Chemical composition and antibacterial activity of two Juniperus species essential oils

Nowadays, the overdose use of antibiotics and the increase of microbial resistances have made researchers to carry out more studies about the new drugs with new chemical composition. In order to produce these new drugs, different species of special plants can be useful by scientists and researchers. Base on this, the leaves and fruits of Juniperus excelsa and Juniperus horizontalis were collected from Iranian National Botanic Garden. Essential oils were isolated by hydro-distillation method. The essential oil components were analyzed by gas chromatography–mass spectrometry (GCMS). The yields of the leaves and fruits of J. excelsa and J. horizontalis essential oils were 0/79 to 4/15% and 1/083 to 2/7%, respectively. There were 15 and 27 components in the essential oil of J. excelsa and J. horizontalis, respectively. The major compound in the essential oils obtained from the leaves and fruits of J. excelsa were α-pinene (79/95 and 89/49%, respectively). The main compound in the essential oils obtained from the leaves and fruits of J. horizontalis were sabinene (30/21 and 38%, respectively). In addition, bornyl acetate (10/66%) and delta-cadinene (3/79%) were identified as major components in the essential oil of the leaves obtained from J. horizontalis. Juniper essential oils were evaluated for the antibacterial activity against thirteen bacterial species by disk diffusion and micro dilution method. Juniper essential oils showed more antibacterial activities against Gram-positive as compared to Gram-negative bacteria species. The antibacterial activity of essential oils may be related to presence of αpinene, limonene, and sabinene which are known to have antibacterial properties.


INTRODUCTION
In recent years, scientists are concerned with the increasing microbial resistance against antimicrobial agents due to the indiscriminative use of commercial antibiotics (Service, 1995;Mukherjee et al., 2002).Synthetic chemicals which are widely used against these micro-organisms unfortunately develop resistance to most of the antibiotics; in addition, some antibiotics *Corresponding author: E-mail: el.ehsani@gmail.com.occasionally cause allergic reaction and immunity suppression (Knobloch et al., 1989).In many cases, people are turning to synthetic drugs, but the vast majority of them are turning to natural products.Antibacterial and antifungal properties of essential oil as well as of oil constituents are documented (Knobloch et al., 1989;Pepeljnjak et al., 2003).Essential oils or their constituents are used as antimicrobial agents for food preservatives, in clinical microbiology or in pharmaceutical preparations.Screening for antimicrobial activity has been the subject of many investigations, and oils with very potent antibacterial and antifungal activity could be promising agents for the future extensive research and in vivo examination.The use of essential oils is less damaging to the human health, because they have generally low toxic and they do not have side effects (Isman, 2000;Misra and Pavlovstathis, 1997).
Among such oils are the essential oils from juniper.Juniperus is the second most diverse genus of the conifers.
The genus Juniperus belonging to Cupresseceae family contains more than 60 species.It is widespread in temperate regions of the northern hemisphere including Europe, Asia, and North America (Adams, 2008).Juniperus excelsa is native to Iranian flora that grows on mountainous regions (Assadi, 1998).But Juniperus horizontalis is non endemic, although, it has been planted in many parks and gardens.
The potential applications of Juniperus oils include aromatherapy, mood scents, scent masks, soaps and candles, cosmetics and fragrances, lotions and remedies (Yesenofski, 1996).In addition, Juniperus species are used for treatment of hyperglycemia, tuberculosis, bronchitis, pneumonia, ulcers, intestinal worms, to heal wounds, and cure liver diseases in traditional medicine (Burits et al., 2001;Loizzo et al., 2007).
The chemical composition of the leaf and wood essential oils from Juniperus excelsa were previously reported (Adams, 1990a(Adams, , 1990b)).There are a number of reports on the composition of the essential oil from berries of Juniperus species and their antimicrobial activities (Angioni et al., 2003;Cosentino et al., 2003;Filipowicz et al., 2003).Topcu et al. (1999) reported antimicrobial activity of hexane and methanol extracts of J. excelsa against Mycobacterium tuberculosis.The antimicrobial activity of J. excelsa essential oil against three standard bacterial strains and Saccharomyces cerevisiae were reported by Aridogan et al. (2002).
The J. excelsa essential oil showed a strong antimicrobial activity against the anaerobic bacterium Clostridium perfingenes, while exhibiting moderate activity against Staphylococcus aureus, Staphylococcus pyogenes, and Candida albicans (Unlu et al., 2008).However, as far as our literature survey could ascertain, there was no report on the antimicrobial activity of J. horizontalis.
The aim of this study was to identify the chemical composition of the oils of J. excelsa and J. horizontalis leaves and fruits and their antibacterial activity in an attempt to contribute to their use as alternative products for microbial control and food preservation.

