|Year : 2015 | Volume
| Issue : 1 | Page : 74-80
Chemical composition and antimicrobial activity of essential oils of Juniperus excelsa Bieb. (Cupressaceae) grown in R. Macedonia
Floresha Sela, Marija Karapandzova, Gjose Stefkov, Ivana Cvetkovikj, Svetlana Kulevanova
Institute of Pharmacognosy, Faculty of Pharmacy, Saints Cyril and Methodius University of Skopje, Republic of Macedonia, Republic of Macedonia
|Date of Submission||06-Apr-2014|
|Date of Acceptance||04-Jun-2014|
|Date of Web Publication||17-Dec-2014|
Str. Majka Tereza 47, 1000 Skopje,Republic of Macedonia
Republic of Macedonia
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: There are no information of the yield, chemical composition and antimicrobial activity of essential oils of berries (EOB) or leaves (EOL) of Juniperus excelsa Bieb. (Cupressaceae) growing wild in R. Macedonia. Materials and Methods: Plant material was collected from two localities during two seasons. Essential oil composition was analyzed by gas chromatography/flame ionization detector/mass spectrometry (GC/FID/MS) and antimicrobial screening was made by disc diffusion and broth dilution method. Results and Discussion: EOB yield ranged from 1.6-9.4 ml/kg and from 8.9-13.9 ml/kg for EOL. Two chemotypes of essential oil were differentiated, α-pinene-type (with 70.81% α-pinene in EOB and 33.83% in EOL), also containing limonene, β-pinene and β-myrcene while the sabinene-type (with 58.85-62.58% sabinene in EOB and 28.52-29.49% in EOL), was rich in α-pinene, β-myrcene, limonene, cis-thujone, terpinolene and α-thujene. The most sensitive bacteria to the antimicrobial activity of EOB was Haemophilus influenzae (MIC = 31 μl/ml). EOL have showed high activity towards: Staphylococcus aureus, Streptococcus pyogenes and Haemophilus influenzae (MIC = 125 μl/ml). The pinene-type of essential oil showed moderate activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Corynebacterium spp. and Campylobacter jejuni (MIC >50%). The sabinene-type of the oil showed moderate activity to Streptococcus pyogenes, Haemopilus influenzae, Campylobacter jejuni and Escherichia coli (MIC >50%). No activity was observed toward Candida albicans. Conclusion : The analysis of EOB and EOL revealed two chemotypes (α-pinene and sabinene type) clearly depended on the geographical origin of the Macedonian Juniperus excelsa which also affected the antimicrobial activity of these oils.
Keywords: Antimicrobial activity, essential oil, gas chromatography/flame ionization detector/mass spectrometry analysis, juniper berries and leaves, Juniperus excelsa
|How to cite this article:|
Sela F, Karapandzova M, Stefkov G, Cvetkovikj I, Kulevanova S. Chemical composition and antimicrobial activity of essential oils of Juniperus excelsa Bieb. (Cupressaceae) grown in R. Macedonia. Phcog Res 2015;7:74-80
|How to cite this URL:|
Sela F, Karapandzova M, Stefkov G, Cvetkovikj I, Kulevanova S. Chemical composition and antimicrobial activity of essential oils of Juniperus excelsa Bieb. (Cupressaceae) grown in R. Macedonia. Phcog Res [serial online] 2015 [cited 2019 Jul 21];7:74-80. Available from: http://www.phcogres.com/text.asp?2015/7/1/74/147212
| Introduction|| |
Juniperus is one of the major genera of Cupressaceae family consisting of approximately 70 species. Juniperus excelsa Bieb. is large shrub or tree, spread mainly throughout the eastern Mediterranean starting from north-eastern Greece and southern Bulgaria across Turkey to the Middle-East countries (Syria and Lebanon) and the Caucasus Mountains. It occurs in Iran, Pakistan and Oman as well.  The plant is known as Greek juniper, which appears as male and female plants, even some individuals could produce both sexes. Two main subspecies of J. excelsa are known, subsp. excelsa (Greek juniper) and subsp. polycarpos (Persian juniper). Only subsp. excelsa is present in Macedonian flora and is spread throughout the whole territory of Republic of Macedonia (RM), but mostly in southern part of the country. 
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 both J. excelsa subspecies, excelsa and polycarpos, 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, L-verbenol and D-verbenol as predominat components,  1,4-cineole and limonene,  myrcene, limonene, caryophyllene, δ-elemene and cedrol,  or cedrol, myrcene and limonene.  The berries essential oil of subsp. polycarpos also contained α-pinene as predominant component, ,, followed by lower amounts of 1,4-cineol, β-ocymene, δ-2-carene, 6-camphenol and sabinene  or δ-3-carene and limonene.  J. excelsa leaves essential oil was reach in cedrol (28.1%), α-pinene (22.5%) and limonene (22.7%),  or α-pinene (29.7%  and 32.34%)  and cedrol (25.3%  and 13.06%)  .
