1 Introduction
The genus Pinus (Pinaceae) comprises 250 species and is widespread in the northern hemisphere, especially in the Mediterranean region, Caribbean area, Asia, Europe, North and Central America [1–4].
The medicinal and aromatic properties of the chemical compounds (e.g., turpentine, resins and essential oil…) of pine make it one of the most popular plants throughout all civilisation. Pine is also still widely used in traditional therapeutic practice in world and has an economic importance [5–7].
The chemical composition of various pine species has been the subject of numerous studies, the majority of the studies focused on North American and Central European species [6,8–10] and only a limited number of chemically oriented reports dealt with Mediterranean pine species [11–13]. Most bibliographical studies of chemical, biological, antimicrobial activities and genetic side of P. halepensis have been reported [11–22].
As part of an extensive phytochemical analysis of P. halepensis growing in Algeria, we have oriented our first investigation towards the chemical composition of the essential oil obtained from the needles of P. halepensis Mill. collected in Algiers region (Sidi Feradj) in Algeria.
2 Experimental
2.1 Plant material
The needles of P. halepensis Mill. were collected in May 2002, at the forest of Sidi Feradj (Algiers). The plant was authenticated by Mr A. Beloued in botanical department, National Agronomic Institute of Algiers (N.A.I), Algeria (Herbarium No. P. 105). The samples were dried in shad ventilated place.
2.2 Oil isolation
The needles were cut into small pieces and separately hydrodistilled for 2 h in a Clevenger-type apparatus with water cooled receiver, in order to reduce hydrodistillation overheating artifacts. The essential oil was taken up in diethyl ether and dried over sodium sulphate and reduced at room temperature under vacuum on rotatory evaporator. The oil obtained was stored at (+4 °C) until analysis.
2.3 Analysis of essential oil
2.3.1 Gas chromatography
GC analysis was performed on a Chrompack CP 9002 chromatograph using fused silica capillary columns with two different stationary phases DB-1 and PEG. The various parameters fixed for DB-1 column are: 30 m × 0.32 mm i.d.; film thickness 0.25 μm column; temp. prog., 50 °C for 3 min then 2 °C/min to 260 °C for 5 min; detector heaters 280 °C; injector heaters 250 °C; nitrogen was used as carrier gas at a flow rate of 1 ml/min in the split mode (Split ratio 1:50), with an injection vol. 0.2 μl. For PEG the parameters are: 30 m × 0.32 mm i.d.; film thickness 0.25 μm column; temp. prog., 50 °C for 3 min then 2 °C/min to 220 °C for 15 min; others parameters as the same for the DB-1 column. Components were quantified as area percentages of total volatiles from the GC–FID system without correction factors.
In order to determine retentions indices (RI) a series of n-alkanes (C5–C28) mixture was analysed under the same operative conditions on DB-1 and PEG columns and the sample indices were calculated following Van den Dool and Kratz [23].
2.3.2 Gas chromatography and mass spectrometry
Mass spectra were obtained from GC–MS analysis on a Trace MS Finnigan chromatograph system equipped with a 30 m × 0.32 mm i.d.; film thickness 0.25 μm DB-1 capillary column it was programmed from 50 °C (3 min) to 260 °C (5 min) at 2 °C/min with helium carrier gas at a flow rate of 1 ml/min and injector heater 250 °C. The mass-spectrometer was operating (full scan-mode) in the EI-mode at 70 eV.
2.3.3 Component identification
Identification of components was made on the basis of their retention indices on non-polar (DB-1) 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 [13,22–25].
3 Results and discussion
The needles from Pinus halepensis collected for the present study were obtained from Algiers region (Sidi Feradj). It's collected from various parts of crowns. Since all of samples were collected in May 2002, the effect of the seasonal variation was diminished [26]. The oil was obtained from the Aleppo pine needles with a yield of 0.52% (v/w).
The chromatographic profile showed a complex mixture of components with a consistent fraction of monoterpenes and sesquiterpenes.
The list of the compounds, in order of elution on DB-1, and the quantitative data (GC–FID peak area percentages without correction factors), are reported in Table 1. More than 41 oil compounds were identified accounting for 67.02% of the total oil, while 32.98% of the oil remained unidentified (Fig. 1).
