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International Journal of Toxicological and Pharmacological Research 2016; 8(4); 281-286

ISSN: 0975-5160
Research Article

Chemical Composition and Antimicrobial Activity of the Essential Oil

of Cupressus sempervirens L. Leaves in Syria
Mohamad Jawad Khubeiz1*, Ghaytha Mansour2 and Bahira Zahraa1
1
Department of Chemistry, faculty of science, Damascus University, Albarameka, Damascus, Syria.
2 Department of Botany, faculty of science, Damascus University, Albarameka, Damascus, Syria.

Available Online: 10th August, 2016

ABSTRACT
In the present research the essential oil contents and compositions of individual plant from Cupressus sempervirens L.
(Cupressaceae), grown in Damascus / Syria have been investigated. The antimicrobial activity of the essential oil
was also discovered. The air-dried leaves were hydro-distilled, and the essential oil analyzed by GC-MS. The essential
oil from C. sempervirens L. sample was tested for antimicrobial activities using agar disc diffusion technique to determine
the diameter of growth inhibition zones. The macro dilution broth susceptibility assay was utilized to determine minimum
inhibitory concentrations (MIC), and minimum bactericide concentration (MBC). Fourteen components were identified in
the essential oil of C. sempervirens L. The main constituents of essential oil leaves were alpha-pinene (36.50%), 3-carene
(22.17%), Germacrene D (12.81%), Terpinolen (5.18%), alpha-tepinnyl acetate (4.76), limonene (3.55%) and beta-
myrcene (3.16%). The results of the antimicrobial activity tests revealed that the essential oil has rather a strong
antimicrobial activity, especially against Streptococcus pyogenes, Staphylococcus aureus and Vi b r i o
parahaemolyticus, with diameter of inhibition zones ranging from (38.9 ±0.16) to (43.2 ± 0.15) mm, it showed a modest
antibacterial effect for Micrococcus luteus, Klebsiella pneumonia and Pseudomonas aeruginosa, with inhibition zones in
the range (8.5± 0.13) to (10.2 ±0.13) mm. Minimum inhibitory concentration (MIC) revealed the lowest activity
against Micrococcus luteus, Klebsiella pneumoniae and Pseudomonas aeruginosa (3.2mg/mL), while the highest
activity was against Streptococcus pyogenes, Vi b r i o parahaemolyticus (1.6 mg/mL) and Staphylococcus aureus (0.4
mg/mL). The Minimum bactericidal concentration (MBC) activity was from (1.6 mg/mL) to (4.6 mg/mL).

Keywords: Cupressus sempervirens L, Essential oil, Antimicrobial activity, MIC, GC-MS.

13
INTRODUCTION . Chemically, these volatile products and their
There is an increasing interest in using medicinal and oxygenated compounds are derived from terpenes14,15.
aromatic plants all over the world1. Bioactive compounds Monoterpenes, sesquiterpenes and diterpenes are natural
of medicinal plants led them to be used in various compounds of essential oils with highly variable structural
industries as botanical drugs, dietary supplements, configurations. Each of these constituents contributes to
functional foods, etc2. Plants also have been used in ethno the beneficial or adverse effects. Many researchers have
pharmacy for various diseases such as hypertension, high also focused on plants in order to discover new drugs.
cholesterol, eczema and diarrhea for centuries, and today Cupressus is considered to be a medicinal tree. It is one of
their scientific validation was provided by identification the most important trees for multipurpose forestry in the
and isolation of bioactive phytochemicals3,4. In the last Mediterranean region, since it is well adapted to calcareous,
decades, the clinical efficacy of many synthetic antibiotics clayish, dry and poor soils16. The Mediterranean
is being threaded by the emergence of a serious problem Cupressus (C. sempervirens L.) grows over a wide natural
which can be defined as multidrug resistant pathogens5. range in diverse environments. It has two main varieties:
Therefore scientists have tried to discover new horizontal is exhibiting abroad crown with wide angles
antimicrobial substances from various sources including between branches and stem and pyramidalis exhibiting a
plants6. Medicinal plants have considerable importance in conical crown form and small angles between branches and
international trade and their clinical, pharmaceutical, and stem. The variety horizontalis grows naturally and can be
economic value is still growing7. Therefore the use of found in northern Iran, Syria, Cyprus, Turkey and Greece.
essential oils is less damaging to the human health8. C. sempervirens L. is only one species native of the genus
Essential oils of plants are of growing interest both in the Cupressus in Syria, whereas C. arizonica is one of the
industry and scientific research9,10. There has been an many species from this genus which has been introduced
increased interest in looking at antimicrobial properties of and cultivated in the country. Cupressus leaves and cones
extracts from aromatic plants particularly essential oils11- have been used as folk remedy in many countries17. With