J. excelsa subsp. excelsa is a medicinal plant that has been used in folk medicine to treat dysmenorrhe, cough, bronchitis and colds, jaundice and tuberculosis and to induce menses and expel fetus.  It is known as a remedy for diarrhea, abdominal spasm, asthma, fever, gonorrhea, headache and leucorrhoea.  Among limited biological and pharmacological properties studied in vitro (cytotoxic,  antioxidant ,,, and antispasmodic activity)  the most investigated was the antimicrobial activity (antibacterial ,,, and antifungal). ,
J. excelsa medicinal properties are also known by people of RM and it is used as pain reliever and for curing cold, asthma, edema or skin diseases. Up to now no chemical investigations were done on this plant and no testing of possible biological and pharmacological activities were performed. Therefore the aim of the present study is analysis of chemical composition of leaves and berries essential oils and evaluation of their antimicrobial activity.
| Materials and Methods|| |
The terminal twigs with leaves and berries were collected from two different localities in R. Macedonia: Lake Dojran (altitude - 140 m) and Lake Ohrid (village Velestovo, National Park Galicica, altitude - 1060 m), in late autumn 2010 and 2011. Plant identity was verified as Juniperus excelsa M. Bieb. and herbarium voucher specimen N ° JE-1/10, N ° JE-3/10 and N ° JE-1/11 were deposited at the Department of Pharmaceutical Botany, Institute of Pharmacognosy, Faculty of Pharmacy, Skopje, RM.
The plant material was dried at room temperature, the berries and the leaves were separated and minced properly.
Dimethylsulfoxide (DMSO) was purchased from Sigma-Aldrich (Steinheim, Germany), sodium chloride and anhydrous sodium sulfate from Merck (Darmstadt, Germany) and from Kemica (Zagreb, Croatia), respectively, while xylene was purchased from Alkaloid (Skopje, RM).
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 gas chromatography/flame ionization detector/mass spectrometry (GC/FID/MS) analysis, the essential oil was dissolved in xylene to obtain 1 μl/ml oil solution.
Gas chromatography and gas chromatography/mass spectrometry
Essential oil samples were analyzed on Agilent 7890ΐ 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-5 ms capillary column (30 m × 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 270°C. One microliter 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
Identification of the components present in essential oils was made by comparison of their mass spectra 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.
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 More Details 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 More Details enteritidis, Acinetobacter spp., Corynebacterium spp., Shigella flexneri and Campylobacter jejuni) were clinical isolates provided from the Institute of Microbiology and Parasitology, Faculty of Medicine, Skopje, R. Macedonia.
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 well-spaced intervals. They were filled with 85 μl of 50% solutions of essential oils in (DMSO (Sigma-Aldrich, Germany) and one disc was filled only with DMSO as a control. The plates were incubated at 37oC, 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 μl of those essential oils were diluted in equal quantities of 0.9% sodium chloride solution, to make them with 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%, 6.2%, 3.1%, 1.5% and 0.7% or 125 μl/ml, 62 μl/ml, 31 μl/ml, 15 μl/ml and 7 μl/ml, respectively. 15 μl 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 MIC.
| Results and Discussion|| |
Both berries and leaves essential oils were transparent, agile, light yellowish liquids with specific and very strong turpentine odor. The berries essential oil (EOB) yield ranged from 1.6-9.4 ml/kg and from 8.9-13.9 ml/kg for leaves essential oil (EOL). [Table 1] shows the GC/FID/MS analysis of the investigated oils isolated from Juniperus excelsa berries and leaves samples collected from two different localities in R. Macedonia: Lake Dojran (from south-eastern part of RM) and Lake Ohrid (from south-western part of RM).
|Table 1: Chemical composition of berries and leaves essential oils from wild growing Juniperus excelsa in R. Macedonia|
Click here to view
Total of 74 components were identified in the analyzed samples of J. excelsa berries and leaves essential oils, representing 82.69-98.13% of the oil. Data analysis of the chemical composition revealed six different classes of components: Monoterpene hydrocarbons (MH), oxygen containing monoterpenes (OM), sesquiterpene hydrocarbons (SH), oxygen containing sesquiterpenes (OS), diterpenes (D) and other, non-terpene components (NT). Among different classes of terpenes present in the berries essential oil from Dojran, the MH fraction was the most abundant with total participation of 80.05%. On the other hand mass part of SH was only 3.54% [Table 1]. The samples of essential oil obtained from the plant material from Ohrid (Velestovo village), for two years of collection, 2010 and 2011, also contained MH as predominant fraction (from 79.92-80.33%) followed by larger amount of OM (from 7.85-10.51%). Fractions of sesquiterpenes, SH and OS, were much lower, ranging from 3.45-6.82% and from 0.12-1.31%, respectively [Table 1].
