Journal of Medicine and Life Volume 7, Special Issue 3, 2014 

Thymus vulgaris essential oil: chemical composition  

and antimicrobial activity 

 

Borugă O*, Jianu C**, Mişcă C**, Goleţ I***, Gruia AT****, Horhat FG***** 

*Department of Ophthalmology, Victor Babeș University of Medicine and Pharmacy, Timișoara, Romania,  

**Department of Food Science, Faculty of Food Processing Technology, Banat’s University of Agricultural Sciences  

and Veterinary Medicine, Timișoara, Romania 

***Department of Management, Faculty of Economics and Business Administration,  

West University of Timișoara, Timișoara, Romania 

****Center for Transplant Immunology, Timișoara County Hospital, Timișoara, Romania 

*****Department of Microbiology, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania  

 
Correspondence to: Călin Jianu, MD, 
Department of Food Science, Faculty of Food Processing Technology,  
Banat's University of Agricultural Sciences and Veterinary Medicine 
119 Calea Aradului, RO-300645, Timisoara, Romania 
Mobile phone: 0040722632199, E-mail: calin.jianu@gmail.com 
 
Abstract 
The study was designed to determine the chemical composition and antimicrobial properties of the essential oil of Thymus vulgaris 
cultivated in Romania. The essential oil was isolated in a yield of 1.25% by steam distillation from the aerial part of the plant and 
subsequently analyzed by GC-MS. The major components were p-cymene (8.41%), γ-terpinene (30.90%) and thymol (47.59%). Its 
antimicrobial activity was evaluated on 7 common food-related bacteria and fungus by using the disk diffusion method. The results 
demonstrate that the Thymus vulgaris essential oil tested possesses strong antimicrobial properties, and may in the future represent 
a new source of natural antiseptics with applications in the pharmaceutical and food industry. 
 

Keywords: thyme, essential oil, GC-MS analysis, antimicrobial activity 

 
Introduction 

The  genus  Thymus,  member  of  the  Lamiaceae  family,  contains  about  400  species  of  perennial  aromatic, 
evergreen  or  semi-evergreen  herbaceous  plants  with  many  subspecies,  varieties,  subvarieties  and  forms  [1].  In 
Romania, the Thymus genus contains one species cultivated as aromatic plant (Thymus vulgaris) and other 18 wild 
species  [2].  T.  vulgaris  (thyme),  locally  known  as  “cimbru”,  is  widely  used  in  the  Romanian  folk  medicine  for  its 
expectorant, antitussive, antibroncholitic, antispasmodic, anthelmintic, carminative and diuretic properties. 

Various studies have aimed to investigate the chemical composition and biological properties of the T. vulgaris 
essential  oil  (EO).  According  to  European  Pharmacopoeia  5.0  (Ph.  Eur.  5.0)  [3],  the  minimum  content  of  EO  in  T. 
vulgaris is 12 mL/kg, but the chemical composition shows variations, six chemotypes being mainly reported, namely 
geraniol, linalool,  gamma-terpineol, carvacrol, thymol  and trans-thujan-4-ol/terpinen-4-ol [4,5]. Both the isolation  yield 
and the chemical composition of the EOs are dependent on a number of factors, such as the environment, growth region 
and cultivation practices [6]. In addition to the flavoring properties determined by the constitutive active ingredients, the 
thyme EO exhibits significant antimicrobial activity [4,7-9] as well as strong antioxidant properties [2,8]. 

The aim of this study is to determine the chemical composition together with the antimicrobial properties of the 
EO of T. vulgaris cultivated in Romania, in order to identify new sources of natural antiseptics with applications in the 
pharmaceutical and food industry. 
 
