Molecules 2009, 14, 4614-4624; doi:10.3390/molecules14114614
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Chemical Composition and Antimicrobial Activity of the
Essential Oils from Two Species of Thymus Growing Wild in
Southern Italy
Laura De Martino 1,*, Maurizio Bruno 2, Carmen Formisano 3, Vincenzo De Feo 1, Francesco
Napolitano 3, Sergio Rosselli 2 and Felice Senatore 3
1
2
3
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, via Ponte don
Melillo, 84084 Fisciano (Salerno), Italy; E-Mail: defeo@unisa.it (V.D.F)
Dipartimento di Chimica Organica, Università di Palermo, Viale delle Scienze, Parco d’Orleans
II, 90128 Palermo, Italy; E-Mails: bruno@dicpm.unipa.it (M.B.); rosselli@unipa.it (S.R.)
Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli, “Federico
II”, Via D. Montesano, 49, 80131 Napoli, Italy; E-Mails: caformis@unina.it (C.F.);
fnapolitano@virgilio.it (F.N.); fesenato@unina.it (F.S.)
* Author to whom correspondence should be addressed; E-Mail: ldemartino@unisa.it;
Tel.: +39089969394; Fax: +39089969602.
Received: 15 October 2009; in revised form: 9 November 2009 / Accepted: 11 November 2009 /
Published: 12 November 2009
Abstract: The volatile constituents of the aerial parts of two samples of Thymus
longicaulis C. Presl, collected in Campania and in Sicily, and two samples of Thymus
pulegioides L. from the same regions, were extracted by hydrodistillation and analyzed.
Considering the four oils together, seventy-eight different compounds were identified: 57
for Thymus longicaulis from Sicily (91.1% of the total oil), 40 for Thymus longicaulis from
Campania (91.5% of the oil), 39 for Thymus pulegioides from Sicily (92.5% of the oil) and
29 for Thymus pulegioides from Campania (90.1% of the oil). The composition of the oils
is different, although the most abundant components are identical in T. pulegioides. The
essential oils showed antibacterial activity against eight selected microorganisms.
Keywords: Thymus longicaulis C. Presl; Thymus pulegioides L.; essential oil composition;
thymol; geraniol; antibacterial activity
Molecules 2009, 14
4615
1. Introduction
Among the aromatic plants belonging to the Lamiaceae family, the genus Thymus is noteworthy for
the numerous species and varieties of wild-growing plants. This genus comprises about 400 species of
perennial aromatic, evergreen or semi-evergreen herbaceous plants with many subspecies, varieties,
subvarieties and forms; the species are endemic and widely distributed, growing in the temperate and
cold regions of the Old World, being native to Southern Europe and diffused in particular in the
Mediterranean area. The plants are extensively used, fresh and dried, as a culinary herb. Their essential
oils are utilized as flavour ingredients in a wide variety of food, beverage and confectionery products,
as well as in perfumery for the scenting of soaps and lotions. Because of their antiseptic, antispasmodic
and antimicrobial properties, they are also used for medicinal purposes [1-5].
In recent years, several reports have been published concerning the composition and/or the
biological properties of Thymus essential oils. These studies have emphasized the existence of marked
chemical differences among oils extracted from different species or varieties. More than 20 essential
oil chemotypes were noticed in different species of Thymus genus [6], but, on the other hand, different
chemotypes can grow in the same habitat, so the study of this genus could be interesting [7]. This
chemical diversity can influence the biological activity of the oils and it is generally a function of three
factors: genetical, physiological conditions and the environment [8]. Moreover, most aspects of
medicinal use of Thymus spp. are related to their essential oil composition, which shows various levels of
thymol and/or carvacrol, phenolic derivatives with strong and wide-spectrum antimicrobial activity [5,8].
T. longicaulis C. Presl is a species with long, somewhat woody, creeping branches, non flowering
or with a terminal inflorescence [9]. This species is important from the ethnobotanical point of view as
a traditional medicinal plant [10-11]: it is reported as an antiseptic, an expectorant and a spasmolytic,
properties probably correlated with the content of essential oils and flavonoids. The composition of
essential oil from T. longicaulis has been studied previously [12-16].
Eight chemotypes were determined for Thymus pulegioides L. [6,17]. This species is widely
distributed in the European continent and in Mediterranean islands. In Portugal, it grows in the
northeast, and it is locally used as an antiseptic. Previous results have demonstrated that this species is
polymorphic [18], and that the thymol/carvacrol chemotype is one of the most abundant in Portugal. In
Italy, it is traditionally used as an expectorant, an anthelmintic, a gastric antispasmodic, and an
astringent [19].
