Phytotoxic Activities of Mediterranean Essential Oils

Molecules 2010, 15, 4309-4323; doi:10.3390/molecules15064309 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Phytotoxic Activities of Mediterranean Essential Oils Luiz Fernando Rolim de Almeida 1, Fernando Frei 2, Emilia Mancini 3, Laura De Martino 3 and Vincenzo De Feo 3,* 1 2 3 Departamento de Botânica, Instituto de Biociências de Botucatu, UNESP - Campu de Botucatu Distrito de Rubião Júnior, S/N, 18.618-000, Botucatu-SP, Brazil; E-Mail: [email protected] (L.F.R.A.) Departamento de Ciências Biológicas, Faculdade de Ciências e Letras, UNESP – Universidade Estadual Paulista, Avenida Dom Antonio, 19806-900, Assis-SP, Brazil; E-Mail: [email protected] (F.F.) Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (Salerno), Italy; E-Mails: [email protected] (E.M); [email protected] (L.D.M.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: + 39 089 969 751; Fax: + 39 089 969 602. Received: 28 April 2010; in revised form: 9 June 2010 / Accepted: 11 June 2010 / Published: 14 June 2010 Abstract: Twelve essential oils from Mediterranean aromatic plants were tested for their phytotoxic activity, at different doses, against the germination and the initial radicle growth of seeds of Raphanus sativus, Lactuca sativa and Lepidium sativum. The essential oils were obtained from Hyssopus officinalis, Lavandula angustifolia, Majorana hortensis, Melissa officinalis, Ocimum basilicum, Origanum vulgare, Salvia officinalis and Thymus vulgaris (Lamiaceae), Verbena officinalis (Verbenaceae), Pimpinella anisum, Foeniculum vulgare and Carum carvi (Apiaceae). The germination and radicle growth of tested seeds were affected in different ways by the oils. Thyme, balm, vervain and caraway essential oils were more active against both germination and radicle elongation. Keywords: essential oils; phytotoxicity; germination; seedling growth; monoterpenes Molecules 2010, 15 4310 1. Introduction Potential damage to human health and to the environment provoked by synthetic herbicides is regarded today as a real problem. It has resulted in an increased interest in alternative strategies leading to the development of biodegradable and non-toxic compounds [1]. In fact, the continued use of synthetic herbicides may threaten sustainable agricultural production and result in serious ecological and environmental problems, such as the increased resistance of weeds and environmental pollution and health hazards [2]. Volatile oils and their constituents are being explored for weed and pest management and are viewed as an important source of lead molecules in agriculture [3]. It is thus pertinent to explore and characterize the phytotoxic properties of aromatic plants and their volatile oils. Bioactive terpenoids constitute an important part of the defensive mechanisms of a large number of organisms and represent a fairly untapped source of active compounds of potential use both in the agricultural and pharmaceutical fields [4]. In fact, a large number of highly phytotoxic allelochemicals are derived from the terpenoid pathway [5] and the phytotoxicity of essential oils has been investigated in various plant tissues which contains or produce these compounds [2,6-8]. The Mediterranean flora is characterized by the abundance of aromatic plants among its components. The feature differentiating these plants from all others, in spite of the fact that they belong to many different families, is the production of chemically related secondary compounds, the low molecular weight and volatile isoprenoids. This remarkable presence of aromatic species is important in determining the allelopathic potential within this ecosystem [9]. Thus, the objective of this study was to evaluate the in vitro possible phytotoxicity of the essential oils from 12 Mediterranean plants, belonging to three different families, Hyssopus officinalis L. (hyssop), Lavandula angustifolia Mill. (lavender), Majorana hortensis L. (marjoram), Melissa officinalis L. (lemon balm), Ocimum basilicum L. (basil), Origanum vulgare L. (oregano), Salvia officinalis L. (sage), Thymus vulgaris L. (thyme) (Lamiaceae), Carum carvi L. (caraway), Foeniculum vulgare Mill. (fennel), Pimpinella anisum L. (anise) (Apiaceae), Verbena officinalis L. (vervain) (Verbenaceae) against the germination and radicle growth of the crop species Raphanus sativus L. cv. Saxa (radish), Lepidium sativum L. (garden cress) and Lactuca sativa L. (lettuce), comparing the effects of the oils in light of their chemical composition. 2. Results and Discussion The yields in essential oil obtained by hydrodistillation of plant species collected at full flowering stage, on a fresh weight basis, were as follows: H. officinalis 0.41%, L. angustifolia 0.49%, M. hortensis 0.26%, M. officinalis 0.25%, O. basilicum 0.42%, O. vulgare 0.21%, S. officinalis 0.46%, T. vulgaris 0.26%, V. officinalis 0.39%, P. anisum 1.80%, F. vulgare 2.30% and C. carvi 2.80%. Table 1 shows the composition of the essential oils. The main constituent of P. anisum and F. vulgare essential oils was cis-anethole, which represented 97.1% and 76.3% of the whole oils, respectively. Our data on anise oil composition agree with the available literature. Tabanca and coworkers [10] reported that anise oil was constituted predominantly by anethole (94.2%). F. vulgare oil is also reported to contain mainly anethole [11]. The dominant components in C. carvi oil were estragole (65.0%), limonene (14.3%), -pinene (7.4%) and trans-pinocamphone (4.3%). Limonene and carvone were reported as the main components [12] of caraway oil and