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Antibacterial activity of essential oils from plants of the genus Origanum at SciVerse ScienceDirect Food Control 34 (2013) 539e546 Contents lists available Food Control journal homepage www elsevier comlocatefoodcont Short communication Antibacterial activity of essential oils from plants of the genus Origanum Michalis K Stefanakis a, Eleftherios Touloupakis a, Elias Anastasopoulos b, Dimitrios Ghanotakis a, Haralambos E Katerinopoulos a,, Pavlos Makridis c a Department of Chemistry, Univers.
Food Control 34 (2013) 539e546 Contents lists available at SciVerse ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Short communication Antibacterial activity of essential oils from plants of the genus Origanum Michalis K Stefanakis a, Eleftherios Touloupakis a, Elias Anastasopoulos b, Dimitrios Ghanotakis a, Haralambos E Katerinopoulos a, *, Pavlos Makridis c a b c Department of Chemistry, University of Crete, Voutes, Heraklion 71003, Crete, Greece Department of Plant Production, Plant Biotechnology Laboratory, Technological and Educational Institute of Larissa, 41110 Larissa, Greece Institute of Aquaculture, Hellenic Centre for Marine Research, P.O Box 2214, 71003 Heraklion, Crete, Greece a r t i c l e i n f o a b s t r a c t Article history: Received 31 December 2012 Received in revised form 15 May 2013 Accepted 22 May 2013 In this study, three plant species, members of the family of Lamiaceae and the genus Origanum, namely, Origanum vulgare subsp hirtum, Origanum onites L., and Origanum marjorana L were studied for their chemical composition and antibacterial activity Essential oils of these plants were received by means of micro-steam distillation and their components were analyzed by gas chromatography and mass spectrometry (GCeEIMS) The major components identified in all three species are carvacrol and thymol The oils were assayed as potential food control antimicrobial agents In vitro studies showed that the essential oils showed strong antimicrobial activity against bacterial and yeast strains Ó 2013 Elsevier Ltd All rights reserved Keywords: Food control Origanum Essential oils Antimicrobial activity Introduction Essential oils (EOs) are aromatic oily liquids obtained from various plants generally localized in temperate to warm countries In nature as secondary metabolites, EOs play an important role in the protection of the plants as antibacterials, antivirals, antifungals, insecticides and also act against herbivores (Bakkali, Averbeck, Averbeck, & Idaomar, 2008) An estimated 3000 essential oils are known, of which about 300 are commercially available and destined mostly for the flavor and fragrances market (Van de Braak & Leijten, 1999, 116 pp.) EOs are very complex natural mixtures containing hydrocarbons (mainly terpenoids) and oxygenated compounds (alcohols, esters, ethers, aldehydes, ketones, lactones, phenols and phenol ethers) Their composition may vary considerably between plant species and varieties, and, within the same variety, from different geographical origin (Zygadlo & Juliani, 2003) EOs are widely used in medicine, in perfumes, cosmetics and bath products, as flavoring agents in food and drink, and in many other manufacturing areas EOs can constitute a powerful tool to reduce the development and dissemination of antimicrobial resistance Nowadays essential oils are recognized as safe substances (ESO, * Corresponding author Tel.: þ30 2810 545026; fax: þ30 2810 545001 E-mail address: kater@chemistry.uoc.gr (H.E Katerinopoulos) 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.foodcont.2013.05.024 GRAS e 182.20) by the Food and Drug Administration (2005) and some contain compounds which can be used as antibacterial additives (Ait-Ouazzou et al., 2011; Cox et al., 