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Essential Oil Characterization of Thymus vulgaris from Various Geographical Locations

Prabodh Satyal
Brittney L. Murray
Robert L. McFeeters
2 and
William N. Setzer
Alchemy Aromatic LLC, 621 Park East Blvd., New Albany, IN 47150, USA
Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA
Author to whom correspondence should be addressed.
Foods 2016, 5(4), 70;
Submission received: 1 September 2016 / Revised: 20 October 2016 / Accepted: 24 October 2016 / Published: 27 October 2016
(This article belongs to the Special Issue Flavour Volatiles of Foods)


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.

1. 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.

2. Materials and Methods

2.1. Plant Material

Thymus vulgaris #1, (linalool chemotype), T. vulgaris #3, (geraniol chemotype), T. vulgaris #4, (sabinene hydrate chemotype), and T. vulgaris #5, (thymol chemotype) were collected in May 2015, from Nyons, France (44°21′37″ N, 5°8′23″ E), Jablanicki, Serbia (42°59′43″ N, 21°55′4″ E), Pomoravje District, Serbia (43°58′0″ N, 21°15′0″ E) and Richerenches, France (44°21′37″ N, 4°54′47″ E), respectively. The plants were identified by Julien Abisset (Natural Essential SA, France). The air-dried aerial parts of each T. vulgaris sample were subjected to steam distillation for 3 h. After decanting and drying of the oils over anhydrous sodium sulfate, they were stored under refrigeration (−4 °C) until analysis. Aerial parts of T. vulgaris #1 produced 1.6% yield of essential oil (EO), T. vulgaris #3 produced 1.2% yield, T. vulgaris #4 produced 1.0% yield, and T. vulgaris #5 produced 1.5% yield.

2.2. 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 CH2Cl2 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.

2.3. 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 CH2Cl2 and then a 0.1 µL sample was injected in a split mode with a split ratio of 1:45.

2.4. Hierarchical Cluster Analysis

A total of 81 T. vulgaris essential oil compositions from the published literature, as well as the four compositions from this study were treated as operational taxonomic units (OTUs). The percentage composition of 33 major essential oil components (thymol, p-cymene, γ-terpinene, linalool, carvacrol, geraniol, β-caryophyllene, cis-sabinene hydrate, borneol, α-pinene, terpinene-4-ol, myrcene, 1,8-cineole, α-terpineol, camphene, α-terpinyl acetate, α-terpinene, camphor, limonene, β-pinene, geranyl acetate, α-thujene, geranyl formate, β-cyclocitral, cis-verbenol, trans-sabinene hydrate, 1-octen-3-ol, thymol methyl ether, caryophyllene oxide, carvacrol methyl ether, bornyl acetate, thymoquinone, and α-humulene) was used to determine the chemical relationship between the various T. vulgaris essential oil samples by agglomerative hierarchical cluster (AHC) analysis using the XLSTAT software, version 2015.4.01. Pearson correlation was selected as a measure of similarity, and the unweighted pair-group method with arithmetic average (UPGMA) was used for cluster definition.

2.5. 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 × 103 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 OD625 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.

