Phytotoxic Activities of Mediterranean Essential Oils

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.


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][7][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

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.  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%), β-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 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. β-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%), α-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.
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].
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 β-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].

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.

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 µm 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 µL were injected manually at 250 ºC and in the splitless mode. Analysis was also run by using a fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm i.d.; 0.20 µm 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 µL 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 µm 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 (C 8 -C 24 ) under the same operating conditions. Relative concentrations of each essential oil component were calculated on the basis of GC peaks without using correction factors.

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 µg/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.

Statistical analyses
Data were ordered in homogeneous sets, and the Student's t test of independence was applied [41].

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 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.