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Vol. 7(16), pp. 1070-1080, 25 April, 2013
DOI: 10.5897/JMPR12.1088
ISSN 1996-0875 ©2013 Academic Journals
http://www.academicjournals.org/JMPR
Journal of Medicinal Plants Research
Full Length Research Paper
Chemical composition, bio-herbicidal and antifungal
activities of essential oils isolated from Tunisian
common cypress (Cupressus sempervirens L.)
Amri Ismail1*, Hamrouni Lamia1, 2, Hanana Mohsen3, Gargouri Samia4 and Jamoussi Bassem5
1Département de Biologie, Faculté des Sciences de Bizerte. Zarzouna, 7021 Bizerte, Tunisie.
2Laboratoire d’Ecologie Forestière, Institut National de Recherches en Génie Rural, Eaux et Forêts. BP 10, 2080 Ariana,
Tunisie.
3Laboratoire de Physiologie Moléculaire des Plantes, Centre de Biotechnologie de Borj-Cédria. BP 901, 2050 Hammam-
lif, Tunisie.
4Laboratoire de Protection des Végétaux, Institut National de la Recherche Agronomique de Tunisie. Rue Hédi Karray,
2080 Ariana, Tunisie.
5Institut Supérieur d’Education et de Formation Continue. Tunis, Tunisie.
Accepted 11 January, 2013
The chemical composition of essential oils obtained by hydrodistillation from leaves, branches and
female cones of Tunisian Cupressus sempervirens L. was determined by gas chromatography (GC) and
CG-mass spectrometry (GC/MS) analysis, 52 compounds were identified; qualitative and quantitative
differences between oils were observed. All oils were rich in monoterpene hydrocarbons, and the major
constituents were α-pinene (27.5 to 35.8%), α-cedrol (7.7 to 19.3%), δ-3-carene (5.8 to 13.2%) and
germacrene D (3.9 to 12.1%). Essential oils of C. sempervirens have shown a significant phytotoxic
effect against the germination and seedling growth of four weeds: Sinapis arvensis L., Trifolium
campestre Schreb (dicots), Lolium rigidum Gaud and Phalaris canariensis L. (monocots). Tested oils
strongly inhibited the germination and seedling growth of all weeds, in a dose dependent manner. The
in vitro antifungal activity of the essential oil samples of C. sempervirens were evaluated against 10
cultivated crop fungi, and all samples have shown a significant antifungal activity against all tested
fungi and it can be suggested to have the potential to be used as a bio-herbicide and alternatives
fungicide.
Key words: Cupressus sempervirens, essential oils, bio-herbicidal activity, antifungal potential, weeds,
allelopathy.
INTRODUCTION
Allelopathy is the science that studies processes in which
secondary metabolites from plants and microorganisms
are involved, affecting growth and development of
biological systems (Qiming et al., 2006). The use of
secondary metabolites implicated in allelopathic
interactions as sources for news agrochemical models
could satisfy the requirements for crop protection and
weeds management (Singh et al., 2003). Weeds may be
defined as plant with little economic value and
possessing the potential to colonize disturbed habitats or
those modified by human activities. Fungi can cause
disasters on the crops; the metabolites of many fungi
*Corresponding author. E-mail: amri_amri@live.fr.
may have adverse or stimulatory effects on plants, such
as suppression of seed germination, malformation, and
retardation of seedling growth. Many crop seeds are
infected by fungi before harvest or during storage. If
conditions are not favourable, the situation is more
serious. According to an estimate, in US alone, weeds
cause a loss on the crop production in the range of 12%
(Pimentel et al., 1991). As per Agrow report, the total
value of world’s agrochemical market was between
US$31 - 35 billion and among the products herbicides
accounted for 48% followed by fungicides (22%) (Agrow,
2007). However, the excessive use of synthetic
pesticides in the croplands, urban environment, and
water bodies to get rid of noxious pests has resulted in an
increased risk of pesticide resistance, enhanced pest
resurgence, toxicological implications to human health
and increased environmental pollution (Gupta et al.,
2008; Hong et al., 2009).
In an attempt to reduce the use of synthetic pesticides,
extensive investigations into the possible exploitation of
plant compounds as natural commercial products, that
are safe for humans and the environment were made.
Indeed, the search of natural compounds and
management methods alternatives to classical pesticides
has become an intense and productive research field
(Zanie et al., 2008; Dudai et al., 1999).
In this regard, greater attention is towards the use of
allelopathic plants and their products for pest
management in a sustainable manner. Therefore, it is
worthwhile to explore the plants as sources of biological
active compounds. Species of Cupressus genus
(Cupressaceae family) are coniferous trees, comprising
12 species which are distributed in the Mediterranean
region, North America and subtropical Asia (Bagnoli et
al., 2009). Common cypress (Cupressus sempervirens
L.) is native to the eastern Mediterranean region. This
tree is mainly used as an ornamental tree due to its
conical crown shape, but it can also be used for timber,
as a privacy screen, and protection against wind as well.
