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The chemical composition of essential oils isolated from the leaves of Juniperus oxycedrus by hydrodistillation was analyzed by GC-MS. 42 compounds, representing 96.73% of total oil, were identified. J. oxycedrus oil was found to be rich in α-pinene (39.63%), manoyl oxide (12.34) and Z-caryophyllene (4.1%) and characterized by relatively high amounts of monoterpenes hydrocarbons and sesquiterpenes. Results of the antifungal testing by in vitro contact assay showed that the oil significantly inhibit the growth of nine plant pathogenic fungi.
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Received: 25th Nov-2011 Revised: 02nd Dec-2011 Accepted: 14th Dec-2012
Research Article
CHEMICAL COMPOSITION AND ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS
ISOLATED FROM JUNIPERUS OXYCEDRUS L.
Ismail Amri1,2, Lamia Hamrouni2, Samia Gargouri3, Mohsen Hanana4, Bassem Jamoussi5
1Faculté des Sciences de Bizerte. Bizerte, 7021, Tunisie.
2Laboratoire d’Ecologie Forestière, Institut National de Recherches en Génie Rural, Eaux et Forêts. BP
10, 2080 Ariana, Tunisie
3Laboratoire de Protection des Végétaux, Institut National de la Recherche Agronomique de Tunisie, Rue
Hédi Karray 2049, Tunisie.
4Laboratoire de Physiologie Moléculaire des Plantes, Centre de Biotechnologie de Borj-Cédria, BP 901,
Hammam-lif 2050, Tunisie.
5Institut Supérieur d’Education et de Formation Continue. Tunis, Tunisie.
ABSTRACT: The chemical composition of essential oils isolated from the leaves of Juniperus oxycedrus by
hydrodistillation was analyzed by GC-MS. 42 compounds, representing 96.73% of total oil, were identified. J.
oxycedrus oil was found to be rich in α-pinene (39.63%), manoyl oxide (12.34) and z-caryophyllene (4.1%) and
characterized by relatively high amounts of monoterpenes hydrocarbons and sesquiterpenes. Results of the
antifungal testing by in vitro contact assay showed that the oil significantly inhibit the growth of nine plant
pathogenic fungi.
Keywords: Juniperus oxycedrus, α-pinene, manoyl oxide, Z-caryophyllene, antifungal activity.
INTRODUCTION
Pests are the largest competitor of agricultural crops and severely reduce the crop production in the range of 25–
50% (Pimentel, et al., 1991; Oerke, 2006). To protect agricultural crops enormous amount of synthetic pesticides
are used. As per Agrow (2007) report, the total value of world’s agrochemical market was between US$31–35
billion and among the products, fungicides accounted for (22%). However, the excessive use of synthetic pesticides
in the crop lands, urban environment, and water bodies to get rid of noxious pests has resulted in an increased risk
of pesticideresistance, enhanced pest resurgence, toxicological implications to human health and increased
environmental pollution. In fact, combating of environmental pollution and its ill-effects on the life is one of the
most serious challenges before the present day world. Efforts are thus being made to replace these synthetic
chemicals with biological products, which are safer and do not cause any toxicological effects on the environment.
The natural pest and disease control either directly or indirectly using natural plant products including essential oils,
holds a good promise ( Isman, 2006; Bakkali, et al., 2008). Juniperus oxycedrus (Cupressaceae) is a shrub or small
tree growing wild in stony places of the Mediterranean and Near East countries. That is one of the most appreciate
plants for its essential oil richness and its plethora of biologically active compounds extensively used in folk
medicine. J. oxycedrus was used for the treatment of various diseases, such as hyperglycemia, obesity, tuberculosis,
bronchitis and pneumonia (Sanchez de Medina, et al., 1994). There are many reports on the chemical composition
of the oils from Juniperus species (Altarejos, et al, 1999; Milaos and Radonic, 2000; Salido et al, 2002. Loizzo, et
al., 2007), most of these reports indicate that α-pinene, manoyl oxide and Z-caryophyllene are the main constituents
of these oils. The chemical composition of Tunisian J. oxycedrus have been reported (Medini, et al., 2010), however
to the best of our knowledge, no report on the antifungal activity of J. oxycedrus essential oils.
