Essential Oil Composition of the Different Parts and In Vitro Shoot Culture of Eryngium planum L

The essential oils obtained by hydrodistillation from the different parts (inflorescence, stalk leaves, rosette leaves and root) as well as from in vitro shoot culture of Eryngium planum L. were analyzed by GC-FID-MS in respect to their chemical composition. The different parts of E. planum and in vitro shoots showed different yields. The part with higher amount was the inflorescences, followed by the stalk leaves and in vitro shoots, rosette leaves and finally roots. The essential oils obtained from rosette leaves and in vitro-derived rosettes had totally different composition. Quantitative differences were also found between compounds of intact plant organs. The main components of stalk leaf oil and rosette leaf oil were monoterpene (limonene, α- and β-pinene) and sesquiterpene hydrocarbons. In inflorescence oil cis-chrysanthenyl acetate (43.2%) was accompanied by other esters (propionate, butanoate, hexanoate and octanoate) and numerous oxygenated sesquiterpenes. Root oil and in vitro shoot oil contained mainly (Z)-falcarinol and 2,3,4-trimethylbenzaldehyde. This is the first report on the chemical composition of this species.


Introduction
The genus Eryngium L. (Sea Holly) belonging to the subfamily Saniculoideae of the Apiaceae is represented by 317 taxa widespread throughout Central Asia, America, Central and Southeast Europe [1]. Some species, such as E. foetidum L., E. maritimum L., E. campestre L. and E. creticum Lam. have been used in traditional medicine worldwide [2]; E. foetidum L. (culantro), known as "spiny coriander" is strongly aromatic and contains essential oil valuable for pharmaceutical, perfumery and flavor industries [3]. The pharmacological activities of Eryngium species depend mainly on high triterpenoid saponin content [4], but presence of flavonoids [5], namely kaempferol and quercetin glycosides [6], and phenolic acids [7] could play an important role. Coumarin derivatives [8], acetylenes [9,10], as well as rosmarinic acid and chlorogenic acid, known as antioxidants [11], have been described for many Eryngium species. Rosmarinic acid accumulation in in vitro E. planum cultures was previously investigated by the authors [12].
Four of the 26 Eryngium species described in Flora Europaea [13] grow in Poland as rare or protected plants. E. planum L. (Flat Sea Holly) is a rare herbaceous perennial species of native flora with restricted distribution in Poland [14]. This erect herb has silvery-blue stems of 40-60 cm in height, basal leaves and bluish inflorescences. E. planum is used in folk medicine in Europe as Eryngii plani herba and Eryngii plani radix. The presence of the active constituents: phenolic acids, triterpenoid saponins, flavonoids, essential oils and coumarins determines their multidirectional pharmacological activity: diuretic, expectorant, spasmolytic, antitussive, antimycotic, stimulant and appetizer [4,7,15]. The chemistry of E. planum has been previously studied, but to our knowledge, no study has dealt with the chemical composition of the essential oil from this species.
Numerous studies have been carried out using plant in vitro cultures as a potential source of valuable constituents. Some difficulties correlated with low or varied concentration of desired compounds in intact plants can be overcome by using plant cell biotechnology. The use of cell and organ cultures, selection of high producing culture and medium optimizations gives the possibility of optimizing the processes of increased secondary metabolites accumulation under controlled conditions. In vitro technology offers following benefits: novel products not found in nature, use of rare, endangered or protected plants, independence from climatic factors, elimination of the geographical and political boundaries, shorter and more flexible production cycles and easier fulfillment of GLP and GMP demands [16]. It was shown that numerous bioactive compounds of medicinal value including essential oil components may be accumulated in in vitro cultures in higher concentration than in intact plants [17,18].

