Juniper essential oil (Juniperi aetheroleum) was obtained from the juniper berry, and the GC/MS analysis showed that the main compounds in the oil were alpha-pinene (29.17%) and betha-pinene (17.84%), sabinene (13.55%), limonene (5.52%), and mircene (0.33%). The juniper essential oil was evaluated for the antimicrobial activity against sixteen bacterial species, seven yeast-like fungi, three yeast and four dermatophyte strains. Juniper essential oil showed similar bactericidal activities against Gram-positive and Gram-negative bacterial species; with MIC values between 8 and 70% (V/V) as well as a strong fungicidal activity against yeasts, yeast-like fungi and dermatophytes, with the MIC values below 10%, V/V. The strongest fungicidal activity was recorded against Candida spp. (MIC from 0.78 to 2 %, V/V) and dermatophytes (from 0.39 to 2%, V/V).Eterično ulje borovice (Juniperi aetheroleum) dobiveno je iz plodova (bobica Juniperus communis L., Cupressaceae) GC/MS analizom dokazane su glavne sastavnice alpha-pinen (29,17%) i beta-pinen (17,84%), sabinen (13,55%), limonen (5,52%) i mircen (0,33%). Ispitan je antimikrobni učinak eteričnog ulja borovice na 16 bakterijskih vrsta, 7 kvasaca i kvascima sličnim gljivicama, te 4 vrste dermatofita. Eterično ulje borovice pokazuje slični baktericidan učinak na Gram-pozitivne i Gram-negativne bakterijske vrste s MIK vrijednostima između 8 i 70% (V/V). Ulje pokazuje snažni protugljivični učinak s MIK vrijednostima ispod 10% (V/V). Najjača protugljivična aktivnost uočena je na vrste roda Candida, s MIK vrijednostima između 0,78 i 2% (V/V) i na dermatofite s MIK vrijednostima između 0,39 i 2% (V/V)

IVAN KOSALEC

NIKOLA BLAŽEVIĆ

STJEPAN PEPELJNJAK

ZDENKA KALOĐERA

Hrčak - Portal of scientific journals of Croatia

Antibacterial and antifungal properties of essential oil as well as of oil constituentsare well documented (1, 2). Essential oils or some of their constituents have found appli-cations as antimicrobial agents for food preservatives, in clinical microbiology or in phar-maceutical preparations. Screening for antimicrobial activity has been the subject of manyinvestigations and oils with very potent antibacterial and antifungal activity could bepromising agents for the future more extensive research and in vivo examination. Amongsuch oils are the essential oils from juniper berries (Juniperus communis L., Cupressaceae).The plant juniper is wide-spread in Croatia and it grows in temperate regions of Europe,Asia, and North America. Juniper berries (female cones) are used commercially for thepreparation of essential oil, gin, and as a spice (3).The essential oil of juniper berry has diuretic properties, gastrointestinal irritant andantiseptic properties. The diuretic action of juniper is primarily due to its essential oil,which contains terpinen-4-ol (4). The content of essential oil in the berry ranges from 0.5to 2.5% (V/m), and its main compounds are terpen hydrocarbons such as - and -pi-nene, myrcene, sabinene, thujone, limonene, etc. Oil also contains sesquiterpene hydro-carbons (caryophyllene, cadinene, elemene) and terpen alcohols (terpinen-4-ol) (4, 5).417Acta Pharm. 55 (2005) 417–422 Short communicationAntimicrobial activity of juniper berry essential oil(Juniperus communis L., Cupressaceae)STJEPAN PEPELJNJAK1*IVAN KOSALEC1ZDENKA KALO\ERA2NIKOLA BLA@EVI]31 Department of MicrobiologyFaculty of Pharmacy and BiochemistryUniversity of Zagreb, Zagreb, Croatia2 Department of PharmacognosyFaculty of Pharmacy and BiochemistryUniversity of Zagreb, Zagreb, Croatia3 Ireks-Aroma, Zagreb, CroatiaReceived February 25, 2005Accepted October 4, 2005Juniper essential oil (Juniperi aetheroleum) was obtainedfrom the juniper berry, and the GC/MS analysis showedthat the main compounds in the oil were -pinene (29.17%)and -pinene (17.84%), sabinene (13.55%), limonene (5.52%),and mircene (0.33%). Juniper essential oil was evaluatedfor the antimicrobial activity against sixteen bacterial spe-cies, seven yeast-like fungi, three yeast and four derma-tophyte strains. Juniper essential oil showed similar bac-tericidal activities against Gram-positive and Gram-negativebacterial species, with MIC values between 8 and 70%(V/V), as well as a strong fungicidal activity against yeasts,yeast-like fungi and dermatophytes, with MIC