The essential oil composition of Thymus vulgaris L. and Rosmarinus officinalis L. endemic to France were determined by GC and GC-MS. Oils were assessed for their cytotoxic, antioxidant and antimicrobial activity. 31 and 37 different compounds were identified representing 99.64% and 99.38% of the thyme and rosemary oils respectively, where oxygenated monoterpenes constituted the main chemical class. Thymol (41.33%) and 1.8-cineole (24.10%) were identified as the main constituents of T. vulgaris L. and R. officinalis L., respectively. Essential oils (EOs) of selected plant species were evaluated for their in vitro cytotoxicity against the human lung adenocarcinoma epithelial cell line (A549). Cytotoxicity was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphynyltetra-zolium bromide] colorimetric assay. Dose-dependent studies revealed IC50 of 8.50 ± 0.01 μg/mL and 10.50 ± 0.01 μg/mL after 72 h on the A549 cells for R. officinalis L. and T. vulgaris L., respectively. Antioxidant activity was determined using a quantitative DPPH (1,1-diphenyl- 2-picryl hydrazyl) assay. Thymus and rosemary EOs exhibited effective radical scavenging capacity with 50% inhibitory concentration (IC50) of 437 ± 5.46 μg/mL and 189 ± 2.38 μg/mL respectively and therefore acts as a natural antioxidant agent. The antimicrobial activity of these species has also been studied against several foodborne pathogens and food isolated Salmonella spp. including S. enteritidis of significant importance. According to the results, T. vulgaris L. showed higher bactericidal effect than those from R. officinalis L. These results suggest that the essential oil from T. vulgaris L. and R. officinalis L. have potential to be used as a natural cytotoxic, antioxidant and antimicrobial agent in food processing.
Microbial contamination is an important factor promoting food deterioration and contributing to food-borne disease incidence. The presence and growth of microorganisms in food may cause spoilage and result in a reduction of quality and quantity [1,2]. Food-borne illnesses associated with Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7 and Salmonella enteritidis present a major public health concern [2,3]. It has been reported that the incidence of foodborne diseases caused by microbial contamination and environmental pollution will globally increase [4,5]. Furthermore, the consumption of foods contaminated with pathogenic microorganisms, such as bacteria, mould, viruses and parasites active the growth of a pathogen. Moreover, new examinations of antimicrobial activity on a wider spectrum of microorganisms, including some new multiresistant strains of bacteria and fungi were necessary [6,7]. In addition, to the increasing demand for safe and natural food in recent years, the great economic costs of deterioration and poisoning of food products by food pathogens have motivated many researchers to explore new alternatives to traditional food preservation practices [
With the growing incidence of infections resistant to antibiotics, an arsenal of either new agents of the supplementation of current antibiotics was needed. According to Daferera et al. (2003) [
Numerous studies have described the chemical composition, the antioxidant and antimicrobial activity of the EOs of several species of the genus Thymus, in the family Lamiaceae [8,17,18] how serves as preservative for foods and as an aromatic ingredient for seasoning various dishes [
In this study, the cytotoxicity, the antioxidant activity and antibacterial effects of essential oils of thyme and rosemary (Thymus vulgaris L. and Rosmarinus officinalis L., Lamiaceae) against several foodborne pathogens, especially the most common causative agent of foodborne salmonellosis, were reported. The chemical characterization of the investigated essential oils was performed by gas chromatography—mass spectrometry (GCMS).
Thymus vulgaris L. and Rosmarinus officinalis L. plants were freshly collected in 2011 during the period of full flowering on the mountain in the south of France (Mediterranean climate country and mountainous region). The specimens of collected plants were identified according to the forester flora of France [
An Agilent Technologies 6890N GC equipped with HP-5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm; Hewlett-Packard) and connected to a FID was used. The column temperature was programmed at 50˚C for 1 min, then 7˚C/min to 250˚C, and finally left at 250˚C for 5 min. The injection port temperature was 240˚C; while that of the detector was 250˚C (split ratio: 1/60).