Moreover, cypress has proved to be very suitable as a
pioneer species for reforestation as it can tolerate poor,
barren, and superficial soils. For all these reasons,
cypress has been introduced in geographic areas that
extend far beyond its natural distribution (Bagnoli et al.,
2009). Phyto-preparation obtained from the core and
young branches of C. sempervirens were reported to
have antiseptic, aroma therapeutic, astringent, balsamic
and anti-inflammatory activities. Cypress is also
described to exert antispasmodic, astringent, antiseptic,
deodorant, and diuretic effects, to promote venous
circulation to the kidneys and bladder area, and finally to
improve bladder tone and as a co-adjuvant in therapy of
urinary incontinence and enuresis (Rawat et al., 2010).
Essential oils and crude extracts of C. sempervirens have
become a subject for a search of natural antioxidants,
antibacterial, insecticidal activities, and inhibition of
glucose-6-phosphatase and glycogen phosphorylase
Ismail et al. 1071
(Rawat et al., 2010). There are many reports on the
chemical composition of essential oils isolated from
various parts of C. sempervirens. Most of these reports
indicate that monoterpene hydrocarbons like α-pinene
and δ-3-carene are the main constituents of these oils
(Chanegriha et al., 1993; Chanegriha et al., 1997; Emami
et al., 2004, 2006; Sacchetti et al., 2005; Mazari et al.,
2010; Milos et al., 2002; Loukis et al., 1991; Chéraif et al.,
2005), but to our knowledge, no study has been reported
on their herbicidal and antifungal activities and knowing
that the chemical composition of essential oils from
aromatic plants depends on several factors such as the
geographical origin and genetic background of plant from
which the oil was obtained, so, the aims of this work
were, in a first step, to assay the main constituents of the
essential oil obtained from the leaves, cones and
branches of C. sempervirens growing in Tunisia. In a
second step, we assessed their antifungal potential
against eight phyto-pathogenic fungi and their herbicidal
effects were tested against germination and seedling
growth of four common weeds in Tunisia, Sinapis
arvensis L., Lolium rigidum Gaud., Trifolium campestre
Schreb. and Phalaris canariensis L.
MATERIALS AND METHODS
Plant material
The leaves, cones and branches of C. sempervirens were collected
from the arboretums of the National Institute of Researches on
Rural Engineering, W ater and Forests in October, 2010 from the
region of Makther. Five samples collected from more than five
different trees were harvested, mixed for homogenization, and used
in three replicates for essential oil extractions. The specimen of the
plant was submitted to the herbarium division of the institute and
identification was confirmed in the Laboratory of Forest Ecology.
Isolation of the essential oils
The essential oils were extracted by hydrodistillation of fresh plant
material (100 g of each s ample in 500 ml of distilled water) using a
Clevenger-type apparatus for 3 h according to the standard
procedure described in the European Pharmacopoeia (2004).
The oils were dried over using anhydrous sodium sulfate (a
pinch/10 ml-1) and stored in sealed glass vials at 4°C before
analysis. Yield was calculated based on dried weight of the sample
(mean of three replications).
Gas chromatography-mass spectrometry
The composition of the oils was investigated by GC and GC/MS.
The analytical GC was carried out on an HP5890-series II gas
chromatograph (Agilent Technologies California USA) equipped
with flame ionization detectors (FID) under the following conditions:
the fused silica capillary column, apolar HP-5 and polar HP
Innowax (30 m × 0.25 mm ID, film thickness of 0.25 mm). The oven
temperature was held at 50°C for 1 min then programmed at rate of
5°C/min-1 to 240°C and held isothermal for 4 min. The carrier gas
was nitrogen at a flow rate of 1.2 ml/min-1; injector temperature:
250°C, detector: 280°C; the volume injected: 0.1 ml of 1% solution
(diluted in hexane). The percentages of the constituents were
1072 J. Med. Plants Res.
calculated by electronic integration of FID peak areas without the
use of response factor correction. GC/MS was performed in a
Hewlette Packard 5972 MSD System. An HP-5 MS capillary column
(30 m × 0.25 mm ID, film thickness of 0.25 mm) was directly
coupled to the mass spectrometry. The carrier gas was helium, with
a flow rate of 1.2 ml/min-1. Oven temperature was programmed
(50°C for 1 min, then 50 to 240°C at 5°C/min-1 ) and subsequently
held isothermal for 4 min. Injector port: 250°C, detector: 280°C, s plit
ratio: 1:50. Volume injected: 0.1 ml of 1% solution (diluted in
hexane); mass spectrometer: HP5972 recording at 70 eV; scan
time: 1.5 s; mass range: 40 to 300 amu. Software adopted to
handle mass spectra and chromatograms was ChemStation. The
identification of the compounds was based on mass spectra
(compared with Wiley 275.L, 6th edition mass spectral library).
Further confirmation was done from Retention Index data
generated from a series of alkanes retention indices (relatives to
C9-C28 on the HP-5 column) (Adams, 2007).