Qualitative and quantitative composition of needles oil of P. halepensis Miller
| Compound a | % on DB-1 | R. I.b on DB-1 | R.I. on PEG | Method of identification |
| Tricyclene | tr | 915 | – | GC, GCMS |
| α-Pinene | 1.23 | 922 | 1036 | GC, GCMS |
| Camphene | tr | 957 | – | GC,GCMS |
| Sabinene | 1.23 | 960 | 1127 | GC, GCMS |
| β-Pinene | 0.23 | 978 | – | GC, GCMS |
| Myrcene | 3.07 | 997 | 1140 | GC, GCMS |
| α-Phellandrene | tr | 1002 | 1296 | GC, GCMS |
| Hexyle acetate | 0.7 | 1008 | 1268 | GC, GCMS |
| δ-3-Carene | 0.15 | 1011 | 1140 | GC, GCMS |
| α-Terpinene | 0.11 | 1021 | 1181 | GC, GCMS |
| Limonene | tr | 1032 | – | GC, GCMS |
| β-Ocimene | 0.21 | 1041 | 1247 | GC, GCMS |
| γ-Terpinene | tr | 1062 | 1231 | GC, GCMS |
| α-Terpinolene | 0.13 | 1088 | – | GC, GCMS |
| α-Pinene oxide | 0.06 | 1095 | 1488 | GC, GCMS |
| Linalool | tr | 1098 | 1566 | GC, GCMS |
| Camphor | tr | 1143 | – | GC, GCMS |
| Borneol | 0.13 | 1165 | 1695 | GC, GCMS |
| Terpinen-4-ol | tr | 1177 | 1591 | GC, GCMS |
| p-Cymen-8-ol | tr | 1183 | 1823 | GC, GCMS |
| α-Terpineol | 0.07 | 1189 | 1684 | GC, GCMS |
| α-Terpinyl acetate | tr | 1350 | – | GC, GCMS |
| α-Cubebene | 0.17 | 1351 | – | GC, GCMS |
| Citronellyl acetate | 0.19 | 1354 | – | GC, GCMS |
| α-Yalangene | 0.64 | 1372 | 1481 | GC, GCMS |
| (Z)-β-Caryophyllene | 40.31 | 1404 | 1591 | GC, GCMS |
| α-Guaiene | 0.1 | 1437 | – | GC, GCMS |
| Aromadendrene | 7.1 | 1439 | 1599 | GC, GCMS |
| α-Humulene | 7.92 | 1454 | – | GC, GCMS |
| allo-Aromadendrene | 0.65 | 1461 | 1685 | GC, GCMS |
| γ-Muurolene | 0.06 | 1477 | 1715 | GC, GCMS |
| Germacrene-D | 0.49 | 1480 | 1702 | GC, GCMS |
| Bicyclogermacrene | tr | 1494 | 1723 | GC, GCMS |
| β-Bisabolene | tr | 1509 | – | GC, GCMS |
| δ-Cadinene | 0.13 | 1513 | 1777 | GC, GCMS |
| β-Sesquiphellandrene | 0.53 | 1524 | 1776 | GC, GCMS |
| Z-Nerolidol | 0.07 | 1534 | – | GC, GCMS |
| Elemol | 0.68 | 1549 | – | GC, GCMS |
| δ-Cadinol | 0.23 | 1622 | 2152 | GC, GCMS |
| γ-Eudesmol | 0.08 | 1630 | – | GC, GCMS |
| Monoyl oxide | tr | 1989 | – | GC, GCMS |
| Monoterpene hydrocarbon | 6.48% | |||
| Oxygenated monoterpene | 0.36% | |||
| Sesquiterpene hydrocarbon | 58.20% | |||
| Oxygenated sesquiterpene | 1.06% |
a Order of elution on DB-1.
b Retention indices. tr: Trace (<0.05%).
Chemical composition groups of needles oil of P. halepensis.