The carrier gas was helium (99.995% purity) with a flow rate of 1.2 ml/min. The analyzed essential oil volume was 2 μl. Percentages of the constituents were calculated by electronic integration of FID peak areas, without the use of response factor correction. Mean percentage of Thymus vulgaris L. and Rosmarinus officenalis L. volatiles compounds represented the average calculated on three individuals. Retention indices (RI) were calculated for separate compounds relative to C9- C16 n-alkanes mixture (Aldrich Library of Chemicals Standards) [
The volatile compounds isolated by HD were analysed by GC/MS, using an Agilent Technologies 6890N GC. The fused HP-5MS capillary column (the same as that used in the GC/FID analysis) was coupled to an Agilent Technologies 5973B MS (Hewlett-Packard, Palo Alto, CA, USA). The oven temperature was programmed as previously (50˚C for 1 min, then 7˚C/min to 250˚C, and then left at 250˚C for 5 min). The injection port temperature was 250˚C and that of the detector was 280˚C (split ratio: 1/100). The carrier gas was helium (99.995% purity) with a flow rate of 1.2 ml/min. The MS conditions were as follow: ionization voltage, 70 eV; ion source temperature, 150˚C; electron ionization mass spectra were acquired over the mass range 50 to 550 m/z.
The volatile compounds of Thymus vulgaris L. and Rosmarinus officinalis L. leaves were identified by comparing the mass spectra data with spectra available from the Wiley 275 mass spectra libraries (software, D.03.00). Further identification confirmations were made referring to RI data generated from a series of known standards of n-alkanes mixture (C8 to C26) [
DPPH radical method. The free-radical scavenging activity of Thymus vulgaris L. and Rosmarinus officinalis L. EOs were measured by 2,2-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich, France) using the method described by Hanato et al. (1988) [
where A0 was the absorbance of the control at 30 min, and A1 was the absorbance of the sample at 30 min. All samples were analyzed in triplicate.
Thymus vulgaris L. and Rosmarinus officinalis L. EOs were screened for cytotoxic activities using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphynyltetrazolium bromide] colorimetric assay against the human lung adenocarcinoma epithelial cell line (A549) as described previously [
After incubation for 24, 48 and 72 hours, the medium in each well was collected and the cytotoxic effect was measured with the MTT colorimetric assay. To determine the cell viability, 20 µl of MTT (5 mg/ml) were added to each well and cells were cultured in additional incubation for 4 h. After washing the supernatant out, the insoluble formazan product was dissolved in acidified isopropanol. Then, optical density (OD) of 96-well culture plates was measured using an enzyme-linked immunosorbent assay (ELISA) reader at 540 nm. The OD of formazan formed in untreated control cells was taken as 100% of viability.
The tested microorganisms included the following Gram-positive bacteria: Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis CIP 106510, Micrococcus luteus NCIMB 8166, Bacillus cereus ATCC 11778, Bacillus cereus ATCC 14579, Listeria monocytogenes ATCC 19115 and Gram negative bacteria: Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Enterococcus feacalis ATCC 29212, Vibrio alginolyticus ATCC 17749, Vibrio alginolyticus ATCC 33787, Salmonella typhimurium ATCC 1408, Salmonella typhimurium LT2 DT104. The antibacterial effect was also tested against 31 strains belonging to Salmonella genus, including 12 species of enteritidis responsible for collective food intoxication isolated in hospital Fatouma Bouguiba Monastir (Tunisia) in June 2000. These microorganisms were kindly provided by Prof. Rhim Amel from the Regional Laboratory of Public Health of Monastir (Tunisia) and the serotyping of the strains was performed at the Pasteur institute, Tunisia.
Antimicrobial activity testing was done according to the Clinical and Laboratory Standards Institute (2006) guidelines [
A sterile filter discs (diameter 6 mm, Whatman Paper No. 3) were impregnated with 10 µl of EO placed on the MH agar mediums. The treated Petri dishes were placed at 4˚C for 1 h - 2 h and then incubated at 37˚C for 18 h - 24 h. The antibacterial activity was evaluated by measuring the growth inhibition diameter zone around the disk. Standard disks of the antibiotic ciprofloxacin (5 µg), served as the positive antibacterial controls according to the Comité de la Société Française de l’Antibiogramme for all strains except L. monocytogenes which standard disks of the antibiotic gentamycin (10 µg/disc), served as the positive antibacterial controls [
The minimal inhibition concentration (MIC) and the minimal bactericidal concentration (MBC) values were determined for all bacterial strains used in this study as described by Güllüce et al. (2007) [
After incubation, bacterial growth was evaluated by the presence of turbidity and a pellet on the well bottom. The MIC was defined as the lowest concentration of the compounds to inhibit the microorganism growth. The MBC values were interpreted as the highest dilution (lowest concentration) of the sample, which showed clear fluid with no turbidity development and without visible growth. All tests were performed in triplicate.
