Volatile compounds from leaf extracts of Juniperus excelsa growing in Syria via gas chromatography mass spectrometry

Abstract

This study reports the use of gas chromatography mass spectrometry (GC-MS) to investigate volatile compounds in leaves of Juniperus excelsa native to Syria. Leaf samples were collected from both 100-year and 10-year old J. excelsa plants. Dried leaf samples were extracted with hexane to obtain essential oil metabolites and other non-polar compounds. GC-MS was used to profile and analyze metabolites in hexane extracts. Mass spectral deconvolution and identification and analysis of KI values allowed us to characterize sixty-nine metabolites, including twenty-four monoterpenes; twenty-nine sesquiterpenes; and sixteen other compounds including alkanes. Among these sixty-nine metabolites, germacrene B, cedrol, γ-elemene, and stenol were produced in leaf extracts of both 100-year and 10-year old trees. Interestingly, we observed that ten monoterpene and nineteen sesquiterpene compounds produced in leaves of the 100-year old trees were not detected in ones of the 10-year old trees investigated. In contrast, junipene was a dominant essential oil component in leaves of the 10-year old trees, but was either just at a detectable level or undetectable in leaves of the 100-year old trees.

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1. Introduction

The genus Juniperus belongs to the family Cupressaceae, which includes ca. 67 species distributed across China, the Middle East, North Africa, Europe, and North America.¹ All juniper species produce essential oils. Eleven species of Juniperus were reported for production of essential oils in the United States of America.² The essential oils of many Juniperus species have been analyzed and quantified by means of several methods of extraction including steam distillation^1,3,4 and hexane or methanol extraction.⁵ More than 80 metabolites have been identified from extracts of the leaves and berries of junipers,⁴ with the dominant essential compounds varying from one species to another. For example, α-pinene, sabinene, and limonene, terpinen-4-ol, γ-cadinene, δ-cadinene, elemol, cubenol, epi-α-cadinol, epi-α-muurolol, α-cadinol, and 4-epi-abietal were major essential metabolites in J. recurva from India,¹ whereas pinene, 3-carene, and trans-totarol were the three major essential oil components in J. procera from East Africa,⁶ and pinene (40–57%) and manoyl oxide (10%) were the two dominant components of essential oils of J. oxycedrus.⁴

The most common native juniper species in Syria is Juniperus excelsa, which is also found in Lebanon, Iran, Turkey, and Greece.⁶ In Syria, J. excelsa is mainly found at high altitude in the mountains (1500–2300 m above sea level) of western and southern Syria through the eastern border of Lebanon.^7,8J. excelsa is evergreen and has two types of leaves. Seedlings develop needle-like leaves 8–10 mm in length, while adult trees grow 0.6–3 mm long scale-shaped leaves. J. excelsa is a dioecious species. The berry-like cones are 6–11 mm in diameter, blue or black and contain 3–6 seeds when mature. One hundred year old trees growing in Syria can be up to 20 m tall with a trunk up to 2 m in diameter and an irregular crown (Fig. S1†). Unfortunately, the woodland populations of J. excelsa in Syria have declined due to soil erosion and human activities, livestock grazing, and fire.^8,9

Gas chromatography mass spectrometry (GC-MS) is a powerful technology for plant metabolites analysis.^10,11 The purpose of this research is to use GC-MS based profiling to analyze hexane-soluble phytochemicals in leaves collected from both 100 year and 10 year old trees of J. excelsa growing in three regions in Syria. This study provides useful data to better understand the arrays of volatile compounds found in this species of juniper.

2. Experimental

2.1. Chemicals

Hexane (100%, HPLC grade) was purchased from VWR (Atlanta, Georgia, USA). Florida TRPH (Total-Recoverable-Petroleum-Hydrocarbons) standard containing 500 μg mL⁻¹ even-numbered chain n-alkenes (C10–C40) was purchased from Restek (Florida, USA, cat#31266).

