Composition of essential oil of Omani Juniperus excelsa fruit and antimicrobial activity against foodborne pathogenic bacteria

Peer review under responsibility of Taibah University

Abstract

Juniperus excelas is an important flowering medicinal plant belonging to the Cupressaceae family. Essential oil was obtained from fresh fruit of J. excelas by hydro-distillation in a Clevenger apparatus and analysed by gas chromatography–mass spectrometry (GC–MS). The chemical constituents of the essential oil were identified by their mass spectra, retention time and retention indices. The yield was 0.27%. We identified 48 chemical compounds accounting for 89.74% of the composition. The major chemical components were α-terpinene (23.85%), limonene (23.42%), fenchene (6.57%), camphene (6%), δ-3-carene (4.17%), 4-terpineol (2.93%), germacrene B (2.21%), myrcene (1.96%), α-pinene (1.77%), β-pinene (1.53%) and abietatriene (1.13%). The antimicrobial activity of the essential oil of J. excelas was determined against one Gram-positive and two Gram-negative foodborne pathogenic bacteria, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. No activity was detected.

Keywords:

• Juniperus excelsa
• Essential oil
• Chemical constituents
• Antimicrobial activity
• GC–MS

1 Introduction

Juniperus excelsa M. Bieb is a flowering medicinal plant belonging to the Cupressaceae family. It grows in the Hajar Mountains of Oman and in Greece, the Islamic Republic of Iran, Lebanon, the Syrian Arab Republic and Turkey. In Oman, it is restricted to the highest areas of woodland, such as the central massif of Al Jebel Al-Akhdar and the outlying mountains of Jebel Qubal and Jebel Al-Khawr [1]. Juniperus excelas is a shrub or tree up to 20 m tall with a trunk as wide as 2 m in diameter. Its crown is conical but later it becomes broader and open [2]. The leaves are evergreen and of two types. Seedlings develop needle-like leaves 8–10 mm in length, while adult trees grow scale-shaped leaves 0.6–3 mm long [3]. The main reported chemical components of the essential oil of the fruit are α-pinene (89.49%) and germacrene B (4.36%) [4,5], and the main chemical constituents of the essential oil of the leaves are cedrol (28.1%), α-pinene (22.5%) and limonene (22.7%) [6].

J. excelas is used in traditional medicine mainly for lowering blood pressure [4]. Its essential oil is also widely used in aromatherapy for mood scents, scent masks, soaps, candles, cosmetics and fragrances, lotions and remedies [5]. In Oman, it is also used traditionally for bronchitis, the common cold, jaundice and tuberculosis [7–14]. Other species of juniper are used for the treatment of hyperglycaemia, tuberculosis, bronchitis, pneumonia, ulcers, intestinal worms, wound healing and liver disease in traditional medicine [5]. In Turkish traditional medicine, juniper species are mainly used as diuretics, stimulants, antiseptics and for wound healing [8,9]. The essential oil of J. excelas is reported to have strong antimicrobial, antioxidant, antifungal, antiviral and antispasmodic activities [9–14]. The aim of this work was to isolate and identify the chemical constituents of the essential oil from fruit of J. excelas and evaluate their antimicrobial activity against foodborne pathogenic bacteria.

2 Materials and methods

2.1 Chemicals

The chemicals and solvents used were of analytical grade from BDH, United Kingdom. The Clevenger apparatus was from Borosil, India. Agar gel petri dishes were prepared at the Department of Biological Sciences, College of Arts and Sciences, University of Nizwa, Oman, which also supplied the bacterial strains, Gram-negative Escherichia coli (ATCC 9637) and Pseudomonas aeruginosa (ATCC 9027) and Gram-positive Staphylococcus aureus (ATCC 29213).

2.2 Plant samples

Fruit samples of J. excelas were collected from Al Jabel Al-Akhdar at Bahla, Oman, on 1 November 2012 between 9:00 and 10.30. The plant was identified morphologically on a website. The samples were then kept in the laboratory at room temperature for further processing.

2.3 Isolation of essential oil

After removal of diseased fruit, the samples were washed with fresh water, and whole fruit (203.89 g) were chopped into small pieces and placed in a round-bottom flask. Water (1 L) was added, a Clevenger apparatus was fitted, and the samples were boiled for 3 h. The essential oil was collected from the receiver, dried over anhydrous sodium sulphate and preserved in a sealed amber-coloured vial at 4 °C until further analysis.

