["Journal of Medicine and Life Volume 7, Special Issue 3, 2014 \n\nThymus vulgaris essential oil: chemical composition  \n\nand antimicrobial activity \n\n \n\nBorugă O*, Jianu C**, Mişcă C**, Goleţ I***, Gruia AT****, Horhat FG***** \n\n*Department of Ophthalmology, Victor Babeș University of Medicine and Pharmacy, Timișoara, Romania,  \n\n**Department of Food Science, Faculty of Food Processing Technology, Banat’s University of Agricultural Sciences  \n\nand Veterinary Medicine, Timișoara, Romania \n\n***Department of Management, Faculty of Economics and Business Administration,  \n\nWest University of Timișoara, Timișoara, Romania \n\n****Center for Transplant Immunology, Timișoara County Hospital, Timișoara, Romania \n\n*****Department of Microbiology, Victor Babes University of Medicine and Pharmacy, Timisoara, Romania  \n\n \nCorrespondence to: Călin Jianu, MD, \nDepartment of Food Science, Faculty of Food Processing Technology,  \nBanat's University of Agricultural Sciences and Veterinary Medicine \n119 Calea Aradului, RO-300645, Timisoara, Romania \nMobile phone: 0040722632199, E-mail: calin.jianu@gmail.com \n \nAbstract \nThe study was designed to determine the chemical composition and antimicrobial properties of the essential oil of Thymus vulgaris \ncultivated in Romania.", 'The essential oil was isolated in a yield of 1.25% by steam distillation from the aerial part of the plant and \nsubsequently analyzed by GC-MS.', 'The major components were p-cymene (8.41%), γ-terpinene (30.90%) and thymol (47.59%).', 'Its \nantimicrobial activity was evaluated on 7 common food-related bacteria and fungus by using the disk diffusion method.', 'The results \ndemonstrate that the Thymus vulgaris essential oil tested possesses strong antimicrobial properties, and may in the future represent \na new source of natural antiseptics with applications in the pharmaceutical and food industry.', 'Keywords: thyme, essential oil, GC-MS analysis, antimicrobial activity \n\n \nIntroduction \n\nThe  genus  Thymus,  member  of  the  Lamiaceae  family,  contains  about  400  species  of  perennial  aromatic, \nevergreen  or  semi-evergreen  herbaceous  plants  with  many  subspecies,  varieties,  subvarieties  and  forms  [1].', 'In \nRomania, the Thymus genus contains one species cultivated as aromatic plant (Thymus vulgaris) and other 18 wild \nspecies  [2].', 'T.  vulgaris  (thyme),  locally  known  as  “cimbru”,  is  widely  used  in  the  Romanian  folk  medicine  for  its \nexpectorant, antitussive, antibroncholitic, antispasmodic, anthelmintic, carminative and diuretic properties.', 'Various studies have aimed to investigate the chemical composition and biological properties of the T. vulgaris \nessential  oil  (EO).', 'According  to  European  Pharmacopoeia  5.0  (Ph.', 'Eur.', '5.0)  [3],  the  minimum  content  of  EO  in  T. \nvulgaris is 12 mL/kg, but the chemical composition shows variations, six chemotypes being mainly reported, namely \ngeraniol, linalool,  gamma-terpineol, carvacrol, thymol  and trans-thujan-4-ol/terpinen-4-ol [4,5].', 'Both the isolation  yield \nand the chemical composition of the EOs are dependent on a number of factors, such as the environment, growth region \nand cultivation practices [6].', 'In addition to the flavoring properties determined by the constitutive active ingredients, the \nthyme 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 \nEO of T. vulgaris cultivated in Romania, in order to identify new sources of natural antiseptics with applications in the \npharmaceutical and food industry.', 'Materials and methods \nRaw  material.', 'Thyme  was  harvested  during  the  flowering  season  (July  2012)  from  the  area  around  the  Broşteni \ncommune – Mehedinţi County, Romania.', 'The plant material was dried in well-ventilated areas, sheltered from direct \nsunlight and then stored in double-layered paper bags at temperatures of 3-5°C until processing.', 'A voucher specimen \n(V.FPT-451) was deposited in the Herbarium of the Faculty of Pharmacy, “Victor Babeș” University of Medicine and \nPharmacy, Timișoara, Romania.', 'Isolation of essential oils.', 'The EO was obtained by hydrodistillation, according to Ph.', 'Eur.', '5.0 [3], by using a modified \nClevenger apparatus (with the EO collection area cooled to prevent the emergence of artifacts).', 'The EO was dried on \nanhydrous sodium sulfate (Sigma-Aldrich Chemie GmbH) and stored in a tightly sealed brown glass bottle at 0-4°C for \ntesting.', '56 \n\n\x0cJournal