2.2. Plant materials

J. excelsa is native to Syria and its herbarium specimen (#Z2L4 2006, Nawras) is at the herbarium in the Biodiversity Garden, Syria–Nabek. This plant grows in the Kalamon Mountains across the areas of Kara (110 km from Damascus), Rass Almarra (80 km from Damascus), and Rankous (50 km from Damascus). The altitude of these mountains is 1500–2300 m above sea level. In these areas, the ages of J. excelsa plants are variable from one year old seedlings to 100 year old trees⁸ (Fig. S1†). In spring 2008, leaves were collected from 5–6 branches of six individual 100-year old trees in each area and then pooled together for analysis of volatile compounds. In addition, leaves from six individual 10-year old trees growing in Kara were collected and pooled together for volatile compound analysis. Fresh leaves were brought back to the University of Damascus and air-dried, then stored at room temperature till extracted.

2.3. Extraction

Air-dried leaf samples of J. excelsa were ground into fine powder in a mortar. Triplicates of two grams of powder contained in glass tubes were extracted with 10 mL of hexane for twelve hours at 4 °C. Tubes were centrifuged at 3000 rpm for 15 min at 4 °C. This extraction step was then repeated once. The two hexane extracts were pooled together. One mL of the 20 mL hexane extract was transferred to a 1.5 mL eppendorf tube and then centrifuged at a speed of 10000 rpm for 10 min to remove turbidity. The supernatant was used for GC-MS analysis as described below.

2.4. GC-MS analysis

GC-MS was performed on a Thermo Trace GC Plus gas chromatograph coupled to a Thermo DSQII single quadrupole mass spectrometer equipped with an ion detector. A positive electron impact ion source (70 EV) was used to ionize compounds and fragment masses were scanned in the range of 40–800 (m/z). Chromatographic separation was achieved with a Restek Rtx5Sil MS (5% phenyl polysiloxane) column (30 m in length; 0.25 mm i.d.; 0.25 μm film thickness). Helium was used as the carrier gas and a constant flow was set at 1 mL per minute (linear gas velocity of ∼40 cm s⁻¹). One μl of extract was injected via using a PTV injector operated in splitless mode. The column oven temperature was programmed for a 60 °C initial temperature with an 8 °C min⁻¹ ramp to a final temperature of 360 °C.

A retention standard containing even-numbered chain n-alkanes (ranging from C-10 to C-40 in chain length) was used to determine Kovats Retention Index (KI) values of compounds (Fig. S2†). The concentration of each n-alkane was 10 μg mL⁻¹ prepared by diluting a Florida TRPH reference standard (Restek) in a solvent mixture consisting of isooctane (60%), tetrahydrofuran (20%), and benzene (20%). Retention times of standards were used to calculate KI values for each metabolite by means of the KI equation formula established by Kovats.^12–15 Compound spectra were matched to the NIST/EPA/NIH Mass Spectral Library, Version 2.0d (built on Apr 26, 2005).

Co-eluting metabolite peaks were deconvoluted by using Automated Mass Spectral Deconvolution and Identification System (AMDIS) (ChemiStation Software). Identification of major compounds was performed by using its KI value¹⁴ and by mass spectrum matching (at least 80% matching) to the Wiley 7th/NIST 05 MS library (Fig. S3 and S4†). In the light of the approach reported by Adams,¹⁴ the average content (percentage) for each peak was obtained by dividing its peak area value with the total TIC peak area value and then timing 100%.