Seed germination and seedling growth experiments
Mature seeds of annual seeds of S. arvensis L., L. rigidum Gaud, T.
campestre Schreb and P. canariensis L. were collected from parent
plants growing in fields in July, 2009. The seeds were sterilized with
15% sodium hypochlorite for 20 min-1. They were then rinsed with
distilled water. Empty and undeveloped seeds were discarded by
floating in tap water and the remaining s eeds were used. Then, the
oil was dissolved in tween-water solution (1%; v/v). The final
concentrations of the treatments were 0 (control), 1, 2, 3, 4 and 5
µl/ml-1. The emulsions of 8 ml were transferred to Petri dish placed
on the bottom two layers of filter paper. Afterward, 20 seeds S.
arvensis, P. canariensis, T. campestre and L. ri gidum were placed
on the filter paper. Petri dishes were closed with an adhesive tape
to prevent escaping of volatile compounds and were kept at 25°C
on a growth chamber supply with 12 h of fluorescent light (Dudai et
al., 1999). The number of germinated seeds and seedling lengths
were measured after 10 days and all tests were arranged in a
completely randomized design with three replications by tr eatment.
Antifungal activity assays
Eight plant pathogenic fungi were obtained from the culture
collection of the Tunisian National Institute of Agronomic Research.
Cultures of each of the fungi were maintained on potato dextrose
agar (PDA) and were stored at 4°C and in 1 ml of glycerol 25% at -
20°C. The fungal species used in this study were: Fusarium
culmorum, Fusarium oxysporum, Fusarium equisiti, Fusarium
verticillioides, Fusarium nygamai, Botrytis cinerea, Microdochium
nivale var. nivale and Alternaria sp. Antifungal activity was studied
by using an in vitro contact assay which produces hyphal growth
inhibition (Cakir et al., 2004). Essential oil was dissolved in 1 ml of
Tween 20 (0.1% v/v) and then added into 20 ml PDA at 50°C to
obtain a final concentration of 4 µl/ml. A mycelia disk of 5 mm in
diameter, cut from the periphery of a 7 day-old culture, was
inoculated in the center of each PDA plate (90 mm diameter), and
then incubated at 24°C for 7 days. PDA plates treated with Tween
20 (0.1%) without essential oil were used as control. Tests were
repeated in triplicate. Growth inhibition was calculated as the
percentage of inhibition of radial growth relative to the control using
the following formula: % Inhibition = [(C – T) / C]*100. Where C is
an average of three replicates of hyphal extension (mm) of controls,
and T is an average of t hree replicates of hyphal extension (mm) of
plates treated with essential oil.
Statistical analysis
Data of germination, seedling growth and fungi inhibition 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 (Sokal and Rohlf, 1995).
RESULTS AND DISCUSSION
Chemical composition of Cupressus sempervirens L.
essential oils
The chemical composition of C. sempervirens oils, the
percentage content of the individual components, the
retention indices and percent yields are summarized in
Table 1. The oil yields were ranged from 0.1 to 0.65%
depending on the part of the plant analyzed. The greatest
yields were in cones and leaves (0.65 and 0.43%,
respectively) and the oil was lowest in the branches
(0.1%). 52 compounds were identified accounting for
93.7, 94.82 and 95.8% of the total oil respectively in
leaves, cones and branches. The monoterpene fraction
amounted (48.1 to 65.9%), sesquiterpenes accounted for
27.3 to 45.01%, while a low amount of diterpenes (less
than 2.6%). In monoterpene fraction, hydrocarbon
compounds represent a great amount, accounting for
43.21 and 42.7% respectively in cones and leaves, and
60.4% in branches. The main monoterpene hydrocarbons
were α-pinene 27.5% in leaves, 28.91% in cones and
35.8% in branches and δ-3-carene (5.8, 7.2 and 13.2%),
respectively in cones, leaves and branches. In
sesquiterpene fraction, sesquiterpene hydrocarbons
varied from 21.9% in leaves, 18.26% in cones and 14.9%
in branches.
The major compounds in this fraction were germacrene
D (3.9 to 12.1%), and some other compounds as (Z)-
caryophyllene, α-humulene and germacrene B. In
oxygenated sesquiterpenes fraction (12.4 to 26.75%), α-
cedrol was the major compound varying from 7.7% in
branches, 18.55% in cones to 19.3% in leaves. So
essential oils of Tunisian C. sempervirens may be
considered as α-pinene, α-cedrol and δ-3-carene
chemotype. In previous studies, essential oils of C.
sempervirens were studied in Iran, Croatia, Italy, Tunisia,
Algeria and Greece (Chanegriha et al., 1993; Chanegriha
et al., 1997; Emami et al., 2004; Emami et al., 2006;
Sacchetti et al., 2005; Mazari et al., 2010; Milos et al.,
2002; Loukis et al., 1991; Chéraif et al., 2005). Obtained
data of these studies are summarized in Table 2 for each
country and each part used for essential oil extraction.
According to these studies, generally α-pinene, α-cedrol,
δ-3-carene, terpinolene and α-terpenyl acetate were
considered the major components on different aerial
parts of C. sempervirens. Differences found between the
main constituents of oils obtained from C. sempervirens
grown in Tunisia and those from the same species but
growing in other countries seem to be related particularly
to dry and extraction methods, climate, soils and genetic
background of trees.
Ismail et al. 1073
Table 1. Essential oils composition of leaves, branches and cones of C. sempervirens L.
S/No. Compounds RI Leaves Cones Branches M. I.
