COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF ARTEMISIA JUDAICA , A . HERBA-ALBA AND A . ARBORESCENS FROM LIBYA

The essential oils obtained by hydrodistillation from the aerial parts of Artemisia judaica L., Artemisia herbaalba Asso. and Artemisia arborescens L. (cultivated) from Libya, were analyzed by GC and GC-MS. The antimicrobial properties were determined using the broth microdilution method against eight bacterial species: Bacillus cereus (clinical isolate), Micrococcus flavus (ATCC10240), Listeria monocytogenes (NCTC7973), Staphylococcus aureus (ATCC6538), Escherichia coli (ATCC35210), Pseudomonas aeruginosa (ATCC27853), Salmonella typhimurium (ATCC13311), Enterobacter cloacae (human isolates) and eight fungal species: Aspergillus niger (ATCC6275), A. ochraceus (ATCC12066), A. versicolor (ATCC11730), A. fumigatus (ATCC1022), Penicillium ochrochloron (ATCC9112), P. funiculosum (ATCC10509), Trichoderma viride (IAM5061) and Candida albicans (human isolate). The major constituents of A. arborescens oil were sesquiterpene hydrocarbons (47.4%). Oxygenated monoterpenes were the dominant constituents in the A. judaica and A. herba-alba oils (54.2% and 77.3%, respectively). Camphor (24.7%) and chamazulene (20.9%) were the major components in the essential oil of A. arborescens, chrysanthenone (20.8%), cis-chrysanthenyl acetate (17.6%) and cis-thujone (13.6%) dominated in the A. herba-alba oil, and the major constituents in the A. judaica oil were piperitone (30.21%) and cis-chrysanthenol (9.1%). The best antimicrobial activity was obtained for A. judaica oil and the lowest effect was noticed in A. arborescens oil. The effect of the tested oils was higher against Gram (+) than Gram (-) bacteria. All three oils showed the best antibacterial activity against Listeria monocytogenes and the lowest against Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, compared to streptomycin and ampicillin. All three oils showed better antifungal activities than ketoconazole, except A. arborescens oil against Aspergillus niger.


INTRODUCTION
Artemisia L. is a large, diverse and economically important genus of the family Asteraceae (Hayat et al., 2009).It has more than 500 species (the number varies depending on the authors (Bremer and Humphries, 1993;Ling, 1982;1991a;1991b;1994;Oberprieler, 2001;Valles and Garnatge, 2005;Jafri and El-Gadi, 1983).The wind-polinated genus Artemisia has a cosmopolitan distribution, displaying highest diversity in temperate areas of the northern hemisphere and in several taxa in the southern hemisphere where it grows in arid and semiarid habitats (Hayat et al., 2009).In the flora of Libya, there are five species of this genus: Artemisia herba-alba Asso, A. judaica L., A. arborescens L. (cultivated), A. monosperma Delile and A. campestris L. (Jafri and El-Gadi, 1983).
Artemisia judaica is a perennial small fragrant shrub with pubescent leaves (Dob and Chelghoum, 2006).Reported medicinal effects of the plant include improved vision, cardiovascular health, capillary strength, connective tissue structure, appearance of skin and enhanced immune system functions, as well as decreased risk of atherosclerosis, cancer, arthritis and gastrointestinal disorders (Jafri and El-Gadi, 1983;Abdalla and Abu-Zagra, 1987;Khafagy et al., 1988;Khafagy and Tosson, 1968).Artemisia herba-alba, commonly known as sheeh, is a herb or shrub distributed in north Africa (Libya), and most of Europe (Jafri and El-Gadi, 1983).Artemisia herba-alba is used in the traditional medicine of the northern Badia region of Jordan, in the form of a decoction against fever and menstrual and nervous problems (Alzweiri et al., 2011).Artemisia arborescens is a morphologically variable species (or aggregate species) with grey-green to silver leaves.It is native to various habitats of the Mediterranean region, where it occurs as a shrub growing up to 1 m in height.According to popular folklore, it is used as an anti-inflammatory remedy (Ballero et al., 2001).A scientific study concerning the aspects of the therapeutic uses of the essential oil of A. judaica, A. arborescens and A. herba-alba from Libya, as well as their chemical composition, remains scarce and incomplete.Because of this, our investigation aimed to determine the chemical composition of the essential oil from the aerial parts of A. judaica, A. arborescens and A. herba-alba from Libya, and to assess their antimicrobial activity.

