Turk J Biol 30 (2006) 65-73 © TÜB‹TAK
Biological Activities of the Essential Oil and Methanol Extract of Achillea biebersteinii Afan. (Asteraceae)
Özlem BARIfi1, Medine GÜLLÜCE1, Fikrettin fiAH‹N2, Hakan ÖZER3, Hamdullah KILIÇ4, Hakan ÖZKAN5, Münevver SÖKMEN6, Tülin ÖZBEK7 1 Department of Biology, Faculty of Arts and Science, Atatürk University, 25240 Erzurum - TURKEY 2 Department of Genetic and Bioengineering, Faculty of Engineering and Architecture, Kay›sdagi, Yeditepe University, 34755 ‹stanbul - TURKEY 3 Department of Crop Science, Faculty of Agriculture, Atatürk University, 25240 Erzurum - TURKEY 4 Department of Chemistry, Faculty of Art and Science, Atatürk University, 25240 Erzurum - TURKEY 5 The Ministry of Health of Turkey, Erzurum Regional Hygiene Institute, 25240 Erzurum - TURKEY 6 Department of Chemistry, Faculty of Arts and Science Cumhuriyet University, 58140 Sivas - TURKEY 7 Department of Biology, Institute of Natural and Applied Sciences, Atatürk University, 25240 Erzurum - TURKEY
Received: 09.03.2006
Abstract: The present work examined the in vitro antimicrobial and antioxidant activities of the essential oil and methanol extract from Achillea biebersteinii Afan. (Asteraceae). The essential oil exhibited antimicrobial activity against 8 bacteria, 14 fungi and the yeast C. albicans, whereas methanolic extract remained inactive. The antioxidative capacity of the samples was evaluated by using DPPH (2,2-diphenyl-1-picrylhydrazyl) and β-carotene/linoleic acid assays. In both assays, the extract showed better antioxidative
capacity than the oil. The extract reduced the stable free radical DPPH with lower IC 50 value (89.90 µg/ml) than the oil (8900 µg/ml). In the β-carotene/linoleic acid assay, the samples were not effectively able to inhibit the linoleic acid oxidation, exhibiting only 22.7% (the extract) and 16% (the essential oil) inhibitions at 2 mg/ml, far below than that of BHT (97.0%). Total phenolic constituent of the extract was 51 µg/mg (5.1%, w/w) as gallic acid equivalent. GC-MS analysis of the essential oil resulted in the identification of 64 components representing 92.24% of the oil. Piperitone, camphor and 1,8-cineole (eucalyptol) were the main constituents.
Key Words: Achillea biebersteinii, Antimicrobial activity, Antioxidant activity, GC-MS analysis, piperitone, camphor, eucalyptol
Achillea biebersteinii Afan. (Asteraceae)'nin Uçucu Ya¤› ve Methanol Ekstrakt›n›n Biyolojik Aktivitesi
Özet: Bu çal›flmada, Achillea biebersteinii Afan. (Asteraceae)'nin metanol ekstrakt› ve uçucu ya¤›n›n laboratuar ortam›nda antimikrobiyal ve antioksidan aktiviteleri araflt›r›ld›. Uçucu ya¤ 8 bakteri, 14 fungus ve C. albicans mayas›na karfl› antimikrobiyal aktivite gösterdi. Metanol ekstrakt›n›n ise antimikrobiyal aktivitesi gözlemlenemedi. Örneklerin antioksidan kapasitesi DPPH (2,2- diphenyl-1-picrylhydrazyl) ve β-karoten/linoleik asit yöntemleri kullan›larak de›erlendirildi. Her iki yöntemde de ekstrakt, uçucu
ya›dan daha iyi antioksidatif kapasite gösterdi. Ekstrakt serbest DPPH radikallerini uçucu ya¤dan (8900 µg/ml) daha düflük IC 50 seviyesi (89.90 µg/ml) ile indirgedi. β-karoten/linoleik asit yönteminde örnekler, linoleik oksidasyonunu etkili bir flekilde engelleyememifltir; 2 mg/ml'de BHT (%97.0)'›n çok alt›nda kalarak, s›ras›yla; sadece %22.7(ekstrakt) ve %16(uçucu ya¤)'l›k inhibisyon gösterdi. Ekstrakt›n toplam fenolik ö¤eleri, gallik asit eflde›eri 51 µg/ml (% 5.1 w/w) olarak tespit edildi. Uçucu y a¤›n GC-MS analizi %92.24'lük de¤erlendirme ile 64 bileflen tan›lanarak sonuçland›. Piperiton, kamfor ve 1,8-sineol (ökaliptol) uçucu ya¤›n ana bileflenleri olarak belirlendi.