1 Tricyclene 926 0.1 - 0.1 RI, MS
2 α-thujene 931 0.1 0.1 - RI, MS
3 α-pinene 939 27.5 28.91 35.8 RI, MS, Co-Inj
4 α-fenchene 950 0.6 0.2 0.7 RI, MS
5 Sabinene 968 0.2 0.6 1.3 RI, MS, Co-Inj
6 β-pinene 976 0.8 0.9 2.5 RI, MS
7 β-myrcene 991 1 1.5 1.9 RI, MS
8 α-phellandrene 1007 1.4 1.8 - RI, MS
9 δ-3-carene 1011 7.2 5.8 13.2 RI, MS, Co-Inj
10 1.8.cineole 1021 1 0.6 - RI, MS
11 p-cymene 1026 0.2 1.7 1.1 RI, MS
12 Limonene 1031 2.2 0.6 1.9 RI, MS, Co-Inj
13 β-phellandrene 1032 0.1 0.2 - RI, MS
14 α-terpinolene 1088 1.3 0.9 1.9 RI, MS
15 linalool 1098 0.1 0.3 - RI, MS
16 α-campholenal 1126 0.2 0.2 0.9 RI, MS
17 Camphre 1142 0.1 - 0.1 RI, MS
18 Borneol 1149 0.2 0.3 - RI, MS
19 δ-terpineol 1163 0.1 0.7 1.7 RI, MS
20 Myrtenal 1168 0.1 - - RI, MS
21 Myrtenol 1176 0.2 - 0.1 RI, MS
22 Terpen-4-ol 1179 1.8 1.9 1.5 RI, MS
23 α-terpineol 1196 1.1 0.8 - RI, MS
24 iso-bornyl acetate 1279 0.3 0.4 0.7 RI, MS
25 α-terpenyl acetate 1337 0.2 0.4 0.5 RI, MS
26 longifolene 1398 0.6 1.2 0.6 RI, MS
27 (Z)-caryophyllene 1420 2.2 1.9 1.1 RI, MS, Co-Inj
28 α-cedrene 1432 0.6 1.8 1.3 RI, MS
29 α-humulene 1448 2.1 2.4 1.9 RI, MS
30 Ermacrene D 1478 12.1 6.36 3.9 RI, MS, Co-Inj
31 β-selinene 1486 0.6 1 1.8 RI, MS
32 α-murrolene 1499 0.5 0.1 0.5 RI, MS
33 epi-zonarene 1501 0.2 0.3 0.6 RI, MS
34 β-bisabolene 1508 0.5 1.1 0.4 RI, MS
35 Cubebol 1510 0.1 0.6 0.3 RI, MS
36 Cis-calmanene 1521 0.2 - - RI, MS
37 δ-cadinene 1524 0.2 0.4 0.6 RI, MS
38 α-copan-11-ol 1540 0.3 0.3 0.1 RI, MS
39 α -calacorene 1542 0.2 0.2 0.1 RI, MS
40 Elemol 1551 0.1 1.4 - RI, MS
41 Germacrene B 1552 1.5 0.9 1.2 RI, MS
42 β-calacorene 1560 0.6 0.8 1 RI, MS
43 Caryophyllene oxide 1576 0.3 0.6 1.1 RI, MS
44 α-cedrol 1592 19.3 18.55 7.7 RI, MS
45 T-cadinol 1616 0.5 1.1 1.3 RI, MS
46 T-murrolol 1627 0.6 1.7 0.1 RI, MS
47 Manoyl oxide 1993 0.9 2.3 1.7 RI, MS
48 Abietatriene 2044 0.4 0.1 0.8 RI, MS
49 Abietadiene 2080 0.4 0.3 0.5 RI, MS
50 Nezukol 2080 0.3 0.2 0.6 RI, MS
51 Sempervirol 2283 0.1 0.4 0.4 RI, MS
1074 J. Med. Plants Res.
Table 1. Contd.
52 (Z)- tartarol 2313 0.2 - 0.3 RI, MS
Yield % (w/w): 0.43 0.65 0.1
Total identified compounds 93.7 94.82 95.8
Monoterpene hydrocarbons 42.7 43.21 60.4
Oxygenated monoterpenes 5.4 5.6 5.5
Sesquiterpene hydrocarbons 21.9 18.26 14.9
Oxygenated sesquiterpenes 22.3 26.75 12.4
Diterpene hydrocarbons 0.8 0.4 1.3
Oxygenated diterpenes 0.6 0.6 1.3
RI, Retention index on apolar HP-5 MS column; MS, mass spectrometry; percentage calculated by GC-FID
on apolar HP-5 MS column; MI, methods of identification; Co-inj, co-injection; -, not detected.
Table 2. Major constituents of essential oils of C. sempervirens from different origins previously reported.
Country
Used part
Major compounds References
Iran Leaves α-pinene (30%), ∆-3-carene (24%), terpinolene (6.6%), α-terpenyl
acetate (6.6%). Emami et al. (2004)
Cones α-pinene (39%), ∆-3-carene (24%), α-terpenyl acetate (5.6%).
Iran Leaves α-pinene (21.4%), ∆-3-carene (16%), germacrene D (13%). Emami et al. (2006)
Cones α-pinene (46%), ∆-3-carene (27%), α-terpinolene (6.4%).