*Author for Correspondence

   Mohamad et al. / Chemical Composition and…

Figure 1: GC/MS Chromatogram of dried C.sempervirens L .leaves essential oil.

Table 1: Chemical compositions of dried C. sempervirens L. leaves essential oil.

S. No. R.I R.IA Compositions Area %
Monoterpene Hydrocarbons 74.23
1. 939 932 Alpha-Pinene 36.50
2. 949 946 Camphene 1.13
3. 976 974 Beta-Pinene 1.04
4. 980 982 Cis-Pinane 1.50
5. 993 988 Beta-Myrcene 3.16
6. 1014 1008 3-Carene 22.17
7. 1030 1024 Limonene 3.55
8. 1090 1086 Terpinolen 5.18
Oxygenated Monoterpenes 4.76
9. 1351 1346 Alpha-Terpinyl acetate 4.76
Sesquiterpene hydrocarbons 19.33
10. 1421 1417 Alpha -Caryophyllene 1.61
1431 1430 β-Copaene 0.72
1449 1449 Alpha -Himachalene 0.45
11. 1455 1455 Alpha-Humulene 1.41
12. 1465 1464 9-epi-Caryophyllene -E 0.84
13. 1484 1484 Germacrene D 12.81
1497 1498 Alpha -Selinene 0.68
14. 1525 1522 delta-Cadinene 1.31
Total 98.82
RI: retention index (Kovalts) relative to n-alkanes (C8–C20) on a non-polar DB-5 column. RIA: retention index in the
literature on a non-polar DB-5 column (Adams, 2007).

respect to these medicinal and pharmacological the taxonomist of Department of Botany. The dirt was
advantages, C. sempervirens L. widely used as a cosmetic removed with tap water. Collected leaves were dried in the
in gradient in perfumery and soap-making, including its shade for about 14 days. The dried leaves of plant parts
essential oil from it18. The aim of this work is to assay the were ground into small powder by a grinder machine.
main constituent of the essential oil obtained from leaves Essential Oil Extraction
of C. sempervirens L. Which is grown in Syria, to compare Dried C. sempervirens L. leaves (100 g) was subjected to
their chemical composition with those extracted from the hydro-distillation in a Clevenger-type apparatus for (3)
same species from different geographical origins, and to hrs. in accordance with the standard procedure described
carry out a comparative evaluation of their antibacterial in the European Pharmacopoeia19. The sample was added
activity. to distilled water (1000 m l) in a 2 L round - bottomed
flask and heated to boiling, after which the essential oil
EXPERIMENTAL was evaporated together with water vapor, and finally
Plant Material collected in a separating funnel. The upper phase that
Fresh leaves of C. Sempervirens L. growing in the contained the essential oil was separated from the lower
campus of Damascus University faculty of science, was one and the essential oil was dried over anhydrous sodium
randomly collected in May 2014, and authenticated by sulfate and preserved in a sealed sample tube and stored

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Table 2: Antibacterial activity of dried C. sempervirens L .leaves essential oil.