As a continuation of our research on the oils of the Lamiaceae growing wild in Southern Italy [2021] and because of this variability of Thymus genus, we examine in this work the composition of the
essential oils of Thymus longicaulis and Thymus pulegioides growing wild in two different regions of
Southern Italy (Campania and Sicily) and their antimicrobial activity on eight selected microorganism.
2. Results and Discussion
2.1. Chemical composition of the essential oils
Considering the four oils together a total of seventy-eight different compounds were identified: 57
for T. longicaulis from Sicily (91.1% of the total oil), 40 for T. longicaulis from Campania (91.5% of
the oil), 39 for T. pulegioides from Sicily (92.5% of the oil) and 29 for T. pulegioides from Campania
Molecules 2009, 14
4616
(90.1% of the oil). The components are listed in Table 1, classified in eight classes on the basis of their
chemical structures.
Table 1. Essential oil composition (% of total) of aerial parts of Thymus longicaulis C.
Presl from Sicily (Tls) and from Campania (Tlc), Thymus pulegioides L. from Sicily (Tps)
and from Campania (Tpc).
Component
LRIa
LRI b
Monoterpene hydrocarbons
α-Thujene
α-Pinene
Camphene
Sabinene
β-Pinene
Myrcene
α-Phellandrene
δ3-Carene
α-Terpinene
o-Cymene
p-Cymene
Limonene
(Z)-β-Ocimene
(E)-β-Ocimene
γ-Terpinene
Terpinolene
Oxygenated monoterpenes
1,8-Cineole
cis-Linalool oxide (furanoid)
trans-Linalool oxide (furanoid)
trans-Sabinene hydrate
Linalool
Camphor
Borneol
Terpinen-4-ol
p-Cymen-8-ol
α-Terpineol
Nerol
Geraniol
Neral
Geranial
Cumin alcohol
Sesquiterpene hydrocarbons
Bicycloelemene
α-Cubebene
α-Copaene
β-Cubebene
β-Bourbonene
β-Elemene
Longifolene
β-Caryophyllene
β-Gurjunene
γ-Elemene
Acoradiene
(Z)-β-Farnesene
(E)-β-Farnesene
α-Humulene
allo-Aromadendrene
Germacrene D
γ-Muurolene
Viridiflorene
Valencene
α-Muurolene
925
938
953
973
978
993
1005
1010
1013
1020
1025
1030
1038
1049
1057
1086
1034
1076
1087
1093
1097
1145
1167
1176
1185
1189
1226
1235
1237
1267
1288
1035
1075
1076
1132
1118
1173
1150
1189
1187
1278
1205
1243
1262
1256
1265
1213
1477
1451
1474
1553
1532
1718
1611
1856
1706
1809
1857
Tls%
Tlc%
Tps%
Tpc%
25.6
1.8
30.2
25.2
0.6
1.3
t
t
t
1.2
1.1
0.8
1.4
0.5
0.1
0.4
4.2
0.1
0.8
t
9.0
3.4
0.1
5.5
0.1
8.2
1.2
0.1
0.3
0.5
3.5
1.3
0.5
0.8
2112
1494
1466
1497
1549
1558
1600
1612
1612
1650
1668
1672
1689
1662
1726
1704
1687
1740
1740
0.9
17.6
1.8
19.9
0.8
44.8
t
5.7
0.1
12.2
1.0
9.4
0.7
0.5
0.6
0.6
3.8
0.4
1.2
0.4
5.6
1.0
2.8
0.8
0.6
4.7
t
1.9
2.1
t
9.9
11.1
0.4
0.5
t
t
0.9
0.9
0.1
20.0
1339
1348
1377
1382
1385
1387
1403
1415
1432
1432
1442
1443
1452
1455
1463
1477
1478
1494
1494
1503
0.1
0.3
0.7
0.1
0.1
1.4
1.4
34.2
0.4
0.7
1.0
4.8
t
t
0.4
0.1
0.7
0.1
5.7
0.4
0.1
1.3
0.7
0.4
0.2
5.3
t
0.1
0.1
0.2
0.3
t
0.1
2.2
t
t
0.5
0.1
0.1
5.9
0.1
t
7.5
0.3
1.2
0.4
0.2
0.2
0.2
t
Identification c
1,2
1,2,3
1,2,3
1,2
1,2,3
1,2,3
1,2,3
1,2
1,2
1,2
1,2,3
1,2,3
1,2,3
1,2
1,2
1,2,3
1,2,3
1,2
1,2
1,2
1,2,3
1,2,3
1,2,3
1,2,3
1,2
1,2,3
1,2,3
1,2,3
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2,3
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
Molecules 2009, 14
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Table 1. Cont.