also our study confirmed Molecules 2010, 15 4311 limonene as one of the most abundant components of this oil. Vervain essential oil was mainly constituted by citral and isobornyl formate. A previous study reported a different composition for vervain oil: Ardakani and coworkers [13] identified 3-hexen-1-ol, 1-octen-3-ol, linalol, verbenone and geranial as its major components. In general, the composition of Lamiaceae oils agrees with the available literature. -Pinene (18.2%), iso-pinocamphone (29.1%), and trans-pinocamphone (11.2%) were the most abundant components of H. officinalis essential oil. This composition agrees with the available literature [14]. Linalol (23.1%) and linalyl acetate (44.4%) represented the main components of the oil of L. angustifolia; also in this case the composition is similar to data reported in literature [15]. Marjoram essential oil was mainly constituted by 1,8-cineole (33.5%), -pinene (9.0%) and limonene (6.4%). The main constituents of M. officinalis essential oil were (-)-citronellal (39.6%), carvacrol (13.3%) and iso-menthone (8.8%); iso-pinocamphone (35.1%) and carvone (39.7%) were the predominant components of O. basilicum essential oil. The compositions of the latter oils agree with literature data [16,17]. In O. vulgare and T. vulgaris oils, o-cymene and carvacrol were the main constituents, accounting, respectively, for 41.9% and 44.0%, in oregano, and 56.2% and 24.4% in thyme oil. The oregano oil appears to be in part different from others reported in literature: in fact, some papers reported p-cymene as the main compound of this oil [18]. Differences were also reported for the composition of thyme oil [2,16]. Sage essential oil was mainly constituted by trans-thujone (37.9%), camphor (13.9%) and borneol (7.6%) and this composition agrees with literature reports [19]. A comparison among the chemical groups present in essential oils was performed in order to verify the similarity of the oil composition among the different plants (Table 2). The components of the oils were divided into 5 chemical groups: alcohols, aldehydes, alkenes, ketones and phenols. Balm and vervain oils were characterized by a high presence of aldehydes, about 39% and 44% of the total oil, respectively. Lavender was characterized by a strong presence of alcohols (41.1%), while marjoram was characterized by a high presence of alkenes (14.8%). Thyme and oregano oils belong to the same group, as determined by the presence of phenols, that represent about 33.1% and 44.8% of the total oil composition, respectively. Hyssop, sage and basil oils belong to the group characterized by the presence of ketones (about 41%, 76% and 52%, respectively). Finally, caraway was characterized by the presence of estragole, while the other two apiaceous oils (fennel and anise) are characterized by the high presence of anethole (77.1% and 97.1%, respectively). Monoterpenes were the most abundant components of all the oils analysed, except for the fennel and anise oils, representing a percentage ranging between 82.3%, in the hyssop oil and 97.4%, in the oil of thyme. Among monoterpenes, oxygenated compounds were in amounts ranging between 47.4% (oregano oil) and 91.2% (vervain). Sesquiterpenes were in lower amounts in all the oils. On the other hand, the oils of anise and fennel were mainly constituted of non terpenes ranging between 97.1%, in the anise oil, and 76.3%, in fennel. In general, a high presence of oxygenated monoterpenes is linked to a potent phytotoxic activity [20]. Vokou and coworkers [21] studied the allelopathic activities of 47 monoterpenoids belonging to different chemical groups, estimating their effects on seed germination and subsequent growth of Lactuca sativa seedlings and found that the most active compounds against both processes belonged to the groups of ketones and alcohols, followed by the group of aldehydes and phenols. Our data agree with this finding: all oils were active against germination and early radicle growth of Lepidium sativum, Raphanus sativus and Lactuca sativa, but at different levels of activity (Tables 3, 4 and 5). Molecules 2010, 15 4312 Table 1. Chemical composition of the studied essential oils. Compound Kia Kib Anise %c Balm % Basil % Caraway % Fennel % Hyssop % Lavender % Marjoram % Oregano % Sage % Thyme % Vervain % Identification d -Thujene -Pinene (-)-Camphene Sabinene Hepten-3-one -Pinene cis-Pinane Verbenene Myrcene -Phellandrene 3-Carene -Terpinene o-Cymene p-Cymene -Phellandrene Limonene 1,8-Cineole (Z)- -Ocimene (E)- -Ocimene -Terpinene cis-Sabinene hydrate cis-Linalol oxide Fenchone Terpinolene Linalol endo-Fenchol cis-Thujone trans-Thujone trans-Pinocarveol (-)-Citronellal iso-Borneol Camphor Menthofuran iso-Pinocamphone trans-Pinocamphone Lavandulol iso-Menthone Pinocarvone Borneol Terpinen-4-ol dihydro-Carveol 930 938 953 973 975 978 980 982 993 995 997 1012 1020 1024 1029 1030 1034 1038 1049 1057 1063 1065 1067 1086 1097 1098 1105 1115 1138 1143 1144 1145 1150 1153 1159 1162 1163 1165 1167 1176 1177 1035 1032 1076 1132 --0.3 ± 0.0 --T ----------0.1 ± 0.0 0.1 ± 0.0 --0.1 ± 0.0 --T ----T --T ----0.2 ± 0.0 T 0.4 ± 0.1 --------------------------------- 0.1±0.0 0.9 ± 0.0 --T T 0.4 ± 0.1 --T 0.1 ± 0.0 T --0.1 ± 0.1 2.3 ± 0.9 0.6 ± 0.0 0.3 ± 0.0 1.4 ± 0.3 0.2 ± 0.0 T T 0.4 ± 0.0 ------0.1 ± 0.0 0.7 ± 0.1 --T ----39.6 ± 0.4 0.5 ± 0.0 1.1 ± 0.0 --T 0.4 ± 0.0 --8.8 ± 0.9 T 0.1 ± 0.0 0.1 ± 0.0 --- T 0.3 ± 0.0 --0.3 ± 0.0 --0.5 ± 0.0 0.1 ± 0.0 T 0.3 ± 0.1 T --T 0.1 ± 0.0 --0.3 ± 0.0 0.4 ± 0.0 0.5 ± 0.1 0.1 ± 0.0 1.2 ± 0.0 T ----0.4 ± 0.1 0.1 ± 0.1 0.7 ± 0.0 0.2 ± 0.0 ----T ----0.6 ± 0.0 --35.1 ± 0.0 T ----0.4 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 --- 0.2 ± 0.0 0.5 ± 0.2 --1.0 ± 0.1 --7.4 ± 0.4 0.1 ± 0.0 T 0.7 ± 0.1 T --T 0.2 ± 0.0 0.1 ± 0.1 0.6 ± 0.2 14.3 ± 0.5 0.1 ± 0.0 0.1 ± 0.0 0.3 ± 0.1 T ------T 0.5 ± 0.1 ----0.1 ± 0.0 T ----T --T 4.3 ± 0.9 --------T --- T 1.8 ± 0.1 --T --0.5 ± 0.1 --T 0.2 ± 0.1 0.3 ± 0.0 0.3 ± 0.1 T 0.7 ± 0.1 0.3 ± 0.0 0.4 ± 0.1 1.5 ± 0.5 T T T 0.1 ± 0.0 ----14.2 ± 0.4 T T ----T T ----T --T ----------T 0.3 ± 0.1 0.4 ± 0.0 1.0 ± 0.0 0.2 ± 0.0 1.4 ± 0.9 --18.2 ± 0.0 --0.1 ± 0.0 1.8 ± 0.2 T --0.2 ± 0.1 0.2 ± 0.0 --1.8 ± 0.2 1.3 ± 0.7 0.2 ± 0.0 0.3 ± 0.0 1.0 ± 0.0 0.2 ± 0.0 ------0.2 ± 0.0 1.0 ± 0.1 --0.1 ± 0.0 --0.1 ± 0.0 ------0.3 ± 0.0 29.1 ± 0.0 11.2 ± 0.9 4.4 ± 0.4 --0.5 ± 0.0 0.1 ± 0.0 0.3 ± 0.1 1.2 ± 0.1 0.2 ± 0.0 --0.7 ± 0.0 T ----0.1 ± 0.0 T 0.3 ± 0.0 0.2 ± 0.0 0.3 ± 0.1 T 0.6 ± 0.1 0.3 ± 0.0 0.1 ± 0.0 0.3 ± 0.0 T 1.7 ± 0.3 0.6 ± 0.1 T 0.3 ± 0.0 0.4 ± 0.1 --T 23.1 ± 0.2 --T --T ----0.9 ± 0.0 --0.1 ± 0.0 ------T 6.3 ± 0.9 0.2 ± 0.0 0.4 ± 0.0 0.1 ± 0.0 9.0 ± 0.1 0.3 ± 0.0 1.1 ± 0.1 T 3.8 ± 0.9 --T 0.7 ± 0.3 0.2 ± 0.0 0.3 ± 0.0 0.1 ± 0.0 2.6 ± 0.9 0.4 ± 0.1 9.1 ± 0.5 6.4 ± 0.5 33.5 ± 0.3 0.1 ± 0.0 0.2 ± 0.1 0.8 ± 0.3 ------0.2 ± 0.1 9.8 ± 0.7 ----T 0.1 ± 0.0 --0.1 ± 0.0 0.2 ± 0.0 --0.2 ± 0.0 T ----T 2.0 ± 0.5 0.4 ± 0.1 0.8 ± 0.1 0.5 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 T --0.2 ± 0.0 0.1 ± 0.0 T 0.5 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 0.5 ± 0.0 41.9 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 0.3 ± 0.0 0.6 ± 0.1 T T 2.8 ± 0.2 0.2 ± 0.0 ----0.1 ± 0.0 0.7 ± 0.3 --T --T ----T --0.1 ± 0.0 T ----T 0.3 ± 0.0 0.4 ± 0.0 --- 0.4 ± 0.0 4.4 ± 0.1 4.1 ± 0.0 0.4 ± 0.0 --2.5 ± 0.1 --T 0.5 ± 0.1 T --T 2.5 ± 0.2 1.2 ± 0.1 1.0 ± 0.0 1.4 ± 0.0 4.2 ± 0.3 T T 0.1 ± 0.0 0.1 ± 0.0 ----T 1.1 ± 0.06 ----37.9 ± 0.1 0.2 ± 0.0 0.2 ± 0.0 --13.9 ± 0.7 --0.1 ± 0.0 0.3 ± 0.0 ----T 7.6 ± 0.4 0.5 ± 0.0 0.2 ± 0.0 T 2.5 ± 0.1 1.0 ± 0.1 T ------T 0.1 ± 0.0 T --0.1 ± 0.0 56.2 ± 0.2 0.1 ± 0.0 0.2 ± 0.1 0.6 ± 0.0 T T T 0.4 ± 0.0 ------0.7 ± 0.1 0.4 ± 0.1 --T --T 0.5 ± 0.1 0.1 ± 0.0 T --T T --0.1 ± 0.0 T 0.2 ± 0.0 T 0.2 ± 0.0 --0.2 ± 0.0 --0.5 ± 0.0 0.2 ± 0.1 T ----------T 0.1 ± 0.0 --0.7 ± 0.2 2.3 ± 0.9 0.4 ± 0.1 T 0.3 ± 0.1 0.1 ± 0.0 ------T 0.1 ± 0.0 ------T --------0.2 ± 0.0 T ----T 0.1 ± 0.0 0.2 ± 0.0 --- 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2 1118 1073 1131 1174 1176 1153 1188 1187 1280 1218 1203 1213 1246 1280 1255 1556 1450 1392 1265 1553 1120 1430 1449 1654 1491 1633 1532 1502 1566 1160 1674 1503 1587 1719 1611 1755 Molecules 2010, 15 4313 Table 1. Cont. p-Cymen-8-ol -Terpineol Myrtenal Estragole Myrtenol Citronellol cis-Carveol Isobornyl formate Carvone Linalyl acetate Geraniol cis-Anethole (E)-Citral Isobornyl acetate Bornyl acetate Cinnamic acid methyl ester Thymol Carvacrol Myrtenyl acetate Terpinyl acetate Eugenol Citronellyl acetate Methyl eugenol -Copaene Geranyl acetate Isoledene -Bourbonene -Elemene -Gurjunene Longifolene -Caryophyllene -Cedrene Aromadendrene -Humulene allo-Aromadendrene Neryl isobutyrate -Gurjunene cis- -Guaiene Bicyclogermacrene cis-Muurola-4(14),5-diene -7-epi-Selinene Caryophyllene oxide -Cadinol Total compounds 1185 1189 1193 1195 1196 1213 1226 1228 1241 1248 1255 1262 1270 1277 1284 1289 1290 1297 1313 1333 1353 1358 1369 1377 1379 1382 1385 1387 1408 1411 1418 1424 1437 1455 1463 1468 1473 1490 1491 1510 1518 1580 1652 1864 1706 1648 1670 1804 1772 1878 1596 1752 1565 1857 1780 1727 1591 2198 2239 1698 1709 2186 1662 2023 1497 1765 1367 1535 1600 1529 1576 1612 1638 1628 1689 1661 1870 1687 1694 1756 1675 1740 2008 2255 --T ------------------97.1 ± 0.4 ------------------------------------T --T --------------------98.3 T 0.1 ± 0.0 ------6.2 ± 0.3 ------2.3 ± 0.3 5.7 ± 0.3 ----T T --0.1 ± 0.0 13.3 ± 0.9 ----0.5 ± 0.0 1.6 ± 0.9 T T 1.7 ± 0.3 T --0.6 ± 0.0 0.4 ± 0.0 0.9 ± 0.1 0.6 ± 0.0 0.3 ± 0.0 T 0.2 ± 0.0 T --0.2 ± 0.0 0.1 ± 0.0 --2.3 ± 0.5 0.6 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 96.3 --1.3 ± 0.3 1.0 ± 0.0 --0.6 ± 0.0 --0.1 ± 0.0 --39.7 ± 0.9 0.4 ± 0.0 ------T T 0.1 ± 0.0 --T 0.5 ± 0.0 ------0.5 ± 0.0 0.1 ± 0.0 --0.1 ± 0.0 1.2 ± 0.3 0.1 ± 0.0 0.4 ± 0.0 0.5 ± 0.0 1.4 ± 0.5 0.5 ± 0.0 T 0.5 ± 0.0 1.2 ± 0.5 --0.5 ± 0.0 --1.5 ± 0.0 3.0 ± 0.9 0.1 ± 0.0 ----97.3 --T 0.1 ± 0.0 65.0 ± 0.9 --------------T --0.1 ± 0.0 0.1 ± 0.0 ------T ------0.6 ± 0.1 T --T --0.2 ± 0.0 ----0.1 ± 0.0 --0.2 ± 0.0 T T ----0.4 ± 0.2 T 0.1 ± 0.0 T --0.6 ± 0.1 98 T --0.1 ± 0.0 0.8 ± 0.1 --------------76.3 ± 0.9 ----------T --------T T --T --T --T T --T T T --T ----T T ----97.8 T 1.2 ± 0.1 1.0 ± 0.3 0.4 ± 0.0 1.3 ± 0.5 --------0.3 ± 0.0 --0.3 ± 0.0 ----T --T T 0.6 ± 0.0 ------0.7 ± 0.0 0.1 ± 0.0 --0.1 ± 0.0 1.3 ± 0.3 T 0.5 ± 0.0 0.5 ± 0.0 1.0 ± 0.5 0.6 ± 0.0 T 0.6 ± 0.0 1.4 ± 0.2 --0.1 ± 0.0 0.4 ± 0.0 3.1 ± 0.5 3.7 ± 0.9 0.1 ± 0.0 --0.3 ± 0.0 96.4 0.3 ± 0.0 0.4 ± 0.0 0.4 ± 0.1 --0.4 ± 0.0 --------44.4 ± 0.7 9.3 ± 0.3 ----0.3 ± 0.0 0.2 ± 0.0 --------------T T --T ------T 1.0 ± 0.9 1.3 ± 0.1 T 0.6 ± 0.0 0.5 ± 0.0 0.1 ± 0.0 ------0.3 ± 0.0 --0.4 ± 0.0 --97.0 0.1 ± 0.0 0.7 ± 0.1 0.7 ± 0.1 0.1 ± 0.0 0.2 ± 0.1 --------3.3 ± 0.6 0.6 ± 0.1 ----0.6 ± 0.1 1.2 ± 0.5 --0.7 ± 0.1 4.1 ± 0.9 T 0.5 ± 0.0 ------0.1 ± 0.0 --T --T --0.1 ± 0.0 0.3 ± 0.1 0.5 ± 0.1 T 0.3 ± 0.1 T --0.1 ± 0.0 --0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 ----97 0.2 ± 0.0 T --0.1 ± 0.0 ----------0.1 ± 0.0 ------T T --0.7 ± 0.0 44.0 ± 0.9 T ------T 0.1 ± 0.0 --0.1 ± 0.0 --T --T 0.2 ± 0.1 0.6 ± 0.0 T 0.1 ± 0.0 T --0.1 ± 0.0 ----T 0.1 ± 0.0 0.2 ± 0.0 --96.9 0.1 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 T 0.2 ± 0.0 --------1.5 ± 0.2 0.3 ± 0.0 ----0.7 ± 0.0 0.88 ± 0.0 --T 0.3 ± 0.0 T --------T --T ------T 1.3 ± 0.0 1.0 ± 0.0 0.1 ± 0.0 5.9 ± 0.9 0.1 ± 0.0 --0.1 ± 0.0 ----T 0.1 ± 0.0 0.8 ± 0.0 --98.7 T 0.3 ± 0.0 0.3 ± 0.0 --0.3 ± 0.0 ----------------T T --8.7 ± 0.9 24.4 ± 0.9 ----------T --T --T --T 0.1 ± 0.0 --T T T --T ----T T ----97.5 T 0.3 ± 0.1 ----------45.4 ± 0.9 ------0.2 ± 0.0 44.5 ± 0.9 T T --------------T 0.2 ± 0.1 --0.1 ± 0.0 --0.2 ± 0.1 --T 0.1 ± 0.1 0.4 ± 0.1 --0.2 ± 0.0 0.1 ± 0.0 --T --0.1 ± 0.0 0.2 ± 0.1 0.2 ± 0.1 ----97.6 1, 2 1, 2, 3 1, 2 1, 2, 3 1, 2 1, 2, 3 1, 2 1, 2 1, 2, 3 1, 2, 3 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2, 3 1, 2, 3 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 Molecules 2010, 15 4314 Table 1. Cont. Monoterpene hydrocarbons Oxygenated Monoterpenes Sesquiterpene hydrocarbons Oxygenated Sesquiterpenes Non terpenes 0.6 0.6 0 0 97.1 6.7 83 6.2 0.4 0 3.7 82.4 11.1 0 0.2 25.5 70.9 1 0.6 0 6.1 15.4 0 0 76.3 28.3 54 13.5 0.3 0.3 5.4 87.2 3.7 0.4 0 35.4 59.9 1.7 0 0 48 47.4 1.3 0.2 0 18.5 70.1 8.6 0.8 0 61.9 35.5 0.1 0 0 4.2 91.2 1.8 0 0.4 The analyses were carried out in triplicate; a: Kovats retention index on HP-5 MS column; b: Kovats retention index on HP Innowax; c --- = absent; t = trace, less than 0.05%; d: Identification based on: 1 = Kovats retention index, 2 = mass spectrum, 3 = coinjection with authentic compound. Table 2. Percentage composition of twelve essential oils on the basis of their chemical groups. Plant Anise Balm Basil Caraway Fennel Hyssop Lavender Marjoram Oregano Sage Thyme Vervain Alcohols Aldehydes 0.4 0 13.9 39.6 3.3 1 0.5 0.1 0.3 0.1 9.6 1 41.1 0.4 14.8 0.7 1.8 0 10.6 0.4 1.5 0.8 0.7 44.5 Alkenes Ketones Phenols 0.5 0.2 97.1 10.2 10.3 13.4 14.6 76.2 0 26.1 4.4 65 5.1 14.2 77.1 41.6 40.9 0.7 8.5 1 0 34 0.4 4.9 7.4 0.1 44.8 24.2 52.2 0.3 5.7 0.1 33.1 5.7 0.4 0.2 Table 3. Effects of different doses of essential oils on germination and radicle elongation of Lepidium sativum. The data are expressed as mean of three replicates ± SE. Germination (number of seeds) Control Anise Balm Basil Caraway ( g/mL) 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 10.0 ± 0.0 9.7 ± 0.6 10.0 ± 0.0 9.7 ± 0.6 9.7 ± 0.6 9.3 ± 1.2 8.7 ± 0.6 9.0 ± 1.0 9.3 ± 0.6 9.0 ± 1.0 9.7 ± 0.6 9.3 ± 0.6 0.06 Fennel Hyssop Lavender Marjoram Oregano Sage Thyme Vervain Molecules 2010, 15 4315 Table 3. Cont. 0.125 10.0 ± 0.0 9.0 ± 0.0 9.3 ± 0.6 8.3 ± 0.6 9.7 ± 0.6 9.7 ± 0.6 9.3 ± 0.6 9.0 ± 1.0 8.3 ± 2.1 9.0 ± 1.0 9.7 ± 0.6 8.3 ± 1.2 0.25 8.7 ± 0.6 9.0 ± 1.7 9.7 ± 0.6 8.3 ± 1.2 9.3 ± 0.6 10.0 ± 0.0 9.3 ± 0.6 8.7 ± 1.2 6.3 ± 4.7 9.7 ± 0.6 7.7 ± 0.6* 0.0 ± 0.0*** 0.625 9.7 ± 0.6 5.3 ± 1.2*** 9.7 ± 0.6 3.7 ± 2.1* 9.3 ± 0.6* 9.3 ± 0.6 9.7 ± 0.6 10.0 ± 0.0 7.3 ± 2.1 9.3 ± 0.6 4.0 ± 1.7** 9.0 ± 1.0 1.25 8.0 ± 1.0 0.3 ± 0.6*** 8.3 ± 1.2 0.7 ± 1.2*** 9.0 ± 1.0 8.7 ± 0.6 8.3 ± 1.2 8.7 ± 0.6 0.0 ± 0.0*** 8.3 ± 1.5 0.0 ± 0.0*** 8.7 ± 0.6 6.3 ± 1.5* 0.3 ± 0.6*** 1.3 ± 1.5** 0.0 ± 0.0*** 0.0 ± 0.0*** 2.5 0.7 ± 1.2*** 0.0 ± 0.0*** 3.3 ± 3.5* 0.0 ± 0.0*** 4.3 ± 2.3* 0.0 ± 0.0*** 1.3 ± 2.3** Radicle growth (length of seeds) Control Anise Balm Basil Caraway Fennel Hyssop Lavender Marjoram Oregano Sage Thyme Vervain ( g/mL) 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 6.1 ± 1.3 0.06 7.6 ± 0.3 4.5 ± 0.4 3.7 ± 0.5* 3.4 ± 0.4* 5.7 ± 0.5 5.9 ± 1.3 4.5 ± 0.3 2.3 ± 0.5* 3.8 ± 0.9 3.9 ± 0.3* 4.3 ± 0.1 3.8 ± 0.4* 0.125 5.3 ± 0.1 4.2 ± 0.5 6.1 ± 0.8 2.3 ± 0.1** 6.0 ± 0.5 4.6 ± 0.4 5.1 ± 0.2 1.6 ± 0.3** 2.6 ± 0.2* 2.9 ± 0.3* 3.0 ± 0.4* 1.9 ± 0.5** 0.25 3.4 ± 0.6* 1.0 ± 0.7** 4.5 ± 0.9 1.9 ± 0.3** 4.9 ± 0.3 3.5 ± 0.3* 2.1 ± 0.4** 1.4 ± 0.5** 1.0 ± 0.8** 2.3 ± 0.4** 1.1 ± 0.2** 0.0 ± 0.0** 0.625 4.4 ± 0.9 0.4 ± 0.2** 5.8 ± 0.7 0.3 ± 0.1** 5.0 ± 0.7 3.7 ± 0.4* 3.7 ± 0.5* 1.9 ± 0.3** 0.9 ± 0.3** 0.9 ± 0.2** 0.2 ± 0.1** 2.8 ± 0.4* 1.25 3.1 ± 0.2* 0.0 ± 0.1** 3.4 ± 0.3* 0.1 ± 0.1** 3.4 ± 1.1 2.8 ± 0.3* 3.1 ± 0.3* 0.9 ± 0.3** 0.0 ± 0.0** 0.4 ± 0.1** 0.0 ± 0.0** 1.1 ± 0.2** The values, followed by * (*p < 0.05; ** p < 0.01, *** p < 0.001), are statistically different according to the Student’s t test. Table 4. Effects of different doses of essential oils on germination and radicle elongation of Raphanus sativus. The data are expressed as mean of three replicates ± SE. Germination (number of seeds) Control ( g/mL) Anise Balm Basil Caraway Fennel Hyssop Lavender Marjoram Oregano Sage Thyme Vervain 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 9.1 ± 1.1 0.06 9.3 ± 0.6 7.7 ± 2.1 8.7 ± 1.5 2.7 ± 2.1*** 8.7 ± 1.2 6.0 ± 0.0*** 8.7 ± 1.5 8.3 ± 0.6 8.3 ± 0.6 6.0 ± 2.6** 8.0 ± 1.0 0.7 ± 1.2*** 0.125 8.7 ± 0.6 7.7 ± 0.6 9.3 ± 1.2 0.0 ± 0.0*** 8.3 ± 0.6 2.3 ± 0.6*** 6.0 ± 1.7** 8.7 ± 0.6 1.0 ± 1.0*** 6.7 ± 1.5** 6.7 ± 1.2** 0.0 ± 0.0*** 0.25 9.0 ± 0.0 2.0 ± 1.0*** 8.0 ± 1.0 3.3 ± 5.8** 7.3 ± 1.2* 0.0 ± 0.6*** 5.0 ± 0.0*** 6.3 ± 2.3** 0.0 ± 0.0*** 4.7 ± 1.2*** 1.3 ± 1.2*** 0.0 ± 0.0*** 0.625 8.7 ± 0.6 0.0 ± 0.0*** 8.3 ± 1.2 0.0 ± 0.0*** 7.0 ± 1.0** 0.7 ± 1.2*** 0.0 ± 0.0*** 5.7 ± 1.5*** 0.0 ± 0.0*** 2.0 ± 2.0*** 0.0 ± 0.0*** 0.0 ± 0.0*** 1.25 9.0 ± 1.0 0.0 ± 0.0*** 8.0 ± 1.7 0.0 ± 0.0*** 5.7 ± 2.3** 0.0 ± 0.0*** 0.0 ± 0.0*** 3.3 ± 2.3*** 0.0 ± 0.0*** 0.7 ± 0.6*** 0.0 ± 0.0*** 0.0 ± 0.0*** 2.5 8.0 ± 1.7 0.0 ± 0.0*** 5.7 ± 2.5** 0.0 ± 0.0*** 5.0 ± 2.0*** 0.0 ± 0.0*** 0.0 ± 0.0*** 0.0 ± 0.0*** 0.0 ± 0.0*** 0.0 ± 0.0*** 0.0 ± 0.0*** 0.0 ± 0.0*** Control ( g/mL) Anise Balm Basil Caraway Fennel Oregano Sage Thyme Vervain 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.9 0.06 3.2 ± 0.2 2.5 ± 0.3 2.9 ± 0.1 1.5 ± 0.6* Radicle growth (length of seeds) Hyssop Lavender Marjoram 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.9 