沙漠研究 22-1, 329 -332 (2012 )"ICAL 1 / DT X"

Journal of Arid Land Studies－Refereed Paper－

Chemical Composition and Biological Potential of Essential Oil from Tunisian
Cupressus sempervirens L.
Maher BOUKHRIS1), Ghayth REGANE1), Thabet YANGUI1), Sami SAYADI1) and Mohamed BOUAZIZ*1)
Abstract: The essential oil was obtained by hydro-distillation of the aerial part of Cupressus sempervirens L. cultivated in Sfax
gardens, Tunisia. The obtained oils were analyzed by gas chromatography-mass spectrometry (GC-MS) and 24 compounds wereidentified. The antioxidant activity of Cupressus sempervirens L. essential oil was evaluated by measuring the radicals-scavenging
effect on 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and by using the 2, 2’-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) assay.
Antimicrobial activity were obtained on ten food-spoilage yeasts Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus,
Bacillus subtilis, Halomonas elongate, Salmonella typhimurium, Enterococcus hirae, Aspergillus niger, Candida albicans andTrichoderma reesei. The results suggest that the essential oil of C. sempervirens might be used as a readily accessible source ofantimicrobial agent and as natural antioxidant.
Keywords: Antimicrobial activity, Antioxidant activity, Cupressus sempervirens L., Essential oil

2. Materials and Methods
1. Introduction
The use of essential oils as functional ingredients in foods,
drinks, toiletries and cosmetics is gaining momentum, both forthe growing interest of consumers in ingredients from natural
sources and also because of increasing concern about
potentially harmful synthetic additives (Reische et al., 1998).
Actually, essential oils and their components are gaining
increasing interest because of their relatively safe status, theirwide acceptance by consumers, and their exploitation forpotential multi-purpose functional use (Ormancey et al., 2001;
Yangui et al., 2009). The genus Cupressus (cupressaceae),
comprising twelve species, is distributed in North America, theMediterranean region and subtropical Asia at high altitudes(Rawat et al., 2010). In Tunisia only one species of the genus
Cupressus, Cupressus sempervirens L. (Cuénod, 1954) was
native. The antimicrobial properties of essential oils havebeen recognised for many years (Yan et al., 2009; Gonzalez
and Marioli 2010). Indeed, phytopreparation obtained fromthe core and young branches of C. sempervirens were reported
to have antiseptic, aromatherapeutic, astringent, balsamic andanti-inflammatory activities (Milos et al., 2002).
In the present paper, we report the results of a study aimed
to define functional antioxidant, antiradical and antimicrobial
properties of cypress oil with some peculiarities related tochemical composition.

* Corresponding Author: mohamed.bouaziz@fsg.rnu.tn
B.P « 1177» 3018 Sfax, Université de Sfax, Tunisie
1) Laboratoire des Bioprocédés Environnementaux, Centre de Biotechnologie de Sfax

