Chemical composition of ultrasonic-assisted n-hexane extracts of Sideritis scardica Grieseb. and Sideritis raeseri Boiss. & Heldr. (Lamiaceae) from Macedonia and Albania

Chemical composition of n-hexane extracts obtained from dried over-ground parts of two species of Sideritis, S. scardica Grieseb. and S. raeseri Boiss. & Heldr. (Lamiaceae) was analyzed using GC/FID/MS. The collection of plants was made on different locations in the western part of Macedonia and the southern part of Albania, comprising twelve different samples of plant material. The ultrasonic-assisted extraction process was used for preparation of the n-hexane extracts yielded 0.73-3.33 % and 9.11-10.44 % of extracts for S. scardica and S. raeseri, respectively. Over one hundred constituents of the extracts were identified, belonging to several classes of components: diterpenes, hydrocarbons, dominantly present in each of the extracts, followed by fatty acids, aliphatic and aromatic alcohols, sterols, triterpene alcohols, and monoterpenes and sesquiterpenes, which were found in much smaller amounts or only in traces. The most abundant constituents of the extracts of both species of Sideritis were two diterpene components, both with M=286, which were not fully identified. Large percentages of nonacosane (1.71-12.22% and 7.46-19.68% for S. scardica and S. raeseri, respectively) and hentriacontane (4.48-20.79% and 8.09-30.31 % for S. scardica and S. raeseri, respectively) were also found in the extracts of both species.

In most of the studies conducted on Sideritis, scientists suggested that phenolic and/or polyphenolic components are responsible mostly for the antioxidant activity of alcohol extracts, while terpene components from the essential oils and even more different diterpeneoids are found to be responsible for the anti-inflammatory, analgesic, antiulcer, antibacterial, antifungal, cytotoxic and antitumor activity (Gomez-Serranillos et. al., 2004). Therefore, preparation of an n-hexane extracts of dried herbs of Sideritis is the most preferable type of extraction if terpenoid components represent research interest (Alcaraz et al., 1989;Gomez-Serranillos et al., 1998;Gomez-Serranillos et al., 1997;Aboutabl et al., 2002;Menghini et al., 2005;Tsaknis and Lalas, 2005;Kostadinova E. et al., 2008). Usually, the process of extraction is carried out via methods of percolation and maceration, characterized by small efficiency and long drawing (Lysyansky and Grebenyuk, 1987). Nowadays, the use of different electro-physical methods (ultrasound, electro-flotation, etc) allows increasing the extraction rate and yield of biologically active compounds (Sulman et al., 1997;Semagina et al., 2000).
Taking into account the consideration mentioned above, the aim of this work was preparation of n-hexane extract of Sideritis scardica and S. raeseri by ultrasonic-assistance extraction and determination of the chemical composition of the extracts using GC/FID/MS method.

Extraction
Plant extracts were obtained using ultrasonic-assisted extraction process at room temperature. n-Hexane was used for the extraction in 2 portions in ratio to plant material 1:20 (1 g plant material was extracted twice with 10 ml solvent). The duration of the extraction was 2 x 30 minutes. The extracts obtained after filtration were concentrated to dryness below 35 o C. The residue were measured as yield of the extraction and then dissolved in n-hexane to obtained solution with concentration of 1 g plant material in 1 ml solution. For GC/FID/MS analysis additional 1 µg/ml dilutions were prepared.

Gas chromatography
n-Hexane extracts were analyzed on Agilent 7890А Gas Chromatography system equipped with FID detector and HP-5ms 5% phenyl 95% dimethylpolysiloxane bonded phase capillary column (30 m x 0.25 mm, film thickness 0.25 µm). Operating conditions were as follows: oven temperature at 60°C for 5 min, then increased to 80°C at rate of 1°C/min and held 2 min and at the end increased to 280°C at rate of 5°C/min and held 5 min; helium as carrier gas at a flow rate of 1ml/min; temperature of the injector 260°C and that of the FID detector 270°C; the GC split ratio 1:1. 1µl of each sample of the essential oil, dissolved in xylene (1: 1000 v/v) was injected per GC run.
The percentage composition of n-hexane extracts were computed by the normalization method from the GC/FID peak areas (relative amount of each compound to the total amount present), calculated by means of three injections from each extract.
The extracts were analyzed on Agilent 7890А Gas Chromatography system interfaced to an Agilent 5975C mass spectrometer. The gas chromatographic conditions were the same as reported for GC analysis and the same column was used. The mass spectrometry conditions were: ionization voltage 70 eV, ion source temperature 230°C and mass range from 50 -500 Da.
Identification of the components present in essential oils was made by comparing mass spectra of components in essential oils with those from Nist, Wiley and Adams mass spectra libraries, by AMDIS (Automated Mass Spectral Deconvolution and Identification System) and by comparing literature and estimated Kovat′s (retention) indices that were determined using mixture of homologous series of normal alkanes from C 9 to C 25 in hexane, under the same above mentioned conditions.