2001; Muyima, Zulu, Bhengu, & Popplewell, 2002; Nerio, Olivero-Verbel, & Stashenko, 2010) They become increasingly popular as natural antimicrobial and antioxidant agents that may be used in food preservation Public concern about the use of antibiotics in livestock feed has increased, because of the emergence of antibiotic resistant bacteria and their possible transmission from livestock to humans In fact, in the European Union the use of synthetic antibiotics, health and growth promoters as additives in livestock feed has been prohibited since 2006 by the European Parliament and Council Regulation (EC No.1831/2003) In this context, one of the possible solutions is the use of EOs such as those found in the genus Origanum and in its species used for this study: Origanum vulgare subsp hirtum, Origanum onites and Origanum marjorana Antimicrobials are used in the food industry for two main reasons: to control natural spoilage processes (food preservation), and to prevent the growth of micro-organisms, including pathogenic micro-organisms (food safety) A large number of reports concerning the antioxidant and the antimicrobial ability of essential oils have already been published (Bagamboula, Uyttendaele, & Debevere, 2003; Bakkali, Averbeck, Averbeck, Zhiri, & Idaomar, 2005; Botelho et al., 2007; Castilho, 540 M.K Stefanakis et al / Food Control 34 (2013) 539e546 Savluchinske-Feio, Weinhold, & Gouveia, 2012; Deba, Xuan, Yasuda, & Tawata, 2008; Gỹlỗin, Elmastasá, & Aboul-Enein, 2007; Kalemba & Kunicka, 2003; Pauli, 2006; Pavela, 2009; Sacchetti et al., 2005; Saidana et al., 2008; Si et al., 2006; Thuille, Fille, & Nagl, 2003; Valero & Salmeron, 2003; Ye, Dai, & Hu, 2013) In these studies, EO exhibited very good insecticide, bactericide and fungicide effects (Kumar, Shukla, Singh, Prasad, & Dubey, 2008; Srivastava, 2008) Burt (2004) gives an overview of the studies of the antibacterial activity of essential oils in foods As typical lipophiles, they cross through the cell wall and cytoplasmic membrane, disrupt the structure of their different layers of polysaccharides, fatty acids and phospholipids and permeabilize them In general, the cytotoxic activity of essential oils is mostly due to the presence of phenols, aldehydes and alcohols (Bruni et al., 2003; Sacchetti et al., 2005) The aims of this study were a) to determine the chemical composition of essential oils extracted from three species of the genus Origanum, and b) evaluate their ability to inhibit in vitro bacterial strains such as Vibrio, isolated from aquaculture facilities, and the common microbial strains Escherichia coli and Saccharomyces cerevisiae Materials and methods 2.1 Materials Samples of leaves, stems and/or seeds from three Origanum species were collected from various regions of Greece, or made available from commercial sources (Table 1) The collection and/or identification of the plant material were carried out by Assistant Professor Ilias Anastasopoulos, at the Plant Biotechnology Laboratory of Larissa’s Technological Educational Institute Voucher specimens have been deposited at the Herbarium of the Institute All non-commercial plants were grown from seeds, except from samples and which were asexually propagated from cuttings of the same plant, and therefore genetically identical Plants were grown in a plastic greenhouse at the Technological Educational Institute, in Larissa, Greece, and received no treatment apart from watering 2.2 Extraction methods After collection, the plant material was air dried in the dark at room temperature (w25 C) for 10 days The dried plant parts underwent hydrodistillation for two hours on a Clevenger apparatus connected to a modified refrigerated essential oil receiver (European Pharmacopoeia 5.0) Refrigeration was used to reduce the byproducts