Essential Oil Composition and Antimicrobial Activity of Achillea biebersteinii Afan . ( Asteraceae ) from Erzincan Region , Turkey

In the present study, chemical composition determination and in vitro antimicrobial effects of essential oil of Achillea biebersteinii plant grown under Erzincan ecological conditions were evaluated. Extractions were carried out with Clevenger apparatus and essential oil composition was determined by Gas Chromatography-Mass Spectrometry (GC-MS). Microorganisms used for the antimicrobial studies were Salmonella enterica serovar. typhimirium ATCC 14028, Staphylococcus aureus subsp. aureus ATCC 25923, Yersinia pseudotuberculosis ATCC 911, Bacillus cereus 702 ROMA, Enterobacter aerogenes CCM 2531, Bacillus subtilis IMG 22 and Proteus vulgaris FMC 1. As a result, a total of 29 components were detected in Achillea biebersteinii. Among them, 1,8-cineole (20.36%), cyclohexanone (8.39%), 2-cyclohexen-1-one (5.38%) and spathulenol (4.19%) were found as the major components. For the in vitro antimicrobial activity determination of essential oil, disc diffusion method was used in our study. Furthermore, 12-14 mm zone diameters were detected in antimicrobial activity assay. The highest resistance zone was detected against B. subtilis with 14 mm diameter while the least resistance zone was detected against Y. pseudotuberculosis, E. aerogenes and P. vulgaris with 12 mm diameter. Consequently, it was concluded that the essential oil extracted from the A. biebersteinii grown under Erzincan ecological conditions had an inhibitory effects on the pathogenic microorganisms in used method.


Introduction
Autoecological studies on economically important plants in Anatolia, which has a rich vegetation cover thanks to its geographical location and climate, are of great significance in order to understand the growth conditions and effective use of these plants (Çelik et al., 2004;Paksoy et al., 2006).The Asteraceae family, which is known to have the greatest number of species in the world, consists of approximately 23000 species and 1535 genera (Öztürk and Çetin, 2013;Nylinder and Anderberg, 2015).It has been reported that a large number of species of the Asteraceae family, expanding widely over the world and in Anatolia, show pharmacological activities.The plants in this family include sesquiterpene lactone metabolites having numerous biological activities such as antibacterial, antifungal, antihelminthic, anti-inflammatory, insecticide and antitumor, in addition to diterpenes and flavonoids (Picman, 1986;Shing et al., 2002;Ertürk, 2003;Bağcı et al., 2008).Achillea L. is a large genus belonging to the family Asteraceae.The genus Achillea L. includes 59 taxa divided into 6 sections.Among them, 31 taxa are endemic to Turkey (53%) (Arabaci, 2012;Aytac et al., 2016;Tabanca et al., 2016).The Achillea genus has a wide expansion range (Çelik and Akpulat, 2008).Differences in oil composition can be due to different environmental factors such as seasonality and developmental stage in addition to plant genetic type, especially in chemically polymorphic and

Materials and Methods
Plant material and GC-MS analysis Achillea biebersteinii samples of the plants were collected as study materials in July 2016 from Erzincan/Turkey surroundings (approximately 1,200 m altitude).Extractions were carried out with Clevenger apparatus (Basaran cam, Turkey and Misung Scientific Co., Korea) using water distillation (Balinova and Dyakov, 1974) and essential oil compositions were determined by Gas Chromatography-Mass Spectrometry (GC-MS).Characterization of essential oil components was based on the library (Wiley and NIST) comparison with the mass spectra of the injected essential oil samples.

Test bacteria and antimicrobial activity
Seven bacterial strains were utilized to detect antimicrobial action of the essential oil.Salmonella enterica serovar typhimirium ATCC 14028 and Staphylococcus aureus subsp.aureus ATCC 25923 were obtained from Giresun Province Control Laboratory, Yersinia pseudotuberculosis ATCC 911 and Bacillus cereus 702 ROMA were obtained from Molecular Biology Department of Rize University.Enterobacter aerogenes CCM 2531, Bacillus subtilis IMG 22 and Proteus vulgaris FMC 1 were obtained from Department of Biology, Fırat University.Bacterial strains were maintained on nutrient agar at 4 °C.Examination of essential oil for antimicrobial efficiency was performed by the disc diffusion method.It was applied using a 24 h culture at 37 °C in Mueller Hinton Broth.The turbidity of bacterial suspensions was adjusted to 108 cfu/mL (turbidity = McFarland standard 0.5).The oil was dissolved in DMSO (dimethyl sulfoxide) 1:2 (v/v) to give stock solution after which they were mixed for total solubilization at 180 rpm for 10 minutes.Dissolved extracts was sterilized by using 0.45 μm pore sized filter (Dobre et al., 2011).Inhibition zones of the essential oil were compared with standard antibiotics (tetracycline and gentamycine).Sterilized nutrient agar medium was poured in petri dishes and was allowed to solidify.The bacterial suspension inoculated into Mueller Hinton agar plates.Sterile discs were put (5 mm diameter) on the agar and 20 μL A. biebersteinii essential oil, and 20 μL DMSO were put on the discs, respectively.The inoculated plates were left in refrigerator for one hour then plates were incubated at 37 °C overnight (Murray et al., 1995;Saric et al., 2009).Diameter of inhibition zones was measured in millimetres.
Beyşehir-Akşehir population, 1,8-cineole (35.5%) and camphor (35.5%); from the Isparta-Yalvaç population, 1,8cineole (34.3%) and camphor (21.7%) had the highest values.Typical for camphor are its antispasmodic, diuretic, antirheumatic and sedative effects.The biological function of the chemical components is not limited by their antimicrobial activity; some of them have antioxidant (limonene, pinene), antitumor (linalool, borneol), antiinflammatory (sabinene, pinene) and analgesic functions (citral) as well (Teneva et al., 2016).Considering the current study reporting essential oils from the A. biebersteinii specimens, the major components were detected as; 1, 8-cineole (20.36%), cyclohexanone (8.39%) and spathulenol (4.19%).In conclusion, the findings obtained from various studies were found to be different from the findings of the present study.This may be explained by the fact that the essential oil composition may have different qualities and quantities under different geographical and environmental conditions as well as at different periods of plant growth (Mazandarani et al., 2013).