Results and discussion
The ultrasonic-assisted extraction yielded different amount of dry extracts (dry residues after evaporation) ranged from 0.73-3.33 % for the samples of S. scardica and from 9.11-10.44 % for the samples of S. raeseri (Table 1.). The yields of n-hexane extracts of S. raeseri were almost the same for different samples of the plant regardless of the collection site. In the case of S. scardica much more variations in the extraction yields appeared. The higher yield of n-hexane extract for S. raeseri against S. scardica, probably due to the larger amounts of components extractible in hexane in S. raeseri. Bulgarian scientists reported 0.50-0.58% yields of the n-hexane extracts for both species, S. scardica and S. raeseri, which is significantly lower from our findings (Kostadinova et al., 2008). The reason of lower yield probably lays in the differences in geographical origin of the samples, but it is worth mentioning that those scientists have used 24 h maceration as extraction procedure while in our case it was ultrasonic-assisted extraction. According to literature data, ultrasonic-assisted extraction has been pointed out as more effective extraction procedure (Sulman et al., 1997, Samagina et al., 2000. Gas-chromatography analysis showed presence of over 100 individual components identified by GC/MS. The results of chemical composition of n-hexane extract of S. scardica are presented in Table 2 and for S. raeseri in Table 3. The chemical composition of the n-hexane extracts of both species showed qualitative and quantitative similarities. Mono and sesquiterpenes were presented in very small amounts, almost in traces, but apart from that, 25 components were identified. In S. scardica the sesquiterpene: trans-caryophyllene, δ-cadinene, caryophyllene oxide and α-cadinol+τmuurolol denoted the main part of this fraction. In S. raeseri additionaly α− and β−pinenes, myrtenol, trans-cadina-1,4diene, viridiflorol, valeranone and α−bisabolole were found. These data differ a lot from the previously reported data of chemical composition of essential oils of S. scardica (Galicnik) and S. raeseri (Galicica) from Macedonia (Kostadinova et. al., 2007). For both species, α-cadinol was defined as the dominant component and elemol acetate and germacron additionaly for the S. raeseri. Interesting results for the chemical composition of essential oils of cultivated S. raeseri subsp. raeseri were recently reported for the plant cultivated in Serbia. The main mono and sesquiterpene components of those oils were bicyclogermacrene and spatulenol (Plevljakisic et al., 2011). In both cases essential oils were obtained by hydro-distillation and besides mono and sesquiterpenes contained fatty acids and their esters, hydrocarbons and several diterpenes. All these components were also found in nhexane extracts of our specimens of Sideritis. The fractions of fatty acids and fatty acid-ester compounds were found in larger amount than fractions of mono and sesqiterpenes. Within these fractions, the main components were methyl, isopropyl hexadecanoate and hexadecanoic acid in S. scardica as well as octadecanoic acid methyl ester and hexadecanoic acid in S. raeseri. Large fraction of the n-hexane extracts belonged to hydrocarbons comprising aliphatic components with long carbon-chains (C5-C33). The hydrocarbons represented 13.33-47.24 % of n-hexane extract of S. scardica ( Fig. 1) and 26.24-68.12 % of n-hexane extracts of S. raeseri (Fig. 2). The most abundant components were heptacosane, nonacosane, hentriacontane and tritriacontane. Among them, hentriacontane was the predominant hydrocarbon in the extracts of both species, with 4.48-20.79 % in S. scardica and 8.09-30.31 in S. raeseri. Very small part of extracts contained oxidized products of hydrocarbons such as different alcohols, rarely some esters. Kostadinova et al. (2008) in n-hexane extracts of S. scardica and S. scardica x S. syriaca hybride also found nonacosane and hentriacontane as predominant hydrocarbons. Seasonal variation in the content of some hydrocarbons, mainly hentriacontane and nonacosane, was found for both species (Table 2 and 3).
In general, diterpenes represented very interesting group of components because of their chemistry and their distribution in plants, the possibilities for their use as markers in order to find or approve some taxonomy solutions, but much more because of their possible biological and pharmacological activity and further use in healing illnesses or other use. For these reasons, diterpenees of Siderits scardica and S. raeseri should be further more investigated, for full identification and determination and assessment of possible biological activity.

Conclussion
The ultrasonic-assisted extraction process with nhexane on dried over-ground parts of two species of Sideritis collected on different locations in western part of Macedonia and south-eastern part of Albania, yielded 0.73-3.33 % and 9.11 -10.44 % of extracts for S. scardica Grieseb. and S. raeseri Boiss. & Heldr., respectively. Over one hundred constituents of the extracts were identified using gas-chromatography (FID) and gas-chromatography/mass spectrometry methods. The components belonged to several classes of components such as diterpenes and hydrocarbons, dominantly presented in each of the extracts, followed by fatty acids, aliphatic and aromatic alcohols, sterols, triterpene alcohols and different monoterpenes and sesquiterpenes, found in much smaller amounts or traces. The most abundant constituents of the extracts of both species of Sideritis were two diterpenee components, both with M=286, which were not fully identified. Large percentages of nonacosane (1.71-12.22% and 7.46-19.68% for S. scardica and S. raeseri, respectively) and hentriacontane (4.48-20.79% and 8.09-30.31 % for S. scardica and S. raeseri, respectively) were also found in the extracts of both species. Season-al variation in the content of some diterpenes and hydrocarbones was found for the both species of Sideritis.