Chemical composition, antioxidant activity and in vitro antibacterial activity of Achillea wilhelmsii C. Koch essential oil on methicillin-susceptible and methicillin-resistant Staphylococcus aureus spp.

The present study investigated the chemical composition of the essential oil (EO) from aerial parts (flowering stage) of Achillea wilhelmsii C. Koch by GC–MS. In addition, the antioxidant activity of the EO as well as its antimicrobial activity against methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MRSA) strains was tested. Antioxidant activity was measured by the ability of the EO to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals while the antimicrobial activity was assessed by the disc-diffusion method. In total, 52 compounds were recognized, accounting for 97.33 % of the EO. The main compounds in the EO were carvacrol (22.49 %), dihydrocarvone (13.23 %), linalool (12 %), 1,8-cineol (11.42 %), camphene (8.31 %), thymol (5.28 %), camphor (3.71 %), pulegone (2.82 %) α-terpineol (2.11 %), bornyl acetate (1.14 %), and farganol (1.01 %). The EC50 value of the EO was 0.01 and 0.08 mg/mL for the antioxidant and DPPH-scavenging ability, respectively. A. wilhelmsii EO affected methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA, but the impact was more effective on MSSA.


Introduction
Herbal medicines are considered to be an important natural medicine for the treatment of health conditions and diseases. The excessive and repeated use of the same drugs used in modern medicine has led to the evolution of antibiotic-resistant microbes, including Staphylococcus aureus whose emergence of antibiotic-resistant strains reduces the number of antibiotics available to treat clinical infections caused by this bacterium (Parker and Jevons 1964). S. aureus is a highly versatile pathogen with considerable importance in human medicine. S. aureus is responsible for a wide range of hospital and communityacquired infections worldwide, from skin infections and food poisoning to life-threatening situations such as toxicshock syndrome, endocarditis, pneumonia, bacteraemia and osteomyelitis (Kim et al. 2006;Akineden et al. 2008). Traditional medicine involving herbs (or the compounds within them) can solve health and medical problems caused by S. aureus. The essential oils (EOs) (primarily from leaves) of Thymus vulgaris and Eucalyptus globulus were tested against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), both EOs to possessing antibacterial activity against MRSA, the former being more potent than the latter (Tohidpour et al. 2010). Daucus crinitus (a medicinal plant) EOs (derived from stems and leaves of wild plants) inhibited S. aureus (Bendiabdellah et al. 2013).
The hygiene industry utilizes A. wilhelmsii EO to make skin tender and soft and to treat skin inflammations using cream formulations (Pieroni et al. 2004). A. wilhelmsii is rich in sesquiterpenes, lactones flavonoids and monoterpenoids which have antioxidant activities (Jaimand and Rezaee 2001;Saeidinia et al. 2005).
The main purpose of the present study was to perform a biological examination on A. wilhelmsii C. Koch from Golmakan Khorasan Razavi, Iran by assessing the antioxidant and antimicrobial activity of the EO.

Plant preparation and procedure
Aerial parts (stems, leaves and flowers) of the flowering stage of A. wilhelmsii C. Koch (Fig. 1a, b) were collected in June 2012 from Golmakan (36°28 0 44 00 N, 59°9 0 17 00 E), Khorasan Razavi, Iran. The plant was taxonomically identified by a botanist at the herbarium of Pharmacognosy, Department of the Faculty of Pharmacy affiliated to Shahid Beheshti University of Medical Sciences of Iran.
Extraction and isolation of the EO Plant parts were air-dried in the shade at ambient temperature (18-25°C) for 12 days. Dried aerial parts (100 g) were cut into small pieces and hydro-distilled for 4 h using Clevenger-type apparatus. The resulting EO was dried over

GC-MS analysis
In this study, an HP 6890 N GC system coupled with an HP MSD5973 N quadruple mass spectrometer was utilized. The extracted compounds were separated on an HP-5MS capillary column (30 m length, 0.25 mm internal diameter, 0.25 mm film thickness). Split injection:sample ratio for distillation was 50:1. The column oven temperature was programmed to rise from an initial 40 to 150°C at 4°C/min, and then to 240°C at 10°C/min. Injection temperature and ion source temperature were 240°C. Helium was used as the carrier gas with a flow rate of 1.2 mL/min. The ionizing energy was 70 eV. All data were obtained by collecting the full-scan mass spectra within the scan range 50-550 amu. Compounds were identified using the Wiley 7n.L Mass Spectral Library (Wiley, New York, NY, USA). Trace compounds were defined as those detected at \0.04 % of the EO.