Plant and oil isolation
The leaves and fruits of J. excelsa and J. horizontalis were collected from the Research Institute of Forests and Rangelands, located in Tehran, Iran.Collected plant materials were air-dried at room temperature.The dried parts of the plants were crushed to Ehsani et al. 6705 small particles.The samples (about 80 g) were hydro distilled for 2 to 2.5 h in a Clevenger-type apparatus to produce the oils.Three distillations were performed for each sample and mixed for analysis.The obtained oils were dried over anhydrous sodium sulfate and stored in sealed vials at 4°C before analysis.

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS analyses were carried out on a Varian 3400 GC-MS system equipped with a DB-5 fused silica column (30 m × 0.25 mm, film thickness 0.25 mm, J&W Scientific Corp).Oven temperature was 50 to 260°C at a rate of 4°C min -1 .Transfer line temperature was 270°C, Helium was used as the carrier gas with a linear velocity of 31.5 cm/s, split ratio was 1:60, ionization energy was 70 eV (electron Volts), scan time was 1 s, and mass range was 40 to 300 amu (Resolution).

Identification of compounds
The constituents were identified by comparison of their mass spectra with those in a computer library (LIBR-TR and Wiley-5 lib.) or with authentic compounds.The identification was confirmed by comparison of their retention indices either with those of authentic compounds or with data in the literature (Adams, 1995).

Microorganisms
Thirteen bacterial species were used to test the antibacterial activity of juniper essential oils.

Disc diffusion method
Disc diffusion method was used to determine of antibacterial activities of the essential oils in question (Lesueur et al., 2007).
Paper discs (6 mm diameter) were impregnated with 30 µl of the oil dissolved in dimethyl sulfoxide (DMSO) (final concentration of 5, 10, and 20% v/w) and transferred onto the Mueller-Hinton agar plates which had been surface spread with 0.5 ml of bacterial suspension adjusted to 3 × 10 8 cfu/ml (1 Mac-Farland's).The DMSO did not have antimicrobial activity and was used as negative control.Commercial standard antibiotics (tetracycline (30 µg/disk) and gentamicin (30 µg/disk)) were used as positive controls.After incubation at 37 ± 1°C for 24 h, the diameter of inhibition zones was measured in millimeters.Tests were carried out in triplicate.
Sensitivity of the bacterial species to the oils was determined comparing the sizes of inhibitory zones (Murray et al., 1995).

Determination of minimum inhibitory and bactericidal concentration (MIC and MBC) by micro dilution method
The minimal inhibitory values were determined by using a 48-well microtiter plates (Murray et al., 1995).The bacterial suspension was adjusted with sterile saline to a concentration of 3 × 10 8 cfu/ml.The essential oils were diluted to the highest concentration (256 µg/ml)
The results of antibacterial activity of the J. excelsa essential oil by the disk diffusion method are presented in Table 2.All tested Gram-positive bacteria species are sensitive to J. excelsa leaves and fruits of essential oil at 20% concentration with the inhibition zones ranging from 13/6 ± 0/58 to 25/3 ± 4/7 mm and 9 to 15/6 ± 3/8 mm, respectively.The inhibition zone for Gram-negative by J. excelsa leaves and fruits essential oil at 20% concentration ranged from 6 to 12 mm and 6 to 11 mm, respectively.According to Table 3, all Gram-positive bacteria species tested are sensitive to J. horizontalis leaves and fruits essential oil at 20% concentration with the inhibition zones ranging from 17 ± 1/73 to 25 ± 2/31 mm. and 11 ± 1 mm to 17 ±3/46, respectively.Gram- Tetracycline for gram positive bacteria and Gentamicin for gram negative bacteria.
Table 3. Antibacterial activity of leave and fruit from J. horizontalis against the bacterial strains based on disc-diffusion method (mm).negative bacteria species have inhibition zones from 10 ± 1 to 12/6 ± 0/58 mm and 10 to 11/3 ± 0/58 mm by J. horizontalis leaves and fruits essential oil at 20% concentration, smaller as compared to Gram-positive bacteria species.

