Characterization of Volatile Compounds of Eleven Achillea Species from Turkey and Biological Activities of Essential Oil and Methanol Extract of A. hamzaoglui Arabacı & Budak

According to distribution of genus Achillea, two main centers of diversity occur in S.E. Europe and S.W. Asia. Diversified essential oil compositions from Balkan Peninsula have been numerously reported. However, report on essential oils of Achillea species growing in Turkey, which is one of the main centers of diversity, is very limited. This paper represents the chemical compositions of the essential oils obtained by hydrodistillation from the aerial parts of eleven Achillea species, identified simultaneously by gas chromatography and gas chromatography-mass spectrometry. The main components were found to be 1,8-cineole, p-cymene, viridiflorol, nonacosane, α-bisabolol, caryophyllene oxide, α-bisabolon oxide A, β-eudesmol, 15-hexadecanolide and camphor. The chemical principal component analysis based on thirty compounds identified three species groups and a subgroup, where each group constituted a chemotype. This is the first report on the chemical composition of A. hamzaoglui essential oil; as well as the antioxidant and antimicrobial evaluation of its essential oil and methanolic extract.

identified Achillea species in Turkey [4]. Antioxidant and antimicrobial activities of essential oil and methanolic extract of A. hamzaoglui were also evaluated. In this paper, we have also investigated essential oil composition of A. biebersteinii Afan., A. coarctata Poir., A. kotschyi Boiss

Chemical Composition
Eleven Achillea essential oils were obtained by hydrodistillation from air dried aerial parts and subsequently analyzed by GC and GC/MS systems. One hundred seventy-six compounds were identified from Achillea oils, which constituted 76.1% to 97.8% of the total oil. Identified compounds in Achillea oils with their relative percentages are listed in Table 1.
Nonacosane (10.6%), heptacosane (9.2%) and pentacosane (6.1%) were main constituents of the oil of A. lycaonica. Forty-eight components were characterized representing 76.1% of the total oil. Previous data on A. lycaonica essential oil is very limited. Thirteen compounds were identified in A. lycaonica essential oil and L-camphor, artemisia alcohol and camphor, were reported as major components of the oil [22]. In another study, trans-sabinene hydrate, terpinen-4-ol and caryophyllene oxide were reported to be main constituent of A. lycaonica essential oil [23].
In the oil of A. setacea, a sum of 31 components were characterized representing 86.6% of the total oil, with α-bisabolon oxide A (27.0%) and hexadecanoic acid (16.4%) as the main constituents. 1,8-cineole and sabinene were previously reported as major components in A. setacea [39]. Noteworthy, α-bisabolon oxide A and hexadecanoic acid were not reported either in major or minor quantities previously.
β-eudesmol (26.4%), hexadecanoic acid (22.7%) and caryophyllene oxide (7.5%) were main constituents of the oil of A. sintenisii. Thirty components were characterized representing 95.8% of the total oil. A literature survey revealed that there is only one report on the essential oil composition of A. sintenisii [40]. According to this study, camphor, 1,8-cineole, β-pinene, borneol and piperitone were reported as main constituents. Minor quantities of caryophyllene oxide were also reported in this study. Our results showed that β-eudesmol and hexadecanoic acid were reported for the first time in A. sintenisii essential oil.
In addition to these findings, we have investigated essential oil composition of A. hamzaoglui, and evaluated antioxidant and antimicrobial activities of both essential oil and methanol extract of the plant. This is the first report on chemical composition of A. hamzaoglui essential oil and data is given in Table 1. Forty-five components were identified representing 93% of the total A. hamzaoglui essential oil. The main components were determined as 1,8-cineole (24.1%), linalool (12.2%) camphor (6.7%) and germacrene D (6.2%).

