Antimicrobial activity of berries and leaves essential oils of Macedonian Juniperus foetidissima Willd. (Cupressaceae)

Chemical composition and antimicrobial activity of leaves and berries essential oils from Juniperus foetidissima Willd. (Cupressaceae) grown in R. Macedonia (RM) was investigated. GC/FID/MS analysis was carried out and 93 components were identified, representing 89.7-96.5% of the oils. The major components of the berries essential oil were α-pinene (19.2%), limonene (24.9%) and cedrol (23.1%), followed by smaller amounts of b-funebrene, trans-caryophyllene, germacrene D and d-cadinene. The composition of the leaves essential oil was variable depending on the region of collection. Accordingly, samples originated from southeastern RM contained essential oil with α-pinene (67.6%) and limonene (10.0%), from central part of RM with limonene (17.9-27.1%) and cedrol (28.8-33.9%), while samples from southwestern RM contained oil with terpinen-4-ol (19.1%), cis-thujone (8.3%), germacrene D (11.0%) and d-cadinene (6.3%) as predominant components in the oil. Antimicrobial screening of the essential oils was made by disc diffusion and broth dilution method against 16 bacterial strains of Gram-positive and Gram-negative bacteria and one strain of Candida albicans. The leaves essential oil showed stronger antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes and Haemophilus influenzae (MIC = 125 ml/ml) and moderate activity against Campylobacter jejuni (MIC > 500 ml/ml). Other investigated bacterial strains and Candida albicans were completely resistant to the antimicrobial activity of J. foetidissima essential oils.


Introduction
Juniperus is one of the major genera of Cupressaceae family consisting of approximately 70 species variable in size and shape, from tall trees to columnar or low spreading shrubs. The plants are evergreen with needle-like or scalelike leaves. Juniperus foetidissima Willd. is a medium-size tree, spread mainly throughout the southeastern Europe and southwestern Asia, starting from southeastern Albania and northern Greece, across Turkey, Syria and Lebanon to the northern Iran and southwestern Turkmenistan. It often occurs together with J. excelsa Bieb., but it could be distinguished by its thicker shoots and green leaves (Marcysiak et al., 2007). The name foetidissima comes from the stinking (fetid) smell of crushed leaves.
J. foetidissima contains essential oil in almost all parts of the plant, with variable composition. Thus, sabinene, a-thujone, terpinen-4-ol and g-terpinene were reported as major components of the leaf essential oil of J. foetidissima from Greece (Adams, 1987;Adams, 1990). The monoterpenes sabinene, a-pinene and limonene were predominant components of the essential oils of fruits and leaves of male and female plant of J. foetidissima from Iran (Asili et al., 2010). Turkish J. foetidissima contained b-thujone and cedrol as major component in the leaf essential oil and sabinene as predominant in berry oil. The major components of seed and seedless cone berry oils were sabinene, b-thujone and abietal while in the oil from branches the most abundant was a-pinene (Tunalier et al., 2002). On the other hand, J. foetidissima heartwood of the root and stem was identified as the new potential source of Cedarwood oil (Tunalier et al., 2004), while in the essential oil obtained from branches of J. foetidissima grown in Syria, citronellol, bornyl acetate and cadalene were found as major constituents (Tayoub et al., 2012). These oils are considered as potential sources of various terpene components as well as biologically active agent.
J. foetidissima occurs in the flora of Republic of Macedonia (RM). Mainly, it grows in southern parts of the country, but could be found in the valleys of the River Crn Drim and the River Treska in western and Karadzica Mtn. in central RM (Micevski, 1998). Up to now only one report on chemical composition, an antioxidant and anti-inflammatory effects of one sample of Macedonian J. foetidissima was published (Lesjak et al., 2013). The present study was aimed to determine the chemical composition and to evaluate the antimicrobial activity of the essential oils obtained from berries and leaves from J. foetidissima collected on several locations in RM.