Amounting to 67.02% of the total oil, the sesquiterpene hydrocarbons had the highest contribution (58.20%), this fraction dominated by β-caryophyllene (40.31%), followed by α-humulene (7.92%) and aromadendrene (7.10%). The monoterpene was relatively poor; it represented (6.50%) in monoterpene hydrocarbons, it is found to contain a significant percent of myrcene (3.07%), followed by α-pinene (1.23%) and sabinene (1.23%). The oil is characterised by 13 compounds could be detected in traces (< 0.05%).
Table 2 summarises previous investigations of authors on the analysis of the volatile oils from several population of P. halepensis. The chemical composition of our P. halepensis Mill. oil was dominated by β-caryophyllene, these results agree with data obtained by Roussis et al. who found that monoterpene (41.8%) was dominated in Greece pine oils with remarkable differences concerning the amounts of component: caryophyllene (19.05%) [11]. Vidrich et al. [27] have reported that β-caryophyllene (26.31%) play an important part of the Italy oil. Macchioni et al. [13] found main compounds of the needles oil of Aleppo pine grown in Italy to be: myrcene (27.9%), α-pinene (18.1%) and β-caryophylene (16.4%), with a 73.2% of monoterpenes and 21.2% of sesquiterpenes. When our results were compared with the chemical composition of essential oils obtained from the leaves of P. halepensis collected in region of Tessimsilt and Djelfa (Algeria) in 1987, remarkable differences were observed: myrcene (8.65%) and α-pinene (17.56%), dominated in later sites, respectively [19].
Chemical composition of needles oils of P. halepensis as reported in the literature
| Constituents | Ouer study | Greece [11] | Italy | Morocco [12] | Algiers [19] | ||
| [13] | [27] | Ta | Db | ||||
| Tricyclene | tr | – | – | – | – | 1.60 | 0.04 |
| α-Thujene | – | 0.16 | 0.1 | – | 0.4 | – | – |
| α-Pinene | 1.23 | 13.4 | 18.1 | 8.54 | 23.3 | 6.66 | 17.56 |
| Camphene | tr. | 0.44 | 0.3 | 0.09 | 0.5 | 0.17 | 0.33 |
| Sabinene | 1.23 | 1.27 | 9.4 | 6.13 | 3.7 | 6.95 | 2.59 |
| β-Pinene | 0.23 | 1.13 | 2.0 | 1.13 | 3.1 | 2.04 | 1.56 |
| Myrcene | 3.07 | 6.62 | 27.9 | 12.48 | 16.3 | 8.65 | 3.22 |
| 3-Octanol | – | – | – | 0.06 | – | – | – |
| α-Phellandrene | tr | 0.05 | – | 0.81 | 1.6 | – | – |
| Hexyl acetate | 0.7 | – | – | – | – | – | – |
| δ-3-Carene | 0.15 | 6.87 | 1.7 | 0.87 | – | 1.88 | 0.13 |
| α-Terpinene | 0.11 | 0.30 | 0.5 | 0.79 | 1.3 | 0.65 | 0.04 |
| p-Cymen | – | – | 1.1 | 11.41 | 0.7 | 0.27 | 3.07 |
| Limonene | tr | 5.03 | 1.1 | 0.98 | 1.3 | 0.8 | 0.14 |
| β-Phllandrene | – | – | 1.0 | – | 1.27 | 0.97 | 0.66 |
| 1,8-Cineole | – | – | – | 1.67 | 1.3 | – | – |
| cis-β-Ocimene | – | – | – | – | – | 0.11 | 0.07 |
| trans-β-Ocimene | 0.21 | – | 0.4 | – | 1.77 | 2.05 | 0.04 |
| γ-Terpinene | tr | 0.42 | 0.8 | 0.24 | 2.4 | 1.18 | 0.02 |
| cis-Sabinene hydrate | – | – | 0.1 | – | – | – | – |
| α-Terpinolene | 0.13 | 3.07 | 9.9 | – | 10.1 | 0.19 | t |