β-Bisabolene
γ-Cadinene
δ-Cadinene
α-Cadinene
Germacrene B
Oxygenated sesquiterpenes
Caryophyllene oxide
Guaiol
t-Cadinol
t-Muurolol
α-Cadinol
(Z,Z)-Farnesol
14-Hydroxy-α-humulene
(Z)-Lanceol
Phenolic compounds
Thymol methyl ether
Carvacrol methyl ether
Thymol
Carvacrol
Thymyl acetate
Carvacryl acetate
Hydrocarbons
Dodecane
Tridecane
Fatty acids
Hexadecanoic acid
Others
1-Octen-3-ol
3-Octanol
Octanol
Nonanol
Total amount of compounds
a
b
1510
1515
1526
1535
1554
1743
1776
1773
1579
1598
1640
1642
1652
1715
1718
1765
2008
2108
2158
2209
2255
1854
1239
1245
1293
1299
1365
1367
1607
1975
2198
2239
1865
1885
1200
1300
1200
1300
1957
2931
980
992
1073
1170
1454
1394
1562
0.5
1.3
0.2
2.2
10.1
0.2
9.2
0.1
0.4
0.1
0.1
26.4
5.5
6.4
1.7
12.8
1.5
0.4
2.6
0.9
0.2
1.3
2.3
1.9
2.2
2.1
0.2
0.1
0.3
0.1
38.7
0.8
0.5
9.3
14.5
0.2
36.4
10.8
21.8
3.1
0.7
39.6
6.0
2.2
26.3
4.7
0.4
13.6
1.0
0.6
0.4
t
0.8
0.6
0.1
0.1
91.1
1,2,3
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2
1,2,3
1,2,3
1,2
1,2
1,2,3
1,2,3
t
1.5
1.3
0.1
0.1
91.5
1,2
1,2
1,2
1,2
1,2
92.5
0.6
0.6
2.0
2.0
1,2,3
1,2
1,2,3
1,2,3
1,2
90.1
c
: HP-5MS column; : HP Innowax column; : 1 = LRI linear retention index; 2 = MS identification based on comparison
of mass spectra; 3 = Co-GC retention time identical to authentic compounds.
For T. longicaulis from Sicily, the monoterpene and sesquiterpene fractions were present in quite
similar amounts, 33.8% and 30.1%, respectively. The monoterpene fraction was almost entirely
constituted by monoterpene hydrocarbons (25.6%), among which the main representatives were pcymene (9.0%), γ-terpinene (5.5%), myrcene (4.2%) and limonene (3.4%). Among oxygen containing
monoterpenes, linalool (3.5%), camphor (1.3%) and 1,8-cineole (1.2%) were the most abundant. τCadinol (9.2%) was the main component of the sesquiterpene fraction of the oil; other abundant
constituents of this fraction were β-caryophyllene (5.7%), germacrene D (5.3%) and germacrene B
(2.2%). Another abundant fraction was constituted by phenolic compounds (26.4%) with a prevalence
of thymyl acetate (12.8%) followed by thymol (6.4%) and thymol methyl ether (5.5%).
The oil from T. longicaulis collected in Campania presented a different composition. In this oil
monoterpenes also constituted the most abundant fraction (46.6%), but with a prevalence of oxygen
containing monoterpenes (44.8%) with a great amount of geraniol (34.2%) and borneol (3.8%). The
phenols carvacrol (14.5%), carvacryl acetate (13.6%) and thymol (9.3%) were also present in a
significant percentages. Among sesquiterpenes, only β-caryophyllene (2.2%) was present in
appreciable amounts.
The two studied oils, even if characterized by a prevalence of monoterpenes, are quite different in
the relative percentages of monoterpene hydrocarbons and oxygenated monoterpenes: the former are
Molecules 2009, 14
4618
predominant in T. longicaulis collected in Sicily, while the oxygenated monoterpenes were only 8.2%.
Moreover, in the T. longicaulis from Campania, the predominant compound is geraniol, which is
absent in the other oil. Our data disagree with those previously reported for T. longicaulis collected in
Mid-Western Turkey [22] in which thymol is the main component. Baser et al. [13-15] also reported
thymol as predominant components of different samples of this species. On the other hand, the
composition of three chemotypes of T. longicaulis subsp. chaubardii from Turkey again, studied by
Tzakou [23], shows the main components were oxygenated monoterpenes (geraniol, linalool and
geranyl acetate). These differences in the essential oils of Thymus longicaulis confirmed that the genus
Thymus is taxonomically and genetically complex [19].