Essential oil extraction and analysis
Plant material of each species, dried at room temperature (150 g), was fragmented, and essential oils were isolated by 3-h hydrodistillation using a Clevenger-type apparatus, according to the procedure described in Ph.Eur. 6 (European Pharmacopoeia 6th Edition, 2007).GC and GC-MS analyses were performed on an Agilent 7890A GC system equipped with a 5975C MSD and flame ionization detector (FID), using an HP-5 MS column (30 m × 0.25 mm × 0.25 μm).Injection volume was 1 μL and injector temperature was 220°C with 200:1 split ratio.Carrier gas (He) flow rate was 1.0 mL/ min at 210°C (constant pressure mode).Column temperature was linearly programmed in a range of 60-300°C at a rate of 3°C/min, with a final 10-min hold.The transfer line was heated at 280°C.The FID detector temperature was 300°C.EI mass spectra (70 eV) were acquired in an m/z range of 40-550.Library search and mass spectral deconvolution and extraction were performed using NIST AMDIS (Automated Mass Spectral Deconvolution and Identification System) software version 2.64 using retention index (RI) calibration data analysis parameters with a "strong" level and 10% penalty for compounds without an RI.The retention indices were experimentally determined using the standard method involving retention times of n-alkanes, injected after the essential oil under the same chromatographic conditions.The search was performed against Adams, NIST05 and Wiley 7 mass libraries.Percentage (relative) of the identified compounds was computed from GC-FID peak area.The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined by the microdilution method (Espinel-Ingroff, 2001).Briefly, fresh overnight bacteria culture was adjusted by spectrophotometer to a concentration of 1×10 5 CFU/mL.Dilutions of inocula were cultured on a solid medium to verify the absence of contamination and check the validity of the inoculum.Different concentrations of essential oils with 0.1% of Tween 80 were carried into the wells containing 100 μL of Tryptic Soy Broth (TSB), and afterwards, 10 μL of inoculum were added to all the wells.The microplates were incubated for 24 h at 37°C.The MIC of the samples was detected following the addition of 40 μL of iodonitrotetrazolium chloride (INT) (0.2 mg/mL) and incubation at 37°C for 30 min.The lowest concentration that produced significant inhibition in the growth of bacteria compared to the positive control was identified as the MIC.The minimum inhibitory concentrations (MICs) obtained from susceptibility testing of various bacteria to tested extracts were determined also by a colorimetric microbial viability assay based on reduction of a INT color and compared with the positive control for each bacterial strain (Tsukatani et al., 2012; Clinical and Laboratory Standards Institute Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 2009).MBC was determined by serial subcultivation of 10 μL into microplates containing 100 μL of TSB.The lowest concentration showing no growth after the subculturing was read as the MBC.Standard drugs, namely streptomycin and ampicillin, were used as positive controls.

Gram
For the antifungal bioassays, eight fungi were used: Aspergillus niger (ATCC 6275), Aspergillus ochraceus (ATCC 12066), Aspergillus fumigatus (ATCC 1022), Aspergillus versicolor (ATCC 11730), Penicillium funiculosum (ATCC 36839), Penicillium ochrochloron (ATCC 9112), Trichoderma viride (IAM 5061), and Candida albicans (human isolate).The organisms were obtained from the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research "Siniša Stanković", Belgrade, Serbia.The micromycetes were maintained on malt agar and the cultures stored at 4°C and subcultured once a month (Booth, 1971).In order to investigate the antifungal activity of the compounds, a modified microdilution technique was used (Espinel-Ingroff, 2001).The fungal spores were washed from the surface of the agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v).The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0×10 5 in the final volume of 100 μL per well.The inocula were stored at 4°C for further use.Dilutions of the inocula were cultured on solid malt agar to verify the absence of contamination and to check the validity of the inoculum.Minimum inhibitory concentration (MIC) determinations were performed by a serial dilution technique using 96-well microtiter plates.The essential oils investigated were dissolved in 0.1% Tween 80 (v/v) and added to broth Malt medium with inoculum.The microplates were incubated in a rotary shaker (160 rpm) for 72 h at 28°C.The lowest concentrations without visible growth (monitored using a binocular microscope) were defined as the MICs.The minimum fungicidal concentrations (MFCs) were determined by serial subcultivation of 2 μL of tested compounds dissolved in medium and inoculated for 72 h in microtiter plates containing 100 μL of broth per well and further incubation 72 h at 28°C.The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum.Commercial fungicides, bifonazole (Srbolek, Belgrade, Serbia) and ke-toconazole (Zorkapharma, Šabac, Serbia), were used as positive controls (1-3500 μg/mL).All experiments were performed in duplicate and repeated three times.

