[['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.'], ['56'], ['\x0cJournal 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).'], [], ['57'], ['\x0cJournal 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'], [], [], [], [], ['58'], ['\x0cTable 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. 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