Chemical Variations in the Leaf Volatile Oils of Two Populations of Juniperus navicularis Gandoger from the Iberian Peninsula (SW Portugal)

Velasco-Negueruela, Arturo


Steam distilled oils obtained from the leaves of two populations ni Juniperus navicularis Gand., gathered in SW Portugal were analyzed by GC and GC/MS in combination with retention indices. The oils contained oc-pinene (27.9-36.1%), α-phellandrene (0.9-11.1%) and limonene + β-phellandrene (24.3-40.7%) as major constituents. Other characteristic compounds were cadinanes + muurolanes (2.4-9.5%) and eudesmanes (t-0.3%). In addition to these components, (E)-nerolidol (t-5.1%) and α-epi-bisabolol (t-1.4%) were also present. From the point of view of morphology and leaf oil chemistry, the populations studied have been shown to be quite homogeneous although a small chemical geographical variation was noted.

Key Word Index

Juniperus navicularis, Cupressaceae, essential oil composition, α-pinene, α-phellandrene, limonene + β-phellandrene.


The genus Juniperus L., belongs to the Cupressaceae family, comprising about 50 species (1) of useful aromatic and medicinal plants, found from the northern hemisphere to the mountains of tropical Africa and West Indians. According to Flora Iberica, two (2) sections can be recognized for the Iberian Peninsula: 1) Section sabina Spach comprising Juniperus phoenicea L. subsp.phoenicea; Juniperus phoenicea L. ssp. turbinata (Guss.) Nyman; J. thurifera L. and J, sabina L.; 2) section juniperus L., comprising Juniperus communis L. ssp. communis; J. communis L. ssp. hemisphaerica (K. Presl) Nyman; J. communis L. ssp. alpina (Suter) Celak; J. oxycedrus L. ssp. oxycedrus; J. oxycedrus L. ssp. badia (H. Gay) Debeaux; J. oxycedrus L. ssp. macrocarpa (Sm.) Ball and J. navicularis Gand.

Recently Adams (3) based on leaf oils and RAPDs (random amplified polymorphic DNAs) established the following species status for the juniperus oxycedrus complex: J, oxycedrus L.; J. badia H. Gay; J. macrocarpa Sibth & Sm. and J. navicidaris Gand.

This last taxon is a dwarf shrub (4) inhabiting the maritime sands (dunes near the coast) and endemic to the Sado District (Sado River Estuary) of SW Portugal. In this work we have examined the leaf oil volatiles of two populations of J. navicularis by GC and GC/MS in combination with retention indices.


Plant material: The sites of collection, sample and voucher specimen numbers and yields of J. navicularis are listed in Table I. Voucher specimens have been deposited at the Herbarium of the Real Jardín Botânico de Madrid, Spain. Plant material was identified by Ginés López González from the Real Jardin Botánico de Madrid, Spain.

Isolation of volatile constituents: Fresh leaves of J. navicularis were steam distilled for 8 h in an all-glass Clevenger-type apparatus, and the plant material was suspended in a chamber above the boiling water. The oils were dried over anhydrous sodium sulfate and stored at 4°C in the dark. The extracted leaves were oven dried 48 h at 100°C for the determination of oil yields.

Analyses: Analytical GC was carried out on a Varian 3300 gas Chromatograph fitted with a Silicone DB-1 capillary column (50 m x 0.25 mm, film thickness 0.25 µm); carrier gas N^sub 2^, flow rate 1.5 mL/min, split mode, temperature programmed 60°0 -240°C at 3°C/min. Injector temperature 250°C, detector used FID, detector temperature 300°C. Injection volume for all samples was 0.1 µL GC/MS analyses were carried out on a Hewlett Packard 5890 gas Chromatograph fitted with a phase bonded poly (5% diphenyl, 95% dimethylsiloxane) silicone PTE5 capillary column (30 m × 0.25 mm, film thickness 0.25 µm). Carrier gas He, flow rate 1.5 mL/min. Temperature program regimen was 70°C, 2 min and then programmed to 25°0C at 2°C/min. Injector temperature 250°C. The Chromatograph was coupled to an HP 5971 A mass selective detector (70 eV).

Component identification: Most constituents were identified by comparing their retention indices with those of authentic standards. The latter we re either purchased, synthesized or identified in oils of known composition. The fragmentation patterns of mass spectra were compared with those stored in the spectrometer data base using the N BS54K. L and WILEY. L built-in libraries and with those reported in the literature (5-8).

Results and Discussion

The components of the oils, the percentage of each constituent and the retention indices are summarized in Table II. The components are arranged in order of GC elution on the Silicone columns. The pinane hydrocarbons of the oils of Juniperus navicularis were dominated by α-pinene (27.9-33.7%) and lower amounts of β-pinene (2.5-3.4%).The total of pinane components was 30.6-38.9%. The major p-menthane constituents were found to be α-phellandrene (0.9-11.1%) and limonene + β-phellandrene (24.3-40.7%) and loweramounts of α-terpinene(t-0.6%),p-cymene(0.2-3.7%), γ-teqjinene (0.1-0.5%), terpinolene (0.8-3.4%), terpinen-4-ol (0.1-1.1%) and αterpineol (1.1-2.0%). The total of p-menthane constituents was as follows: p-menthane hydrocarbons (39.1-48.0%), p-menthane aromatics (0.3-4.1%) and p-menthane alcohols (1.5-2.3%). The most important sesquiterpene components were shown to be γ-muurolene (0.1-0.6%), β-caryophyllene (0.1-0.6%), α-muurolene (0.1-0.6%), γ-cadinene (0.2-0.9%), δ-cadinene (0.6-3.0%), (E)-nerolidol (t-5.1%), 1-epi-cubenol (0.1-0.5%), T-muurolol + T-cadinol (0.6-2.2%), α-cadinol (0.6-2.0%) and epi-α-bisabolol (t-1.4%). Manoyl oxide was found as 0.1% in all samples.