| cis-3-Hexene-1-ol | – | – | – | – | – | t | 0.09 |
| 3,5-Dimethyl-styrene | – | – | – | – | – | t | t |
| α-Pinene oxide | 0.06 | – | – | – | – | t | t |
| Linalool | tr | 0.78 | 0.3 | 0.41 | – | 0.39 | 2.01 |
| Fenchol | – | 0.11 | – | – | – | – | – |
| Camphor | tr | – | – | – | – | t | 0.05 |
| Borneol | 0.13 | 0.02 | – | – | – | – | – |
| Umbellulone | – | – | 0.1 | – | – | – | – |
| Terpinen-4-ol | tr | 0.7 | – | – | 3.8 | – | 0.59 |
| p-Cymen-8-ol | tr | – | – | – | – | t | 0.14 |
| α-Terpineol | 0.07 | 0.54 | 0.2 | – | 0.6 | 0.29 | 1.48 |
| cis-piperitol | – | – | – | – | – | t | – |
| Methyl chavicol | – | – | – | 5.06 | – | – | – |
| Decanale | – | – | – | 0.83 | – | – | – |
| Fenchyl acetate | – | 0.28 | – | – | – | – | – |
| Nerol | – | – | – | – | – | – | 0.04 |
| d-Citronellolo | – | – | – | 1.26 | – | – | – |
| l-Citronellolo | – | – | – | 0.41 | – | – | – |
| Methyl thymyl ether | – | 0.10 | – | – | – | – | – |
| Geraniol | – | – | – | – | – | t | t |
| 2-Phenyl ethyl acetate | – | – | – | – | 2.5 | – | 0.12 |
| Bornyl acatate | – | – | – | – | – | t | 0.16 |
| Carvacrol | – | – | – | 1.72 | – | – | – |
| δ-Elmene | – | 0.03 | – | – | – | – | 0.20 |
| α-Terpenyl acetate | tr | 0.01 | – | – | – | – | – |
| α-Cubebene | 0.17 | – | 0.1 | – | – | t | t |
| Citronellyl acetate | 0.19 | – | – | – | – | – | – |
| Eugenol | – | – | – | 0.60 | – | – | – |
| Neryl acetate | – | 0.36 | – | – | – | – | – |
| α-Yalangene | 0.64 | – | – | – | – | 0.24 | 0.21 |
| α-Copaene | – | – | 0.4 | – | 0.5 | t | 0.03 |
| Z-3-Hexenyl-isovalerate | – | – | – | – | – | t | t |
| Geranyl acetate | – | 0.19 | 0.3 | 0.86 | 5.3 | t | 0.29 |
| β-Elemene | – | – | – | – | – | t | 0.20 |
| Methyl eugenol | – | – | – | – | – | t | t |
| (Z)-β-Caryophyllene | 40.31 | – | – | – | – | – | – |
| (E)-β-Caryophyllene | – | – | – | – | – | 7.07 | 2.69 |
| β-Caryophyllenec | – | 19.05 | 16.4 | 26.31 | 14.2 | – | – |
| α-Gurjunene | – | 1.18 | – | – | – | – | – |
| α-Guaiene | 0.1 | – | – | – | – | – | – |
| α-Cedrene | – | – | – | 0.08 | – | – | – |
| Aromadendrene | 7.1 | – | – | – | – | – | – |
| cis-carane-trans 2-ol | – | – | – | – | – | t | 0.54 |
| α-Elemene | – | – | – | – | – | t | 0.58 |
| α-Humullene | 7.92 | 3.36 | 2.9 | – | 3.2 | 2.77 | 1.38 |
| Allo-aromadendrene | 0.65 | – | – | – | – | – | – |
| Calarene | – | 0.39 | – | – | – | – | – |
| Methyl iso-eugenol-2 | – | – | – | – | – | 0.27 | t |
| (E)-β-Farnesene | – | – | 0.2 | – | – | t | 0.03 |
| 9-epi-(E)-Caryophyllene | – | – | 0.1 | – | – | – | – |
| γ-Muurolene | 0.06 | – | – | – | – | 0.29 | 0.19 |
| Germacrene D | 0.49 | 0.5 | 0.1 | – | – | 0.21 | 0.03 |
| δ-Selinene | – | – | – | – | – | t | t |
| Phenyl ethyl-3-methyl butanoate | – | – | 1.2 | – | – | – | – |
| Epi-Cubebol | – | – | 0.2 | – | – | – | – |
| Bicyclogermacrene | tr | – | – | – | – | – | – |
| Methyl iso-eugenol-l | – | – | – | 5.06 | – | 0.17 | t |