Results and Discussion
Some species of Apiaceae have been studied for its high essential oils yield, however Pala-Paul et al. [14] reported that the genus Eryngium did not contain large amounts of essential oil. The hydrodistillation of the dried aerial parts (inflorescences, leaves and rosette leaves), roots and in vitro-derived shoot culture of E. planum gave essential oils in yields ranging from 0.05% (roots), 0.07% (rosette leaves), 0.10% (stalk leaves and in vitro shoots) to 0.23% (inflorescences). This fact can be explained by the process of essential oil distribution from roots through the plant during the vegetative season [19]. The chemical composition of the oils were analyzed by GC-FID-MS. More than one hundred constituents were identified according to their retention indices (RI) and mass spectra. The identified compounds are presented in Table 1. Similarities were observed in the qualitative composition and some significant differences in the quantitative composition of the oils obtained from stalk leaves and rosette leaves. The main constituents of both oils were monoterpene hydrocarbons (42.0% and 28.4%, respectively) with limonene, α-and β-pinene, β-phellandrene and camphene predominating. The second important group was numerous sesquiterpene hydrocarbons (20.0% and 24.4%, respectively). The main difference was high content of terpinen-4-ol (10.9%) and bornyl acetate (18.1%) in rosette leaf oil.
Samples of rosette leaves of intact plants and in vitro-derived rosettes (shoot culture) were gathered at the same regeneration phase from different type of soil (ground and in vitro culture medium). It was surprising that essential oils obtained from these two populations had totally different composition. Quantitative similarities in oil components were found in two different organs -roots of intact plants and in vitro regenerated shoot culture. The major constituents of essential oil in shoot in vitro cultures and root were (Z)-falcarinol (49.1% and 64.4 % respectively) and 2,3,4-trimethylbenzaldehyde (6.2% and 17.4% respectively). These observations could be explain by vegetation phase of plant, location and type of soil [27,28].
Polyacetylenes such as falcarinol and falcarindiol are wide spread among the Apiaceae plant family [10]. They are common in carrots and related vegetables such as parsley, celery, parsnip and fennel as well as in medicinal plants such as ginseng [29]. They show a wide variety of different pharmacological effects including anti-inflammatory, antiplatelet-aggregatory, cytotoxic and antitumor activity [29,30]. Moreover these aliphatic C 17 -polyacetylenes of the falcarinol-type exhibit anti-bacterial, antifungal and antimycobacterial activities [31]. Falcarinol (heptadeca-1,9-dien-4,6-diyn-3-ol) appears to be the most bioactive compound in the falcarinol-type polyacetylenes group. It has shown a pronounced cytotoxic activity against human tumor cells in vitro and it also seems to possess in vivo anti-tumor activity [10]. These polyacetylenes have also been shown to be responsible for allergic skin reactions [10]. The beneficial effects of falcarinol-type polyacetylenes occur at nontoxic concentrations and thus represent pharmacologically useful properties indicating that polyacetylenes may be important nutraceuticals.
Overall the results suggest that oil from different parts of in vivo as well as in vitro shoots could be a source of falcarinol and polyacetylenes which are important health promoting compounds.

Intact Plant
Plants of E. planum were collected at the full flowering stage in natural site near Torun, in the Kujawy region of Central Poland. The fruits were gathered from the same place. The voucher specimens from the Department of Pharmaceutical Botany and Plant Biotechnology, K. Marcinkowski University of Medical Sciences in Poznan were deposited in the Herbarium of Institute of Natural Fibres and Medicinal Plant in Poznan. Plants were divided into parts (inflorescence, stalk leaves, rosette leaves and roots) and were air-dried.

In Vitro Shoot Culture
Seedlings of E. planum were obtained from the seeds, which were isolated from the ripened fruits after their stratification and scarification. For initiation of in vitro cultures, the seeds isolated from fruits were washed with distilled water and dipped in 70% ethanol for 30 s followed by rising in 20% Clorox (5% sodium hypochloride) solution containing two drops of Tween 80 for 5 min. They were finally rinsed three times in sterilized double-distilled water. Shoot tips of axenic seedlings (30-day old) were used for induction of shoot culture and establishment on MS [32] basal medium supplemented with 3% sucrose and plant growth regulators: BAP 1.0 mg·L −1 , and IAA 0.1 mg·L −1 . Media were solidified with 0.8% agar and adjusted to pH 5.7-5.8, autoclaved at 121 °C for 20 min (105 kPa). Shoot cultures were maintained in 250 cm 3 Erlenmeyer flasks with 50 cm 3 of culture medium, subcultured to fresh medium every 6-8 weeks and incubated in growth chamber under a 16/8 h photoperiod at 55 μmol m −2 s −1 light provided by cool-white fluorescent lamps and a temperature of 23 ± 2 °C. The 'shoot culture', a type of in vitro cultures, in case of E. planum is a rosette of leaves (like a juvenile stadium of intact plant). For isolation of essential oil the multiplied in vitro shoots were washed from medium and air dried.

Isolation and Analysis of Essential Oil
The essential oils were obtained by hydrodistillation for three hours of dried plant material using a glass Clevenger-type apparatus, according to European Pharmacopoeia 5.0. GC-FID-MS analyses were performed using a Trace GC Ultra apparatus (Thermo Electron Corporation) equipped with FID and MS DSQ II detectors and FID-MS splitter (SGE). Operating conditions: apolar capillary column Rtx-1ms (Restek), 60 m × 0.25 mm i.d., film thickness 0.25 µm; temperature program, 50-300 °C at 4 °C/min; SSL injector temperature 280 °C; FID temperature 300 °C; split ratio 1:20; carrier gas helium at a regular pressure 200 kPa. Mass spectra were acquired over the mass range 30-400 Da, ionization voltage 70 eV; ion source temperature 200 °C.
Identification of components was based on the comparison of their MS spectra with those of a laboratory-made MS library, commercial libraries (NIST 98.1, Wiley Registry of Mass Spectral Data, 8th Ed. and MassFinder 4.1, laboratory-made list) and with literature data [33,34] along with the retention indices (Rtx-1, MassFinder 4.1) associated with a series of alkanes with linear interpolation (C 8 -C 26 ). A quantitative analysis (expressed as percentages of each component) was carried out by peak area normalization measurements without correction factors.

Conclusions
The results suggest that oil from different parts of in vivo E. planum plants as well as in vitro shoots could be a source of falcarinol, polyacetylene which is an important health promoting compound. Our studies have shown that the yield of the oil isolated from different parts of E. planum and in vitro shoots is low and the essential oils contain complex mixtures of up to 111 different compounds.