Journal of Saudi Chemical Society (2014) 18, 972–976 King Saud University Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com ORIGINAL ARTICLE Essential oil from Rhaponticum acaule L. roots: Comparative study using HS-SPME/GC/GC–MS and hydrodistillation techniques Batoul Benyelles a, Hocine Allali a, Mohamed El Amine Dib Nassim Djabou a,b, Boufeldja Tabti a, Jean Costa b a,* , a Laboratoire des Substances Naturelles et Bioactives (LASNABIO), Universite´ Abou Bekr Belkaı¨d, BP 119, Tlemcen 13000, Algeria b Laboratoire Chimie des Produits Naturels, Universite´ de Corse, UMR CNRS 6134, Campus Grimaldi, BP 52, 20250 Corte, France Received 14 September 2011; accepted 1 December 2011 Available online 8 December 2011 KEYWORDS Rhaponticum acaule L.; Essential oil; GC; GC/MS; HS-SPME Abstract The composition of essential oil extracted from Rhaponticum acaule L. roots growing wild in Algeria was studied by hydrodistillation (HD) and by Head-Space Solid Phase Micro-Extraction (HS-SPME). Quantitative but not qualitative differences have been found in the chemical composition of both analysed samples depending on the extraction method. However, the oil obtained from R. acaule roots shows that aliphatic alcohols were found to be the major class (69.2%), followed by the terpenes (5.5%), alkenes (5.2%) and alkynes (4.0%). In both cases the analysis were carried out using Gas Chromatography (GC) and Gas Chromatography–Mass Spectrometry (GC–MS). Our study shows that HS-SPME extraction could be considered as an alternative technique for the isolation of volatiles from plant. 25 components were identified in oil vs. 39 in the HS-SPME. However the oil composition of roots was mainly represented by a variety of aliphatic hydrocarbons (alcohols, aldehydes and ketones) and terpenes which are known for their antimicrobial activities. ª 2011 Production and hosting by Elsevier B.V. on behalf of King Saud University. 1. Introduction * Corresponding author. Address: Laboratoire des Substances Naturelles et Bioactives (LASNABIO), Département de Chimie, Université Aboubekr Belkaid, BP 119, Universite de Tlemcen, Algeria. Tel./fax: +213 43286530. E-mail address: a_dibdz@yahoo.fr (M. El Amine Dib). Peer review under responsibility of King Saud University. Production and hosting by Elsevier For many years, secondary metabolism was virtually ignored. Today, the situation is different. The wide structural variability of these compounds has attracted the curiosity of chemists and the biological activities possessed by natural products have inspired the pharmaceutical industry to search for new structures. In relation to pathogenic bacteria, a growing and worrisome problem is the increase in bacterial resistance to antibiotics (Nostro et al., 2004; Georgopapadakou, 2001). For patients, antimicrobial resistance increases morbidity and mor- http://dx.doi.org/10.1016/j.jscs.2011.12.001 1319-6103 ª 2011 Production and hosting by Elsevier B.V. on behalf of King Saud University. Essential oil from Rhaponticum acaule L. roots: Comparative study tality, while there is a significant increase in costs for health care institutions (Coutinho et al., 2005; Dancer, 2001). With respect to the growing clinical importance given to the hospital community and bacterial infections and the progressive development of antimicrobial resistance, considerable scientific research has focused on the antibacterial properties of plant products (Coutinho et al., 2008; Simos et al., 2008). Aromatic plants and culinary herbs have long been the basis of traditional medicine in many countries. Therefore, evaluation of the pharmacological activities of plant