The aims of this study were to determine the chemical composition of essential oils extracted from the aerial parts
of J. oxycedrus L. and to determine their antifungal activity against nine plant pathogenic fungi.
International Journal of Applied Biology and Pharmaceutical Technology Page: 227
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Amri et al
MATERIALS AND METHODS
Plant material
The leaves of J. oxycedrus were collected from the INRGREF arboretum (Tunisia) in December 2009.
Identification was performed in the Laboratory of Genetic of INGREF. A voucher specimen is deposited in the
Herbarium of this laboratory.
Isolation of the essential oils
The essential oils were extracted by hydrodistillation of dried plant material (100 g of leaves in 500 mL of distilled
water) using a Clevenger-type apparatus for 5 h. The oils were dried over anhydrous sodium sulphate and stored in
sealed glass vials at 4°C prior to analysis. Yield based on dried weight of the sample was calculated.
Analysis of the essential oils
Gas chromatography analysis/mass spectrometry analysis conditions
Gas chromatography analysis
The essential oils were analysed using a Hewlett Packard 5890 II GC equipped with Flame Ionization Detector
(FID) and HP-5 MS capillary column (5% phenyl/95% dimethylpolysiloxane: 30 m×0.25 mm id, film thickness
0.25 µm). Injector and detector temperature were set at 250 °C and 280 °C, respectively. Oven temperature was kept
at 50 °C for 1 min then gradually raised to 250 °C at 5 °C/min and subsequently, held isothermal for 4 min.
Nitrogen was the carrier gas at a flow rate of 1.2 ml/min. Diluted samples (1/100 in hexane, v/v) of 1.0 µl were
injected manually and in the splitless mode. Quantitative data were obtained electronically from FID area percent
data without the use of correction factors.
Gas chromatography analysis/mass spectrometry analysis
Analysis of the oils was performed using a Hewlett Packard 5890 II GC, equipped with a HP 5972 mass selective
detector and a HP-5 MS capillary column (30 m×0.25 mm id, film thickness 0.25 µm). For GC/ MS detection, an
electron ionization system, with ionization energy of 70 eV, a scan time of 1.5 s and mass range 40–300 amu, was
used. Helium was the carrier gas at a flow rate of 1.2 ml/min. Injector and transfer line temperatures were set at 250
and 280 °C, respectively. Oven program temperature was the same with GC analysis. Diluted samples (1/100 in
hexane, v/v) of 1.0 µl were injected manually and in the splitless mode. The identification of the compounds was
based on mass spectra (compared with Wiley 275.L, 6th edition mass spectral library) or with authentic compounds
and confirmed by comparison of their retention indices either with those of authentic compounds or with data
published in the literature as described by Adams (2001). Further confirmation was done from Kovats Retention
Index data generated from a series of n-alkanes retention indices (relative to C9–C28 on the HP-5 MS capillary
column). (Davies, 1990).
Antifungal activity assays
Nine plants pathogenic fungi were obtained from the culture collection (INRAT). Cultures of each of the fungi were
maintained on potato dextrose agar (PDA) and were stored at 4 °C. The fungal species used in this study were: F.
equisiti, F. culmorum, F. oxysporum, F. solani, F. verticillioides, F. nygamai, Botritys cinerea, Microdochium
nivale var nivale, Alternaria sp.
Antifungal activity was studied by using a contact assay (in vitro), which produces hyphal growth inhibition (Cakir,
et al., 2004). Briefly, potato dextrose agar (PDA) plates were prepared using 9 cm diameter glass Petri dishes. The
essential oil was dissolved in tween water solution (1%, v/v) and required amounts of the solutions were added to
each of the PDA plates containing 20 ml of agar at 50 °C.
A disc (5mm diameter) of the fungal species was cut from 1-week-old cultures on PDA plates and then the mycelia
surface of the disc was placed upside down on the centre of a dish with fungal species in contact with growth
medium on the dish. Then, the plates were incubated in the dark at 25 °C. The extension diameter (mm) of hyphae
from centers to the sides of the dishes was measured after 6 days. Mean of growth measurements were calculated
from three replicates of each of the fungal species. PDA plates containing tween–water solution (1%, v/v), without
essential oil were used as negative control. The percentage of growth inhibition by treatment was calculated using
the following equation:
% Inhibition = (C-T)/Cx100.
