South Asian J Exp Biol; 7 (2): 74-83; 2017

ISSN: 2230-9799 Vol. 7, Issue 2, Page 74-83 http://www.sajeb.org

REGULAR ARTICLE

Chemical composition, antibacterial and antioxidant activities of

Juniperus oxycedrus essential oils from North Western Algeria

Cherifa Zahaf1,*, Djilali Achour1, Meriem Mokhtar2, Moussa Brada3

1
Laboratory of Vegetal Chemistry-Water- Energy, University of Hassiba Benbouali, Chlef, 02000, Algeria
2
Laboratory of Beneficial Microorganisms, Functional Food and Health (LMBAFS), University of Abdelhamid Ibn Badis,
27000 Mostaganem, Algeria
3
Laboratory of Natural Substances Valorization, University of Djilali Bounaama, Khemis-Miliana, Theniet El Had
44225, Algeria
ARTICLE INFO ABSTRACT

Article History: The aim of the current study was to identify the essential oils of Juniperus
Received: 14 Oct 2017 oxycedrus from North Western Algeria (Mostaganem) by gas chromatog-
Revised: 16 Nov 2017 raphy coupled to mass spectrometer (GC-MS), and to evaluate their antibac-
Accepted: 20 Nov 2017 terial and antioxidant activities. Extraction was carried out by two processes,
hydro-distillation (HD) and steam distillation (SD). Analysis by GC-MS led to
*Corresponding Author: the identification of 38 components using both methods. HD was found to
Email:1984cherifazahaf@gmail.com be the best process for the extraction of Juniperus essential oil, and the ma-
Telephone: 00213 776 02 00 62 jor identified compounds were: germacrene-D (38.28%), followed by α.-
Fax: 021327664863 pinene (17.66%), and δ-cadinene (5.91%). In the antibacterial test, SD oil had
a higher inhibitory effect. The strain Staphylococcus aureus was the most
Keywords: Juniperus oxycedrus L; sensitive with an inhibition zone of 15.25 mm and a minimum inhibitory con-
essential oils; extraction; GC-MS; centration (MIC) of 0.25 mg/mL. The antioxidant activity of J. oxycedrus es-
antibacterial; antioxidant sential oils obtained by HD was more important than SD (IC50= 19.52 mg/mL
and IC50= 38.62 mg/mL) with DPPH and β-carotene bleaching tests respec-
tively. Essential oils of J. oxycedrus could therefore represent good candi-
dates to be used as antioxidant and antibacterial agents.