2.4 Gas chromatography–mass spectroscopy

Gas chromatograph–mass spectrometer (GC–MS) analysis of the essential oil was performed in a Perkin Elmer Clarus 600 GC system equipped with a Rtx^®-5MS fused silica capillary column (30 m × 0.25 id, film thickness 0.25 μm) coupled with a Perkin Elmer Clarus 600C MS. Helium gas was used as the carrier at a constant flow rate of ±1 ml/min. The mass transfer line and injector temperatures were set at 250 °C and 300 °C, respectively. The oven temperature was programmed from 40 °C (hold 2 min) to 270 °C at 2 °C/min, then held isothermal for 10 min and finally raised to 300 °C at 10 °C/min. Diluted samples (1/100, v/v, in dichloromethane) of 1 μl were injected in split mode with a split ratio of 120:1. The percentages of essential oil constituents were derived from peak areas.

2.5 Identification of essential oil chemical constituents

The chemical constituents of the essential oil were identified from their GC retention time and calculation of their retention indices based on n-alkanes (C₆–C₂₄). Individual compounds were identified by comparison of their mass spectra (NIST 2005 v.2.0 and Wiley Access Pak v.7, 2003 of GC–MS systems) [15,16].

2.6 Antibacterial activity assay

The disc diffusion method was used to determine the antibacterial activity of the essential oil [16]. Concentrations of 2.0, 1.0, 0.5 and 0.25 mg/ml were prepared in dimethyl sulphoxide, impregnated on filter paper discs (5 mm diameter) and placed on agar plates inoculated with bacteria. Amoxicillin (0.5 mg/ml) was used as a positive control and dimethyl sulphoxide as a negative control. The plates were incubated microaerobically at 37 °C for 24 h. Antibacterial activity was evaluated by measuring the diameter of inhibition zones. Tests were carried out in triplicate.

3 Results

3.1 Chemical composition of essential oil

The yield of essential oil from the fruits of J. excelas by hydro-distillation was 0.56 g (0.27%). The oil was pale yellow with a very strong odour. GC–MS revealed the presence of 48 compounds accounting for 89.74% (Table 1), which included high concentrations of monoterpene hydrocarbons, sesquiterpenes hydrocarbons and their oxygenated mono- and sesquiterpenes (Fig. 1 and Table 1).

Fig. 1 Chromatogram of essential oil obtained from fruits of JE.

Table 1 Percentage of essential oil composition of JE grown in Oman.

Sl. No	Retention time (min)	Compound	Yield (%)	KI
1	3.41	Furfuryl methyl ether	0.035	855.37
2	4.79	Bornylene	0.343	908.64
3	5.19	Tricyclene	0.053	924.09
4	5.30	α-Thujene	0.383	928.34
5	5.59	α-Pinene	1.771	939.53
6	5.83	Fenchene	6.572	948.64
7	5.88	Camphene	6.215	950.57
8	6.51	Sabinene	0.063	974.90
9	6.64	β-pinene	1.533	979.92
10	7.00	Myrcene	1.965	993.82
11	7.67	δ-3-Carene	4.177	1014.74
12	7.80	α-Terpinene	0.097	1018.49
13	8.45	Limonene	23.428	1037.28
14	9.23	γ-Terpinene	0.288	1059.82
15	10.32	σ-Terpinolene	23.859	1091.32
16	11.21	α-Fenchol	0.012	1114.87
17	11.70	α-Campholenal	0.010	1127.31
18	12.20	Trans-Pinocarveol	0.024	1139.69
19	12.42	Camphor	0.074	1145.27
20	13.56	Abietatriene	1.130	1173.86
21	13.73	4-Terpineol	2.930	1178.21
22	14.26	α-Terpineol	0.149	1191.45
23	14.51	Myrtenal	0.008	1197.81
24	15.40	trans-Carveol	0.102	1219.54
25	16.42	Carvone	0.081	1244.05
26	16.84	Piperitone	0.259	1254.32
27	18.17	Bornyl acetate	0.896	1286.23
28	20.27	δ-Elemene	0.058	1306.70
29	20.75	α-Cubebene	0.018	1308.80
30	21.83	α-Copaene	0.023	1313.53
31	22.47	β-Elemene	0.053	1316.33
32	23.57	β-Caryophyllene	0.409	1418.43
33	24.12	γ-Elemene	0.092	1432.32
34	24.92	α-Humulene	0.390	1452.52
35	25.82	α-Amorphene	0.042	1475.25
36	25.99	Germacrene D	0.255	1479.54
37	26.75	α-Muurolene	0.032	1498.73
38	27.28	-Cadinene	1.153	1512.77
39	27.31	4-Cubebol	0.083	1513.45
40	27.65	δ-Cadinene	0.228	1522.53
41	28.62	Elemol	0.128	1547.49
42	28.60	Germacerene D	2.217	1547.59
43	29.57	Germacrene D-4-ol	0.015	1573.08
44	32.04	γ-Eudesnol	0.066	1640.05
45	32.31	β-Eudesmol	0.030	1647.51
46	32.48	α-Eudesmol	0.061	1650.00
47	32.48	β-Cadinol	0.061	1652.21
48	44.29	Biformene	0.244	2006.68

KI: Retention indices relative to (C₆–C₂₄) n-alkanes on the Rtx^®-5MS silica capillary column.

