Chemical composition and antimicrobial activity of berry essential oil of Juniperus oxycedrus L . ( Cupressaceae ) grown wild in Republic of Macedonia

Chemical composition and antimicrobial activity of essential oil isolated from berries from 2 different samples of Juniperus oxycedrus L. (Cupressaceae), growing wild in Republic of Macedonia was investigated. Performing GC/FID/MS analysis, one hundred components were identified, representing 96.0-98.95% of the oil. The major components were α-pinene (22.5427.12%), myrcene (11.2615.13%) and limonene (2.78-18.06%). Antimicrobial screening of the J. oxycedrus 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. The most sensitive bacteria was Haemophilus influenzae (MIC = 125 ml/ml). The essential oils showed moderate antimicrobial activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Corynebacterium spp., Escherichia coli and Campilobacter jejuni (MIC > 500 ml/ml) and no activity against Candida albicans, Staphylococcus epidermidis, Acinetobacter spp., Salmonella enteritidis, Shigella flexnery, Klebsiella pneumonia, Pseudomonas aeruginosa, Enterococcus and Proteus mirabilis.


Introduction
Juniperus oxycedrus L. (Cupressaceae) is a hardy spreading plant, that vary in size from a low shrub to a tree of about 6 m.The leaves are tiny, narrow and prickly and are reason for people' name of the plant:"Prickly Juniper".The cones are yellow and rounded, and the fruits are berry-like, growing in clusters, and black when ripe.The plant is known as Cade juniper as well, mainly because of the oil that is obtained through destructive distillation of the wood.Cade oil is dark, aromatic oil with a strong smoky smell which is used in some cosmetics and traditional skin treatment drugs, as well as аn incense (Lamnauer, 2005).
J. oxycedrus is known as Mediterranean equivalent of the common juniper, native across the Mediterranean region from Morocco and Portugal, southern France and east to Iran, Lebanon and Israel.It grows on rocky sites from sea level up to 1600 m altitude.Among J. oxycedrus interesting subspecies are J. oxycedrus var.badia H.Gay (syn.J. oxycedrus subsp.badia (H.Gay) Debeaux), distinguished on the basis of larger cones and described from northern Algeria, but also reported for Portugal and Spain (Salido et al., 2002;Velasco-Negueruela et al., 2003) and J. oxycedrus subsp.macrocarpa (S. et Sm.) Ball., confined to Mediterranean coastal sands, which differs in the broader leaves 2-3 mm wide and larger cones (Stassi et al., 1995).
Aromatical oils from different parts of J. oxycedrus have been used since antiquity for fragrance, flavoring, medicinal, antibacterial, insecticidal, and cosmetic purposes.In folk medicine, they have been recommended as a mouth analgesic and for stomach disorders.Berries of this plant are used as a spice, particularly in European cuisine, and essential oil is used for production of beverages such as gin, to which the oil gave distinguishing flavor.The composition of leaf essential oil of J. oxycedrus from Morocco (Derwich and Chabir, 2011), Tunisia (Medini et al., 2010), Greece (Adams et al., 1999), Corsica (Boti et al., 2006), and Allgeria (Dob et al., 2006) has been reported in varying details.The oil is characterized by large amount of α-pinene followed by smaller amounts of sabinene, limonene, β-pinene, caryophyllene oxide and myrcene (Derwich et al., 2010;Derwich and Chabir, 2011;Adams et al., 1999).Berry essential oil of J. oxycedrus was examined in Italy and similar composition to that of the leaf essential oil was found with predominant amounts of α-pinene followed by smaller amounts of β-pinene, δ-3-carene, sabinene, myrcene, α-phellandrene, limonene and germacrene D (Angioni et al., 2003).Additionally, authors found that ripeness of the berries does not have large influence on the oil composition.Boti et al. (2006) found a-pinene, b-phellandrene and d-3-carene as the most abundant constituents in the berry essential oil from Corsica.The berry essential oil of J. oxycedrus ssp.badia from Spain contained a-pinene and myrcene as the predominant components (Velasco-Negueruela et al., 2003), while the oil of J. oxycedrus ssp.macrocarpa contained α-pinene, germacrene D, myrcene, abietadiene and cis-calamenene as major constituents (Medini et al., 2012).
In Macedonian flora, only J. oxycedrus L. subsp.oxycedrus is present (Micevski, 1989).This species is spread throughout the whole territory of Republic of Macedonia, except on very high altitudes on mountains in the continental part.Up to now, J. oxycedrus was not chemically investigated and no testing of possible biological and pharmacological activities was performed.Therefore the aim of the present study was analysis of chemical composition of berry essential oil of this plant and evaluation of its antimicrobial activity.