of Medicine and Life Volume 7, Special Issue 3, 2014 \n\nGas  chromatography-mass  spectrometry.', 'Samples  were  analyzed  by  gas  chromatography  using  a  HP6890 \ninstrument coupled with a HP 5973 mass spectrometer.', 'The gas chromatograph is equipped with a split-splitless injector \nand a Factor FourTM VF-35ms 5% fenil-methylpolysiloxane, 30 m, 0.25 mm, 0.25 μm film thickness capillary column.', 'Gas \nchromatography conditions include a temperature range of 50 to 250°C at 40°C/min, with a solvent delay of 5 min.', 'The \ninjector was maintained at a temperature of 250°C.', 'The inert gas was helium at a flow of 1.0 mL/min, and the injected \nvolume in the splitless mode was 1 μL.', 'The MS conditions were the following: ionization energy, 70 eV; quadrupole \ntemperature, 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 \npercent area of each peak of the sample compounds.', 'The mass spectrum of each compound was compared with the \nmass 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: \nStaphylococcus  aureus  (ATCC  25923),  Pseudomonas  aeruginosa  (ATCC  27853),  Salmonella  typhimurium  (ATCC \n14028), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13882), Enterococcus faecalis (ATCC 29212) \nand Candida albicans (ATCC 10231), using the disk diffusion method as previously described [10].', 'Briefly, a suspension \nof the tested microorganism (106 cells/mL-1) was spread on the solid media plates (Mueller-Hinton agar for bacteria and \nSabouraud  cloramphenicol  agar  for  fungi).', 'The  paper  discs  (Whatman  No  1  filter  paper  -  6  mm  diameter)  were \nimpregnated with 5, 10, 15 and 20µL EO and placed on the inoculated agar.', 'The plates inoculated with bacterial strains \nwere incubated for 24 h at 37°C and 48 h at 30°C for fungi, respectively.', 'As positive controls, ciprofloxacin (30 µg/disk) \nand cephalexin (10 µg/disk) were used for bacterial strains and fluconazole (10 µg/disk) for fungi.', 'After incubation, the \ndiameter of the zone of inhibition was measured in millimeters.', 'Each test was performed in triplicate on at least three \nseparate experiments.', 'Statistical analysis.', 'The statistical analysis was performed by using SPSS Version 21 (IBM Corp., NY).', 'The mean \ninhibition zone for each group of nine observations was compared with the value of the disc diameter (6 mm) using the t-\ntest.', 'The GLM procedure was used to conduct a two-way analysis of variance (ANOVA) on the inhibition zones.', 'The \ntype of microorganism and amount of essential oil were used as factors in the full factorial model.', 'Post-hoc tests for each \namount of essential oil were conducted by using Tukey’s HSD method, in order to compare the effect on different types \nof microorganisms.', 'Results and Discussion \n\nThe isolation yield was 1.25% (v/w), based on dry plant material and confirmed that the plant analyzed meets \nthe requirements of pharmaceutical quality for thyme as EO source [3].', 'The chemical composition determined by GC/ \nMS 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 \nanalyzed  belongs  to  the  thymol  chemotype  in  agreement  with  those  previously  reported  in  Romania  [2].', 'The  other \ncomponents were present in a total amount of less than 13.01%.', 'The chemical composition of the EO analyzed by us is \nvery different from that previously reported in Morocco and Spain for the same species of thyme [11,12].', 'Similar studies \nin Poland, Iran, Spain and Italy, respectively, reported as major compounds in the T. vulgaris EO p-cymene, γ-terpinene \nand thymol [4,13-15].', 'These differences can be attributed to a large extent to the different chemotypes mentioned above \n[4,5,13].', 'The antimicrobial activity of thyme oil against 7 common food-related bacteria and fungus tested is presented in \nTable  2.', 'The  null  hypothesis  that  the  inhibition  zone  is  equal  to  the  disc  diameter  (6  mm)  was  rejected  for  each \nmicroorganism at every amount of essential oil with a high significance level (p = 0.00).', 'The main finding of the ANOVA \nanalysis is a strong interaction effect between the type of microorganism and the amount of essential oil (p = 0.00).', 'The \nhighly significant interaction effect adds difficulty in drawing general conclusions on the main effects, even if the two \nfactors are also highly significant (p = 0.00).', 'For example, K. pneumoniae has the highest inhibition zone overall but for \nthe amount of 20 [μL], where E. coli