Antioxidant activity
Antioxidant activity was measured by the paired diene method (Lingnert et al. 1979). The antioxidant activity measured is the ability of the EO to inhibit the peroxidation of linoleic acid in which the double bond is altered to a paired diene. Each EO sample (0.01-30 mg/mL) in methanol (100 lL) was mixed with 3 mL of 10 mM linoleic acid (Sigma Chemical Co., St. Louis, MO, USA) to form an emulsion in 0.2 M sodium phosphate buffer (pH 6.6) in test tubes and placed in the dark at 37°C to quicken oxidation. After incubation for 17 h, 7 mL of 70 % methanol in deionized water was added, and the absorbance of the mixture was measured at 234 nm against a blank in a Hitachi U-2001 spectrophotometer (Tokyo, Japan). Antioxidant activity was quantified as follows: Antioxidant activity (%) = [(DA 234 of control -DA 234 of sample)/DA 234 of control] 9 100. Analyses were repeated three times. a-Tocopherol, butylated hydroxyanisole (BHA) and ascorbic acid (Sigma) were used as standard controls.
Scavenging ability on 1, 1-diphenyl-2-picrylhydrazyl radicals The scavenging ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma) radicals, which was measured by the method of Shimada et al. (1992), is the ability of the EO to react quickly with DPPH radicals and to decrease most DPPH radical molecules. The assay was repeated three times. a-Tocopherol, BHA and ascorbic acid were used as standard controls. Each EO sample (0.5-30 mg/mL) in methanol (5 mL) was mixed with 1 mL of methanolic solution containing DPPH radicals, resulting in a final concentration of 0.2 mM DPPH. The mixture was shaken vigorously, left to stand for 45 min in the dark, and the absorbance was then measured at 517 nm against a blank. The scavenging ability was calculated as follows: Scavenging ability (%) = [(DA 517 of control -DA 517 of sample)/DA 517 of control] 9 100.
EC 50 value (mg/mL) is the efficient concentration at which the antioxidant activity was inhibited by 50 % and DPPH radicals were scavenged by 50 %, and was gained by interpolation from linear regression analysis.
Antibacterial study

Preparation of microorganisms
The S. aureus strains utilized in this study were clinical isolates from patients with S. aureus, obtained from the microbiological laboratory of the central hospital in Shiraz, Iran. This study was approved by the ethics committees of Zabol and Shiraz Universities of Medical Sciences. MRSA that were isolated were identified by screening tests on Mueller-Hinton agar (MHA, Torlak, Berlin, Germany) supplemented with 5 % NaCl and 1 mg/mL oxacillinimpregnated disc to isolate MRSA (Roberts et al. 2002). Finally, 10 MRSA strains and 5 methicillin-susceptible S. aureus (MSSA) strains were isolated from patients. In this study, two standards strains, ATTC 25923 (MRSA) and PTCC 1341 (MSSA), were used.

Disc-diffusion assay
Antimicrobial tests were performed by the disc-diffusion method using 100 lL of suspension (containing 2.0 9 10 8 CFU/mL of bacteria) dispersed evenly on MHA in sterilized Petri dishes (80 mm in diameter). To the discs (6 mm in diameter, HiMedia Laboratories Pvt. Ltd., Mumbai, India), 20, 50, 100 and 200 lL of EO and placed on the inoculated agar. The inoculated plates were maintained at 4°C for 2 h and incubated at 37°C for 24 h. Antimicrobial activity was evaluated by measuring the zone of inhibition (mm) against the test bacterial (MRSA and MSSA) strains.

Statistical analysis
The EO was prepared in triplicate for chemical characterization and for antioxidant and antibacterial assays. Data was subjected to analysis of variance following a completely random design to determine the least significant difference (LSD) at P \ 0.05 using SPSS v. 11.5.