Values were expressed as means ± standard deviation. Analysis of variance was conducted and differences between variables were tested for significance by one-way ANOVA with a SPSS 11 (Statistical Package for the Social Sciences) programme. Differences at p < 0.05 were considered statistically significant.
GC-MS analyses of the thyme and rosemary oils led to the identification of 31 and 37 different components, representing 99.64% and 99.38% of the total oil respectively. The identified compounds of the volatile constituents of the essential oils (percentage content of each compound, retention index (RI), and structural subclass) are listed in
by 1,8-cineole, α-pinene, camphene, formed the major group. Ketones constitute 20.67% and camphor was the major compound of this class (
The antioxidant activity of Thymus vulgaris L. and Rosmarinus officinalis L. EOs was assessed by DPPH assay, evaluating the H-donating or radical scavenging ability of the oils using the stable radical 2,2-diphenyl-1- picrylhydrazyl (DPPH) as a reagent. The concentrations that led to 50% inhibition (IC50) for thyme and rosemary oil are 437 ± 5.46 µg/mL and 189 ± 2.38 µg/mL respectively. In this study, IC50 of both used oil were less potent than the reference antioxidants butylated hydroxytoluene (BHT) and quercetin (IC50 values of 4.21 ± 0.08 µg/mL and 1.07 ± 0.01 µg/mL respectively) [
Cell viability was performed after 24, 48 and 72 h exposure to R. officinalis L. and T. vulgaris L. for their anticancer activity using the MTT colorimetric assay.
The EOs were prepared and screened for their in vitro cytotoxic effects against human respiratory epithelial cell line (A549). A concentration and time dependent inhibitory effect on A549 cell were observed. After 24 h of incubation, rosemary and thymol oil cytotoxicity were considered whenever cell survival percent were less than 50. The extracts were not cytotoxic towards A549 cell line in all tested concentrations. But after 48 and 72 h essential oil exposure, R. officinalis L. was strongly inhibited the proliferation of the A549 cells and IC50 is 80.00 ± 0.02 µg/mL and 8.50 ± 0.01 µg/mL respectively (
The in vitro antimicrobial activity of T. vulgaris L. and R. officinalis L. EOs estimated by the diameter of inhibittion varied according to essentials oils and bacteria strains were summarised in
The bacteriostatic and bactericidal effectiveness of the thyme and Rosemary EOs estimated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) respectively are shown in
(
In the antimicrobial action of essential oil componentsis as follows: phenols > aldehydes > ketones > alcohols > ethers > hydrocarbons [
Regarding the susceptibility of different bacteria to the EOs tested, it was verified that Gram-negative bacterial strain is known to have a high level of intrinsic resistance to many antimicrobials and antibiotics because of a very restrictive outer membrane barrier, and it is highly resistant even to synthetic drugs [
In summary, the results presented here contribute to the knowledge of antimicrobial activities and chemical composition of the tested EOs obtained from aromatic plants growing in the mountain in the south of France. Our data also support the possible use of EOs of T. vulgaris and R. officinalis, in particular the EO of T. vulgaris, as potential natural agents for food preservation. Despite the moderate activity of R. officinalis, the data presented in this study are also significant given that this is the first time its bacteriostatic and bactericidal effects against the bacteria strains assayed have been reported. The case of L. monocytogenes, which has shown in most cases a significant sensitivity to the both EOs tested, is also noteworthy. L. monocytogenes is able to multiply under refrigerated conditions and so is a pathogen of great concern to the food industry. Because of that, the use of EOs as an additional barrier of food preservation should be considered. In general, the use of food presservation methods conjointly with the use of EOs could enhance the antimicrobial activity of these EOs; therefore, more research into the biological activities of these EOs, alone or combined with food preservation techniques, is recommended.
We are grateful to Prof. Rhim Amel from the Regional Laboratory of Public Health of Monastir (Tunisia) for her help to collect the microorganisms.