| α-Muurolene | – | 0.53 | 0.4 | – | 0.5 | 0.29 | 0.19 |
| Bicycloelemene | – | – | – | – | – | t | t |
| β-Bisabolene | tr | – | – | – | – | – | – |
| trans-γ-Cadinene | – | – | 0.3 | – | – | – | – |
| Calamenene | – | – | – | – | – | t | 0.18 |
| δ-Cadinene | 0.13 | 0.55 | 0.3 | – | 1.0 | 0.87 | 0.47 |
| β-Sesquiphellandrene | 0.53 | – | – | – | – | – | – |
| Levomenol | – | – | – | – | – | t | 0.03 |
| Ethyl laurate | – | – | – | – | – | t | t |
| Terpenyl-n-butyrate | – | – | – | – | – | t | 1.48 |
| β-Phenyl ethyl-isobutyrate | – | – | – | – | – | t | t |
| Phenyl ethyl 2-methyl-butyrate | – | – | – | – | – | 0.97 | 10.29 |
| Phenyl ethyl-isovalerate | – | – | – | – | – | 7.37 | 8.38 |
| Elemol | 0.68 | 0.36 | – | – | – | t | – |
| Nerolidol | 0.07 | – | – | – | – | t | – |
| Caryophyllene oxide | – | – | 0.1 | – | 1.2 | – | – |
| Globulol | – | 0.01 | – | – | – | – | – |
| Phenyl ethyl-tiglate (E) | – | – | – | – | – | t | t |
| Guaiol | – | 1.05 | 0.3 | – | – | 0.20 | 0.17 |
| Phenyl ethyl n-hexanoate | – | – | – | – | – | t | t |
| Humulene epoxyde II | – | – | – | – | – | 0.17 | 0.17 |
| Cubenol | – | – | – | – | – | t | – |
| β-Eudesmol | – | – | – | – | – | – | t |
| α-Eudesmol | – | 0.41 | 0.1 | – | – | – | – |
| Cadinol | – | – | – | – | 1.1 | t | 0.17 |
| α-Cadinol | – | – | – | – | – | 0.18 | 0.15 |
| δ- Cadinol | 0.23 | – | – | – | – | – | – |
| trans-Cadinol | – | – | – | – | – | t | 0.17 |
| γ-Eudesmol | 0.08 | – | – | – | – | – | – |
| Phenyl ethyl-tiglate (Z) | – | – | – | – | – | t | t |
| Torreyol | – | – | – | – | – | t | t |
| Aristolene | – | 1.09 | – | – | – | – | – |
| – | 6.57 | – | – | – | – | – |
| Kaureuol | – | – | – | – | – | t | t |
| Diethyl-phatalate | – | – | – | – | – | – | 0.08 |
| Cembrene | – | 7.62 | – | – | – | – | – |
| (11E,13Z) Labdadien-8-ol | – | 0.30 | – | – | – | – | – |
| Neocembrene | – | – | – | – | – | – | – |
| Manoyle oxide | tr | – | – | – | – | t | – |
a T: Tissemsilt.
b D: Djelfa.
c Correct isomer not identified.
Our results differ from those obtained by Hmamouchi et al. [12] who studied the oil composition of needles of the same specie sample in Morocco, in which α-pinene (23.3%) was revealed to be dominant and β-caryophylene (14.2%) present. Several reports on the composition of the needle oils of other Pinus species revealed that monoterpene hydrocarbons were the major constituents in the most of the oil, they often constituted 50% or more of the oil [4,12,13].
The comparison of our result with literature (Table 2) shows some qualitative and quantitative differences in compositions of P. halepensis oils. The variability in oil composition is present even in P. halepensis and these variations, sufficient to allow the distinction of different chemotypes, are the results of an adaptative process to particular ecologic conditions.
Acknowledgements
We are very grateful to P. Roland-Gosselin Thermo-Finnigan, France for her technical assistance.