1. Introduction produce an empyreumatic oil, or juniper oil, used

in dermatology, cosmetology (Chalchat et al., 1990;
Juniperus oxycedrus (Cupressaceae) is one of seven- Marongiu et al., 2003), against skin problems, pso-
ty species in the genus Juniperus worldwide (Akkol
riasis, and eczema (Achour et al., 2011). It is essen-
et al., 2009). It is a shrub or small tree growing in tially a product to treat diseases such as hypergly-
stony places of the Near East and Mediterranean cemia, obesity, tuberculosis, and bronchitis
countries. According to Klimko et al. (2007), J. ox- (Sanchez de Medina et al., 1994). Essential oils are
ycedrus could be divided into four subspecies: highly concentrated substances generally used for
subsp. macrocarpa, subsp. transtagana, subsp. ba- their flavor, therapeutic or odoriferous properties.
dia, and subsp. oxycedrus. These subspecies differ Extraction of these compounds remains one of the
in habit, width of needles and dimension of cones most time- and effort-consuming processes. The
(Chaouche et al., 2015; Roma-Marzio et al., 2017). choice of extraction method is very important as it
J. oxycedrus has been used since ancient times to can affect the quality of the oils, thus, the thera-
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 Zahaf et al., South Asian J Exp Biol; 7 (2): 74-83; 2017
peutic properties. 2.3. Antibacterial activity
Several studies have been conducted in different The antibacterial properties of the essential oils of
parts of the world on the extraction of these oils J. oxycedrus berries were tested on four pathogenic
from berries and on the determination of their bacteria (Escherichia coli ATCC 25922, Pseudomo-
chemical composition (Loizzo et al., 2007). These nas areuginosa ATCC 27853, Staphylococcus aureus
studies have shown that the quality and quantity of ATCC 25923 and Salmonella typhimurium ATCC
essential oils not only depend on the population 13311), using the disk diffusion method. Bacterial
but also between the organs of the same plant inocula were prepared by 16-h culture in Muller–
(Medini et al., 2010). These oils are generally char- Hinton broth (MHB, Oxoid) at 37 °C. The overnight
acterized by a high content of α-pinene, whatever cultures were adjusted to 108 CFU/L prior to use
the subspecies and the origin (Valentini et al., and inoculated onto 20 ml MHB. Ten microliters of
2003). In the recent years, essential oils uses have the tested sample was dropped on sterile filter pa-
increased to reach different aspects of human life per disks (6 mm) and deposited on the surface of
(pharmacy, cosmetics, pesticide and food industry), the plate. After incubation at 37°C for 24 hours, the
which made this field very interesting for chemical diameter of the clear zone was measured. The ex-
and biological investigations. No previous study periment was carried out in triplicate.
was conducted on the essential oils of J. oxycedrus
berries growing in Mostaganem (Western Algeria). The minimum inhibitory concentration (MIC) of the
Therefore, this research was carried out to identify tested samples was determined by the broth mi-
these oils extracted by two processes (steam- crodilution method (CLSI, 2008). A series of essen-
distillation and hydro-distillation), and evaluate tial oil concentrations ranging from 0.1-40 mg/mL
their biological properties (antioxidant and antibac- were prepared. MIC was defined asthelowest con-
terial). centration at which no bacterial growth wasob-
served afterincubation.
2. Material and Methods
2.4. Antioxidant activity
2.1. Extraction of volatile compounds
2.4.1. Radical scavenging assay (DPPH)
J. oxycedrus berries were collected during the last
week of November 2013 in Mostaganem (North DPPH (1,1-diphenyl-di-picrylhydrazyl) is a stable
Western Algeria). Two samples, each made of a radical that loses its purple color when it accepts an
mixture of 150 g of dried plant and 1200 ml of dis- electron from an antioxidant molecule. In the DPPH
assay, the ability of the examined extract to act as a
tilled water were introduced separately in Cleveng-
er type device for 3 hours (Adams, 1991). Extrac- donor of hydrogen atoms or electrons in the trans-
tion by hydro-distillation (HD) and steam distillation formation of DPPH˙ into its reduced form DPPH-H
(SD) was repeated several times to obtain a suffi- was investigated. To assess the antioxidant activity
cient amount of essential oil. of the tested essential oil, we used the DPPH meth-
od (Chen et al., 2004). Measurements were done at
2.2. GC–MS analyses 514 nm using a UV/Vis spectrophotometer. The
DPPH solution was obtained by dissolving 2.4 mg of
A sample of 10 mg of J. Oxycedrus essential oil was the powder in 100 mL of ethanol (EtOH). Essential
dissolved in 5 ml of hexane and was analyzed by oil solutions were prepared by dissolution in etha-
gas chromatography coupled to mass spectrometer nol (EtOH) to obtain the following concentrations:
(GC-MS) using an Agilent 5973 GC-MS coupled to 2.5, 5, 10, 20, 40, 80 and 120 mg/mL. To each 25 µL
an Agilent 6800 gas chromatograph fitted with a of the prepared solution, 975 µL of the DPPH solu-
split-splitless injector at 250°C (Splitless mode). The tion were added.
analytical conditions were fixed as follows: Ag-
ilentHP-5MS capillary column (30 m x 0.25 mm, df = The reference antioxidant or the positive control
0.25µm), temperature program: from 70°C to 250° (BHT) was also prepared according to the same
C at 10°C/minute. The carrier gas was helium at protocol. The percentages of inhibition were calcu-
1mL/min. Mass spectra was recorded in EI mode lated using the following formula (Wang, 2002):
(70 eV), scanned mass range: 34 to 550 amu. The
source and quadruple temperatures were fixed at I (%) = 100× (A control-A test)/A control
230°C and 150°C, respectively (Adams, 1995). A control: the absorbance of the control (containing