of the thermal treatment The essential oils were then diluted with mL of ether and filtered through anhydrous sodium sulfate to remove water traces The resulting essential oils were stored at C The oil content was estimated in mL/100 g (dry weight of the plant material) 2.3 Chemical analysis 2.3.1 GCeEIMS analysis GCeEIMS analysis of the extracts was performed on a Shimadzu GC-17A gas chromatograph coupled with a Shimadzu GCMS-QP 5050 mass-selective detector with the appropriate data system The GC was equipped with a Grob-type split-splitless injector The fused silica capillary column (Supelco, SBP-5 with 0.25 mm film thickness, 30 m  0.25 mm i.d.) was directly coupled to the ion source Helium was used as a carrier gas with a back pressure of 0.8 Atm The injector temperature was 250 C and the oven temperature program started at 50 C for and then increased at a rate of C/min up to 150 C, retained at this temperature for 10 and increased again at a rate of C/min up to 280 C, where it remained for 20 2.3.2 Identification The scanning range was 30e700 m/z The quantification of the components was based on the total number of fragments (total ion count) of the metabolites, as detected by the mass spectrometer The identification of the chemical components was carried out based on the retention time of each component (Rt) compared with those of commercially available compounds, by analysis of their mass spectra, by the use of the NIST21, NIST107 and PMW_TOX2 mass spectra libraries (NIST, 2010) as well as by comparison with literature data (Adams, 2007) Calculation of retention indices was performed in accordance to the work of Van den Dool and Kratz (1963), in comparison to the retention times of standard hydrocarbons (C9eC25) Also, when necessary, co-injection with standard compounds was carried out 2.4 Antimicrobial screening The microbial strains used as test organisms were as follows: E coli, S cerevisiae, Listonella anguillarum (CECT 522), Vibrio splendidus DMC-1 (kindly provided by Prof T.H Brikbeck, University of Glaskow), Vibrio alginolyticus, isolated from seabream larvae (Sparus aurata) (kindly provided by Dr P Katharios, Institute of Aquaculture, Hellenic Center for Marine Research) and Vibrio sp isolated from enriched Artemia metanauplii homogenate by Dr P Makridis at the Institute of Aquaculture, Hellenic Center for Marine Research L anguillarum, V splendidus, V alginolyticus and Vibrio sp were grown on tryptic soy agar dishes S cerevisiae were grown on YPD agar dishes and E coli were grown on LB agar dishes The agar disc diffusion method was employed for the determination of antimicrobial activity of the essential oil (NCCLS, 1997) For this purpose, Watmann number paper disks with mm diameter soaked with mL of the essential oil were laid on top of the agar culture medium plate previously inoculated (50 mLe108 CFU/mL) with the different micro-organisms tested in this work In addition Table Origanum samples used in the study Sample Date Species Oct/2007 Aug/2006 Aug/2006 Aug/2006 Aug/2006 Oct/2007 Oct/2007 Aug/2006 Aug/2006 Oct/2007 O O O O O O O O O O vulgare subsp vulgare subsp vulgare subsp marjorana vulgare subsp vulgare subsp vulgare subsp onites onites vulgare subsp hirtum hirtum hirtum hirtum hirtum hirtum hirtum Place of collection or purchasing Geographical coordinates (altitude) Pilio, Greece CC Botanicals Ltd (Warwick, UK) Agrafa, Greece Pieterpikzonen b.v (Luinjeberd, NL) CC Botanicals Ltd (Warwick, UK) Pilio, Greece Sisses, Greece Naxos island, Greece Naxos island, Greece Agrafa, Greece 39 260 N 23 20 E (700 m) e 39 80 N 21 380 E (700 m) e e 39 260 N 23 20 E (700 m) 35 220 N 24 280 E (500 m) 37 50 N 25 280 E (700 m) 37 50 N 25 280 E (700 m) 39 80 N 21 380 E (700 m) M.K Stefanakis et al / Food Control 34 (2013) 539e546 541 