Antimicrobial activity of needle essential oil of Pinus peuce Griseb . ( Pinaceae ) from Macedonian flora

Chemical composition and antimicrobial activity of needle essential oil, obtained by hydrodistillation from wild Pinus peuce Griseb. (Pinaceae), growing on three different locations in R. Macedonia were investigated in period 2008/2009. Carried out GC/FID/MS analysis, one hundred and three constituents were identified belonging to the six different classes of components: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, diterpenes and other non-terpene components, representing 88.61/94.04% of the entire oil. The most abundant constituents were α-pinene (12.89/27.34%), β-pinene (6.16/13.13%), limonene + β-phellandrene (2.09/6.64%) and bornyl acetate (2.92/11.67%) as well as trans-(E)-caryophyllene (4.63/7.13%) and germacrene D (8.75/20.14%). Antimicrobial screening of Pinus peuce needle essential oil was made by hole-plate diffusion and broth dilution method against 13 bacterial isolates of Gram positive and Gram negative bacteria and one strain of Candida albicans. The most sensitive bacteria against tested Pinus peuce essential oils were Streptococcus pneumonia encompassing Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Acinetobacter spp. and Streptococcus pyogenes. Minimal inhibitory concentrations (MICs) of the oils ranged from 7.5 62.5 μl/ml.

Pinus peuce, known as "Molica" or "Macedonian pine" is an endemic species of Balkan Peninsula mostly spread in southern and western parts of R. Macedonia (Micevski, 1985), northern part of Greece and south-eastern parts of Bulgaria, as well as in parts of Albania, Serbia and Montenegro.Up to present, there are a lack of data that are related to the essential oil composition of Pinus peuce population from Macedonian flora and its antimicrobial activity, thus the aim to this study was to investigate the chemical composition of the needle essential oil of Pinus peuce from Macedonian flora and to assess its antimicrobial activity against certain types of microorganisms that affect respiratory, gastrointestinal and urogenital system and provoke pathological conditions on skin.

Plant material
Plant material was collected from four different localities in R. Macedonia: Baba Mtn.(Pelister), Nidze Mtn., Shara Mtn. andKaradzica Mtn. in July, 2008 and2009, and was dried at room temperature and on draft for two weeks.Just before hydrodistillation, the needles were separated from the branches and were properly minced.

Determination of water content
Determination of water content was made by distillation according to Ph.Eur.7 regulations (2.2.13.) from 20 g dried plant material.

Essential oil isolation
Essential oil isolation was made by hydrodestillation in all-glass Clevenger apparatus (Ph.Eur 7, 2.8.12.).For that purpose, 20 g of minced needles were distilled for 4 hours.For purification purpose, anhydrous sodium sulfate was added to the isolated essential oil to remove residual water.For GC/FID/MS analysis, the essential oil was dissolved in xylene (Alkaloid, R. Macedonia) to obtain 1 µl/ ml oil solution.

Gas chromatography
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 µm) 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 detector 270 °C.1µl 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 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 and average values were taken into further consideration (n=3).Hole-plate diffusion method was used for screening the antimicrobial activity of all essential oils (determination of growth inhibition zones of studied microorganisms that occur around certain essential oil).This investigation was followed by broth dilution method (determination of minimal inhibitory concentration MIC of the particular oil that had revealed good antimicrobial activity by hole-plate diffusion method).

Hole-plate diffusion method
A nutrient agar (Mueller Hinton) was prepared by dissolving agar (28 g) in distilled water (1000 ml).The mixture was heated to dissolve and autoclaved at 121 °C for 15 minutes.The nutrient agar was poured into sterile Petri dishes at uniform depth of 3 mm and allowed to solidify.Blood agar (Oxoid) was used for testing the antimicrobial activity against Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae and Enterococcus, while Sabouraund agar (bioMerieux) was used for testing the antimicrobial activity against Candida albicans.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, holes of 6 mm in diameter were made at wellspaced intervals.They were filled with 85 µl of 50% solutions of essential oils in dimethylsulfoxide (DMSO, Sigma-Aldrich, Germany) and one hole was filled only with DMSO as a control.The plates were incubated at 37 °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.If the diameter of the growth inhibition zones was in the range between 0 and 14 mm, those species were accepted like resistant; if the diameter was between 14 and 19 mm, the species were accepted like moderate susceptible and in the cases where the diameter was above 19 mm, species were accepted like susceptible.