Anahtar Sözcükler: Achillea biebersteinii, Antimikrobiyal aktivite, Antioksidan aktivite, GC-MS analiz; piperiton, kamfor ve ökaliptol
65 Biological Activities of the Essential Oil and Methanol Extract of Achillea biebersteinii Afan. (Asteraceae)
Introduction voucher specimen has been deposited at the Herbarium of Plant-derived substances/drugs have recently become of the Department of Biology, Atatürk University (ATA HERB great interest owing to their versatile applications. In 9788). particular, herbal medicine is as old as the history of Preparation of the extracts humankind. As can be traced by many plants, species The air-dried and finely ground sample was extracted as belonging to the genus Achillea (Asteraceae) are such plants; described elsewhere (9). Briefly, the sample weighing about the genus is said to be named after Achilles, who used 100 g was extracted in a Soxhlet apparatus with methanol Achillea spp. for healing soldiers wounded during the Trojan (MeOH) at 60 °C for 6 h. The extract was filtered and War (1). Their herbal parts are known to be used in folk concentrated in vacuo at 45 °C, yielding a waxy material medicine for the treatment of several diseases, disorders (19.38% w/w), which was then lyophilized and kept in the and ailments (2). These species have some interesting dark at +4 °C until tested. properties and are used in cosmetics, fragrances and Isolation of the essential oils agriculture, e.g., plant protection (3). The air-dried and ground herbal parts were submitted The genus Achillea is represented by about 85 species to water-distillation for 3 h using a Clevenger-type throughout the world and 42 of them are found in the flora apparatus (yield 0.63% v/w). The obtained essential oil of Turkey; 23 of these are endemics (4,5) (EO) was dried over anhydrous sodium sulfate and, after A. biebersteinii Afan. (Asteraceae, Section: Filipendulinae filtration, stored at +4 ºC until tested and analyzed. (D.C.) Boiss) (syn. A. micrantha) is a perennial herb, villose, GC-MS analysis conditions stems erect, simple or branched from the base; 30-60 cm high; leaves up to 10 cm, oblong-lanceolate in outline, The analysis of the essential oil was performed using a pinnatisect into numerous narrow segments, segments Thermofinnigan Trace GC/Trace DSQ /A1300 (E.I divided into minute linear-lanceolate mucronate lobes; the Quadrapole) equipped with a SGE-BPX5 MS capillary heads are radiate, in large dense compound corymbs; column (30 m x 0.25 µm i.d., 0.25 mm). For GC-MS involucre 4–5 mm, oblong-ovoid; flowering period, detection an electron ionization system with ionization April–May. A. biebersteinii is locally named ‘Sarıçiçek’ in energy of 70 eV was used. Helium was the carrier gas, at a Turkish, and is used as a folk remedy to treat abdominal flow rate of 1 ml/min. Injector and MS transfer line pain, wounds and stomachache (6,7). Reports concerning temperatures were set at 220 and 290 °C, respectively. The the antimicrobial and antioxidant properties of this species program used was 50-150 °C at a rate of 3 °C/min, held are also available in the literature (8). isothermal for 10 min and finally raised to 250 °C at 10 °C/min. Diluted samples (1/100, v/v, in methylene chloride) The objectives of this study are: i) to analyze the of 1.0 µl were injected manually and in the splitless mode. chemical composition of a hydrodistilled essential oil of A. The components were identified based on the comparison of biebersteini plants collected from Erzurum, in the eastern their relative retention time and mass spectra with those of Anatolia region of Turkey, by a GC/MS system in order to standards, Wiley7N, TRLIB library data of the GC-MS determine the essential oil chemotype; ii) to investigate the system and literature data (10). The results were also in vitro antimicrobial and antioxidant activities of methanol confirmed by the comparison of the compounds’ elution extract and the essential oil isolated from A. biebersteinii. order with their relative retention indices on non-polar phases reported in the literature. Materials and Methods Antimicrobial activity Collection of plant material Microbial strains Achillea biebersteinii was collected at the flowering stage The methanolic extracts and the essential oil of A. from Erzurum. The taxonomic identification of plant biebersteinii was individually tested against a panel of materials was confirmed by a senior plant taxonomist, microorganisms including total 55 microbial cultures Meryem fiengül, at the Department of Biology, Atatürk belonging to 35 bacteria and 19 fungi and a yeast species. University, Erzurum. Collected plant materials were dried in The list of microorganisms used in this study is given in the shade, and the leaves were separated from the stem, Tables 1 and 2. Microorganisms were provided by the and ground in a grinder with a mesh 2 mm in diameter. The Department of Biology, Atatürk University.