essential oils commonly used in traditional medicine and aromatherapy has received considerable interest due to their presumed safety and therapeutic effects. In biological systems, studying the mechanisms by which essential oils demonstrate their activities (including anti-microbial) are complex and dependent on the overall composition of the essential oil (Yanishlieva et al., 2006; Tomaino et al., 2005; Mimica-Dukic et al., 2003; Souza et al., 2007; MImica-Dukic et al., 2004). A wide variety of essential oils are known to possess antimicrobial properties and in many cases this activity is due to the presence of active constituents, mainly attributable to isoprenes such as monoterpenes, sesquiterpenes and related alcohols, other hydrocarbons and phenols (Griffin et al., 1999; Dorman and Deans, 2000). Rhaponticum is a genus belonging to the Asteraceae family and consists of about 20000 species that are widely distributed around the world (Tardif, 2003). However, many species of the genus Rhaponticum have long been used in traditional medicine. Indeed, the root of Rhaponticum uniflorum has been used against intoxication and for the treatment of fever. It has been demonstrated that this species inhibits peroxidation of membrane lipids and possesses anti-atherosclerotic activity (Zhang et al., 2002). A survey conducted by herbalists identified that, in folk medicine, the roots of R. acaule was crushed mixed with honey and are used as aperitif, cholagogue, depurative, digestive, stomachic and tonic. The only work published on chemical composition oils of aerial parts of R. acaule analysed by GC and GC–MS showed that this plant is richer in diterpenoids (23.7%), aromatic compounds (23.6%) and oxygenated sesquiterpenes (21.3%) (Boussaada et al., 2008). During the course of our study on discovery of new plant products antimicrobial, we examined the constituents of the roots of R. acaule widely used in Algeria. Hence, the aim of this present study has been made to investigate the chemical composition essential oil of roots of R. acaule extracted by hydrodistillation and HS-SPME. 2. Plant material Roots of the plant material R. acaule were collected from Ain Fezza forest area (at about 12 km east of Tlemcen, Algeria) [1000 m, 3450 N 1170 O] during the month of March 2007. The plant was authenticated by Prof. N. Benabadji, of the Botanical Laboratory, Biology, Tlemcen (Algeria). The voucher specimen, As 09.11, has been deposited in the Department of Biology, Abou bekr Belkaı̈d University, Tlemcen, Algeria. 2.1. Essential oil extraction The oil used in this study was isolated by hydrodistillation (250 g) for 5 h using a Clevenger-type apparatus (Conseil de 973 l’Europe, 1996) according to the European Pharmacopoeia and yielded 0.025% w/w of oil. 2.2. HS-SPME conditions The roots of R. acaule were cut roughly with scissors (1–2 cm long) before subjection to HS-SPME. The SPME device (Supelco) coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 30 lm) was used for extraction of the plant volatiles. Optimization of conditions was carried out using fresh roots of the plant (1 g in a 20 mL vial) and based on the number and the sum of total peak areas measured on GC-FID. Temperature, equilibration time and extraction time were selected after nine experiments combining four temperatures (30, 50, 70 and 90 C), four equilibration times (20, 40, 60 and 80 min) and three extraction times (15, 30 and 45 min). HS-SPME and subsequent analyses were performed in triplicate. After sampling, SPME fibre was inserted into the GC and GC–MS injection ports for desorption of volatile components (5 min), both using the splitless injection mode. Before sampling, each fibre was reconditioned for 5 min in the GC injection port at 260 C. The coefficient of variation (1.6% < CV < 17.8%) calculated on the basis of total area obtained from the FID-signal for the sample indicated that the HS-SPME method produced reliable results. 