values bel-ow 10% (V/V). The strongest fungicidal activity was re-corded against Candida spp. (MIC from 0.78 to 2%, V/V)and dermatophytes (from 0.39 to 2%, V/V).Keywords: Juniperus communis, juniper, essential oil, anti-bacterial, antifungal activity* Correspondence, e-mail: spep33@yahoo.comThe use of juniper berry has been approved for dyspepsia and, with other plants forbladder and kidney conditions. The berry has digestive, stomachic, and antirheumaticproperties.Because of these antiseptic properties, we decided to investigate the antimicrobialactivity of the essential oil from juniper berry (Juniperi aetheroleum) against some Gram--positive and Gram-negative bacterial species as well as against fungi.EXPERIMENTALPlant materialEssential oil of dried juniper berry (Juniper communis L.) was obtained from Bioaro-matica d.o.o. (Croatia)Gas-chromatography/mass spectrometry (GC/MS) analysisThe GC/MS analysis were carried out with a Pye Unicam PU 4550 GC apparatus(Pye Unicam, UK), equipped with a FID coupled to a PU 4810 integrator, column: WCOTfused silica CP-Sil 8CB (length 25 m x 0.32 mm i. d., coating thickness 0.13 µm). Workingconditions: injector temperature 220 °C, detector temperature 250 °C, oven temperatureprogram 60 °C (1 min), 60–240 °C at 4 °C min–1, carried gas H2, 0.3 mL min–1, split ratio1:50. GC/MS analyses were performed on a Shimadzu QP 1000-GC-MS-EI 70 eV instru-ment (Shimadzu, Japan) connected to a data station. The components were identified bycomparing their retention times to those of authentic samples, as well as by comparingtheir mass spectra with those of Wiley and NBS Libraries described by Masada (6). Quan-titative data were obtained by the peak normalization technique using integrated FIDresponse.MicroorganismsSixteen bacterial species, seven yeast-like fungi, three yeast and four dermatophytespecies were used to test the antimicrobial activity of juniper essential oils.Microorganisms used were from the Collection of Microorganisms of the Depart-ment of Microbiology, Faculty of Pharmacy and Biochemistry, Zagreb. The microorgan-isms included bacterial species Bacillus cereus ATCC 11778, Bacillus subtilis NCTC 8236,Micrococcus flavus MFBF, Micrococcus luteus ATCC 8341, Staphylococcus aureus ATCC 6538,Staphylococcus aureus MFBF, Staphylococcus epidermidis MFBF, Enterococcus faecalis MFBF,Serratia spp. MFBF, Citrobacter freundiiMFBF, Salmonella enteritidisMFBF, Proteus mirabilisMFBF, Shigella sonnei MFBF, Klebsiella oxytoca MFBF, Escherichia coli MFBF, Yersinia entero-colitica MFBF; yeasts and yeast-like fungi: Candida albicans MFBF, C. krusei MFBF, C. tro-picalis MFBF, C. parapsilosis MFBF, C. glabrata MFBF, C. kefyr MFBF, C. lusitaniae MFBF,Cryptococcus neoformans MFBF, Geotrichum candidum MFBF, Hansenula anomala MFBF anddermatophytes Microsporum gypseum MFBF, M. canis MFBF, Trichophyton mentagrophytesMFBF, and T. rubrum MFBF.418S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.Antimicrobial activity – diffusion and dilution methodsAntimicrobial activity by the diffusion method was determined using the methodsdescribed in European Pharmacopoeia (7). Inoculum was prepared with fresh cultures ofbacterial strains, cultured on tryptic-soy agar (Merck, Germany) for 18 h at 37 ± 1C withphysiological saline, with 3 x 106 cells mL–1. Inoculum density was compared with Mac-Farland’s standard solution of BaSO4 (0.1 mL of 1% BaCl2 + 9.9 mL of 1% H2SO4) (8).Yeasts and dermatophytes were cultivated on Sabouraud 2% (m/V) dextrose agar (Bio-life, Italy) with the addition of 50 mg L–1 chloramphenicol (Sigma, Germany) for 5 daysfor yeasts and 10 days for dermatophytes at 25 ± 1 C. One mL of inoculum was mixedwith 22 ± 5 mL of Müller-Hinton agar (Merck, Germany) for bacterial strains, and thesame amount of Sabouraud agar for fungi. After cooling the inoculated agars at roomtemperature for 25 min, holes (d = 6 mm) were made with stainless steel cylinders. 