Essential Oil Composition of Juniperus Oxycedrus. Growing in Algeria

Abstract

The chemical composition of the essential oil of Juniperus oxycedrus. from Algeria was studied for the first time. The oil was obtained from the leaves of the plant by hydrodistillation and analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) techniques. The yield of essential oil is 0.1%, based on dry weight. Eighty-nine components representing 82.3% of the total oil were identified. The results show that oxygenated monoterpene (41.0%) composes the major part of oil and the main components are trans.-pinocarveol (7.0%), cis.-verbenol (6.3%), and manoyl oxide (6.0%). A comparison with oils from J. oxycedrus. complex (subspecies and varieties) of other countries reported in literature is discussed briefly.

Keywords:

• cis.-verbenol
• Cupressaceae
• essential oil composition
• Juniperus oxycedrus
• manoyl oxide
• trans.-pinocarveol

Introduction

The genus Juniperus. belongs to the Cupressaceae family, comprising about 70 species all over the world (Nakanishi et al., 2004). The genus is divided into three sections: Caryocedrus., Juniperus. (=Oxycedrus.), and Sabina. (Adams et al., 1998). The species status for the J. oxycedrus. complex are J. oxycedrus. L. subsp. oxycedrus., J. oxycedrus. L. subsp. badia. (H. Gay) Debeaux, J. oxycedrus. L. subsp. macrocarpa. (Sm.) Ball, J. navicularus. Gand (=J. oxycedrus. L. subsp. transtagana. Franco), J. cedrus. Webb & Berth. (=J. oxycedrus. L. subsp. madrensis. Menezes; J. oxycedrus. L. var. grandifolia. Link in Buch.; J. webbi. Car.), and J. brevifolia. (Seub.) Antoine (=J. oxycedrus. L. var. brevifolia.) (Adams 2000; da Silva et al., 2000; Cavaleiro et al., 2002). In Algerian flora, five species are present (Quezel & Santal, 1963). Among them, J. oxycedrus. grows on the steppe and is commonly known as taga.. This species has been used in Algerian folk medicine as diuretic, stimulative, tonic of estomac, and pulmonary and depurative disinfectant (Baba Aïssa, 1991). Moreover, the extracts from this plant (wood and branches) oil of cade are used by the pharmaceutical industry against skin diseases (eczemas, psoriasis) and as a vermifuge (Baba Aïssa, 1991; Salido et al., 2002; Karaman et al., 2003).

Over the past 30 years, this species has been extensively studied. Previous investigation on the nonvolatile constituents of J. oxycedrus. revealed the presence of polyphenolic compounds such as flavonol diglycosides, biflavones, and coumarins (Stassi et al., 1998). However, numerous studies in the literature have reported the composition of essential oil isolated from leaves (Horster, 1974; Stassi et al., 1995; Adams, 199819992000; da Silva et al., 2000; Milos & Radonic, 2000; Salido et al., 2002; Cavaleiro et al., 20022003; Angioni et al., 2003; Marongiu et al., 2003), berries (Guerra Hernadez, 1987; Stassi et al., 1995; da Silva et al., 2000; Milos & Radonic, 2000; Salido et al., 2002; Angioni et al., 2003; Cavaleiro et al., 2003; Marongiu et al., 2003; Velasco-Negueruela et al., 2005), and wood (Chalchat et al., 1988; Uçar & Balaban, 2002; Marongiu et al., 2003) from different parts of world. Thorough investigation of the biological activity of this species has been reported (Stassi et al., 1996; Angioni et al., 2003; Karaman et al., 2003; Marongiu et al., 2003).

To the best of our knowledge, there is no previous phytochemical work that has been recorded for J. oxycedrus. from Algeria. As part of ongoing work on the characterization of Algerian aromatic and medicinal plants, in this paper we report the chemical composition of essential oil obtained from J. oxycedrus. collected from Djelfa (steppe region) City of Algeria.