Figure 2.
Dendrogram obtained by HCA based on the Euclidean distance between groups of the essential oils of eleven Achillea species growing in Turkey. A. setacea essential oil was characterized by the highest percentages of two main components in group A: α-bisabolon oxide A and hexadecanoic acid (27% and 16.4%, respectively) and a moderate content of α-bisabolol (4.8%). The essential oil of A. millefolium subsp. millefolium essential oil was characterized by the highest percentages of α-bisabolol and muurola-4,10(14)-dien-1-ol (11.7% and 6.8%, respectively) as well as its caryophyllene oxide and hexadecanoic acid content (7.7% and 4.6%, respectively) while A. hamzaoglui essential oil was characterized by the highest levels of 1,8-cineol, linalool, germacrene D (24.1%, 12.2% and 6.2%, respectively) in group A.
In the genus Achilea, composition of the essential oil is highly variable due to some biotic and abiotic factors, such as ontogenic and morphogenic differentiations, environmental factors and applied method of oil extraction [47]. Previous studies on the oil composition of Achillea species revealed that 1,8-cineole was the most abundant compound, ranging from trace levels to 47.7% in essential oils of Balkan Achillea, while camphor and borneol were the second and third repeatedly detected compounds, respectively. Moreover, caryophyllene oxide and β-caryophyllene were reported to be frequently identified sesquiterpenoids [5,15,16]. According to our results, all Achillea species, except A. vermicularis, investigated in this study contain 1,8-cineole from 0.2% to 24.1%. Contrary to Balkan Achillea, camphor, which was detected in all species between 0.5% and 41.3%, was found to be the most abundant compound in this study. In addition to camphor, caryophyllene oxide and spathulenol were detected in all species. 1,8-cineole, borneol, tricosane and pentacosane were the second most detected compounds (Table 1). Oxygenated sesquiterpenes such as β-eudesmol, viridiflorol, spathulenol, nerolidol, caryophyllene oxide, caryophylladienol II, α-bisabolon oxide, α-bisabolol and muurola-4,10(14)-dien-1-ol were found to be major components of investigated species. Among sesquiterpenes, chamazulene was regarded as characteristics of the members of Millefolium group (Syn: sect. Achillea) by Radulovic et al. [16]. However, according to some other researchers, this is not accepted as a universal phenomenon in the group because some species belonging Millefolium group was reported most of the cases as chamazulene free, moreover, some species outside the group contain chamazulene [15,47]. In paralel to this agreement, in the present study, among five species belonging Millefolium group, A. biebersteinii, A. coarctata, A. kotschyi subsp. kotschyi, A. millefolium subsp. millefolium and A. setacea, chamazulene was detected only in A. millefolium subsp. millefolium (0.4%). Futhermore, chamazulene was reported as one of the major components of essential oil of A. millefolium subsp. millefolium [30,31], while in many cases, the species was chamazulene free [24,25,32]. Cubebene, which was reported previously from Achillea species [15], was not identified in this study. Besides sesquiterpenes, in this study, some fatty acid derived compounds such as 15-hexadecanolide, hexadecanoic acid, nonacosane, heptacosane and pentacosane were found to be as major compounds in nine species (in minor quantities in A. schischkinii and A. millefolium subsp. millefolium) ( Table 1).
Noteworthy, major compounds of A. lycaonica investigated in this study were consists of fatty acid derived compounds.