40 Lof juniper essential oil was dropped into holes. In order to accelerate diffusion of the es-sential oil into agar, plates were incubated at 4 C for 1 h and were then incubated at 37± 1 °C for 18 h under aerobic conditions. The diameter of the inhibition zone aroundeach hole was measured and recorded.Minimum inhibitory concentration (MIC) and minimum microbicidal concentration(MMC) were determined by the broth twofold macro dilution method in Müller-Hintonbroth (Merck, Germany) for bacterial strains and in Sabouraud 2% (m/V) broth (Biolife,Italy) for fungal strains, as described by Pepeljnjak et al. (2) MIC was defined as the low-est concentration of juniper essential oil that allows no more than 20% growth of thebacteria, which is seen as the decreased number of colonies after removing the loop with10 L of each dilution on tryptic-soy agar and incubation at 37 ± 1 C for 18 h.MMC wasdefined as the lowest concentration of the juniper essential oil that allows no growth ofmicroorganisms.RESULTS AND DISCUSSIONThe preliminary GC/MS analysis of the juniper essential oil showed that there wereapproximately 35 compounds present in the essential oil with -pinene (29.17%), -pinene(17.84%), sabinene (13.55%), limonene (5.52%) and mircene (0.33%) as the most abund-ant ones.The results of antimicrobial activity of the juniper essential oil by the diffusion me-thod are presented in Table I. All Gram-positive bacterial species tested are sensitive tothe juniper essential oil with the inhibition zones ranging from 10 mm to 16 mm.The juniper essential oil possesses bactericidal activity against six of the eight strainstested, with Citrobacter freundii and Escherichia coli resistant to the essential oil. Suscepti-ble Gram-negative bacterial strains have inhibition zones from 8 mm to 17 mm, smallercompared to Gram-positive bacterial strains.All yeasts and yeast-like fungi were sensitive to the juniper essential oil, and the in-hibition zones varied from 8 to 17 mm. Among the dermatophyte species tested, onlyMicrosporum canis was resistant, while M. gypseum and Trychophyton spp. showed inhibi-tion zones between 10 and 14 mm.419S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.The results of the antimicrobial activity of the juniper essential oil by the dilutionmethod are given in Table II. Minimum inhibitory concentrations for the bacterial speciesranged from 18 to 70%, and the lowestMIC values were found for the fungal strains tested.All fungi showed MIC below 10%, and Candida spp. had the lowest MIC values (C. kefyr0.78%, C. albicans 1%, C. krusei 2% and C. tropicalis 3.12%, respectively). Yeasts of theCryptococcus neoformans and Hansenula anomala species had MIC values of 8 and 10%.Dermatophytes were very sensitive to the juniper berry essential oil, and MIC valuesranged from 0.39 (against T. rubrum strain) and 2% against theMicrosporum gypseum strain.420S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.Table I. Antimicrobial activity of the juniper essential oil by the diffusion methodInhibition zones (mm)Gram-positivebacterial speciesBacillus cereus ATCC 11778 16Bacillus subtilis NCTC 8236 10Micrococcus flavus MFBF 15Micrococcus luteus ATCC 9341 12Staphylococcus aureus ATCC 6538 11Staphylococcus aureus MFBF 14Staphylococcus epidermidis MFBF 13Enterococcus faecalis MFBF 11Gram-negativebacterial speciesSerratia spp. MFBF 8Citrobacter freundii MFBF 0Salmonella enteritidis MFBF 8Proteus mirabilis MFBF 13Shigella sonnei MFBF 17Klebsiella oxytoca MFBF 16Escherichia coli MFBF 0Yersinia enterocolitica MFBF AEøYeasts andyeast-like fungiCandida albicans MFBF 9Candida krusei MFBF 11Candida tropicalis MFBF 10Candida parapsilosis MFBF 17Candida glabrata MFBF 11Candida kefyr MFBF 10Candida lusitaniae MFBF 15Cryptococcus neoformans MFBF 8Geotrichum candidum MFBF 10Hansenula anomala MFBF 8Dermatophytes Microsporum gypseum MFBF 11Microsporum canis MFBF 0Trichophyton mentagrophytes MFBF 10Trichophyton rubrum MFBF 14ATCC – American Type Culture Collection, Rockville, USA.NCTC – National Collection of Type Cultures, London, Great Britain.MFBF – number of strains from the Collection of microorganisms of the Department of Microbiology,Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.AEø – microbicidal influence of aerosol.Our results on the antifungal activity of the juniper essential oil are similar to the re-sults of Hammer et al. (9), who found a MIC against C. albicans of 2% (V/V). The samegroup of authors found low MIC values against Staphylococcus aureus and Salmonella ty-phimurium (2%, V/V), in contrast to our high values for this strains. Differences in MICvalues are probably due to the different percentage of essential oil