2.3. Gas chromatography GC analyses were carried out using a Perkin Elmer Autosystem GC apparatus (Walhton, MA, USA) equipped with a single injector and two flame ionization detectors (FID). The apparatus was used for simultaneous sampling of two fusedsilica capillary columns (60 m · 0.22 mm, film thickness 0.25 lm) with different stationary phases: Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol). Temperature programme: 60–230 C at 2 C min 1 and then held isothermal at 230 C (30 min). Carrier gas: helium (1 mL min 1). Injector and detector temperatures were held at 280 C. Split injection was conducted with a ratio split of 1:80. Injected volume: 0.1 lL. For HS-SPME-GC analysis, only Rtx-1 (polydimethylsiloxane) column was used and volatile components were desorbed in a GC injector with a SPME inlet liner (0.75 mm. I.D., Supelco). 2.4. Gas chromatography–mass spectrometry The oil and the volatile fraction were investigated using a Perkin Elmer TurboMass quadrupole detector, directly coupled to a Perkin Elmer Autosystem XL equipped with two fused-silica capillary columns (60 m · 0.22 mm, film thickness 0.25 lm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol). Other GC conditions were the same as described above. Ion source temperature: 150 C; energy ionization: 70 eV; electron ionization mass spectra were acquired with a mass range of 35–350 Da. Oil injected volume: 0.1 lL, fraction injected volume: 0.2 lL. GC–MS analysis of the volatile fractions sampling by HS-SPME was carried out only on a Rtx-1 capillary column, for the desorption GC injector was equipped with a SPME inlet liner (0.75 mm. 1.D., Supelco). 974 2.5. Component identification Identification of the components was based (i) on the comparison of their GC retention indices (RI) on non polar and polar columns, determined relative to the retention time of a series of n-alkanes with linear interpolation, with those of authentic compounds or literature data (Jennings and Shibamoto, 1980; Joulain and König, 1998; Mc Lafferty and Stauffer, 1989; Mc Lafferty and Stauffer, 1988; Adams, 2001; National Institute of Standards and Technology, 1999) and (ii) on computer matching with commercial mass spectral libraries (Lafferty and Stauffer, 1994; National Institute of Standards and Technology, 2005; Adams, 2001) and comparison of spectra with those of our own library of authentic compounds or literature data. (Jennings and Shibamoto, 1980; Joulain and König, 1998). Relative amounts of individual components were calculated on the basis of their GC peak areas on the capillary Rtx-1 column, without FID response factor correction. 2.6. Statistical analysis All the values of volatile components were expressed as mean ± SE. Data were analysed statistically using the Student’s t-test. In all cases, p < 0.05 was used as the criterion of statistical significance. 