Materials and Methods

Plant material

Juniperus oxycedrus. was collected in May 2003 from Djelfa (City of Algeria) (longitude: 2°45′; latitude: 34°30′; elevation: 1143 m; annual precipitation: 308 mm; climate type: semiarid). The plant was authenticated in the Botanical Department, National Institute of Agronomic (NIA; Algeria), where a voucher specimen of the plant was deposited in the herbarium (HNIA/FA/N°: P105bis) of this school.

Extraction and isolation

The shade-dried and finely powdered leaves of the plant were exhaustively extracted by hydrodistillation for 3 h using a Clevenger-type apparatus with a water-cooled receiver, in order to reduce hydrodistillation overheating artifacts. The oil was extracted from the distillate with diethyl ether and then dried over anhydrous sodium sulfate. After filtration of the sodium sulfate, the solvent was removed by distillation under reduced pressure in a rotary evaporator. The oil was obtained in a yield of 0.1% based on the dried weight of sample. The oil was stored in a sealed glass vial in the dark at +4°C until analysis.

Gas chromatography

Gas chromatography (GC) analysis was performed on a Trace Carlo Erba CE Thermo-Finnigan (Italy-USA) chromatograph using a fused silica capillary column with a stationary phase of DB-5. The various parameters fixed for a DB-5 column were 30 m × 0.32 mm i.d., 0.25-µm film thickness, column, oven temperature 60°C for 3 min then 3°C/min to 240°C for 5 min; FID-detector heaters 250°C; injector heaters 250°C; nitrogen was used as carrier gas at a flow rate 1 ml/min in the split mode 1:50, with an injection vol. of 0.2 µl. Quantitative data were obtained by electronic integration of area percents without the use correction factors.

Gas chromatography/mass spectrometry

Gas chromatography/mass spectrometry (GC/MS) analysis was performed on a Trace MS Thermo-Finnigan chromatograph apparatus equipped with a DB-5 column (30 m × 0.32 mm i.d, with 0.25-µm film thickness), with helium as carrier gas at a flow rate of 1 ml/min. The GC oven temperature was kept at 60°C for 3 min and programmed to 240°C for 3 min at a rate 3°C (injector 250°C). The mass spectrometer was operating in the EI-mode at 70 eV. The source temperature was set at 200°C. Acquisition mass range, m/z. 40–450.

Identification

In order to determine retention indices (RI), a series of a mixture of n.-alkanes (C₅–C₂₈) was analyzed under the same operative conditions on the DB-5 column, and the sample indices were calculated following Van den Dool and Kratz (1963). Identification of components was made on the basis of their retention indices on a nonpolar (DB-5) column and by computerized matching of the acquired mass spectra with those stored in the spectrometer database using the NIST mass spectral library and literature (Chalchat et al., 1988; Davies, 1990; Adams et al., 1995199819992000; Stassi et al., 1996; da Silva et al., 2000; Milos & Radonic, 2000; Cavaleiro et al., 20022003; Salido et al., 2002; Angioni et al., 2003; Marongiu et al., 2003).

Results and Discussion

The oil yield was 0.1% based on dried weight of sample. The chemical composition of the oil was investigated using both GC and GC/MS techniques. Table 1 shows the components identified according to their elution order on a DB-5 capillary column, their relative concentrations, and their relative indices. Eighty-nine components representing 82.3% of total oil were identified. Preliminary GC and GC/MS examinations of the oil indicated that it consists mainly of oxygenated monoterpenes (41.0%). Our results show that the oil contains about (13.4%) sesquiterpene hydrocarbons and (12.8%) oxygenated sesquiterpenes. The concentration of monoterpene hydrocarbons (4.7%) is relatively low (see Table 2).

Table 1 Percentage composition of essential oil of J. oxycedrus..