constituents.CONCLUSIONSIn this study, the antimicrobial activity of the essential oil of juniper berries was deter-mined. The oil showed activity against all eight Gram-positive bacterial strains, but twoGram-negative strains (Citrobacter freundii and Escherichia coli) were resistant among theeight Gram-negative bacterial strains tested. MIC for the bacterial strains were very high,ranging from 8 to 70% (V/V) of essential oil. Yeasts, yeast-like fungi and dermatophytes werefound to be very sensitive to the juniper essential oil, withMIC values below 10% (V/V).The lowest values of MIC of the essential oil against fungal strains indicate that themain compounds present in the oil-terpen hydrocarbons (pinenes, sabinene, mircene andlimonene) had a stronger antifungal than antibacterial activity.Acknowledgements. – We thank Boris Filipaj (Bioaromatica, Zagreb) for the donation of the juni-per essential oil.421S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.Table II. Antimicrobial activity of the juniper essential oil by the dilution methodMicroorganisms tested MIC (%, V/V) MMC (%, V/V)Bacterial species Bacillus cereus ATCC 11778 8 12.5Bacillus subtilis NCTC 8236 50 55Sarcina flava MFBF 15 25Staphylococcus aureus ATCC 6538 40 50Staphylococcus aureus MFBF 15 25Staphylococcus epidermidis MFBF 40 50Citrobacter freundii MFBF 60 65Salmonella enteritidis MFBF 70 75Shigella sonnei MFBF 55 60Klebsiella oxytoca MFBF 70 75Yersinia enterocolitica MFBF 40 50Fungal species Candida albicans MFBF 1 1.56Candida krusei MFBF 2 3.12Candida tropicalis MFBF 3.12 6.25Candida kefyr MFBF 0.78 1.56Cryptococcus neoformans MFBF 10 12.5Hansenula anomala MFBF 8 12.5Microsporum gypseum MFBF 2 3.12Trichophyton mentagrophytes MFBF 1 1.56Trichophyton rubrum MFBF 0.39 0.78For symbols see Table I.REFERENCES1. K. Knobloch, A. Pauli, B. Iberl, H. Weigand and N. Weis, Antibacterial and antifungal proper-ties of essential oil components, J. Essent. Oil Res. 1 (1989) 119–128.2. S. Pepeljnjak, I. Kosalec, Z. Kalo|era and D. Ku{trak, Natural Antimycotics from Croatian Plants,in Plant-derived Antimycotics – Current Trends and Future Prospects (Eds. M. Rai, D. Mares),Haworth Press, New York 2003, pp. 41–84.3. Herbal Medicine – Expanded Commission E Monographs (Eds. M. Blumenthal, A. Goldberg and J.Brinckmann), American Botanical Council, Austin 2000, 178–181.4. R. Hänsel, O. Sticher and E. Steinegger, Pharmakognosie – Phytopharmazie, 6th ed., Springer Ver-lag, Berlin 1999, pp. 860–861.5. A. Y. Leung and S. Foster, Encyclopedia of Common Natural Ingredients Used in Food, Drugs, andCosmetics, 2nd ed., John Wiley & Sons, New York 1996.6. Y. Masada, Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry, John Wiley &Sons, New York 1976.7. European Pharmacopoeia, 4th ed., Council of Europe, Strasbourg 2001, pp. 93–99.8. J. F. MacFaddin, Biochemical Tests for Identification of Medical Bacteria, 3rd ed., Lippincott Williams& Wilkins, Philadelphia 2000, pp. 825–826.9. K. A. Hammer, C. F. Carson and T. V. Riley, Antimicrobial activity of essential oils and otherplant extracts, J. Appl. Microbiol. 86 (1999) 985–990.S A @ E T A KAntimikrobni u~inak eteri~nog ulja borovice (Juniperus communis L., Cupressaceae)STJEPAN PEPELJNJAK, IVAN KOSALEC, ZDENKA KALO\ERA i NIKOLA BLA@EVI]Eteri~no ulje borovice (Juniperi aetheroleum) dobiveno je iz plodova (bobica Juniperuscommunis L., Cupressaceae) GC/MS analizom dokazane su glavne sastavnice -pinen (29,17%)i -pinen (17,84%), sabinen (13,55%), limonen (5,52%) i mircen (0,33%). Ispitan je anti-mikrobni u~inak eteri~nog ulja borovice na 16 bakterijskih vrsta, 7 kvasaca i kvascimasli~nim gljivicama, te 4 vrste dermatofita. Eteri~no ulje borovice pokazuje sli~ni bakte-ricidan u~inak na Gram-pozitivne i Gram-negativne bakterijske vrste sMIK vrijednostimaizme|u 8 i 70% (V/V). Ulje pokazuje sna`ni protugljivi~ni u~inak s MIK vrijednostimaispod 10% (V/V). Najja~a protugljivi~na aktivnost uo~ena je na vrste roda Candida, s MIKvrijednostima izme|u 0,78 i 2% (V/V) i na dermatofite s MIK vrijednostima izme|u 0,39 i2% (V/V).Klju~ne rije~i: Juniperus communis, borovica, kleka, eteri~no ulje, protubakterijsko, protugljivi~no djelovanjeFarmaceutsko-biokemijski fakultet Sveu~ili{ta u Zagrebu, ZagrebIrex-Aroma, Zagreb422S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.