Monoterpenes constituted the most abundant fraction of Thymus pulegioides from Sicily (42.4%),
with a prevalence of monoterpene hydrocarbons (30.2%) among which p-cymene (17.6%) and
γ-terpinene (5.7%) predominated. Among seven oxygen containing monoterpenes (12.2%), linalool
(5.6%) and borneol (2.8%) were the most abundant. Ten sesquiterpene hydrocarbons (with two
compounds in trace amounts) accounted for the 9.9% of the total oil, with β-caryophyllene (5.9%) as
main compound, while caryophyllene oxide (1.9%) was the only abundant oxygenated sesquiterpene.
Thymol (21.8 %) and thymol methyl ether (10.8 %) represented the main components of the phenolic
fraction (36.4%).
The essential oil of Thymus pulegioides from Campania was mainly constituted by phenolic
compounds (39.6%), being thymol (26.3%) the main one. The monoterpene fraction (34.6%)
comprised mainly hydrocarbons (25.2%) with p-cymene (19.9%) as predominant component. Among
oxygen containing monoterpenes, linalool (4.7%), terpinen-4-ol (2.1%) and borneol (1.9%) were the
most abundant compounds. β-Caryophyllene (7.5%) represented the main sesquiterpene hydrocarbon.
Moreover, the two samples studied of T. pulegioides have a similar composition pattern and both
belong to a thymol chemotype. Our results are in agreement with previous data on the essential oils of
the Italian wild growing T. pulegioides, which were defined as belonging to the thymol chemotype
according to the high content of thymol, p-cymene, and γ-terpinene [19]. On the other hand, previous
papers on the essential oils of different varieties of T. pulegioides, concluded that there is no clear
chemical relation between the varieties and chemotypes [24].
2.2. Antimicrobial activity
The in vitro antibacterial activity of Thymus longicaulis and T. pulegioides against eight bacterial
species was evaluated by the in vitro paper-disk diffusion method [25]. The microorganism selected
are representative of the Gram positive and Gram negative classes and known to cause respiratory,
gastrointestinal, skin and urinary disorders in humans. The results obtained in the antibacterial assay
are shown in Tables 2a and 2b. Our samples were more active against Gram positive bacteria but
generally are less active than gentamycin and tetracyclin. This finding totally agrees with the
observations derived from studies with essential oils from other thyme species. Nedorostova et al. [26]
reported, in particular, the antimicrobial activity in vapour phase of essential oils of Thymus
pulegioides; instead, Ložiene et al. [27] reported the antibacterial activity of extracts of this species.
Molecules 2009, 14
4619
Table 2a. Antibacterial activity (diffusion method) of different amounts of the essential oil of T. longicaulis. Inhibition is expressed in
mm and include the diameter of paper disc (6 mm). Results are shown as mean ± standard deviation (SD) of the inhibition zone (n = 3).
Bacteria
S. aureus
S. faecalis
Thymus longicaulis from Sicily
Concentration tested (mg/ml)
10
5
2.55
1.25
21 ± 1.3 18 ± 0.3 13 ± 1.1 10 ± 1.0
18 ± 0.9 14 ± 1.3 10 ± 1.0 7 ± 0.3
B. subtilis
B. cereus
P. mirabilis
E. coli
S. typhi Ty2
P. aeruginosa
15 ± 1.0
16 ± 0.3
12 ± 1.0
17 ± 0.6
13 ± 0.3
15 ± 0.0
12 ± 0.8
13 ± 1.1
9 ± 0.7
13 ± 1.0
10 ± 1.1
11 ± 1.2
9 ± 0.6
10 ± 0.3
n.a
10 ± 0.2
7 ± 0.4
8 ± 0.9
a
n.a
7 ± 0.9
n.a
7 ± 0.8
n.a
n.a
0.62
7 ± 0.8
n.a.
n.a
n.a
n.a
n.a
n.a
n.a
Thymus longicaulis from Campania
Concentration tested (mg/ml)
10
5
2.55
1.25
0.62
18 ± 1.1 15 ± 1.2 10 ± 0.3 8 ± 1.1 n.a.
