Essential Oil Characterization of Thymus vulgaris from Various Geographical Locations

Thyme (Thymus vulgaris L.) is a commonly used flavoring agent and medicinal herb. Several chemotypes of thyme, based on essential oil compositions, have been established, including (1) linalool; (2) borneol; (3) geraniol; (4) sabinene hydrate; (5) thymol; (6) carvacrol, as well as a number of multiple-component chemotypes. In this work, two different T. vulgaris essential oils were obtained from France and two were obtained from Serbia. The chemical compositions were determined using gas chromatography–mass spectrometry. In addition, chiral gas chromatography was used to determine the enantiomeric compositions of several monoterpenoid components. The T. vulgaris oil from Nyons, France was of the linalool chemotype (linalool, 76.2%; linalyl acetate, 14.3%); the oil sample from Jablanicki, Serbia was of the geraniol chemotype (geraniol, 59.8%; geranyl acetate, 16.7%); the sample from Pomoravje District, Serbia was of the sabinene hydrate chemotype (cis-sabinene hydrate, 30.8%; trans-sabinene hydrate, 5.0%); and the essential oil from Richerenches, France was of the thymol chemotype (thymol, 47.1%; p-cymene, 20.1%). A cluster analysis based on the compositions of these essential oils as well as 81 additional T. vulgaris essential oils reported in the literature revealed 20 different chemotypes. This work represents the first chiral analysis of T. vulgaris monoterpenoids and a comprehensive description of the different chemotypes of T. vulgaris.


Introduction
Thymus vulgaris L. (Lamiaceae) is an evergreen herb native to the southern Europe and the Mediterranean [1]. The plant has been used since ancient times as a culinary ingredient, to add flavor to cheeses [2,3] and liqueurs [4,5], and to flavor meats such as rabbit, boar, and lamb [6]. Today it is a common component of bouquet garni [7] and of herbes de Provence [8]. In addition to its use in foods, T. vulgaris is a well-known herbal medicine that has been used for thousands of years to treat alopecia, dental plaque, dermatophyte infections, bronchitis, cough, inflammatory skin disorders, and gastrointestinal distress [9]. The major constituents of commercial T. vulgaris essential oil are thymol (23%-60%), γ-terpinene (18%-50%), p-cymene (8%-44%), carvacrol (2%-8%), and linalool (3%-4%) [10]. T. vulgaris oil as well as thymol have shown antibacterial, antifungal, and anti-inflammatory effects, accounting for the medicinal uses of T. vulgaris [9]. There are, however, numerous varieties and cultivars of T. vulgaris; Tropicos lists nine subspecies and varieties of T. vulgaris [11]. As many as 13 different chemotypes of T. vulgaris, based on the predominance of particular monoterpenoids in the essential oils, have been identified [12][13][14][15]. In this work, we present the essential oil compositions, including monoterpenoid enantiomeric compositions, of four different chemotypes of T. vulgaris essential oils from Europe. In addition, a hierarchical cluster analysis has been carried out to elucidate/delineate the various chemotypes of T. vulgaris, and we have examined the antifungal properties of three essential oils from Europe.

Gas Chromatography-Mass Spectrometry (GC-MS)
The essential oils of T. vulgaris chemotypes were analyzed by GC-MS using a Shimadzu GCMS-QP2010 Ultra operated in the electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 amu, scan rate = 3.0 scans/sec, and GC-MS solution software. The GC column was a ZB-5 fused silica capillary column with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm. The carrier gas was helium with a column head pressure of 80 psi and flow rate of 1.37 mL/min. Injector temperature was 250 • C and the ion source temperature was 200 • C. The GC oven temperature program was programmed for 50 • C initial temperature, temperature increased at a rate of 2 • C/min to 260 • C. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared and 0.1 µL was injected with a splitting mode (30:1). Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes, and by comparison of their mass spectral fragmentation patterns with those reported in the literature [16], and stored in our in-house MS library.

Chiral Gas Chromatography-Mass Spectrometry
Chiral analysis of the essential oils was performed on a Shimadzu GCMS-QP2010S operated in the EI mode (electron energy = 70 eV), scan range = 40-400 amu, scan rate = 3.0 scans/s. GC was equipped with a Restek B-Dex 325 capillary column (30 m × 0.25 mm ID × 0.25 µm film). Oven temperature was started at 50 • C, and then gradually raised to 120 • C at 1.5 • C/min. The oven was then raised to 200 • C at 2 • C/min and held for 5 min. Helium was the carrier gas and the flow rate was maintained at 1.8 mL/min. Samples were diluted 3% w/v with CH 2 Cl 2 and then a 0.1 µL sample was injected in a split mode with a split ratio of 1:45.