Where C is the mean of four replicates of hyphal extension (mm) of controls and T is the mean of four replicates of
hyphal extension (mm) of plates treated with essential oil and the compound solutions.
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Amri et al
Statistical analysis
Data of antifungal activity assays were subjected to one-way analysis of variance (ANOVA), using the SPSS 13.0
software package. Differences between means were tested through Student-Newman-Keuls (SNK) and values of p
0.05 were considered significantly different.
RESULTS AND DISCUSSION
Chemical composition
The oil yield obtained from hydrodistillation of the Junipers oxycedrus leaves was 0.18 % (v/w), considerable
differences were observed in the essential oil yield obtained in Croatia (0.05%) (Milaos and Radonic, 2000) and
Spain (0.27%) (Salido, et al., 2002). Table 1 show the constituents identified in the essential oil with their area
percentage and retention indices. 42 compounds, representing 96.73% of the essential oil were identified, the
chemical composition of our J. oxycedrus oil was dominated bymonoterpene hydrocarbons (51.73%), oxygenated
sesquiterpenes (21.55 %) and sesquiterpene hydrocarbons (16.05%). while the monoterpene hydrocarbons and
diterpenes were present by low percentage, respectively, 1.5 and 5.9%. Like the essential oil from other Juniperus
species, the major constituents of the oil were α-pinene (39.63%), manoyl oxide (12.34%), Z-caryophyllene (4.1%),
δ-3-carene (3.9%), geranyl acetone (3.69%) and caryophyllene oxide (2.67%). The essential oil of J. oxycedrus was
previously investigated in other countries, and in agreement with our result, generally it was shown that α-pinene
and manoyl oxide were the major components in the oil but with different levels (Altarejos, et al., 1999; Milaos and
Radonic, 2000; Salido, et al., 2002; Loizzo, et al., 2007, Medini, et al., 2010). These differences in the oil
composition and yield may be due to several factors such as time of collection, geographic and climatic conditions.
Antifungal activity of essential oils
The essential oils isolated from the leaves of the J. oxycedrus were tested for their antifungal activity against nine
important agriculturally fungal species. These results showed that the oil significantly reduced the growth of the
fungal species over a very broad spectrum (table 2). The obtained results confirm the antifungal activity of conifer
essential oils reported by others reports (Lis- Balchin, et al., 1998). As seen table 2, and according the statistical
analysis, the oils exhibited different degrees of inhibition on the growth of tested fungi; Microdochium nivale, F.
culmorum and F.equisiti were the most sensitive to the action of the oil, while Botrytis cinerea was the most
resistant. Generally, Juniperus species are known to possess an antifungal activity (Parajuli, et al., 2005;
Pepeljnjak, et al., 2005). In our study, J. oxycedrus oil was considered rich monoterpenes and oxygenated
sesquiterpenes, in addition, there was a correlation between the antifungal activity and percentage of some
components, Table 1 indicated that J. oxycedrus essential oils were characterized by the relatively high content of α-
pinene, manoyl oxide, Z-caryophyllene and geranyl acetone, which are known to possess an important antifungal
activity (Sokovic and Griensven, 2006; Hui-Ting, et al., 2008). Indeed, many authors have attributed the antifungal
capacity of essential oils from different Juniperus, Calocedrus, Pistacia and Cupressus species to the presence of
α-pinene, Z-caryophyllene and other sesquiterpenes (Duru, et al., 2003; Pepeljnjak, et al., 2005; Hui-Ting, et al.,
2008; Mazari, et al., 2010).
