential oil from a sample
collected in Italy (150 m a.s.l., La Spezia Province [30]
was characterized by very high content of the carvacrol,
while the oil collected from Sannine Mountain in Lebanon
(1800 m) had no any phenols but was characterized by
very high amounts of alcohols 1,8-cineole (8.87%), linalool
(11.41%) and γ-terpineol (12.66%) [12]. Decrease of the
phenol content, followed by the increase of alcohols was
also recorded in the chemical compositions of the oils
from two different localities in Bosnia and Herzegovina,
Trebinje - Konjic (carvacrol 23.3-10.6%; thymol 31.7-3.8%;
geraniol 0.1-22.3%, terpinen-4-ol 0.8-10.3%, borneol 2.9-
4.8%) [4].
Satureja montana essential oils showed very high
antimicrobial activity against all tested strains. Our
results concur with previous investigations studying
the antimicrobial activity of S. montana against
P. aeruginosa, wherein the said strain showed
higher resistance when compared to other tested
microorganisms [4,7]. The study by Nedorostova et al.
[10] demonstrated complete resistance of this strain to
S. montana essential oil. In our studies, the obtained
activity of essential oils from the S2 and S3 samples
showed much higher effect against P. aeruginosa while
S1 was not as effective. This difference in activity can
be attributed to the higher alcohol content in the of
these oils [31]. The activity of S. montana essential oil
(carvacrol-50.2% and thymol-11.0%) against clinical
enteropathogens Escherichia coli, Plesiomonas
shigelloides, Shigella exneri, Salmonella enterica
serovar typhimurium, Yersinia enterocolitica and
Vibrio parahaemolyticus was previously investigated
and conrmed to be in the range of MIC/MBC=0.02-
0.32/0.04-0.64% (w/v), as well as against much more
resistant E. coli O157:H7 (thymol (43.0%); MIC/MBC
= 0.05/0.013 (vol/vol)) [8]. In the case of gram positive
bacteria, previous investigations of S. montana essential
oil against S. aureus were conrmed by our results,
showing high sensitivity of this strain to the tested
essential oils [4,7,8,10]. Altogether our results conrmed
the previously reported data on the ecacy of S. montana
essential oil in controling growth and survival of Listeria
monocytogenes [9] as well as against food contaminant
B. cereus [7]. During the last decades, the main issue in
microbiology has been growing number of multiresistant
strains and, at the same time, decline in the number
of the useful antibiotics without toxic or carcinogenic
effect. All three tested oils exhibited activity against
all tested multiresistant strains, with the exception of
P. aeruginosa, Citrobacter sp. and K. pneumoniae.
Among multiresistant strains tested, K. pneumoniae was
also the more resistant strain in the study performed by
Skočibušić and Bežić [7]. Satureja montana essential
oils generaly exhibited very high antifungal activity,
regardless of the individual components’ percentage
values, which conrms previous results on this subject
[7]. Beside this, the oil from the material collected in
Italy (Malba Mt.) was active against 46 species from 23
genera of pathogenic fungi at very low concentrations
from 0.10 to 0.25 µL mL-1 [11]. Among the tested essential
oils, the S1 showed the most prominent antimicrobial
activity, where it is very clear that high phenol and
alcohol content in the chemical composition were the
most efcient antimicrobial combinations. Generally
high antimicrobial activity of the S. montana ssp
montana essential oil, reported in previous and present
studies can be contributed to its major compounds
carvacrol, thymol, terpinen-4-ol and linalool [8,32,33].
Mentioned results not only suggest synergistic activity
of the individual oil components, but also points to the
675
Chemical composition, antimicrobial, antioxidative and anticholinesterase activity
of
Satureja montana
L. ssp
montana
essential oil
signicance of their percentage ratio in the antimicrobial
mixture. Antioxidant activity testing showed relatively
low antioxidant potential of the tested essential oils. One
can clearly note that antioxidant activity of all samples
showed maximum activity at the medium altitude of
the collection, which is in accordance with oxygenated
monoterpenes content in the S2, conrmed by
correlation analysis. In the case of sesquiterpenes, the
situation is reversed, so we can state as a conclusion
that total reducing power may in general be attributed to
the monoterpene fraction of the examined essential oils.
Many neurological disorders, associated with increased
activity of cholinesterase could be prevented by its
inhibition. In that way, examination of widely applied
natural products such as essential oils as a potential
source of cholinesterase inhibitors, can play an important
role. All three essential oils, especially S1, qualify in that
way as potent cholinesterase inhibitors with prospective
application in treatment of neural diseases.
