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Department of Medicinal and Aromatic Plants Research, National Research Center, Dokki, Giza, Egypt
Department of Pharmacy, University of Pisa, Via Bonanno 33, 56126 Pisa, Italy
c
Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy
b
a r t i c l e
i n f o
Article history:
Received 22 September 2014
Received in revised form 18 January 2015
Accepted 19 January 2015
Available online 5 February 2015
Keywords:
Origanum syriacum
Essential oil
Carvacrol
Antibacterial
Antifungal activity
a b s t r a c t
The main constituents of essential oil (EO) of Origanum syriacum L. collected from cultivated and growing
wild in Sinai, Egypt were identied and determined by GCMS. The antimicrobial activity of these essential oils against Gram positive and Gram negative bacterial strains was studied. The antimycotic activity
of these EOs was also evaluated against eight fungal strains isolated from different sources. Forty-six
compounds were identied in the EO of both samples, dominated by carvacrol in cultivated type. Thymol, -terpinene, linalool and 4-terpineol were the most represented constituents in O. syriacum plants
collected from wild populations. Both EOs showed antibacterial activity with varying magnitudes, while
EO from cultivated O. syriacum showed high antibacterial activity against Staphylococcus aureus with an
inhibition zone of 32 4.0 mm. Both EOs showed good antifungal activity against all fungal strains. O.
syriacum EO from cultivated plants showed the lowest MIC 0.25 mg/L with Aspergillus fumigates clinical
strain isolated either with Aspergillus avus.
2015 Elsevier B.V. All rights reserved.
1. Introduction
Medicinal plants and culinary herbs have long been the basis of
traditional medicine in many countries. Some important medicinal wild plants suffered from unwise human manipulation which
resulted in the extinction of some. Projects to recover the genetic
diversity of wild plants are carried out in Egypt as well as scientic
research of their use based on traditional medicine in Bedouin communities. Indeed, there are a great number of rural jobs dependent
on this sector (Viuda-Martos et al., 2010, 2011). Cultivation of these
species is considered a mean to conserve these species within our
outside their habitats (in-sito or ex-sito conservation). Cultivation
of medicinal and aromatic plants in Egypt is of great socioeconomic
interest as they are mainly produced for export.
Essential oils of herbs and their components, products of a secondary metabolism of the plant, have many applications in folk
medicine, food avoring and preservation as well as in cosmetic
and pharmaceutical industries. The antimicrobial and antioxidant
properties of essential oils have been known for a long time, and
a number of investigations have been conducted on antimicrobial activities using various bacteria, viruses and fungi (Alma et al.,
2003).
Oregano is the common name for the general aroma and avor of the plants derived from the Lamiaceae family (Ibrahim et al.,
2012). The genus Origanum is well known for its volatile oil and
constituents and is characterised by a large morphological and
chemical diversity. Forty-nine taxa divided into 10 sections belong
to this genus, most of them having a very local distribution around
the Mediterranean basin (Loizzo et al., 2009). In folk medicine, Origanum species are used as powerful disinfectants, avoring agents,
in perfumes and in scenting soaps (Kamel et al., 2001). They are
also useful as a source of antimicrobial compounds (Sokovic et al.,
2007). Due to these properties, spices and herbs have been added
to food since ancient times, not only as avoring agents but also as
preservatives (Kalemba and Kunicka, 2003).
Origanum syriacum L. is an aromatic, herbaceous and perennial plant growing wild in the Sinai Desert of Egypt (Tackholm,
1974). Oregano is also used as a stimulant, analgesic, antitussive,
expectorant, sedative, antiparasitisc and antihelminthic, but
mostly for gastrointestinal complaints (Baser et al., 2003).
202
Table 1
Geographical and environmental conditions and some soil composition from cultivated and wild O. syriacum.