Antioxidant and Antimicrobial Activity of Essential Oil and Methanol Extract of A. hamzaoglui
Free radicals, which are continuously produced in human body as normal products of cellular metabolism, are essential for several physiological processes in low concentrations. However, in higher amounts they can react with membrane lipids, nucleic acids, proteins, enzymes and other small molecules and cause human diseases, including cancer, diabetes, atherosclerosis, failures in immunity and endocrine functions [48]. Antioxidants act as safeguard against the accumulation of free radicals and their elimination from the system. In addition, due to their important role in living systems, antioxidants have been widely used in cosmetics and foods [49]. In the present study, free radical scavenging activity of characterized essential oil (AH-EO) and methanolic extract (AH-ME) of A. hamzaoglui was evaluated by DPPH method in comparison with that of a synthetic antioxidant, tert-butylhydoxy-toluene (BHT), at different concentrations [50]. Radical scavenger activity was expressed as the amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (median effective concentration value, EC50) ( Table 2). Beside DPPH method, total antioxidant capacity (TAC) of methanol extracts and essential oil was also evaluated. The TAC assay, which is a single assay sufficient for reliable determination of antioxidant potential of a complex sample, is based on the reduction of copper (II) to copper (I) by antioxidants [51]. TAC values were shown as mM reducing equivalents to uric acid (UAE) and as μM copper reducing equivalents (CRE) in Table 2. As far as antioxidative potency of the samples is concerned, AH-ME was more effective in both DPPH and TAC assays. AH-ME reduced the stable free radical DPPH with a very low EC50 value (32.09 ± 1.98 μg/mL), which was very similarto that of reference compound BHT (EC50 = 29.83 ± 1.23 μg/mL). EC50 value of AH-EO could not be calculated because of lower values of inhibition than 50%. Total antioxidant capacity of AH-ME and AH-EO was measured as 2.038 ± 0.011 UAE and 0.082 ± 0.003 URE, respectively, showing that the AH-ME is 25-fold stronger than AH-EO.
In vitro antimicrobial activity of AH-EO and AH-ME against common Gram-positive and Gram-negative bacteria and standard Candida strains associated with human infections was assessed by using broth microdilution method [52,53]. Minimum inhibitory concentrations (MIC) of the test samples and standards are summarized in Table 3. According to the microdilution assay, AH-ME showed relatively weak antimicrobial effects against all tested bacteria (MIC; 0.625 mg/mL) except Pseudomonas aeruginosa (MIC; 0.15625 mg/mL). In comparison to Chloramphenicol, Clarithromycin and Tetracycline, AH-EO showed moderate to weak inhibitory effects (MIC; 0.15625-0.625 mg/mL) against the tested bacteria, except Staphylococcus aureus (MIC; 0.07812 mg/mL), which was strongly inhibited by AH-EO. Both AH-ME and AH-EO also showed moderate inhibitory effects on the Candida species (MIC; 0.15625-0.3125 mg/mL).
Previous investigations of Achillea species essential oils demonstrated mostly weak or moderate antimicrobial activity. As there is no data available about the activity of essential oil of A. hamzaoglui in the literature, antimicrobial activity can only be directly compared with chemically similar oils, composing at least the same major compounds. However, apart from chemical variations of essential oils, differences in methods applied and diversity of microorganisms used, make results incomparable. In light of these facts, antimicrobial activity of essential oils of some Achillea species containing 1,8-cineol as major component were given. Tzakou et al., reported that the best inhibitory effect of essential oils of the inflorescences and leaves of A. coarctata, which were characterized by the abundance of oxygenated monoterpenes 1,8-cineole (26.9% and 29.1%, respectively), camphor (22.1%, 9.2%) and borneol (5.0% and 6.8%, respectively), was detected against Micrococcus flavus, Enterococcus faecalis and C. albicans (MIC; 3.25 mg/mL) [19]. According to literature survey, essential oils of A. setaceae and A. teretifolia, whose major oil was 1,8-cineol (18.5% and 19.9%, respectively), exhibited inhibitory effects on Clostridium perfringens, Acinetobacter lwoffii and C. albicans with a range of minimum inhibitory concentration values extended from 0.28 to 2.25 mg/mL [39]. In another study, water-soluble and water-insoluble fractions of methanol extract and essential oil of A. millefolium, which contain 1,8-cineol (24.6%), camphor (16.7%), α-terpineol (10.2%), β-pinene (4.2%), and borneol (4.0%) as principal components, were tested against various microorganism. The essential oil possessed stronger antimicrobial activity than the extracts tested. The oil exhibited moderate activity against Streptococcus pneumoniae, Clostridium perfringens and C. albicans, and weak activity against Mycobacterium smegmatis, Acinetobacter lwoffii and C. krusei [25]. In addition, in contrary to our results, same authors also reported that the oil strongly reduced the DPPH radical (IC50 = 1.56 μg/mL) while water-soluble part of methanol extract of the plant showed weaker radical scavenging ability (IC50 = 45.60 μg/mL). In another study, it was reported that essential oils of A. teretifolia and A. vermicularis, which were investigated at various concentrations and incubation time, also showed considerable DPPH scavenging activity [18]. DPPH radical scavenging capacity of the floral infusions of A. biebersteinii, A. coarctata, A. kotschyi subsp. kotschyi, A. schischkinii, A. setacea, and A. teretifolia, which are growing in Turkey, were reported to be 33.5%, 23.9%, 27.4%, 33.6%, 27.4% and 28.7%, respectively, while total antioxidant capacity of infusions, based on the reduction of Mo (VI) to Mo (V), were determined to be 8.419, 4.671, 5.599, 8.419, 6.999 and 6.928 mMα-Tocopherol/100 mL, respectively [54]. In another study, ethanol extracts of aerial parts of Iranian A. millefolium, A. vermicularis and A. wilhelmsii exhibited DPPH radical scavening activity with EC50 = 49.43, EC50 = 85.28 and EC50 = 118.90 [55]. There are no previous data on the antioxidant potential of A. lycaonica and A. sintenisii extracts.

