Essential oil of Thymus vulgaris L. and Rosmarinus officinalis L.: Gas chromatography-mass spectrometry analysis, cytotoxicity and antioxidant properties and antibacterial activities against foodborne pathogens

Hanene Miladi, Rihab Ben Slama, Donia Mili, Sami Zouari, Amina Bakhrouf, Emna Ammar

Laboratory of Analysis, Treatment and Valorisation of Environment Polluants and Products, Faculty of Pharmacy, Monastir, Tunisia.
Laboratory of Biochemistry, Faculty of Medicine, Monastir, Tunisia.
Range Ecology Laboratory, Arid Land Institute of Medenine, Medenine, Tunisia.
UR Study & Management of Urban and Coastal Environments, LARSEN—National Engineering School, Sfax, Tunisia.

DOI: 10.4236/ns.2013.56090                

Abstract

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.

Keywords

Thymus vulgaris L.; Rosmarinus officinalis L.; GC–MS; Cytotoxicity; Antioxidant Activity; Antimicrobial Activity

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Soliman, K.M. and Badeaa, R.I. (2002) Effect of oil extracted from some medicinal plants on different my cotoxigenic fungi. Food Chemical Toxicology, 40, 1669-1675. doi:10.1016/S0278-6915(02)00120-5
[2]	Vivek, K.B., Atiqur, R. and Sun C.K. (2008) Chemical composition and inhibitory parameters of essential oil and extracts of Nandina domestica Thunb. to control food borne pathogenic and spoilage bacteria. International Journal of Food Microbiology, 125, 117-122. doi:10.1016/j.ijfoodmicro.2008.03.011
[3]	Hall, R.L. (1997) Food-borne illness: Implications for the future. Emerging Infectious Diseases, 3, 555-559. doi:10.3201/eid0304.970421
[4]	Loizzo, M.R., Tundis, R., Chandrika, U.G., Abeysekera, A.M., Menichini, F. and Frega, N.G. (2010) Antioxidant and antibacterial activities on foodborne pathogens of Artocarpus heterophyllus Lam. (Moraceae) leaves extracts. Journal of Food Science, 75, 291-295.
[5]	Zeng, W.C., Zhang, Z., Gao, H., Jia, L.R. and He, Q. (2012) Chemical composition, antioxidant, and antimicrobial activities of essential oil from pine needle (Cedrus deodara). Journal of Food Science, 77, 824-829. doi:10.1111/j.1750-3841.2012.02767.x
[6]	Cowan, M.M. (1999) Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12, 564-582.
[7]	Bozin, B., Mimica-Dukic, N., Samojlik, I. and Jovin, E. (2007) Antimicrobial and antioxidant properties of rosemary and sage (Rosmarinus officinalis L. and Salvia officinalis L., Lamiaceae) essential oils. Journal of Agricultural Food Chemistry, 55, 7879-7885. doi:10.1021/jf0715323
[8]	Ait-Ouazzou, A., Loran, S., Bakkali, M., Laglaoui, A., Rota, C., Herrera, A., Pagana, R. and Conchello, P. (2011) Chemical composition and antimicrobial activity of essential oils of Thymus algeriensis, Eucalyptus globulus and Rosmarinus officinalis from Morocco. Journal of the Science of Food and Agriculture, 91, 2643-2651. doi:10.1002/jsfa.4505
[9]	Tepe, B., Donmez, E., Unlu, M., Candan, F., Daferera, D., Vardar-Unlu, G., Polissiou, M. and Sokmen, A. (2004) Antibacterial and antioxidative activities of the essential oils and methanol extracts of Salvia cryptantha (Montbret et Aucher ex Benth.) and Salvia multicaulis p (Vahl). Food Chemistry, 84, 519-525. doi:10.1016/S0308-8146(03)00267-X
[10]	Daferera, D.J., Ziogas, B.N. and Polissiou, M.G. (2003) The effectiveness of plant essential oils on the growth of Botrytis cinerea, Fusarium sp. and Clavibacter michiganensis subsp. michiganensis. Crop Protection, 22, 39-44. doi:10.1016/S0261-2194(02)00095-9