Latitude (N)
Longtude (E)
Altitude (m)
Distance (km)a
Directiona
Max. temperatureb
Min. temperatureb
Max.Min. temp.b
Average temperatureb
Relative humidity (%)b
Average precipitation mm
Soil type
EC (1:5)
pH (1:2.5)
TSS %
O.M %
CaCO3%
Saint Katherine
cultivated O.
syriacum
Wadi-Firan wild
O. syriacum
38 3319.2
33 5652.9
1574
450
NorthEast
23.8
10.38
13.42
18.68
27.67
21
Sandy
7.6
7.2
2.12
0.50
2.20
28 4242.5
33 3946.9
1362
400
NorthEast
23.8
10.38
13.42
18.68
27.67
21
Sandy
8.3
7.7
2.66
0.15
2.34
203
concentrations (4, 2, 1 and 0.5 l EOs per disc) was put on the
discs.
- The plates were then incubated at optimum temperature 37 C
for 24 h, followed by the measurement of the diameter of growth
inhibition zone expressed in millimetres (mm). All tests were
performed in triplicate.
- The reference bacterial strains were tested by Kirby-Bauer
method to evaluate their in vitro sensitivity to 5 Antibiotics:
Tetracycline (30 g), Ceftazidime (30 g), Rifampicin (30 g),
Cephalexin (30 g), and Cefotaxime (30 g). The results were
interpreted on the basis of the indications suggested by the
National Committee for Clinical Laboratory Standards, 2002.
2.5.1.3. Determination of concentration effect. The concentration
effect (CE) was studied to ascertain which dose of EOs had an
inhibitory effect on bacterial growth in the disc diffusion assay. The
culture techniques used were those described in the previous paragraph (Agar disc diffusion method), but adding 4, 2, 1 and 0.5 l of
each EO, which mean doses of 100, 50, 25 and 12.5% of the initial volume (Viuda-Martos et al., 2010). All tests were performed in
triplicate.
2.5.2. Antifungal activity
2.5.2.1. Fungal isolates. Eight fungal isolates belonging to 3
Aspergillus species (Aspergillus fumigatus, Aspergillus avus and
Aspergillus niger) were examined. More in detail 3 isolates of A. fumigatus were cultured from birds died from invasive aspergillosis, the
other was obtained from soil, 2 isolates of A. avus were isolated
from Pinus pinea and bovine litter, respectively, and 2 of A. niger isolated from crops and soil (one isolate each). All fungal strains were
isolated in the Mycology Laboratory of Department of Veterinary
Sciences, University of Pisa, Italy.
2.5.2.2. In vitro antifungal testing. EOs were diluted in sweet
almond oil (Prunus dulcis, Mill) to obtain a 20% stock solution.
Serial dilutions in RPMI were made to achieve nal concentrations ranging from 10% to 0.25%. To avoid bias due to differing
amounts of sweet almond oil, each test was carried out using
a concentration equal to 40% of this oil. This concentration was
also added to the medium in control cultures. Before testing, each
isolate was subcultured at least twice on potato dextrose agar
(PDA) to ensure viability and purity. EUCAST broth microdilution
method testing (BMD) was performed as detailed by EUCAST with
slight modication (2008 EUCAST technical note on the method
for the determination of broth dilution minimum inhibitory
concentrations of antifungal agents for conidia-forming moulds
http://www.eucast.org/documents/technical notes) by using RPMI
1640 medium with 2.0% glucose, at-bottom microdilution trays,
inocula of 2 105 to 5 105 CFU/ml, and incubation at 25 C
(EUCAST, 2008). The MIC values were determine visually as the lowest concentration of EO that caused complete inhibition of growth
(rst clear well) relative to that of the growth control after 48 h of
incubation. A conventional drug (itraconazole) was also tested, following the above described procedure. To assess MFC value inocula
which did not grow were washed with distilled water, subcultured
onto PDA and incubated at 25 C for 4 weeks, to evaluate if the lack
of growth was due to a fungistatic or fungicidal effect of EOs.