Isolation of Essential Oil and Extraction
The air-dried aerial parts of the plant material were hydrodistilled for 3 h using a Clevenger-type apparatus to produce a small amount (<0.01%) of volatiles, which was trapped in n-hexane. Air-dried and ground plant material of A. hamzaoglui were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus to obtain essential oil in 0.07% yield. All samples were stored at 4 °C in the dark until analyzed. Twenty grams of plant material was also extracted with methanol three times at 40 °C and methanol was evaporated under reduced pressure (yield 11.6% w/w).

Gas Chromatography Analysis
The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300 °C. To obtain the same elution order with GC/MS, simultaneous auto injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms.

Gas Chromatography-Mass Spectrometry Analysis
The GC/MS analysis was carried out with an Agilent 5975 GC/MSD system. Innowax FSC column (60 m × 0.25 mm, 0.25 μm film thickness) was used with helium as carrier gas (0.8 mL/min). GC oven temperature was kept at 60 °C for 10 min and programmed to 220 °C at a rate of 4 °C/min, and kept constant at 220 °C for 10 min and then programmed to 240 °C at a rate of 1 °C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250 °C. Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450.

Identification of Components
Identification of the oil components was carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley and MassFinder 3) [56,57] and in-house "Başer Library of Essential Oil Constituents" built up by genuine compounds and components of known oils, as well as MS literature data [58] were also used for the identification.

DPPH Radical Scavenging Assay
The free radical scavenging activity of the fractions was measured in vitro by 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assay according to the method described earlier [50]. The stock solution was prepared by dissolving 24 mg DPPH (Sigma-Aldrich, St. Louis, MO, USA) with 100 mL methanol and stored at 20 °C until required. The working solution was obtained by diluting DPPH solution with methanol to attain an absorbance of about 0.98 ± 0.02 at 517 nm using the spectrophotometer. A 3 mL aliquot of this solution was mixed with 100 μL of the sample at various concentrations (1-150 μg/mL). The reaction mixture was shaken well and incubated in the dark for 30 min at room temperature. Then the absorbance was taken at 517 nm. The scavenging activity was estimated based on the percentage of DPPH radical scavenged as the following equation: DPPH radical scavenging activity (%) = [(A0 − A1)/A0] × 100 (1) A0: Absorbance of the control at 30 min (517 nm); A1: Absorbance of the sample at 30 min (517 nm). BHT (Sigma-Aldrich) was used as a positive control. Tests were carried out in triplicate. Afterwards, a curve of % DPPH scavenging capacity versus concentration was plotted and EC50 values were calculated. EC50 denotes the concentration of sample required to scavenge 50% of DPPH free radicals.