Plant materials
The terminal plant twigs were collected from four different localities in RM (Table 1). Plant identity was verified as Juniperus foetidissima Willd. and herbarium vouch-er specimens were deposited at the Department of Pharmaceutical Botany, Institute of Pharmacognosy, Faculty of Pharmacy, Skopje, RM. The plant material was dried at room temperature. Just before hydroisolation, leaves and berries were separated from the branches and minced properly.
The plant material was collected from 4 localities from central (Veles), southern (Udovo), southeastern (Valandovo) and southwestern (Ohrid) RM. According to this the samples of leaves oils were marked as Ve-LEO, U-LEO, Val-LEO and Oh-LEO, respectively. The sample of berries essential oil was marked as Val-BEO (Table 1).

Essential oil isolation
The essential oils were obtained from dried plant material through distilled stem using all glass Clevenger-type apparatus. For that purpose, 20 g of minced plant material was distilled for 4 hours. After isolation, anhydrous sodium sulfate was added to remove residual water from the oil. The essential oil yield was calculated on dried plant material and was expressed in ml/kg. For GC/FID/MS analysis, the essential oil was dissolved in xylene to obtain 1 ml/ml oil solution.

Gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS)
Essential oil samples were analyzed on Agilent 7890А Gas Chromatography system equipped with FID detector and Agilent 5975C Mass Quadrupole detector as well as capillary flow technology which enables simultaneous analysis of the samples on both detectors. For that purpose, HP-5ms capillary column (30 m x 0.25 mm, film thickness 0.25 mm) was used. Operating conditions were as  (2015) follows: oven temperature at 60 °C (5 min), 1 °C/min to 80 °C (2 min) and 5 °C/min to 280 °C (5 min); helium as carrier gas at a flow rate of 1ml/min; injector temperature 260 °C and that of the FID 270 °C. 1ml of each sample was injected at split ratio 1:1. The mass spectrometry conditions were: ionization voltage 70 eV, ion source temperature 230 °C, transfer line temperature 280 °C and mass range from 50 -500 Da. The MS was operated in scan mode.

Identification of the components
Identification of the components present in essential oils was made by comparison of their mass spectra with those from Nist, Wiley and Adams mass spectra libraries, by AMDIS (Automated Mass Spectral Deconvolution and Identification System) and by comparing literature and estimated Kovat's (retention) indices that were determined using mixture of homologous series of normal alkanes from C 9 to C 25 in hexane, under the same above mentioned conditions.
The percentage ratio of essential oils components was computed by the normalization method of the GC/FID peak areas without any correction factors. A nutrient (Mueller Hinton) agar from Merck (Darmstadt, Germany), blood agar (Oxoid, Basingstoke, UK) and Sabouraund agar (bioMerieux, Durham, NC) were used for growing of the microbial strains.

Disc diffusion method
Disc diffusion method was used for screening the antimicrobial activity of all essential oils in order to determine the growth inhibition zones of studied microorganisms that occur around certain essential oil. In this regard, microorganisms were suspended in sterile broth with turbidity corresponding to 0.5 and 1 Mc Farland (approximate by 10 7 -10 8 CFU/ml) for all bacteria and for Candida albicans, respectively. The microbial suspensions were streaked over the surface of the agar media using a sterile cotton swabs to ensure uniform inoculation. After inoculation of microorganisms, discs of 6 mm in diameter were made at wellspaced intervals. They were filled with 85 ml of 50% solutions of essential oils in dimethylsulfoxide (DMSO, Sigma-Aldrich, Germany) and one disc was filled only with DMSO as a control. The plates were incubated at 37 0 C, aerobically for 24 hours. Only, Campylobacter was incubated in microaerophilic atmosphere, at 42°C for 48 hours. The growth inhibition zones were measured after incubation of the isolates under their optimal growth conditions and were ranged between 6 mm and 30 mm in diameter. The antimicrobial activity was determined according to the diameters of the inhibition zones (0-14 mm resistant (R), 14-19 mm moderate susceptible (M) and 19-30 mm susceptible microorganisms (S)).