2.6. Statistical analysis
Conventional statistical methods were used to calculate means
and standard deviations of three simultaneous assays carried
out with the different methods. Statistical analysis (ANOVA) was
applied to the data to determine differences (P < 0.05). To discover
where there were signicant differences between the levels of the
204
RT
KI
Cultivated
Wild
-Thujene
-Pinene
Camphene
Sabinene
1-Octen-3-ol
-Myrcene
-Phellandrene
-3-Carene
-Terpinene
-Cymene
Limonene
-(Z)-ocimene
-(E)-ocimene
-Terpinene
cis sabinene hydrate
Terpinolene
Linalool
-Thujone
cis-p-Menth-2-en-1-ol
trans-p-Menth-2-en-1-ol
Menthone
Borneol
4-Terpineol
p-Cymen-8-ol
-Terpineol
cis-Dihydrocarvone
trans-Dihydrocarvone
iso-Dihydrocarveol
cis-Carveol
Thymol methyl ether
Carvone
Carvacrol methyl ether
Carvenone
Bornyl acetate
Thymol
Carvacrol
Eugenol
-Caryophyllene
Aromadendrene
-Humulene
Bicyclogermacrene
-Cadinene
Spathulenol
Caryophyllene oxide
1,10-di-epi-Cubenol
t-Cadinol
Monoterpene hydrocarbons
Oxyg. Monoterpenes
Sesquiterpene Hydroc.
Oxig. Sesquiterpenes
Others
Total
4.625
4.792
5.153
5.772
5.877
6.226
6.63
6.816
7.012
7.269
7.402
7.687
8.04
8.41
8.76
9.475
9.905
10.463
10.736
11.456
12.044
12.511
12.996
13.344
13.556
13.835
14.154
14.24
15.155
15.853
16.031
16.19
16.524
16.871
18.001
18.672
20.777
23.355
24.169
24.78
26.608
27.332
29.87
30.071
31.365
32.372
930
939
953
978
979
991
1003
1011
1017
1025
1031
1041
1050
1064
1072
1088
1101
1114
1122
1141
1153
1169
1177
1183
1189
1193
1201
1215
1229
1235
1243
1245
1258
1263
1290
1299
1359
1419
1441
1456
1495
1513
1578
1581
1614
1640
0.06 0.01
0.72 0.01
0.12 0.02
tr
0.22 0.01
1.32 0.01
0.17 0.02
0.09 0.01
1.24 0.01
8.48 0.05
0.37 0.02
tr
tr
1.98 0.02
0.11 0.02
0.21 0.01
0.22 0.01
tr
tr
tr
tr
0.19 0.01
0.89 0.01
tr
0.15 0.01
0.18 0.03
0.06 0.03
tr
tr
tr
tr
0.11 0.01
tr
tr
0.35 0.02
81.38 0.08
tr
0.11 0.01
tr
tr
tr
tr
tr
0.09 0.01
tr
0.12 0.03
14.68 0.08
83.83 0.14
0.11 0.01
0.09 0.04
0.22 0
98.93 0.26
2.1 0.15
0.65 0.03
0.05 0.01
1.24 0.06
0.31 0.02
1.76 0.06
0.3 0.01
0.04 0.01
5.5 0.19
3.88 0.16
1.77 0.03
0.03 0.02
0.06 0.01
14.32 0.75
2.34 0.05
1.37 0.03
9.44 0.33
0.20 0.01
0.69 0.03
0.37 0.02
0.05 0.01
0.09 0.01
7.68 0.11
tr
5.51 0.11
0.14 0.01
tr
0.18 0.01
tr
tr
tr
tr
0.19 0.01
0.1 0.02
31.73 0.93
3.9 0.18
0.04 0
1.92 0.11
0.22 0.01
0.09 0
0.52 0.03
0.06 0
0.17 0.02
0.14 0.02
tr
0.28 0.03
33.07 1.51
62.61 1.83
2.81 0.16
0.58 0.06
0.35 0.02
99.42 3.59
205
Table 3
Antibacterial activity (inhibition zone, mm) of cultivated and wild O. syriacum EOs against the selected bacterial strains tested by disc diffusion method.