2.5. Principal component analysis

We used principal component analysis (PCA) to reveal the correlation of 100- and 10-year old trees grown in three areas, which impacted metabolite composition. Sixty-nine identified compounds were used as attributes. Three areas and ages of trees were used as cross-product matrix. The software used was PC-ORD, which was developed for multivariate analysis of ecological studies (McCune and Mefford, 1990).¹⁶

3. Results and discussion

3.1. Results

The goal of this work was to perform a survey study on volatile compounds of leaves of J. excelsa growing in Syria. Leaf samples were collected from both 100-year old trees (in Kara, Rass ALMarra, and Rankous) and 10-year old trees (growing in Kara). Gas chromatography analysis showed nearly 120 significant peaks in the hexane extracts of leaves of both 100-year old trees and 10-year old shrubs of J. excelsa. Analysis of the corresponding MS and analysis of KI values allowed us to characterize 69 metabolites in total, including 24 monoterpenes, 29 sesquiterpenes, and 16 other compounds including alkanes from these leaves (Table 1).

Table 1 Volatile compounds identified in leaf extracts of J. excelsa from Syria

No.	RI	Compound name	Relative Percentage (%)^a
			100-year old trees			10-year old trees
			Rass Almarra	Kara	Rankous	Kara
a Bold numbers mean the percentage higher than 2% to show main components. b mt: monoterpene. c st: sesquiterpene. d —: not detected. e The order of RI differs from theoretical ones.
1	991	β-Pinene (mt, C10H16)^e	0.55	1.66	1.23	—^d
2	996	β-Myrcene (mt, C10H16)	1.35	0.26	1.27	0.55
3	1005	Camphene (mt, C10H16)	1.85	0.21	0.09	—
4	1014	α-Pinene (mt^b, C10H16)^e	1.65	0.22	0.65	—
5	1019	δ-3-Carene (mt, C10H16)	—	0.22	—	0.5
6	1037	Phenanthrene (C14H10)	—	0.23	0.37	0.5
7	1039	D,L-Limonene (mt, C10H16)	0.52	0.3	0.27	0.11
8	1045	L-Limonene (mt, C10H16)	2.15	2.01	0.89	—
9	1048	Limonene oxide (mt, C10H16O)	—	—	—	0.42
10	1068	γ-Terpinene (mt, C10H16)	0.22	—	0.32	0.3
11	1095	α-Terpinolene (mt, C10H16)	0.36	0.28	0.19	—
12	1107	Linalool (mt, C10H18O)	0.18	1.54	—	—
13	1138	Allo-Ocimene (mt, C10H16)	0.84	1.54	0.65	—
14	1153	Trans-Pinocarveol (mt, C10H16O)	—	—	—	1.02
15	1163	Trans-Carveol (mt, C10H16O)	—	—	—	0.45
16	1166	Carvone (mt, C10H14O)	—	—	—	0.23
17	1170	Camphor (mt, C10H16O)	0.12	0.21	0.06	0.52
18	1183	Thymol (mt, C10H14O)	2.5	—	1.66	—
19	1222	Pinocarvone (mt, C10H14O)	—	—	—	0.43
20	1229	p-Cymene 8-ol (mt, C10H14O)	—	—	—	0.45
21	1233	d-Verbenol (mt, C10H16O)	—	0.17	—	1.64
22	1235	Verbenone (mt, C10H14O)	0.09	0.17	0.05	1.02
23	1248	Hexyl isovalerate (mt)	0.14	0.12	0.19	—
24	1250	Myrtenol (mt, C10H16O)	—	—	—	0.16
25	1255	Myrtenal (mt, C10H14O)	—	—	—	0.37
26	1257	Benzen, 1,3-bis-dimethylethyl (C14H22)	0.21	0.19	0.21	0.75
27	1302	Bornyl acetate (C12 H20 O2)	—	—	—	1.46
28	1317	Tetradecane (C14H30)	—	0.23	—	0.21