Table Results of qualitative and quantitative (%v/v) analysis of the Essential Oil Origanum (O vulgare subsp hirtum), in different samples Season Summer Autumn Components Molecular formula Ret time R.I.b R.I.c Oil Oil Oil Oil Oil Oil Oil a-Thujene a-Pinenea C10H16 C10H16 C10H16 C10H16 C8H16O C8H16O C10H16 C10H16 C10H16 C10H16 C10H14 C10H16 C10H18O C10H14 C10H18O C10H16 C10H18O C10H18O C10H18O C10H18O C10H14O C10H18O C10H16O C10H16O C11H16O C10H14O C11H16O C10H14O C10H14O C12H16O2 C10H12O2 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H22 C15H24O C15H24O C15H26O C15H24O C15H26O C15H26O C15H26O C15H26O C15H18 C15H26O 11.591 11.793 12.259 13.299 13.358 13.466 13.606 13.949 14.083 14.266 14.482 14.582 14.663 15.311 15.537 15.988 16.245 16.737 17.638 17.796 18.297 18.379 18.441 18.644 18.866 18.902 18.956 19.848 20.067 21.178 21.231 21.519 21.615 21.844 22.772 23.014 23.295 23.720 24.301 24.769 24.960 25.003 25.171 25.472 25.674 25.676 27.476 27.617 27.881 28.365 28.954 29.181 29.575 29.791 30.148 30.294 930 937 952 977 980 987 990 1002 1008 1017 1024 1028 1033 1059 1071 1090 1096 1127 1169 1177 1184 1190 1210 1218 1235 1244 1245 1290 1302 1352 1359 1376 1377 1388 1422 1435 1442 1454 1479 1485 1502 1508 1512 1526 1531 1531 1578 1583 1590 1622 1643 1648 1661 1672 1682 1685 930 939 954 979 979 987 990 1002 1011 1017 1024 1029 1031 1059 1070 1088 1096 1128 1169 1177 1182 1188 1210 1218 1235 1243 1244 1290 1299 1352 1359 1375 1376 1388 1419 1434 1441 1454 1479 1485 1500 1505 1512 1526 1531 1534 1578 1583 1590 1623 1643 1648 1662 1675 1682 1685 n.d.e trd n.d n.d 0.43 0.28 0.13 n.d n.d n.d 1.82 n.d n.d tr 0.05 n.d 0.05 tr 0.09 0.67 0.07 0.17 n.d 0.16 n.d n.d 0.06 9.58 79.36 n.d 0.08 n.d n.d n.d 0.60 n.d n.d 0.09 0.06 n.d n.d 0.05 0.77 0.15 0.38 n.d 0.23 3.12 tr 0.35 tr 0.28 0.05 n.d 0.07 0.16 n.d n.d n.d n.d n.d n.d 0.07 n.d n.d n.d 0.40 0.20 n.d tr 0.13 n.d 0.05 n.d tr 0.31 n.d 0.08 0.08 tr n.d tr tr 9.11 85.52 n.d 0.07 tr 0.07 tr 0.89 n.d 0.07 0.07 0.19 0.05 n.d 0.05 0.63 0.17 0.52 n.d 0.16 0.54 n.d 0.05 tr 0.09 0.08 n.d n.d 0.08 0.17 0.19 n.d 0.11 0.28 0.10 1.18 0.33 0.12 0.67 3.33 0.28 tr 2.01 0.39 0.17 0.18 tr 0.21 2.09 tr 0.34 n.d 0.13 2.30 0.29 n.d 6.30 73.83 0.07 0.39 tr n.d tr 1.09 n.d n.d 0.14 0.20 0.05 n.d 0.09 0.71 0.16 0.49 n.d 0.09 0.92 n.d 0.13 0.05 n.d 0.08 n.d n.d 0.16 0.08 0.19 0.05 n.d 0.30 0.16 0.55 tr tr 0.49 6.47 0.36 0.07 0.54 1.29 0.18 0.31 n.d 0.80 1.76 0.14 0.50 0.15 n.d n.d 0.05 0.09 67.43 9.10 n.d n.d n.d n.d n.d 1.14 0.06 0.10 0.26 n.d n.d n.d n.d 3.14 tr 0.20 n.d 0.47 2.75 n.d 0.52 n.d n.d n.d n.d n.d 0.21 0.39 0.23 0.06 0.40 0.21 0.10 2.33 0.20 0.10 1.33 6.79 0.49 n.d 3.46 0.36 0.26 0.25 n.d 0.23 1.13 0.11 0.37 0.15 n.d n.d n.d 0.05 56.26 8.87 n.d 0.05 n.d n.d n.d 6.83 n.d 0.18 1.56 n.d n.d 0.11 n.d 1.43 0.11 n.d n.d 0.34 3.41 n.d 0.60 n.d n.d n.d 1.12 n.d 0.11 0.67 0.44 0.15 0.31 0.37 0.10 2.41 0.15 0.16 1.56 8.00 0.57 n.d 1.63 0.62 0.46 0.30 0.21 0.47 1.60 0.19 0.79 n.d 0.17 n.d n.d n.d 55.30 10.45 n.d 0.05 n.d n.d n.d 3.98 0.12 n.d 0.47 n.d n.d tr n.d 4.24 n.d n.d 0.14 0.35 2.84 n.d 0.31 n.d n.d n.d n.d n.d 0.30 0.39 0.11 0.05 n.d 0.19 0.10 0.53 n.d n.d n.d 4.31 0.34 n.d 2.41 1.70 0.08 0.27 1.17 0.34 0.63 n.d 0.25 0.05 n.d n.d tr n.d 72.43 7.19 n.d n.d n.d n.d n.d 1.45 tr 0.11 0.36 tr n.d tr n.d 2.60 tr n.d n.d 0.30 1.62 0.33 0.33 n.d n.d n.d n.d n.d 0.12 Total identified (%) 99.36 99.73 99.82 99.91 99.98 99.88 99.76 Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Essential oil content (mL/100 g dry weight) 2.76 90.24 2.17 4.19 3.25 Ỉ 0.3 0.67 95.17 2.71 1.18 3.56 Ỉ 0.3 8.56 85.95 2.93 2.38 4.08 Æ 0.1 9.91 80.02 5.46 4.52 2.36 Æ 0.2 16.96 67.22 10.22 5.58 3.09 Ỉ 0.3 16.51 69.23 8.95 5.19 1.69 Ỉ 0.1 9.48 82.06 4.52 3.70 1.40 Ỉ 0.2 a Camphene b-Pinenea 1-Octen-3-ol 3-Octanone Myrcenea a-Phellandrene d-3-Carene