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 Zahaf et al., South Asian J Exp Biol; 7 (2): 74-83; 2017
all the reactants without sample) only in steam distilled oils (table 1). The results also
indicated that concentration of individual compo-
A test: the absorbance of the tested sample. nent in the EOs obtained by different extraction
The graph of the variation of the inhibition percent- methods was significantly different (P≤0.05).
age for essential oil concentration was used to de- The major compounds of the hydro-distillation oil
termine the IC50. were: germacrene-D (38,28%), α.-pinene (17.66%),
2.4.2. β-carotene bleaching test δ-cadinene (5.91%), isopimaradiene (3.17%), trans-
caryophyllene (2.73%) and α-caryophyllene (2.47).
In this analysis, the antioxidant capacity was deter- for the steam distillation, there were some qualita-
mined by measuring the inhibition of volatile or- tive and quantitative differences; the major com-
ganic compounds and conjugated dienehydroper- pounds were germacrene-D (39,22%), α.-pinene
oxides resulting from the oxidation of linoleic acid (29.54%), δ-cadinene (5.96%), trans-caryophyllene
(Tepe et al., 2006). Two milligrams of β-carotene (2.87%), α-caryophyllene (2.44%) and β-myrcene
were dissolved in 1mL of chloroform, and then the (2.42%). Besides that, the two methods also
obtained solution was introduced into a flask con- differed regarding the extracted groups. Higher
taining 2 mg of linoleic acid and 200 mg of Tween percentages of sesquiterpene and monoterpenes
40. After evaporation of chloroform, 100 mL of dis- were obtained with steam distillation, while in the
tilled water saturated with oxygen were added un- hydro-distillation higher levels of oxygenated mon-
der vigorous stirring. 2.5 mL of this solution were oterpenes, oxygenated sesquiterpenes and diter-
added to 350 μL of each of the essential oil solu- penes.
tions (2.5-120 mg/mL) and were incubated for 48 h
at room temperature.Absorbance was measured at 3.2. Antibacterial activity
490 nm. Antioxidative capacities of the extracts The results pertaining to antibacterial activity of
were compared with those of BHT and blank. Juniperus essential oils are presented in table 2.
AAR (%) = (Abs sample / Abs BHT) × 100(2) According to the results, all the bacteria were sen-
sitive to the essential oils of both processes but the
AAR: Relative antioxidant activity; inhibition zones obtained by steam distillation were
Abs Sample: sample absorbance; slightly bigger than the ones obtained by hydro-
distillation. Also the minimum inhibition concentra-
Abs BHT: BHT absorbance. tions (MIC) values were less important with the SD
process.
2.5. Statistical analysis
The strain Staphylococcus aureus was the most
The statistical analysis was performed using statbox
sensitive (table 2) with an inhibition zone of 15.25
pro (6.40). Results are expressed as means ± SD,
mm and a MIC value of 0.25 mg/mL, followed by
and differences between the means were evaluat-
Escherichia coli (13.75 mm, 0.5 mg/mL), Pseudomo-
ed using one-way ANOVA. The difference was con-
nas aeruginosa (12.5 mm, 0.5 mg/mL) and Salmo-
sidered significant at P≤ 0.05.
nella typhimurium (12 mm, 0.75 mg/mL).
3. Results
3.3. Antioxidant activity
3.1. Essential oils composition
Antioxidant activity of berry essential oil was inves-
The extracted oils of J. oxycedrus L berries of Mo- tigated using the DPPH and β-carotene as an oxi-
staganem are very aromatic viscous liquids with dant agents. The results, expressed as percentage
colors from light yellow to dark yellow. The ob- of inhibitory activity (figure 2, 3), showed that the
tained extraction yields were 0.29% (± 0.01%) for oils obtained by the two methods present an anti-
hydro-distillation and 0.22% (± 0.02%) for steam radical activity. The antioxidant activity of the es-
distillation. Some differences were also noticed in sential oils obtained by HD was higher than the one
the identified compounds for both extraction of SD (IC50= 19.52 mg/mL and IC50= 66.81mg/mL)
methods (figure 1). for DPPH test and (IC50= 38.62 mg/mL and IC50=
116.18 mg/mL) for β-carotene bleaching method
Among the identified essential oils, thirteen com- respectively. The obtained result for the standard
pounds were found just in hydro-distilled samples. BHT was higher than both of essential oils (IC50=
On the other hand, four components were present

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Figure 1: Juniperus oxycedrus essential oil profile obtained by (A): Hydro-distillation, (B): Steam distillation.