29	1320	Cyclohexene, 2-ethenyl-1, 3, 3-trimethyl- (C11H18)	0.12	0.1	0.14	—
30	1347	α-Terpinene (mt, C10H16)	2.58	0.78	0.36	—
31	1359	α-Cubebene (st^c, C15H24)	0.92	0.19	—	—
32	1403	β-Elemene (st, C15 H24)	2.67	1.33	0.45	—
33	1480	α-Caryophyllene (st, C15H24)	—	—	—	1.87
34	1438	Trans-Caryophyllene (st, C15H24)	0.38	2.19	—	—
35	1441	β-Caryophyllene (st, C15H24)	1.11	—	1.32	—
36	1449	γ-Elemene (st, C15 H24)	5.66	1.32	2.53	0.36
37	1466	Germacrene D (st, C15H24)	1.91	1.13	1.23	—
38	1479	α-Humulene (st, C15H24)	0.51	—	0.25	—
39	1495	Copaene (st, C15H24)	0.91	—	0.45	—
40	1499	Cedrene (st, C15H24)	0.13	—	—	—
41	1515	β-Selinene (st, C15H24)	0.81	0.5	0.56	—
42	1519	α-Selinene (st, C15H24)	—	0.64	0.72	—
43	1520	Aromadendrene (st, C15H24)	—	0.17	0.67	2.24
44	1540	δ-Cadinene (st, C15H24)	1.52	0.64	2.6	—
45	1542	Epoxy caryophyllene (st, C15H24O)	4.77	2.18	1.4	—
46	1554	γ-Cadinene (st, C15H24)	2.56	—	—	—
47	1559	α-Muurolene (C15H24)	0.23	0.24	0.26	—
48	1562	γ-Selinene (st, C15H24)	0.35	0.21	—	—
49	1564	Germacrene B (st, C15H24)	7.63	3.34	5.5	0.45
50	1573	Elemol (st, C15 H26 O)	2.2	2.21	1.42	0.35
51	1597	Hexadecane (C16H34)	1.77	5.45	0.73	5.47
52	1599	α-Cadinol (st, C15 H26 O)	0.63	0.25	1.35	—
53	1615	Junipene (C15H24)	—	0.41	0.65	30.2
54	1621	Isospathulenol (st, C15 H24 O)	0.14	—	—	0.41
55	1632	β-Farnesene (st, C15H24)	—	—	—	0.23
56	1644	Caryophyllene oxide (st, C15H24O)	—	1.23	2.24	0.75
57	1656	α-Farnesene (st, C15H24)	—	1.99	1.23	0.41
58	1712	Heptadecane (alkane, C17H36)	—	—	—	1.17
59	1713	α-Amorphene (st, C15H24)	0.64	2.1	0.46	—
560	1735	Ledol (st, C15H26O)	0.13	2.23	0.15	—
61	1804	Cedrol (st, C15H26O)	3.4	1.66	2.16	1.24
62	1902	Nonadecane (C19H40)	—	0.53	1.03	0.45
63	1965	α-Copaene-8-ol (C15H24O)	—	2.86	2.24	—
64	1998	Eicosane (C20H42)	0.32	0.34	0.22	7.65
65	2196	Docosane (C22H46)	4.49	0.49	3.2	—
66	2398	Tetracosane (C24H50)	—	0.67	3.3	—
67	2696	Heptacosane (C27H56)	0.13	—	—	3.11
68	2906	Nonacosane (C29H60)	0.21	—	—	0.14
69	3205	Stenol (C18H38O)	4.84	6.3	4.4	4.38
Total percentage out of total TIC values			66	53	51	72

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Thirty-seven constituents accounting for nearly 72% of the total level of all detected compounds were identified from the hexane extract of leaves of 10-year old trees (Table 1). The six principal constituents were junipene (30%), eicosane (7%), hexadecane (5.47%), stenol (4.38%), heptacosane (3.11%), and aromadendrene (2.24%).

Forty-seven metabolites accounting for nearly 53% of the total level of all detected compounds were identified from leaves of 100-year old trees in Kara (Table 1). Ten most abundant compounds were stenol (6.3%), hexadecane (5.45%), germacrene B (3.34%), α-copaene-8-ol (2.86%), ledol (2.23%), elemol (2.21%), trans-caryophyllene (2.19%), epoxy caryophyllene (2.18%), α-amorphene (2.1%), and L-limonene (2.01%).

