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BIHAREAN BIOLOGIST 5 (1): pp.4-7 Biharean Biologist, Oradea, Romania, 2011 Article No.: 101102 http://biologie-oradea.xhost.ro/BihBiol/index.html  Antimicrobial activity of Rosemary, Fennel and Galbanum essential oils
against clinical isolates of Staphylococcus aureus
Mohaddese MAHBOUBI1,*, Nastaran KAZEMPOUR1 and Mona MAHBOUBI2 1. Department of Microbiology, Biology Center of JundiShapour, 87135-1178 Kashan, Iran 2. Department of Chemistry, School of Sciences, Payame Noor University, Ardakan, Iran. * Corresponding Author, M. Mahboubi, Tel: +98 866436 2112, Fax: +98 866436 2187, E- Mail: [email protected]
Received: 16. August 2010 / Accepted: 29. November 2010 / Available online: 12. December 2010 Abstract. The essential oils from rosemary (Rosmarinus officinalis L.), galbanum (Ferula gummosa), and fennel (Foeniculum vulgare)
were obtained from Barij Essence Pharmaceutical Company and were analyzed by GC and GC-MS. The essential oils were evalu-
ated for their anti-staphylococcal activities against Methicillin Sensitive (MSSA) and Resistant S. aureus (MRSA) by disc diffusion
and micro broth dilution assays. GC-MS analysis of oils showed that β-pinene, α-pinene and trans-anethole as the major compo-
nents of galbanum, rosemary and fennel oils, respectively. The Inhibition Zone diameters (IZ) of essential oils in disc diffusion assay
increased in a dose dependent manner and in different concentrations of oils, the IZs were compatible with vancomycin (30 µg). Al-
together, antimicrobial evaluations exhibited that galbanum oil had the best antimicrobial activity against MRSA and MSSA, fol-
lowed by fennel and rosemary oil, respectively.

Keywords: Rosemary; Fennel; Galbanum; Essential oil; Methicillin; Staphylococcus aureus.

Introduction
female climacteric syndrome and dysmenorrheal and in- Staphylococcus aureus, a gram positive, non motile, catalase Rosmarinus officinalis L. (Labiatae) commonly known as and coagulase positive, facultative anaerobe coccus is a rosemary, traditionally used as antispasmodic and for common type of bacteria that normally lives on the skin and treatment of dysmenorrhea, respiratory disorders, nervous nasal passages of healthy people. When it enters the body ailments and to stimulate growth of the hair (Zargari 1995). through a cut or other medical devices, it can cause local or The aim of this study was to evaluate the antistaphylo- serious infections (Franklin 1998). Methicillin Resistant S. coccal activities of rosemary, galbanum and fennel oils aureus (MRSA) has become one of the major causes of noso- against clinical isolates and identifying the chemical compo-comial and community pathogens causing significant mor- sition of the essential oils related to it. bidity and mortality because there are multi drug resistant pathogens that are resistant to all penicillins, so the option antibiotics for treatment of MRSA infections are limited to Materials and Methods
antibiotics such as vancomycin, tigecycline, lincozolid and Essential oils and identification of chemical compositions of the oils mupirocin (Simor et al. 2007). The patterns of antimicrobial The essential oils from aerial parts of rosemary, seeds of fennel and the susceptibility of S. aureus have been changed worldwide and resin of galbanum were prepared from Barij Essence Pharmaceutical it has been reported increasingly to be less effective. Devel- The oil analysis was carried out using GC and GC/MS. The GC ap- opment of mupirocin (dos Santos et al. 2007) and vancomy- paratus was Agilent technology (HP) 6890 system, capillary column of cin (Appelbaum 2006) microbial resistance in MRSA has in- HP-5MS (60 m × 0.25 mm, film thickness 0.25 µm). The oven temperature creased in settings with extensive use of these agents. Micro- program was initiated at 40 °C, held for 1 min then raised up to 230 °C at bial resistances to conventional antibiotics and adverse ef- a rate of 3 °C /min held for 10 min. Helium was used as the carrier gas at a flow rate 1.0 ml/min. The detector and injector temperatures were 250 fects of these agents have led to find new sources as antim- and 230 °C respectively. GC/MS analysis was