3.0 ± 0.9 2.7 ± 0.8 1.6 ± 0.4* 2.7 ± 0.3 2.3 ± 0.4 1.6 ± 0.3* 2.2 ± 0.4 1.7 ± 0.7* 0.2 ± 0.3*** Molecules 2010, 15 4316 Table 4. Cont. 0.125 3.1 ± 0.1 1.8 ± 0.4 2.8 ± 0.6 0.0 ± 0.0*** 2.3 ± 0.3 1.4 ± 0.7* 2.5 ± 0.2 2.8 ± 0.3 0.5 ± 0.5*** 2.1 ± 0.3 0.8 ± 0.2** 0.0 ± 0.0*** 0.25 2.9 ± 0.2 0.3 ± 0.2*** 2.6 ± 0.1 0.3 ± 0.6*** 2.9 ± 1.4 0.9 ± 1.3** 2.3 ± 0.2 2.1 ± 0.6 0.0 ± 0.0*** 1.6 ± 0.4* 0.7 ± 0.6*** 0.0 ± 0.0*** 0.625 2.7 ± 0.2 0.0 ± 0.0*** 2.3 ± 0.4 0.0 ± 0.0*** 2.3 ± 0.4 0.4 ± 0.7*** 0.0 ± 0.0*** 1.7 ± 0.2* 0.0 ± 0.0*** 0.9 ± 0.8** 0.0 ± 0.0*** 0.0 ± 0.0*** 1.25 2.0 ± 0.1 0.0 ± 0.0*** 2.7 ± 0.5 0.0 ± 0.0*** 1.9 ± 0.3 0.0 ± 0.0*** 0.0 ± 0.0*** 1.3 ± 0.4** 0.0 ± 0.0*** 0.5 ± 0.5*** 0.0 ± 0.0*** 0.0 ± 0.0*** The values, followed by * (*p < 0.05; ** p < 0.01, *** p < 0.001), are statistically different according to the Student’s t test. Table 5. Effects of different doses of essential oils on germination and radicle elongation of Lactuca sativa. The data are expressed as mean of three replicates ± SE. Germination (number of seeds) Control ( g/mL) Anise Balm Basil Caraway Fennel Hyssop Lavender Marjoram Oregano Sage Thyme Vervain 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 5.6 ± 1.5 0.06 6.7 ± 1.2 1.7 ± 0.6** 5.3 ± 2.5 0.0 ± 0.0*** 5.3 ± 1.2 5.3 ± 1.5 4.3 ± 2.5 3.0 ± 1.7 7.7 ± 0.6 4.0 ± 2.0 0.0 ± 0.0*** 0.3 ± 0.6** 0.125 6.3 ± 1.2 1.0 ± 1.0** 5.7 ± 1.5 2.3 ± 0.6* 7.7 ± 2.3 3.7 ± 1.2 5.0 ± 2.6 5.0 ± 1.7 3.7 ± 2.9 1.3 ± 1.5** 0.0 ± 0.0*** 0.7 ± 1.2** 0.25 8.7 ± 1.2* 0.0 ± 0.0*** 3.0 ± 1.7 0.3 ± 0.6** 6.7 ± 2.1 3.7 ± 1.5 2.7 ± 1.2* 4.7 ± 0.6 0.3 ± 0.6** 2.0 ± 1.0* 0.0 ± 0.0*** 1.0 ± 1.7** 0.625 5.7 ± 0.6 0.0 ± 0.0*** 4.0 ± 1.0 0.3 ± 0.6** 7.7 ± 1.5 3.3 ± 3.2 0.3 ± 0.6** 1.3 ± 0.6** 0.0 ± 0.0*** 0.3 ± 0.6** 0.0 ± 0.0*** 0.0 ± 0.0*** 1.25 8.7 ± 0.6 0.0 ± 0.0*** 4.7 ± 0.6 0.0 ± 0.0*** 6.0 ± 2.6 4.7 ± 2.1 0.7 ± 0.6** 0.0 ± 0.0*** 0.0 ± 0.0*** 1.0 ± 1.7** 0.0 ± 0.0*** 0.0 ± 0.0*** 2.5 1.0 ± 1.0* 0.0 ± 0.0*** 1.7 ± 2.1* 0.0 ± 0.0*** 5.3 ± 3.1 0.0 ± 0.0*** 0.7 ± 0.6** 0.0 ± 0.0*** 0.0 ± 0.0*** 0.3 ± 0.6** 0.0 ± 0.0*** 0.0 ± 0.0*** Radicle growth (length of seeds) Control ( g/mL) Anise Balm Basil Caraway Fennel Hyssop Lavender 1.2 ± 0.2 1.2 ± 0.2 0.06 2.0 ± 0.3** 0.7 ± 0.9 0.125 1.3 ± 0.1 Marjoram Oregano 1.2 ± 0.2 1.2 ± 0.2 1.2 ± 0.2 1.2 ± 0.2 1.4 ± 0.3 0.0 ± 0.0*** 1.6 ± 0.3 0.9 ± 0.3 0.2 ± 0.2*** 0.7 ± 0.1** 0.3 ± 0.1*** 1.4 ± 0.3 0.3 ± 0.1*** 0.5 ± 0.2** 0.2 ± 0.1*** 1.2 ± 0.2 1.2 ± 0.2 1.2 ± 0.2 1.2 ± 0.2 0.9 ± 0.6 0.5 ± 0.1** 1.3 ± 0.4 0.9 ± 0.2 0.0 ± 0.0*** 0.1 ± 0.1*** 0.3 ± 0.1*** 0.4 ± 0.1*** 0.3 ± 0.3** 0.0 ± 0.0*** 0.1 ± 0.1*** Thyme Vervain 1.2 ± 0.2 1.2 ± 0.2 0.4 ± 0.2** 0.6 ± 0.3* 0.0 ± 0.0*** 0.1 ± 0.1*** 0.25 1.3 ± 0.4 0.0 ± 0.0*** 0.4 ± 0.2** 0.1 ± 0.1*** 0.7 ± 0.1** 0.4 ± 0.2** 0.625 1.1 ± 0.3 0.0 ± 0.0*** 0.6 ± 0.2** 0.1 ± 0.1*** 0.4 ± 0.1*** 0.1 ± 0.1*** 0.4 ± 0.1*** 0.0 ± 0.0*** 0.1 ± 0.2*** 0.0 ± 0.0*** 0.0 ± 0.0*** 1.25 1.8 ± 0.3* 0.0 ± 0.0*** 0.5 ± 0.1** 0.0 ± 0.0*** 0.7 ± 0.1** 0.2 ± 0.1*** 0.2 ± 0.2*** 0.2 ± 0.1*** 0.0 ± 0.0*** 0.1 ± 0.1*** 0.0 ± 0.0*** 0.0 ± 0.0*** 0.9 ± 0.1 0.2 ± 0.3*** Sage The values, followed by * (*p < 0.05; ** p < 0.01, *** p < 0.001), are statistically different according to the Student’s t test. Molecules 2010, 15 4317 The germination of Lepidium sativum was drastically affected by a 2.5 g/mL dose of the essential oils of balm, caraway, hyssop, thyme and vervain, with a 100% inhibition (Figure 1). Figure 1. Percent inhibition of germination of Lepidium sativum seeds treated with different doses of essential oils. Germination of cress seeds 100.0 60.0 0.06 0.125 0.25 0.625 1.25 2.5 40.0 20.0 vervain thyme sage oregano marjoram lavender fennel hyssop -20.0 caraway balm basil 0.0 anise % inhibition vs control 80.0 -40.0 Essential oils Thyme and oregano oils inhibited both germination and radicle elongation at a dose of 1.25 g/mL. Caraway, vervain, sage and marjoram essential oils affected, in a significative way, the radicle elongation of this seed, at all doses. Anise oil was the less active on germination, whereas fennel oil was less active on radicle elongation of garden cress. Moreover, some oils (anise, basil), at the lowest dose, promoted the germination and/or radicle elongation of garden cress. Generally, garden cress is the less sensitive seed. Almost all oils, except anise, basil and fennel, inhibited by 100% the germination of R. sativus, at the highest dose tested (Figure 2). Figure 2. Percent inhibition of germination of Raphanus sativus seeds treated with different doses of essential oils. Germination of radish seeds 100.0 % inhibition vs control 80.0 60.0 0.06 0.125 0.25 0.625 1.25 2.5 40.0 20.0 0.0 anise balm basil caraway fennel hyssop lavender -20.0 Essential oils marjoram oregano sage thyme vervain Molecules 2010, 15 4318 Vervain oil inhibited by 100% the germination of radish, at almost all doses tested. In addition, caraway, hyssop and sage oils inhibited, in a significative way, the germination of radish, at all doses tested. The radicle growth of the same seeds was affected by 100% by vervain, caraway, oregano, thyme, hyssop and lavender essential oils, at the three highest doses assayed (0.625, 1.25 and 2.5 g/mL). Moreover, all oils cited above, except lavender, were active towards radicle elongation, at all doses. The germination and radicle elongation of Lactuca sativa were affected by vervain, balm, caraway and oregano oils, resulting in a maximum inhibitory activities (100% inhibition), at doses of 1.25 and 2.5 g/mL (Figure 3). Figure 3. Percent inhibition of germination of Lactuca sativa seeds treated with different doses of essential