15 ± 0.9 12 ± 1.3 8 ± 0.6
n.a
n.a
Ga
Tb
29 ± 1.4
30 ± 1.0
n.t
n.t
11 ± 1.0
14 ± 1.9
9 ± 1.8
14 ± 0.3
11 ± 1.2
13 ± 1.4
28 ± 1.2
28 ± 1.3
n.t
n.t
n.t
n.t
n.t
n.t
29 ± 1.1
26 ± 1.5
21 ± 0.9
28 ± 1.3
7 ± 1.0
10 ± 0.3
7 ± 0.4
10 ± 0.5
10 ± 1.2
9 ± 1.5
n.a
7 ± 0.4
n.a
7 ± 0.7
7 ± 0.8
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
Gentamycin (10 μg); b Tetracyclin (15 μg), n.a.= not active; n.t.= not tested.
Table 2b. Antibacterial activity (diffusion method) of different amounts of the essential oil of T. pulegioides. Inhibition is expressed in
mm and include the diameter of paper disc (6 mm). Results are shown as mean ± standard deviation (SD) of the inhibition zone (n = 3).
Bacteria
S. aureus
S. faecalis
B. subtilis
B. cereus
P. mirabilis
E. coli
S. typhi Ty2
P. aeruginosa
10
17 ± 1.0
16 ± 0.3
12 ± 0.9
15 ± 1.2
11 ± 1.5
15 ± 1.4
13 ± 1.2
13 ± 1.1
Thymus pulegioides from Sicily
Concentration tested (mg/ml)
5
2.55
1.25
14 ± 1.2 11 ± 1.1 8 ± 1.0
13 ± 1.4 10 ± 1.2 7 ± 0.9
9 ± 1.5
n.a.
n.a.
11 ± 1.6
8 ± 1.0
n.a.
7 ± 1.2
n.a.
n.a
12 ± 1.4
9 ± 1.4
7 ± 0.7
9 ± 1.0
n.a.
n.a
10 ± 1.3
7 ± 0.9
n.a.
a
0.62
n.a
n.a.
n.a.
n.a
n.a
n.a
n.a
n.a
Thymus pulegioides from Campania
Concentration tested (mg/ml)
10
5
2.55
1.25
0.62
20 ± 0.9 17 ± 1.1 14 ± 1.2 19 ± 1.3 7 ± 0.9
16 ± 1.3 13 ± 1.2 9 ± 1.3 7 ± 1.2
n.a
16 ± 1.2 13 ± 1.4 10 ± 1.0 7 ± 1.0
n.a
15 ± 1.4 11 ± 1.1 8 ± 0.3
n.a
n.a
11 ± 1.1 7 ± 0.5
n.a
n.a
n.a
16 ± 1.2 12 ± 1.1 9 ± 0.6 7 ± 1.1
n.a
12 ± 1.1 8 ± 1.3
n.a
n.a
n.a
13 ± 1.5 10 ± 1.5 7 ± 0.5
n.a
n.a
Gentamycin (10 μg); b Tetracyclin (15 μg), n.a.= not active; n.t.= not tested.
Ga
Tb
29 ± 1.4
30 ± 1.0
28 ± 1.2
28 ± 1.3
n.t
n.t
n.t
n.t
n.t
n.t
n.t
n.t
29 ± 1.1
26 ± 1.5
21 ± 0.9
28 ± 1.3
Molecules 2009, 14
4620
Our result agree with the study of Thymus longicaulis L. of Chorianopoulos and coworkers [11]
who reported the antimicrobial activity of this species’ essential oil against five common foodborne
pathogens. Generally, the chemical composition of the oils determine their antimicrobial activity;
Valero and Giner reported the effects of antimicrobial activity of essential oil components, particularly
of phenols, such as carvacrol and thymol [28]. The antimicrobial activity of Thymus species and
Thymus pulegioides, in particular, was attributed to the presence of thymol [29,30]; in reality, even if
antimicrobial activity of an essential oil is often attributed mainly to its major components, today it is
known that the synergistic or antagonistic effect of one compound in minor percentage of mixture has
to be considered [31].
3. Experimental Section
3.1. Plant material
Aerial parts of Thymus longicaulis were collected, at the full flowering stage in Sicily at Madonie a
high mountain group of Palermo province, and in Campania, near the town of Agerola, Naples
province; aerial parts of Thymus pulegioides L. were collected at the full flowering stage, at Madonie
and near Agerola. Plants were identified by the Prof. V. De Feo. Voucher specimens of the plants are
deposited in the Herbarium of the Medical Botany Chair, at the University of Salerno.
