study of biogenic volatile organic compounds emitted ...

Proceedings of the Global Conference on Global Warming-2009, July 5-9, 2009, Istanbul, Turkey

STUDY OF BIOGENIC VOLATILE ORGANIC COMPOUNDS EMITTED FROM JUNIPERUS COMMUNIS AND JUNIPERUS OXYCEDRUS GROWING IN ALGERIA Y.Foudil-Cherif1*; N. Yassaa1,2, N. Boutarene1, B.Y.Meklati1,2 1

Université des Sciences et de la Technologie Houari Boumediène , U.S.T.H.B, Faculté de Chimie, EL-Alia BP 32, Bab-Ezzouar 16111, Alger, ALGERIE. 2 C.R.A .P.C., BP 248 Alger RP 16400, ALGERIE. * Corresponding author: [email protected]

ABSTRACT The analysis of biogenic volatile organic compounds (BVOCs) was performed both in the essential oils and in the atmospheric emissions of some Juniperus communis and Juniperus oxycedrus growing in Algeria. While the essential oils were obtained by steam distillation of plant materials and then analysed by gas chromatography equipped by either flam ionization detector or mass spectrometer, the biogenic emission from detached plant materials were studied using a technique of static headspace solid-phase microextraction combined gas chromatography-mass spectrometery. Tricyclene with circa 26 % was the major compound followed by β-caryophyllene with 11 % and αpinene with 10% in the atmospheric emissions of the needles of J. communis. However in the emissions of the berries of the same species, β-caryophyllene dominated by 22 % followed by αpinene with 17 %. In the essential oils obtained from the needles of J. communis, tricyclene was the dominant monoterpene species (circa 30 %) followed indeed by α-pinene (circa 20%). The berries of Juniperus oxycedrus showed a peculiar pattern with high predominance of α-pinene both in the essential oils (circa 58 %) and in the gaseous emission (circa 76 %). INTRODUCTION The volatile and semi-volatile substances, other than CO and CO2, produced by plants and other live species are collectively known as biogenic volatile organic compounds (BVOCs). They comprise a large number of organic substances, which includes isoprene and terpenoid compounds, alkanes, alkenes, carbonyl compounds, alcohols, and esters and are typically present in the atmosphere at concentration in the parts-per-trillion and parts-per-billion range. Isoprene and terpenoid compounds are the principal components usually found (Kesselmeier and Staudt, 1999). These volatile compounds are responsible for multiple interactions between plants and other organisms, such as pollinating animals and predators of herbivore creatures. Determination of the chemical composition of the BVOC mix is essential for studies of the biological processes involved in the production emission, and effects of such substances. Identification, characterization, and quantification of BVOCs are also relevant because of their key role in atmospheric chemistry. Terpenes emcompass several wide classes of compounds, including monoterpenes, sesquiterpenes and oxygenated terpenes. These compounds are emitted from conifers as well as broad-leaved trees as a function of temperature, or both temperature and light (Kesselmeier and Staudt, 1999). They react with OH, O3, and NO3, the common atmospheric oxidants, with lifetimes that range from minutes to days (Atkinson and Arey, 2003). Several monoterpene oxidation products are known to undergo gas-particle portioning and have been found in ambient air in the gas and particle phases. The genus Juniperus belongs to the family Cupressaceae and comprises about 70 species distributed in the Northern Hemisphere. Among them, Juniperus communis is a familiar species. It is a shrub or small tree, very variable and often a low spreading shrub, but occasionally reaching 10 m tall. Common Juniper has needle-like leaves in whorls of three; the leaves are green, with a single white stomatal band on the inner surface. The seed cones are berry-like. Juniperus communis is commonly used in horticulture as an ornamental shrub, but is too small to have any general wood usage.