[11]	Helenicy, N.H., Veras Fabíola, F.G.R., Colares, A.V., Menezes, I.R.A., Coutinho Henrique, D.M., Botelho, M.A. and Costa, J.G.M. (2012) Synergistic antibiotic activity of volatile compounds from the essential oil of Lippia si doides and thymol. Fitoterapia, 83, 508-512. doi:10.1016/j.fitote.2011.12.024
[12]	Schelz, Z., Molnar, J. and Hohmann, J. (2006) Antimicrobial and antiplasmid activities of essential oils. Fitoterapia, 77, 279-285. doi:10.1016/j.fitote.2006.03.013
[13]	Burt, S. (2004) Essential oils: Their antibacterial proper ties and potential application in foods e a review. International Journal of Food Microbiology, 94, 223-253. doi:10.1016/j.ijfoodmicro.2004.03.022
[14]	Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (2008) Biological effects of essential oils—A review. Food Chemistry and Toxicology, 46, 446-475. doi:10.1016/j.fct.2007.09.106
[15]	Zhang, C., Li, H., Yun, T., Fu, Y., Liu, C., Gong, B. and Neng, B. (2008) Chemical composition, antimicrobial and antioxidant activities of the essential oil of Tibetan herbal medicine Dracocephalum heterophyllum Benth. Natural Product Research, 22, 1-11. doi:10.1080/14786410701619076
[16]	Jeong-Ho, L., Byung-Kyu, L., Jong-Hee, K., Sang Hee, L. and Soon-Kwang, H. (2009) Comparison of chemical compositions and antimicrobial activities of essential oils from three conifer trees; Pinus densiflora, Cryptomeria ja ponica, and Chamaecyparis obtuse. Journal of Microbiology and Biotechnology, 19, 391-396. doi:10.4014/jmb.0803.191
[17]	Rota, C., Carrami Nana, J.J., Burillo, J. and Herrera, A. (2004) In vitro antimicrobial activity of essential oils from aromatic plants against selected foodborne patho gens. Journal of Food Protection, 67, 1252-1256.
[18]	Hazzit, M., Baaliouamer, A., Verissimo, A.R., Faleiro, M.L. and Miguel, M.G. (2009) Chemical composition and biological activities of Algerian Thymus oils. Food Chemistry, 16, 714-721. doi:10.1016/j.foodchem.2009.03.018
[19]	Zarzuelo, A. and Crespo, E. (2002) The medicinal and non medicinal uses of thyme. In thyme. The genus Thymus. In: Stahl-Biskup, E. and Saez, F., Eds. Medicinal and Aromatic Plants Industrial Profiles, New York, Taylor and Francis, 263-292.
[20]	Gachkar, L., Yadegari, D., Rezaei, M.B., Taghizadeh, M., Astaneh, S.A. and Rasooli, I. (2007) Chemical and bio logical characteristics of Cuminum cyminum and Rosmarinus officinalis essential oils. Food Chemistry, 102, 898-904. doi:10.1016/j.foodchem.2006.06.035
[21]	Chizzola, R., Michitsch, H. and Franz C. (2008). Anti oxidative properties of Thymus vulgaris leaves: Comparison of different extracts and essential oil chemotypes. Journal of Agricultural Food Chemistry, 56, 6897-6904. doi:10.1021/jf800617g
[22]	Lee, S.E., Lee, H.S. and Ahn, Y.J. (1999) Scavenging Effect of plant-derived materials on free radicals and active oxygen species. Agricultural Chemistry and Biotechnology, 42, 40-44.
[23]	Bicchi, C., Binello, A. and Rubiolo, P. (2000) Determination of phenolic diterpene antioxidants in rosemary (Rosmarinus officinalis L.) with different methods of extraction and analysis. Phytochemical Analysis, 11, 236-242. doi:10.1002/1099-1565(200007/08)11:4<236::AID-PCA503>3.0.CO;2-B
[24]	Yesil Celiktas, O., Hames Kocabas, E.E., Bedir, E., Vardar Sukan, F., Ozek, T. and Baser, K.H.C. (2007) Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. Food Chemistry, 100, 553-559. doi:10.1016/j.foodchem.2005.10.011