oils. Germination of lattuce seeds 100.0 80.0 40.0 0.06 0.125 0.25 0.625 20.0 vervain thyme sage oregano marjoram lavender hyssop fennel caraway basil -20.0 balm 0.0 anise % inhibition vs control 60.0 1.25 2.5 -40.0 -60.0 -80.0 Essential oils Thyme oil inhibited by 100% germination and radicle elongation of lettuce seeds, at all assayed doses. Moreover, vervain, balm and caraway oils inhibited, significantly, germination of lettuce seeds, at all doses. Marjoram and vervain, and caraway again, inhibited, in a significative way, the radicle growth of the seeds. Also in this case, fennel and anise oils were among less active ones, against both germination and radicle elongation. As reported in this paper, some essential oils possess strong phytotoxic effect; this opens the door to their use as herbicides. Although few studies have addressed this herbicidal activity, the authors in [22] demonstrated that some essential oils, including thyme oil, are highly phytotoxic [23]. Kordali and coworkers [24] reported that the herbicidal effects of oregano oil can be attributed to its major component, carvacrol. Moreover, it has been documented that some essential oils isolated and their phenolic compounds, carvacrol and thymol, possess potent herbicidal effects on weed germination and seedling growth of various plant species [2,25]. Some doses of the same oil are inhibitory, other stimulatory. The concept of a generalized “lowdose stimulation - high-dose inhibition” or “hormesis” was gradually supported by field observations [26]. There is evidence that exposure to novel environments or a toxic substance increases the variance of phenotypic traits such as enzyme activity [27], morphological features [28] and growth [29]. However, the reasons for such increases and their adaptive implications remain unclear [30]. Molecules 2010, 15 4319 Dudai and coworkers [31] reported that monoterpenes act on seeds at very low levels, and that their content in various parts of wheat seeds differs. In particular, among the Lamiaceae family, many species release phytotoxic monoterpenes that hinder the development of herbaceous species among which -pinene, limonene, p-cymene, 1,8-cineole [2]. In previous studies, plants exposed to monoterpene vapour have shown severe internal damage. The absence of a variety of intact organelles and the presence of membrane fragments indicate that structural breakdown and decomposition occur within inhibited roots [32]. Some of the most inhibitory compounds have been repeatedly reported as phytotoxic against a number of target species [33,34], though not always with the same level of activity. Reynolds [34], also working with L. sativa, compared a large number of compounds belonging to different chemical groups as to their effect on seed germination and early seedling development. Moreover, it is well known that monoterpenes in the essential oils have phytotoxic effects that may cause anatomical and physiological changes in plant seedlings leading to accumulation of lipid globules in the cytoplasm, reduction in some organelles such as mitochondria, possibly due to inhibition of DNA synthesis or disruption of membranes surrounding mitochondria and nuclei [35,36]. 3. Experimental Section 3.1. Plant material Plants of Hyssopus officinalis, Lavandula angustifolia, Majorana hortensis, Melissa officinalis, Ocimum basilicum, Origanum vulgare, Salvia officinalis and Thymus vulgaris, Verbena officinalis, Pimpinella anisum, Foeniculum vulgare and Carum carvi were grown at the Garden of Medicinal Plants in Salerno, State University Campus. Samples from the above plant species were collected at full flowering stage, in July-August 2008. Vouchers specimens of each plant were deposited in the herbarium of the Medical Botany Chair, Faculty of Pharmacy, Salerno University. 3.2. Oil isolation Five-hundred g of freshly picked aerial parts of each lamiaceous species, aerial parts of vervain, and fruits of each apiaceous species, were cut into small pieces and then subjected to hydrodistillation for 3 h, following the procedure described in the European Pharmacopoeia [37]. Extraction procedure was repeated three times, on three samples of the same drug. 3.3. GC and GC-MS analyses Essential oils were analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). GC analyses were performed using a Perkin-Elmer Sigma-115 gas chromatograph with a data handling system and a FID. Analyses were carried out using a DB-1 fusedsilica column (30 m × 0.25 mm i.d; 0.25 m film thickness). The operating conditions were as follows: injector and detector temperatures, 250 and 280 ºC, respectively; oven temperature programme, 5 min isothermal at 40 ºC, then at 2 ºC/min up to 250 ºC and finally held isothermally for 20 min. Aliquots of 1 L were injected manually at 250 ºC and in the splitless mode. Analysis was also run by using a Molecules 2010, 15 4320 fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm i.d.; 0.20 m film thickness). In both cases, helium was used as the carrier gas (1 mL/min). Diluted samples (1/100 v/v, in n-hexane) of 1 L were injected manually at 250 ºC and in the splitless mode. GC–MS analyses were carried out using a Hewlett-Packard 5890A gas chromatograph connected on line to a HP Mass Selective Detector (MSD 5970 HP), equipped with a HP-1 fused-silica column (25 m × 0.25 mm i.d.; 0.33 m film thickness); GC and GC-MS analyses: ionization voltage 70; electron multiplier energy 2000 V. Gas chromatographic conditions were as reported above; transfer line was kept at 295 ºC. Most components of the essential oils were identified on the basis of their GC retention indices or of their MS spectra that were compared either with those reported in literature [38,39] either with those stored in NBS and Wiley5 libraries or with those of standard compounds available in our laboratories and purchased from Sigma Aldrich, Co. Milan, Italy. The retention indices were determined in relation to a homologous series of n-alkanes (C8-C24) under the same operating conditions. Relative concentrations of each essential oil component were calculated on the basis of GC peaks without using correction factors. 