Chemical composition of Marrubium vulgare L . essential oil from Algeria

The chemical constituants of the essential oil from aerial parts of Marrubium vulgare, collected in Algeria, were analyzed by GC and GC/MS. The oil yield of the dried plant aerial parts, obtained by hydrodistillation, was 0.04 % (w/w). 50 compounds, accounting for 82.46 % of the oil, were identified. The major constituants were: 4,8,12,16-Tetramethyl heptadecan-4-olid (16.97 %), Germacrene D-4-ol (9.61 %),α- pinéne (9.37 %) Phytol (4.87 %), Dehydro-sabina ketone (4.12 %), Piperitone (3.27 %), δ – Cadinene (3.13 %), 1-Octen-3-ol (2.35 %) and Benzaldehyde (2.31 %).


INTRODUCTION
In recent years, essential oils obtained from vegetative parts of plants and their by products are highly demanded by the manufacturers of foods flavoring, perfumes, cosmetics, and pharmaceutical industries due to the growing interest of consumers in ingredients from natural sources. Lamiaceae is composed of more than 240 genera, most of them are highly aromatic due to the presence of external glandular structures, namely peltate and capitate trichomes that produce essential oils. According to Lawrence [1], it is possible to distinguish beteween the Lamiaceae oil-rich and oil-poor species. The latter being characterized by hydrocarbon-rich oils, such as germacrene D,β-caryophyllene, (E)-β-farnesene, δ-cadinene and α-humulene, among others. The Marrubium genus is represented by about 30 species [2]. Considered oil-poor species [1], little is known about their essential oils since more importance has been given to their maceration extract, which is consisted of the known and dominant active component marrubiin [3]. Marrubium vulgare, commonly known as horehound or hoarhond, is native in Europe, Western Asia and North Africa, and is cultivated worldwide as a source for food flavoring and for medicinal purposes [4,5]. The name ''marrubium'' refers to the bitter taste of the herb and ''hoar'' to the white pubescence covering the plant [6]. Under Polish climatic conditions, Marrubium vulgare L. is aperennial plant. Medicinal properties of horehound have been long known and the origin of its use goes back to ancient Egypt. The medicinal raw material is the herb of horehound (Marrubi herba) [7]. The herb consists of whole or crushed flowering aerial parts of Marrubium vulgare L. [8], and it shows multiple effects on human organism [9][10][11]. The essential oil of Marrubium vulgare L. has a relaxant and expectorant effect as well as avasodilator [12]. In Algeria, Marrubium vulgare is used in folk medicine to cure several diseases of the digestive tract, such as diarrhoea, as well as diabetes, rheumatism, cold and respiratory pains [13,14].
Pursuing our studies on the Algerian flora, this work reports the morphology and distribution of the glandular trichomes of M. vulgare growing spontaneously in Algeria, and the composition of its oil during the flowering and vegetative phases.

1. Plant material
Marrubium vulgare yielded during the spring in May 2009, in the zone of Nigrine district of El-Ater in the wilaya of Tebessa, north east of Algeria.

Distillation of essential oil
Marrubium vulgare: samples were dried in the shade in natural air far from moisture and all pollutants for a fortnight in the room temperature. The dried aerial parts were ground prior to the operation and then 100 g of ground marrubium were submitted to water distillation for 4 hrs using a Clevenger apparatus. The distilled essential oils were dried over anhydrous sodium sulfate, filtered and stored at 4 °C.

3. Gas chromatography
The gas chromatographic analyzes were performed using a Hewlett Packard 6890 chromatograph equipped with a nonpolar column HP5MS (30 x 0.25 mm d.i., Film thickness 0.25 microns) and a flame ionization detector. The procedures conditions were as follow: carrier gas: nitrogen, flow rate 0.8 ml/min, injector temperature: 250 °C, detectors temperature: 300 °C, temperature program: from 60 to 250 at 2 °C / min, with two levels: 8 minutes at 60 °C and 15 min at 280 °C, injection of 0.4 μl of pure essential oil and 1μl of absolute mode: mode split 1: 20. In order to determine retentions indices (RI) a series of nalkanes (C5-C28) mixture was analysed under the same operative conditions on HP-5 columns and the sample indices were calculated following Van den Dool and Kratz [15].

4. Gas chromatography and mass spectrometry
The essential oils were analyzed on an apparatus of gas chromatography coupled to mass spectrometry brand Hewlet Packard 5973A, equipped with an apolar capillary column (HP5MS, 30 m x 0.25 mm, phase thickness: 0.25 μm). The detection mode: electronic impact, ionization current: 70 eV, carrier gas: helium, flow rate: 0.7 ml/mn, the source pressure: 10-7mbar, interface temperature: 280 °C, injection: 250 °C, the programming of the oven: 2 °C / min from 60 °C to 280 °C, with isothermal: 8 min at 60 °C. and 15 minutes at 280 °C. 0.1 to 0.2 μl of pure essential oil and 1μl absolutely were injected in split mode 1: 20.
International Letters of Chemistry, Physics and Astronomy Vol. 13 Table 1

5. Component identification
Identification of components was made on the basis of their retention indices on nonpolar (HP-5) and/or on polar (PEG) columns and by computerised matching of the acquired mass spectra with those stored in the spectrometer data base using Willey mass spectral library and with the literature [16][17][18].

RESULTS AND DISCUSSION
The study showed that the essential oil content in the dry herb of Marrubium vulgare L. was on average 0.05 % [19]. The results obtained through our study are recorded in Table1. Concerning the dominant components, which rates were more than 1 % of the total composition of the oil, nine components among fifty were at least determined. These former represent 56 % of 82.42 % the total rate of the collected volatil oil. Table 1 illustrates also the nine components with a supremacy of three major compononents: 4,8,12,16-Tetramethyl heptadecan-4-olid (16.97 %), Germacrene D-4-ol (9.61 %), α-pinene (9.37 %). They represnt about 36 % of 56 %. Table 1 shows the different chemical groups with a dominance of other compounds with 41.64 % of the total rate of volatil oil, followed by Oxygenated sesquiterpene with a lower rate (13.17 %) and, Monoterpene hydrocarbon (12.61 %) Oxygenated monoterpene (9.46 %), Sesquiterpene hydrocarbon (5.58 %) respectively.