Broth dilution method
This method was used for some particular essential oil (50% solution in DMSO) that had revealed good antimicrobial activity by hole-plate diffusion method.For that purposes, 25 µl 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 µl of each bacterial or fungal suspension, thus the final concentrations were: 12.5%, 6.25%, 3.125%, 1.562% and 0.75% or 125 µl/ml, 62.5 µl/ml, 31.25 µl/ml, 15.26 µl/ml and 7.5 µl/ml, respectively.15µl 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 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
Essential oil yield, calculated on anhydrous needles of Pinus peuce is given in Table 1.The obtained essential oils were transparent, agile, light yellowish liquids with specific and very strong turpentine odor.Table 2 shows components that were identified in the Pinus peuce needle essential oils with their percentage amount and Kovat′s retention indices.Given percentage values are averages of percentages of the components that are present in the essential oils obtained from plant material collected from various altitudes on four different localities: Pelister, Nidze, Shara and Karadzica Mtn.
Total of one hundred and three components were identified in the investigated samples of Pinus peuce needle essential oils.Analysis of the essential oils composition shows seasonal variations in the content of components that are present, probably due to the influence of weather conditions throughout the year.The most abundant components in all samples of isolated essential oils were monoterpenes: α-pinene, β-pinene, limonene + β-phellandrene and bornyl acetate and sesquiterpenes: trans-(E)-caryophyllene and germacrene D. The content of each of these 6 components varies in relatively wide range but generally they are present in amounts from 2% to 28% and are likely to determine the physico-chemical characteristics as well as chemical profile of the oils.Large amount of monoterpenes camphene and α-terpenil acetate and sesquiterpenes δ-cadinene and α-cadinol was observed in some essential oils samples (   Data analysis of the chemical composition of Pinus peuce needle essential oils revealed six different classes of components: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, diterpenes and other non-terpene components.Among different classes of components that are present in the essential oil isolated from plant material from Baba (Pelister), Nidze, Shara and Karadzica Mtn, the major constituents were terpene hydrocarbons (62.44%, 76.59%, 69.86% and 54.46%, respectively) while oxygenated terpenes were present in smaller amount (Fig. 1.).The most abundant fraction among the total terpene amount were monoterpenes (47.2% in essential oil samples from Pelister, 62.16% in essential oil samples from Nidze Mtn. and 49.35% in samples from Shara Mtn.) (Fig. 2.).Dominant sesquiterpene fraction (45.23%) despite monoterpenes (23.96%) is present only in the needle essential oils isolated from cultivated Pinus peuces from Karadzica Mtn.This is probably due to the environmental conditions that affect terpene biosynthesis of Pinus peuce in this region.
The most sensitive bacteria against tested needle essential oils were Streptococcus pneumoniae.Streptococcus agalactiae showed similar sensitivity as previous bacteria and was susceptible to the effects of needle essential oils isolated from Pinus peuce from Shara and Nidze Mtn. as well as moderate susceptible to the effects of the needle essential oil from Pelister.Streptococcus pyogenes was susceptible to the effects of essential oils isolated from Minimal inhibitory concentration (MIC) values of Pinus peuce needle essential oils for tested microorganisms determined by broth dilution method are given in Table 3.The lowest MIC (less than 7.5 µl/ml) is determined in essential oil isolated from Pinus peuce from Shara Mtn. against Streptococcus pyogenes, while the highest MIC value (62.5 µl/ml) is determined in essential oil obtained from plant material collected on Baba Mtn.(Pelister) toward Staphylococcus aureus.Although the biological activity of essential oils is well known, the mode of their action is not completely understood.Generally, the activity of essential oils is the result of the combined effect both of their active and inactive components.Inactive components may affect resorption, speed of reaction and bioavailability of active components (Colgate, 1993;Svoboda, 1995), while the active components can have a synergistic effect.Essential oils are familiar with antibacterial, antifungal and antiviral activity.Many studies have explained antibacterial activity of essential oils against Gram-positive and Gram-negative bacteria (Reichling, 1999a;Weseler, 2005;Reichling, 1999b;Youesf, 1980;Dorman, 2000;Takarada, 2004) and have confirmed their antifungal activity (Hammer, 1998).There are differences in the biological activity of essential oils, especially when it comes to different strains of Gram-positive and Gram-negative bacteria.The different susceptibility of bacteria to the action of essential oils are not only due to a different chemical composition of the oils, but the differences in the composition and structure of the cell wall, lipid and protein composition of cytoplasmic membrane and specific physiological processes in different bacterial species.The way and mechanism of antibacterial activity of essential oils are not sufficiently explained yet.Nowadays, it is accepted that some essential oils and their components mainly act on bacterial cytoplasmic membrane (Ulrich, 2008).According to the literature data, there are only three references that are related to the antimicrobial screening of pine needle essential oils.Oluwadayo et al. (2008) reported that the major constituents of essential oils isolated from the needles of Pinus caribaea Morelet are β-phellandrene (67.9%), β-caryophyllene (10.2%) and α-pinene (5.4%).This essential oil exhibited moderate activity against Pseudomonas aeruginosa at minimum inhibitory concentration of 1000 µl/ml and no activity against other investigated microorganisms as Candida albicans, Bacillus subtilis, Staphylococcus typhi, Bacillus aureus and Proteus mirabilis.According to Sacchetti et al. (2005), the most abundant components of Pinus radiata D. Don are β-pinene (35.21%), α-pinene (21.9%), β-phellandrene (12.6%), iso-silvestrene (8.42%), α-terpineol (3.01%), terpinolene (2.21%), estragole (2.07%) and citronellol (1.87%).This essential oil showed a moderate inhibiting activity against tested yeasts as Candida albicans, Rhodotorula glutinis, Schizosaccharomyces pombe, Saccharomycea cerevisiae and Yarrowia lypolitica at minimum inhibitory concentrations of 0.14, 0.09, 0.02, 0.06 and 0.29 mg/ml, respectively.The antimicrobial effects of volatile components extracted from needles of Pinus densiflora S. and Z. was examined by Youg-Suk et al. (2005).α-Ocimene (29.3%), sabinene (10.9%), β-myrcene (9.6%), β-caryophyllene (8.0%), β-cadinene (7.3%), α-terpinolene (4.9%), 2-hexanal (4.5%) and β-pinene (4.3%) were considered as dominant components.Antimicrobial activity was screening against six different microorganisms: Bacillus cereus, Salmonella typhimurium, Vibrio parahaemolyticus, Listeria monocytogenes, Staphylococcus aureus and Escherichia coli.Among tested microorganisms, the growth of Bacillus cereus, Salmonella typhimurium and Staphylococcus aureus was completely inhibited.
Compared to our results, composition of Pinus peuce needle essential oil differs a lot from above tested pine oils.Antimicrobial screening was performed on different bacterial and fungi strains.All mentioned results indicate that the antimicrobial activity of pine needle essential oils depend on the type of microorganism as well as chemical composition of the oil.For example, antimicrobial activity of Pinus radiata essential oil against Candida albicans is probably due to the higher amount of α-pinene (21.9%) and β-pinene (35.21%) in comparison with Pinus peuce needle essential oil which contains lower amounts of these two components and hasn't showed antimicrobial effect against tested fungi.On the other hand, the mechanism of antimicrobial activity of needle essential oil from different pine species has not been clarified yet thus the action of essential oils on bacterial cytoplasmic membrane may be considered as a superior mechanism of antimicrobial activity of Pinus peuce needle essential oil.