3. Results 3.1. Essential oil composition The yield of the essential oil from the roots of R. acaule was 0.025%. The essential oil was pale white and a total of 42 different volatile and semi-volatile compounds were identified in R. acaule root oil, distributed by distinct chemical classes: fourteen alcohols (1–14), six ketones (15–20), seven aldehydes (21–27), three terpenic compounds (28–30), four alkenes (31– 34), five alkynes (35–39), one furan (40), one alkane (41) and one ether (42). The oil extracted by hydrodistillation was characterized by a large amount of alcohol compounds (69.2%) made up of octadeca-9,12,15-triene-1-ol (60.3%), dodeca9,11-diene-1-ol (7.6%) and but-3-ene-2-ol (1.3%). Terpenic compounds were presented only by the camphor with percentage of 5.5% of the oil. Other compounds identified in this oil were pentadecene (5.2%) and hexadecyne (3.8%), while bis(3,5,5-trimethyl hexyl)-ether (1.5%) and nonanal (1.1%) were minor constituents of the oil (Table 1). B. Benyelles et al. Table 1 Chemical composition of R. acaule roots essential oil and volatile components. No. 01 02 03 04 05 06* 07 08 09 10 11* 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35* 36 37 38 39 40 41 3.2. HS-SPME analysis of volatiles constituents 42 The volatiles emitted from the R. acaule root were investigated using HS-SPME under optimized parameters. The optimization of the HS-SPME sampling parameters was carried out using fresh plant material based on the sum of the total peak areas obtained using GC-FID. The maximum sum of the total peak area was acquired for a temperature of 70 C, an equilibrium time of 60 min, and an extraction time of 30 min. The oil vapour adsorbed by headspace SPME also showed higher amounts of alcohol compounds (65.3%) represented by octadeca-9, 12,15-triene-1-ol (60.4%), a-santolina alcohol (1.6%) Compounds Hexanol cis-Hept-3-ene-1-ol O-Guicol Cresol cis-Oct-5-ene-1-ol But-3-ene-2-ol Trans-non-2-ene-1-ol (4Z)-Decen-1-ol (E)-Anethol Carvacrol Dodeca-9,11-diene-1-ol Octadeca-9,12,15-triene-1-ol a-Santolina alcool Pentadec-2-yne-1-ol P of alcohol compounds Trans-oct-3-ene-2-one Nonan-2-one Octa-3,5-dien-2-one (E)-Nona-3,8-diene-2-one Carvone Undecan-2-one P of ketone compounds Hexanal 3-Furancarboxaldehyde cis-Heptan-4-al Benzaldehyde (E, E)-Heptadienal Nonanal Cuminaldehyde P of aldehyde compounds Trans isolimonene Camphre a-Curcumene P of terpenic compounds Dodecatriene Pentadecene (Z)-Hexadecene Heptadec-1-ene P of alkene compounds Hexadecyne Octadec-5-yne Tetradec-5-ene-3-yne Tetradec-3-ene-5-yne (E)-Hexadec-4-en-6-yne P of alkyne compounds 2-Pentylfurane P of furane compounds Dodecane P of alkane compounds Bis-(3,5,5-trimethyl hexyl)-ether P of ether compounds Total Roots Irlitt Oil SPME ± SE 852 942 1025 1027 1043 1148 1153 1239 1260 1277 1654 1657 1705 1807 – – – – – 1.3 – – – – 7.6 60.3 tr tr 69.2 – tr tr tr – tr – – – – – – 1.1 tr 1.1 – 5.5 tr 5.5 tr 5.2 tr tr 5.2 3.8 tr tr 0.1 0.1 4.0 tr – tr – 1.5 1.5 86.5 0.2 ± 0.01 0.3 ± 0.01 0.2 ± 0.01 0.2 ± 0.01 1.3 ± 0.08 – 0.4 ± 0.02 0.1 ± 0.01 tr tr – 60.4 ± 0.65 1.6 ± 0.25 0.6 ± 0.01 65.3 0.1 ± 0.01 tr 0.7 ± 0.02 0.1 ± 0.01 0.2 ± 0.02 tr 1.1 0.1 ± 0.01 0.1 ± 0.01 0.2 ± 0.02 0.2 ± 0.01 tr 0.1 ± 0.02 0.1 ± 0.01 0.8 tr 0.1 ± 0.01 tr 0.1 0.4 ± 0.03 12.1 ± 0.16 2.3 ± 0.09 2.9 ± 0.12 17.7 – 6.3 ± 0.24 0.4 ± 0.02 0.7 ± 0.11 0.4 ± 0.01 7.8 0.6 ± 0.01 0.6 0.1 ± 0.01 0.1 1.1 ± 0.16 1.1 94.6 1015 1058 1066 1205 1215 1273 775 809 870 932 987 1083 1212 994 1120 1468 1168 1488 1591 1697 1663 1681 1700 1715 1795 980 1100 1568 Each value is presented as mean of triplet treatments. and cis-oct-5-ene-1-ol (1.3%). As in oil, alkene compounds (17.7%) were found in higher amounts than the alkyne compounds (7.8%) and ethers (1.8%) (Fig. 1). Quantitative but not qualitative differences have been found in the chemical composition of both analysed samples. Octadeca-9,12,15-tri- Essential oil from Rhaponticum acaule L. roots: Comparative study 975 ene-1-ol (60.3–60.4%) was the principal component of this species. Other compounds found at average concentrations in SPME extract were pentadecene (5.2–12.1%), octadec-5-yne (tr – 6.3%), heptadec-1-ene (tr – 2.9%) and (Z)-hexadecene (tr – 2.9%) (Table 1). group has been confirmed (Dorman and Deans, 2000; Lawrence, 1993) and the relative position of the hydroxyl group on the phenolic ring does not appear to strongly influence the degree of antibacterial activity. Aldehydes are known to possess powerful antimicrobial activity. It has been proposed that an aldehyde group conjugated to a carbon to carbon double bond is a highly electronegative arrangement, which may explain their activity (Moleyar and Narasimham, 1986), suggesting an increase in electronegativity that increases the antibacterial activity (Kurita et al., 1979, 1981). The ketones such as 2-undecanone and undecanone were reported to have antimicrobial and nematicidal activity (Benhadj et al., 2007; Nikoletta et al., 2011). Aliphatic alcohols were reported to possess strong to moderate activities against several bacteria. The activity increased with the length of the carbon chain (Kabelitz et al., 2003). Terpenes have also shown antimicrobial properties that appear to have strong to moderate antibacterial activity against Gram positive bacteria and against pathogenic fungi, but in general weaker activity was observed against Gram-negative bacteria (Hada et al., 2003; Tepe et al., 2004). 4. Discussion 5. Conclusion HS-SPME is a simpler and more rapid procedure for extraction of the volatile fraction from aromatic plants (Belliardo et al., 2006; Demirci et al., 2005) in comparison with hydrodistillation, which is time consuming and needs a large amount of sample. HS-SPME analysis allowed a qualitative estimate of volatile compounds using a small quantity of material (Paolini et al., 2008). The HS-SPME has also been used for the characterization of chemical variability of aromatic plants and for the study of volatile fractions emitted by species without essential oil. Thus, the chemical differences observed between the essential oil and the volatile fractions extracted of roots from R. acaule using hydrodistillation and HS-SPME, respectively, can be explained by the fact that the first technique is based on the liquid quasi-total extraction of plant volatiles and the latter technique is controlled by a solid/gas equilibrium step. During hydrodistillation, the most volatile compounds and water-soluble compounds are lost in the gaseous phase and in the hydrolate phase, respectively, whereas, with HS extraction, it is the fibre affinity of each compound that monitors the sampling of the volatiles. As a consequence, it should be noted that 18 compounds (1, 2, 3, 4, 5, 7, 8, 9, 10, 15, 19, 21, 22, 23, 24, 25, 27 and 28) were identified only in the volatile fractions extracted using HS-SPME and three compounds (identified by an asterisk in Table 1) were identified only in the essential oil. Various chemical compounds isolated by hydrodistillation and HS-SPME of roots from R. acaule have direct activity against many species of bacteria, such as terpenes and a variety of aliphatic hydrocarbons (alcohols, aldehydes and ketones). The lipophilic character of their hydrocarbon skeleton and the hydrophilic character of their functional groups are of main importance in the antimicrobial action of essential oils components. Therefore, a rank of activity has been proposed as follows: phenols > aldehydes > ketones > alcohols > esters > hydrocarbons (Kalemba and Kunicka, 2003). For example, some essential oils containing phenolic structures are highly active against a broad spectrum of microorganisms (Kalemba and Kunicka, 2003; Güllüce et al., 2003). The importance of the hydroxyl The present study is the first