INVESTIGATION ON ANTIBACTERIAL SYNERGISM OF ORIGANUM VULGARE AND THYMUS VULGARIS ESSENTIAL OILS

Essential oils are well known as strong antimicrobial agents of plant origin. In spite of this, the antimicrobial synergism of essential oils isolated from different plant species is poorly investigated. The following study examines the synergism of the essential oils of Origanum vulgare L. and Thymus vulgaris L against pathogenic bacteria, Staphylococcus aureus and Salmonella typhimurium. First, the antibacterial effect of the oils was tested, and the minimal inhibitory concentrations (MIC) of both oils were determined using the microdilution method. To test whether the oils act synergistically, every possible combination of essential oil concentrations was used in a dynamic checkerboard method. The results indicated that the oils indeed acted synergistically with fractional inhibitory concentration indexes of 0.45 and 0.50.


INTRODUCTION
Despite progress in medical care and treatment, infectious diseases caused by pathogen microorganisms are still a major threat to public health (WHO, 2002).New drugs are introduced but they are expensive and relatively unavailable in developing countries (Okeke et al., 2005).Emerging resistance to synthetic products also poses a public concern.New strategies are needed, and in the last two decades a growing interest in the use of essential oils as safe alternatives is increasing.
Essential oils (EO) are products of plant secondary metabolites.They are a mixture of compounds, mainly mono-and sesquiterpenes, carbohydrates, alcohols, aldehydes and ketones.The biological activity is in direct dependence of the genetically induced specific chemical composition of an EO.
Origanum vulgare L. (oregano) is a spice herb from the family Lamiaceae, growing from 20-80 cm in height, with opposite leaves and purple flowers produced in erect spikes.It is native to western and southwestern Eurasia and the Mediterranean region.The main components of its EO are phenolic compounds: carvacrol and thymol.Chemical compositions vary depending on geographical region and session of collecting (Faleiro et al., 2002).Thymus vulgaris L. (thyme) is a low-growing herbaceous plant that hardly grows above 20 cm, forming a small shrub.A member of the Lamiaceae family, it is native to southern Europe.The main components of its EO are thymol, linalool or carvacrol depends on chemotype.The EOs of oregano and thyme are known to possess a wide range of biological activities (Adam et al., 1998;Sartoratto et al., 2004).Both plants are commonly used in foods, for flavor and aroma, in pharmaceutical and cosmetic industry.
There have been reports of that EOs are more strongly antimicrobial than their major components individually (Lachowicz et al., 1998) which indicates that the minor components play a important role and may have a synergistic effect.The synergistic effect of the major components of EOs has been reported in several studies (Ipek et al., 2005, Ettayebi et al., 2000, Hummelbrunner and Isman, 2001), however, examinations of whole oil are rare.
The concept of synergisms appears to have a great potential and influence on the biological activity of EO.For this reason, we have evaluated the synergistic potential of O. vulgare and T. vulgaris EO against pathogens.

Essential oil isolation
Essential oils were isolated by hydrodistillation in a Clevenger type apparatus for 3 h.The essential oils obtained were stored at +4 °C until further tests.

Gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS)
Qualitative and quantitative analyses of the oils were performed using GC and GC/MS.The GC analysis of the oil was carried out on a GC HP-5890 II apparatus, equipped with a split/splitless injector, attached to an HP-5 column (25 m x 0.32 mm, 0.52 μm film thickness) and fitted to a flame ionization detector (FID).Carrier gas flow rate (H2) was 1 ml/min, split ratio 1:30, injector temperature was 250°C, detector temperature 300°C, while column temperature was linearly programmed from 40-240°C (at rate of 4°/ min).The same analytical conditions were employed for GC/MS analysis, where HP G 1800C Series II GCD system equipped with an HP-5MS column (30 m x 0.25 mm, 0.25 μm film thickness) was used.The transfer line was heated at 260°C.Mass spectra were acquired in EI mode (70 eV), in an m/z range of 40-400.Electron impact identification of individual constituents was made by comparison of their retention times with those of analytical standards of available terpenoids, and by computer searching, matching mass spectra with those held in the Wiley 275 library of mass spectra.Confirmation was done using a calibrated AMDIS program for the determination of experimental values for retention indices of recorded constituents and comparing them with those from the literature (Adams, 2001).For quantification purpose, area percent data obtained by FID were used.

Microorganisms and culture conditions
For the bioassays, Gram (-) bacterial species Salmonella typhimurium (ATCC 13311), and Gram (+) bacterial species Staphylococcus aureus, were used.The organisms tested were from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research "Siniša Stanković", Belgrade, Serbia.Bacteria were maintained on Mueller-Hinton agar (MHA).