a-Terpinenea p-Cymenea Limonenea 1,8-Cineolea g-Terpinenea cis-Sabinene hydrate Terpinolene Linaloola trans-p-Menth-2-en-ol Borneola Terpinen-4-ol p-Cymen-8-ol a-Terpineola cis-Dihydro carvone trans-Dihydro carvone Thymol methyl ether Carvone Carvacrol methyl ether Thymola Carvacrola Thymyl acetate Eugenol a-Ylangene a-Copaene b-Bourbonene b-Caryophyllene trans-a-Bergamotene Aromandendrene a-Humulene g-Muurolene Germacrene-D Leden a-Muurolene b-Bisabolene g-Cadinene d-Cadinene cis-Calamenene Spathulenol Caryophyllene oxide Globulol Humulene epoxide Cubenol s-Cadinol a-Cadinol Eudesm-7(11)-en-4-ol Cadalene a-Bisabolol a b c d e Identification by comparison of retention times and co-injection with authentic compound R.I (Retention Indices) from experimental using a SBP-5 column using a homologous series of n-alkanes (C9eC25) R.I (Retention Indices) from literature data and Adams (2007) for a DB-5 column tr: Traces of substances ( 0.05 were considered to be significant The Principal Coordinate Analysis (PCA) GenAlEx was Table Results of qualitative and quantitative (%v/v) analysis of the Essential Oil Origanum (O onites), in different samples Components Molecular formula Ret time R.I.b R.I.c Oil Summer Oil Summer a-Thujene a-Pinenea C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H16 C10H14 C10H16 C10H18O C10H14 C10H18O C10H16 C10H18O C10H18O C10H18O C10H14O C10H18O C10H16O C10H16O C11H16O C12H22O2 C10H14O C10H14O C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24 C15H24O C15H24O C15H24O C15H26O C15H26O C15H26O C15H26O C15H26O 11.591 11.793 12.259 13.299 13.606 13.949 14.083 14.266 14.482 14.582 14.663 15.311 15.537 15.988 16.245 17.638 17.796 18.297 18.379 18.441 18.644 18.956 19.375 19.848 20.067 21.615 21.844 22.772 23.014 23.295 23.720 24.301 24.769 24.960 25.171 25.472 25.674 27.476 27.617 28.365 28.954 28.965 29.181 29.526 30.294 930 937 952 981 990 1002 1008 1017 1026 1030 1033 1059 1071 1090 1098 1172 1177 1184 1190 1210 1218 1245 1272 1290 1302 1377 1388 1422 1435 1442 1454 1478 1485 1502 1512 1526 1531 1578 1583 1622 1643 1644 1648 1651 1685 930 939 954 979 990 1002 1011 1017 1024 1029 1031 1059 1070 1088 1098 1169 1177 1182 1188 1210 1218 1244 1275 1290 1299 1376 1388 1419 1434 1441 1454 1479 1485 1500 1512 1526 1531 1578 1583 1623 1643 1646 1648 1650 1685 0.11 0.18 0.07 0.07 0.59 tr tr 0.79 5.63 0.55 0.14 0.89 0.25 0.06 0.19 0.38 1.50 n.d 0.31 0.09 n.d n.d n.d 34.62 46.65 tr tr 2.35 n.d 0.07 0.45 0.07 n.d tr 0.45 0.05 0.15 0.30 2.57 0.24 n.d n.d n.d n.d n.d n.d.e trd tr n.d 0.20 n.d n.d 0.27 1.99 0.19 n.d 0.52 tr 0.11 0.14 0.18 1.77 tr 0.19 0.05 tr 0.10 0.11 20.21 66.18 n.d n.d 1.42 tr 0.17 0.21 0.07 tr 0.18 1.50 0.08 0.30 0.88 1.53 0.18 0.22 0.08 0.34 0.13 0.34 Total identified (%) 99.77 99.84 Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Essential oil content (mL/100 g dry weight) 8.98 84.13 3.55 3.11 0.72 Ỉ 0.1 3.28 88.93 3.93 3.7 1.97 Æ 0.1 Camphenea b-Pinenea Myrcenea a-Phellandrene d-3-Carene a-Terpinenea p-Cymenea Limonenea 1,8-Cineolea g-Terpinenea cis-Sabinene hydrate Terpinolene Linaloola Borneola Terpinen-4-ol p-Cymen-8-ol a-Terpineola cis-Dihydro carvone trans-Dihydro carvone Carvacrol methyl ether Dihydro-Linalool acetate Thymola Carvacrola a-Copaene b-Bourbonene b-Caryophyllene trans-a-Bergamotene Aromadendrene a-Humulene g-Muurolene Germacrene-D Leden b-Bisabolene g-Cadinene d-Cadinene Spathulenol Caryophyllene oxide Humulene epoxide Cubenol g-Eudesmol s-Cadinol b-Eudesmol a-Bisabolol a b c d e Identification by comparison of retention times and co-injection with authentic compound R.I (Retention Indices) from experimental using a SBP-5 column using a homologous series of n-alkanes (C9eC25) R.I (Retention Indices) from literature data and Adams (2007) for a DB-5 column tr: Traces of substances (