conducted on a HP 6890 icrobial agents. Medicinal plants have a long history of use GC system coupled with 5973 network mass selective detector with a cap-as traditional medicines for treatment of different kinds of illary column the same as above, carrier gas helium with flow rate 1 ailments especially for infectious diseases. ml/min with a split ratio equal to 1/50, injector and oven temperature programmed was identical to GC. The compounds of the oil were identi- Galbanum, is one of the most important resins from fied by comparison of their retention indices (RI), mass spectra fragmen- roots and aerial parts of Ferula gummosa (Apiaceae) and is tation with those on the stored Wiley 7n.1 mass computer library, and one of the most important rangeland products of Iran with NIST (National Institute of Standards and Technology)(Adams 2001). high export demand (Nadjafia et al. 2006). Galbanum is used Staphylococcal isolates traditionally as food flavor for treatment of some gastroin- Twelve clinical isolates of S. aureus cultured from patients and S. aureus testinal disorders such as stomach pain, and as antileptic ATCC 25923 were used in all experiments. Methicillin resistant S. aureus remedy for epilepsy, cholera and as wound healing remedy directed detected on CHROMagarTM MRSA (CHROMagar Paris, France). (Zargari 1995). Galbanum oil can be applied to neck or Bacterial suspensions were made in Brain Heart Infusion (BHI) broth to concentration of approximately 108 CFU/ml using standard routine spec- drunk for improving the memory (Adams et al. 2007). trophometrical method. Subsequent dilutions were prepared from the Foeniculum vulgare (Apiaceae) is a well-known Umbelli- above suspensions, which were then used in the tests. ferous plant, commonly known as fennel. It is a perennial herb that grows all over the world and is used traditionally Disc diffusion method The disc diffusion method was employed for determination of antimicro- from ancient times as carminative, antiseptic, expectorant, bial activity of essential oil. Briefly, using a sterile cotton swab, above mi- digestive and diuretic agents. The seeds of fennel have been crobial suspensions was spread on the Mueller Hinton Agar (MHA) used to regulate menstruation, alleviate the symptoms of plates. Sterile paper discs (6 mm in diameter) were impregnated with 10, 15, 20 µl of each oil and were placed on the inoculated plates. After re- Antimicrobial activity of Rosemary, Fennel and Galbanum essential oils against clinical isolates of Staphylococcus aureus maining at 4 °C for 2 h, plates were incubated for 24 h at 37 °C. The followed by verbenone (8.6%), camphor (6.8%) and cam- diameters of the inhibition zones were measured in millimeters. All tests phene (6.3%) (Table 2). were performed in triplicate (NCCLS 2009). Among twenty six identified components of fennel oil, Determination of minimum inhibitory and bactericidal concentrations representing 99.9% of total composition, trans-anethole The minimum inhibitory concentration (MIC) and minimum bactericidal (56.6%), β-thujone (13.2%) were found as the major com- concentration (MBC) values of oil were determined by micro broth dilu- pounds. p-anisaldehyde (8.7%), fenchone (7.42%) were other tion assay. The oil was twofold serially diluted with 10% DMSO contain-ing 32-0.0125 µl/ml of oil. These dilutions were prepared in a 96-well mi- main components of fennel oil (Table 3). cro titter plate. Cation adjusted Muller Hinton broth was used as broth The antistaphylococcal activity of essential oils by disc media. After shaking, 100 µl of oil was added to each well. The above mi- diffusion method exhibited that this effect increased in a crobial suspensions were diluted (1-5 x106 CFU/ml) and then 100 µl was dose dependent manner. Inhibition zone diameters of galba- added to each well and incubated at 35 °C. MIC was defined as the lowest num, fennel and rosemary oils were in compatible with van- concentration of oil that inhibits bacteria after 24. MBC value was the first well that showed no growth on Manitol Salt Agar (NCCLS 2009). comycin (30 μg) at 10, 15, >20 μl of oils, respectively. Table2. Chemical composition of R. officinalis L. essential oil.