3.4. Biological assay A bioassay based on germination and subsequent radicle growth was used to study the phytotoxic effects of the essential oils on seeds of Raphanus sativus, Lactuca sativa and Lepidium sativum L. The seeds were surface-sterilized in 95% ethanol for 15 s and sown in Petri dishes (Ø = 90 mm), containing five layers of Whatman filter paper, impregnated with distilled water (7 mL, control) or tested solution of the essential oil (7 mL), at the different assayed doses. The essential oils, in water–acetone mixture (99.5:0.5), were assayed at the follow doses: 2.5, 1.25, 0.625, 0.25, 0.125 and 0.06 g/mL. Controls performed with water–acetone mixture alone showed no appreciable difference in comparison with controls in water alone. The germination conditions were as follow: for radish and garden cress seeds, 20 ± 1 ºC, and for lettuce seeds, 24 ± 1 ºC, with natural photoperiod. Seed germination process was observed directly in Petri dishes, each 24 hours. A seed was considered germinated when the protrusion of the root became evident [40]. After 96 h (on the fourth day), the effects on radicle elongation were measured (the lengths were measured in centimeters). Each determination was repeated three times, using Petri dishes containing 10 seeds each. Data are expressed as the mean ± SE of both germination and radicle elongation. 3.5. Statistical analyses Data were ordered in homogeneous sets, and the Student’s t test of independence was applied [41]. 4. Conclusions Monoterpenes were the most abundant components of all the oils analysed, except for the fennel and anise oils. In particular, a high presence of oxygenated monoterpenes is related to a potent phytotoxic activity. Vokou and coworkers [21] studied the allelopathic activities of 47 monoterpenoids belonging to different chemical groups, estimating their effects on seed germination and subsequent Molecules 2010, 15 4321 growth of Lactuca sativa seedlings and found that the most active compounds against both processes belonged to the groups of ketones and alcohols, followed by the group of aldehydes and phenols. Our data agree with this finding: all oils were active against germination and early radicle growth of Lepidium sativum, Raphanus sativus and Lactuca sativa, but at different levels of activity. On the other hand, it appears confirmed, by our data in vitro, the potent biological activities of essential oils from aromatic plants of the Mediterranean ecosystem [9]. Future experiments, involving both essential oils and each of their components, could focus on the possible effects of the length of time during which such compounds are present in soil, possible structural modifications with consequent loss or acquisition of activity, and biological action on weed seeds in field conditions. However, the specific structural factors, that operate and determine the activity of monoterpenoid and essential oils, remain still obscure. References and Notes 1. Vyvyan, J.R. Allelochemicals as leads for news herbicides and agrochemicals. Tetrahedron 2002, 58, 1631-1646. 2. Angelini, L.G.; Carpanese, G.; Cioni, P.L.; Morelli, I.; Macchia, M.; Flamini, G. Essential oils from Mediterranean Lamiaceae as weed germination inhibitors. J. Agric. Food Chem. 2003, 51, 6158-6164. 3. Singh, H.P.; Kaur, S.; Mittal, S.; Batish, D.R.; Kohli, R.K. Essential oil of Artemisia scoparia inhibits plant growth by generating reactive oxygen species and causing oxidative damage. J. Chem. Ecol. 2009, 35, 154-162. 4. Tellez, M.R.; Kobaisy, M.; Duke, S.O.; Schrader, K.K.; Dayan, F.E.; Romagni, J. Terpenoidbased defense in plants and other organisms. In Lipid Technology; Kuo, T.M., Gardner, H.W., Eds; Marcel Dekker: New York, NY, USA, 2002; p. 354. 5. Duke, S.O.; Dayan, F.E.; Romagni, J.G.; Rimando, A.M. Natural products as sources of herbicides: current status and future trends. Weed Res. 2000, 40, 99-111. 6. Arminante, F.; De Falco, E.; De Feo, V.; De Martino, L.; Mancini, E.; Quaranta, E. Allelopathic activity of essential oils from Mediterranean Labiatae. Acta Hortic. 2006, 723, 347-352. 7. Azirak, S.; Karaman, S. Allelopathic effect of some essential oils and components on germination of weed species. Acta Agric. Scand. Sect. B 2008, 58, 88-92. 8. Duke, S.O.; Oliva, A. Mode of Action of Phytotoxic Terpenoids. In Allelopathy. Chemistry and Mode of Action of Allelochemicals; Macias, F.A., Galindo, J.C.G., Molinillo, J.M.G., Cutler, H.G., Eds.; CRC Press: Boca Raton, FL, USA, 2004; pp. 201-206. 9. Vokou, D. The Allelophatic Potential of Aromatic Shrubs in Phryganic (East Mediterranean) Ecosystems. In Allelopathy: Basic and Applied Aspects; Rizvi, S.J.H., Rizvi, V., Eds.; Chapman & Hale: London, UK, 1992; pp. 303-320. 10. Tabanca, N.; Demirci, B.; Ozek, T.; Kirimer, N.; Can Baser, K.H.; Bedir, E.; Khand, I.A.; Wedge, D.E. Gas chromatographic-mass spectrometric analysis of essential oils from Pimpinella species gathered from Central and Northern Turkey. J. Chromatogr. A 2006, 1117, 194-205. Molecules 2010, 15 4322 11. Singh, G.; Maurya, S.; de Lampasona, M.P.; Catalan C. Chemical constituents, antifungal and antioxidative potential of Foeniculum vulgare volatile oil and its acetone extract. Food Control 2006, 17, 745-752. 