According to bibliography, the chemical pattern in the oils of J. oxycedrus was to produce pinane hydrocarbons as major components (3,9-11,13), in the oils of J, baclia the main compounds were found to be pinane hydrocarbons, germacrene D and manoyl oxide (11), in an oil of J, macrocarpa gathered in Spain (3,11,13) pinane and sabinane hydrocarbons were present as principal constituents and in the oil of this last species gathered in Greece (12) the major components were shown to be α-pinene, cedrol and p-cymen-8-ol (9). Lastly, in the oil of J, navicularis gathered in Lisbon, Portugal (3,13) the main constituents were found to be α-pinene (22.9%), sabinene (8.2%), myrcene (8.6%) α-phellandrene (8.00%) and Iimonene (14.3%). According to our results, the leaf oils of J, navicularis were characterized by the production of pinane hydrocarbons and the p-menthanes Iimonene + β-phellandrene and αphellandrene. Our results are very similar to those of Adams (3,13) and these have been included for comparison in Table II.

The populations of J, navicularis studied have been shown to possess homogeneous morphology and oil chemistry although a small chemical geographical variation has been noted. This Juniper is an endemic species inhabiting a small area (the so-called “Sado River Estuary”) in SW Portugal. Both populations gathered had small quantitative differences in their oil composition. Samples gathered near Comporta (see Table I) 10751 female plants and 10750 male plants, have oils respectively dominated by α-pinene (28.4-31.1%), α-phellandrene (8.9-10.3%) and Iimonene + β-phellandrene (34.2-25.8%) together with two minor characteristic components (E)-nerolidol (1.1-5.0%) and epi-α-bisabolol (0.5-1.0%), whereas samples collected near Troia (see Table I) 10753 female plants and 10752 male plants, had respectively as major components α-pinene (31.7-34.9%), Iimonene + β-phellandrene (40.2-39.5%) and moderate amounts of α-phellandrene (3.7-2.4%) and small quantities of (E)-nerolidol (0.2-0.1%) and epi-α-bisabolol (


1. DJ. Mabberley, The Plant-Book. Cambridge University Press, Cambridge, UK (1998).

2. J. do Amaral Franco, Juniperus in Flora lberica. Edits., S. Castroviejo, M. Lainz, G. López Gonzalez, P. Monserrat, F. Muñoz Garmendia, J. Paiva and L. Villar, pp 181-188, Real Jardin Botónico, Madrid, Spain (1986).

3. P.P. Adams, Systematics of Juniperus section Juniperus based on leaf essential oils and random amplified polymorphic DNAs (RAPDs). Biochem. Syst. Ecol., 28, 515-528 (2000).

4. S. Rivas Martínez, M. Lousa, T.E. Díaz, F. Fernández González and J.C. Costa, La vegetación del sur de Portugal (Sado, Alentejo y Algarve). ltinera Geobot., 3, 5-126 (1990).

5. LM. Libey, A Paradox data base for GC/MS data on Components of Essential oils and Other Volatiies. J. Essent. oil Res., 3,192-194 (1991 ).

6. R.P. Adams, Identification of Essential oils Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Co., Illinois (1995).

7. A.A. Swigar and R.M. Silverstein, Monoterpenes. Aldrich Chem. Co., Milwaukee, Wisconsin (1981).

8. D. Joulain and W.A. König, The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. EB.-Verlag, Hamburg (1998).

9. D.V. Banthorpe, H.S. Davies, C. Gatford and S.R. Williams, Monoterpene patterns in Juniperus and Thuja species. Planta Med., 23,54-69 (1973).

10. V.H. Horster, Vergleigh der monoterpenfraktionen von Juniperus drupacea and Juniperus oxycedrus. Planta Med., 26, 113-118 (1974).

11. R.P. Adams, J. Altarejos, C. Fernandez and A. Camacho, The leaf essential oils and Taxonomy ofJuniperus oxycedrus L. subsp. oxycedrus, subsp. badia (H. Gay) Debeaux and subsp. macrocarpa (Sibth. & Sm.) Ball. J. Essent oil Res., 11, 167-172(1999). 12. V. Stassi, E. Verykokidou, A. Loukis, A. Harvala and S. Philianos, Essential oil of Juniperus oxycedrus L. subsp. macrocarpa (Sm.) Ball. J. Essent. oil Res., 7, 675-676 (1995).

13. R.P. Adams, The leaf essential oils and chemotaxonomy of Juniperus sect. Juniperus. Biochem. Syst. Ecol., 26, 637-645 (1998).

Arturo Velasco-Negueruela,* María José Pérez-Alonso, Jesús Palá-Paúl and Ana Íñgo

Departamento de Biología I (Botánica), Facultad de Biología, Universidad Complutense, 28040-Madrid, Spain

Ginés López

Real Jardín Botánico de Madrid CSIC, Plaza de Murillo 2, 28014- Madrid, Spain

* Address for correspondence

Received: November 2001

Revised: February 2002

Accepted: March 2002

1041-2905/04/0006-0608$6.00/0-© 2004 Allured Publishing Corp.

Copyright Allured Publishing Corporation Nov/Dec 2004

Provided by ProQuest Information and Learning Company. All rights Reserved

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