Antimikrobni učinak eteričnog ulja borovice (Juniperus communis L., Cupressaceae)

HRČAK - Portal of Croatian Scientific and Professional Journals

Antibacterial and antifungal properties of essential oil as well as of oil constituentsare well documented (1, 2). Essential oils or some of their constituents have found appli-cations as antimicrobial agents for food preservatives, in clinical microbiology or in phar-maceutical preparations. Screening for antimicrobial activity has been the subject of manyinvestigations and oils with very potent antibacterial and antifungal activity could bepromising agents for the future more extensive research and in vivo examination. Amongsuch oils are the essential oils from juniper berries (Juniperus communis L., Cupressaceae).The plant juniper is wide-spread in Croatia and it grows in temperate regions of Europe,Asia, and North America. Juniper berries (female cones) are used commercially for thepreparation of essential oil, gin, and as a spice (3).The essential oil of juniper berry has diuretic properties, gastrointestinal irritant andantiseptic properties. The diuretic action of juniper is primarily due to its essential oil,which contains terpinen-4-ol (4). The content of essential oil in the berry ranges from 0.5to 2.5% (V/m), and its main compounds are terpen hydrocarbons such as - and -pi-nene, myrcene, sabinene, thujone, limonene, etc. Oil also contains sesquiterpene hydro-carbons (caryophyllene, cadinene, elemene) and terpen alcohols (terpinen-4-ol) (4, 5).417Acta Pharm. 55 (2005) 417–422 Short communicationAntimicrobial activity of juniper berry essential oil(Juniperus communis L., Cupressaceae)STJEPAN PEPELJNJAK1*IVAN KOSALEC1ZDENKA KALO\ERA2NIKOLA BLA@EVI]31 Department of MicrobiologyFaculty of Pharmacy and BiochemistryUniversity of Zagreb, Zagreb, Croatia2 Department of PharmacognosyFaculty of Pharmacy and BiochemistryUniversity of Zagreb, Zagreb, Croatia3 Ireks-Aroma, Zagreb, CroatiaReceived February 25, 2005Accepted October 4, 2005Juniper essential oil (Juniperi aetheroleum) was obtainedfrom the juniper berry, and the GC/MS analysis showedthat the main compounds in the oil were -pinene (29.17%)and -pinene (17.84%), sabinene (13.55%), limonene (5.52%),and mircene (0.33%). Juniper essential oil was evaluatedfor the antimicrobial activity against sixteen bacterial spe-cies, seven yeast-like fungi, three yeast and four derma-tophyte strains. Juniper essential oil showed similar bac-tericidal activities against Gram-positive and Gram-negativebacterial species, with MIC values between 8 and 70%(V/V), as well as a strong fungicidal activity against yeasts,yeast-like fungi and dermatophytes, with MIC values bel-ow 10% (V/V). The strongest fungicidal activity was re-corded against Candida spp. (MIC from 0.78 to 2%, V/V)and dermatophytes (from 0.39 to 2%, V/V).Keywords: Juniperus communis, juniper, essential oil, anti-bacterial, antifungal activity* Correspondence, e-mail: spep33@yahoo.comThe use of juniper berry has been approved for dyspepsia and, with other plants forbladder and kidney conditions. The berry has digestive, stomachic, and antirheumaticproperties.Because of these antiseptic properties, we decided to investigate the antimicrobialactivity of the essential oil from juniper berry (Juniperi aetheroleum) against some Gram--positive and Gram-negative bacterial species as well as against fungi.EXPERIMENTALPlant materialEssential oil of dried juniper berry (Juniper communis L.) was obtained from Bioaro-matica d.o.o. (Croatia)Gas-chromatography/mass spectrometry (GC/MS) analysisThe GC/MS analysis were carried out with a Pye Unicam PU 4550 GC apparatus(Pye Unicam, UK), equipped with a FID coupled to a PU 4810 integrator, column: WCOTfused silica CP-Sil 8CB (length 25 m x 0.32 mm i. d., coating thickness 0.13 µm). Workingconditions: injector temperature 220 °C, detector temperature 250 °C, oven temperatureprogram 60 °C (1 min), 60–240 °C at 4 °C min–1, carried