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 Zahaf et al., South Asian J Exp Biol; 7 (2): 74-83; 2017

N Compounds HD% SD% KI

1 α.-Pinene 17.66 29.54 936
2 Camphene - 0.26 947
3 Sabinene 0.93 1.92 973
4 β-Pinene 1.29 2.01 976
5 β-Myrcene 1.68 2.42 993
6 β-Phellandrene 0.81 1.06 1029
7 γ-Terpinene 0.23 0.28 1059
8 α-Terpinolene 0.54 0.69 1088
9 Terpinene-4-ol 1.56 0.31 1181
10 α-Terpineol 0.35 - 1196
11 α-Cubebene - 0.37 1348
12 β-Bourbonen 0.61 0.57 1382
13 trans-Caryophyllene 2.73 2.87 1418
14 β -Cubebene 0.41 0.46 1427
15 α-Caryophyllene 2.47 2.44 1453
16 Germacrene-D 38.28 39.22 1490
17 α-Amorphene 1.40 1.51 1496
18 α-Muurolene 0.98 1.05 1500
19 γ-Cadinene 2.11 2.30 1516
20 δ-Cadinene 5.91 5.96 1527
21 4,4-Dimethyl-3-(3-methylbut3-enylidene)-2- 0.32 - 1558
methylenebicyclo [4.1.0] heptane
22 Germacrcne-D-4-ol 1.2 - 1578
23 Bicyclo[2.2.2]oct-2-ene, 1,2,3,6-tetramethyl- 0.35 - 1590
24 Guaia-1(10),11-diene 0.41 - 1616
25 tau-Muurolol 1.46 1647
26 α-Copaene 0.35 - 1651
27 α-Cadinol 2.27 0.78 1659
28 Isoaromadendrene epoxide - 0.46 1690
29 Alloaromadendrene oxide-(1) 1.52 - 1692
30 1,5,9,13-Tetradecatetraene 0.48 - 1929
31 13-Isopimaradiene 0.92 0.38 1960
32 Isopimaradiene 3.17 1.35 2000
33 PIMARA-8(9),15-DIENE 0.66 - 2011
34 Androst-5-en-4-one 2.94 0.73 2083
35 1,2-Dimethyl-3,5 divinylcyclohexane 0.26 - 2136
36 N-Benzyl-2-(4-methoxyphenoxy)acetamide - 0.50 2182
37 Cembrene 1.96 - 2184
38 6-(1'-Oxo-2'-propenyl)-1,3-cis, cis cyclooctadien 1.40 - 2220
Monoterpenes 23.14 38.18
Oxygenated monoterpenes 1.91 0.31
Sesquiterpenes 55.66 56.75
Oxygenated sesquiterpenes 6.77 1.24
Diterpenes 6.05 1.73
Oxygenated diterpenes 0.66 -
Others 5.43 1.23
Total identified 99.62 99.44
Table 1: Essential oils of Juniperus oxycedrus identified by GC-MS.