Cultivated
Strains
S. aureus
E. faecalis
E. coli
P. aeruginosa
S. aureus
E. faecalis
E. coli
P. aeruginosa
4 l/disc
100%
2 l/disc
50%
1 l/disc
25%
0.5 l/disc
12.5%
Mean
St. dev.
Mean
St. dev.
Mean
St. dev.
Mean
St. dev.
32 a
19 a
25 a
NA
Wild
24.5 abc
11.0 c
17.0 b
NA
4.0
1.0
1.0
27.5 ab
13.5 b
18.5 b
NA
0.5
1.5
0.5
23 bc
10.5 c
18 b
NA
5.0
0.5
1.0
22 bc
9 cd
12 c
NA
4.0
0.0
0.0
2.5
1.0
3.0
18 cd
8d
11 cd
NA
1.0
1.0
1.0
13.0 de
NA
8.5 cd
NA
2.0
9.0 e
NA
8.0 d
NA
0.0
0.5
0.0
Values followed by the same small letter within the same line have not signicant differences (P > 0.05) according to Tukeys multiple range test. N.A. = non-active.
Since both cultivated and wild O. syriacum EOs were rich in oxygenated monoterpenes and their precursors, they were tested for
antimicrobial activities against fungal and bacterial pathogens.
3.2. Antibacterial activity
The diameter of inhibition zones of the tested essential oils from
both cultivated and wild O. syriacum measured by disk agar diffusion method is presented in Table 3. The results revealed that
both essential oils showed signicance antibacterial activity with
varying magnitudes, depending on the essential oil concentration. The inhibition zone of EO from cultivated plant was more
signicant than EO from wild plants. Inhibition zone of EO from
cultivated plant ranged from 9 0.01 to 32 4.0 mm. The largest
inhibition zone was obtained against S. aureus (32 4.0 mm) with
4 l/disc (100%) and the lowest against E. faecalis (9 0.01 mm)
with 0.5 l/disc (12.5%) concentration. Similarly, the antibacterial
activity of EO from wild plant varied from 8 0.01 to 24 4.0 mm,
where the largest zone of inhibition was obtained against S. aureus
(24.5 2.5 mm) with 4 l/disc (100%) concentration, while the lowest against E. faecalis and E. coli (8 0.01 mm) with 2 and 0.5 l/disc
(50 and 12.5%) concentration, respectively.
Either cultivated or wild plant essential oils had activity against
P. aeruginosa in all tested concentrations. Furthermore wild plant
had no activity against E. faecalis with lower concentrations (25 and
12.5%).
Cultivated O. syriacum EO showed a good activity against the
bacterial strains tested, in particular against S. aureus, where the
inhibition zone was ranged between 32 4.0 and 22 4.0 mm with
4 and 0.5 l/disc, respectively. A good inhibition was also obtained
against E. coli with a concentration of 4 l/disc, while wild O. syriacum EO resulted less effective than cultivated EO.
These results are very similar to those reported by other
researchers on the antimicrobial activity of O. syriacum EOs as well
as for other origanum species. Viuda-Martos et al. (2010), Lakis et al.
(2012), and Ibrahim et al. (2012) reported that the essential oils of O.
syriacum can be considered as effective antistaphylococcal natural
products.
Table 4
The inhibition zones resulted from the application of different antibiotics against bacteria strains.
S. aureus
E. faecalis
E. coli
P. aeruginosa
Tetracycline
(30 g/disc)
Rifampicin
(30 g/disc)
21 (S)
22 (S)
19 (S)
11 (R)
16 (S)
NA
23 (S)
7 (R)
32 (S)
21 (S)
15 (R)
11 (R)
32 (S)
NA
18 (S)
NA
24 (S)
18 (I)
30 (S)
NA
206
Table 5
Minimum inhibitory concentration (MIC) values of the essential oil of cultivated and
wild Origanum syriacum against some fungal strains.