Antifungal Screening
Antifungal activity was carried out using Candida albicans (ATCC #18804), Cryptococcus neoformans 24067 (serotype D or var. neoformans), and Aspergillus niger (ATCC #16888). Qualitative assessment of antifungal activity utilized broth macrodilution (for C. albicans and C. neoformans), whereas minimum inhibitory concentrations (MIC) were determined using microdilution methods. Initially cultures were grown on potato dextrose agar for 48-72 h before a single colony was isolated and grown in potato dextrose broth for 48-72 hours to create initial liquid cultures. Cells were diluted to a final concentration of 2 × 10 3 cells/mL using MOPS (3-(N-morpholino)propanesulfonic acid) buffered RPMI (Roswell Park Memorial Institute) medium and 900 µL were aliquoted into sterile 12 × 75 mm tubes. Each T. vulgaris essential oil (100 µL of 1% DMSO solution) was added to each tube, which were then incubated at 37 • C for 72 h in a shaking incubator (175 rpm). For determination of the MIC for C. albicans and C. neoformans, microdilution in 96-well plates was performed in triplicate. Briefly, serial dilution of the T. vulgaris samples was performed by adding 50 µL of RPMI to each well then an equal volume of sample to be tested to the first row. After mixing, 50 µL was removed and added to the next row. The procedure was repeated for each row. To this mixture, 50 µL of cells diluted to 2000 cells/mL in RPMI were added to each well. The plates were incubated for 48 hours at 37 • C before growth was quantitated visually based on turbidity.
For the mold-like Ascomycota A. niger, disk diffusion was used to characterize each T. vulgaris essential oil. Initial cultures were grown on malt extract agar for 5-7 days before conidia were collected and suspended in potato dextrose broth. The suspension was then filtered into a sterile test tube using cheesecloth to remove hyphae. Conidia suspension was then diluted until it reached an OD 625 of 0.1-0.2. The suspension (100 µL) was plated on malt extract agar before a sterile filter paper disk was placed in the center and 50 µL of T. vulgaris essential oil was added. The culture was grown for 4-5 days at 25 • C before zones of inhibition were determined.

Antifungal Activity
Three of the four T. vulgaris chemotypes in this study were tested for inhibition of Aspergillus niger, Cryptococcus neoformans var. neoformans, and Candida albicans. Macrodilution was utilized for the yeast-like Ascomycota, C. albicans, and Basidiomycota C. neoformans, to determine antifungal minimum inhibitory concentrations (MICs, Table 2). The T. vulgaris linalool and geraniol chemotypes both demonstrated some degree of inhibition against these pathogens. For the sporulating mold-like Ascomycota, A. niger, a larger surface area was required for hyphal growth. Thus, it was grown on malt extract agar plates with a filter disk impregnated with the T. vulgaris chemotype of interest. Disk diffusion showed only slight inhibition of A. niger with the only clear zone of inhibition for T. vulgaris sample #1. The remaining chemotypes showed growth over the filter disk, indicating no significant antifungal activity against A. niger. Because it has been shown that linalool is biotransformed to non-pathogenic compounds and that linalyl acetate increases A. niger hyphal growth [64], it is speculated that camphor in sample #1 is responsible for A. niger inhibition. Significant levels of camphor are not found in the other T. vulgaris samples. The differential antifungal activities observed in this study mirror those previously reported by Giordani and co-workers [13]. That is, the sabinene hydrate chemotype showed the lowest antifungal activity, the linalool chemotype was next, then the geraniol chemotype. Giordani and co-workers had found that the thymol chemotype showed much stronger antifungal activity [13]. The T. vulgaris thymol chemotype was also found to be the most larvicidal against Culex quinquefasciatus [15] and exhibited the most antioxidant properties [14].

Conclusions
This work has presented the most comprehensive analysis of Thymus vulgaris chemotypes, revealing at least 20 different types based on essential oil composition. In addition, this is the first analysis to characterize the enantiomeric distributions of T. vulgaris monoterpenoids. Enantiomers are well known to elicit different odorant responses in insects [65,66] as well as humans [67], and it is reasonable to assume that different enantiomers will have different medicinal biological activities [68]. Thus, for example L-linalool has shown anticonvulsant activity in a mouse model whereas D-linalool was inactive [69]. Similarly, both D-α-pinene and D-β-pinene showed antifungal activity whereas the L-enantiomers were inactive [70], while L-α-pinene was more active than the D-enantiomer against Listeria monocytogenes [71]. Therefore, not only is the particular chemotype of a culinary and medicinal herb such as T. vulgaris an important consideration, but the enantiomeric distribution may also have a profound influence on its bioactivity, flavor, and aroma profile.

Conflicts of Interest:
The authors declare no conflict of interest.