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The juniper berries, are too bitter to eat raw and are usually sold dried and used to flavour meats, sauces, and stuffings. They are generally crushed before use to release their flavour. The cones are used to flavour gin. Juniper berries have long been used as medicine by many cultures. Juniper berries act as a strong urinary tract disinfectant if consumed and were used by American Indians as a herbal remedy for urinary tract infections. Juniperus oxycedrus is a species of juniper, native across the Mediterranean region from Morocco and Portugal, north to southern France, and east to westernmost Iran, growing on a variety of rocky sites from sea level up to 1600 m altitude. The seed cones are berry-like, green ripening in 18 months to orange-red with a variable pink waxy coating. An additional variety or subspecies J. oxycedrus var. badia H.Gay (syn. J. oxycedrus subsp. badia (H.Gay) Debeaux) is distinguished on the basis of larger cones (10–13 mm diameter), tinged purple when mature; it is described from northern Algeria, and also reported from Portugal and Spain. Cade oil is the essential oil obtained through destructive distillation of the wood of this shrub. It is a dark, aromatic oil with a strong smoky smell which is used in some cosmetics and (traditional) skin treatment drugs, as well as incense. In this study, the organic compounds were characterised both in the essential oils and in the atmospheric emissions of some Juniperus communis and Juniperus oxycedrus growing in Algeria. The results obtained are discussed in the context of plant-atmopshere interactions. EXPERIMENTAL 1) Essential oils Plant Materials: The needles and berries of leaves of the Juniperus communis and Juniperus Oxycedrus were collected from the trees located in Tala Guilef and Bouira (National Park of Djurdjura). Oil isolation: The oils were isolated from the fresh leaves by steam distillation for 2.5 hours using a modified Clevenger-type apparatus and stored at low temperature. GC-MS analysis: samples were analyzed by GC/MS (EI) on gas chromatograph (Agilent Technologies a GC 6890A) coupled to a mass-selective detector (MSD 5973 inert) from the same company. Two different columns were used: HP5 (30m x 0.25mm) and HPWAX 20M (30m x 0.32mm) fused silica columns. The GC condition for the two columns: 60°C (6min) to 240°C at 3°C/min. The identification of components was established by combining the comparison of mass spectra of components with the published spectra (Adams, 1995) and the retention indices with the published index data (Jennings and Shibamoto, 1980). The quantitative composition was obtained by peak area normalisation, the response factor for each component was supposed to equal to one. The quantitative composition was obtained by peak area normalization, the response factor for each component was supposed to equal to one. 2) Atmospheric emission 2.1. Materials A manually operated SPME holder was used throughout these experiments. The three beds 50/30 µm divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS) fibre from Supelco (Taufkirchen, Germany) has been used in this study. Each new fibre was thermally conditioned before use, according to the manufacturer recommendations. 2.2. Headspace SPME Headspace-SPME was carried out by weighing 200 mg of detached needles of each juniper species into a 20 ml vial fitted with a Teflon/silicone septum (Supelco) and the SPME holder needle was inserted through the septum and (DVB-CAR-PDMS) was exposed to the headspace for a given time. Each extraction was repeated 5 times. 2.3. Chromatographic analysis Immediately after SPME sampling (headspace SPME) the needle was introduced into the split/splitless injector of the gas chromatograph. A glass inlet liner with a narrow internal diameter (0.75 mm I.D., Supelco) was used in order to improve the GC resolution and the peak shape. Desorption was achieved in splitless mode at 250 °C for 2 min. These settings were found to be

