Chemical composition , antimicrobial and antileishmanial activity of essential oil of Juniperus excelsa M . Bieb . from Ziarat , Balochistan

Essential oils isolated from leaves, barriers and from their mixture of Juniper (Juniperus excelsa M.Bieb) growing in Ziarat district. Essential oils were obtained by Clevenger-type apparatus, and components identified by gas chromatography mass spectroscopy (GC-MS). A total 27 components were identified which were inconsistent in their composition in the essential oils of J.excelsa, where five major variable components were: limonene (15.5-28.59%), followed by αpinene (5.01-14.04%), cedrol (4.68-14.5%), δ-elemen (6.9-13.01%) and δ-cadinene (5.212.06%). Essential oils were evaluated for their in vitro antimicrobial activity against five bacterial species by disk diffusion method. J. excelsa essential oils showed antibacterial activity against tested organisms whilst the highest inhibition zone was observed for E. coli. Antileishmanial activity of essential oils isolated from juniper leaves, barriers and their mixture were also determined by using different concentration from (0.01ng/ml to 100 μg/ml). The IC50 value of all three essential oils were 0.0065 μg/ml, 0.0088 μg/ml and 0.0093 μg/ml respectively and demonstrated almost 100 times more antileishmanial activity in comparison to the standard Amphotericin B.


Introduction
Juniperus forests along with certain plants and animals forms an ecosystem found in the Balochistan province southwest of Pakistan, which is unique in its qualities due to existence in dry and unfavorable weather conditions.Juniperus forests are not only scarce genetic asset but also a strong heritage of the province as considered in the most ancient species of trees due to which they are often named as "Living Forest Fossil" [1].The elevation where Juniperus forests are abundant ranges from 2000-3350 meters.Their geographic location extends between latitude 30 degrees 18 N -30 degrees 30 N and longitude ranging from 67 degrees 54 E-67 degrees 67 E [2].Koh I Khlifat 3475m is the highest peak falling under Ziarat district.Physical composition of the area includes asymmetrical hard ridges, upright terrain in addition to small valleys usually commencing from the east and ending towards west.Dry temperate climate is usually experienced in Ziarat district [3].Few herbariums have classified Juniperus into 54 different species that are scattered all over the world which includes South Asia, West Asia, Central Asia, North Africa, Europe and North America [4].Six species out of above mentioned 54 species including J. excelsa exists in Pakistan, a major portion of which lies in the northern areas of Pakistan and Balochistan province.Open canopy is one of the attributes of juniper forests.Their regeneration is usually poor and their growth rate is considered to be slowest as compared to others.They are spread out in an area of 141,000 hectares.The large area covering 100,000 hectares are spotted in Ziarat district and Zarghun area adjacent to the provincial capital Quetta [5].Herboi hills located in Kalat district also offer a fair sum of area of junipers.Juniper forests in addition to the mountains cover almost 35,325 hectares of area of the whole Ziarat district which constitutes 54% of the total area of the district [5].Personnel working with the field of forestry find these forests of great significance to their study which includes the age, sex, size and growth rates of different species.
Male and ovulate strobili are born at ends of short twigs or along the twigs, mostly on different trees (dioecious) or in a few species on the same tree (monoecious).Male flowers are visible in January while female flowers appear in the mid of March.Depending upon climatic conditions, the male flowers become mature up to April.In the meantime the female flowers also become ready to receive the pollen, which are very rapidly shed by the male flowers.Abundant pollen is produced and can be seen as yellowish powder in the air.Female flowers are greenish in colour and are found at the tips of new branches like the male flowers.The formation of small greenish globose berries takes place soon after pollination and can be observed in the month of May, very minute in size, growing progressively and become fully mature in November-December next year [1, 6, 7].The Juniperus species are characterized by large amount of essential oil in berries and needles as well as in wood and seed.In the last decade the composition of the berries essential oils of J. excelsa was investigated and some data were published recently.The berries essential oil of subsp.excelsa is characterized by presence of very high amounts of α-pinene, followed by cedrol, Lverbenol and D-verbenol and/or as predominant components: Leishmaniasis is amongst the major abandoned diseases.According to the World Health Organization data, which affects 350 million people worldwide and Pakistan is also among the endemic regions.Some studies have already conducted on antileishmanial activity of leaf, fruit and their organic solvents fraction of leaf of J. excelsa on L. major.Crude methanolic extract in addition to its portions chloroform fraction and di-ethyl ether fraction of J. excelsa berries were evaluated biologically and significant antileishmanial activity was depicted by chloroform fraction [20].The antileishmanial activity of leaf and fruit and their organic solvents extracts were studied and the extracts of leaf showed significant activity on in vitro and in vivo model [27].
Leaves extract and its solvent fraction showed maximum antileishmanial activity as compared to fruits and its fraction of Greek juniper [28].Essential oil of Juniperus species was also studied for its antileishmanial activity and the result was satisfactory [29].The aims of the study were to identify compounds present in essential oils from leaves, berries and their mixture of Juniperus excelsa M.Bieb., to assess their antimicrobial activity and to evaluate their antileishmanial activity on Leishmania major.