The results of the present study show a correlation
between the chemical composition of the S. montana
ssp montana essential oil and altitude of growth of the
plant material. With increase of the altitude, the content
of phenol compounds (carvacrol and thymol) are either
decreased, or their ratio is changed. The same change
in altitude is related to the increase in the alcohol content
in the essential oil’s composition. High inhibitory and
microbicidal effect of S. montana ssp montana essential
oil on human pathogens and food contaminants
together with moderate antioxidative potential, conrms
traditional usage of this plant species for the treatment
of the respiratory and intestinal infections. Beside
this, the tested oils were active against multiresistant
clinical isolates from wounds, which is very signicant
considering their general resistance and difculties in
the treatment of these infections. This fact can be used
for further researches with focus on the development
of the drugs that contain active constituents from these
oils. Finally, the investigated oils could be used for the
preparation of aerosols and disinfectants of the closed
areas for the purpose of reduction and prevention
of the spore germination from spoilt food or allergic
fungal species. All tested samples proved themselves
to be a cholinesterase inhibitors, justifying their
wide usage in folk medicine and nutrition. As a nal
recommendation derived from the present study,
commercial cultivation of the species S. montana ssp
montana should be done at lower altitudes. In this way,
higher content of antibacterial and antifungal compounds
in the essential oil isolated from this plant material will
be achieved, while at the same time, the other benets
such as antioxidative properties and anticholinesterase
action would not be diminished.
Acknowledgments
The authors are very grateful to the Ministry of Education
and Sciences of the Republic of Serbia for the nancial
support of Grant No. III-41018. Also, the authors are
thankful for nancial support from the Provincial Secretariat
for Science and Technological Development of the
Autonomous Province of Vojvodina, Republic of Serbia,
in the frame of project “Molecular and phenotypic diversity
of taxa of economic and epidemiological importance, and
endangered and endemic species in Europe“.
References
[1] Redžić S., Wild edible plants and their traditional
use in the human nutrition in Bosnia-Herzegovina,
Free Radic. Res., 2006, 45, 189 – 232
[2] Leporatti M.L., Ivancheva S., Preliminary
comparative analysis of medicinal plants used in
the traditional medicine of Bulgaria and Italy, J.
Ethnopharmacol., 2003, 87, 123–142
[3] Zavatti M., Zanoli P., Benelli A., Rivasi M., Baraldi
C., Baraldi M., Experimental study on Satureja
montana as a treatment for premature ejaculation,
J. Ethnopharmacol., 2011, 133, 2629–633
[4] Ćavar S., Maksimović M., Šolić M., Jerković-Mujkić
E., Bešta R., Chemical composition and antioxidant
and antimicrobial activity of two Satureja essential
oils, Food Chem., 2008, 111, 648–653
[5] Radonić A., Miloš M., Chemical Composition and
In Vitro Evaluation of Antioxidant Effect of Free
Volatile Compounds from Satureja montana L.,
Free Radic. Res., 2003, 37, 673–679
[6] Skočibušić M., Bežić N., Chemical composition
and antidiarrhoeal activities of winter savory
(Satureja montana L.) Essential Oil, Pharm. Biol.,
2003, 41, 622-626
[7] Skočibušić M., Bežić N., Chemical composition
and antimicrobial variability of Satureja montana
L. essential oils produced during ontogenesis, J.
Essent. Oil. Res., 2004, 16, 387–391
[8] Oussalah M., Caillet S., Saucier L., Lacroix M., Inhibitory
effects of selected plant essential oils on the growth of
four pathogenic bacteria: E. coli O157:H7, Salmonella
typhimurium, Staphylococcus aureus and Listeria
monocytogenes, Food Control, 2007, 18, 414–420
[9] Carramiñana J.J., Rota C., Burillo J., Herrera A.,
Antibacterial efciency of spanish Satureja montana
676
T. Mihajilov-Krstev
et al.
essential oil against Listeria monocytogenes among
natural ora in minced pork, J. Food Protect, 2008,
71, 502-508
[10] Nedorostova L., Kloucek P., Kokoska L., Stolcova
M., Pulkrabek J., Antimicrobial properties of selected
essential oils in vapor phase against foodborne
bacteria, Food Control, 2009, 20, 157–160
[11] Ciani M., Menghini L., Mariani F., Pagiotii R.,
Menghini A., Fatichenti F., Antimicrobial properties
of essential oil of Satureja montana L., on
pathogenic and spoilage yeasts, Biotechnol. Lett.,
2000, 22, 1007-1010
[12] Lampronti I., Saab A.M., Gambari R.,
Antiproliferative activity of essential oils derived
from plants belonging to the Magnoliophyta
division, Int. J. Oncol., 2006, 29, 989-995
[13] Prieto J.M., Iacopini P., Cioni P., Chericoni S., In vitro
activity of the essential oils of Origanum vulgare,
Satureja montana and their main constituents in
peroxynitrite-induced oxidative processes, Food
Chem., 2007, 104, 889–895
[14] Slavkovska V., Jančić J., Bojović S., Milosavljević
S., Đoković D., Variability of essential oils of
Satureja montana L. and Satureja kitaibelii Wierzb.