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 Zahaf et al., South Asian J Exp Biol; 7 (2): 74-83; 2017
2.56 mg/mL and IC50 = 3.09 mg/mL) with DPPH ygenated monoterpenes (0.2-2.7%) and oxygenat-
and β-carotene bleaching test. ed diterpenes (0.0-1.7%).
4. Discussion According to Rajeswara Rao et al. (2014), the condi-
tions of hydro-distillation (water quality, pH, tem-
The purpose of our study was to identify the main perature etc.) might contributed in the hydrolysis,
essential oils extracted by two popular methods: isomerization, racemization and oxidative altering
hydro-distillation and steam distillation; and also of the structures of the essential oil components
evaluate their antioxidant and antimicrobial activi- (Lee and Shibamoto, 2001), producing differences
ties. After extraction of essential oils, the yield ob- in essential oil composition relative to steam distil-
tained with hydro-distillation method was higher lation.
than the steam distillation.
In recent years, there is an interest in natural bioac-
Nowadays, the major concern for EOs producers is tive compounds able to solve the problem of antibi-
to obtain oils with better quality and quantity, and otic resistant bacteria. Beside that; a lot of re-
with known and stable biological properties. How- searches are conducted on the development of
ever, the quantity (yield) and the quality (chemical natural food preservatives due to the increasing
composition) of EOs are influenced by the interac- consumer pressure on the food industry aims to
tion among several factors such as genotype, envi-
avoid the use of synthetic preservatives (Mokhtar
ronment, plant age, season and time of harvest and et al., 2015).
extraction method (Yesil Celiktas et al., 2007; Zan-
tar et al., 2015). According to literature, a higher oil In the literature, it is reported that an essential oil
yield in the hydro-distillation method was due to has bacteriostatic action if its inhibition diameter is
controlled distillation and continuous cohobation greater than 12 mm (Baudoux, 2001). The oils ob-
of the distilled water (Rajeswara Rao et al., 2014). tained by both processes have a strong inhibition
The superiority of this method over field distillation activity against all bacteria which was demonstrat-
has been reported (Rajeswara Rao et al., 2006). ed by the inhibition zones that were all superior to
12 mm with the exception of Salmonella typhimuri-
The number of compounds identified in the hydro- um with the HD process. The essential oils of Juni-
distillation method was more important and con- perus berries extracted by steam- distillation exhib-
tain high levels of oxygenated monoterpenes, oxy- ited a higher antibacterial activity than hydrodistil-
genated sesquiterpenes and diterpenes, while big- lation. Steam distillation essential oil had a higher
ger percentages of sesquiterpene and monoter- value of both monoterpenes and sesquiterpenes
penes were obtained with steam distillation. The which can explain these results. According to Cakir
major identified compound was germacrene-D,
et al. (2004), the inhibitory action of the essential
followed by α.-pinene and δ-cadinene. oil could be attributed to the occurrence of high
These results are in agreement with those obtained proportions of monoterpenes and sesquiterpenes
in a study conducted by Marongiu et al. (2003); the in the oil (Cakir et al., 2004). Olajuyigbe and Ashafa
essential oil of berries from Sardinia (Italy) was also (2014) indicated the importance of α-pinene, β-
dominated by germacrene D. In another research, pinene, p-cymene and 1, 8-cineole in the antibacte-
α.-pinene was the major compound both in nee- rial activity of essential oil.
dles and berries of J. oxycedrus with 92% and 85%, The essential oil of J. communis growing wild in
respectively. β-myrcene and β-pinene compressed Kosovo, showed moderate to high activ-ities
only 3%and 2% of total monoterpenes, respectively against Staphylococcus aureus, Escherichia coli and
(Foudil-Cherif and Yassaa, 2012). Hafnia alvei, while Pseudomonas aeruginosa was
Hajdariet al. (2014) determined the essential oil resistant to the antimicrobial effects of the oil
composition of J. oxycedrus from different part of (Haziri et al., 2013). These results allowed us to
Kosovo obtained by hydro-distillation. The main conclude that the antibacterial properties of essen-
components were β-myrcene (45.5-56.9%), α.- tial oil depend on their chemical composition that
pinene (10.2-36.6%), DL-limonene (3.6-13.8%) and varies between the different samples with the
germacrene-D (1.7-8.7%). Monoterpenes repre- same species. One of the responsible factors of the
sentted the highest percentage of all compounds variation of the chemical composition is the extrac-
(70.24-88.22%), followed by oxygenated sesquiter- tion method (Shu et al., 1997).
penes (4.9-11.4%), sesquiterpenes (3.5-11.0%), ox-
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 Zahaf et al., South Asian J Exp Biol; 7 (2): 74-83; 2017

Strain SD HD
Inhibition zone MIC (mg/ Inhibition zone MIC (mg/
(mm) mL) (mm) mL)
Escherichia coli 13.75±0.15 0.5 12.62±0.53 0.75
Pseudomonas areuginosa 12.50±0.22 0.5 12.30±0.14 0.75
Staphylococcus aureus 15.25±0.3 0.25 14.5±0.12 0.5
Salmonella typhimurium 12.00±0.12 0.75 09.87±0.53 1.25
Table 2: Antibacterial activity of Juniperus oxycedrus essential oils.

Figure 2: Antioxidant activity of Juniperus oxycederus essential oils extracted by hydro-distillation (HD)
and steam distillation (SD) on 2,2-diphenyl-2-picryl-hydrazyl free radical (DPPH) radicals.

Figure 3: β-carotene bleaching test of Juniperus oxycederus essential oil extracted by hydro-distillation
(HD) and steam distillation (SD). RAA: relative antioxidant activity = (sample absorbance/BHT absorb-
ance) × 100.

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 Zahaf et al., South Asian J Exp Biol; 7 (2): 74-83; 2017
Different assays are reported in the literature de- J.oxycedrus exhibit good antibacterial and antioxi-
scribing the capability of redox molecules to scav- dant activities which encourage their uses in both
enge free radicals to measure the antioxidant ca- food and pharmaceutical industries.
pacity (Floegel et al., 2010). The most popular spec-
trophotometric assays are ABTS and DPPH, even Conflicts of interest: The authors declare no con-
though others like ORAC and FRAP have been used flicts of interest.
(Thaipong et al., 2006; Mokhtar et al., 2015). In this
study, and in order to evaluate the antioxidant of
essential oils of Juniperus berries, DPPH and β- References
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