Test Organisms Different concentrations of essential oil Amoxicillin
25% 50% 75% 100% 30mg/mL
gram-positive bacteria
Bacillus subtilis NA NA NA NA 40 ± 0.11
Micrococcus luteus 7.40 ± 0.16 7. 70 ± 0.14 8.10 ± 0.17 8.50 ± 0.13 17 ± 0.21
Streptococcus pyogenes 13.50 ± 0.13 15.60 ± 0.16 18.40 ± 0.13 38.90 ± 0.16 NA
Staphylococcus aureus 14.40 ± 0.12 19. 50 ± 0.11 27.40 ± 1.15 42.30 ± 0.18 45± 0.13
gram-negative bacteria
Klebsiella pneumoniae 7.60 ± 0.12 8.10 ± 0.17 9.10 ± 0.15 10.20 ± 0.13 NA
Vi b r i o parahaemolyticus 19.20 ± 0.13 25. 20 ± 0.17 39.18 ± 0.19 43.20 ± 0.15 30± 0.32
Proteus vulgaris NA NA NA NA 45± 0.12
Salmonella Typhimurium NA NA NA NA 16± 0.31
Escherichia coli NA NA NA NA NA
Pseudomonas aeruginosa 7. 40 ± 0.11 8. 20 ± 0.10 8.70 ± 0.14 9.40 ± 0.16 NA
Data are mean ± SD of three independent experiments. NA: Not active

Table 3: MIC and MBC (mg/mL) of dried C. with those obtained from the Wiley Libraries spectra,
sempervirens L. leaves essential oil. stored in the GC-MS database. Further confirmation was
Microorganism MIC (mg/mL) MBC done from Retention Index data generated from a series
(mg/mL) of alkane's retention indices (relatives to C8-C20 on the
Bacillus subtilis NA NA DB-5MS column) and (Adams, 2007).
Micrococcus luteus 3.2 6.4 Screening for Antimicrobial Activity
Streptococcus 1.6 1.6 The essential oil from C. sempervirens L. sample was
pyogenes tested for antimicrobial activities using agar disc diffusion
Staphylococcus aureus 0.4 1.6 technique to determine the diameter of growth inhibition
Klebsiella 3.2 6.4 zones while broth macro-dilution method was used to
pneumoniae determine the MIC and MBC. The biological assays were
Vi b r i o 1.6 3.2 carried out on the essential oil against ten local human
parahaemolyticus bacteria isolates. These microbes were four gram-positive
Proteus vulgaris NA NA bacteria (Bacillus subitus, Staphylococcus aureus,
Salmonella NA NA Streptococcus pyogenes, Micrococcus luteus) and six
Typhimurium gram-negative bacteria (Escherichia coli, Salmonella
Escherichia coli NA NA typhimurium, Pseudomonas aeruginose, Klebsellia
Pseudomonas 3.2 6.4 pneumonia, Proteus vulgaris, Vibrio parahaemolyticus)
aeruginosa obtained from the Department of Medical Microbiology
NA: Not active and Parasitology, Faculty of Medicine, Damascus
university. The bacteria cultures were maintained on
in the fridge at 4oC until analysis20. nutrient agar slant at 37oC for 24 hrs. The organisms were
Analysis of the Essential Oil (GC/MS) then sub cultured and preserved at 4oC in sterile bottles
Essential oil composition was studied with gas containing both nutrient broth and 15% sterile glycerol.
chromatography mass spectrometry (GC/MS). Gas The essential oil of the dried leaves was tested for
chromatography analysis was carried out with an Agilent antimicrobial activity against the tested organism using the
6890 N gas chromatograph (GC) equipped with Agilent agar disc diffusion method21. Each of the organisms was
5973 mass selective detector (MSD), Agilent Auto sampler transferred into a separate test- tube containing nutrient
7683 and Agilent DB-5MS capillary column (30 m, 0.25 broth to reactivate them by culturing overnight at 37oC. A
i.d., 0.25 µm film thickness) (Agilent Technologies, Santa suspension of the tested bacteria (0.1 ml of 106 CFU/mL)
Clara, CA, USA). The MS detector was operated in was spread on the solid media plates. Sterile apery discs (5
electron impact (EI) mode at 70eV with interface mm diameter) prepared from Whatman Number 1. Filter
temperature of 280oC; the scan range was 50–550amu. The paper discs were loaded with 20 µL of the essential oil and
injection port temperature was set at 250 °C. But GC was placed on the inoculated plates and, after staying at 4oC for
performed in split less mode; carrier gas was helium at a 2 hrs, the plates were incubated at 37oC for 24 hrs.
constant flow rate of 1 ml /min. The column temperature Antimicrobial activities studied with four concentrations
was programmed as follows: an initial temperature of 60 100%, 75%, 50% and 25% of the essential oil, which was
0
C increased to 2800C at rate of 3oC/min. The injection dissolved in dimethyl sulphoxide (DMSO). The zones of
volume was 1.0 µL. inhibition were measured at the end of the incubation
Identification of Components period. Antimicrobial activities of the essential oil leaves
Relative percentage amounts were calculated from peak were expressed by mm in diameter. The result recorded for
area by apparatus software. The identification of individual each bioassay was the average of triplicate test. Negative
compounds was based on comparison of their mass spectra control was prepared using DMSO solvent. And