Compound	RI^a.	%	Identificationmethods^b.
Tricyclene	920	1.8	1, 2
α.-Thujene	924	0.1	1, 2
α.-Pinene	939	0.2	1, 2
Camphene	944	0.1	1, 2
Thuja-2,4(10)-diene	958	tr	1, 2
Sabinene	974	tr	1, 2
1-Octen-3-ol	978	tr	1, 2
β.-Pinene	981	tr	1, 2
Myrcene	990	tr	1, 2
δ.-3-Carene	1010	tr	1, 2
α.-Terpinene	1014	0.4	1, 2
p.-Cymene	1026	tr	1, 2
Limonene	1029	tr	1, 2
cis.-β.-Ocimene	1038	0.2	1, 2
γ.-Terpinene	1059	0.3	1, 2
n.-Octanol	1070	tr	1, 2
p.-Mentha-3,8-dien	1076	0.6	1, 2
Terpinolene	1087	0.5	1, 2
trans.-Linalool oxide	1093	tr	1, 2
Linalool	1095	0.1	1, 2
n.-Nonanal	1102	tr	1, 2
cis.-p.-Menth-2-en-1-ol	1110	1.2	1, 2
3-Methyl-2-isobutyl pyrazine	1114	3.4	1, 2
Terpin-1-ol	1120	tr	1, 2
trans.-Pinocarveol	1129	7.0	1, 2
cis.-Verbenol	1136	6.3	1, 2
trans.-Verbenol	1139	1.6	1, 2
Sabina ketone	1147	tr	1, 2
trans.-Pinocamphone	1152	1.0	1, 2
Pinocarvone	1158	5.1	1, 2
Borneol	1164	0.1	1, 2
p.-Mentha-1,5-dien-8-ol	1166	0,5	1, 2
Terpinen-4-ol	1176	0.9	1, 2
p.-Cymen-8-ol	1182	1.8	1, 2
α.-Terpineol	1188	2.1	1, 2
Myrtenol	1196	0.5	1, 2
Verbenone	1202	3.6	1, 2
trans.-Carveol	1210	2.4	1, 2
Fenchyl acetate	1217	tr	1, 2
cis.-Carveol	1230	tr	1, 2
Thymol, methyl ether	1233	0.2	1, 2
Carvacrol, methyl ether	1241	0.4	1, 2
Piperitone	1248	0.2	1, 2
Bornyl acetate	1273	1.3	1, 2
Undec-10-en-1-al	1279	0.2	1, 2
Carvacrol	1300	0.4	1, 2
trans.-Carvyl acetate	1325	tr	1, 2
trans.-Piperitol acetate	1330	tr	1, 2
α.-Terpinyl acetate	1334	0.4	1, 2
α.-Cubebene	1346	0.1	1, 2
α.-Copaene	1375	tr	1, 2
trans.-β.-Damascenone	1380	tr	1, 2
β.-Bourbonene	1384	tr	1, 2
β.-Elemene	1389	tr	1, 2
α.-Gurjunene	1411	0.1	1, 2
trans.-Caryophyllene	1420	0.1	1, 2
β.-Gurjunene	1428	0.1	1, 2
γ.-Elemene	1437	0.8	1, 2
α.-Humulene	1451	tr	1, 2
γ.-Muurolene	1467	5.4	1, 2
Germacrene D	1480	1.1	1, 2
α.-Muurolene	1501	2.9	1, 2
γ.-Cadinene	1510	1.1	1, 2
δ.-Cadinene	1524	0.1	1, 2
trans.-Calamenene	1529	tr	1, 2
Cadina-1,4-diene	1533	0.1	1, 2
α.-Cadinene	1541	1.3	1, 2
β.-Calacorene	1555	tr	1, 2
trans.-Nerolidol	1565	0.1	1, 2
Spathulenol	1575	0.4	1, 2
Caryophyllene oxide	1580	0.4	1, 2
cis.-Sesquivandulol	1598	1.5	1, 2
trans.-Sesquivandulol	1627	0,7	1, 2
epi-α.-Cadinol	1637	0.5	1, 2
Cubenol	1641	1.0	1, 2
7-epi-α.-Eudesmol	1651	tr	1, 2
α.-Cadinol	1657	5.5	1, 2
Juniper camphor	1688	tr	1, 2
(Z,Z.)-Farnesol	1708	2.1	1, 2
(E,E.)-Farnesol	1728	0.3	1, 2
(E,Z.)-Farnezol	1745	tr	1, 2
β.-Bisabolenal	1765	tr	1, 2
(Z,E.)-Farnesyl acetate	1824	tr	1, 2
(E,E.)-Farnesyl acetate	1846	tr	1, 2
Cembrene	1939	tr	1, 2
Manoyle oxide	1979	6.0	1, 2
Kaurene	2041	1.9	1, 2
Abietatriene	2052	0.1	1, 2
Abietadiene	2067	2.1	1, 2