Analyses of galbanum oil revealed forty four components which accounted for 100% of the total composition of oil, β- pinene (43.1%) and α-pinene (5.4%) were the main compo- nent of essential oil followed by β-cubebene (4.9%), epi- bicyclosesquiphellandrene (4.4%), p-cymene and 4-terpineol Table1. Chemical composition of F. gummosa essential oil
Table3. Chemical composition of F. vulgare L. essential oil.
Thirty three components were identified from rosemary essential oil representing 89.8% of the total oil. The major The diameter of growth inhibition zones ranged from components were α-pinene (21.5%) and 1,8-cineole (15.2%) 11.6-34.2 mm at 10-20 μl of galbanum oil with the highest in- hibition zone value observed 25.6 mm at 10 μl of essential Discussion
oil.
Fennel oil had antistaphylococcal activity with IZ of 7.2- Three tested essential oils exhibited different degrees of an- 24.1 mm at 20 μl of essential oil, the highest IZ was related to timicrobial activities against clinical isolates of S. aureus. The 2 clinical isolates of S. aureus in the ranges of 21.9-24.1 mm. maximum anti-staphylococcal activity was shown by galba-10 μl of Galbanum oil against under study isolates showed num oil, followed by fennel and rosemary oils. Eftekhar et al that seven isolates had IZ upper than 15 mm. 15 μl of fennel (2004) reported the antibacterial activity of 25 μl of galba-oil on 13 isolates of S. aureus showed that 3 isolates had IZ num oil with β-pinene (50.1%) and α-pinene (18.3%) as main upper than 15 mm. Rosemary oil had IZs in the ranges of components by disc diffusion test against S. aureus ATCC 7.9-15.2 mm. At 20 μl of oil, the IZs of all isolates were lower 25923. Galbanum oil containing α-pinene (14.3%), β-pinene than that of 15 mm, disc diffusion assay exhibited that gal- (14.1%) and sabinene (40.1%) inhibited the growth of S. banum oil had the best antistaphylococcal activity followed aureus at MIC value of 3.125 μl/ml (Abedi et al. 2008). In this by fennel and rosemary oil, respectively (Table 4). study, the chemical composition of galbanum oil is like as In quantitatively test, the MIC, MBC values of galbanum, Eftekhar et al study (2004) and sabinene was not found in fennel, rosemary oils were in the ranges of 8-32, 4-32, 8-32 chemical composition of under study galbanum oil. μl/ml. nine, eight, nine out of 13 isolates of S. aureus had the The antimicrobial activity of galbanum oil can be ex- MIC values of 8, 16, 16 μl/ml for galbanum, fennel, rose- plained by its main components. β-pinene and α-pinene are mary oils, respectively. The almost MBC values for galba- bicycle monoterpene hydrocarbon and are precursors of num, fennel, rosemary oils were 16, 32, 32 μl/ml (Table 5). many flavors and fragrances. α-pinene finds in sage, signi- Table 4. Antistaphylococcal activity of essential oils by disc diffusion method.
