ANALYSIS OF ESSENTIAL OILS IN LEAF EXTRACTS FROM BRACKEN FERN, PTERIDIUM AQUILINUM (L.) KUHN. SUB. AQUILINUM

Aleksandra Halarewicz¹, Antoni Szumny^2
1 Department of Botany and Plant Ecology, Wrocław University of Environmental and Life Sciences, Poland
² Department of Chemistry, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

In this study aimed at comparing the content of volatile oils in the leaves of Pteridium aquilinum sub. aquilinum samples were taken from two natural habitats, on serpentinous soil and non-serpentinite mable in Lower Silesia, Poland. Biochemical analyses were performed on samples taken on 2 dates within the vegetative period. Extracts from the sampled leaves, were prepared in a Dering apparatus and were analysed using GC/MS. The voltaile oils qualitative composition was independent of the habitat type: the main constituent of all extracts was benzaldehyde whereas limonene, linalool, terpineol and cytral were represented in low quantities. The quantitative composition of the oils varied less between the sites at the beginning of the season but differences were greater by the end of it.

Key words: volatile, compounds, bracken fern, GC/MS.

INTRODUCTION

Ferns are commonly seen as a group of plants infested by a relatively small number of insect herbivores. They are considered by some authors as not attacked by insects at all [17]. In order to avoid massive herbivore attack, the plant undoubtedly uses biochemical defences. In case of ferns, the high effectiveness of these defences is probably the result of their long coevolution with herbivorous insects since their appearance in the Upper Devonian period [7]. In spite of research accomplished so far, the role of secondary plant metabolites in insect-fern relationships remains unclear [4]. Tannins and terpenes are listed among many substances probably responsible for ferns' chemical defences [3]. Other chemicals named in the literature include low molecular phenolics [1], tiaminase factors (compounds altering tiaminase activity), as well as low-quality plant proteins and late-season lignins, deposited in plant tissues, both of which are detrimental for insect development on ferns [6]. Essential oils are also compounds primarily affecting herbivore behavior at the host plant selection stage by inhibiting infestation, feeding, oviposition or hiding [14]. Their concentrations in leaf extracts from the common fern species, Pteridium aquilinum sub. aquilinum, was determined based on samples from two genets. Analyses therefore allowed determination of the extent to which the qualitative and quantitative composition of the essential oils might represent a species-specific trait.

Essential oil concentrations in plant vary considerably and are affected by a variety of factors including climate [15] and the availability of nutrients, especially N [5]. In some species, habitat may influence the proportional composition of essential oils so intensively, that a number of distinct chemotypes of the species may be recognized at different sites [13]. In order to identify and, eventually to compare the chemotypes of Pteridium aquilinum, two populations were selected, growing at two separate sites: one in a serpentinous soil and the second in non-serpentinous soil. Serpentinite-derived soils are well known for their low content of macronutrients such as N and P, and at the same time, for their high concentrations of Al, Si and heavy metals [18]. Individuals of Pteridium aquilinum growing in such substrates take up heavy metals and accumulate them in their stems [9,12]. It is supposed that high heavy metals concentrations are the reason for the dwarfish growth of the bracken fern's above-ground parts [8]. However, as yet, there is no experimental data available relating concentrations of ferns secondary metabolites, and essential oils in particular, to the different edaphic conditions in which the plants develop.

STUDY AREA

Plant material was sampled from two natural locations of Pteridium  aquilinum within  Ślęża Massif (Lower Silesia, Poland). Site I is located in spruce forest on serpentinous soil (N 50°50'30, E 16°42'11), whereas site II is on acid brown soil in submontane acidophilous oak forest (N 50°52'17, E 16°41'28). Both sites were designated for the study for their equal altitude (400 m above MSL), southern exposure of the slopes, the presence of the windows in the tree canopy and the absence of other shade-tolerant species in the shrub layer.