^a.Retention indices in elution order from DB-5 column.

^b.1, GC; 2, GC/MS; tr, trace amounts (<0.1%).

Table 2 Group composition of J. oxycedrus. oil.

Monoterpene hydrocarbons	4.7%
Oxygen-containig monoterpenes	41.0%
Sesquiterpene hydrocarbons	13.4%
Oxygen-containing sesquiterpenes	12.8%
Diterpenes	10.2%
Other	0.2%
Total amount	82.3%

The main constituents were trans.-pinocarveol (7.0%), cis.-verbenol (6.3%), and manoyl oxide (6.0%). On the other hand, α.-cadinol (5.5%), γ.-muurolene (5.4%), and pinocarvone (5.1%) were other notable constituents of the oil. Five constituents were diterpenes: cembrene (<0.1%), manoyl oxide (6.0%), kaurene (1.9%), abietatriene (0.1%), and abietadiene (2.9%).

Thirty-one of the 98 identified compounds such as sabinene, δ-3-carene, terpin-1-ol, α-copaene, β-elemene, and juniper camphor were found in trace amounts (<0.1%). It is worth noting the presence of three compounds (3.2% of total oil) not found in the NIST database.

According to the published literature, the chemical pattern in berry oils of J. oxycedrus. produced α.-pinene, myrcene, and limonene as major components (Horster, 1974; Adams, 1998; da Silva et al., 2000; Cavaleiro et al., 20022003; Salido et al., 2002; Angioni et al., 2003). In the wood oils of J. oxycedrus., the main compounds were found to be thujopsene and γ.-cadinene (Horster, 1974; Uçar & Balaban, 2002).

Table 3 summarizes previous studies on the essential leaf oils from several populations of J. oxycedrus. complex (subspecies and varieties) from different countries (only the main compounds are considered). Several investigations (Horster, 1974; Adams et al., 199819992000; da Silva et al., 2000; Milos & Radonic, 2000; Cavaleiro et al., 20022003; Salido et al., 2002; Angioni et al., 2003) of J. oxycedrus. leaf oils showed that α.-pinene, limonene, and sabinene are a standard character of most varieties of this plant. Spanish and Italian oils contain large amounts of α.-pinene (20.7–85.6%) and sabinene (26.5%) (Adams et al., 199819992000; Salido et al., 2002; Angioni et al., 2003). The major compounds identified in oils from Portugal, Croatia, and Greece were α.-pinene (6.3–70.7%) and limonene (7.0–79.0%) (Stassi et al., 1995; Adams et al., 19981999; da Silva et al., 2000; Milos & Radonic, 2000; Cavaleiro et al., 20022003). In this study J. oxycedrus. leaf oil was found to be rich in trans.-pinocarveol (7.0%). This finding differentiates the Algeria plant from those described in literature (see Table 3).

Table 3 The three main components dominant in the essential oils from leaves of Juniperus oxycedrus. complex (subspecies and varieties), as reported in the literature.