Forty-five constituents were identified from the hexane extract of leaves of 100-year old trees in Rass Almarra (Table. 1). These 45 metabolites accounted for nearly 66% of the total level of all detected compounds. The major constituents were germacrene B (7.63%), γ-elemene (5.66%), stenol (4.89%), epoxy caryophyllene (4.77%), docosane (4.49%), cedrol (3.4%), β-elemene (2.67%), α-terpinene (2.58%), γ-cadinene (2.56%), thymol (2.5%), elemol (2.22%), and L-limonene (2.15%).

Forty-five constituents were identified from leaves of 100-year old trees in Rankous. These forty-five components formed nearly 51% of the total content of all detected compounds (Table. 1). Nine major constituents were germacrene B (5.5%), stenol (4.4%), tetracosane (3.3%), docosane (3.2%), δ-cadinene (2.6%), γ-elemene (2.53%), α-copaene-8-ol (2.24%), caryophyllene oxide (2.24%), and cedrol (2.16%).

3.2. Discussion

In our study, we observed that the RI value of α-pinene was higher than β-pinene. This observation was different from the reported RI value order for the two compounds recorded in the reference book edited by R. P. Adams (2001).¹⁴ We checked all samples and found that the retention time of β-pinene was identically earlier than that of α-pinene (Fig. S4†). This different RI order likely resulted from the Restek Rtx5Sil MS (5% phenyl polysiloxane) column used in our study, which differed from capillary column products such as J & W DB-5 (Adams 2001).¹⁴

We observed dramatic differences in the arrays of volatile compounds between 10-year and 100-year old trees in Kara. We found that junipene was the most dominant component (30%) in the hexane extract of leaves from the 10-year old trees, but was a minor component (only 0.41%) in leaves of the 100-year old trees in the same area. In addition, junipene was not detected from leaves of the 100-year old trees in Rass Almarra (Table 1). Furthermore, pinocarvone, myrtenol, myrtenal, and p-cymene-8-ol produced in leaves of the 10-year old trees were not detected in any of those studied 100-year old trees. In contrast, ten monoterpenes and nineteen sesquiterpene compounds were identified in leaves of the 100-year old trees, but were not detected in leaves of the 10-year old trees investigated (Table 1). To characterize the correlation between ages of trees and volatile composition, we performed principal component analysis (PCA) and cluster analysis. It was obvious that those 100-year old trees from three different areas were clearly separated from 10-year trees (Fig. 1). In addition, 100-year old trees from Kara and Rankous areas showed close correlations in identified volatile compounds in leaves. We suggest that these variations in volatiles may result from differences in environment, ages of trees, sampling time, and germplasm etc. Variable essential oil compositions have been commonly observed in different species of Juniperus, in the same species growing in different regions, and in the same tree collected from different seasons.^1,4,6 Salido et al. (2002) reported that the percentage of α-pinene was higher in leaves of J. oxycedrus collected in September than in October.⁴ Furthermore, potential genetic differences can also result in dramatic variation of chemical compositions of essential oils in Juniperus species.³

Fig. 1 a: Dendrogram obtained from the cluster analysis of identified 69 compounds from leaves of both 100-year old trees in three regions and 10-year old trees in Kara. Six-nine metabolites were used as cross-products matrix for PCA analysis to reveal their correlation in two different ages' trees and in three different areas. b: An ordination 2D graph of PCA analysis shows separation of metabolites in samples collected from 10-year trees from 100-year trees. Kara 10 and 100: 10- and 100-year old trees growing in Kara; Rank100: 100-year old trees growing Rankous; Ras100: 100-year old trees growing in Rass Almarra.