66 Ö. BARIfi, M. GÜLLÜCE, F. fiAH‹N, H. ÖZER, H. KILIÇ, H. ÖZKAN, M. SÖKMEN, T. ÖZBEK
Table 1. Antimicrobial activities of the essential oil Achillea biebersteinii against the bacterial strains tested.
Achillea biebersteinii Antibioticsa Test microorganisms DDb MICc DDa MICa (max)
Acinetobacter baumanii-A8 8 500 18 (OFX) 31.25 Acinetobacter lwoffi F1 - - 24 (OFX) 62.50 Bacillus macerans-A199 12 250 19 (OFX) 15.62 Bacillus megaterium – A59 - - 9 (SCF) 15.62 Bacillus subtilis-ATCC-6633 - - 28 (OFX) 62.50 Bacillus subtilis-A57 12 250 28 (OFX) 125 Brucella abortus A77 - - 12 (SCF) 62.50 Burkholdria cepacia A225 - - 22 (SCF) 125 Clavibacter michiganense-A227 - - 25 (SCF) 16.62 Cedecea davisae F2 - - 14 (OFX) 62.50 Enterobacter cloacae-A135 - - 20 (NET 31.25 Enterococcus faecalis-ATCC-29122 - - 18 (SCF) 31.25 Escherichia coli-A1 13 250 - (OFX) 62.50 Klebsiella pneumoniae F3 - - 12 (OFX) 125 Klebsiella pneumoniae-A137 - - 12 (OFX) 125 Morgonella morganii F4 - - 14 (OFX) 125 Proteus vulgaris-A161 - - 12 (OFX) 125 Proteus vulgaris-KUKEM1329 - - 13 (OFX) 125 Pseudomonas aeruginosa-ATCC9027 - - 22 (NET) 31.25 Pseudomonas aeruginosa-ATCC27859 - - 22 (NET) 15.62 Pseudomonas aeruginosa F5 - - 18 (NET) 125 Pseudomonas pseudoalkaligenes F6 - - 18 (NET) 125 Pseudomonas syringae pv.tomato A35 - - 24 (OFX) 125 Salmonella cholerasuis arizonae F7 - - 14 (NET) 250 Salmonella enteritidis-ATCC-13076 - - 27 (SCF) 62.50 Serratia plymuthica F8 - - 16 (NET) 125 Shigella sonnei F9 - - 24 (NET) 31.25 Staphylococcus aureus-A215 10 500 22 (SCF) 31.25 Staphylococcus aureus-ATCC-29213 10 500 22 (SCF) 62.50 Staphylococcus epidermis-A233 - - - (SCF) 15.62 Staphylococcus hominis F10 - - - (SCF) 15.62 Streptococcus pyogenes-ATCC-176 - - 10 (OFX) 62.50 Streptococcus pyogenes-KUKEM-676 - - 13 (OFX) 31.25 Xanthomonas campestris-A235 - - 20 (SCF) 31.25 Yersinia enterocolitica F11 - - 16 (OFX) 62.50 aDD = OFX = Ofloxacin (10 µg/disc); SCF = sulbactam (30 µg)+ cefoperazona (75 µg) (105 µg/disc) and NET = Netilmicin, (30 µg/disc) were used as positive reference standards antibiotic discs (Oxoid); a MIC= Maxipine (µg/ml) was used as reference antibiotic in micro well dilution assay (Sigma). b Inhibition zone in diameter (mm) around the discs impregnated with 300 µg/disc of methanol extract. c Minimal Inhibitory concentrations (µg/ml)
67 Biological Activities of the Essential Oil and Methanol Extract of Achillea biebersteinii Afan. (Asteraceae)
Table 2. Anticandidal and antifungal activities of the essential oil Achillea biebersteinii and the yeast and fungi isolates.