3.2. Isolation of the volatile components
The air-dried samples (50 g) were ground in a Waring blender and then subjected to
hydrodistillation for 3 h using n-hexane as a solvent, according to the standard procedure previously
described [21]. The extracts were dried over anhydrous sodium sulphate and then stored in sealed
vials, at -20 °C, ready for the GC and GC-MS analyses. The samples yielded 0.8% (v/w) Thymus
longicaulis from Campania, 0.7% (v/w) T. longicaulis from Sicily, 0.9% (v/w) T. pulegioides from
Campania and 1.1% (v/w) T. pulegioides from Sicily as yellow oils, with a pleasant smell.
3.3. GC and GC/MS analyses
GC analyses were carried out on a Hewlett Packard Sigma 115 gas chromatograph equipped with a
FID and a 30 m × 0.25 mm i.d.. HP 5MS fused silica capillary column (film thickness: 0.25 μm).
Column temperature: 40 °C, with 5 min initial hold, and then to 270 °C at 2 °C/min, 260 °C (20 min);
injection mode splitless (1 µL of a 1:1,000 n-pentane solution). Injector and detector temperatures
were 250 °C and 290 °C, respectively. Analysis was also run by using a fused silica HP Innowax
polyethylenglycol capillary column (50 m × 0.20 mm, 0.25 µm film thickness). In both cases carrier
gas was He, with flow rate of 1 mL/min. GC-MS analyses were performed. Analysis on an Agilent
6850 Ser. II apparatus, fitted with a fused silica DB-5 capillary column (30 m × 0.25 mm i.d.; 0.33 μm
film thickness), coupled to an Agilent Mass Selective Detector MSD 5973; ionization energy 70 eV;
electron multiplier voltage 2000 V. Mass spectra were scanned in the range 40-500 amu, scan time
5 scans/s. Gas chromatographic conditions were as reported above; transfer line temperature, 295 °C.
Molecules 2009, 14
4621
3.4. Identification of components
Most constituents were identified by gas chromatography by comparison of their linear retention
indices (LRi) with either those of the literature [32,33] or with those of authentic compounds available
in our laboratories. The linear retention indices were determined in relation to a homologous series of
n-alkanes (C8-C28) under the same operating conditions. Further identification was made by
comparison of their mass spectra on both columns with either those stored in NIST 02 and Wiley 275
libraries or with mass spectra from the literature [32,34] and a home-made library. Components
relative concentrations were obtained by peak area normalization. No response factors were calculated.
3.5. Antimicrobial activity
The antimicrobial activity of the oil was evaluated by the in vitro paper-disk diffusion method [25]
against eight selected Gram+ and Gram– bacteria: Staphylococcus aureus (ATTC 25923),
Streptococcus faecalis (ATTC 29212), Bacillus subtilis (ATCC 6633), Bacillus cereus (PCI 213),
Proteus mirabilis (ATCC 12453), Escherichia coli (ATCC 25922), Salmonella typhi Ty2 (ATCC
19430), Pseudomonas aeruginosa (ATCC 27853). Aliquots of each oil were dissolved in dimethyl
sulfoxide to give solutions containing from 10 to 0.62 mg/mL. Sterile 6 mm discs (Whatman N.1) were
impregnated with 20 μL of each solution and placed on the surface of agar plates so prepared: 25 mL
of Mueller-Hinton agar medium [NCCLS, 1984] and a standardized inoculum of the correspondent test
microorganism (standard 0.5 McFarland). Plates were incubated at 37 °C for 24 h. The analyses were
carried out in triplicate and the results are expressed as mean ± SD. Control disks with 10 μL of
dimethyl sulfoxide showed no inhibition in a preliminary test. Gentamycin (10 μg) and Tetraciclyn
(15 μg) served as positive control on Gram– and Gram+ bacteria, respectively.
4. Conclusions
Considering the remarkable variations reported for the essential oil of T. pulegioides from different
localities, it was of interest to continue research on its chemical polymorphism. Our data reveals that
the essential oils studied, from two different localities, belong to a thymol chemotype with an high
percentage of p-cymene. The two studied oils of Thymus longicaulis, even if characterized by a
prevalence of the monoterpene fraction, are quite different with regards to the relative percentages of
monoterpene hydrocarbons and oxygenated monoterpenes and the data obtained serve to highlight the
chemical polymorphism of T. longicaulis. Essential oils are extensively used as flavour ingredient in a
wide variety of food, beverage and confectionary products. This use and the antibacterial activity of
these substances make them potential natural preservatives in food industry.
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© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).