Collection of plant materials
Leaves and barriers from several trees of J. excelsa were collected from Ziarat, Balochistan and air dried at room temperature in the shade for few weeks.

Extraction of essential oil
Plants dried parts were crushed and each specimen (100g) leaves, Barriers and their mixture was subjected to hydro distillation for four hours in a Clevenger-type apparatus.The obtained oils were dried and stored separately at 4 ⁰C for further analysis.The extraction of essential oil was carried out in triplicate.

Gas chromatography-mass spectrometry (GC-MS) analyses
GC-MS analyses were carried out with a Varian 3400 GC-MS framework outfitted with a DB-5 combined silica section (30 m × 0.25 mm, film thickness 0.25 mm, J&W Scientific Corp.).The oven temperature was set at 50 to 260°C at a rate of 4°C /min.Exchange line temperature was 270°C, Helium was utilized as the transporter gas with a straight speed of 31.5 cm/sec., split proportion was 1:60, vitality of ionization was 70 eV, check time was 1 sec., and mass extent was from 40

Disc diffusion method
Paper discs of 6 mm in diameter were impregnated with 30 µl of essential oil dissolved in dimethyl sulphoxide (DMSO) at a final concentration of 5, 10, and 20% v/w and transferred onto the Mueller-Hinton agar (Oxide UK) plates, the surface was spread with 0.5 ml of bacterial suspension adjusted to 3 × 10 8 CFU/ml (1 Mac-Farland's standard).For negative control DMSO was used.Antibiotics Tetracycline (30 µg/disk) and Gentamicin (30 µg/disk) of commercial standard were used as positive controls.The diameter of inhibition zone was measured in millimeters (mm) after incubation at 37 ± 1°C for 24 hrs.Tests were carried out in triplicate.Sensitivity of the bacterial species to the essential oils was determined comparing the sizes of inhibitory zones [31].

Antileishmanial activity
The antileishmanial screening of the essential oil was performed in flat-bottomed plastic 96-well tissue plates.An amount 130 µl of 199 medium was added in wells of 96 well micro-titer plates (SPL Company).70 µl of J. excelsa essential oil was added in the first well and then serially diluted.70µl was discarded from the last well to keep the final volume 130µl.100µl parasite culture was added in each well and 2 rows were left for positive and negative control.DMSO was taken as negative control and successively diluted in the 199 medium.Glucantime was taken as positive control and was also serially diluted in 199 medium.Micro-titer plates were kept in incubator (DNP-9022) at 24°C for 72 hours.Assay was performed in triplicate.After 72 hours, 20µl was picked from each dilution and placed on Neubauer counting chamber and live parasites were counted under microscope.IC50 values of J. excelsa essential oil having anti-promastigotes activity were calculated by Graph Pad Prism® software.