Italy Leaves α-pinene (19.3%), sabinene (39.6%), limonene (7.31%), zingibirene
(6.9%), δ-terpinene (6.14%), δ-cadinene (5.45%). Sacchetti et al. (2005)
Greece Cones α-pinene (39.54%) and γ-terpinene (11.56%). Loukis et al. (1991)
Croatia Leaves α-pinene (28.4 - 79.2%), γ-3-carene (9.1 - 32.6%), α-cedrol (1.2 - 12.9%),
limonene (1.4 - 8.7%) Milos et al. (2002)
Algeria Leaves
α-pinene (47.00 - 52.76%), δ-3-carene (19.35 - 21.13%), α-terpinyl
acetate (4.10 - 6.47%), cedrol (2.03 - 3.92%), myrcene (3.11 - 3.48%)
and limonene (2.28 - 3.31%).
Chanegriha et al. (1993)
Algeria Leaves α-pinene (2.8 - 44.9%), δ-3-carene (31 - 10.6%) and α-terpinyl acetate
(5.5 - 12.0%) Chanegriha et al. (1997)
Algeria Leaves α-pinene (60.5%), cedrol (8.3%), Mazari et al. (2010)
Tunisia Branches α-pinene (20%), δ-3-carene (22.9%), α-terpinolene (9.4%), α-terpinyl
acetate (7.5%), limonene (5.1%) Chéraif et al. (2005)
Herbicidal activity of essential oils from Cupressus
sempervirens
Phytotoxic effects of essential oils obtained from aerial
parts of C. sempervirens were tested on germination and
seedling growth of S. arvensis, T. campestre, L. rigidum
and P. canariensis which are very invasive weeds in
cultivated areas. Providing statistical analysis, phytotoxic
effects of tested oils were significantly influenced by
doses, tested weeds and the sample oils.
The results (Tables 3, 4 and 5) show that all oils
completely inhibited the emergence of these four weeds
Ismail et al. 1075
Table 3. Inhibitory effects of essential oils of C. sempervirens on weeds germination.
Weed Doses (µl/ml-1) Germination %
Leaves Cones Branches
S. arvensis
0 95 ± 5a 95 ± 5a 95 ± 5a
1.25 60 ± 5b 61.66 ± 5.77b 58.33 ± 2.88b
2.5 23.33 ± 7.63c 30 ± 5c 50 ± 5b
3.75 0.0 ± 0.0d 8.33 ± 2.88d 38.33 ± 5.77c
5 0.0 ± 0.0d 0 ± 0e 15 ± 0d
T. campestre
0 88.33 ± 2.88a 88.33 ± 2.88a 88.33 ± 2.88a
1.25 73.33 ± 10.4b 66.66 ± 2.88b 65 ± 8.66b
2.5 40 ± 13.22c 46.66 ± 2.88c 48.33 ± 2.88c
3.75 13.33 ± 5.77d 13.33 ± 7.63d 31.66 ± 2.88d
5 0 ± 0d 0 ± 0e 5 ± 5e
L. rigidum
0 81.66 ± 7.63a 81.66 ± 7.63a 81.66 ± 7.63a
1.25 73.33 ± 7.63a 71.66 ± 2.88a 61.66 ± 2.88b
2.5 45 ± 8.66b 51.66 ± 2.88b 35 ± 5c
3.75 16.66 ± 2.88c 18.33 ± 10.4c 26.88 ± 2.88c
5 0 ± 0 d 0 ± 0d 26.66 ± 2.88c
P. canariensis
0 81.66 ± 2.88a 81.66 ± 2.88a 81.66 ± 2.88a
1.25 53.33 ± 5.77b 63.33 ± 10.4b 58.33 ± 7.63b
2.5 31.66 ± 2.88c 36.66 ± 5.77c 43.33 ± 12.58c
3.75 11.66 ± 2.88d 18.33 ± 5.77d 38.33 ± 5.7c
5 0 ± 0e 0 ± 0e 15 ± 0d
Means in the same column by the same letter are not significantly different of the test Student-Newman-
Keuls (p ≤ 0.05). (Mean of three replicates).