(MR=Methicillin Resistance; MS=Methicillin Sensitive; *= millimeter) MR 12.9±0.1 14.5±0.7 18.4±0.8 10.9±0.1 16.3±0.4 24.1±0.1 11.7±0.4 13.4±0.2 14.1±0.1 17.65±0.21 MR 25.6±0.1 35.5±0.7 34.2±1.7 12.1±0.1 16.2±0.5 21.9±0.2 8.6±1.9 9.9±1.5 12.1±0.1 19.1±0.14 MR 16.4±0.6 16.8±0.2 17.5±0.6 10.0±0.6 10.8±0.3 12.3±0.4 7.9±0.1 8.1±0.1 10.1±0.1 18.5±0.14 MR 10±0.3 14.3±0.4 15.7±0.5 8.7±0.1 11.4±0.4 13.1±0.1 8.1±0.4 9.3±0.3 10.7±0.5 17.95±0.35 MR 15.4±0.1 16.4±0.1 20.4±0.0 9.45±0.1 10.2±0.4 12.0±0.0 9.4±0.9 10.3±1.3 11.6±0.5 18.8±0.14 MS 14.6±0.6 18.6±0.4 21.5±0.7 11.1±0.5 12.3±0.4 13.1±0.1 10.0±0.0 11.2±0.1 12.4±0.1 17.95±0.07 MS 20.2±0.3 23.8±0.3 26.7±0.5 14.4±1.3 14.4±0.6 16.5±0.6 7.8±0.3 8.8±0.3 9.7±0.42 19.1±0.77 MS 15.8±0.4 19.4±0.2 22.7±0.4 8.7±0.6 10.9±0.1 12.3±0.4 8.5±0.7 8.8±0.3 10.1±0.1 20.2±0.28 MS 11.6±0.5 13.9±0.0 16.2±0.3 13.3±0.1 14.0±0.3 16.1±0.1 8.0±0.1 9.3±0.6 9.5±0.71 19.6±0.28 MR 13.0±0.0 16.3±0.4 17.9±0.1 9.9±0.8 14.1±0.1 16.0±0.1 9.7±1.2 13.3±0.4 15.2±0.3 24.6±0.98 MR 12.4±0.7 16.4±0.7 17.8±0.4 8.5±0.4 10.9±0.7 12.5±0.3 11.6±0.4 13.2±0.3 14.1±0.1 21.8±0.28 MS 17.7±0.9 22.9±0.1 25.0±0.1 12.9±1.2 15.6±0.1 18.1±0.1 9.0±0.7 9.9±0.1 10.7±0.4 22.2±0.56 MR 20.0±0.0 24.2±0.3 26.7±0.5 9.9±0.8 13.4±0.0 14.5±0.1 11.0±0.2 10.9±0.9 12.7±0.6 21.9±0.99 Table 5. The antistaphylococcal activity of essential oils by micro broth dilution assay.
[MIC = Minimal Inhibitory Concentration; MBC=minimal bactericidal cincentration] Antimicrobial activity of Rosemary, Fennel and Galbanum essential oils against clinical isolates of Staphylococcus aureus rosemary oils that have been shown to have very different Dorman, H.J.D., Deans, S.G. (2000): Antimicrobial agents from plants: spasmogenic effects (Lis-Blachin et al. 1999) and is able to antibacterial activity of plant volatile oils. Journal of Applied Microbiology 88: 308-316. inhibit significantly the growth and cell viability of gram dos Santos, K.R.N., de Souza Fonseca, L., Filho, P.P.G. (1996): Emergence of positive bacteria (Leite et al. 2007). The MIC value of 13.6 high-level mupirocin resistance in methicillin-resistant Staphylococcus aureus μl/ml for α-pinene against S. aureus is reported (Pichette et isolated from Brazilian university hospitals. Infection Control and Hospital Epidemiology 17: 813-816. al. 2006). β-pinene is well known monoterpene with antim- Eftekhar, F., Yousefzadi, M., Borhani, K. (2004): Antibacterial activity of the icrobial activity (Dorman & Deans 2000). So, the antimicro- essential oil from Ferula gummosa seed. Fitoterapia 75:758–759. bial activity of galbanum oil could be due to the Pinene-type Franklin, D.L. (1998): Staphylococcus aureus infection. The New England Journal monoterpene hydrocarbons (α-Pinene & β -Pinene). Hammer, K.A., Carson, C.F., Riley, T.V. (1999): Antimicrobial activity of Trans-anethole is the most component of sweet fennel oil essential oils and other plant extracts. Journal of Applied Microbiology 86(6): and reaches 84-90%, in this study the content of fenchone is about 7.4% and the amount of trans-anethole is about 57%. Leite, A.M., Lima, E.D.O., de Souza, E.L., Melo Diniz, M.F.F., Trajano, V.N., de Medeiros, I.M. (2007): Inhibitory effect of β-pinene, α-pinene and eugenol on The antimicrobial activity of fennel oil investigated in differ- the growth of potential infectious endocarditis causing