Achillea biebersteinii Antibioticsa Test yeast and fungi DDb MICc DDa MICa (Amp B)
yeast Candida albicans-A117 12 250 - (NET) 31.25 Fungi Alternaria alternate 21 62.50 - (NET) 31.25 Alternaria solani - - - (NET) 31.25 Aspergillus flavus 21 62.50 - (NET) 15.62 Aspergillus niger - - - (NET) 15.62 Aspergillus variecolor 23 31.25 - (NET) 15.62 Fusarium acuminatum 35 15.62 - (NET) 62.50 Fusarium oxysporum 35 15.62 - (NET) 62.50 Fusarium solani 12 125 - (NET) 62.50 Fusarium tabacinum - - - (NET) 62.50 Microsporum canis - - - (NET) 62.50 Moniliania fructicola 22 31.25 - (NET) 15.62 Mortieraula alpine - - - (NET) 62.50 Penicillum spp. 15 62.50 - (NET) 31.25 Rhizopus spp. 24 31.25 - (NET) 125 Rhizoctonia solani 35 15.62 - (NET) 31.25 Sclorotinia minor 35 15.62 - (NET) 125 Sclorotinia sclerotiorum 35 15.62 - (NET) 62.50 Trichophyton rubrum 8 500 - (NET) 31.25 Trichophyton mentagrophytes 35 15.62 - (NET) 15.62
a DD = NET = Netilmicin (30 µg/disc) was used as positive reference standards antibiotic discs (Oxoid). a MIC = AmpB = Amphotericin B (µg/ml) was used as reference antibiotic in micro well dilution (Sigma). b Inhibition zone in diameter (mm) around the discs impregnated with 300 µg/disc of methanol extract. c Minimal Inhibitory concentrations (µg/ml)
Disc-diffusion assay using the same solvents employed to dissolve the plant The dried plant extracts were dissolved DMSO to a final extracts. Ofloxacin (10 µg/disc), sulbactam (30 µg) + concentration of 30 mg/ml and sterilized by filtration by cefoperazona (75 µg) (105 µg/disc) and/or netilmicin (30 0.45 µm Millipore filters. Antimicrobial tests were then µg/disc) were used as positive reference standards to carried out by the disc diffusion method (11) using 100 µl determine the sensitivity of each strain/isolate of microbial of suspension containing 10 8 CFU/ml of bacteria, 10 6 species tested. The inoculated plates were incubated at 37 CFU/ml of yeast and 10 4 spore/ml of fungi spread on °C for 24 h for clinical bacterial strains, 48 h for yeast and nutrient agar (NA), Sabouraud dextrose agar (SDA), and 72 h for fungi isolates. Plant associated microorganisms potato dextrose agar (PDA) medium, respectively. The were incubated at 27 °C. Antimicrobial activity was discs (6 mm in diameter) were impregnated with 10 µl of evaluated by measuring the zone of inhibition against the essential oil or the 30 mg/ml extracts (300 µg/disc) placed test organisms. Each assay in this experiment was on the inoculated agar. Negative controls were prepared repeated twice.
68 Ö. BARIfi, M. GÜLLÜCE, F. fiAH‹N, H. ÖZER, H. KILIÇ, H. ÖZKAN, M. SÖKMEN, T. ÖZBEK
Micro-well dilution assay solutions were immediately poured into petri plates after 4 The minimal inhibition concentration (MIC) values vortexing. The plates were spot inoculated with 5 µl (10 were studied for the bacterial strains sensitive to the spore/ml) of each fungi isolate. Amphotericin B (Sigma A essential oil and/or extracts in the disc diffusion assay. 4888) was used as a reference antifungal drug. The The inocula of the bacterial strains were prepared from inoculated plates were incubated at 27 and 37 °C for 72 12 h broth cultures, and suspensions were adjusted to h for plant and clinical fungi isolates, respectively. At the 0.5 McFarland standard turbidity. The essential oil A. end of the incubation period, the plates were evaluated biebersteinii dissolved in 10% dimethylsulfoxide (DMSO) for the presence or absence of growth. MIC values were were first diluted to the highest concentration (500 determined as the lowest concentration of the essential µg/ml) to be tested, and then serial 2-fold dilutions were oil where absence of growth was recorded. Each test made in order to obtain a concentration range from 7.8 repeated at least twice. to 500 µg/ml in 10 ml sterile test tubes containing Antioxidant activity nutrient broth. MIC values of A. biebersteinii essential oil The antioxidative capacity of the samples was against bacterial strains were determined based on a evaluated by using DPPH and ß-carotene/linoleic acid micro-well dilution method (11). assays as given elsewhere (11). In both assays, butylated The 96-well plates were prepared by dispensing into hydroxytoluene (BHT) was used as the positive control. each well 95 µl of nutrient broth and 5 µl of inoculum. A DPPH assay 100 µl aliquot from the stock solutions of plants essential oil initially prepared at the concentration of 500 µg/ml This spectrophotometric assay uses stable free radical was added into the first wells. Then 100 µl from their