Chemical Composition and Antimicrobial Activity of the Essential Oil of the Leaves of Cupressus macrocarpa Hartweg . ex Gordon

1 Department of Medicinal Chemistry, Theodor Bilharz Research Institute, Kornish El-Nile, 12661 Warrak El-Hadar, Giza, Egypt. 2 Pharmacognosy Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki-12622, Cairo, Egypt. 3 Department of Chemistry, College of Science and Arts, Sajir, Shaqra University, Kingdom of Saudi Arabia. 4 Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki12622, Cairo, Egypt.


INTRODUCTION
Cupressus macrocarpa (Hartweg.ex Gordon) as a medicinal plant belongs to the family Cupressaceae and commonly known as Monterey Cypress (Cool, 2005;Thukral et al., 2014).It is widely distributed throughout the tropical and temperate regions around the world i.e., Mexico, North America Asia, and North Africa (El-Ghorab et al., 2007).C. macrocarpa was used traditionally for decades for the treatment of various ailments, e.g., styptic problem, eliminates fluid retention, whooping cough and rheumatism (Thukral et al., 2014).C. macrocarpa received little concern regarding its phytochemical constituents; Cool (2005) reported the isolation of ten sesquiterpenes from its foliage.Furthermore, Al-Sayed and Abdel-Daim (2014) investigated the protective role of the isolated cupressuflavone from C. macrocarpa against CCl 4 induced hepatoand nephrotoxicity in mice.
Several Cupressus species have been investigated for their essential oil content and evaluated for biological activity.El-Ghorab et al. (2007) identified 43 components from the fresh and dried leaves of the Egyptian C. macrocarpa that showed remarkable antimicrobial and antioxidant activities.In addition, thirteen components from the Iranian C. sempervirens L. were identified ( Emami et al., 2004) and investigated for activity as fungistatic and bacteriostatic activity.Allo-ocimene along with other fourteen components were identified from the Egyptian C. sempervirens (Ibrahim et al., 2009), which exhibited promising antimicrobial and antiviral activities.
Furthermore, Boukhris et al. (2012) identified 24 components from the Tunisian C. sempervirens essential oil and evaluated their antioxidant and antimicrobial activities.The aims of the current study were to analyze the essential oil composition of the Egyptian C. macrocarpa fresh leaves qualitatively by GC-MS and quantitatively via GC-FID, as well as the evaluation of the antimicrobial activity against eight pathogenic microbial strains.

Plant material
Fresh leaves of Cupressus macrocarpa Hartweg.ex Gordon (Cupressaceae) were collected from the Zoo Garden, Giza, Egypt in June 2014.The plant was identified by Mrs. Threase Labib, consultant of plant taxonomy at the Ministry of Agriculture; formerly, the head of taxonomist specialists at the garden, a voucher specimen (No.C10/1/4) was kept at the herbarium of the garden.

Essential oil isolation
The fresh leaves of C. macrocarpa (2 kg) were fragmented into small pieces and subjected to hydrodistillation using Clavenger apparatus (Ibrahim et al., 2015) to extract the essential oil which was determined as mean of triplicate.The chemical composition of the oil was determined quantitatively via GC-FID and qualitatively via GC-MS by comparing their retention times and mass spectral fragmentation patterns with previously reported data (Adams, 1989(Adams, , 2001(Adams, , 2012)).

Gas chromatography-mass spectrometry (GC-MS) analysis
The GC-MS analysis was performed using a Thermo Scientific, Trace GC Ultra/ISQ Single Quadrupole MS and TGSMS Fused Silica Capillary Column (30m, 0.25mm, 0.1mm Film thickness).For GC-MS detection, an electron ionization system with ionization energy for 70 ev was used as the carrier gas at a constant flow rate of 1mL/min.The injector and MS transfer line temperature was set at 280°C.The oven temperature was programmed at an initial temperature 40°C (hold 3 min.)to 280°C was a final temperature at an increasing rate of 5°C/min (hold 5 min).The identified components were investigated using a percent relative peak area.A tentative identification of the volatile compounds was performed based on the comparison of their relative retention time and mass spectra with those of the NIST08s, WILLY8, Adams and Library data of the GC/MS system (Adams, 1989(Adams, , 2001(Adams, , 2012;;Ghareeb et al., 2016).