Bacteria
The results of the antibacterial activity of the J. excelsa and J. horizontalis essential oil by the dilution method are given in Table 4. MIC for the bacteria species ranged from 32 to 256 µg/ml and 16 to 256 µg/ml for J. excelsa leaves and fruits essential oil, respectively.The lower MIC values were found for the tested Gram-positive bacteria as compared to the tested Gram-negative bacteria.The lowest MIC belonged to B. anthracis with 32 and 16 µg/ml values for leaves and fruits essential oil of J. excelsa, respectively.MIC ranged from 64 to greater than 256 µg/ml for J. excelsa leaves and fruits essential oil.Gram-positive bacteria showed the lower MBC.The lowest MBC belonged to B. anthracis with 64 µg/ml value.
As shown in Table 4, MIC ranged from 32 to 256 µg/ml and 128 to 256 µg/ml for J. horizontalis leaves and fruits essential oil, respectively.In Table 4, all Gram-positive bacteria had lower MIC except E .coli.MBC ranged from 32 to greater than 256 µg/ml for J. horizontalis leaves and fruits essential oil.Gram-positive bacteria showed the lowest MBC.

DISCUSSION
In this study, the essential oil compositions of J. excelsa and J. horizontalis leaves and fruits were collected from Iranian National Botanic Garden and were analyzed by GC-MS.
α-Pinene as major components in both leaf and fruit oil of J. excelsa was reported by Adams (2011).In contrast to our results, Narasimhachari and von Rudloff (1961) reported that alpha-cedrene, thujopsene, cuparene, cedrol, and widdrol are determined as the main components of J. horizontalis essential oil.These differences in essential oil composition may be related to the climatic and geographical condition of growth.
Our results indicated more susceptibility of the Grampositive bacteria which have been reported by others (Ouattara et al., 1997;Shelef et al., 1980).The weak antibacterial activity against Gram-negative bacteria was related to the presence of an outer membrane (Mann et al., 2000;Gaunt et al., 2005).Hydrophilic polysaccharide chains act as a barrier to hydrophobic essential oils and do not allow them to enter and have antibacterial activity.In addition, the resistance in the Gram-negative bacteria may be related to the possible resistance genes on plasmids (Shelef et al., 1980) that may inactivate essential oil components with antimicrobial potential.Leaves of J. excelsa and J. horizontalis showed more antibacterial activity as compared to the fruits.According to MBC and MIC, the leaves essential oil of J. horizontalis exhibited better antimicrobial activity against all tested bacteria except C. freundii.More activity of J. horizontalis essential oil may be related to more amounts of different components such as limonene and α-pinene.The antibacterial activity of limonene (Bevilacqua et al., 2010) and sabinene (Sandra et al., 2007) has been evaluated.Terpene hydrocarbons (including α-pinene) which are known to possess strong antibacterial activities (Aligianis et al., 2001;Couladis et al., 2000;Unlu et al., 2008).
All terpene hydrocarbons are antiseptic, antiinflammatory, and antibacterial.They are also pain killers, sedative, stimulators, and media for the excommunication of excrete mucus (Barjaktarevic et al., 2005).In addition, terpenes retard the retention of toxins in human organisms, they increase the abstraction of aggregated toxic material from the veins and liver and act as antispasmodic agents (media for mitigating convulsions) (Damnjanovic, 2000).Certainly, α-pinene is an acute antiseptic and it was found to act as a rubefaciens, while cadinene, caryophyllene, terpinene, and sabinene pronounced anti-inflammatory and antibacterial properties (Damnjanovic, 2000).Myrcene acts as a sedative, an anti-inflammatory agent and as a pain-killer for peripheral organs (Damnjanovic, 2000).Furthermore, myrcene stimulates the recovery of liver and it is known as a strong