Conclusion
The yield of needle essential oils obtained by hydrodistillation of Pinus peuce from Pelister, Nidze, Shara and Karadzica Mtn.ranged from 5.04, 9.24, 9.93 and 2.86 ml/ kg, respectively.The obtained essential oils were transparent, agile, light yellowish liquids with specific and very strong turpentine odor.
Using GC/FID/MS, one hundred and three components were identified in the Pinus peuce needle essential oils.The most abundant components in all samples of isolated oils were monoterpenes: α-pinene, β-pinene, limonene + β-phellandrene and bornyl acetate and sesquiterpenes: trans-(E)-caryophyllene and germacrene D.
The most sensitive bacteria against tested Pinus peuce needle essential oils were Streptococcus pneumoniae, while Streptococcus agalactiae, Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis and Acinetobacter spp.showed different sensitivity to the antimicrobial effects of the essential oils deepening on the area of collection of plant material.Essential oil obtained from plant material collected from Nidze Mtn.showed the highest antimicrobial activity against tested microorganism.
As the most promising activity of Pinus peuce needle essential oil was found against Streptococcus pneumoniae, this oil could be recommend for inhalation for infections of upper respiratory tract or for treatment of different skin condition provoked by Staphylococcus aureus.Further examination of safety of Pinus peuce needle essential oil should be done before their final recommendation.
n.a.-no activity found