MATERIALS AND METHODS

The leaves of Mentha piperita, Mentha spicata, Anethum graveolens and Foeniculum vulgare have been collected during March-April 2010 in Iran (Ilam: Elevation 1339 m, Latitude East 33.638, Longitude North 46.431) then the plants were isolated from the other specimen and conserved for extraction. The essential oils were extracted by hydrodistillation using an apparatus of Clevenger. For this, mixing 250 g of plants was used in 1600 mL of distilled water the extraction took 3 h. After filtration the solvent was eliminated by reduced pressure distillation in rotary evaporator and pure oil was stored at 4°C in obscurity till the beginning of analysis. GC analysis was performed, using a Shimadzu GC-9A gas chromatograph equipped with a DB-5 fused silica column (30 mx0.25 mm i.d., film thickness 0.25 μm). The oven temperature was held at 50°C for 5 min and then programmed to 250°C at a rate of 3°C min^-1. Injector and detector (FID) temperatures were 290°C; helium was used as carrier gas with a linear velocity of 32 cm sec^-1. The percentages were calculated by electronic integration of FID peak areas without the use of response factors correction. Linear retention indices for all components were determined by co injection of the samples with a solution containing homologous series of C₈-C₂₂ n-alkanes. GC-MS analyses were carried out on a Varian 3400 GC-MS system equipped with a DB-5 fused silica column (30 mx0.25 mm i.d.); oven temperature was 40-240°C at a rate of 4°C. Transfer line temperature was 260°C. Carrier gas was helium with a linear velocity of 31.5 cm sec^-1, split ratio 1/60. In addition, ionization energy was 70 eV, scan time 1 sec and mass range 40-300 amu. Identification of components in the oil was based on retention indices relatives to n-alkanes and computer matching with the WILLEY 275. L library, as well as by comparison of the fragmentation patterns of mass spectra with those reported in the literature (Adams, 2001). The chromatographic conditions were identical to those used for GC analysis.

Tests for antibacterial activity: The microorganisms used in the present study were 6 g positive (Staphylococcus aureus, Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Micrococcus luteus and Streptococcus-β-haemolyticus) and 9 g negative (Salmonella typhi, Shigella dysenteriae, Shigella shiga, Shigella sonnei, Shigella boydii, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa and Proteus sp.) human pathogenic bacteria as well as eight pathogenic fungi (Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Vasin factum, Mucor sp., Candida albicans, Fusarium oxysporum and Colletotrichum falcatum). The antibacterial assays were carried out by the disc-diffusion (Verpoorte et al., 1983) and microdilution method (Daouk et al., 1995; Hanel and Raether, 1988; Espinel-Ingroff, 2001) in order to determine the antibacterial activity of oils and their components against the human pathogenic bacteria. The bacterial suspensions were adjusted with sterile saline to a concentration of 1.0x10⁵ CFU mL^-1. The inocula were prepared daily and stored at +4°C until use. Dilutions of the inocula were cultured on solid medium to verify the absence of contamination and to check the validity of the inoculum.

Disc-diffusion test: Compounds were investigated by the disc diffusion using 4 mm filter discs. Bacteria were cultured overnight at 28°C in LB medium and then adjusted with sterile saline to a concentration of 1.0x10⁵ CFU mL^-1. The suspension was added to the top of agar (6 mL) and dissolved in Petri dishes (2 mL agar^-1 plate) with solid peptone agar. Filter discs with essential oils and main components (1.0 μg mL^-1) were placed on agar plates (1 disc per agar plate). The fungi were grown in Potato Dextrose Agar (PDA) and/or Nutrient agar media. After 24 h of incubation at 28°C for bacteria or at 25°C for fungi the diameter of the growth inhibition zones was measured. Streptomycin was used as a positive control and 1 μL was applied to the discs from stock solution (1 mg mL^-1), whereas, antifungal drug is Nystatin at 50 μg disc^-1. All tests were done in duplicate; Three replications were done for each oil and for each component (Sokovic et al., 2008).

Microdilution test: The minimum inhibitory and bactericidal and fungicidal concentrations (MICs and MBCs) were determined using microtitre plates. The bacterial suspension was adjusted with sterile saline to a concentration of 1.0x10⁵ CFU mL^-1. Compounds to be investigated were dissolved in broth LB medium (100 μL) with bacterial inoculum (1.0x10⁴ CFU per well) to achieve the wanted concentrations (0.02-15.0 μg mL^-1). The microplates were incubated for 24 h at 28°C. The lowest concentrations without visible growth (at the binocular microscope) were defined as concentrations that completely inhibited bacterial growth (MICs). The MBCs were determined by serial sub-cultivation of 2 μL into microtitre plates containing 100 μL of broth per well and further incubation for 72 h. The lowest concentration with no visible growth was defined as the MBC, indicating 99.5% killing of the original inoculum. The optical density of each well was measured at a wavelength of 655 nm by microplate manager 4.0 (Bio-Rad Laboratories) and compared with a blank and the positive control. Streptomycin was used as a positive control using the same concentrations as in the disc diffusion test, whereas antifungal drug is Nystatin at 50 μg disc^-1. Three replications were done for each oil and each component (Sokovic et al., 2008).