Several studies have demonstrated the antifungal proprieties of these compounds; for example, Sokovic and
Griensven (2006) showed that α-pinene and limonene posses an important antifungal activity against Verticillium
fungicola and Trichoderma harzianum (MIC 4.0–9.0 µl/ml). Other studies have tested the antifungal activity of
some sesquiterpenes and they showed that oxygenated sesquiterpene were more effective than sesquiterpene
hydrocarbons which are more effective than hydrocarbon monoterpenes components of the oil (Hui-Ting, et al.,
2008), for these reasons the antifungal activity of our oil was attributed to the presence of both sesquiterpenes and
monoterpenes and the synergism between components does play an important role. But the exact mechanism of
action of essential oils and its components on fungi remains unclear however, a number of effects and hypothesis
have been reported by many authors. In general, the majority of reports agree that essential oils result in significant
morphological changes to the hyphae, most noticeably a reduction in hyphae wall thickness, possibly related to
interference by essential oil components in the enzymatic reactions of cell wall synthesis leading to incorrect
assembly of wall components, such as chitin, glucans and glycoproteines (Helal, et al., 2006; Sharma and Triphati,
2006).
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Amri et al
Table 1: Chemical composition of Juniperus oxycedrus L. Essential oil.
Peaks RIa Compounds Area % Identification
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
926
931
939
953
953
956
976
980
991
1005
1011
1018
1026
1031
1062
1088
1125
1143
1177
1189
1372
1384
1390
1391
1402
1418
1428
1454
1455
1460
1461
1480
1508
1524
1538
1542
1565
1581
1684
1990
2054
2080
Tricyclene
α-thujene
α-pinene
camphene
α-fenchene
thuja-2,4(10)-diene
sabinene
β-pinene
myrcene
α-phellandrene
δ-3--carene
α-terpinene
p-cymene
limonene
δ-terpinene
α-terpinolene
α-compholenal
camphor
terpinen-4-ol
α-terpineol
α-ylangene
β-bourbonene
β-cubebene
β-elemene
langiofolene
Z-caryophyllene
β-copaene
α-humulene
geranyl acetone
bornyl iso-butanoate
α-muurolene
Germacrene D
β-bisabolene
δ-cadinene
α-cadinene
α-calacorene
Nerolidol
Caryophyllene oxide
Eudesma-4(15),7-diene-1-β-ol
Manoyl oxyde
Abietariene
Abietadiene
0.15
0.03
39.63
0.33
0.12
0.01
0.41
0.55
2.37
0.22
3.9
1.21
0.22
1.65
0.83
0.1
0.19
0.1
1.1
0.11
0.31
0.19
1.3
0.9
1.87
4.1
0.73
1.35
3.69
0.49
1.18
2.99
0.1
0.21
0.21
0.61
0.26
2.67
2.1
12.34
3.38
2.52
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
MS RI
Total Identified Compounds (%): 96,73
Monoterpene hydrocarbons (%): 51,73
Oxygenated monoterpenes (%): 5.19
Sesquiterpene hydrocarbons (%): 16.05
Oxygenated Sesquiterpenes (%): 17.86
Diterpenes (%): 5.9
International Journal of Applied Biology and Pharmaceutical Technology Page: 230
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Amri et al
Table 2 : Antifungal activity of J. oxycedrus essential oil.
Fungi Control Growth
(mm) Essential oil (4µL/mL)
Growth (mm) Inhibition%
F. nygamai 62±1.52 24.66±0.33 60.18±0.73b
Alternaria sp 60.66±0.66 26.66±0.88 56.00±1.89ab
F. solani 64±0.66 24±2.08 62.39±3.65b
Microdochium nivale 68.66 ±1.76 19±0.57 72.30±0.89c
F. culmorum 71.66±0.88 20±0.57 72.07±0.96c
Botrytis cinerea 84±0.33 42±1.52 50.40±1.68a
F.equisiti 72±1.15 19.66±0.33 72.67±0.64c
F.oxysporum 71.33±0.88 31.33±0.88 56.09±0.68ab
F.verticilloides 73±0.57 30.66±1.2 57.99±1.60b
Means in the same column by the same letter are not significantly different of the test Student-Newman-Keuls (p0.05).