Materials and methods 
Raw  material.  Thyme  was  harvested  during  the  flowering  season  (July  2012)  from  the  area  around  the  Broşteni 
commune – Mehedinţi County, Romania. The plant material was dried in well-ventilated areas, sheltered from direct 
sunlight and then stored in double-layered paper bags at temperatures of 3-5°C until processing. A voucher specimen 
(V.FPT-451) was deposited in the Herbarium of the Faculty of Pharmacy, “Victor Babeș” University of Medicine and 
Pharmacy, Timișoara, Romania. 
Isolation of essential oils. The EO was obtained by hydrodistillation, according to Ph. Eur. 5.0 [3], by using a modified 
Clevenger apparatus (with the EO collection area cooled to prevent the emergence of artifacts). The EO was dried on 
anhydrous sodium sulfate (Sigma-Aldrich Chemie GmbH) and stored in a tightly sealed brown glass bottle at 0-4°C for 
testing. 

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Journal of Medicine and Life Volume 7, Special Issue 3, 2014 

Gas  chromatography-mass  spectrometry.  Samples  were  analyzed  by  gas  chromatography  using  a  HP6890 
instrument coupled with a HP 5973 mass spectrometer. The gas chromatograph is equipped with a split-splitless injector 
and a Factor FourTM VF-35ms 5% fenil-methylpolysiloxane, 30 m, 0.25 mm, 0.25 μm film thickness capillary column. Gas 
chromatography conditions include a temperature range of 50 to 250°C at 40°C/min, with a solvent delay of 5 min. The 
injector was maintained at a temperature of 250°C. The inert gas was helium at a flow of 1.0 mL/min, and the injected 
volume in the splitless mode was 1 μL. The MS conditions were the following: ionization energy, 70 eV; quadrupole 
temperature, 100°C; scanning velocity, 1.6 scan/s; weight range, 40-500 amu. 

The percent composition of the volatile compounds was calculated. The qualitative analysis was based on the 
percent area of each peak of the sample compounds. The mass spectrum of each compound was compared with the 
mass spectrum from the NIST 98 spectrum library (USA National Institute of Science and Technology software). 
Determination  of  antimicrobial  activity.  Thyme  EO  was  tested  on  7  common  food-related  bacteria  and  fungus: 
Staphylococcus  aureus  (ATCC  25923),  Pseudomonas  aeruginosa  (ATCC  27853),  Salmonella  typhimurium  (ATCC 
14028), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13882), Enterococcus faecalis (ATCC 29212) 
and Candida albicans (ATCC 10231), using the disk diffusion method as previously described [10]. Briefly, a suspension 
of the tested microorganism (106 cells/mL-1) was spread on the solid media plates (Mueller-Hinton agar for bacteria and 
Sabouraud  cloramphenicol  agar  for  fungi).  The  paper  discs  (Whatman  No  1  filter  paper  -  6  mm  diameter)  were 
impregnated with 5, 10, 15 and 20µL EO and placed on the inoculated agar. The plates inoculated with bacterial strains 
were incubated for 24 h at 37°C and 48 h at 30°C for fungi, respectively. As positive controls, ciprofloxacin (30 µg/disk) 
and cephalexin (10 µg/disk) were used for bacterial strains and fluconazole (10 µg/disk) for fungi. After incubation, the 
diameter of the zone of inhibition was measured in millimeters. Each test was performed in triplicate on at least three 
separate experiments. 
Statistical analysis. The statistical analysis was performed by using SPSS Version 21 (IBM Corp., NY). The mean 
inhibition zone for each group of nine observations was compared with the value of the disc diameter (6 mm) using the t-
test. The GLM procedure was used to conduct a two-way analysis of variance (ANOVA) on the inhibition zones. The 
type of microorganism and amount of essential oil were used as factors in the full factorial model. Post-hoc tests for each 
amount of essential oil were conducted by using Tukey’s HSD method, in order to compare the effect on different types 
of microorganisms. 
 