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sufficient for a quantitative desorption of all the analytes studied. This was established by subjecting the analysed fibre to a second desorption and observing no carry-over peaks. The analysis of the samples collected by SPME were conducted using gas chromatograph (Agilent Technologies a GC 6890A) coupled to a mass-selective detector (MSD 5973 inert) from the same company. The MS system was operated in electron impact mode with the following conditions: ionization potential 70 eV; source temperature 230°C. The MS system was operated in scan mode (20-250 u) for the identification of compounds and in SIM mode for their quantification. The enantiomeric and non-enantiomeric monoterpenes were separated using a Cyclodex-B capillary column (30 m-long, 0.256 mm I.D., 0.25 µm film thickness) supplied by J & W Scientific (Folsom, CA, USA). With a helium (Messer Griesheim 6.0) gas carrier flow rate of 1 ml min-1, the column temperature was maintained at 40°C for 5 minutes, then increased to 160°C at 1.5°C per minute as previously established by Yassaa et al. (2001). For each plant species and each fibre coating, SPME/GC/MS was performed at least in triplicate to obtain a mean peak area. RESULTS AND DISCUSSION The total ion current (TIC) chromatographic profiles recorded from the GC/MS analysis in scan mode (20-250 u) of the gaseous emissions of J. communis berries is shown in Fig. 1. Rather very good separation has been obtained using HS-SPME for sampling the volatile fraction and beta-cyclodextrin capillary GC-MS for separation and analysis.

Sesquiterpenes Abundance 4500000

Monoterpenes

4000000 3500000 3000000 2500000 2000000 1500000 1000000 500000 15.00

Time-->

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

65.00

Fig. 1. Total ion current profiles of terpenes present in the headspace above the J. communis berries. Table 1 reports the percentage composition of various classes of compounds contained in the essential oils of the Juniperus communis needles and the Juniperus oxycedrus berries. More than 87 and 85 % of the detected chromatographic peaks were identified in the Juniperus communis needles and the Juniperus oxycedrus berries, respectively by comparing the Kovats retention indexes of compounds contained in the essential oils with those reported in the literature. Retention time confirmation of individual BVOC was performed by analyzing pure standards under the same conditions. The elution order of terpenes was further confirmed by comparison with our previous work (Yassaa et al., 2001; Yassaa and Williams, 2005) and from the literature. Monoterpene hydrocarbons were the dominant species in both J. communis needles and J. oxycedrus berries with 65 and 76 %, respectively. Sesquiterpenes accounted for 6 % in J. oxycedrus berries whereas alcohols with 12 % were the second highest compound class in J. communis needles.

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Table 1. Classes of compounds contained in the essential oils of the needles of Juniperus communis and in the berries of Juniperus oxycedrus. Needles of J. Communis

Classes of Compounds

Berries of J. Oxycedrus

Monoterpenes

65.1

76.3

Ketones

2.0

1.4

Sesquiterpenes

2.1

6.1

Aldehydes

0.4

---

Esters

4.6

---

Alcohols

11.8

1.3

Oxides Percentage of the identified compounds compiunds

1.6

0.4

87.7

85.6

The percent composition of the major compounds identified in the essential oils of the Juniperus communis needles and in the Juniperus oxycedrus berries and the percent composition of the major terpenes present in the gaseous emissions of the Juniperus communis needles and berries and in the Juniperus oxycedrus berries are given in Tables 2 and 3, respectively . As can be seen from Tables 2 and 3, Tricyclene with circa 26 % was the major compound in the atmospheric emissions of the J. communis needles followed by β-caryophyllene and α-pinene with 11 and 10%, respectively. However in the gaseous emissions of the berries of the same species, βcaryophyllene dominated by 22 % followed by α-pinene with 17 %. In the essential oils obtained from the needles of J. communis, tricyclene was the dominant monoterpene species (circa 30 %) followed indeed by α-pinene (circa 20%). The berries of Juniperus oxycedrus showed a peculiar pattern with high predominance of α-pinene both in the essential oils (circa 58 %) and in the gaseous emission (circa 76 %). Overall the compositions of the gaseous emissions of juniper species resembled those of their respective essential oils. This interesting finding implies that the emitted organic compounds and those containing in the essential oils are produced from the same photosynthesis pathway. Table 2. Composition of the major compounds identified in the essential oils of the needles Juniperus communis and in the berries of Juniperus oxycedrus. Compounds