and S. typhimurium have higher values.', 'In order to compare more thoroughly the \neffect of T. vulgaris on each microorganism (Fig.', '1), the results of multiple comparisons, at each oil amount, has to be \nconsidered.', 'Tukey’s HSD test reveals that the only microorganisms with non-significant differences in the antimicrobial \neffect are S. typhimurium and E. coli at all oil amounts, and S. typhimurium, E. coli and C. albicans at 10 [μL].', 'The \nobserved  p-value  for  the  pairwise  differences  in  the  above-mentioned  cases  does  not  pass  acceptable  significance \nlevels, being larger than 0.4.', 'All the other pairwise differences are highly significant (p = 0.00).', '57 \n\n\x0cJournal of Medicine and Life Volume 7, Special Issue 3, 2014 \n\nTable 1.', 'Chemical composition of thyme EO \n\nRT (min) \n5.39 \n5.63 \n6.89 \n6.97 \n7.53 \n7.77 \n8.04 \n8.26 \n8.46 \n8.96 \n9.48 \n12.55 \n16.17 \n17.32 \n19.03 \n\nNo.', '1 \n2 \n3 \n4 \n5 \n6 \n7 \n8 \n9 \n10 \n11 \n12 \n13 \n14 \n15 \nTotal \n*Constituents presented in the order of elution from the VF 35 MS column.', 'Area % of total \n1.06 \n1.07 \n0.37 \n1.53 \n0.33 \n3.76 \n0.29 \n0.21 \n8.41 \n30.90 \n0.47 \n0.46 \n47.59 \n2.68 \n0.78 \n99.91% \n\nConstituents* \nalpha-Thujene \nalpha-Pinene \nbeta-Pinene \nbeta-Myrcene \nalpha-Phellandrene \nCarene<δ-2-> \nD-Limonene \nbeta-Phellandrene \npara-Cymene \ngamma-Terpinene \nTerpineol \nTerpinen-4-ol \nThymol \nCaryophyllene \nCyclohexene, 1-methyl-4-(5-methyl-1-methylene-4-hexenyl) \n \n\nThe inhibition of the growth of E. coli, K. pneumoniae, S. aureus, P. aeruginosa and E. faecalis was previously \nreported [4,7,9] along with the efficacy against C. albicans [9,16,17] and S. typhimurium [4,9], respectively.', 'In contrast, \nsome 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  \n\nzone for each of the nine repeated measurements \n\n \n\n \n\n58 \n\n\x0cTable 2.', 'Effects of thyme oil against bacteria expressed by the mean sizes of the inhibitory zones \n\nJournal of Medicine and Life Volume 7, Special Issue 3, 2014 \n\nTest microorganism \nStaphylococcus aureus \nATCC 25923 \nSalmonella typhimurium \nATCC 14028 \nPseudomonas aeruginosa \nATCC27853 \nE. coli \nATCC 25922 \nKlebsiella pneumoniae \nATCC 13882 \nEnterococcus faecalis \nATCC 29212 \nCandida albicans \nATCC 10231 \n \n\nAmount of essential oil [μL] \n5 \n23.93 ± 0.33 \n\n10 \n29.2 ± 0.6 \n\n15 \n29.9 ± 0.35 \n\n20 \n31.4 ± 0.47 \n\n14.49 ± 0.34 \n\n19.71 ± 0.39 \n\n30.68 ± 0.33 \n\n34.94 ± 0.22 \n\n11.82 ± 0.27 \n\n13.34 ± 0.33 \n\n14 ± 0.22 \n\n14.13 ± 0.19 \n\n14.63 ± 0.36 \n\n19.82 ± 0.41 \n\n30.67 ± 0.31 \n\n34.99 ± 0.19 \n\n30.21 ± 0.12 \n\n31.02 ± 0.31 \n\n32.79 ± 0.24 \n\n33.93 ± 0.14 \n\n8.99 ± 0.15 \n\n15.06 ± 0.15 \n\n15.99 ± 0.18 \n\n24.06 ± 0.15 \n\n15.14 ± 0.38 \n\n19.43 ± 0.55 \n\n25.74 ± 0.24 \n\n30.2 ± 0.17 \n\nThe inhibitions are expressed in mm and include the diameter of the paper disc (6 mm).', 'Data distributions were \nexpressed as mean values and standard deviations (SD) (n = 9).', 'Ciprofloxacine and cephalexine (for bacterial strains) \nand fluconazole (for fungi), respectively, were used as positive controls.', 'The antimicrobial activity of EOs depends on their chemical constituents.', 'Apparently, the antimicrobial activity \nof the EO analyzed is related to the presence of phenolic compounds (thymol) and terpene hydrocarbons (γ-terpinene), \nrespectively [4,7,18].', 'p-Cymene, the third major element according to percentage, does not show antibacterial efficacy \nwhen used alone [7], synergistic effects being however attributed to it in relation to thymol and γ-terpinene, respectively \n[19,20], which might represent another cause of the antimicrobial activity recorded.', 'On the other hand, a number of \nstudies have shown that EOS exhibit stronger antimicrobial activity than that of their major constituents or their mixtures, \nrespectively  [21,22],  which  suggests  synergistic  effects  of  the  minor  components,  but  also  the  importance  of  all \ncomponents in relation to the biological activity of EOs.', 'Conclusions \n\nThe 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 \nelucidate  the  mechanisms  underlying  their  biological  activity,  for  the  purpose  of  accessing  new  natural  antiseptics \napplicable in the pharmaceutical and food industry.', 'References \n1.', 'De Martino L, Bruno M, Formisano C, De Feo V, Napolitano F, Rosselli S, Senatore F. 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