Strain
Source
Cultivated (%)
Wild (%)
Itraconazole (mg/L)
A. fumigatus
A. fumigates
A. fumigatus
Aspergillus
A. avus
A. avus
A. niger
A. niger
Clinical isolatea
Clinical isolateb
Clinical isolatec
Soil
P. pinea
Bovine litter
Food
Soil
0.25
2.5
1.25
1.25
0.25
2.5
1.25
1.25
0.25
5.0
5.0
2.5
0.25
5.0
5.0
5.0
1.0
1.0
1.0
1.0
0.25
0.5
1.0
0.5
a
b
c
has been shown to cause disruption of the cellular membrane, inhibition of ATPase activity, and release of intracellular ATP and other
constituents (Viuda-Martos et al., 2010, 2011). Both EOs showed
a good activity against the bacterial strains tested, compared to
standard antibiotics as shown in Table 4.
3.3. Antifungal activity
The minimum inhibitory concentration MIC values of both cultivated plants and wild plants EOs against several fungal strains
are shown in Table 5. The MICs of both EOs were corresponding for
A. fumigatus clinical strain (isolated from lung of Psittacus erithacus) and A. avus (isolated from P. pinea). The MICs of cultivated O.
syriacum EOs ranged from 0.25 to 2.5 mg/L and those of wild O. syriacum ranged from 0.25 to 5 mg/L. All tested isolates were sensitive
to itraconazole.
Our results are similar to those reported by different authors on
the antifungal activity of EO of O. syriacum as well as those of similar species (Ibrahim et al., 2012; Turkolmez and Soylu 2014). Some
researchers reported antifungal activity of the plant oil against different phytopathogenic and medically important fungi (Tolouee
et al., 2010). The antifungal activity of essential oils is mainly
attributable to their major components although the possibility
of other phenomena, such as synergy or antagonism with minor
components, must also consider (Daferera et al., 2003).
In general, the antifungal activity of oregano EOs is mostly due to
the presence of phenols such as thymol and carvacrol (Portillo-Ruiz
et al., 2012). Possible action mechanisms by which mycelia growth
may be reduced or totally inhibited have been proposed. The effect
of phenolic antioxidants on microbial growth and toxin production
could be the result of the ability of phenolic compounds to alter
microbial cell permeability, permitting the loss of macromolecules
from the interior. They could also interact with membrane proteins,
causing a deformation in their structure and functionality (Fung
et al., 1977).
Conner and Beuchat (1984) suggested that the antimicrobial
activity of the essential oils of herbs and spices or their constituents
such as thymol, carvacrol, and eugenol, etc., could be the result
of damage to enzymatic cell systems, including those associated
with energy production and synthesis of structural compounds.
Davidson (2001) reported that the exact cause effect relation for
the mode of action of phenolic compounds, such as thymol, eugenol
and carvacrol, has not been determined, although it seems that they
may inactivate essential enzymes, react with the cell membrane or
disturb genetic material functionality.
4. Conclusions
The main constituents of Origanum essential oils were determined as carvacrol in cultivated plant and thymol in wild plant.
Both EOs showed antibacterial activity with varying magnitudes,
The tested EOs were active against all tested fungi, in particular
cultivated plants appeared to show lower MIC values, probably
due to its high carvacrol content. These products could therefore
be considered as promising candidates to control the overgrowth
of Aspergillus spp. in food stuffs. These EOs could be used as natural
food ingredients and could represent a useful alternative for the
food industry to reduce the quantity of synthetic additives. These
analyses need to be extended to other Egyptian populations of O.
syriacum, in order to obtain more possibility for clinical, nutritional
or pharmacological applications.
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