Total Antioxidant Capacity (TAC) Assay
The assay was carried out using commercial TAC assay kit (OxiSelect™ Total Antioxidant Capacity (TAC) Assay Kit, Cell Biolabs, Inc., San Diego, CA, USA). Upon reduction, the copper (I) ion further reacts with a coupling chromogenic reagent that produces a color with a maximum absorbance at 490 nm. The net absorbance values of antioxidants are compared with a known uric acid standard curve. Absorbance values are proportional to the sample's total reductive capacity. Results are expressed as μM copper reducing equivalents or mM uric acid equivalents. A fresh uric acid standard was prepared by weighing out the uric acid powder for a 10 mg/mL solution in 1 N NaOH. This 10 mg/mL is equivalent to a concentration of 60 mM. The 60 mM uric acid solution was used to prepare a 2 mM solution of uric acid (e.g., add 100 μL of the 60 mM uric acid standard to 2.9 mL of deionized water). Each sample was prepared using the stock solution of 100 mg/mL concentration. An initial reading was taken at 490 nm. Then, 50 μL of the 1× copper ion reagent was added and incubated for 5 min on an orbital shaker. Then, 50 μL of the stop solution was added to terminate the reaction and the plate was read again at 490 nm. All determinations were performed in triplicate and results were averaged.

Test Microorganisms
Microorganisms were obtained from ATCC, NRRL or clinical isolates (Eskişehir Osmangazi University, Turkey; Anadolu University, Faculty of Science, Department of Biology, Eskişehir, Turkey) and were stored in 15% glycerol containing micro-test tubes at −86 °C (strain numbers of microorganisms were given in Table 3). All microorganism strains were inoculated on Mueller Hinton Agar (MHA) or Sabouraud Dextrose Agar (SDA) prior the experiments at 37 °C. After sufficient growth, the microorganisms were transferred to Mueller Hinton Broth (MHB) for further incubation at the same conditions for another 24 h [52,53].

Broth Microdilution Assay
Test samples as well as standard antimicrobial controls were first dissolved in DMSO (25%) at an initial concentration. Serial dilution series were prepared in 100 µL Mueller Hinton Broth (MHB) with an equal amount of the test samples. Overnight grown microorganism suspensions at appropriate conditions were first diluted in double strength MHB and standardized turbitometrically to 1 × 5 10 6 -10 8 CFU/mL (McFarland No: 0.5) under sterile conditions. Then each microorganism suspension was pipetted into each well and incubated at 37 °C for 24 h. Sterile distilled water and medium served as a positive growth control. The first well without turbidity was assigned as the minimum inhibitory concentration (MIC, in µg/mL). To visualize the antimicrobial activity Tetrazolium Violet 1% (w/v, EtOH) (2,5-diphenyl-3-[αnaphthyl] tetrazolium chloride, TTC, Sigma) was also used. Average results of separately performed three experiments were given [52,53].

Statistical Analysis
The essential oils components useful in reflecting chemotaxonomic and biological relationships, compounds detected in oil samples with an average concentration of greater than 0.6% were selected. The components were subjected to a principal component analysis (PCA) and to hierarchical cluster analysis (HCA) using IBM SPSS software version 22.0.

Conclusions
In this study, we investigated essential oil compositions of A. hamzaoglui along with ten Achillea species growing in Turkey, which is one of the main diversity centers of the genus Achillea. With regard to their essential oil composition, PCA and HCA analysis enabled to identify three groups and a subgroup of Achillea species, where each group constituted a chemotype.
There is a widespread agreement that synthetic antioxidants need to be replaced with natural ones because some synthetic antioxidants have shown potential health risks and toxicity. Therefore, in order to find new sources of safe antioxidants of natural origin, a great interest has been given to the antioxidant and radical scavenging properties of plant extracts. In the present study, A. hamzaoglui methanol extract exhibited very strong radical scavenging activity and high antioxidant capacity. The findings indicate that methanol extract of A. hamzaoglui can be considered a rich natural source of antioxidants that could be used in pharmaceutical preparations, cosmetics and foods. On the other hand, essential oil of the plant exhibited much more prominent antimicrobial activity against tested microorganisms than that of methanol extract. Because of its strong antibacterial activity against Staphylococcus aureus (ATCC BAA-1026) and moderate antibacterial activity against Pseudomonas aeruginosa (ATTC 10145) and Propionibacterium acnes (ATCC 6919), the essential oil could be effectively used for skin infections. This study is the first report on essential oil composition and antioxidant and antimicrobial activities of A. hamzaoglui, and calls for further investigations to elucidate its effect on other biological activities.