[25]	Barbosa, L.N., Mores Rall, V.L., Henrique Fernandes, A.A., Ushimaru, P.I., da Silva Probst, I. and Ary Fernan des, J. (2009) Essential oils against foodborne pathogens and spoilage bacteria in minced meat. Foodborne Patho gens and Disease, 6, 725-728. doi:10.1089/fpd.2009.0282
[26]	Alaoui Jamalia, C., El Bouzidia, L., Bekkouchea, K., Lahcenb, H., Markouka, M., Wohlmuthc, H., Leachc, D. and Abbad A. (2012) Chemical composition and antioxidant and anticandidal activities of essential oils from different wild moroccan thymus species. Chemistry and Bio diversity, 9, 1188-1197.
[27]	Rameau, J.C., Mansion, D., Dumé, G., Gauberville, C., Bardat, J., Bruno, E. and Keller, R. (2008) Flore forestière Francaise: Guide ecologique illustré. Tome 3, région méditerranéenne. Institut Pour le Developpement For estier, Paris & Ministere de l’Agriculture et de la Peche, Paris.
[28]	European Pharmacopoeia (1975) Volume 3. Maisonneuve, Sainte-Ruffine.
[29]	Kovàts, E. (1958) Characterization of organic compounds by gas chromatography. Part 1. Retention indices of aliphatic halides, alcohols, aldehydes and ketones. Helvetica Chimica Acta, 41, 1915-1932.
[30]	Adams, R.P. (2001) Identification of essential oil components by gas chromatography/quadrupole mass spectrometry. Allured Publishing Corporation, Carol Stream.
[31]	Sibanda, S., Chigwada, G., Poole, M., Gwebu, E.T., No letto, J.A., Schmidt, J.M., Rea, A.I. and Setzer, W.N. (2004) Composition and bioactivity of the leaf essential oil of Heteropyxis dehniae from Zimbabwe. Journal of Ethnopharmacology, 92, 107-111. doi:10.1016/j.jep.2004.02.010
[32]	Zouari, S., Zouari, N., Fakhfakh, N., Bougatef, A., Ayadi, M.A. and Neffati, M. (2010) Chemical composition and biological activities of a new essential oil chemotype of Tunisian Artemisia herba alba Asso. Journal of Medicinal Plants Research, 4, 871-880.
[33]	Hanato, H., Kagawa, T., Yasuhara, J. and Okuda, T. (1988) Two new flavonoids and other constituents in licorice root: Their relative astringency and radical scavenging effect. Chemical and Pharmaceutical Bulletin, 36, 1090-1097.
[34]	Mossman, B.T., Jean, L. and Landesman, J.M. (1983) Studies using lectins to determine mineral interactions with cellular membranes. Environmental Health Perspectives, 51, 23-25. doi:10.1289/ehp.835123
[35]	Clinical and Laboratory Standards Institute (2006) Per formance standards for antimicrobial susceptibility testing. 16th Informational Supplement, M100-S16, Clinical and Laboratory Standards Institute, Wayne.
[36]	Cavallo, J.D., Chardon, H., Chidiac, C., Choutet, P., Cour valin, P., Dabernat, H., Drugeon, H., Dubreuil, L., Gold stein, F., Jarlier, V., Leclerq, R., Nicolas-Chanoine, M.H., Philippon, A., Quentin, C., Rouveix, B., Sirot, J. and Soussy, C.J. (2006) Antibiogram committee of the French mi crobiology society. Pathology Biology, Paris.
[37]	Güllüce, M., Sokmen, M., Daferera, D., Agar, G., Ozkan, H., Kartal, N., Polissiou, M., Sokmen, A. and Sahin, F. (2003) In vitro antibacterial, antifungal, and antioxidant activities of the essential oil and methanol extracts of herbal parts and callus cultures of Satureja hortensis L. Journal of Agricultural and Food Chemistry, 51, 3958-3965. doi:10.1021/jf0340308