Gas Chromatography-Flame Ionization Detector (GC-FID) analysis
The GC-FID analyses were carried out with a Varian 3400 apparatus (Varian GmbH, Darmstadt, Germany) equipped with a FID detector and a DB-5 fused-bonded capillary column (30m x 0.25mm i.d., film thickness 0.25µm; Ohio Valley, Ohio, USA).The oven temperature was programmed isothermal at 45°C for 2 min., then rising from 45°C to 300°C (4°C/min.),and finally held isothermal at 300°C for 20 min.; injector temperature was 250°C; detector temp.was 300°C; Helium gas was used as carrier gas (2.0mL/min.);split ratio, 1:20.Peak Simple ® 2000 chromatography software (SRI Instruments, California, USA) was used for recording and integrating the chromatograms.Average areas under the peaks of three independent chromatographic runs were used for calculating the % composition of each component.

Test organisms
The bacteria used in this study were Gram-positive bacterial strains Bacillus subtilis NRRL 543 and Staphylococcus aureus NRRLB-313 and Gram-negative bacterial strains, Escherichia coli NRRL B-210 and Pseudomonas aeruginosa NRRL B-23.Fungi, Aspergillus niger NRRL 599, Fusarium oxysporum NRRL 28184, Candida albicans NRRL Y-477 and Fusarium solani.These micro-organisms were obtained from Northern Utilization Research and Development Division, United State Department of Agriculture, Peoria, Illinois, USA.

Screening test
The bacterial strains were revived for bioassay by subculturing in fresh nutrient broth medium for 24 hours before test, while fungi were cultured on potato dextrose agar (PDA) (2.5% w/v agar) for 7 days at 28°C before the experiment was carried out.

Agar diffusion method
Cup plate agar diffusion method has been employed for the determination of the antimicrobial activity of some essential oils as well as paraffin oil.Several test microbes including G+ve bacteria (B.subtilis and S. aureus), G-ve bacteria (P.aeruginosa and E. coli) and fungi (C.albicans, F. oxysporum, A. niger and F. solani) were applied. 1 mL of cell suspension of 24h-old bacterial cultures (10 7 -10 8 colonies/ml) in sterile distilled water was added to 250 mL of sterile solidified nutrient agar medium.For fungal strains, 1 mL spore suspension of seven days cultures fungal cultures (10 6 -10 7 colonies/mL) was added to 250 mL sterile solidified PDA (potato dextrose agar) medium.Holes of 9 mm in diameter were made using a cork borer.Aliquots of 0.1mL of the diluted essential oil with paraffin oil were poured inside the holes.A hole filled with paraffin oil only was also used as control.The plates were left for 2h at 4 o C as a period for diffusion.The diameter of each inhibition zone was measured and compared with that of the standard.Plate cultures were kept in an incubator at 28°C for 48h for fungi and at 37°C for 24h for bacteria (Linday, 1962).Streptomycin and Nystatin were used as control antibiotics at a concentration of 50 and 100 µg/mL respectively.Following incubation, the zone of inhibition for each sample was recorded in mm (including the hole).
Microorganisms with inhibition zone diameter ≥ 28 mm were classified as strongly sensitive, while that of < 28-16 mm were moderately sensitive, and with < 16-12 mm assorted as weakly sensitive and isolates with zone diameter of <12 mm as resistant (Bauer et al., 1996;Elgayyar et al., 2001).

The minimum inhibitory concentration (MIC)
Stock solutions of the EO were diluted and transferred into the first tube, and serial dilutions were prepared with concentrations ranged from 0.001-0.02μL/mL.Spore suspension (10 μL) of each strain was inoculated in nutrient medium and incubated for 24-72 hours at 37°C.The control tubes containing the same medium were inoculated only with bacterial strains suspension.The minimal concentrations at which no visible growth was observed were defined as the MICs and expressed in (v/v %) (Carson et al., 1995).