Microdilution method
In order to investigate the antimicrobial activity of the isolated essential oils and the main constituents, the modified microdilution technique was used (Daouk et al., 1995).Bacterial species were cultured overnight at 37ºC in Tryptic Soy Broth (TSB) medium The bacterial cell and fungal suspension was adjusted with sterile saline to a concentration of 1.0 x 10 5 units in a final volume of 100 μl per well.The inocula were stored at +4ºC for further use.Dilutions of the inocula were cultured on solid MHA for bacteria to verify the absence of contamination and to check the validity of the inoculum.
Minimum inhibitory concentration (MIC) determination was performed by a serial dilution technique using 96-well microtiter plates.The investigated essential oils (0.01-10.00 μl/ml) were added in TSB medium with inoculum.The microplates were incubated for 24 h at 37ºC for bacteria.Bacterial growth was detected by the addition of 30 μl of a 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) water solution (0.5 mg/ml), (Sigma).The bacteria were again incubated at 37ºC for 30 min, and in the wells where bacterial growth occurred, INT changed from yellow to purple.MIC values were defined as the lowest concentration of essential oil to completely inhibit microbial growth.

Checkerboard method
This method was carried out using 96-well microplates containing Tryptic Soy Broth (TSB) with essential oil concentrations ranging from 1/16 -4 × MIC and combined with each other on the plate in a checkerboard style.The bacterial inoculum was 1.0 x 10 5 colony-forming unit (CFU) per well.The microplates were incubated for 24 h at 37ºC (Jacqueline et al., 2005).The fractional inhibitory concentration (FIC) index was calculated as follows: drug A (O. vulgare essential oil) FIC (drug A MIC combined/ drug A MIC alone) + drug B (T. vulgaris essential oil) FIC (drug B MIC combined/drug B MIC alone).Synergism was defined as FIC index < 0.5; additivity FIC index > 0.5-< 2; indifference FIC index > 2-< 4 and antagonism FIC index > 4 (Lorian, 2005).The strains that had MIC > 32 μg/ml and MIC > 1024 μg/ ml were considered to be MICs of 64 μg/ml and 2048 μg/ml, respectively.

RESULTS AND DISCUSSION
Results on the chemical composition of the essential oils of Origanum vulgare and Thymus vulgaris are presented in Table 1.The essential oil of O. vulgare was rich in carvacrol (64.50%), while p-cymene and γ-terpinene were also dominant constituents with slightly lower percentage (10.90%and 10.80%).T. vulgaris essential oil was abundant with thymol (48.92%), followed by p-cymene (18.99%).The other constituents were present in amounts less than 5%.Chemical analysis of O. vulgare by other authors (Tian and Lai, 2006;Masood et al., 2007) also showed that the main constituents of this oil are phenols: carvacrol and thymol.However, some reports (Bussata et al., 2007) revealed monoterpene hydrocarbons terpinen-4-ol and γ-terpinen as the main components in chemical analysis.The results of chemical analysis of T. vulgaris are in accordance with previous studies (Rota et al., 2008, Hudaib et al., 2002) with major constituents being thymol and p-cymene.The high content of phenol compounds from this oil is responsible for its strong antimicrobial and antioxidant activity.
Investigating the antimicrobial synergism of a combination of thyme and oregano essential oils we obtained the results presented in Table 1.These results indicated that oregano and thyme essential oils indeed expressed antimicrobial synergism.As for the S. aureus MIC results, it can be seen from Table 1 that it was more sensitive than S. typhimurium to the effect of oregano essential oil.The MIC for oregano essential oil on S. aureus was 0.5 μl/ml, and for S. typhimurium was 1.0 μl/ml.The essential oil of thyme expressed the same MICs on both bacteria tested (1.0 μl/ml).The combined effect of thyme and oregano essential oils on S. aureus was synergistic in a combination of MIC 0.2 μl/ml for oregano oil and 0.25 μl/ml for thyme oil.The FIC index was calculated to be 0.45, which is <0.5 and according to the literature (Lorian, 2005) this value indicates compound synergism.A synergistic effect of combined essential oils on S. typhimurium was also obtained with combined MICs of 0.5 μl/ml and 0.05 μl/ml, for oregano and thyme oils, respectively.The value for the FIC index was calculated to be 0.505, which is approximately <0.5 and the effect is also considered to be synergistic.
Plant essential oils are a potentially useful source of antimicrobial compounds.The results of different studies are difficult to compare, most probably because of the different test methods, bacterial strains and sources of antimicrobial samples used.Natural products demonstrate different modes of action (McAllister et al., 2000), and in order to achieve the desired inhibition effect, products belonging to different plants should be combined as shown in this study.
By mixing different essential oils, their concentration can be significantly reduced, which possibly eliminates their undesired sensorial impact on antimicrobial preparation.The antibacterial properties of terpenic compounds are due to the presence of de-localized electrons in the molecule, which according to Ultee (2002) allows for easier deprotonation, playing an important role in their antibacterial activity.It is reported that terpenoid compounds have a greater inhibiting effect on Gram-positive than on Gramnegative bacteria (Nascimento et al., 2000) and our results confirm these previous findings.
In conclusion, O. vulgare and T. vulgaris are valuable sources of secondary metabolites with biologi-cal activities.This study demonstrates the synergistic effect of a natural mixture of two popular plant essential oils, O. vulgare and T. vulgaris.This potential should be further evaluated for application in modern medicine and industry against pathogen microorganisms.
Acknowledgments -The authors are grateful to the Ministry of Education and Science of Serbia for financial support (Grant № 173032).

Table 1 .
Chemical composition of essential oils