Chemical Composition and Antimicrobial Activity of the Essential Oils from Two Species of Thymus Growing Wild in Southern Italy

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.


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][2][3][4][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][13][14][15][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 [20][21] 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.
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 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][14][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].
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].

Thymus pulegioides from Sicily
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].

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.

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.

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.

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 (C 8 -C 28 ) 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.

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.

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.