Adams (1990) reported that both α-pinene and limonene were the dominant essential oils in J. excelsa collected from Greece. In addition, Topcu et al. (2005) reported that the two compounds together with cedrol were three dominant components of essential oils isolated from leaves and berries of J. excelsa grown in Turkey.¹⁷ Recently, Asili et al. (2008) also found that leaves of J. excelsa grown in Iran produced the three compounds forming principal components of essential oils.¹⁸ Although Syria is neighboring to the two countries, in our analyses, α-pinene and limonene identified from extracts were not dominant components of essential oils of leaves. These different observations from our experiment likely resulted from seasonal distinction of sampling, different ages of trees, geographical separation, and other factors. Here, we particularly suggest that the ages of trees likely impact volatile composition. In those studies completed by Adams (1990) and Topcu et al. (2005),^17,18 the ages of those sampled trees were not identified. In our study, we collected leaves from 100- and 10-year old trees respectively. Although, for 10-year old trees, we could only sample 10-year old trees from the area of Kara to investigate chemical composition in leaves due to being unable to identify 10-year old ones from the areas of Rass Almarra and Rankous, those young trees served as solid examples to reveal potential impacts of tree ages on volatile composition (Table 1 and Fig.1). Our study indicates that volatile compounds from leaves is impacted by ages of trees and suggests that when sampling tissues for anti-bacterial and other medicinal tests, estimation of ages of trees will be necessary.

These variations of constituents may explain the different effects of leaf extracts on inhibition of bacterium growth. We previously observed that the inhibitory activity of 10-year old tree leaf extracts on the growth of bacterium was more effective than that of extracts from the 100-year old trees, when tested at the same dosage.¹⁹ In another preliminary study, we observed that the concentrations of 10 mg mL⁻¹ leaf extracts of the 10-year old trees growing in Kara could inhibit the growth of Staphylococcus aureus-Gram-positive bacterium and Pseudomonas aeruginosa- Gram-negative bacterium. In contrast, extracts of leaves of 100-year old trees growing in the same area did not show any inhibitory activity against these two bacteria until the concentration was increased to 20 mg mL⁻¹.¹⁹ These differences in inhibitory activity against bacterium growth most likely are due to the variation in essential oil composition. A few experiments had been performed to test antimicrobial activity for juniper essential oils. Observations similar to ours were found in other species, e.g., J. oxycedrus.^1,20–22 Up to date, only a few studies have tested the effects of other juniper essential oil components on bacterium growth. α-Pinene was identified as one of the major juniper oil constituents.^6,23 In spite of the slight anti-bacterial activity observed by Dorman and Deans (2000),²⁴ Angioni et al. (2003) reported that α-pinene did not show antimicrobial activity.²³ In recent studies, essential oils of J. excelsa were shown to have strong antimicrobial activity against Clostridium perfinges and moderate inhibitory activity against the growth of S. aureus, S. pyogenes, S. pneumoniae, Mycobcaterium smegmatis, Candida albicans, and C. krusei.²⁵ Preliminary tests of individual components of J. excelsa oils showed that δ-3-carene was more effective than α-pinene against bacterium growth. In addition, the component selinene was found to inhibit the growth of S. aureus.²⁵ These tests provided evidence for the medicinal activity of juniper essential oils. In order to better understand the mechanism of the anti-bacterial activity and other medicinal uses of juniper oils from different species, further tests are to be performed in the future.

4. Conclusion

GC-MS based profiling reveals different volatile compositions in leaves of 100-year old trees grown in three areas. More importantly, this study shows different volatile arrays in leaves of 100- and 10-year old trees, indicating that ages of trees greatly impact biosynthesis of metabolites in J. excelsa.

Acknowledgements

The analysis of volatile compounds was financially supported by North Carolina State University. We thank Drs. Marc Johnson and Dr David Danehower for critical reading and suggestions.

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Footnote

† Electronic supplementary information (ESI) available: Supplementary Fig. S1–S4. See DOI: 10.1039/b9ay00256a

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