gas H2, 0.3 mL min–1, split ratio1:50. GC/MS analyses were performed on a Shimadzu QP 1000-GC-MS-EI 70 eV instru-ment (Shimadzu, Japan) connected to a data station. The components were identified bycomparing their retention times to those of authentic samples, as well as by comparingtheir mass spectra with those of Wiley and NBS Libraries described by Masada (6). Quan-titative data were obtained by the peak normalization technique using integrated FIDresponse.MicroorganismsSixteen bacterial species, seven yeast-like fungi, three yeast and four dermatophytespecies were used to test the antimicrobial activity of juniper essential oils.Microorganisms used were from the Collection of Microorganisms of the Depart-ment of Microbiology, Faculty of Pharmacy and Biochemistry, Zagreb. The microorgan-isms included bacterial species Bacillus cereus ATCC 11778, Bacillus subtilis NCTC 8236,Micrococcus flavus MFBF, Micrococcus luteus ATCC 8341, Staphylococcus aureus ATCC 6538,Staphylococcus aureus MFBF, Staphylococcus epidermidis MFBF, Enterococcus faecalis MFBF,Serratia spp. MFBF, Citrobacter freundii MFBF, Salmonella enteritidis MFBF, Proteus mirabilisMFBF, Shigella sonnei MFBF, Klebsiella oxytoca MFBF, Escherichia coli MFBF, Yersinia entero-colitica MFBF; yeasts and yeast-like fungi: Candida albicans MFBF, C. krusei MFBF, C. tro-picalis MFBF, C. parapsilosis MFBF, C. glabrata MFBF, C. kefyr MFBF, C. lusitaniae MFBF,Cryptococcus neoformans MFBF, Geotrichum candidum MFBF, Hansenula anomala MFBF anddermatophytes Microsporum gypseum MFBF, M. canis MFBF, Trichophyton mentagrophytesMFBF, and T. rubrum MFBF.418S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.Antimicrobial activity – diffusion and dilution methodsAntimicrobial activity by the diffusion method was determined using the methodsdescribed in European Pharmacopoeia (7). Inoculum was prepared with fresh cultures ofbacterial strains, cultured on tryptic-soy agar (Merck, Germany) for 18 h at 37 ± 1C withphysiological saline, with 3 x 106 cells mL–1. Inoculum density was compared with Mac-Farland’s standard solution of BaSO4 (0.1 mL of 1% BaCl2 + 9.9 mL of 1% H2SO4) (8).Yeasts and dermatophytes were cultivated on Sabouraud 2% (m/V) dextrose agar (Bio-life, Italy) with the addition of 50 mg L–1 chloramphenicol (Sigma, Germany) for 5 daysfor yeasts and 10 days for dermatophytes at 25 ± 1 C. One mL of inoculum was mixedwith 22 ± 5 mL of Müller-Hinton agar (Merck, Germany) for bacterial strains, and thesame amount of Sabouraud agar for fungi. After cooling the inoculated agars at roomtemperature for 25 min, holes (d = 6 mm) were made with stainless steel cylinders. 40 Lof juniper essential oil was dropped into holes. In order to accelerate diffusion of the es-sential oil into agar, plates were incubated at 4 C for 1 h and were then incubated at 37± 1 °C for 18 h under aerobic conditions. The diameter of the inhibition zone aroundeach hole was measured and recorded.Minimum inhibitory concentration (MIC) and minimum microbicidal concentration(MMC) were determined by the broth twofold macro dilution method in Müller-Hintonbroth (Merck, Germany) for bacterial strains and in Sabouraud 2% (m/V) broth (Biolife,Italy) for fungal strains, as described by Pepeljnjak et al. (2) MIC was defined as the low-est concentration of juniper essential oil that allows no more than 20% growth of thebacteria, which is seen as the decreased number of colonies after removing the loop with10 L of each dilution on tryptic-soy agar and incubation at 37 ± 1 C for 18 h. MMC wasdefined as the lowest concentration of the juniper essential oil that allows no growth ofmicroorganisms.RESULTS AND DISCUSSIONThe preliminary GC/MS analysis of the juniper essential oil showed that there wereapproximately 35 compounds present in the essential oil with -pinene (29.17%), -pinene(17.84%), sabinene (13.55%), limonene (5.52%) and mircene (0.33%) as the most abund-ant ones.The results of antimicrobial