Results and discussion
The composition of A. wilhelmsii essential oil The mass spectra and retention indices (RI) were used in this study to determine the EO composition of A. wilhelmsii. In total, 52 compounds were identified accounting for 97.33 % of the EO components (Table 1). The main compounds of the EO were (in decreasing order) carvacrol (22.49 %), dihydrocarvone (13.23 %), linalool (12 %), 1,8-cineol (11.42 %), camphene (8.31 %), thymol (5.28 %), camphor (3.71 %), pulegone (2.82 %), a-pinene (2.2 %), terpineol (2.11 %), bornyl acetate (1.14 %) and farganol (1.01 %). Some compounds were detected in trace amounts (not listed in Table 1): heptanal, isopentyl isovalerate, neo-3-thujanol, cis-jasmone, elemol. The main compound in the EO of A. wilhelmsii from the Golmakan Khorasan Razavi (Iran) area was carvacrol (22.42 %). Javidnia et al. (2004) also found 25.1 % carvacrol in A. wilhelmsii oil as the main compound. The major constituent of the EO of the flowers and leaves of A. wilhelmsii from Mazandaran (Iran) province was camphor, 21.2 % and 24.1 %, respectively (Azadbakht et al. 2003). Carvacrol and camphor have no harmful effects on humans and environment (Rajendran and Sriranjini 2008;Khani and Asgari 2012). The amount of camphor in A. wilhelmsii EO collected from Kerman was 9.0 % (Afsharypuor et al. 1996) and in the EO of aerial parts from Kazeroon (Iran, Fars) province was 2.2 % (Javidnia et al. 2004). Afsharypuor et al. (1996) reported that main compound in the EO of A. wilhelmsii was caryophyllene oxide (12.5 %), much higher than that reported in our study (0.08 %). 1,8-cineol, which was found at 3.32 % of the stem EO in this study was the major constituent of the oil of A. wilhelmsii from Egypt and Turkey (Javidnia et al. 2004;Baris et al. 2006). The variations in the qualitative and quantitative composition of the EOs from different locations within the same country or from different countries are likely caused by genetic variation, growth conditions, geographic variation and analytical protocols used to assess the EOs. Previous studies showed that monoterpenes, the main part of the A. wilhelmsii EO from Golmakan, have influential insecticidal effects against stored product insects (Papachristos et al. 2004;Rajendran and Sriranjini 2008). Consequently, the A. wilhelmsii EO from Golmakan could be an important optional phytochemical control strategy without undesirable effects such as direct toxicity to humans and environmental pollution (Rajendran and Sriranjini 2008;Khani and Asgari 2012). In addition, A. wilhelmsii EO contains sesquiterpenes, lactones and flavonoids, which have in the ability to lower blood lipid levels and hypertension (Asgary et al. 2000).

Antioxidant activity and scavenging ability
The results for antioxidant activity and scavenging ability on DPPH radicals of the EOs assayed are summarized in Table 2. The efficiency of antioxidant activity and scavenging ability is inversely related with their EC 50 values. The antioxidant activity EC 50 values were 0.08, 0.05, 4.07 and 0.01 mg/mL for a-tocopherol, BHA, ascorbic acid and A. wilhelmsii EO, respectively. The scavenging ability EC 50 values was 0.11, 0.07, 9.09 and 0.58 mg/mL for a-tocopherol, BHA, ascorbic acid and A. wilhelmsii EO, respectively. The antioxidant activity of A. wilhelmsii EO was stronger than three standards tested (Table 2), which could be used for the treatment of human diseases to remove free radicals (Dharmendra et al. 2009).

Antibacterial activity
The results of antibacterial activity of EOs (Table 3) showed that the maximum level of EO (200 lL) was inhibitory (largest zone of inhibition) against MSSA (22.56 mm) and MRSA (14.22 mm). The inhibitory activity against MSSA was greater than against MRSA. The EOs of A. wilhelmsii had a more negative impact on MSSA than MRSA (Table 3). Monoterpenes, which are rich in the EO of A. wilhelmsii, have powerful antibacterial effects (Unlu et al. 2002;Sokmen et al. 2004;Prabuseenivasan et al. 2006). In addition, phenolic and flavonoid compounds, also present in the EO of A. wilhelmsii, have antimicrobial activity (Stojanovic et al. 2005;Eleyinmi 2007;Yaghoubi et al. 2007;Mothana et al. 2009).

Conclusion
In this study, chemical composition, antioxidant activity and in vitro antibacterial activity of A. wilhelmsii L. Essential oil on methicillin-susceptible and methicillinresistant S. aureus spp. was investigated. A. wilhelmsii C. Koch has emerged as an important medicinal plant. Its EO could be commercialized for its antioxidant, insecticidal and antibacterial applications, or used in the pharmaceutical, cosmetic or perfume industries.