relative to the control. In general, a dose-response
relationship was observed and the emergence declined
with the increase amount of cypress oils. At the doses of
1.25, 2.5 and 3.75 µl/ml-1, weeds germination was
partially reduced by all oils, and totally inhibited at 5 µl/ml-
1, while the germination of S. arvensis was totally
inhibited by leaves oil at the dose 3.75 µl/ml-1. When
germination was partially inhibited, not only emergence,
even the seedling growth measured as roots and shoots
lengths were significantly reduced, the reduction was
greater with increasing amount of cypress oil. In the
literature, herbicidal effects of essential oils from
Lamiaceae, Anacardiaceae, Verbenaceae, Rutaceae,
Asteraceae, Cupressaceae, Myrtaceae and other family
against weeds have been previously reported (Barney et
al., 2005; Ens et al., 2009; Angelini et al., 2003; Amri et
al., 2012a, b, c; Batish et al., 2008; De Feo et al., 2002;
Verdeguer et al., 2009); on the other hand, nothing was
reported on the phytotoxic effects of C. sempervirens. In
recent reports, we have demonstrated the herbicidal
effects of essential oils obtained from Cupressaceae
family that Juniperus oxycedrus and Juniperus
phoniceae, the chemical analysis of these oils indicate
their richness in monoterpenes hydrocarbons like α-
pinene (Amri et al., 2011a, 2012a), which is consistent
with obtained results in this study. Based on previous
reports, we can conclude that phytotoxic effects of
essential oils were attributed to individual components,
while synergism and antagonism does play an important
role on the biological activity. Previous studies have
reported that essential oils and individual monoterpenes,
such as α-pinene, limonene, terpinen-4-ol, camphor, 1,8-
cineole, thymol and carvacrol strongly inhibit seed
germination and seedling growth of some agricultural
crops and weeds (Ens et al., 2009; De Feo et al., 2002;
Singh et al., 2006; Scrivanti et al., 2003; Tworkoski et al.,
2002; Wang et al., 2009; Kil et al., 2000; De Martino et
al., 2010; Bulut et al., 2006). Looking at the chemical
composition of the oil of C. sempervirens, more than 14
compounds are known to have herbicidal activity; α-
pinene, β-pinene, β-myrcene, limonene, δ-3-carene and
p-cymene are six hydrocarbonated monoterpenes that
are present in our oil, indeed, these compounds have
been reported to have herbicidal activities (Vokou et al.,
2003; De Martino et al., 2010). Linalool, terpen-4-ol,
myrtenal, α-terpineol borneol, 1.8-cineoole and bornyl
acetate are 7 oxygenated monoterpenes; these
compounds are present in the oil of C. sempervirens with
1076 J. Med. Plants Res.
Table 4. Inhibitory effects of essential oils of C. sempervirens on roots growth of weeds.
Weed Doses (µl/ml-1) Germination %
Leaves Cones Branches
S. arvensis
0 13.13 ± 0.66a 13.13 ± 0.66a 13.13 ± 0.66a
1.25 8.2 ± 1.1b 9.93 ± 1.8b 8.66 ± 1.7b
2.5 2.93 ± 0.6c 5.73 ± 0.64c 6.03 ± 0.89c
3.75 0 ± 0d 2.23 ± 0.25d 4.13 ± 0.41d
5 0 ± 0d 0 ± 0e 1.8 ± 0.72e
T. campestre
0 10.55 ± 1a 10.55 ± a 10.55 ± 1a
1.25 9.4 ± 1.44a 8.53 ± 1.16b 8.46 ± 0.47b
2.5 5.33 ± 0.7b 6.16 ± 0.15c 5.1 ± 0.26c
3.75 2.5 ± 0.5c 2.33 ± 0.65d 5.1 ± 0.36c
5 0 ± 0d 0 ± 0e 1.5 ± 0.45d
L. rigidum
0 13.56 ± 0.6a 13.56 ± 0.6a 13.56 ± 0.6a
1.25 9.96 ± 1.19b 7.4 ± 1.05b 8.03 ± 0.55b
2.5 5.73 ± 0.58c 5.3 ± 0.75c 5.43 ± 1.4c
3.75 3.9 ± 0.85d 2.43 ± 0.45d 3.7 ± 0.62d
5 0 ± 0e 0 ± 0e 0.93 ± 0.11e
P. canariensis
0 13.03 ± 0.47a 13.03 ± 0.47a 13.03 ± 0.47a
1.25 8.6 ± 1.65b 7.96 ± 2.21b 9.96 ± 1.26b
2.5 4.83 ± 0.76c 4.7 ± 0.7c 5.4 ± 0.55c
3.75 1.63 ± 0.41d 1.56±0.45d 3.53 ± 0.47d
5 0 ± 0e 0 ± 0d 2.03 ± 0.55e
Means in the same column by the same letter are not significantly different of the test Student-
Newman-Keuls (p ≤ 0.05). (Mean of three replicates).
different percentages and they are known for their
potential herbicide (Vokou et al., 2003). In addition, in our
study, the oil was rich in sesquiterpenes that (Z)-
caryophyllene which are known for their phytotoxic
effects (Kil et al., 2000; De Feo et al., 2002; Singh et al.,
2006; wang et al., 2009). The exact mechanism by which
germination and seedling growth are affected by C.