Plant materials
The terminal twigs with berries were collected from two different localities in R. Macedonia: Velestovo village near Ohrid Lake and Vodno Mtn., near Skopje, in late autumn 2010 and 2012.Plant identity was verified as Juniperus oxycedrus L., and herbarium voucher specimen N o JO-1/10, N o JO-1/12, were deposited at the Department of Pharmaceutical Botany, Institute of Pharmacognosy, Faculty of Pharmacy, Skopje, R. Macedonia.
The plant material was dried at room temperature.Just before essential oil isolation, the berries were separated from leaves and branches and minced properly.

Essential oil isolation
Essential oil isolation from plant material was made by steam distillation in special 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 °C, transfer line temperature 280 °C and mass range from 50 -500 Da.The MS was operated in scan mode.
Chemical composition and antimicrobial activity of berry essential oil of Juniperus oxycedrus L. (Cupressaceae) grown...

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 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.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 microorganisms -S).

Broth dilution method
This method was used in order to determine minimal inhibitory concentration (MIC) of the particular essential oil (50% solution in DMSO) that had revealed antimicrobial activity by disc diffusion method.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 (Mueller-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).

Results and discussion
The obtained essential oils of J. oxycedrus were transparent, agile, light yellowish liquids with specific and very strong turpentine odor.Using GC/FID/MS a total of 100 components were identified, representing 96.01-98.95% of the oils.Among different classes of components, the most abundant fraction for Velestovo and Vodno oils were the monoterpene hydrocarbons (MH) (59.23% and 60.43%, respectively), followed by the sesquiterpene hydrocarbons (SH) (21.58% and 28.72%, respectively) (Table 1).Both samples of essential oils, contained smaller amounts of oxygen-containing monoterpenes (OM) (5.63% and 3.67%, respectively) as well as oxygen-containing sesquiterpenes (OS) (9.13% and 5.75%, respectively) (Table 1).
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.The most sensitive bacteria to antimicrobial effects of Juniperus oxycedrus berry essential oils was Haemophilus influnzae (MIC = 125 ml/ml) (Table 2).
According to literature data, essential oils of J. oxycerdrus, J. phoenicea and J. communis were tested against Candida albicans, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.The results obtained led to a non-significant inhibitory effect, although essential oil of J. phoenicea and leaf oil of J. oxycerdrus 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 found to be 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., 2012).J. oxycerdrus leaf essential oil provided to be an emergent alternative as antifungal agent against dermatophyte strains.d-3-carene was shown to be a fundamental compound for this activity (Cavaliero et al., 2006).

Conclusion
The major components in the essential oil of two samples of berries of Juniperus oxycedrus L. (Cupressaceae), growing wild in R. Macedonia were α-pinene, myrcene and limonene.β-pinene, caryophyllene, a-humulene and d-cadinene were determinate in smaller amounts while larger amount of germacrene D was found only in one sample of the oil.Antimicrobial screening of the essential oils showed that the most sensitive bacteria was Haemophilus influenzae (MIC = 125 ml/ml).The J. oxycedrus essential Chemical composition and antimicrobial activity of berry essential oil of Juniperus oxycedrus L. (Cupressaceae) grown... oil showed moderate antimicrobial activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Corynebacterium spp., Escherichia coli and Campilobacter jejuni (MIC > 500 ml/ml) and no activity against other investigated bacteria and Candida albicans.
Antimicrobial activity: microbial 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, Haemophylus influenzae, Proteus mirabilis, Salmonella enteritidis, 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 2 .
Antimicrobial activity of the berry essential oil of Juniperus oxycedrus.