3. Results and Discussion

3.1. Essential Oil Compositions

The chemical compositions of T. vulgaris essential oils from this study are listed in Table 1. T. vulgaris sample #1, a linalool chemotype collected from Nyons, France, was dominated by linalool (76.15%) and linalyl acetate (14.26%). The enantiomeric ratios of these two compounds were 1:99 d/l. Other compounds in the oil were β-caryophyllene (2.27%), camphor (1.79%, 100% d enantiomer), and camphene (1.17%, d/l ratio = 99:1). T. vulgaris #3 was a geraniol chemotype collected from Jablanicki, Serbia, and was rich in geraniol (59.75%) and esters of geraniol, geranyl acetate (16.72%) and geranyl propionate (1.26%). Oil #3 also had sizable concentrations of linalool (7.15%, d/l ratio = 97:3) and β-caryophyllene (3.67%). T. vulgaris #4 was collected from Pomoravje District, Serbia, and had sabinene hydrates as defining compounds with cis-sabinene hydrate (30.77%, d/l ratio = 97:3) and trans-sabinene hydrate (4.98%). Other major components in oil #4 were terpinene-4-ol (9.50%, d/l = 30:70), linalool (7.89%, d/l = 3:97), γ-terpinene (4.58%), and myrcene (4.09%). T. vulgaris #5, representing the thymol chemotype, was collected from Richerenches, France. The composition of oil #5 was rich in thymol (47.06%) and p-cymene (20.07%) with lesser quantities of linalool (5.00%, d/l = 1:99) and carvacrol (3.24%). Interestingly, the l-enantiomer dominated linalool in samples #1, #4, and #5, but was reversed in sample #3. Özek and co-workers have found l-linalool to be the predominant enantiomer in several Thymus species, but apparently they did not examine T. vulgaris [17]. Similarly, d-terpinen-4-ol was the major enantiomer in sample #3, but a minor one in samples #4 and #5. An enantiomeric composition analysis of Melaleuca alternifolia revealed this essential oil to be composed of mostly d-cis-sabinene hydrate and d-terpinen-4-ol [18]. d-α-Thujene was the dominant enantiomer in samples #3 and #4, while l-α-thujene predominated in sample #5. The l-enantiomer of α-thujene was found to be predominant in various Citrus essential oils [19]. l-α-Pinene dominated samples #1 and #3, but d-α-pinene was the major enantiomer in #4. Sample #5 showed a nearly racemic mixture of α-pinenes. l-Camphene dominated samples #1 and #3, but d-camphene dominated #4 and #5. Both d-α-pinene and d-camphene were found to be the predominant enantiomers in Ocimum canum and O. kilimandscharicum essential oils [20]. Remarkably, both d-camphor and d-borneol were the only enantiomers in sample #1, and d-borneol was also the only enantiomer found in samples #3 and #4. However, only l-camphor was found in essential oils #3 and #4. This is in contrast to the results of Tabanca and co-workers, who had exclusively observed the l-enantiomers of borneol along with 99% l-camphor in Micromeria cristata subsp. phrygia [21]. Thus, not only are the chemical profiles different between the different chemotypes, but the enantiomeric distributions are also profoundly different.

3.2. Chemotypes of Thymus vulgaris

A total of 85 T. vulgaris essential oil compositions were used to carry out a hierarchical cluster analysis (Figure 1). The cluster analysis revealed as many as 20 different chemotypes. The chemotype with the most samples (39) was the thymol chemotype, which had been recognized previously [12,13,14,15]. Other previously recognized chemotypes were the geraniol chemotype, with three representative samples (including sample #3 from Serbia in this study); the linalool chemotype, with four samples (including sample #1 from France in this study); the carvacrol chemotype, with two samples; the borneol chemotype, only one sample; the sabinene hydrate/terpinene-4-ol chemotype, with two samples (including sample #4 from Serbia in this study); and the cyclocitral/verbenol chemotype, with only one sample. The second most populated chemotype in the cluster analysis was a p-cymene/thymol type represented by 18 samples. The α-terpineol chemotype [12] was not found in the present study; only three T. vulgaris samples (all from Austria) showed α-terpineol concentrations around 10%, but all three had higher concentrations of other components [14]. Other chemotypes identified include carvacrol/γ-terpinene/thymol, which was previously described as carvacrol and thymol/carvacrol [14]; linalool/carvacrol/p-cymene, previously labeled carvacrol/linalool [14]; sabinene hydrate/geraniol/geranyl acetate/α-terpineol, previously described as geraniol/α-terpineol/sabinene hydrate [14]; sabinene hydrate/α-terpinyl acetate/thymol, previously described as α-terpineol/sabinene hydrate/thymol [14]; sabinene hydrate/linalool [14]; α-terpinyl acetate/carvacrol, previously described as α-terpineol/carvacrol [14]; carvacrol/α-terpineol/borneol; sabinene hydrate/ terpinen-4-ol, which includes one of the samples from France in this study; geranyl formate/geraniol, previously called geraniol [13]; p-cymene/thymol/carvacrol; terpinene-4-ol/p-cymene; camphor/camphene; and 1,8-cineole/α-terpinyl acetate.