Antimicrobial activity
The antimicrobial activity of the essential oil was tested against G +ve and G -ve bacteria and fungi.Generally, it has been found that the activity was decreased with increasing the dilution titer of the tested microbes.EO at dilution (1:5, v/v) showed higher antifungal activity against F. oxysporum followed by A. niger and F. solani (17, 16 and 15 mm), respectively, and it recorded activity against C. albicans with (19 mm).The potent antibacterial activity resulted at dilution of 1:5, v/v against P. aeruginosa followed by E. coli with 21 and 15 mm for G-ve, and B. subtilis followed by S. aureus with 20 and 14 mm for G +ve, respectively (Table 2).The MIC values proved that the oil inhibited the growth of the tested fungi and bacteria at dilution 1:12.5-50(v/v), with Escherichia coli being the most sensitive (Table 3).
EOs exhibited promising antimicrobial activity against a wide range of bacteria, and are very useful in food industry and clinical practice (Nakatani, 1994).Usually, Gram +ve organisms are highly susceptible than Gram -ve (Burst, 2004) The activity of the EOs (i.e., antimicrobial) may be attributed to its major components (Lahlou, 2004), which may act synergistically (Peschel et al., 2006) to provide the activity.El-Ghorab et al., (2007) reported that the EO of Egyptian C. macrocarpa strongly inhibited the growth of S. aureus, P. aeruginosa, E. coli, A. niger and C. albicans, and concluded that, this may be attributed to the presence of neral, geraniol, eugenol dihydro, carvacrol acetate and phenol (2,6-dimethoxy).Moreover, Manimaran et al. (2007) reported the potent antimicrobial activity of the EO of the Indian C. macrocarpa and concluded that, such activity mainly due to the EO components i.e., caryophyllene, αterpineol etc.In addition, a comparative antimicrobial study was done on the EOs content of Cupressus glauca, C. funebris, C. lawsonia, C. macrocarpa and C. sempervirens.The results showed that C. macrocarpa possessed the potent antibacterial and antifungal activities at concentration of 100 mcg/mL while against C. albicans was 50 mcg/mL (Manivannan et al., 2005).
In the current study, -terpineol was reported to be the major component of the EO of C. macrocarpa, which was suspected to be responsible for the antimicrobial activity.Previous studies are in full agreement with our findings which correlated the antimicrobial activity to one or several major constituents.Yang et al. (2014) concluded that α-terpineol and p-cymene are the reason for the antimicrobial activity of the EO of Glossogyne tenuifolia.Moreover, Krist et al. (2008) investigated the antimicrobial effect of five aroma compounds, among them α-terpineol showed the highest activity.Kubo et al. (1991) proved that α-terpineol has high antimicrobial activity against S. aureus, and P. aeruginosa.
In fact, the oxygenated monoterpenes (e.g.monoterpene alcohols) are promising antimicrobial agents due to the presence of the alcoholic part, which increase their water solubility ( Hammer et al., 2003).Their mode of action may be attributed to protein denaturation or dehydration on the vegetative cells (Dorman and Deans, 2000).In addition, α-terpineol as a member of the oxygenated monoterpenes can act as antimicrobial agent via the cell barrier destruction, and initiate seepage of proteins and lipids (Oyedemi et al., 2009).On the other hand, some studies have concluded that the antimicrobial activities of the whole essential oil were greater than the activities ascribed to certain individual constituents, and the synergistic effect should be taken in our minds due to a complex interaction between such individual constituents (Gill et al., 2002;Savelev et al., 2003).
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Table 1 :
Chemical compositions of the essential oil of C. macrocarpa leaves.

Table 2 :
Antimicrobial activities of the essential oil from C. macrocarpa leaves.

Table 3 :
MICs of the essential oil from C. macrocarpa leaves.