Essential oils composition
The essential oil of the aerial parts of A. judaica, yield 0.62% (w/w), was lemon yellow in color and had a strong smell.GC and GC-MS analyses resulted in the identification of 75 compounds, making up 92.5% of the oil (22 components were found in traces).The essential oil of the aerial parts of A. herba-alba, yield 0.90% (w/w), was golden yellow in color and had a strong smell.GC and GC-MS analyses resulted in the identification of 74 compounds, making up 97.5% of the oil (23 components were found in traces).The essential oil of the aerial parts of A. arborescens, yeld 0.75% (w/w), was ink blue and with a somewhat milder smell than the previous two species.GC and GC-MS analyses resulted in the identification of 48 compounds, making up 95.6% of the oil (15 components were found in traces).All the components are listed in Table 1, in order of elution.The results showed that sesquiterpene hydrocarbons dominate the essential oil of A. arborescens (47.4%), while oxygenated monoterpenes were dominant in the essential oils of A. judaica and A. herba-alba (54.2% and 77.3%, MICs and MBCs (mg/mL), mean value of two measurements; Str, Streptomycin was used as stock solution 0.1 mg/mL; Amp, Ampicillin was used as stock solution 0.1 mg/mL respectively).The investigated essential oil of A. arborescens from Libya was characterized by an exceptionally high percentage of camphor (24.7%) and chamazulene (20.9%), followed by aromatic hydrocarbon isomers C 14 H 18 (14.9%− three compounds), linalool (6.0%), C 21 H 34 (5,8%), bornyl acetate (4.9%) and germacrene D (4.4%).The essential oil of the aerial parts of A. arborescens from Italy was characterized by camphor (35.7%), β-thujone (23.9%) and chamazulene (7.6%) as the main components (Lai et al., 2007), while the essential oil of the aerial part of plants from Algeria was characterized by a high percentage of chamazulene (30.2%) and β-thujone (27.8%) (Abderrahim et al., 2010).The essential oil of A. herba-alba from Libya was characterized by an exceptionally high percentage of chrysanthenone (20.8%), cischrysanthenyl acetate (17.6%) and cis-thujone (13.6%), followed by filifolone (4.8%), transthujone (4.0%), trans-pinocarveol (3.3%) and trans-sabinyl acetate (3.1%).The main components of the essential oil from the aerial parts of A. herba-alba from Jordan were trans-sabinyl acetate (5.4%), germacrene D (4.6%), α-eudesmol (4.2%) and caryophyllene acetate (5.7%) (Hudaib and Aburjai, 2006).The essential oil of A. herba-alba from Libya lacks germacrene D and α-eudesmol, while chrysanthenone (20.8%), cischrysanthenyl acetate (17.6%) and cis-thujone (13.6%) emerged as the dominant components.