3.3. 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].

5. 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.


We thank Julien Abisset for plant identification, Joshua Thomerson for assistance with steam distillation of the essential oils, and Erin McClelland (Middle Tennessee State University) for C. neoformans cells. B.L.M. was supported by a GEM University Fellowship.

Author Contributions

P.S. conceived and designed the experiments; P.S. carried out the GC-MS analysis; B.L.M. did the antifungal assays; W.N.S. performed the cluster analysis; R.L.M. and W.N.S. analyzed the data; all authors contributed to writing the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.


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Figure 1. Dendrogram obtained from the agglomerative hierarchical cluster analysis of 85 Thymus vulgaris essential oil compositions.
Figure 1. Dendrogram obtained from the agglomerative hierarchical cluster analysis of 85 Thymus vulgaris essential oil compositions.
Foods 05 00070 g001
Table 1. Chemical compositions and enantiomeric distributions of Thymus vulgaris essential oils.
Table 1. Chemical compositions and enantiomeric distributions of Thymus vulgaris essential oils.
783Methyl α-methyl butyrate0.10 0.07 tr tr
850(3Z)-Hexenol 0.05
922Tricyclene0.05 tr 0.09
924α-Thujenetr tr91:90.7187:130.4310:90
947α-Fenchene tr
948Camphene1.1799:1 0.38 99:10.250:1001.1910:90
971Sabinenetr 0.0598:22.0378:22
9771-Octen-3-ol0.43 0.42
9771-Octen-3-ol + β-Pinene 0.9720:800.5420:80
9836-Methylhept-5-en-2-one 0.05
9833-Octanone tr tr
988Myrcene0.09 0.44 4.09 1.59
9963-Octanoltr 0.07 tr
1004(3Z)-Hexenyl acetatetr
1004p-Mentha-1(7),8-diene tr
1006α-Phellandrene tr55:450.11
1008δ-3-Carene tr
1016α-Terpinene tr 2.65 1.30
1019o-Cymene 0.06
1024p-Cymene 0.09 0.18 1.09 20.07
1028Limonene0.05 0.3985:152.8586:140.3980:20
1030β-Phellandrene tr 0.37 0.09
10311,8-Cineole0.37 0.31 0.30 0.72
1033m-Cymene tr
1034Lavender lactone + (Z)-β-Ocimenetr
1045(E)-β-Ocimenetr 0.05
1057γ-Terpinene 0.09 4.58 9.03
10633-Methylbut-2-enyl butanoatetr
1069cis-Sabinene hydrate 0.31 30.7797:30.17
1070cis-Linalool oxide (furanoid)0.28
1071Pinol tr
1084Terpinolene 0.97 0.07
1085trans-Linalool oxide (furanoid)0.23 tr
1089p-Cymenene tr
1101trans-Sabinene hydrate tr 4.98 tr
1104Nonanal0.05 tr
1106α-Pinene oxide tr
11071-Octen-3-yl acetatetr tr
1124cis-p-Menth-2-en-1-ol 0.65
1136trans-Limonene oxide tr
1142trans-p-Menth-2-en-1ol 0.25
1148α-Cyclogeraniol tr
1154β-Pinene oxide 0.05
1162Lavanduloltr tr tr
1169cis-Linalool oxide (pyranoid)tr
1174trans-Linalool oxide (pyranoid) tr
1178p-Mentha-1,8-dien-4-ol tr
1180Terpinen-4-ol0.06 0.1770:309.5030:701.2540:60
1184(3Z)-Hexenyl butanoatetr
1186p-Cymen-8-ol tr
1196cis-Piperitol 0.14
1197cis-Dihydrocarvonetr tr tr