[38]	Flamini, G., Cioni, P.L., Morelli, I., Macchia, M. and Ceccarini, L. (2002) Main agronomic productive characteristic of two ecotypes of two Rosmarinus officinalis L. and chemical composition of their essential oils. Journal of Agricultural and Food Chemistry, 50, 3512-3517. doi:10.1021/jf011138j
[39]	Lawrence, B.M. (1997) Progress in essential oils: Rosemary oil. Perfum Flavor, 22, 71.
[40]	Pintore, G., Usai, M., Bradesi, P., Juliano, C., Boatto, G. and Tomi, F. (2002) Chemical composition and antimicrobial activity of Rosmarinus officinalis L. oils from Sardinia and Corsica. Flavor and Fragrance Journal, 17, 15-19. doi:10.1002/ffj.1022
[41]	Zaouali, Y., Bouzaine, T. and Boussaid, M. (2010) Essential oils composition in two Rosmarinus officinalis L. varieties and incidence for antimicrobial and antioxidant activities. Food and Chemical Toxicology, 48, 3144-3152. doi:10.1016/j.fct.2010.08.010
[42]	Ruberto, G. and Baratta, M.T. (2000) Antioxidant activity of selected essential oil components in two lipid model systems. Food Chemistry, 69, 167-174. doi:10.1016/S0308-8146(99)00247-2
[43]	Madsen, H.L. and Bertelsen, G. (1995) Spices as antioxidants. Trends Food Science Technology, 6, 271-277. doi:10.1016/S0924-2244(00)89112-8
[44]	Bai, N., He, K., Roller, M., Lai, C.S., Shao, X., Pan, M.H. and Ho, C.T. (2010) Flavonoids and phenolic compounds from Rosmarinus officinalis. Journal of Agricultural and Food Chemistry, 58, 5363-5367. doi:10.1021/jf100332w
[45]	Yesil-Celiktas, O., Sevimli, C., Bedir, E. and Vardar-Sukan, F. (2010) Inhibitory effects of rosemary extracts, carnosic acid and rosmarinic acid on the growth of various human cancer cell lines. Plant Foods for Human Nutrition, 65, 158-163. doi:10.1007/s11130-010-0166-4
[46]	Lopez, P., Sanchez, C., Batle, R. and Nerin, C. (2005) Solid and vaporphase antimicrobial activities of six essential oils: Susceptibility of selected food borne bacterial and fungal strains. Journal of Agricultural and Food Chemistry, 53, 6939-6946. doi:10.1021/jf050709v
[47]	Nowak, A., Kalemba, D., Krala, L., Piotrowska, M. and Czyzowska, A. (2012) The effects of thyme (Thymus vulgaris) and rosemary (Rosmarinus officinalis) essential oils on Brochothrix thermosphacta and on the shelf life of beef packaged in high-oxygen modified atmosphere. Food Microbiology, 32, 212-216. doi:10.1016/j.fm.2012.05.001
[48]	Rosato, A., Vitali, C., De Laurentis, N., Armenise, D. and Milillo, M. (2007) Antibacterial effect of some essential oils administered alone or in combination with norfloxacin. Phytomedicine, 14, 727-732. doi:10.1016/j.phymed.2007.01.005
[49]	Rota, M.C., Herrera, A., Martinez, R.M., Sotomayor, J.A. and Jordan, M.J. (2008) Antimicrobial activity and chemical composition of Thymus vulgaris, Thymuszygis and Thymushyemalis essential oils. Food Control, 19, 681-687. doi:10.1016/j.foodcont.2007.07.007
[50]	Angioni, A., Barra, A., Cereti, E., Barile, D., Cosson, J.D., Arlorio, M., Dessi, S., Coroneo, V. and Cabras, P. (2004) Chemical composition, plant genetic differences, antimicrobial and antifungal activity investigation of the essential oil of Rosmarinus officinalis L. Journal of Agricultural Food Chemistry, 52, 3530-3535. doi:10.1021/jf049913t
[51]	Skocibusic, M., Bezic, N. and Dunkic, V. (2006) Phytochemical composition and antimicrobial activities of essential oils from Satureja subspicata Vis. growing in Croatia. Food Chemistry, 96, 20-28. doi:10.1016/j.foodchem.2005.01.051