sempervirens volatile oil is unknown and not prospected
in our study. However, such inhibitory effects could be
caused by allelochemicals interfering with physiological
and biochemical processes in target species (Singh et al.,
2006; Scrivanti et al., 2003; Kaur et al., 2010). Indeed, it
has been reported that the inhibition of germination may
be the consequence of the inhibition of water uptake,
increased abscisic acid content, decreased indole-3-
acetic acid and zeatin riboside contents and disruption of
the activity of metabolic enzymes that are involved in
glycolysis and oxidative pentose phosphate pathway
(Yang et al., 2008; Muscolo et al., 2001). On the other
hand, previous studies showed that essential oils have
phytotoxic effects that may cause anatomical and
physiological changes in plant seedlings, leading to
accumulation of lipid globules in the cytoplasm, reduction
in some organelles such as mitochondria, possibly due to
inhibition of DNA synthesis or disruption of membranes
surrounding mitochondria and nuclei (Koitabashi et al.,
1997). Muscolo et al. (2001) reported that the inhibition of
seed germination in Pinus laricio was attributed to a
disruption of the activity of metabolic enzymes that are
involved in glycolysis and the oxidative pentose
phosphate pathway. Another suggested mechanism for
the inhibition of seed germination and radicle elongation
is the disruption of dark or mitochondrial respiration. At
this point, it has been shown that some volatile
constituents such as α-pinene strongly affected the
respiratory activity by interfering with the electron flow in
the cytochrome pathway, resulting in decreased
adenosine triphosphate (ATP) production and hence,
alteration of other cell processes which are energy-
demanding (Abrahim et al., 2001). In contrast, due to the
difficulties to measure the allelochemicals effects on
mitochondrial respiration in intact plants because many of
these effects are masked by photorespiration, it has been
hypothesized that the ability of monoterpenes to act as
allelochemicals on intact seeds was probably directly
related to their ability to permeate intracellular
compartments (Abrahim et al., 2001; Zunino et al., 2004;
Xu et al., 2006). Concerning the negative effects of
Ismail et al. 1077
Table 5. Inhibitory effects of essential oils of C. sempervirens on shoots growth of weeds.
Weed Doses (µl/ml-1) Germination %
Leaves Cones Branches
S. arvensis
0 12.93 ± 1.77a 12.93 ± 1.77a 12.93 ± 1.77a
1.25 7.56 ± 0.6b 6.96 ± 0.4b 6.7 ± 0.81b
2.5 4.56 ± 0.6c 4.8 ± 0.76c 4.16 ± 0.76c
3.75 0 ± 0d 2.7 ± 0.49d 3.1 ± 0.52c
5 0 ± 0d 0 ± 0e 1.56 ± 0.4d
T. campestre
0 9.23 ± 0.75a 9.23 ± 0.75a 9.23 ± 0.75a
1.25 9 ± 1.32a 6.9 ± 0.55b 8.56 ± 0.66ab
2.5 6.3 ± 0.9b 5.96 ± 0.45b 7.23 ± 1.12b
3.75 3.93 ± 0.4c 3.46 ± 0.85c 4 ± 0.86c
5 0 ± 0d 0 ± 0d 3.53 ± 0.89c
L. rigidum
0 12.83±1.6a 12.83 ± 1.6a 12.83 ± 1.6a
1.25 7.96±0.55b 6.6 ± 0.36b 8.83 ± 0.58b
2.5 6.2±0.91c 4.8 ± 0.2c 7.16 ± 1.25b
3.75 4.6±0.45c 3.83 ± 0.2c 4.36 ± 1.19c
5 0±0d 0 ± 0d 3.1 ± 0.36c
P. canariensis
0 15.5±0.86a 15.5 ± 0.86 a 15.5 ± 0.86a
1.25 9.43±1.1b 7.5 ± 1.37b 9.1 ± 1.34b
2.5 6.2±1.31c 6.7 ± 0.26 b 5.1 ± 0.52c
3.75 4.33±0.8d 4.6 ± 0.65c 3.13 ± 1.2d
5 0±0e 0 ± 0d 2.16 ± 0.58d
Means in the same column by the same letter are not significantly different of the test Student-
Newman-Keuls (p ≤ 0.05). (Mean of three replicates).
volatile oils on seedling growth, Nishida et al. (2005) and
Singh et al. (2009) have reported that the exposure to α-
pinene, β-pinene, 1,8-cineole and camphor inhibited root
growth of Brassica campestris by inhibiting cell
proliferation in root apical meristems, and decreased the
mitotic index. Beside these manifestations, the latter
authors also found that α-pinene disrupts membrane
permeability resulting in solute leakage and bio-energetic
failure which induce a cell death by apoptosis and
necrosis (Singh et al., 2003; Kaur et al., 2010). The data
obtained by Abrahim et al. (2003) indicate that α-pinene
affects energy metabolism of isolated mitochondria from
maize coleoptiles and primary roots by two mechanisms:
uncoupling of oxidative phosphorylation and inhibition on
the electron transfer chain which result the uncoupling of
mitochondrial energy metabolism and inhibition of the
mitochondrial ATP production. In the same report it
demonstrates that the actions of α-pinene on isolated
mitochondria are consequences of unspecific
disturbances in the inner mitochondrial membrane.
According to Weir et al. (2004), the decrease in
membrane permeability was attributable to the
accumulation of reactive oxygen species (ROS). The
latter components such as singlet oxygen (1-O2) and
superoxide (O2-), hydroxyl (OH) as well as hydroperoxyl
(HO2) radicals can affect membrane permeability, cause
damage to DNA and proteins, and generate lipid peroxide
signaling molecules. Moreover, it has been shown that
the increased ROS generation following the exposure of
Cassia occidentalis roots to α-pinene, was concomitant to
enhanced activity of anti-oxidant enzymes mainly
superoxide dismutase, ascorbate peroxidase, guaiacol
peroxidase, glutathione reductase, peroxidase and
catalase (Singh et al., 2006). Despite the absence of
comprehensive and systemic investigations in functional
mechanism of allelopathy of cypress volatile oils, we can
conclude that the strong inhibitory effects on seed
germination and radicle elongation in weeds are
attributable to one or more of the above-mentioned
mechanisms. Deep physiological and biochemical
investigations should be performed.