anti-inflammatory substance (Damnjanovic, 2000).Monoterpenes hydrocarbons, such as sabinene (Sokovic et al., 2004;Staniszewska et al., 2005;Skocibusic and Bezic, 2004;Delaquis et al., 2002;Jirovetz et al., 2003) also showed antimicrobial properties that appear to have strong to moderate antibacterial activity against Gram-positive bacteria and against pathogenic fungi, but in general, weaker activity was observed against Gram-negative bacteria (Tepe et al., 2005;Haznedaroglu et al., 2001).Some studies indicate that whole essential oils have a higher antibacterial activity than the combination of the major isolated components, indicating that minor components are critical to the activity, probably by producing a synergistic effect (Burt, 2004;Mastelic et al., 2005).For example, myrcene synergizes the antibiotic potency of other essential oil components (Onawunmi et al., 1984).Toxic effects on membrane structure and function have been generally used to explain the antimicrobial action of essential oils and their monoterpenoid components.In fact, as a result of their lipophilic character, monoterpenes will preferentially partition from an aqueous phase into membrane structures (Sikkema et al., 1994(Sikkema et al., , 1995)).This result in membrane expansion, increased membrane fluidity and permeability, disturbance of membraneembedded proteins, inhibition of respiration, and alteration of ion transport processes.Andrews et al. (1980) studied the toxic effects of α-pinene and some other terpenes produced by the Douglas fir on some Bacillus strains and on S. cerevisiae.It was shown that αpinene, limonene, camphene, and isobornyl acetate inhibited microorganisms.The presence of such terpenes in the diet of these insects was found to strongly influence the infectivity of Bacillus thuringiensis spores.αpinene destroyed the cellular integrity and inhibited respiratory activity in yeast mitochondria.Helander et al. (1998) described the effects of selected essential oil components on outer membrane permeability in Gramnegative bacteria, evidencing that monoterpene uptake is also determined by the permeability of the outer envelope of the target microorganism.However, specific mechanisms involved in the antimicrobial action of monoterpenes remain poorly characterized.
The antimicrobial efficacy of three monoterpenes (linalyl acetate, menthol and thymol) was examined (Domenico et al., 2005).The results were related to the Ehsani et al. 6709 relative lipophilicity and water solubility of the compounds examined.The antimicrobial effect of three monoterpenes may result from a perturbation of the lipid fraction of microorganism plasma membrane, resulting in alterations of membrane permeability and in leakage of intracellular materials.This effect also seems to be dependent on lipid composition and net surface charge of microbial membranes.Furthermore, the drugs might cross the cell membranes, penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity (Domenico et al., 2005).

Conclusion
The results of this work showed that the J. excelsa and J. horizontalis essential oils possess antibacterial properties (mainly J. horizontalis leave essential oil, because of its more components like limonene, myrcene, and sabinene).This showed their potential to be use as natural antibacterial agents to treat infectious diseases and to preserve food.Furthermore, the development of natural antibacterial agents will help to decrease negative effects (pollution in environment, resistance) of synthetic chemicals and drugs.

Table 1 .
Constituents (Area %) of the oil of J. excelsa and J. horizontalis.

Table 2 .
Antibacterial activity of leave and fruit from Juniperus excelsa against the bacterial strains based on disc-diffusion method (mm).

Table 4 .
The MIC and MBC values of leaves and fruits from J. excelsa and J. horizontalis against the microorganism tested in microdilution assay.