activity of the juniper essential oil by the diffusion me-thod are presented in Table I. All Gram-positive bacterial species tested are sensitive tothe juniper essential oil with the inhibition zones ranging from 10 mm to 16 mm.The juniper essential oil possesses bactericidal activity against six of the eight strainstested, with Citrobacter freundii and Escherichia coli resistant to the essential oil. Suscepti-ble Gram-negative bacterial strains have inhibition zones from 8 mm to 17 mm, smallercompared to Gram-positive bacterial strains.All yeasts and yeast-like fungi were sensitive to the juniper essential oil, and the in-hibition zones varied from 8 to 17 mm. Among the dermatophyte species tested, onlyMicrosporum canis was resistant, while M. gypseum and Trychophyton spp. showed inhibi-tion zones between 10 and 14 mm.419S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.The results of the antimicrobial activity of the juniper essential oil by the dilutionmethod are given in Table II. Minimum inhibitory concentrations for the bacterial speciesranged from 18 to 70%, and the lowest MIC values were found for the fungal strains tested.All fungi showed MIC below 10%, and Candida spp. had the lowest MIC values (C. kefyr0.78%, C. albicans 1%, C. krusei 2% and C. tropicalis 3.12%, respectively). Yeasts of theCryptococcus neoformans and Hansenula anomala species had MIC values of 8 and 10%.Dermatophytes were very sensitive to the juniper berry essential oil, and MIC valuesranged from 0.39 (against T. rubrum strain) and 2% against the Microsporum gypseum strain.420S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.Table I. Antimicrobial activity of the juniper essential oil by the diffusion methodInhibition zones (mm)Gram-positivebacterial speciesBacillus cereus ATCC 11778 16Bacillus subtilis NCTC 8236 10Micrococcus flavus MFBF 15Micrococcus luteus ATCC 9341 12Staphylococcus aureus ATCC 6538 11Staphylococcus aureus MFBF 14Staphylococcus epidermidis MFBF 13Enterococcus faecalis MFBF 11Gram-negativebacterial speciesSerratia spp. MFBF 8Citrobacter freundii MFBF 0Salmonella enteritidis MFBF 8Proteus mirabilis MFBF 13Shigella sonnei MFBF 17Klebsiella oxytoca MFBF 16Escherichia coli MFBF 0Yersinia enterocolitica MFBF AEøYeasts andyeast-like fungiCandida albicans MFBF 9Candida krusei MFBF 11Candida tropicalis MFBF 10Candida parapsilosis MFBF 17Candida glabrata MFBF 11Candida kefyr MFBF 10Candida lusitaniae MFBF 15Cryptococcus neoformans MFBF 8Geotrichum candidum MFBF 10Hansenula anomala MFBF 8Dermatophytes Microsporum gypseum MFBF 11Microsporum canis MFBF 0Trichophyton mentagrophytes MFBF 10Trichophyton rubrum MFBF 14ATCC – American Type Culture Collection, Rockville, USA.NCTC – National Collection of Type Cultures, London, Great Britain.MFBF – number of strains from the Collection of microorganisms of the Department of Microbiology,Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.AEø – microbicidal influence of aerosol.Our results on the antifungal activity of the juniper essential oil are similar to the re-sults of Hammer et al. (9), who found a MIC against C. albicans of 2% (V/V). The samegroup of authors found low MIC values against Staphylococcus aureus and Salmonella ty-phimurium (2%, V/V), in contrast to our high values for this strains. Differences in MICvalues are probably due to the different percentage of essential oil constituents.CONCLUSIONSIn this study, the antimicrobial activity of the essential oil of juniper berries was deter-mined. The oil showed activity against all eight Gram-positive bacterial strains, but twoGram-negative strains (Citrobacter freundii and Escherichia coli) were resistant among theeight Gram-negative bacterial strains tested. MIC for the bacterial strains were very high,ranging from 8 to 70% (V/V) of essential oil. Yeasts, yeast-like fungi and dermatophytes werefound to be very sensitive to the juniper essential oil, with MIC values below 10% (V/V).The lowest