Results and Discussion 

The isolation yield was 1.25% (v/w), based on dry plant material and confirmed that the plant analyzed meets 
the requirements of pharmaceutical quality for thyme as EO source [3]. The chemical composition determined by GC/ 
MS is presented in Table 1. Fifteen components representing 99.91% of the total detected constituents were identified. 
The major components were p-cymene (8.41%), γ-terpinene (30.90%) and thymol (47.59%), which suggests that the EO 
analyzed  belongs  to  the  thymol  chemotype  in  agreement  with  those  previously  reported  in  Romania  [2].  The  other 
components were present in a total amount of less than 13.01%. The chemical composition of the EO analyzed by us is 
very different from that previously reported in Morocco and Spain for the same species of thyme [11,12]. Similar studies 
in Poland, Iran, Spain and Italy, respectively, reported as major compounds in the T. vulgaris EO p-cymene, γ-terpinene 
and thymol [4,13-15]. These differences can be attributed to a large extent to the different chemotypes mentioned above 
[4,5,13]. 

The antimicrobial activity of thyme oil against 7 common food-related bacteria and fungus tested is presented in 
Table  2.  The  null  hypothesis  that  the  inhibition  zone  is  equal  to  the  disc  diameter  (6  mm)  was  rejected  for  each 
microorganism at every amount of essential oil with a high significance level (p = 0.00). The main finding of the ANOVA 
analysis is a strong interaction effect between the type of microorganism and the amount of essential oil (p = 0.00). The 
highly significant interaction effect adds difficulty in drawing general conclusions on the main effects, even if the two 
factors are also highly significant (p = 0.00). For example, K. pneumoniae has the highest inhibition zone overall but for 
the amount of 20 [μL], where E. coli and S. typhimurium have higher values. In order to compare more thoroughly the 
effect of T. vulgaris on each microorganism (Fig. 1), the results of multiple comparisons, at each oil amount, has to be 
considered. Tukey’s HSD test reveals that the only microorganisms with non-significant differences in the antimicrobial 
effect are S. typhimurium and E. coli at all oil amounts, and S. typhimurium, E. coli and C. albicans at 10 [μL]. The 
observed  p-value  for  the  pairwise  differences  in  the  above-mentioned  cases  does  not  pass  acceptable  significance 
levels, being larger than 0.4. All the other pairwise differences are highly significant (p = 0.00).  
 

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Journal of Medicine and Life Volume 7, Special Issue 3, 2014 

Table 1. Chemical composition of thyme EO 

RT (min) 
5.39 
5.63 
6.89 
6.97 
7.53 
7.77 
8.04 
8.26 
8.46 
8.96 
9.48 
12.55 
16.17 
17.32 
19.03 

No. 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
Total 
*Constituents presented in the order of elution from the VF 35 MS column. 
 

Area % of total 
1.06 
1.07 
0.37 
1.53 
0.33 
3.76 
0.29 
0.21 
8.41 
30.90 
0.47 
0.46 
47.59 
2.68 
0.78 
99.91% 

Constituents* 
alpha-Thujene 
alpha-Pinene 
beta-Pinene 
beta-Myrcene 
alpha-Phellandrene 
Carene<δ-2-> 
D-Limonene 
beta-Phellandrene 
para-Cymene 
gamma-Terpinene 
Terpineol 
Terpinen-4-ol 
Thymol 
Caryophyllene 
Cyclohexene, 1-methyl-4-(5-methyl-1-methylene-4-hexenyl) 
 

The inhibition of the growth of E. coli, K. pneumoniae, S. aureus, P. aeruginosa and E. faecalis was previously 
reported [4,7,9] along with the efficacy against C. albicans [9,16,17] and S. typhimurium [4,9], respectively. In contrast, 
some studies report the inefficiency of thyme EO against E. coli [16,17], S. aureus [16] and K. pneumoniae [16]. 
 