The major components were α-terpinene (23.85%), limonene (23.42%), fenchene (6.57%), camphene (6.21%), δ-3-carene (4.17%), 4-terpineol (2.93%), germacerene D (2.21%), myrcene (1.96%), α-pinene (1.77%), β-pinene (1.53%) and abietatriene (1.13%). Minor components were identified at concentrations less than 6% (Table 1). The predominant compounds in the essential oil of J. excelas fruit were α-terpinene, limonene, fenchene and camphene.

3.2 Antimicrobial activity

The essential oil did not show any activity against E. coli, P. aeruginosa or S. aureus (Table 2)

Table 2 Antimicrobial activity of essential oil against food borne pathogenic bacteria.

	Concentration (mg/ml)	E. coli^a (mm)	S. aureus (mm)	P. aeruginosa (mm)
Essential oil	2	nd	nd	nd
	1	nd	nd	nd
	0.5	nd	nd	nd
	0.25	nd	nd	nd
Standard (amoxicillin)	0.5	24 ± 0.10	29 ± 0.21	nd

a Values are represented as the mean ± S.D. of three experiments; nd = not detected

4 Discussion

A total of 79 chemical components were detected in J. excelas essential oil, but only 48 chemical compounds were identified (Table 1), equivalent to 89.74% of the content. The yield and chemical composition of volatile oils is influenced by genetic constitution and environmental conditions, and correlations between chemotype polymorphism, sexual polymorphism and the environment have been detected [17]. The oil contained α-terpinene, limonene, fenchene, camphene, δ-3-carene, 4-terpineol, germacerene D, myrcene, α-pinene, β-pinene and abietatriene, with α-terpinene and limonene as the predominant components. Several studies have identified the chemical constituents of the essential oil of J. excelas fruit [18–20]. According to Ehsani et al. [14], α-pinene, myrcene and germacerene B are the main chemical components, and Adams reported that α-pinene is a major component of both the fruit and leaves of the essential oil [16]. Bakkour et al. [17] reported 32 compounds (86%) and 30 compounds (86.81%) in essential oils from unripe and ripe berries of J. excelas growing in Lebanon. Trans-nerolidol, (Z,E)-farnesol and α-pinene have been reported as major compounds of essential oil from unripe berries and α-pinene, β- myrcene, (E,E)-farnesol, and β-pinene as the major compounds of oil from ripe berries [15,16]. Unlu et al. [18] reported that the main chemical components of essential oil of J. excelas grown in Turkey [17] were α-pinene, α-cedrol and verbenone. Atas et al. [19] reported 25 compounds, representing 97.2% of the essential oil, with α-pinene as the main component. The difference in the chemical constituents of the essential oil in the present study and that of studies on J. excelas collected elsewhere might be due to temperature, climate and geographical location. The samples used in this study were collected in a hot desert climate.

We found no antibacterial activity against the tested organisms, in contrast to several studies that have reported that the essential oil of fruits of J. excelas have antibacterial activity [18–20]. The results of the present study are, however, similar to those of Atas et al. [19], who found no activity against B. subtilis, C. albicans, E. coli, P. aeruginosa or S. aureus. The lack of activity might be due to decomposition of the essential oil during the antimicrobial assay.

5 Conclusion

Forty-eight compounds were identified in essential oil from the fruit of on J. excelas. α-Terpinene and limonene were major constituents. The oil was inactive against the tested bacteria.

Acknowledgements

The authors are grateful to University of Nizwa, Nizwa, Oman, for providing chemicals and laboratory facilities. The authors are thankful to Professor Dr Nafsiah Binti Shamsudin for her continuous encouragement during the work. The authors would like to thank the staff of the Pharmaceutical Chemistry Laboratory, Pharmacognosy Laboratory and Organic Chemistry Laboratory for their assistance. The authors wish to express sincere gratitude to the Central Instrument Laboratory, College of Agriculture and Marine Sciences, Sultan Qaboos University, Oman, where the test were confirmed (Grant no. 507/SOP/OB/1/2013).

Notes

Peer review under responsibility of Taibah University