2.1. Plant materials
The aerial parts of Cupressus sempervirens L. werecollected from the random gardens in Sfax (Tunisia) on April
2010. C. sempervirens is largely used as windbreaks andornamentals throughout northern and center of Tunisia, andused as traditional medicines to treat cough, influenza, andrheumatism by local people. Identification of the species was
carried out by Professor Mekki Boukhris from Botanic
Ecology of Faculty of Sciences of the University of Sfax,
Tunisia. A voucher specimen was deposited at the herbariumof this laboratory under the code LBPE.11 C.
2.2. Essential oils extraction
The essential oils were extracted by hydrodistillation of
fresh aerial part of C. sempervirens. Material (1000 g ofsample in 4L of distilled water) was subjected to hydro-distillation during approximately 4 h in a Clevenger-typeapparatus. The oils were dried over anhydrous sodium
sulphate and stored in sealed glass vials at 4-6°C prior to
analysis.
2.3. GC/MS analysis
The analysis of the essential oils was performed on a
GC-MS HP model 5975B inert MSD (Agilent Technologies, J&W Scientific Products, Palo Alto, CA, USA), equipped withan Agilent Technologies capillary DB-5MS column (30 m
length; 0.25 mm i.d.; 0.25 mm film thickness), and coupled toa mass selective detector (MSD5975B, ionization voltage 70
eV; all Agilent, Santa Clara, CA). The carrier gas was He andwas used at 1 mL.min-1 flow rate. The oven temperature
program was as follows: 1 min at 100°C ramped from 100 to
260°C at 4°C.min-1 and 10 min at 260°C.
The
chromatograph was equipped with a split/splitless injector usedin the split mode. The split ratio was 1:100. Identificationof components was assigned by matching their mass spectrawith Wiley and NIST library data, standards of the main
components and comparing their Kovats retention indices withreference libraries (Bouaziz et al., 2009) and from the literature.
The component concentration was obtained bysemi-quantification by peak area integration from GC peaks
and by applying the correction factors.
2.4. Antioxidant Potential of Essential oil
2.4.1. DPPH assay
DPPH assay was carried out as described by Bouaziz et al(2010). Various concentrations of essential oil in ethanol (50µL) were mixed with methanolic solution containing DPPH
radicals (6 µM). The mixtures were shaken vigorously andleft to stand for 30 min in the dark. Decrease in colorization
was measured spectrophotometrically at 517 nm using a
UV-1800PC, Japan spectrophotometer.
The radical
scavenging activity (RSA) was calculated using the equation:% RSA = [(ADPPH – AE) / ADPPH] × 100
Where AE was the absorbance of solution containing
antioxidant extract and ADPPH was the absorbance of DPPH
solution. The extract concentration providing 50% inhibition(IC50 µg.mL-1) was calculated from the graph of RSA
percentage against extract concentration. 2, 6-di-tert-butyl-4-hydroxy-boxylic acid (BHT) was used as referencecompound.
2.4.2. ABTS assay
The free-radical scavenging capacity was measured using
the ABTS decoloration method (Re et al., 1999) with somemodifications. Briefly, ABTS was dissolved in water to get a
7 mM concentration. ABTS radical (ABTS.+) was produced
by reacting this stock solution with a 2.45 mM K2S2O8 solution
and allowing the mixture to stand in the dark at room
temperature for 12-16 hours before use. The ABTS.+ solution
was diluted with ethanol to an absorbance of 0.70 ± 0.02 at
730 nm. After the addition of 100 µL of sample, ethanol as a
blank, or Trolox standard to 2.9 mL of diluted ABTS.+ solution,
absorbance readings were taken after 6 min. ethanolic solutionsof known concentrations of Trolox were used forcalibration. This standard curve was linear between 0.2 mM
and 0.8 mM of Trolox (y = 0.6792 x, r² = 0.9965). The
Results were expressed in TEAC (mM).