RESULTS AND DISCUSSION

The results of the chemical analyses of essential oils are presented in Table 1. In Mentha spicata, 25 volatile constituents, representing 96.45% of the total composition, were identified in the leaves oils (Table 1). The most abundant components found in the leaf oil were β-myrcene (4.01%), followed by limonene (10.1%), 1,8-cineole (2.16%), menthon (24.14%), menthol (6.12%) and carvone (40.11%). The yield of Mentha spicata oil is 2.8% (v/w). The essential oils yield of collected Mentha piperita from region of Iran (Ilam) was 3.02% and main components are sabinene (3.01%), limonene (8.16%), 1,8-cineole (6.31%), menthon (14.12%), menthofuran (4.12%), menthol (33.02%) and menthyl acetate (20.21%). Other components were presents in amounts less than 2% (Table 1). The chemical compositions revealed that these leaves had compositions similar to those of other Mentha piperita essential oils analyzed in Morocco by (Derwich et al., 2010) which the major component was menthol (29.1%), Menthol (5.58%), Menthyl acetate (3.34%) and Menthofuran (3.01%). Menthanol (36.24%) and menthone (32.42%). Also, menthanol (36.24%) and menthone (32.42%) were the major compounds of the Mentha piperita essential oil studied in Iran (Behnam et al., 2006).

Table 1:	Chemical composition of essential oils investigated

The chemical compositions of Mentha piperita L. essential oil studied in Iran, contained α-terpinene (19.7%), isomenthone (10.3%), trans-carveol (14.5%), pipertitinone oxide (19.3%) and β-caryophyllene (7.6%) as the major compounds (Yadegarinia et al., 2006). The chemical compositions revealed that the leaves had compositions similar to those of other Mentha spicata essential oils analyzed by (Sokovic et al., 2008), the major components was limonene (5.77%), Menthol (21.92%) and carvone (49.52%). Menthol and carvone were the main components of Mentha piperita (Derwich et al., 2010). Abbaszadeh et al. (2009), studying of essential oil compounds variations in leaves of Mentha species, indicated that was significant difference between essential oil yields in leaves of mint species. In the essential oil of Anethum graveolens leaves, 22 compounds were identified. Monoterpenic hydrocarbons were found predominant in the leaves oil representing 31.62% of the total content and main components are Myristicin (3.31%), α-Phellandrene (18.36%), Dill ether (5.02%) and β-Phellandrene (3.38%) which α-phellandrene (18.36%) constituted the major compound. Present result showed that in the leaves oil, the ketonic compound carvone was not present (Table 1). Principle components of fennel leaves oil were t-anethole (17.19%), estragole (3.18%), fenchone (4.3%) and terpin-4-ol (4.8%) (Table 1). The obtained results about antibacterial and antifungal activity of the leaves essential oils of Mentha piperita and Mentha spicata, Anethum graveolens and Foeniculum vulgare from Iran are shown on Table 2 and 3. All the tested oils in the disc-diffusion method showed bacteriostatic activity in concentration of 1 μg disc^-1, except of fennel (Anethum graveolens) and dill (Foeniculum vulgare) leaves oil. The tested essential oil of Mentha piperita and Mentha spicata by disc-diffusion method, showed very strong antibacterial and antifungal activity. A concentration of 1 μg disc^-1 of oil inhibited Staphylococcus aureus, all of Bacillus species, Salmonella typhi, all of Shigella species, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Proteus sp., all of Aspergillus species, Vasin factum, Mucor sp., Candida albicans, Fusarium oxysporum and Colletotrichum falcatum. The essential oils which showed the best antibacterial and antifungal activity in disc-diffusion method were Mentha piperita (15.0-30.0 mm) and Mentha spicata (20.0-35.0 mm). Streptomycin at 1 μg disc^-1 showed inhibition zones in the range of 9.0-22.0 mm (Table 2). Nystatin at 50 μg disc^-1 showed inhibition zones in the range of 15-25.0 mm (Table 2). Good inhibition zones were also obtained for Mentha spicata and Mentha piperita oils. It can be seen that essential oils from Mentha spicata and Mentha piperita possess a higher antibacterial and antifungal effects than streptomycin and Nystatin. Table 2 shows that essential oil of Mentha piperita possesses greater antibacterial and fungistatic activity than Mentha piperita oil.