Results and discussion
The yield of the obtained essential oils by hydro distillation of leaves, berries and mixture of J. excelsa was 1.15, 1.17 and 1.14% respectively.The individual color of the essential oil was light yellowish transparent liquids with characteristic turpentine like odor.The GC/MS analysis of the juniper essential oil is detailed in Table 1 which showed that there were 27 compounds present in three essential oils.The components might be assigned to three diverse classes: monoterpene, diterpene, and sesquiterpene hydrocarbons which were present in all samples, while an alcoholic compound was also present.In J. excelsa essential oils of leaves, berries and mixture ten common constituents were identified, all analyzed samples were rich in limonene (15.5 -28.59%), followed by α -Pinene (5.01-14.04%),cedrol (4.68-14.5%),δelemen (6.9-13.01%),δ -cadinene (5.2-12.06%),6-epi-α-cubebene (2.85-8.31%),terpine-4-ol (4.71-5.91%),bornyl acetate (2.79-3.7%),cembrene A (1.29-1.58%)and cembrene C (0.36-0.53%).Two common constituents identified in leaves and berries essential oils were: β-pinene (10.15%, 8.46%) and isoaromadendrene epoxide (1.07%, 1.09%) respectively.Three common constituents identified in essential oils of berries and mixture were: cis-β-terpinyl acetate (7.607%, 15.14%), γ -terpineol (4.52%, 5.64%) and β -eudesmol (1.83%, 1.51%) respectively.In essential oils of leave & mixture only one common component β-elemen (3.6%) was identified.The dissimilarities between leaves, berries and mixture essential oils exist.In essential oil of leaves six components: 2-carene (2.07%), perillene (3.26%), caren-4-ol (2.81%), cymene-8-ol (2.67%), spathulenol (1.4%) and 1-heptatriacotanol (0.43%) were identified, which were absent in essential oils of berries and mixture.In essential oil of berries 13-epi manoyl oxide (0.28 %) was identified which was not present in essential oils of leaves and mixture.Three components cis-verbenol (1.97%), ɤmuurolene (2.33%), α-cadinol (1.09%) were identified in essential oil of mixture and they were absent in essential oils of leaves and berries.The composition of the essential oils of J. excelsa described in this study, numerous similarities and/or differences could be found.The overall composition of the essential oils is comparable to previously reported study by Khoury et  which is almost similar and terpinen-4-ol (1.15%) was a bit low to that of present study.In Iranian samples it was found that the amount of α-pinene, limonene and cedrol were (22.5%, 28% and 22.7%) respectively [33], by comparing the obtained components and their amount presented in Table 1, dissimilarities with respect to components percentage and their abundance were observed.Major and minor components present in Omani J. excelsa berries are almost same as reported in this study while their percentages are not same [43].The components present in essential oil of mixture (leaves and berries) of J. excelsa the amount of the components limonene, cedrol and α-pinene followed a bit same pattern as that of essential oils of leaves and berries.Variation among the identified components in all three essential oils could be due to the interaction between different oil components that can be of four different kinds: antagonistic, synergistic, additive and/or indifferent.Antagonism means that the effects of one or both components are less when combined together than used separately.Synergism gives a better effect when two components are used together than used individually.Additive interaction means that the effect is same when the two components are put together than apart.While indifferent means that there is no interaction at all [44].As mentioned above when two components interact with each other, for example in the essential oil limonene was in leaves (15.5%), berries (25.35%) and in mixture (28.59%) respectively, according to the interaction between different essential oil components it is synergistic component because when leaves and barriers combined together for essential oil extraction the extracted mixture oil gives the better effect in term of percentage of a component as compare to essential oil of berries.The result of the antibacterial activity of the j.excelsa essential oil by the disc diffusion method is given in the Table 3 displays IC50 values of J. excelsa essential oil studied for antileishmanial activity.The essential oil of leaves, berries and mixture were checked for their activity and possible mode of action against Leishmania major parasites at different concentrations, i.e., from (0.01ng/ml to 100 μg/ml).The IC50 of J. excelasa leaves essential oil was in the range of 0.0065μg/ml, while the IC50 value of berries was 0.0088μg/ml and the IC50 value of mixture was in the range of 0.0093μg/ml.The J. excelasa mixture essential oil was10 times more active than the oil extracted from barriers and leaves (Table3).Further, all the three essential oils (leaves, berries and mixture) were found more active than the standard drug Amphotericin B (0.34 μg/ml). A