report which describes the volatiles compounds and essential oil of roots from R. acaule. Concerning the plant chemistry, we conclude that the root’s oil is mainly represented by alcohol compounds and that can be use against multidrug-resistant bacteria. However, further studies are in progress in our laboratory to confirm the antimicrobial activity of this plant. Figure 1 Abundance of the chemical classes identified in R. acaule roots’ oil. Acknowledgement The authors would like to express thanks to N. Benabadji of Botanical Laboratory, Biology Department, Aboubekr Belkaı̈d University, for identification of the vegetable matter. References Adams, R.P., 2001. Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured Publishing, Carol Stream, IL, USA. Belliardo, F., Bicchi, C., Cordero, C., Liberto, E., Rubiolo, P., Sgorbini, B., 2006. Headspace-solid-phase microextraction in the analysis of the volatile fraction of aromatic and medicinal plants. J. Chromatogr. Sci. 44, 416–429. Benhadj, F.M., Marzouk, B., Chraif, I., Boukef, K., 2007. Analysis of Tunisian Ruta graveolens L. oils from Jemmel. Int. J. Food Agric. Environ. 5, 52–55. Boussaada, O., Ammar, S., Saidana, D., Chriaa, J., Chraif, I., Daami, M., Helal, A.N., Mighri, Z., 2008. Chemical composition and antimicrobial activity of volatile components from capitula and aerial parts of Rhaponticum acaule DC growing wild in Tunisia. Microbiol. Res. 163, 87–95. Conseil de l’Europe, 1996. Pharmacopée Européenne. Sainte Ruffine, Maisonneuve, SA. Coutinho, H.D.M., Cordeiro, L.N., Bringel, K.P., 2005. Antibiotic resistance of pathogenic bacteria isolated from the population of Juazeiro do Norte Ceara. Rev. Bras. Cienc. Saude 9, 127–138. Coutinho, H.D.M., Costa, J.G.M., Siqueira Jr, J.P., Lima, E.O., 2008. In vitro anti-staphylococcal activity of Hyptis martiusii Benth against methicillin-resistant Staphylococcus aureus MRSA strains. Rev. Bras. Farmacogn. 18 (Suppl.), 670–675. 976 Dancer, S.J., 2001. The problem with cephalosporins. J. Antimicrob. Chemother. 48, 463–478. Demirci, B., Demirci, F., Baser, K.H.C., 2005. Headspace-SPME and hydrodistillation of two fragrant Artemisia sp. Flavour Fragrance J. 20, 395–398. Dorman, H.J.D., Deans, S.G., 2000. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88, 308–316. Georgopapadakou, N.H., 2001. Infectious disease: drug resistance, new drugs. 2005. Drug Resist. Updat. 5, 181–191. Griffin, S.G., Wyllie, S.G., Markham, J.L., Leach, D.N., 1999. The role of structure and molecular properties of terpenoids in determining their antimicrobial activity. Flavour Fragrance J. 14, 322–332. Güllüce, M., Sökmen, M., Daferera, D., Agar, G., Özkan, H., Kartal, N., Polissiou, M., Sökmen, A., 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. J. Agric. Food Chem. 51 (14), 3958–3965. Hada, T., Shiraishi, A., Furuse, S., Inoue, Y., Hamashima, H., Masuda, K., Shiojima, K., Shimada, J., 2003. Inhibitory effects of terpene on the growth of Staphylococcus aureus. Nat. Med. 57, 464–467. Jennings, W., Shibamoto, T., 1980. Qualitative Analysis of Flavour and Fragrance Volatiles by Glass-Capillary Gas Chromatography. Academic Press, New York. Joulain, D., König, W.A., 1998. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. EB-Verlag, Hamburg. Kabelitz, N., Santos, P.M., Heipieper, H.J., 2003. Effect of aliphatic alcohols on growth and degree of saturation of membrane lipids in Acinetobacter calcoaceticus. FEMS Microbiol. Lett. 220, 223–227. Kalemba, D., Kunicka, A., 2003. Antibacterial and antifungal properties of essential oils. Curr. Med. Chem. 10, 813. Kurita, N., Miyaji, M., Kurane, R., Takahara, Y., Ichimura, K., 1979. Antifungal activity and molecular orbital energies of aldehyde compounds from oils of higher plants. Agric. Biol. Chem. 43, 2365– 2371. Kurita, N., Miyaji, M., Kurane, R., Takahara, Y., Ichimura, K., 1981. Antifungal activity of components of essential oils. Agric. Biol. Chem. 45, 945–952. Lawrence, B.M., 1993. A planning scheme to evaluate new aromatic plants for the flavor and fragrance industries, pp. 620–627. Mc Lafferty, F.W., Stauffer, D.B., 1994. Wiley Registry of Mass Spectral Data, sixth ed. Mass Spectrometry Library Search System Bench-Top/PBM version 3.10d. Palisade, Newfield. Mc Lafferty, F.W., Stauffer, D.B., 1988. The Wiley/NBS Registry of Mass Spectral Data, fourth ed. Wiley-Interscience, New York. Mc Lafferty, F.W., Stauffer, D.B., 1989. The Wiley/NBS Registry of Mass Spectra Data, vols. 1–7. Wiley-Interscience Publication, New York. B. Benyelles et al. Mimica-Dukic, N., Bozin, B., Sokovic, M., Mihajlovic, B., Matavulj, M., 2003. Antimicrobial and antioxidant activities of three Mentha species essential Oils. Planta Med. 69, 413–419. Mimica-Dukic, N., Bozin, B., Sokovic, M., Simin, N., 2004. Antimicrobial and antioxidant activities of Melissa officinalis L. (Lamiaceae) essential Oil. J. Agric. Food Chem. 52, 2485–2489. Moleyar, V., Narasimham, P., 1986. Antifungal activity of some essential oil components. Food Microbiol. 3, 331–336. National Institute of Standards and Technology, 1999. PC version 1.7 of the NIST/EPA/NIH Mass Spectral Library, Perkin Elmer Corporation, Norwalk, CT. National Institute of Standards and Technology, 2005. NIST Chemistry WebBook, NIST Standard Reference Database. Gaithersburg, MD. <http://webbook.nist.gov/chemistry>. Nikoletta, G., Francesca, N., Manconi, Leonti, M., Maxia, A., Caboni, P., 2011. Aliphatic ketones from Ruta chalepensis (Rutaceae) induce paralysis on root knot nematodes. J. Agric. Food Chem. 59 (13), 7098–7103. Nostro, A., Blanco, A.R., Cannatelli, M.A., Enea, V., Flamini, G., Morelli, I., 2004. Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol. FEMS Microbiol. Lett. 230, 191–195. Paolini, J., Nasica, E., Desjobert, J.M., Muselli, A., Bernardini, A.F., Costa, J., 2008. Essential oil composition and volatile constituents of Adenostyles briquetii Gamisans (syn Cacalia briquetii). Phytochem. Anal. 19, 266–276. Simos, C.C., Araujo, D.B., Araujo, R.P.C., 2008. Study, in vitro and ex vivo, of the action of different concentrations of propolis extracts against microorganisms present in human saliva. Rev. Bras. Farmacogn. 18, 84–89. Souza, E.L., Stamford, T.L.M., Lima, E.O., Trajano, V.N., 2007. Effectiveness of Origanum vulgare L. essential oil to inhibit the growth of food spoiling yeasts. Food Control 18, 409–413. Tardif, J., 2003. Société des amis de jardin Vaan Den Hende, Université Laval, Une visite de jardin Roger-Van Den. un parcours de l’évolution des végétaux, Ed. Illustrated, p. 137. Tepe, B., Daferera, D., Sokmen, M., Polissiou, M., Sokmen, A., 2004. In vitro antimicrobial and antioxidant activities of the essential oils and various extracts of Thymus eigii M. Zohary et P.H. Davis. J. Agric. Food Chem. 52, 1132–1137. Tomaino, A., Cimino, F., Zimbalatti, V., Venuti, V., Sulfaro, V., Pasquale, A., Saija, A., 2005. Influence of heating on antioxidant activity and the chemical composition of some spice essential oils. J. Agric. Food Chem. 89, 549–554. Yanishlieva, N.V., Marinova, E.M., Pokorny, J., 2006. Natural antioxidants from herbs and spices. Eur. J. Lipid Sci. Technol. 108, 776–793. Zhang, Y.H., Cheng, J.K., Yang, L., Cheng, D.L., 2002. Triterpenoids from Rhaponticum uniflorum. J.Chin. Chem. Soc. 49, 117–124.