Antimicrobial activities of essential oils
Essential oils of the aerial parts of A. herba-alba, A. arborescens and A. judaica were tested for antibacterial and antifungal activity.The antimicrobial potential was investigated against eight bacterial and eight fungal species.Antibacterial activity is presented in Table 2.The MIC of all the tested oils ranged between 0.05-2.5 mg/mL and the MBC 0.125-2.5 mg/mL.The effect of the tested oils was higher against Gram (+) than Gram (-) bacteria.The most resistant bacteria was Escherichia coli (MIC/MBC -1.25-2.5 mg/ mL), while L. monocytogenes (MIC/MBC -0.05-0.125 mg/mL) was the most susceptible.
The essential oil of A. arborescens displayed lower activity than the other two oils tested, while the oil of A. judaica exhibited the best activity among all of them.The commercial antibiotic streptomycin, used as a control, displayed antibacterial activity at 0.05-0.5 mg/mL (MIC) and 0.1-0.3mg/mL (MBC), while ampicillin exhibited MIC at 0.10-0.30mg/mL and MBC at 0.15-0.50mg/mL.The essential oils of A. herba-alba and A. judaica showed the same antibacterial activity as streptomycin, but higher than ampicillin, against S. aureus.The same oils exhibited stronger antibacterial activity than both antibiotics against B. cereus, while in the case of S. typhimurium these oils showed better activity only against ampicillin.The essential oil of A. judaica showed higher antibacterial activity than both antibiotics against M. flavus.All three oils showed better antibacterial potential than streptomycin and ampicillin against L. monocytogenes.The oils possessed lower antibacterial capacity than both antibiotics against P. aeruginosa, E. coli and A. cloacae.
The results of the antifungal activity of the tested oils are presented in Table 3.The MIC ranged between 0.03-0.25 mg/mL, while the MFC was in the range of 0.06-0.5 mg/mL.The commercial antifungal agent bifonazole showed MIC at 0.10-0.2mg/mL and MFC at 0.20-0.25 mg/mL.Ketoconazole displayed fungistatic activity at 0.20-2.50mg/mL and fungicidal effect at 0.30-3.50mg/mL.The essential oil of A. judaica exhibited higher antifungal potential than bifonazole against A. versicolor, A. ochraceus, A. niger and Penicillium species.The other two oils showed almost the same or slightly lower antifungal activity than bifonazole.All the oils tested showed better antifungal effect than ketoconazole, except in the case of A. arborescens oil against A. niger.
The obtained results showed that the essential oil of A. arborescens possessed lower antimicrobial activity than the other two oils.Considering that the essential oil A. judaica has the highest content of oxygenated compounds, it is expected to have the best antibacterial and antifungal effect.Oxygenated monoterpenes exhibit high antimicrobial activity on whole cells.In contrast, hydrocarbon derivatives have lower antimicrobial activity because of their lower solubility and diffusion through the medium (Knobloch et al., 1986).According to their structural type, hydrocarbons are relatively inactive in relation to their hydrogenbonding capacity and solubility in water (Griffin et al., 2000).Ketones, aldehydes and alcohols are active, but with differing specificity and levels of activity, which is related to the present functional MICs and MFCs (mg/mL), mean value of two measurements; Bif, Bifonazole was used as a stock solution 1 mg/mL; Ketoc, Ketoconazole was used as a stock solution 1 mg/mL group, but also associated with hydrogen-bonding parameters in all cases.Previous results have shown that greater antimicrobial potential could be ascribed to oxygenated terpenes, especially phenolic compounds (Soković et al., 2002;Couladis et al., 2004;Soković et al., 2005;Soković and van Griensven, 2006;Soković et al., 2010).

CONCLUSIONS
The presented results support future research into the antimicrobial properties and synergistic effects of the components (piperitone, camphor, chamazulene, chrysanthenone, cis-chrysanthenyl acetate, cis-thujone, etc.) of essential oils belonging to different groups of compounds (monoterpenes, sesquiterpenes) found in species of the genus Artemisia, for their potential application in medicine, agriculture and food industry.

Table 1 .
Composition of essential oils of the aerial parts ofA.arborescens, A. herba-alba and A. judaica.

Table 2 .
Antibacterial activity of essential oils of the aerial parts of Artemisia arborescens, A. herba-alba, A. judaica, Streptomycin and Ampicillin.

Table 3 .
Antifungal activity of essential oils of the aerial parts of Artemisia arborescens, A. herba-alba, A. judaica, Bifonazole and Ketoconazole.