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Table 4: Main constituents of dried C. sempervirens L. leaves essential oil from different origins.
No. α-pinene β-myrcene 3-carene α-terpinylacetate α-terpinolene Germacrene D α-cedrol
Our study 36.50 3.16 22.17 4.76 5.18 12.81 -
24 30.00 4.10 24.00 6.60 6.60 4.00 -
25 21.40 5.00 16.00 5.60 5.60 13.00 3.30
26 60.50 3.90 0.20 - 2.00 2.30 8.30
27 28.40-79.20 - 9.10-32.60 0.80-4.50 0.60-7.00 - 1.20-12.90
28 54.1 4.6 10.8 5.5 4.1 2.4 4.9
29 48.60 4.10 22.10 - 4.50 1.60 3.50
30 37.10 3.60 19.60 - 4.70 1.30 1.69
31 27.5 1.0 13.2 0.2 7.2 12.1 19.3
32 6.9 - 17.85 - 9.17 2.75 21.29

Amoxicillin was used as a positive control. exhibited a potent inhibitory effect against Streptococcus
Determination of the MIC and MBC pyogenes, Staphylococcus aureus and Vi b r i o
The MIC values of essential oil were tested by two-fold parahaemolyticus, with diameter of inhibition zones
serial dilution method22. The test samples of essential oil ranging from 38.9 ± 0.16 to 43.2 ± 0.15 mm, as shown in
were first dissolved in 5% DMSO, and incorporated into Table 2.
nutrient broth medium in a tube to obtain a concentration On the other hand, essential oil exhibited the modest
of 12.8 mg/mL, and serially diluted to achieve 6.4, 3.2,1.6 antibacterial effect for Micrococcus luteus, Klebsiella
and 0.8 mg/mL, respectively. Ten microliters of pneumonia and Pseudomonas aeruginosa, with inhibition
standardized suspension of each test organism (106 zones in the range 8.5± 0.13 to 10.2 ± 0.13 mm, whereas
CFU/mL) was transferred to the tubes, and incubated at it showed no activity against the Bacillus subtilis (gram
37oC for 24 hrs. The MIC was determined as the lowest positive) and Proteus vulgaris, Salmonella Typhimurium
concentration (mg/mL) of the essential oil where no visible and Escherichia coli (gram negative). The antimicrobial
growths of test organisms occur. The microorganism activity of the C. sempervirens L. essential oil was more
growth was indicated by turbidity. MBC is usually an pronounced against Gram-positive than Gram-negative
extension from the MICs, where the organisms bacteria. The results of the tests for MIC and MBC
quantitatively indicate the minimum concentration revealed that the MIC and MBC values for Micrococcus
when no viable organism appears in the culture23. luteus 3.2, 6.4 mg/ mL, Klebsiella pneumonia 3.2, 6.4
Statistical Analysis mg/ mL and Pseudomonas aeruginosa 3.2, 6.4 mg/ mL
All determinations in this article were carried out in respectively, were bigger than that of Streptococcus
triplicates and SPSS Statistics 19.0 Software was used to pyogenes 1.6, 1.6 mg/ mL and Vi b r i o
evaluate one-way analysis of variance (ANOVA) at p ≤ parahaemolyticus 1.6, 3.2 mg/ mL respectively, while no
0.05. Canonical Discriminate Analysis was also used to active MIC and MBC f o r Bacillus subtilis, Proteus
establish differences between samples, and to evaluate the vulgaris, Salmonella Typhimurium and Escherichia coli as
importance of different variables on discrimination. shown in Table 3. It is difficult to compare between