Plasma membrane disruption , mitochondrial structure disorganization, decreases in both lipid and saturated fatty
acid content , increases in insatured fatty acids and Mg2+, Ca2+ and K+ leakage from exposed cells have been
reported(Zamboneli, et al., 1996; Helal, et al., 2006; Sharma and Triphati, 2006) . Other reports suggested that the
components of the essential oils cross the cell membrane, interacting with the enzymes and proteins of the
membrane such as the H+-ATPase pumping membrane, so producing a flux of protons towards the cell exterior
which induces changes in the cells and, ultimately, their death. Besides, (Lucini et al., 2006; Cristani, et al., 2007;
Viuda-Martos, et al., 2008; Tatsadjieu, et al., 2009) reported that the antimicrobial activity is related to ability of
terpenes to affect not only permeability but also other functions of cell membranes, these compounds might cross
the cell membranes, thus penetrating into the interior of the cell and interacting with critical intracellular sites. Other
reports, showed that the essential oils would act on the hyphae of the mycelium, provoking exit of components from
the cytoplasm, the loss of rigidity and integrity of the hypha cell wall, are resulting in its collapse and death of the
mycelium (Sharma and Tripathi, 2008).
These findings need to be explored as a viable, alternative source to commercially available agrochemicals for plant
pathogenic fungi. Further studies are under way to isolate and characterize the major active principles of the oil and
test the compounds on different microorganisms and against various plant diseases, where in the information
procured would further serve as a strong evidence for the plant as potent antimicrobial agent.
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... Less investigated are the polar extracts. Among literature studies, some investigations on J. oxycedrus revealed the presence of polyphenols such as biflavones, flavonols, and coumarins and antioxidant, hypoglycaemic, anti-inflammatory, antimicrobial, analgesic, anti-nociceptive, and antifungal activities [5,6,[22][23][24][25]. ...
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In this work, we conducted a comparative phytochemical, chemotaxonomic, and biological study of essential oils (EOs) and extracts (ethyl acetate and methanol) obtained from the leaves of Juniperus macrocarpa and J. oxycedrus. The dominant compounds of J. macrocarpa EO, analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), are α-pinene, sabinene, manoyl oxide, and germacrene D, whereas α-pinene, limonene, (Z,E)-farnesol, β-pinene, and γ-cadinene are the most representative volatiles of J. oxycedrus EOs. A multivariate analysis of EOs, included a selection of literature data comparing our samples to samples of J. oxycedrus/macrocarpa/deltoides from the Mediterranean area, was performed. As evident by high-performance liquid chromatography (HPLC) analyses, apigenin, (-)-epicatechin, and luteolin were abundant in J. oxycedrus extracts, while gallic acid, kaempferol-3-O-glucoside, and protocatechuic acid were the dominant constituents of J. macrocarpa extracts. EOs and extracts have been investigated for their potential antioxidant properties and anti-proliferative activity against lung adenocarcinoma (A549), breast cancer (MCF-7 and MDA-MB-231), and lung large cell carcinoma (COR-L23) human cell lines. The methanol and ethyl acetate extracts of J. oxycedrus exerted the most valuable antioxidant activity and exhibited the most promising activity against the COR-L23 cell line with an IC50 of 26.0 and 39.1 μg/mL, respectively, lower than that obtained with the positive control (IC50 of 45.5 μg/mL). To the best of our knowledge, this is the first report highlighting the anti-proliferative activity of J. oxycedrus and J. macrocarpa extracts against this lung cancer cell line. Our results indicate that J. oxycedrus may be considered a source of natural compounds with antioxidant and anti-proliferative effects that could be suitable for future applications.
... Aqueous and methanol extracts of J. oxycedrus leaves showed in vitro antimicrobial activity (Karaman et al., 2003) while methanol and dichloromethane extracts of leaves and stems have been found to reduce the blood pressure of normotensive rats (Bello et al., 1997), to inhibit the response to histamine, serotonin and acetylcholine (Moreno et al., 1998) and possess analgesic properties (Moreno et al., 1998). The methanolic extracts of fruits and leaves exhibited antinociceptive and anti-inflammatory activities (Akkol et al., 2009), antioxidant (Lizzo et al., 2007) and antifungal activities have been also reported (Amri et al., 2013). ...
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Adams, R. P. 2007. Identification of essential oil components by gas chromatography/ mass spectrometry, 4th Edition. Allured Publ., Carol Stream, IL Is out of print, but you can obtain a free pdf of it at www.juniperus.org
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