Table 2:	Antibacterial activity of essential oils (1.0 μg mL-1) in disc-diffusion method, inhibition zones (mm)

Anethum graveolens and Foeniculum vulgare oil showed the lowest MIC (8.0-10.0 μg mL^-1) and MBC (7.0-12.0 μg mL^-1) in the microdilution method. The essential oils from Mentha spicata and Mentha piperita inhibited all the bacteria and fungi in very small concentrations. Oils from Mentha spicata (MIC (0.5-1.5 μg mL^-1) and MBC (1-1.0 μg mL^-1) and Mentha piperita (MIC (1.0-1.0 μg mL^-1) and MBC (1.5-1.0 μg mL^-1) exhibited much higher antibacterial and antifungal activity. Streptomycin showed MIC at 4.0-6.0 μg mL^-1 and MBC at 5-3.0 μg mL^-1 while, Nystatin showed MIC at 3.0-3.0 μg mL^-1 and MBC at 4.0-2.0 μg mL^-1. From the obtained results it can be noticed that oils from Anethum graveolens and Foeniculum vulgare possessed lower antibacterial and antifungal activity than streptomycin and Nystatin while oils Mentha spicata and Mentha piperita showed almost the same antibacterial potential as the antibiotic (Table 3). The results of antibacterial activity of essential oil components are presented in Table 4 and 5. limonene inhibited bacterial growth of all bacteria and fungi and inhibition zones were 10.0-22.0 mm, 1,8-cineole reacted slightly better (inhibition zones 10.0-25.0 mm) while streptomycin showed inhibition with zones of 9.0-25.0 mm. Strong antibacterial and antifungal activity was noticed for menthol (20.0-35.0 mm), menthon (20.0-30.0 mm) and especially for carvone (20.0-38.0 mm). Streptomycin was inactive against all Aspergillus species, Vasin factum, Mucor sp., Candida albicans, Fusarium oxysporum and Colletotrichum falcatum. It can be seen that menthol, carvone and menthon showed higher antibacterial and antifungal activity than streptomycin and Nystatin. The monoterpenic hydrocarbons 1,8-cineole and linalool showed similar activity with MIC of 5.0-7.0 μg mL^-1 and MBC of 6.0-10.0 μg mL^-1.

Table 3:	Antibacterial activity of essential oils (MIC and MBC μg mL-1), microdilution method

Menthon and menthol showed very strong activity with MIC at 1.5-3.5 μg mL^-1 and 0.5-2.0 μg mL^-1, respectively while bactericidal effect were achieved at 1.5-1.0 μg mL^-1 for menthon and 0.5-2.0 μg mL^-1 for menthol. Carvone showed the strongest antibacterial and antifungal activity with MIC at 0.5-0.5 μg mL^-1 and MBC at 0.25-1.0 μg mL^-1.

Table 4:	Antibacterial activity of essential oils components (1.0 μg mL-1) in disc-diffusion method, inhibition zones (mm)

Only menthon, menthol and carvacrol showed higher antibacterial activity than streptomycin (MIC 3.0-3.0 μg mL^-1 and MBC 5-4.0 μg mL^-1) and Nystatin (MIC 4.0-6.0 μg mL^-1 and MBC 3-5.0 μg mL^-1) (Table 5). Carvone showed higher antifungal activities than previous components. It can be seen that essential oils from Mentha spicata possess a higher antibacterial and antifungal effect than Mentha piperita. The differences between antibacterial and antifungal activities of these two essential oils could be due to different chemical composition of essential oils. The greater antibacterial and antifungal potential of Mentha piperita essential oil could be explained by the presence of carvone which possesses very strong antifungal activity (Adam et al., 1998; Knobloch et al., 1988; Sokovic et al., 2009). The essential oil of Mentha piperita and Mentha piperita have menthol and 1,8-cineole as main components which also exhibited very good antifungal properties but lower than carvone. Carvone has better antifungal properties because of its high water solubility. One of the reasons for lower antifungal activity of Mentha piperita essential oil could be the large amount of menthyl acetate which causes a decrease of antifungal properties (Griffin et al., 2000; Sokovic et al., 2009). Both tested Mentha oils showed strong antibacterial activity against a variety of bacteria (Sokovic et al., 2008). According to present results, volatile oil extracted from leaves of both Fennel and Dill did not show any inhibition of the growth of the above mentioned test organisms. In a study by Lo Cantore et al. (2004) however, essential oil extracted from fennel showed a lesser antibacterial effect compared to coriander oil in inhibition of Escherichia coli and Bacillus megaterium.

Table 5:	Antibacterial activity of essential oils components (MIC and MBC μg mL-1), microdilution method

In agreement with present result Abed (2007) reported that essential oil and leaves extracts of fennel and dill did not show any inhibition antimycobacterial and anticandidal activity. The results obtained by both methods suggested that menthon, menthol and carvacrol possessed greater antibacterial and antifungal activity than other investigated compounds.