Broth dilution method
This method was used in order to determine minimal inhibitory concentration (MIC) of the particular essential oil that had revealed antimicrobial activity by disc diffusion method. For that purposes, 25 ml of those essential oils (50% solution of essential oil in DMSO = 500 ml/ml) were diluted in equal quantities of 0.9% sodium chloride solution, to make them with concentration of 25% (250 ml/ml). This concentration was decreased five times, subsequently, by adding 25 ml of each bacterial or fungal suspension, thus the final concentrations were: 12.5%, 6.2%, 3.1%, 1.5% and 0.7% or 125 ml/ml, 62 ml/ml, 31 ml/ml, 15 ml/ml and 7ml/ml, respectively. 15 ml of each bacterial or fungal suspensions with these particular concentrations were inoculated on solid media (Miller-Hinton agar, blood agar, Sabouraund agar), depending on the type of microorganism. The growth of any microorganism was evaluated after its incubation under the optimal growth conditions. The lowest concentration of essential oil which was able to inhibit the growth of the particular microorganism was considered as its minimal inhibitory concentration (MIC).
Comparing to literature data, similarities and differences could be noticed. Adams found sabinene (19.6%), a-thujone (18.6%), terpinen-4-ol (17.6%) and g-terpinene (6.5%) being the major components of the leaf essential oil of J. foetidissima collected in Greece (Adams, 1990). At the same time, the minor compounds of this essential oil were a-terpinene (4.3%), b-thujone (3.5%), cedrol (3.2%), myrcene (2.7%) and a-pinene (2.6%). According to the Iranian researchers, the major components of the essential oils of fruits, leaves of male and leaves of female plant of J. foetidissima were sabinene (37.1, 19.9 and 16.8%), a-pinene (29.9, 22.2 and 18.6%) and limonene (11.8, 20.9 and 13.6%), respectively (Asili et al., 2010). Tunalier et al. found b-thujone and cedrol as predominant components of the essential oil of leaf and sabinene as major component in the berry essential oil of J. foetidissima from Turkey (Tunalier et al., 2002). Considering essential oil composition of J. foetidissima from Balkans, only one article was published. Lesjak et al. examined one sample from region of Prespa Lake from RM and found sabinene (39.9%), g-terpinene (10.1%) and terpinen-4-ol (17.0%) as major monoterpenes and germacrene D (0.7%) and g-cadinene (2.9%) as major sesquiterpene (Lesjak et al., 2013). Although the mentioned region of collection was geographically close to Ohrid Lake, where our samples were collected, the leaves oil composition differed mainly in the percentage amounts of trans-thujone (2.6%) and cis-thujone (8.3%) that were present in our oil samples, but were not identified by Lesjak et al. tained led to a non-significant inhibitory effect, although essential oil of J. phoenicea and J. oxycedrus exhibited rather good or weak activity against Candida albicans and Staphylococcus aureus (Angioni et al., 2003).
The study of the antimicrobial activity of leaf essential oil of J. oxycedrus from Tunisia showed that Escherichia coli was extremely resistant to this oil while Staphylococcus aureus was the most sensitive strain with MIC ranged from 600 to 650 mg/ml (Medini et al., 2010). Regarding antifungal activity, J. oxycedrus leaf essential oil provided to be an emergent alternative as antifungal agent against dermatophyte strains. Delta-3-carene was shown to be a fundamental compound for this activity (Cavaleiro et al., 2006). The evaluation of the biological activity of berry essential oil of Iranian J. foetidissima showed no antimicrobial activity against Candida albicans, Escherichia coli and Pseudomonas aeruginosa. The leaf essential oil of the same plant showed moderate activity against Bacillus subtilis, Candida albicans, Escherichia coli, Pseudomonas aeruginosa (MIC values between 3.125 and 6.25 mg/ml) and Staphylococcus aureus (MIC = 25 mg/ml) (Asili et al., 2010).
shown stronger antimicrobial activity against Staphylococcus aureus and Candida albicans, rarely against other bacterial or fungal strains. Only few authors found susceptible antimicrobial activity against Escherichia coli, Streptococcus pyogenes and Streptococcus pneumonia. Comparing to our findings, leaves essential oil of Macedonian J. foetidissima showed similar antimicrobial activity against Staphylococcus aureus and Streptococcus pyogenes. Promising antimicrobial activity of leaves essential oil against Haemophilus influenzae was found for the first time. It is worth to mention that besides good antifungal activity presented in the literature (Asili et al., 2010;Haziri et al., 2013;Unlu et al., 2008), essential oils from Macedonian J. foetidissima have shown no activity against Candida albicans.