values of MIC of the essential oil against fungal strains indicate that themain compounds present in the oil-terpen hydrocarbons (pinenes, sabinene, mircene andlimonene) had a stronger antifungal than antibacterial activity.Acknowledgements. – We thank Boris Filipaj (Bioaromatica, Zagreb) for the donation of the juni-per essential oil.421S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.Table II. Antimicrobial activity of the juniper essential oil by the dilution methodMicroorganisms tested MIC (%, V/V) MMC (%, V/V)Bacterial species Bacillus cereus ATCC 11778 8 12.5Bacillus subtilis NCTC 8236 50 55Sarcina flava MFBF 15 25Staphylococcus aureus ATCC 6538 40 50Staphylococcus aureus MFBF 15 25Staphylococcus epidermidis MFBF 40 50Citrobacter freundii MFBF 60 65Salmonella enteritidis MFBF 70 75Shigella sonnei MFBF 55 60Klebsiella oxytoca MFBF 70 75Yersinia enterocolitica MFBF 40 50Fungal species Candida albicans MFBF 1 1.56Candida krusei MFBF 2 3.12Candida tropicalis MFBF 3.12 6.25Candida kefyr MFBF 0.78 1.56Cryptococcus neoformans MFBF 10 12.5Hansenula anomala MFBF 8 12.5Microsporum gypseum MFBF 2 3.12Trichophyton mentagrophytes MFBF 1 1.56Trichophyton rubrum MFBF 0.39 0.78For symbols see Table I.REFERENCES1. K. Knobloch, A. Pauli, B. Iberl, H. Weigand and N. Weis, Antibacterial and antifungal proper-ties of essential oil components, J. Essent. Oil Res. 1 (1989) 119–128.2. S. Pepeljnjak, I. Kosalec, Z. Kalo|era and D. Ku{trak, Natural Antimycotics from Croatian Plants,in Plant-derived Antimycotics – Current Trends and Future Prospects (Eds. M. Rai, D. Mares),Haworth Press, New York 2003, pp. 41–84.3. Herbal Medicine – Expanded Commission E Monographs (Eds. M. Blumenthal, A. Goldberg and J.Brinckmann), American Botanical Council, Austin 2000, 178–181.4. R. Hänsel, O. Sticher and E. Steinegger, Pharmakognosie – Phytopharmazie, 6th ed., Springer Ver-lag, Berlin 1999, pp. 860–861.5. A. Y. Leung and S. Foster, Encyclopedia of Common Natural Ingredients Used in Food, Drugs, andCosmetics, 2nd ed., John Wiley & Sons, New York 1996.6. Y. Masada, Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry, John Wiley &Sons, New York 1976.7. European Pharmacopoeia, 4th ed., Council of Europe, Strasbourg 2001, pp. 93–99.8. J. F. MacFaddin, Biochemical Tests for Identification of Medical Bacteria, 3rd ed., Lippincott Williams& Wilkins, Philadelphia 2000, pp. 825–826.9. K. A. Hammer, C. F. Carson and T. V. Riley, Antimicrobial activity of essential oils and otherplant extracts, J. Appl. Microbiol. 86 (1999) 985–990.S A @ E T A KAntimikrobni u~inak eteri~nog ulja borovice (Juniperus communis L., Cupressaceae)STJEPAN PEPELJNJAK, IVAN KOSALEC, ZDENKA KALO\ERA i NIKOLA BLA@EVI]Eteri~no ulje borovice (Juniperi aetheroleum) dobiveno je iz plodova (bobica Juniperuscommunis L., Cupressaceae) GC/MS analizom dokazane su glavne sastavnice -pinen (29,17%)i -pinen (17,84%), sabinen (13,55%), limonen (5,52%) i mircen (0,33%). Ispitan je anti-mikrobni u~inak eteri~nog ulja borovice na 16 bakterijskih vrsta, 7 kvasaca i kvascimasli~nim gljivicama, te 4 vrste dermatofita. Eteri~no ulje borovice pokazuje sli~ni bakte-ricidan u~inak na Gram-pozitivne i Gram-negativne bakterijske vrste s MIK vrijednostimaizme|u 8 i 70% (V/V). Ulje pokazuje sna`ni protugljivi~ni u~inak s MIK vrijednostimaispod 10% (V/V). Najja~a protugljivi~na aktivnost uo~ena je na vrste roda Candida, s MIKvrijednostima izme|u 0,78 i 2% (V/V) i na dermatofite s MIK vrijednostima izme|u 0,39 i2% (V/V).Klju~ne rije~i: Juniperus communis, borovica, kleka, eteri~no ulje, protubakterijsko, protugljivi~no djelovanjeFarmaceutsko-biokemijski fakultet Sveu~ili{ta u Zagrebu, ZagrebIrex-Aroma, Zagreb422S. Pepeljnjak et al.: Antimicrobial activity of juniper berry essential oil (Juniperus communis L., Cupressaceae), Acta Pharm. 55 (2005) 417–422.

Antimikrobni učinak eteričnog ulja borovice (Juniperus communis L., Cupressaceae)

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