1205β-Cyclogeraniol tr
1208trans-Piperitol 0.16
12177-Ethylidenebicyclo[3.3.0]octan-3-one 0.90
12206,7-Epoxyneral tr
1223Neroltr 0.97
12237-Methylenebicyclo[3.3.1]nonan-3-ol 6.07
1225Citronelloltr 0.35
1228Thymol methyl ether 0.19
12306,7-Epoxygeranial tr
12344-t-Amylcyclohexanone 0.08
1237Neraltr 0.61 tr
1237Carvacrol methyl ether 0.32
1243Carvone tr 0.09
1249Linalyl acetate14.261:99 3.400:100
1249Geraniol 59.75 0.24 0.05
1266Geranialtr 1.25 tr tr
1279Isothymol 0.07
1282Lavandulyl acetatetr
1284Bornyl acetate0.21 0.14 0.07
1284neo-iso-3-Thujanol acetate tr
1288Thymol 0.42 tr 47.06
1296Carvacrol0.05 3.24
1296Geranyl formate 0.09
13312,3-Epoxygeraniol 0.06
1341cis-p-Menthadienyl acetate 0.19 4.75
1344Citronellyl acetate 0.28
1346α-Terpinyl acetate 0.05
1348Citronellyl acetate 0.07
1349Eugenol 0.08
1357Neryl acetatetr 0.18 tr
1372trans-p-Menthadienyl acetate 0.05
1375α-Copaene tr
1377Geranyl acetate0.06 16.72 0.48
1383α-Bourbonene 0.20 tr 0.09
1403Isocaryophyllenetr tr
1419β-Caryophyllene2.27 3.67 2.03 1.79
1429cis-Carvyl propanoate 0.30
14347-Methyl-3-methylene-7-octen-1-yl propanoate tr
1437Aromadendrene tr
1454α-Humulene0.06 0.12 0.06 0.05
1468Geranyl propanoatetr 1.26
1473trans-Cadina-1(6),4-diene 0.06
1480Germacrene D0.26 0.05 0.58
1489Viridiflorene tr
1494Bicyclogermacrene tr 0.09
1497α-Muurolene tr
1506Geranyl isobutyrate 0.15
1506β-Bisabolene tr
1511δ-Amorphene 0.07
1516δ-Cadinene tr 0.16
1531trans-Cadina-1,4-diene tr
1547Elemol 0.96
1554Geranyl butanoate0.05 0.74
1575Germacrene-d-4-ol 0.10 tr
1575Spathulenol tr
1581Caryophyllene oxide0.41 0.50 0.10 0.13
1595Geranyl isovalerate 0.06
163110-epi-γ-Eudesmol tr
1654α-Eudesmol 0.17
Table 2. Antifungal activities (minimum inhibitory concentrations, MIC) of Thymus vulgaris essential oils.
Table 2. Antifungal activities (minimum inhibitory concentrations, MIC) of Thymus vulgaris essential oils.
Essential OilMIC (μg/mL)
C. albicansC. neoformans
#1 (linalool chemotype)1250313
#3 (geraniol chemotype)625156
#4 (sabinene hydrate chemotype)>2500>2500

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Satyal, P.; Murray, B.L.; McFeeters, R.L.; Setzer, W.N. Essential Oil Characterization of Thymus vulgaris from Various Geographical Locations. Foods 2016, 5, 70.

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Satyal P, Murray BL, McFeeters RL, Setzer WN. Essential Oil Characterization of Thymus vulgaris from Various Geographical Locations. Foods. 2016; 5(4):70.

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Satyal, Prabodh, Brittney L. Murray, Robert L. McFeeters, and William N. Setzer. 2016. "Essential Oil Characterization of Thymus vulgaris from Various Geographical Locations" Foods 5, no. 4: 70.

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