Chemical composition and antimicrobial activity of leaves essential oil of Juniperus communis ( Cupressaceae ) grown in Republic of Macedonia

Chemical composition and antimicrobial activity of essential oils isolated from leaves of three different samples of wild growing Juniperus communis L. (Cupressaceae) from R. Macedonia was investigated. Essential oil yield ranged from 7.3 to 9.0 ml/kg. Performing GC/ FID/MS analysis, ninety components were identified, representing 86.07-93.31% of the oil. The major components of the leaves essential oil (LEO) were α-pinene (21.37-28.68%) and sabinene (2.29-16.27%), followed by limonene, terpinen-4-ol, β-elemene, trans-(E)-caryophyllene, germacrene D and δ-cadinene. Antimicrobial screening of the LEO was made by disc diffusion and broth dilution method against 16 bacterial isolates of Gram positive and Gram negative bacteria and one strain of Candida albicans. Two bacteria, Staphylococcus aureus and Streptococcus pyogenes were sensitive to antimicrobial activity of LEO (MIC = 125 μl/ml). Additionally, LEO showed moderate antimicrobial activity against Streptococcus agalactiae, Haemophilus influnzae, Corynebacterium spp. and Campylobacter jejuni (MIC > 500 μl/ml). Candida albicans, Staphylococcus epidermidis, Acinetobacter spp., Salmonella enteritidis, Shigella flexneri, Klebsiella pneumonia, Pseudomonas aeruginosa and Proteus mirabilis were completely resistant to the antimicrobial effects of this.


Introduction
The common juniper, Juniperus communis L. (Cupressaceae), is an evergreen shrub or small coniferous tree, wide spread through the cool temperate Northern Hemisphere.The above-ground parts, especially leaves and berries of juniper are rich in essential oil that has characteristic aromatic flavour and bitter taste.Due to its diuretic and gastrointestinal properties, common juniper is used as me-dicinal plant for centuries.Juniper oil is used in the pharmaceutical, food and cosmetic industries, as well as for the production of perfumes.Certain beverages (gin) are made with distillation from fermented juniper berries.
According to the literature data, juniper essential oil can be obtained from berries, leaves, wood and seeds by hydrodistillation (Orav et al., 2010a;Chatzopoulou and Katsiotis, 1993;Kumar et al., 2007).The average oil yield varies from 0.5 to 2.5% (for berries) and from 0.2 to 1.0% (for needles) (Orav et al., 2010a).Dissimilarities in the oil's yield and the chemical composition can vary from the geo-graphical location, age and degree of plant ripeness, harvesting methods, distillation techniques and other factors.
In vitro antimicrobial (antibacterial and antifungal) activity of the berries essential oil was studied and the results showed strong to moderate antimicrobial activity (Filipowitz et al. 2003;Stassi et al. 1995).Other results from the antimicrobial assessment of the leaves essential oil (LEO) demonstrate no or weak antimicrobial activity against various tested microbial strains (Asili et al., 2008;Angioni et al., 2003).
Common juniper, Juniperus communis, is widely spread shrub throughout the territory of Republic of Macedonia (Micevski, 1998).The berries of this plant are extensively utilized in production of blended teas and other herbal medicinal products, in food industry, as a spice, in production of alcoholic beverages, etc.For years, the juniper berries and the juniper essential oil are exported from R. Macedonia.On the other hand the juniper leaves are used in folk medicine for various purposes.Up to date there is no information of the composition and antimicrobial activity of the leaves' essential oil from Macedonian juniper.Therefore the aim of the present study was to investigate the chemical composition and the antimicrobial activity of the leaves essential oil of Juniperus communis grown wild in R. Macedonia.

Plant materials
The terminal twigs of Juniperus communis were collected from tree different localities in R. Macedonia in late autumn 2010 and 2011.Plant identity was verified as Juni-perus communis L. and herbarium voucher specimen were deposited at the Department of Pharmaceutical Botany, Institute of Pharmacognosy, Faculty of Pharmacy, Skopje, R. Macedonia (Table 1).
The plant material was dried at room temperature.Just before essential oil isolation, the juniper leaves were separated and minced properly.

Essential oil isolation
The essential oils were obtained from dried plant material through steam distillation 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 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

Identification of the components
Identification of the components present in essential oils was made by comparing mass spectra of components in essential oils 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 (Merck, Darmstadt, Germany), blood agar (Oxoid, Basingstoke, UK) and Sabouraud agar (bioMerieux, Durham, NC) were used for growing of the microbes.

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.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 susceptible -S microorganisms).

