Chemical Composition of Essential Oil from Italian Populations of Artemisia alba Turra (Asteraceae)

The use of essential oils as chemotaxonomic markers could be useful for the classification of Artemisia species and to caracterize biodiversity in the different populations. An analysis of the chemical composition of four essential oils from Italian populations of Artemisia alba Turra (collected in Sicily, Marche and Abruzzo) was investigated. In this paper an in depth study of the significant differences observed in the composition of these oils is reported.


Results and Discussion
Hydrodistillation of the aerial parts of A. alba Turra collected in Madonie (A), Marche (B), Majella (C) and Mt. Velino (D) yielded 1.5%, 0.4%, 0.16% and 0.03% (w/w) of essential oils, respectively, all characterized by a pale yellow colour. In Table 1 the compounds identified are listed according to their retention indices on a HP-5MS column, and are classified in seven classes on the basis of their chemical structures. The composition of the oils is different, both qualitatively and quantitatively. The oil obtained from Artemisia alba from Madonie (A) is characterized by a high concentration of sesquiterpenes that represents more than 60% of the composition of the oil, while in the oils of other populations the presence of monoterpenes and sesquiterpenes is roughly equivalent. All oil extracts from the populations of Marche, Majella and Monte Velino have a content of monoterpenes (43.3%, 38.6% and 41.6%, respectively), which is about twice as high compared with the same class of compounds identified in the oil from Madonie (24.1%).
Among the monoterpenic hydrocarbons in the oil from Madonie, santolinatriene (7.3%), an irregular monoterpene, predominates and it is also present in low concentrations in B, but absent in C and D. On the other hand in the oil from Marche irregular oxygenated monoterpenes are found in higher concentrations. In fact, santolina alcohol, artemisia alcohol, artemisia ketone and chrysanthenone represent about one third (13.9%) of the fraction while in the oil from Madonie santolina alcohol, despite being the most abundant oxygenated monoterpene, accounts for only 2.6%, the remaining (10.2%) of this fraction being constituted by regular oxygenated monoterpenes. The most abundant oxygenated monoterpenes of oil from Marche are borneol (9.3%), artemisia alcohol (6.0%) and artemisia ketone (4.6%); the last two being absent in A, C and D. In the oils from Abruzzo (Majella, C and Monte Velino, D) monoterpenic ketones (cis-pinocamphone, piperitone) are prevalent instead and they account for more than half of the content of monoterpenes.
According to the literature [17] α-thujone and camphor are two markers allowing a distinction of Artemisia in two groups. Our four oils are characterized by the absence of α-thujone, whereas camphor and its biogenetic precursor, borneol are present in A, B and C.

Plant Material
The aerial parts of the four populations of Artemisia alba Turra, were collected from blooming plants in Sicily

Isolation of the Essential Oil
The air-dried samples were ground in a Waring blender and then subjected to hydrodistillation for 3 h using n-hexane as solvent, according to the standard procedure of the European Pharmacopoeia [26]. The extracts were dried over anhydrous sodium sulphate and then stored in sealed vials, at −20 °C, ready for the GC and GC-MS analyses. The samples yielded 1.5% (A), 0.40% (B), 0.16% (C) and 0.03% (D) (w/w) of pleasant smelling yellow oils.

Gas Chromatography-Mass Spectrometry
Analytical gas chromatography was carried out on a Perkin-Elmer Sigma 115 gas chromatograph (Napoli, Italy) equipped with a HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness), a split-splitless injector heated at 250 °C and a flame ionization detector (FID) at 280 °C. Column temperature was initially kept at 40 °C for 5 min, then gradually increased to 250 °C at 2 °C/min, held for 15 min and finally raised to 270 °C at 10 °C/min. The injection volume was 1.0 μL (split ratio 1:20). A fused silica HP Innowax polyethylene glycol capillary column (50 m × 0.20 mm, 0.25 μm film thickness) was also used for analysis. In both cases helium was the carrier gas (1 mL/min). GC-MS analysis was performed on an Agilent 6850 Ser. II apparatus (Napoli, Italy), fitted with a fused silica DB-5 capillary column (30 m × 0.25 mm, 0.33 μm film thickness), coupled to an Agilent Mass Selective Detector MSD 5973; ionization voltage 70 eV; electron multiplier energy 2000 V; source temperature 250 °C. Mass spectra were scanned in the range 35-450 amu, scan time 5 scans/s. Gas chromatographic conditions were the same as those for GC; transfer line temperature, 295 °C.

Identification of Components
Most of the constituents were identified by GC by comparison of their retention indices (K i ) with either those in the literature [27,28] or with those of authentic compounds available in our laboratories. Retention indices were determined in relation to a homologous series of n-alkanes (C 8 -C 28 ) under the same conditions. Whenever possible, co-injection with authentic substances was also performed. Component-related concentrations were calculated based on GC peak areas without using correction factors. Further identification of oil components was achieved by comparing their mass spectra on both columns, either with those stored in NIST 02 and Wiley 275 libraries or with mass spectra from the literature [28,29] and our personal library.

Conclusions
The differences in composition between the four oils makes it possible to hypothesize that the Italian populations of Artemisia alba Turra growing on the Madonie (Sicily), in the Marche region, on the Majella and Monte Velino (Abruzzo)-in part related to different cytotypes [19]-surely express from climatic as well as genetic differences. Furthemore, the differences of the oil of the population of the Artemisia alba Turra from Madonie-the most southerly of the species-let us consider that this belongs to a different chemotype from the other ones.