Eugenol-rich essential oil of Anthemis mazandranica and its antibacterial activities

The hydro-distilled volatile oil obtained from aerial parts of Anthemis mazandranica was analyzed by gas chromatography and mass spectrometry (GC-MS). Seventeen compounds were identified, representing 93.35% of the total oil composition, with eugenol (35.5%) being the main component. Oil antimicrobial activity was carried out using the disk diffusion and minimal inhibitory concentration (MIC). The best antibacterial activity was observed against Salmonella Typhi with ZI = 19 ± 0.5 mm and MIC value of 32 μg/mL.


INTRODUCTION
The genus Anthemis, comprises 130 species widespread in the Mediterranean, South West Asia and South Africa (Kudryashev, 1932). Present in Iran are 39 species growing wild, among which 15 are endemic (Mozaffarian, 1996). From the Roman times up to now, Anthemis taxa have been commonly used as folk remedies, insecticides and dyes (Niko et al., 2009). Water-distilled essential oils from the leaves and flowers of A. altissima (L.) Var. Altissima. was analyzed by GC-MS. β-Thujone (33.7 and 19.7%, respectively) was found as the major constituent in the leaf and flower oil (Rustaiyan et al., 2004). Over the last two decades, Anthemis volatile compounds have received more attention (Vuckovic et al., 2006;Williams et al., 2001;Vajs et al., 1999).

Plant material and isolation procedure
The aerial parts of A. mazandranica growing wild in Shiraz (Provincial capital of Fars) was collected at the flowering stage in May, 2011. Their identities were confirmed by Dr. Valiollah Mozaffarianand and a voucher specimen (no. VS-21-13) was deposited at the Herbarium of Science and Research Branch, Islamic Azad University (Tehran, Iran).

Hydrodistillation
The air-dried aerial parts (leaves, petals and stems) (100 g) were dried, powdered and the volatile fraction was isolated by hydrodistillation for 3 h using a Clevenger-type apparatus. The essential oil had a bright yellow color and yielded 0.59% w/w.

GC and GC/MS analysis
GC analysis of the oil was performed using a Shimadzu 15 A gas chromatograph equipped with flame ionization detector (FID) and a DB-5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 μm). Temperature program: 60°C (3 min), 60 to 220°C at 5°C/min, 220°C (5 min); injector and detector temperatures, 260°C; the carrier gas was N2 (1 ml/min). The sample was injected in splitsplitless mode, using a split ratio of 1:50. The percentages of each component were reported as raw percentages without standardization. GC-MS analysis was carried out on a Hewlett-Packard 6890/5973 using an HP-5MS column (30 m × 0.25 mm i.d., film thickness 0.25 μm). The oven temperature was as above, *Corresponding author. E-mail: aboeef@yahoo.com. interface temperature, 260°C; mass range was 40 to 300 amu; scan time, 1 s. Retention indices (RI) of compounds were determined relative to the retention times of a series of n-alkanes (C6 to C25) with linear interpolation. Identification of the oil components was done by comparison of their mass spectra with Wiley 275 GC-MS library, by comparing them with those reported in the literature and confirmed by comparison of its retention index either with those of authentic compounds or with data in the literature (Jenning and Shibamoto, 1980;Adams,1995).

Antimicrobial activity
All test microorganisms were obtained from the Persian type culture collection (

Assessment of antimicrobial activity
The antibacterial activity of the A. mazandranica essential oil was screened against Gram-positive and Gram-negative bacteria, with two methods:

Disc diffusion assay
Antimicrobial tests were carried out by the disc diffusion method reported by Murray and his co-worker in 1999 (Wayne, 2006). The dried A. mazandranica essential oil was dissolved in Dimethyl sulfoxide (DMSO) to a final concentration of 30 mg/ml and filtered through 0.45 µm Millipore filters, using 100 µl of suspension containing 108 CFU/ml of bacteria and 104 spore/ml of fungi spread on the nutrient agar (NA) and potato dextrose agar (PD) mediums, respectively. The discs (6 mm in diameter) impregnated with 10 µl of the essential oil solution (300 µg/disc) and DMSO (as negative control) were placed on the inoculated agar. The inoculated plates were incubated for 24 h at 37°C for bacterial strains and 48 and 72 h at 30°C for mould isolates, respectively. Gentamicin (10 µg/disc) and ampicillin (5 µg/disc) were used as positive controls for bacteria. The diameters of inhibition zones were used as a measure of antimicrobial activity and each assay was repeated twice.

MIC agar dilution assay
The lowest concentration of the compounds that prevented visible growth was considered as the minimal inhibitory concentration (MIC). MIC value of the plant essential oil against standard bacterial strains was evaluated based on the agar dilution method. Appropriate amounts of the A. mazandranica oil were added aseptically to sterile molten Sabouraud dextrose agar (SDA) medium added with Tween 20 (0.5%, v/v) to produce the concentration range of 8 to 500 µg/ml. The resulting SDA agar solutions were immediately mixed and poured into Petri plates. The plates were spot inoculated with 5 µl (104 spore/ ml) of each fungus isolate. At the end of incubation period, the plates were evaluated for the presence or absence of growth. Ampicillin and tetracycline were used as references for gram-positive and negative bacteria, respectively. The MIC was defined as the lowest concentration of the oil needed to inhibit the growth of microorganisms. Each test was repeated at least twice. ZI: Zone of Inhabitation. Including the diameter of the filter paper disc (6 mm); mean value of four experiments ± SD. Campicillin (5 µg/disc), gentamicin (10 µg/disc) were used as references for Gram-positive and negative bacteria, respectively.

RESULTS AND DISCUSSION
The oil of the aerial part contained 19 compounds with a yield of 0.59% (w/w), representing 93.35% of the total oil composition (Table 1), where main component was eugenol (35.5%). In particular, Oxygenated monoterpenes (59.11%) were the most abundant group of compounds. In this study, the antimicrobial activities of the aerial parts essential oil of A. mazandranica were investigated against six bacterial standard strains in laboratory situation (in vitro). Antimicrobial activity (inhibition zone and MIC) of the oil against standard microorganisms is shown in Table 2. The best antibacterial activity was observed against S. Typhi (ZI and MIC value 19 ± 0.5 mm and 32 μg/ml, respectively). From a medical point of view, the antimicrobial activity of A. mazandranica oil against S. Typhi is particularly interesting because of the role of this microorganism as a pathogen agent responsible for severe typhoid fever infection.