2.5. Antimicrobial activity
2.5.1. Test organisms and culture media
The most of the test organisms used were from theinternational culture collection strains of Gram-positivebacteria Staphylococcus aureus ATCC 6538, Enterococcus
hirae ATCC 10541, Bacillus subtilus ATCC 6633,
Gram-negative bacteria Pseudomonas aeruginosa ATCC
15442, Escherichia coli ATCC 10536, Salmonella
typhimurium TA 97, the yeast mould Candida albicans ATCC
10231, and filamentous fungi Aspergillus niger ATCC 16404,
Trichoderma reesei Rut C30.
The bacteria were cultivated in Tryptic Soy Broth (TSB) or
Agar (TSA) (Sigma) at the appropriated temperature (30°C or
37°C) of the strain. Fungi and yeasts were cultured on Malt
Extract Broth (MEB) or Agar (MEA) (Fluka) at 28°C.
Inocula were prepared by adjusting the turbidity of eachbacterial and yeast cultures to reach an optical comparison tothat of a 0.5 McFarland standard, corresponding toapproximately 1-5 108 cfu.mL-1. The concentration of spore
suspensions was determined using a hematocytometer (Thomacell) and adjusted to 1-5 108 spores mL-1.
2.5.1. Minimal Inhibitory Concentration (MIC), Minimal
Cidal Concentration (MCC)
This test was used to determine MIC and MCC of theessential oil of C. sempervirens against the test organisms asrecommended by the National Committee for ClinicalLaboratory Standard (NCCLS, 2002).
This test was
performed in a sterile 96-well microplate. The inhibitory
activity of the essential oil was properly prepared andtransferred to each microplate well in order to obtain a twofold
serial dilution of the original sample. To obtain stablediffusion, a stock solution of the essential oil was prepared in
0.1% ethanol. The inocula (100 µL) containing 104 cfu ofbacteria were added to each well. Control wells contained
bacteria or fungi only, adequate medium only andchloramphenicol or gentamycin antibiotics (for bacteria andfungi respectively) (10 µg.L-1). Thereafter, 30 µL of 0.02%
resazurin and 12.5 µL of 20% Tween 80 were added. Plates
were aerobically incubated at 30°C for 16-20 h. Afterincubation, the wells were observed for a color change fromblue to pink. MIC was defined as the lowest concentration atwhich no growth was observed (blue colored) after incubation.
Ethanol at 0.1% had no inhibition effect.
To determine MCC values, 10 µL of each culture medium
with no visible growth were removed of each well andinoculated in TSA or MEB plates. After aerobic incubation at
30°C during 16-20 h and the number of surviving organisms
was determined.

Abundance

3. Results and Discussion

TIC: 290408_10.D\data.ms
4000000

3.1. Chemical composition
In this study, a total of 24 compounds were identified,
which accounted for 92.95% of the essential oil (Table 1).
The major components of different aerial parts of cypress were
α-pinene (37.14%), δ-3-carene (19.67%), limonene (5.43%)
and α-terpinolene (4.69%). The monoterpenic hydrocarbons
were found to be high in this oil. The dominant compounds
in cypress oil were monoterpenes hydrocarbons (84.38%),
followedbyoxygenatedmonoterpenes(3.81%),
sesquiterpenes hydrocarbons (3.07%) and oxygenatedsequiterpenes (1.69%) (Table 1).
3.2. Antioxidant Potential of essential oil
To assess the antioxidant potential of essential oil, theapplication of at least two different assays varying in theirmechanisms of antioxidant action has been recommended.
Table 1. Chemical composition of essential oil of Cupressus
sempervirens L.
N°
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

TR (min)
8.52
8.71
9.01
9.28
9.33
10.18
10.65
11.27
11.41
11.65
11.78
11.86
12.69
13.60
15.27
16.25
19.31
19.72
21.04
22.74
23.74
24.42
25.42
27.33

Compounds
Tricyclene
α-thujene
α-pinene
α-fenchene
Camphene
β- pinene
β-myrcene
δ-3-carene
1,3-p-menthadieneo-cymene
β-limonene
1,8-cineol
ν-terpinene
α-terpinolene
Camphor
4-Terpineol
Bronyl acetate
Carvacrol
R(+)-limonenetrans-Caryophyllene
α-humulene
Germacrene-Dδ-cadinene
α-cedrol
Total
Monoterpenes
Oxygenated monoterpenes
Sesquiterpenes
Oxygenated sesquiterpenes

a

KIa
915
922
930
945
947
980
998
1012
1015
1021
1024
1026
1044
1064
1104
1161
1289
1304
1355
1421
1459
1485
1527
1610

%
0.35
1.66
37.14
1.31
0.49
2.11
3.63
19.67
0.71
1.22
4.42
1.18
1.20
4.69
0.37
2.26
0.25
0.35
5.43
0.49
0.36
1.36
0.61
1.69
92.95
84.38
3.81
3.07
1.69

KI, retention index (Kovalts) relative to n-alkanes (C9–C26) on a polar HP-5
column.