Component analysis of the J. excelsa EOB showed existence of two chemotypes of the oils (pinene- and sabinene-type). The sample from south-east RM (around Lake Dojran) was rich in α-pinene (70.81%), while the most abundant component in the samples from south-west RM (around Lake Ohrid) was sabinene (from 54.85-62.58%). Both "chemotypes" of berries oils had more than 80% of MH + OM fractions. Pinene-type of the essential oil was rich in: Limonene (4.00%), β-pinene (2.54%) and β-myrcene (1.58%), while sabinene-type contained following monoterpenes: α-pinene (4.51-7.09%), β-myrcene (5.06-5.65%), limonene (2.20-2.23%), cis-thujone (1.72-3.01%), terpinolene (1.52-1.75%) and α-thujene (1.03-1.17%). On the other hand, the sesquiterpene fraction was very low in both types of essential oils. Only germacrene D was presented in larger amounts in sabinene-type of the oil (1.21-3.12%).
Furthermore, in the essential oil from leaves can be distinguish the same two chemotypes (pinene and sabinene) as with EOB. The differences between berries and leaves essential oil exist in the sesquiterpenes fractions which is higher in the oils distilled from leaves. This can be seen in the samples originated from south-east RM where the SH and OS fractions are accounted for 16.11% and 25.28%, respectively. However, these fractions were lower in the EOL from south-west RM, ranging from 6.91%-15.03% (for SH) and from 0.67%-3.71% (for OS). Despite the large amount of α-pinene (33.83%), leaves essential oil from Dojran was the only investigated oil that contains large amount of cedrol (24.44%). Additionally, this oil contains the following components: limonene 6.14%, caryophyllene 3.3%, germacrene D 2.26% and g-cadinene 2.67%. There is difference in the composition of the leaves essential oil among samples from south-western part of RM and those from south-east part, which can be noticed in the EOB samples as well. The EOL was rich in: Sabinene (28.52-59.25%), cis-thujone (2.91-26.20%) and trans-thujone (0.47-12.86%), followed by large amounts of α-pinene (1.76-6.48%), β-myrcene (2.09-4.81%), g-terpinene (1.51-3.53%), terpinen-4-ol (2.58-5.87%) and germacrene D (1.21-4.16%). No cedrol was identified in this oil.
Further, comparing to literature data a lot of similarities and differences could be found. Many authors found α-pinene as the most abundant component of J. excelsa berries essential oil, ,,,, presented in amounts of 46.1%,  34%  or 47.64%.  Turkish authors found that despite α-pinene, cedrol (12.3%) and verbenol (L-verbenol 5.4% and D-verbenol 4.4%) were important constituents of the oil.  In the samples of oil that originated from Iran, except α-pinene, the dominant constituents were: β-myrcene (5.91%), limonene (4.5%), caryophyllene (3.6%), g-elemene (5.5%) and cedrol (12.01%).  Opposite to this, data for sabinene-type of J. excelsa berries essential oil is rarely reported. Serbian authors found very high amounts of sabinene (75.40%) in berries essential oil of J. excelsa from Serbia, followed by: α-pinene (5.2%), β-myrcene (4.96%), limonene (2.25%), terpinene (1.39%), terpinen-4-ol (1.15%) and germacrene D (2.15%).  Iranian authors reported completely different berries oil composition characterized by large amounts of cedrol (28%), α-pinene (22.5%) and limonene (22.7%). 
Considering J. excelsa leaves essential oil, Adams reported that the oil from wild growing J. excelsa from Greece contained cedrol (28.1%), α-pinene (22.5%) and limonene (22.7%) as predominant constituents.  Some former data point out on J. excelsa leaves essential oil as a natural source of cedrol, aroma component valuable for perfume production.  Turkish authors found that J. excelsa leaves essential oil consists mainly of α-pinene (29.7%) and cedrol (25.3%).  Furthermore, Iranian researchers confirmed that these two components are predominant in the leaves essential oil from wild growing J. excelsa from Iran (α-pinene-32.34% and cedrol-13.06%).  These data are similar with our findings for leaves essential oil composition of J. excelsa growing in south-east of RM. On the contrary, the results that we present for J. excelsa samples from south-western part of RM have sabinene and thujone as predominant compounds of the leaves oil [Table 1], a combination of constituents which is reported for the first time. This characteristic essential oil composition is most likely due to the specific geographic origin where the samples were collected. Some authors pointed out on seasonal variations of J. excelsa berries and leaves essential oils, putting stress on harvest season as very important factor that influence the oil composition.  Adams et al., revealed presence of moderate geographical variations in the volatile leaves oil of J. excelsa, comparing the samples from Greece, Bulgaria, Turkey and Cyprus.  Cedrol was found as the most abundant constituent of the oils, ranged continuously from 11.3-35.8%.