Antimicrobial activity
The most commonly studied dimension of essential oils is related to antimicrobial activities.These oils have antimicrobial effects against a variety of microorganisms including gram (-) and gram (+) bacteria.As the essential oils are complex mixtures comprising different components, action levels thereof vary depending on diversity and amount of the active substances.Despite having limited information relating to their mechanisms of action, it seems to be associated with lipophilic features and chemical structures of the oils (Bayaz, 2014).For the in vitro antimicrobial activity determination of essential oil, disc diffusion method was used in our study.The results were compared to the standard antimicrobial agents such as gentamycine and tetracycline.Zone diameters of 12 mm -14 mm were detected in disc diffusion methods.Achillea biebersteinii essential oil was effective against Salmonella enterica serovar typhimurium Staphylococcus aureus subsp.aureus, Yersinia pseudotuberculosis, Bacillus cereus, Enterobacter aerogenes, Bacillus subtilis and Proteus vulgaris.The widest resistance zone was against Bacillus subtilis with 14 mm diameter while the smallest resistance zone was detected against Y. pseudotuberculosis, E. aerogenes and P. vulgaris with 12 mm diameter (Table 2).The essential oil exhibited lower activity than that of gentamycine, but it exhibited similar activity to that of tetracycline.Barış et al. (2006) examined antimicrobial activity of essential oil and methanol extract from A. biebersteinii.The methanol extract was not active against any studied microorganisms.On the other hand, essential oil inhibited the growth of 6 bacterial species tested.The essential oils also inhibit antifungal action as reported by Sökmen et al. (2004) who assessed antimicrobial activity of essential oil from A. biebersteinii.In their study, the essential oil displayed strong activity against Candida albicans, Clostridium perfringens, Streptococcus pneumoniae, Staphylococcus aureus, Bacillus cereus, Escherichia coli and Mycobacterium smegmatis (Sökmen et al., 2004).Likewise, we observed activity against S. aureus and B. cereus in our study.Kotan et al. (2010) examined chemical composition of the essential oil of A. biebersteinii by GC-MS and tested antimicrobial effect of essential oil against 25 plant pathogenic bacteria.We also studied antimicrobial efficiency of A. biebersteinii, but it was obtained different results when compared to previous studies.These different results may have been obtained due There are many studies related to antimicrobial activity of different Achillea species.For example, Bezic et al. (2003) investigated volatile constituents and antimicrobial potentials of Achillea clavennae, and they found important decline of bacterial growth against Escherichia coli and Proteus mirabilis.On the other hand, Bacillus cereus, Bacillus subtilis and Streptococcus faecalis were resistant to the essential oil with inhibition zones between 4 mm and 6.5 mm.In a study which was carried out by Candan et al. (2003), essential oil of Achillea millefolium subsp.millefolium manifested antimicrobial activity against Streptococcus pneumoniae, Clostridium perfringens, Candida albicans, Mycobacterium smegmatis, Acinetobacter lwoffi and Candida krusei.Başer et al. (2002) worked out composition and antimicrobial activity of the essential oil of Achillea multifida.Plant pathogenic bacteria inhibition potential of Achillea millefolium was investigated by et al. (2006).The strength of the antimicrobial property has been reported to vary according to the used extract type, collecting plant materials from different locations, used extract concentration, and microorganisms tried (Srivastava et al., 2013).

Conclusions
Considering the current study with the essential oils from the of A. biebersteinii plants, the major compnents were detected as, 1,8-cineole (20.36%), cyclohexanone (8.39%), 2-cyclohexen-1-one (5.38%) and spathulenol (4.19%).According to the results obtained in the hereby study, it is possible to conclude that antimicrobial activity of the essential oil is slightly lower in comparison antibiotics (tetracycline and gentamycine) effect on tested seven different bacteria genus (S. enterica serovar typhimirium, Y. pseudotuberculosis, E. aerogenes, B. subtilis, S. aureus subsp.aureus, P. vulgaris and B. cereus).These findings may be a valuable resource for further biotechnological, biodiversity, pharmaceutical and medical studies.It will also help to understand the importance of the biological diversity and conservation biology efforts.

Table 1 .
Essential oil composition of Achillea biebersteinii

Table 2 .
Antimicrobial activity (inhibition zones) of the essential oils from Achillea biebersteinii