Fig. 1 The antimicrobial activity of thyme oil, at different amounts, expressed as a mean inhibition  

zone for each of the nine repeated measurements 

 

 

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Table 2. Effects of thyme oil against bacteria expressed by the mean sizes of the inhibitory zones 

Journal of Medicine and Life Volume 7, Special Issue 3, 2014 

Test microorganism 
Staphylococcus aureus 
ATCC 25923 
Salmonella typhimurium 
ATCC 14028 
Pseudomonas aeruginosa 
ATCC27853 
E. coli 
ATCC 25922 
Klebsiella pneumoniae 
ATCC 13882 
Enterococcus faecalis 
ATCC 29212 
Candida albicans 
ATCC 10231 
 

Amount of essential oil [μL] 
5 
23.93 ± 0.33 

10 
29.2 ± 0.6 

15 
29.9 ± 0.35 

20 
31.4 ± 0.47 

14.49 ± 0.34 

19.71 ± 0.39 

30.68 ± 0.33 

34.94 ± 0.22 

11.82 ± 0.27 

13.34 ± 0.33 

14 ± 0.22 

14.13 ± 0.19 

14.63 ± 0.36 

19.82 ± 0.41 

30.67 ± 0.31 

34.99 ± 0.19 

30.21 ± 0.12 

31.02 ± 0.31 

32.79 ± 0.24 

33.93 ± 0.14 

8.99 ± 0.15 

15.06 ± 0.15 

15.99 ± 0.18 

24.06 ± 0.15 

15.14 ± 0.38 

19.43 ± 0.55 

25.74 ± 0.24 

30.2 ± 0.17 

The inhibitions are expressed in mm and include the diameter of the paper disc (6 mm). Data distributions were 
expressed as mean values and standard deviations (SD) (n = 9). Ciprofloxacine and cephalexine (for bacterial strains) 
and fluconazole (for fungi), respectively, were used as positive controls. 

The antimicrobial activity of EOs depends on their chemical constituents. Apparently, the antimicrobial activity 
of the EO analyzed is related to the presence of phenolic compounds (thymol) and terpene hydrocarbons (γ-terpinene), 
respectively [4,7,18]. p-Cymene, the third major element according to percentage, does not show antibacterial efficacy 
when used alone [7], synergistic effects being however attributed to it in relation to thymol and γ-terpinene, respectively 
[19,20], which might represent another cause of the antimicrobial activity recorded. On the other hand, a number of 
studies have shown that EOS exhibit stronger antimicrobial activity than that of their major constituents or their mixtures, 
respectively  [21,22],  which  suggests  synergistic  effects  of  the  minor  components,  but  also  the  importance  of  all 
components in relation to the biological activity of EOs. 
 
Conclusions 

The results demonstrate the effectiveness of thyme EO against the food-related bacteria and fungus tested. 
The  synergism,  antagonism  and  additive  effects,  respectively,  of  the  EOs  components  require  further  research  to 
elucidate  the  mechanisms  underlying  their  biological  activity,  for  the  purpose  of  accessing  new  natural  antiseptics 
applicable in the pharmaceutical and food industry. 
 
References 
1.  De Martino L, Bruno M, Formisano C, De Feo V, Napolitano F, Rosselli S, Senatore F. Chemical Composition and Antimicrobial Activity 

of the Essential Oils from Two Species of Thymus Growing Wild in Southern Italy. Molecules. 2009; 14(11): 4614-4624. 

2.  Grigore A, Paraschiv I, Colceru-Mihul S, Bubueanu C, Draghici E, Ichim M. Chemical composition and antioxidant activity of Thymus 

vulgaris L. volatile oil obtained by two different methods. Romanian Biotechnological Letters. 2010; 15(4): 5436-5443. 

3.  European pharmacopoeia. 5 ed., 2004, Strasbourg: Council of Europe, E.P.C., 217-218. 
4.  Rota MC, Herrera A, Martínez RM, Sotomayor JA, Jordán MJ. Antimicrobial activity and chemical composition of Thymus vulgaris, Thymus 

zygis and Thymus hyemalis essential oils. Food Control. 2008; 19(7): 681-687. 
Thompson JD, Chalchat JC, Michet A, Linhart YB, Ehlers B. Qualitative and quantitative variation in monoterpene co-occurrence and 
composition in the essential oil of Thymus vulgaris chemotypes. Journal of Chemical Ecology. 2003; 29(4): 859-880. 

6.  Hudaib M, Aburjai T. Volatile components of Thymus vulgaris L. from wild-growing and cultivated plants in Jordan. Flavour and Fragrance 

5. 