Species	Countries	Main components	References
J. oxcedrus. L.	Djelfa (Algeria)	trans.-Pinocarveol (7.0%)	(Dob et al., 2006)
		cis.-Verbenol (6.3%)
		Manoyl oxide (6.0%)
	Near Split Central Dalmatia (Croatia)	α.-Pinene (41.4%)	(Milos & Radonic, 2000)
		Manoyl oxide (12.3%)
		Farnesol (8.6%)
J. oxycedrus. L. subsp.oxycedrus.	El pennon (Spain)	α.-Pinene (41.3%)	(Adams, 1998)
		α.-Phellandrene (8.2%)
		p.-Cymene (6.2%)
	Crysortis (Greece)	α.-Pinene (42.7%)	(Adams et al., 1999)
		Limonene (17.1%)
		δ.-3-Carene (13.7%)
	Sardinia (Italy)	α.-Pinene (85.6%)	(Angioni et al., 2003)
		δ.-3-Carene (2.8%)
		γ.-Terpinene (1.4%)
J. oxycedrus. L. subsp. macrocarpa.	Island of ElHaphonissos (Greece)	α.-Pinene (26.9%)	(Stassi et al., 1995)
		Cedrol (13.9%)
		Dihydro-p.-cymen-8-ol (8.5%)
J. oxycedrus. L. subsp. badia.	Jean City (Spain)	α.-Pinene (20.7%)	(Adams, 2000)
		Manoyl oxide (10.9%)
		Germacrene D (8.5%)
		α.-Pinene (39.8–57.0%)	(Salido et al., 2002)
		Manoyl oxide (4.8–10.2%)
		Germacrene D (0.4–2.6%)
J. oxycedrus. L. subsp. transtagana. Franco^a.	Lispon (Portugal)	α.-Pinene (22.9%)	(Adams, 1998)
		Limonene (14.3%)
		Myrcene (8.6%)
	Many populations (Portugal)	α.-Pinene (6.3–38.0%)	(Cavaleiro et al., 2003)
		Limonene (7.0–34.6%)
		α.-Phellandrene (2.2–13.1%)
J. oxycedrus. L. subsp. maderensis. Menezes^b.	Tenerife Canary Islands (Portugal)	α.-Pinene (70.7%)	(Adams, 1998)
		Myrcene (6.3%)
		β.-Phellandrene (4.6%)
	Carreiras-Madeira (Portugal)	α.-Pinene (19.6–55.3%)	(Cavaleiro et al., 2002)
		Limonene (17.3–32.7%)
		δ.-3-Carene (5.5–15.7%)
J. oxycedrus.. var. brevifolia..(Seub.) Antoine^c.	San Miguel Island, Azores Islands (Portugal)	Limonene (43.4%)	(Adams, 1998)
		Sandaracopimara-8(14), 15-diene (19.5%)
		Myrcene (6.3%)
	Five Azorean Islands (Portugal)	Limonene (66.6–79.0%)
		α.-Pinene (6.0–16.6%)	(Da Silva et al., 2000)
		Sandaracopimara-8(14), 15- diene (0.8–5.1%)
J. oxycedrus. L. subsp. oxycedrus.	Lemo (Greece)	Limonene (27.7%)	(Adams et al., 1999)
		α.-Pinene (25.3%)
		Myrcene (3.8%)
J.oxycedrus. L. subsp. macrocarpa.	Tarifa (Spain)	Sabinene (26.5%)
		α.-Pinene (22.6%)
		Terpinen-4-ol (7.3%)

^a.J. navicularus. Gand.

^b.J. cedrus. Webb & Berth.(=J. oxycedrus. L. var. grandifolia. Link in Buch.; J webbi. Car.).

^c.J. brevifolia. Antoine.

Briefly, the conclusions drawn from the current study and the previous studies (see Table 3) the presence of a large geographical diversity in J. oxycedrus. complex (subspecies and varieties) with respect to the chemical composition and the main components of essential leaf oils, which appears to be divided into four chemo types: α.-pinene type, limonene type, sabinene type, and trans.-pinocarveol type.

Acknowledgment

We are very grateful to P. Roland-Gosselin Thermo-Finnigan, France, for her technical assistance.