MATERIAL AND METHODS

The season of bracken fern vegetation on Ślęża Massif usually starts late, around mid-May, and it terminates already at the beginning of August, when the physiological senescing fronds can be observed. Biochemical analyses were performed on two dates within the plant growth season (in June and August 2009). On each sampling occasion, five fully expanded fronds were cut from the same rhizome, assuming that a single genet has been sampled. The volatile fractions from all the leaf blades of any individual frond, excluding that frond's rachis, were prepared in Dering apparatus by simultaneous distillation-extraction (SDE) in 1 ml hexane or in diisopropyl ether for 12 h.

The oil was analyzed by GC/MS using Saturn GC/MS/MS 2000 apparatus. Operating parameters were as follows: carrier gas: helium, with a flow rate of 2 ml/min; column: Chrompack capillary CP-sil 5 (CB, 25m x 0.25µm x 0.25mm), temperature: 50°C – 5 min, next 50–200, 3°C/min, finally 300°C – 10 min. Volume injected: 5 µL, split ratio 1:50. The MS parameters were as follows: ionization potential 70 eV, ionization current 2 A, ion source temperature 200°C.  Identification of components in oil was based on computer matching with the NIST Spectral Library (NIST 05), Standard Saturn Mass Spectral Libraries and retention index. Commercial samples of some terpenes were used as standards.

In the statistical analysis the concentrations found in extracts from the individual fronds were treated as sample replicates. For their relatively low number (five) and as the data distribution is far from normal the non-parametric Kolmogorov-Smirnov two sample test was used in order to compare the compounds' concentrations from the two habitats of P. acquilinum. The procedure is available in Statistica package.

RESULTS AND DISCUSSION

Volatile oils compositions obtained from the leaves of Pteridium aquilinum didn't dependend on the habitat conditions. Fig. 1 shows an example of a typical GC-MS analysis carried out in June 2009 using plant material from a serpentinous soil.

Throughout the entire growth season extracts were characterized by a very low content of volatile oils and shows remarkable crude similarities (Table 1). The main constituent of all extracts was benzaldehyde (44–50%). All analyses demonstrated significantly higher content of this compound in extract from Pteridium aquilinum samples from site I, compared to site II. Benzaldehyde is the simplest representative of the aromatic compounds analysed and is also the primary component of bitter almond oil extract.

The presence of benzeneacetaldehyde (13–18%) and 2-phenylethyl isothiocyanate (8–11%) in extracts is the result of hydrolysis of cyanogenic glycosides. Cyanogenesis, which is a polymorhpic trait in bracken fern populations, results in reduced damage from chewing insects compared to that observed in acyanogenic individuals [10,16].

The synthesis and accumulation of phenolic derivatives such as 2-methoxy-4-vinylphenol in the bracken fronds is known to be regulated by total amount of solar radiation reaching the plant [2]. It therefore seems likely, that the change of light conditions, resulting from tree defoliation observed in August, was responsible for the significant difference in the concentration of 2-methoxy-4-vinylphenol in the analysed oils from the plants at the site II, compared to the site I.

Throughout the growth season the following monoterpenes and monoterpenoids: limonene, linalool, terpineol, cytral and ionones were present in extracts from bracken leaves. These secondary metabolites are also commonly found in other plant species and play a defensive role in protecting plants against herbivores [11]. Their concentration in extracts from both the bracken fern sites was similar in June, but in August the samples from the site I contained significantly more linalool and terpinol compared to the site II samples. Therefore, the study indicates to a possible relationship between the production of at least some secondary compounds in the bracken fern and the edaphic conditions in which the plants develop.

CONCLUSIONS

1. The qualitative composition of the volatile oils in the leaves of Pteridium aquilinum sub. aquilinum is independent of the site or the season of the year.

2. The main volatile component of the bracken leaf extract is benzaldehyde.

3. The terpene fraction of the extract is represented by limonene, linalol, terpineol and cytral but all of these compounds are present in low quantities.

4. The most apparent quantitative variation of the chemical composition of the bracken fern volatile oils is observed by the end of the plant vegetation.

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Accepted for print: 17.11.2010

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