7.  Dorman HJD, Deans SG. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology. 2000; 

8.  Sacchetti G, Maietti S, Muzzoli M, Scaglianti M, Manfredini S, Radice M, Bruni R. Comparative evaluation of 11 essential oils of different 

origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chemistry. 2005; 91(4): 621-632. 

9.  Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology. 1999; 

Journal. 2007; 22(4): 322-327. 

88(2): 308-316. 

86(6): 985-990. 

10.  Jianu C, Misca C, Pop G, Rusu LC, Ardelean L, Gruia AT. Chemical Composition and Antimicrobial Activity of Essential Oils Obtained from 

Dill (Anethum graveolens L.) Grown in Western Romania. Revista de Chimie (Bucharest). 2012; 63(6): 641-645. 
Imelouane B, Amhamdi H, Wathelet JP, Ankit M, Khedid K, El Bachiri A. Chemical Composition and Antimicrobial Activity of Essential Oil 
of Thyme (Thymus vulgaris) from Eastern Morocco. International Journal of Agriculture and Biology. 2009; 11(2): 205-208. 

12.  Ballester-Costa C, Sendra E, Fernández-López J, Pérez-Álvarez JA, Viuda-Martos M. Chemical composition and in vitro antibacterial 

11. 

properties of essential oils of four Thymus species from organic growth. Industrial Crops and Products. 2013; 50: 304-311. 

59 

Journal of Medicine and Life Volume 7, Special Issue 3, 2014 

13.  De Lisi A, Tedone L, Montesano V, Sarli G, Negro D. Chemical characterisation of Thymus populations belonging from Southern Italy. 

Food Chemistry. 2011; 125(4): 1284-1286. 

14.  Pirbalouti AG, Hashemi M, Ghahfarokhi FT. Essential oil and chemical compositions of wild and cultivated Thymus daenensis Celak and 

Thymus vulgaris L. Industrial Crops and Products. 2013; 48: 43-48. 

15.  Kowalski R, Wawrzykowski J. Essential oils analysis in dried materials and granulates obtained from Thymus vulgaris L., Salvia officinalis 

L., Mentha piperita L. and Chamomilla recutita L. Flavour and Fragrance Journal. 2009; 24(1): 31-35. 

16.  Nascimento  GGF,  Locatelli  J,  Freitas  PC,  Silva  GL.  Antibacterial  activity  of  plant  extracts  and  phytochemicals  on  antibiotic-resistant 

bacteria. Brazilian Journal of Microbiology. 2000; 31(4): 247-256. 

17.  Sartoratto A, Machado ALM, Delarmelina C, Figueira GM, Duarte MCT, Rehder VLG. Composition and antimicrobial activity of essential 

oils from aromatic plants used in Brazil. Brazilian Journal of Microbiology. 2004; 35(4): 275-280. 

18.  Skočibušić M, Bezić N, Dunkić V. Phytochemical composition and antimicrobial activities of the essential oils from Vis. growing in Croatia. 

Food Chemistry. 2006; 96(1): 20-28. 

19.  Delgado B, Fernández PS, Palop A, Periago PM. Effect of thymol and cymene on Bacillus cereus vegetative cells evaluated through the 

use of frequency distributions. Food Microbiol. 2004; 21(3): 327-334. 

20.  Gallucci MN, Oliva M, Casero C, Dambolena J, Luna A, Zygadlo J, Demo M. Antimicrobial combined action of terpenes against the food-

borne microorganisms Escherichia coli, Staphylococcus aureus and Bacillus cereus. Flavour and Fragrance Journal. 2009; 24(6): 348-354. 

21.  Gill AO, Delaquis P, Russo P, Holley RA. Evaluation of antilisterial action of cilantro oil on vacuum packed ham. International Journal of 

Food Microbiology. 2002; 73(1): 83-92. 

22.  Mourey A, Canillac N. Anti-Listeria monocytogenes activity of essential oils components of conifers. Food Control. 2002; 13(4-5): 289-292. 

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