different studies that concerning essential oil composition
RESULTS AND DISCUSSION of C. sempervirens L . due to the difference in
The yield of C. sempervirens L. leaves essential oil from compounds. In previous studies, essential oil of C.
the hydro distillation of Sample was 1.28% v/w. Table sempervirens L. was studied in Iran24,25, Algeria26,
1 shows the identified constituents of this essential oil Croatia27, Greece28, Saudi Arabia29, Tunisia30,31 and
percentage and fig 1 shows their GC/MS Chromatogram. Egypt32. The major components were α-cedrol, 3-carene,
A total of fourteen compounds amounting to 98.82% in the terpinolene, Germacrene D and α-terpenyl acetate.
C. sempervirens L. leaves essential oil were identified. According to these studies, α-pinene is the first major
Among these 74.23% were monoterpene hydrocarbons, component of leaves essential oil, these findings are in
4.76% were oxygenated monoterpenes, and it also general agreement with our reports, but α-pinene is the
contained 19.33% sesquiterpene hydrocarbons and no fourth major component in the previous research33.
oxygenated sesquiterpenes found. The major constituents 3-carene is the second major component in the
in the C. sempervirens L. leaf essential oil were alpha- reported investigation for researches24-30, whereas it was
pinene 36.50%, 3-carene 22.17%, Germacrene D 12.81%, in third major component in (31), and the second in (32).
Terpinolen 5.18%, alpha-terpinnyl acetate 4.76, limonene In addition, Germacrene D is the third component in
3.55% and beta-myrcene 3.16%. The in vitro antibacterial the previous research (29), and the fourth in (31).
activities of essential oil of C. sempervirens L. leaves Ultimately, α-cedrol is the first major component in
against the bacteria used were qualitatively assessed by (32), but in (26, 31) the second major component,
the presence or absence of inhibition zones. A total of ten when it is third in (27) and fourth in (28, 29). In
bacterial strains, including four Gram-positive and six comparison with our results, the major components
Gram-negative bacteria were tested. The essential oil are α-pinene, 3-carene, Germacrene D, α-terpinyl acetate,
exhibited antibacterial activity against three Gram- α-terpinolene respectively, while no α-cedrol included.
positive and three Gram-negative bacteria. They The study of essential oil of C. sempervirens L . grows

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in Mediterranean countries, as Syria (this study), Algeria26, 2. Grzanna R., Lindmark K L., Frandozo. C. G., J.
Greece28, Tunisia30,31, and Egypt32. It showed dissimilarity Med.Food. 2005. 8(2):125-132. doi:10.
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of synthetic chemicals and drugs. The quality of natural 19. European Pharmacopoeia. 2004. 5th ed. EDQM
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ACKNOWLEDGMENTS 22. Chandrasekaran, M., Venkatesalu, V., J.
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Prof. M. J. Alkhateeb for his keen interest in this work. 23. Sovcíková A, Mikulásová M, Horáková K, Floch L,
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