Needles of J. Communis

Berries of J. Oxycedrus

α -Pinene

20.0

Tricyclene

33.3

63.4 ---

p-Cymene

5.5

1,6

β-Phellandrène

---

Limonene

2.4 ---

Linalol

0.4

0.4

4-Terpineol

4.9

0.2

α -Terpineol

0.5

0.1

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---


Table 3. Percent composition of the major terpenes identified in the emissions of the needles and berries of Juniperus communis and in the berries of Juniperus oxcedrus. Terpenes

Rt

α-Thujene α-Pinene β-Myrcene Tricyclene Sabinene Camphene δ-3-Carene α-Phellandrene 4-Carene β-Pinene Cis-b-Ocimene Limonene β-Phellandrene γ-Terpinene α-Terpinolene 1,8-Cineol 4-Terpineol α-Terpineol Camphor Neomenthol Thujopsene α-Cubebene Bornyl acetate Ylangene Copaene Borneol β-Caryophyllene

20.766 24.317 26.638 27.577 28.809 28.328 30.088 30.151 30.366 30.537 32.119 32.507 34.179 34.852 36.845 38.293 47.89 51.883 55.311 56.871 58.134 58.423 59.882 60.291 60.819 63.263 68.068

Needles of J. Communis 4.97 10.01 7.68 25.82 0.15 0.15 --1.15 2.63 1.43 3.92 4.51 6.35 4.96 3.09 --2.64 2.36 --5.33 --------1.72 0.39 10.74

Berries of J. Communis 5.46 17.27 14.65 13.57 0.08 0.15 --0.23 2.23 1.47 2.14 2.08 1.52 3.77 1.64 --1.15 0.85 0.07 5.45 --0.60 0.27 0.09 3.05 --22.23

Berries of J. Oxycedrus 0.17 76.05 4.90 0.09 0.05 0.38 0.01 0.05 0.18 1.77 0.68 1.75 2.43 0.34 1.83 0.01 ----0.44 0.42 0.12 5.44 0.71 0.17 1.41 --0.59

CONCLUSION More than 133 compounds were identified in the essential oils of juniper species namely : J. communis and J. oxycedrus. The major compounds were α-pinene and Tricyclene. Interestingly, the major volatile organic compound, α-pinene, was also the predominant one in the atmospheric emission of juniper species. This result could provide an important insight about the origin of 1150 TgC (Tg=1012g) of biogenic volatile organic compounds emitted each year into our atmosphere (Guenther et al., 1995) by terrestrial trees. REFERENCES Adams, R.P.. Identification of Essential Oils Components by Gas Chromatography/Mass Spectrometry. Allured Publishing Corporation, Carol Stream, Illinois, U.S.A (1995). Atkinson, A., Arey, J.. Gas-phase tropospheric chemistry of biogenic volatile organic compounds : A review. Atmos. Environ. 37, S197-S219, 2003. Guenther, A. et al.. A global-model of natural volatile organic compound emissions, J. Geophys. Res., 100, 8873-8892, 1995. Jennings, W., Shibamoto, T.. Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Chromatography. Academic Press, NewYork (1980). Kesselmeier, J., Staudt, M.. Biogenic volatile organic compounds (VOC): An overview on emission, physiology, and ecology. J. Atmos. Chem. 33, 23-88, 1999. Lelieveld, J. et al., Atmospheric oxidation capacity sustained by a tropical forest. Nature 452, 737-740, 2008.

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Yassaa, N., Brancaleoni, E., Frattoni, M.,Ciccioli, P.. Trace level determination of enantiomeric monoterpenes in terrestrial plant emission and in the atmosphere using a -cyclodextrin capillary column coupled with thermal desorption and mass spectrometry, J. Chromatogr. A, 915, 185-197, 2001. Yassaa, N., Williams, J..Analysis of enantiomeric and non-enantiomeric monoterpenes in plant emissions using portable dynamic air sampling/solid-phase microextraction (PDAS-SPME) and chiral gas chromatography/mass spectrometry, Atmos.Environ.,39,4875-4884, 2005.

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