3500000
3000000
2500000
2000000
1500000
1000000
5000000

6.00 8.0010.0012.0014.0016.0018.0020.0022.0024.0026.0028.00

Time-->

Fig. 1. GC Chromatogram of Cupressus sempervirens L.
Table 2. Antioxydant potential of Cupressus sempervirens L. essential oil.

Compounds
Cypress oil
BHT

DPPH (µg/mL)
7.70±0.70
8.13±1.07

ABTS.+ (TEAC, mM Trolox)
2.14±0.51
2.41±0.10

The antioxidant capacity of C. sempervirens essential oil was
determined applying the DPPH and ABTS assays.
On the other hand, essential oil showed high antioxidant
activity (7.7 µg.mL-1 and 2.14 mM Trolox for DPPH and ABTS
assays, respectively) when compared to BHT. The antioxidant
activity of essential oil could be assigned to the synergistic
effects of two or more compounds present in the oil. In thiscontext, Lu and Foo (2001) reported that most natural
antioxidative compounds often work synergistically with eachother to produce a broad spectrum of antioxidative properties
that create an effective defense system against free radicals.
3.3. Antimicrobial activity of essential oil
The antimicrobial activity of the essential oil of C.
sempervirens was examined by broth microdilution
susceptibility assay. The results presented in table 3, revealthat the oil of C. sempervirens inhibited the growth ofsusceptible bacteria, filamentous fungi and yeasts. The MIC
and MCC values indicate that C. sempervirens essential oil was
highly effective. In addition, MIC/MCC ratio confirmed abactericidal and fungicidal activity of the essential oil.
The antimicrobial activity of the C. sempervirens essential
oils was more pronounced against Gram-positive thanGram-negative bacteria. This is a general observation derived
from studies with essential oils from many other species (Burt,
2004; Delamare et al., 2007). Generally, the higher resistanceamong Gram-negative bacteria could be ascribed to thepresence of their outer phospholipidic membrane, almost
impermeable to lipophilic compounds (Rattanapitigorn et al.,2006). The absence of this barrier in Gram-positive bacteria
allows the direct contact of the essential oils hydrophobic
constituents with the phospholipids bilayer of the cell
membrane, where they bring about their effect, causing either
an increase of ion permeability and leakage of vitalTable 3. Antimicrobial activity of essential oil of Cupressus
sempervirens L.
MIC
MCC
MCC/MIC(µg mL-1)(µg mL-1)
Pseudomonas aeruginosa
0.62-0. 31
0.62-0. 31
1Escherichia coli

0.62-0. 31

0.62-0. 31

1Staphylococcus aureus

0.15-0. 07

0.15-0. 07

1Bacillus subtilis

0.62-0. 31

0.62-0. 31

1Halomonas elongate

0.62-0. 31

0.62-0. 31

1Salmonella typhimurium

0.62-0. 31

0.62-0. 31

1Enterococcus hirae

0.15-0. 07

0.15-0. 07

1Aspergillus niger

2.5-1. 25

2.5-1. 25

1

Candida albicans

0.15-0. 07

0.15-0. 07

1Trichoderma reesei

0.62-0. 31

0.62-0. 31

1

MIC: Minimal Inhibitory Concentration, MCC: Minimal Cidal Concentration

intracellular constituents, or impairment of the bacteria enzyme(Dorman and Deans, 2002; Burt, 2004). The essential oil
exhibited a broad antifungal spectrum, with higher activity(2.5-1.25 µL.mL-1) against yeasts strains (0.07-0.15 µL.mL-1)(Table 3). However, Pinto et al. (2007) reported thatfilamentous fungi are more susceptible than yeasts to theessential oils of C. sempervirens.

4. Conclusion
Essential oil extracted from aerial part of C. sempervirens.
growing in Tunisia, comparing with the same species
cultivated in other countries, was found to be rich in α-pinene,
δ-3-carene, limonene and α-terpinolene. These differences
could be due to climatic conditions. Composition of theTunisian essential oil showed remarkable antioxidant andantimicrobial activities.

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