Antifungal activity of essential oil
Essential oils isolated from leaves, cones and branches
of C. sempervirens L. were tested for their antifungal
activity against eight plant pathogenic fungal species.
1078 J. Med. Plants Res.
Table 6. Antifungal activity of essential oil extracted from aerial parts of C. sempervirens L.
Fungi Inhibition of fungi growth %.
Leaves Cones Branches
F. nygamai 60.91 ± 4.02bcB 78.56 ± 3.81aA 54.39 ± 4.02aB
Alternaria sp 75.21 ± 6.1aA 75.43 ± 5.37aA 51.9 ± 7.33aB
M. nivale 71.11 ± 6.81bcA 78.33 ± 6.17aA 58.49 ± 3.8aB
F. culmorum 72.06 ± 3.78bcA 71.11 ± 10.87abA 53.73 ± 9.02aA
B. cinerea 70.46 ± 2.99bcB 82.46 ± 2.03aA 56.76 ± 6.58aC
F. equisiti 71.63 ± 4.53bcA 58.49 ± 6.2bcA 53.45 ± 12.63aA
F. oxysporum 58.51 ± 2.79bcB 69.35 ± 5.01abA 63.51 ± 3.83aAB
F. verticilloides 66.73 ± 8.5bcB 79.16 ± 2.18aA 52.81 ± 5.63aC
Small letters c ompare means in the lines and capital letters in the columns. Means in the same c olumn by the same letter
are not significantly different of the test Student-Newman-Keuls (p ≤ 0.05). (Mean of three replicates). Means in the same
line by the same letter are not significantly different of the test Student-Newman-Keuls (p ≤ 0.05).
According to obtained results in Table 6, essential oils
of C. sempervirens showed significant inhibition of fungal
growth, this study also indicated that the antifungal
activity is variable depending on the dose, fungal strain
and tested oils. According to statistical analysis, the
highest inhibitions were obtained with cones and leaves,
while weak activities were obtained with branches oils.
Different degrees of sensitivity were recorded as
Alternaria sp was the most sensitive to the oil of leaves,
whereas, Alternaria sp, F. verticilloides, F. nygamai and
M. nivale were the most sensitive to cones oil, however,
all fungi showed the same sensitivity behavior to
branches oil. Essential oils of C. sempervirens showed a
significant inhibition of the growth of all fungi, in general,
there was a correlation between the antifungal activity
and percentage of some major components. As
mentioned above, cypress oils were characterized by
relatively high content of monoterpenes hydrocarbons
(40.2 to 60%) as α-pinene, δ-3-carene and oxygenated
sesquiterpenes like α-cedrol which could be responsible
for the antifungal activity observed in this study. Indeed,
several authors have attributed the antifungal capacity of
essential oils to the presence of these components (Amri
et al., 2011a, b, 2012a, b; Sokovic et al., 2006). Besides,
Sokovic and Van Griensven (2006) showed that limonene
and α-pinene have a strong antifungal activity against
Verticillium fungicola and Trichoderma harzianum
(Sokovic et al., 2006). Moreover, Chang et al. (2008)
showed the fungicide activity of limonene, α- and β-
pinene against Fusarium solani and Colletotrichum
gloeosporioides. Thus, the antifungal activity of the oil in
this study is not attributed only to the high proportions of
the monoterpenes, however, other major or trace
components in the oil could give rise to its antifungal
activity. Indeed, there are synergistic and antagonistic
interactions between oil components. The mode of action
of essential oils was investigated by many authors who
suggested that the antimicrobial activity is produced by
interactions provoked by terpenes in the enzymatic
systems related with energy production and in the
synthesis of structural components of the microbial cells
(Omidbeygi et al., 2007). 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 toward the cell,
exterior which induces changes in the cells and ultimately
their death. Besides, several authors (Cristani et al.,
2007; Lucini et al., 2006; 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. In addition, Daferera et al. (2000) reported that the
fungitoxic activity of essential oils may have been due to
formation of hydrogen bonds between the hydroxyl group
of oil phenols and active sites of target enzymes. These
components would increase the concentration of lipidic
peroxides such as hydroxyl, alkoxyl, and alkoperoxyl
radicals and so bring about cell death (Daferera et al.,
2000). 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 hyphae cell wall, resulting in its collapse
and death of the mycelium (Daferera et al., 2000; Sharma
et al., 2006). Even though the inhibitory effect of the
essential oils was lower than those obtained by the
chemical fungicide, however, essential oils could reduce
significantly the growth of all fungi tested.
Conclusion
Our study could give the solution, which in its first part
had focused on the correlation between the chemical
composition and the effectiveness as antifungal and
herbicidal agents of three essential oils extracted from
common Tunisian cypress (leaves, cones and branches
of C. sempervirens). Results of essential oils bioactivities
showed that C. sempervirens exhibited stronger
phytotoxic and antifungal effects. Based on our
preliminary results, the essential oils of C. sempervirens
could be suggested as alternative herbicides and
fungicides. However, further studies are required to
determine the cost, applicability, safety and phytotoxicity
against the cultured plants of these agents as potential
bio-pesticide.
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