Broth dilution method
This method was used in order to determine minimal inhibitory concentration (MIC) of the particular essential oil prepared as 50% solution in DMSO).For that purposes, 25 ml of those essential oils were diluted in equal quantities of 0.9 % sodium chloride solution, to make them with the concentration of 25%.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.15ml of each bacterial or fungal suspensions with these particular concentrations were inoculated on solid media (Miller-Hinton agar, blood agar, Sabouraud 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).

Antimicrobial activity
Antimicrobial screening of the essential oils was made by disc diffusion and broth dilution method against 16 bacterial isolates of Gram positive and Gram negative bacteria and one strain of Candida albicans (Table 3).The highest MIC (125 µl/ml) of LEOs were towards Staphylococcus aureus and Streptococcus pyogenes, and moderate antimicrobial activity against Streptococcus agalactiae, Haemophillus influnzae, Corynebacterium spp.and Campylobacter jejuni (MIC > 500 µl/ml).Candida albicans, Staphylococcus epidermidis, Acinetobacter spp., Salmonella enteritidis, Shigella flexneri, Klebsiella pneumonia, Pseudomonas aeruginosa and Proteus mirabilis were completely resistant to the antimicrobial activity of juniper oil.
Antimicrobial activity of juniper essential oils was previously investigated so the available literature pointed out no activity to some antimicrobial effects against vari-Chemical composition and antimicrobial activity of leaves essential oil of Juniperus communis (Cupressaceae) grown in R. Macedonia  ous tested microbial strains.Essential oil (Juniperi aetheroleum) obtained from the juniper (J.communis) berries was evaluated for the antimicrobial activity against sixteen bacteria, seven yeast-like fungi, three yeasts and four dermatophyte strains.Juniper essential oil showed similar bactericidal activities against Gram-positive and Gram-negative bacterial strains, with MIC values between 8 and 70% (V/V), as well as a strong fungicidal activity against yeasts, yeast-like fungi and dermatophytes, with MIC values below 10% (V/V).The strongest fungicidal activity was recorded against Candida spp.(MIC from 0.78 to 2%, V/V) and dermatophytes (MIC from 0.39 to 2%, V/V).GC/MS analysis of the essential oil showed that predominant constituents in this oil were α-pinene (29.17%), β-pinene (17.84%), sabinene (13.55%), limonene (5.52%) and β-myrcene (0.33%) (Pepelnjak et al., 2005).The essential oil of J. communis growing wild in Kosovo, showed moderate to high activities against Staphylococcus aureus, Escherichia coli and Hafnia alvei, while Pseudomonas aeruginosa was resistant to the antimicrobial effects of the oil (Haziri et al., 2013).
The leaves essential oils of J. communis subsp.hemisphaerica and J. oblonga from Iran did not show noticeable activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans (Asili et al., 2008).Angioni et al. reported similar results concerning the antimicrobial activity of the essential oils from ripe and unripe berries and leaves of Italian J. communis against the most of the above mentioned microbial.
Antimicrobial activity: мicrobial strains and cultures 16 bacterial isolates representing both Gram positive and Gram negative bacteria and one strain of Candida albicans were used for antimicrobial screening.Five isolates were standard strains (Staphylococcus aureus ATCC 29213, Escherichia coli 25927, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231).The remaining 12 bacterial strains (Staphylococcus epidermidis, Enterococcus, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Haemophilus influenzae, Proteus mirabilis, Salmonella enteritidis, Corynebacterium spp., Salmonella enteritidis, Shigella flexneri, Campylobacter jejuni and Acinetobacter spp.) were clinical isolates provided from the Institute of Microbiology and Parasitology, Faculty of Medicine, Skopje, R. Macedonia.

Table 1 .
Plant samples of Juniperus communis from R. Macedonia Chemical composition and antimicrobial activity of leaves essential oil of Juniperus communis (Cupressaceae) grown in R. Macedonia °C, transfer line temperature 280 °C and mass range from 50 -500 Da.The MS was operated in scan mode.

Table 2 .
The chemical composition of the leaves essential oil (LEO) of Juniperus communis from R. Macedonia

Table 3 .
Antimicrobial activity of the leaves essential oil of Juniperus communis DD -Disc diffusion (zone of inhibition including the diameter of disc 6 mm), R -resistant with zone of inhibition 0 -14 mm, M -moderate susceptible with zone of inhibition 14 -19 mm and S -susceptible microorganism with zone of inhibition 19 -30 mm); MIC -minimum inhibitory concentration (µl/ ml); n.m. -not measured.