ex Heuff. from the central part of the Balkan
peninsula, Phytochem., 2001, 57, 71-76
[15] Miloš M., Radonić A., Bežić N., Dunkić V.,
Localities and seasonal variations in the chemical
composition of essential oils of Satureja montana
L. and S. cuneifolia Ten., Flavour. Fragr. J., 2001,
16, 157–160
[16] Ibraliu A., Dhillon B.S., Faslia N., Stich B., Variability
of essential oil composition in Albanian accessions
of Satureja montana L., J. Med. Plants. Res., 2010,
4, 1359-1364
[17] Mastelić J., Jerković I., Gas chromatography–mass
spectrometry analysis of free and glycoconjugated
aroma compounds of seasonally collected Satureja
montana L., Food Chem., 2003, 80, 135–140
[18] Šilić Č., Monograph of the genera Satureja L.
Calamintha Miller, Micromeria Bentham, Acinos Miller
and Clinopodium L. in ora of Yugoslavia [Monograja
rodova Satureja L. Calamintha Miller, Micromeria
Bentham, Acinos Miller i Clinopodium L. u ori
Jugoslavije], Zemaljski Muzej BiH, Sarajevo, 1979
[19] Bežić N., Šamanić I., Dunkić V., Besendorfer V.,
Puizina J., Essential Oil Composition and internal
transcribed spacer (ITS) sequence variability of
four south-Croatian Satureja species (Lamiaceae),
Molecules, 2009, 14, 925-938
[20] Clevenger J.P., Content o essential oil in plants,
American Perfumer and Essential Oil Review,
1928, 23, 467-503
[21] Adams R.P., Identication of essential oil
components by gas hromatography/mass
spectrometry. 4th Ed., Allured Publishing
Corporation, Carol Stream, IL, 2007
[22] Mihajilov-Krstev T., Radnović D., Kitić D., Zlatković
B., Ristić M., Branković S., Chemical composition
and antimicrobial activity of Satureja hortensis L.
essential oil., Cent. Eur. J. Biol., 2009, 4, 411-416
[23] Kulišić T., Radonić A., Katalinić V., Miloš M., Use of
different methods for testing antioxidative activity of
oregano essential oil, Food Chem., 2004, 85, 633–640
[24] Stojanović G., Stojanović I., Stankov-Jovanović
V., Mitić V., Kostić D., Reducing power and radical
scavenging activity of four Parmeliaceae species,
Cent. Eur. J. Biol., 2010, 5, 808-813
[25] Re R., Pellegrini N., Proteggente A., Pannula
A., Yang M., Rice-Evans C., Antioxidant activity
applying an improved abts radical cation
decolorization assay, Free Radic. Biol. Med., 1999,
26, 1231-1237
[26] Sanchez-Moreno C., Methods used to evaluate the
free radical scavenging activity in foods and biological
systems, Food Sci. Technol. Int., 2002, 8, 121–137
[27] Stankov-Jovanović V.P., Nikolić-Mandić S.D.,
Mandić Lj.M., Mitić V.D., Modication of the kinetic
determination of pancuronium bromide based on
its inhibitory effect on cholinesterase, J. Clin.Lab.
Anal., 2007, 21, 124-131
[28] Hadian J., Tabatabaei S.M.F., Naghavi M.R.,
Jamzad Z., Ramak-Masoumi T., Genetic diversity
of Iranian accessions of Satureja hortensis L.
based on horticultural traits and RAPD markers,
Sci. Hortic., 2008, 115, 196–202
[29] Dunkić V., Bezić N., Vuko E., Cukrov D.,
Antiphytoviral Activity of Satureja montana L. ssp.
variegata (Host) P. W. Ball Essential Oil and Phenol
Compounds on CMV and TMV, Molecules, 2010,
15, 6713-6721
[30] Angelini L.G., Carpanese G., Cioni P.L., Morelli
I., Macchia M., Flamini G., Essential oils from
Mediterranean Lamiaceae as weed germination
inhibitors, J. Agric. Food Chem., 2003, 51, 6158-6164
[31] Nikaido H., Prevention of drugs access to bacterial
targets: permeability barriers and active efux,
Science, 1994, 264, 382-388
[32] Dorman H.J.D., Deans S.G., Antimicrobial agents
from plants: antibacterial activity of plant volatile
oils, J. App. Microbiol., 2000, 88, 308-316
[33] Zhou F., Ji B., Zhang H., Jiang H., Yang Z., Li J., Li J.,
Yan W., The antibacterial effect of cinnamaldehyde,
thymol, carvacrol and their combinations against
the foodborne pathogen Salmonella typhimurium,
J. Food Safety, 2007, 27, 124–133
677
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