Gram-positive ent studies and the antimicrobial activity of fennel oil has bacteria. Brazilian Journal of Pharmaceutical Sciences 43 (1):121-126. been shown against S. aureus (Sagdic & Yasar 2005). Ham- Lis-Balchin, M., Ochoka, R.J., Deans, S.G., Asztemborska, M., Hart, S., (1999): Differences in bioactivity between the enantiomers of α-pinene. Journal of mer et al (1999) found that fennel oil is active against S. aureus at concentration above 1%. Our study showed this oil Nadjafia, F., Bannayana, M., Tabrizia, L., Rastgoo, M. (2006): Seed germination is effective against S. aureus at 4-16 μl/ml. Patra et al. (2002) and dormancy breaking techniques for Ferula gummosa and Teucrium polium. Journal of Arid Environments 64: 542-547. reported that anethole and its isomer are responsible for an- NCCLS (2009): Methods for dilution Antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard M7-A8, Eighth Edition, The efflux inhibitory activity of rosemary oil is demon- NCCLS (2009): Performance standard antimicrobial disc susceptibility testing, strated (Oluwatuyi et al. 2004). Major constituents of rose- mary oil were α-pinene and 1,8-cineole. Oluwatuyi, M., Kaatz, G.W., Gibbons, S.G. (2004): Antibacterial and resistance 1,8-cineole is a monocyclic terpene alcohol that does not modifying activity of Rosmarinus Officinalis. Phytochemistry 65: 3249-3254. induce autolysis of S. aureus but were found to cause leak- Patra, M., Shahi, S.K., Midgely, G., Dikshit, A. (2002): Utilization of essential oil as natural antifungal against nail infective fungi. Flavour and Fragrance age. It may permeabilize bacterial membranes and facilitate the entry of others (Carson et al. 2006). The percent of α- Pichette, A., Larouche, P.L., Lebrun, M., Legault, J. (2006): Composition and pinene in rosemary oil is higher than that of galbanum oil antibacterial activity of Abies balsamea essential oil. Phytotherapy Research 20 (5): 371-373. but the antimicrobial activity of rosemary is lower than that Simor, A.E., Phillips, E., McGeer, A., Konvalinka, A., Loeb, M., Devlin, H.R., of galbanum oil, so, other components in the oil such as 1,8- Kiss, A. (2007): Randomized controlled trial of chlorhexidine gluconate for cineole or β-pinene can affect on antibacterial activity of α- washing, intranasal mupirocin, and rifampin and doxycycline versus no treatment for the eradication of methicillin-resistant Staphylococcus aureus colonization. Clinical Infectious Diseases 44: 178-185. Among three studied oils, galbanum oil is a suitable case Sagdic, O., Yasar, A.N.K. (2005): Antibacterial effects of single or combined for treatment of infectious diseases that are caused by S. plant extracts. Annals of Microbiology 55: 67-71. Simor, A.E., Stuart, T.L., Louie, L., Watt, C., Ofner-Agostini, M., Gravel, D., aureus, some in vivo and clinical studies can be done for Mulvey, M., Loeb, M., McGeer, A., Bryce, E., Matlow, A. (2007): Mupirocin- demonstration of the antistaphylococcal activity of galba- Resistant, Methicillin-Resistant Staphylococcus aureus Strains in Canadian Hospitals. Antimicrobial Agents and Chemotherapy 51 (11): 3880-3886. Zargari, A. (1995): Medical Plants, 5th Edition, Tehran. University Press.
Acknowledgement. This study is supported by Barij Essence
Pharmaceutical Company.
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