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 berries essential oil was Haemophilus influenzae (MIC = 31 μl/ml) [Table 2]. The pinene-type of essential oil showed moderate antimicrobial activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Corynebacterium spp. and Campylobacter jejuni with MIC >50%. On the other hand, the sabinene type of the oil showed moderate activity toward four bacteria: Streptococcus pyogenes, Haemophilus influenzae, Campylobacter jejuni and Escherichia coli (MIC >50%). Both type of berries essential oil, pinene and sabinene types, showed no activity against Candida albicans. J. excelsa leaves essential oil has activity against: Staphylococcus aureus, Streptococcus pyogenes and Haemophilus influenzae with MIC values of 125 μl/ml [Table 2]. Candida albicans was resistant to the antimicrobial activity of the tested oils.
|Table 2: Antimicrobial activity of berries and leaves essential oils of J. excelsa|
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Antimicrobial activity of berries and leaves essential oil of J. excelsa was previously investigated and literature data showed wide range of antimicrobial activity against various tested microbial strains. ,, J. excelsa essential oil have shown strong antimicrobial effects against anaerobic bacterium Clostridium perfringens and moderate activity toward Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumonia, Mycobacterium smegmatis, Candida albicans and Candida krusei.  α-Pinene has been considered as antimicrobial active component responsible for the activity of J. excelsa essential oils.  Besides moderate to no activity against Candida, berries essential oil of J. excelsa have shown strong antifungal activity against 10 strains of pathogenic fungi such as Aspergillus niger, A. flavus, Fusarium tricinctum, Penicillium ochrochloron, P. funiculosum, Phomopsis helianthi, Trichoderma viride, Trichophyton mentagrophytes, Microsporum canis and Epidermophyton floccosum. The MIC and MFC (minimal fungicidal concentration) values were very low, ranging between 6-35 μl/ml and 8-45 μl/ml, respectively.  In this regard, authors suggested that this essential oil could be used in production of food as natural preservative of fungal contamination.
| Conclusion|| |
Chemical composition of the essential oil of Macedonian J. excelsa was variable depending on the regions of collection, therefore two different chemotypes of berries and leaves essential oils were distinguished: Pinene-type, originated from south-eastern part of RM and sabinene-type, from south-western part of the country. The most abundant components in both chemotypes were monoterpenes. The pinene chemotype contained 70.81% (in EOB) and 33.83% (in EOL) α-pinene, followed by: limonene, β-pinene and myrcene, while the sabinene-type of oils despite sabinene (EOB = 58.85-62.58%; EOL = 28.52-29.49%), were rich in α-pinene, β-myrcene, limonene, cis-thujone, terpinolene and α-thujene. However the chemotype did not influenced the sesquiterepenes which were present in smaller amounts in the berries essential oils compared to the leaves essential oils.
The leaves essential oil from south-eastern part of RM was rich in cedrol (24.44%), a component absent from the EOLs from south-western part and has tujones as one of the predominant constituents.
The most sensitive bacteria to the antimicrobial activity of berries essential oil was Haemophilus influenzae (MIC = 31 μl/ml). The pinene-type of EOB showed moderate antimicrobial activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Corynebacterium spp. and Campylobacter jejuni (MIC > 50%). The sabinene type of the oil showed moderate activity to Streptococcus pyogenes, Haemophilus influenzae, Campylobacter jejuni and Escherichia coli (MIC > 50%). However, Staphylococcus aureus, Streptococcus pyogenes and Haemophilus influenzae (MIC = 125 μl/ml) were sensitive to J. excelsa leaves essential oil. Both chemotypes of berries and leaves essential oils showed no activity against Candida albicans.
| Acknowledgement|| |
The plant material was collected and analyzed within the project No. 13-3582/1 financed by the Ministry of Education and Science of Republic of Macedonia. The microbial strains as well as the tests for determination of the antimicrobial activity were supplied and performed by Elena Trjakovska-Dokikj and Ana Kaftandzieva from the Institute of Microbiology and Parasitology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, R. Macedonia.
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[Table 1], [Table 2]