12. Bailer, J.; Aichinger, T.; Hackl, G.; de Hueber, K.; Dachler, M. Essential oil content and composition in commercially available dill cultivars in comparison to caraway. Ind. Crop Prod. 2001, 14, 229-239. 13. Ardakani, M.S.; Mosaddegh, M.; Shafaati, A. Volatile constituents from the aerial parts of Verbena officinalis L. (vervain). Iran. J. Pharm. Res. 2003, 2, 39-42. 14. Mazzanti, G.; Battinelli, L; Salvatore, G. Antimicrobial properties of the linalol-rich essential oil of Hyssopus officinalis L. var decumbens (Lamiaceae). Flavour Fragrance J. 1998, 13, 289-294. 15. Morgan, T.J.; Morden, W.E.; Al-Muhareb, E.; Herod, A.A.; Kandiyoti, R. Essential oils investigated by size exclusion chromatography and gas chromatography-mass spectrometry. Energy Fuels 2006, 20, 734-737. 16. Lee, S.J.; Umano, K.; Shibamoto, T.; Lee, K.G. Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem. 2005, 91, 131-137. 17. Chalchat, J.C.; Öezcan, M.M. Comparative essential oil composition of flowers, leaves and stems of basil (Ocimum basilicum L.) used as herb. Food Chem. 2008, 110, 501-503. 18. Veres, K.; Varga, E.; Dobos, A.; Hajdú, Z.S.; Máthé, I.; Németh, E.; Szabó, K. Investigation of the composition and stability of the essential oils of Origanum vulgare ssp. vulgare L. and O. vulgare ssp. hirtum (Link) Ietswaart. Chromatographia 2003, 57, 95-98. 19. Lima, C.F.; Carvalho, F.; Fernandes, E.; Bastos, M.L.; Santos-Gomes, P.C.; Fernandes-Ferreira, M.; Pereira-Wilson, C. Evaluation of toxic/protective effects of the essential oil of Salvia officinalis on freshly isolated rat hepatocytes. Toxicol. In Vitro 2004, 18, 457-465. 20. Kotan R.; Cakir, A.; Dadasoglu, F.; Aydin, T.; Cakmakci, R.; Ozer, H.; Kordali, S.; Mete, E.; Dikbas, N. Antibacterial activities of essential oils and extracts of Turkish Achillea, Satureja and Thymus species against plant pathogenic bacteria. J. Sci. Food Agric. 2010, 90, 145-160. 21. Vokou, D.; Douvli, P.; Blionis, G.J.; Halley, J.M. Effects of monoterpenoids, acting alone or in pairs, on seed germination and subsequent seedling growth. J. Chem. Ecol. 2003, 29, 2281-2301. 22. Tworkoski, T. Herbicide effects of essential oils. Weed Sci. 2002, 50, 425-431. 23. Isman M.B.; Machial C.M.; Miresmailli S.; Bainard L.D. Essential Oil-Based Pesticides: New Insights from Old Chemistry. Pestic. Chem. 2007, 201-209. 24. Kordali, S.; Cakir, A.; Ozer, H.; Cakmakci, R.; Kesdek, M.; Mete, E. Antifungal, phytotoxic and insecticidal properties of essential oil isolated fromTurkish Origanum acutidens and its three components, carvacrol, thymol and p-cymene. Bioresour. Technol. 2008, 99, 8788-8795. 25. Dudai, N.; Poljakoff-Mayber, A.; Mayer, A.M.; Putievsky, E.; Lerner, H.R. Essential oils as allelochemicals and their potential use as bioherbicides. J. Chem. Ecol. 1999, 25, 1079-1089. 26. Stebbing, A.R.D. Hormesis-the stimulation of growth by low levels of inhibitors. Sci. Total Environ. 1982, 22, 213-234. 27. Holloway, G.J.; Crocker, H.J.; Callaghan, A. The effects of novel and stressful environments on trait distribution. Funct. Ecol. 1997, 11, 579-584. Molecules 2010, 15 4323 28. Hoffmann, A.A.; Parsons, P.A. Extreme Environmental Change and Evolution; Cambridge University Press: Cambridge, UK, 1997. 29. Forbes, V.E.; Depledge, M.H. Environmental stress and the distribution of traits within populations. In Ecotoxicology: Ecological Dimensions; Baird, D.J., Maltby, L., Greig-Smith, P. W., Douben, P.E.T., Eds.; Chapman & Hall: London, UK, 1996; pp. 71-86. 30. Forbes, V.E. Sources and implications of variability in sensitivity to chemicals for ecotoxicological risk assessment. Arch. Toxicol. 1998, 20 (Suppl.), 407-418. 31. Dudai, N.; Larkov, O.; Putievsky, E.; Lerner, H.R.; Ravid, U.; Lewinsohn, E.; Mayer, A.M. Biotransformation of constituents of essential oils by germinating wheat seed. Phytochemistry 2000, 55, 375-382. 32. Scrivanti, L.R.; Zunino, M.P.; Zygadlo, J.A. Tagetes minuta and Schinus areira essential oils as allelopathic agents. Biochem. Syst. Ecol. 2003, 31, 563-572. 33. Muller, W.H.; Muller, C.H. Volatile growth inhibitors produced by Salvia species. Bull. Torrey Bot. Club 1964, 91, 327-330. 34. Reynolds, T. Comparative effects of alicyclic compounds and quinones on inhibition of lettuce fruit germination. Ann. Bot. 1987, 60, 215-223. 35. Zunino, M.P.; Zygadlo J.A. Effect of monoterpenes on lipid oxidation in maize. Planta 2004, 219, 303-309. 36. Nishida, N.; Tamotsu, S.; Nagata, N.; Saito, C.; Sakai, A. Allelopathic effects of volatile monoterpenoids produced by Salvia leucophylla: inhibition of cell proliferation and DNA synthesis in the root apical meristem of Brassica campestris seedlings. J. Chem. Ecol. 2005, 31, 1187-1203. 37. European Pharmacopoeia, 5th ed.; Council of Europe: Strasbourg Cedex, France, 2004; Volume I, 2.8.12, pp. 217-218. 38. Davies, N.W. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phases. J. Chromatogr. 1990, 503, 1-24. 39. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Corporation: Carol Stream, Illinois, IL, USA, 2001. 40. Bewley, D.; Black, M. Seeds. In Physiology of Development and Germination; Plenum Press: New York, NY, USA, 1985. 41. Sokal, R.R.; Rohlf, F.J. Biometry, 2nd ed.; WH Freeman and Company: New York, NY, USA, 1981. Sample Availability: Samples of the compounds of the essential oils are available from the authors. © 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).