diphenylpicrylhydrazyl (DPPH) as a reagent (13). Fifty serial dilutions were transferred into 6 consecutive wells. microliters of various concentrations of the samples in The last well, containing 195 µl of nutrient broth without methanol was added to 5 ml of a 0.004% methanol compound and 5µl of the inoculum on each strip was used solution of DPPH. After a 30 min incubation period at as a negative control. The final volume in each well was room temperature the absorbance was read against a 200 µl. Maxipime (Bristol-Myers Squibb) at the blank at 517 nm. Inhibition of free radical DPPH in concentration range of 500-7.8 µg/ml was prepared in percent (I %) was calculated in following way: nutrient broth and used as standard drug for the positive I %= ( A blank– Asample / Ablank) x 100 control. The plate was covered with a sterile plate sealer. where A blank is the absorbance of the control reaction Contents of each well were mixed on plate shaker at 300 (containing all reagents except the test compound), and rpm for 20 s and then incubated at appropriate Asample is the absorbance of the test compound. Extract temperatures for 24 h. Microbial growth in each medium concentration providing 50% inhibition (IC 50) was was determined by reading the respective absorbance calculated from the graph plotted of inhibition percentage (Abs) at 600 nm using the ELx 800 universal microplate against extract concentration. Tests were carried out in reader (Biotek Instrument Inc., Highland Park, Vermont, triplicate. USA) and confirmed by plating 5 µl samples from clear β wells on nutrient agar medium. The essential oil tested in -Carotene-linoleic acid assay this study was screened twice against each organism. The A stock solution of β–carotene-linoleic acid mixture MIC values were defined as the lowest concentration was prepared as follows: 0.5 mg β–carotene was dissolved required for inhibiting the growth of microorganisms. in 1 ml of chloroform (HPLC grade) and 25 µl of linoleic MIC agar dilution assay acid and 200 mg Tween 40 was added. Chloroform was completely evaporated using a vacuum evaporator. Then MIC values for the fungal isolates were determined by 100 ml of distilled water saturated with oxygen (30 min the agar dilution method as described previously (12). 100 ml/min) was added with vigorous shaking. After that, The essential oil A. biebersteinii was added aseptically to 2500 µl of this reaction mixture was dispersed to test sterile molted PDA medium containing Tween 20 (Sigma tubes and 350 µl portions of the extracts prepared at 2 g/l 0.5%, v/v) in appropriate volumes to produce the concentrations were added and the emulsion system was concentration range of 7.8-500 µg/ml. The resulting PDA incubated for up to 48 h at room temperature. The same
69 Biological Activities of the Essential Oil and Methanol Extract of Achillea biebersteinii Afan. (Asteraceae)
procedure was repeated with positive control BHT and a activity against only 6 bacteria strains tested (Table 1). blank. After this incubation period absorbance of the Compared to the antibiotics employed, the activity was mixtures was measured at 490 nm. Antioxidant capacities weak. As the essential oils of A. biebersteinii have not of the extracts were compared with those of BHT and been fully evaluated against bacteria in a broad spectrum, blank (14). our results could be evaluated as the first report on the Assay for total phenolics antibacterial features of A. biebersteinii . Furthermore, the oil was found to have strong antifungal activities Total phenolic constituents of the extracts were against some of the fungi species listed in Table 2. The determined by employing the methods given in the maximal inhibition zones and MIC values for fungi strains, literature involving Folin-Ciocalteu reagent and gallic acid which were sensitive to the essential oil of A. as standard (11,15). biebersteinii, were 19-35 mm and 15.62-125 µl/ml respectively (Table 2). Results and Discussion As far as antioxidative capacity of the samples is The chemical composition of A. biebersteinii essential concerned, the extract was more effective in the DPPH oil was analyzed by employing GC-MS, leading to a assay, as it reduced the stable free radical DPPH with a comparison of the relative retention times and the mass lower IC 50 value (89.90 mg/ml) than the oil (8900 spectra of oil components with those of authentic samples mg/ml), showing that the extract is 100-fold stronger and mass spectra from the data library. As shown in than the oil. Nevertheless, both samples had weaker Table 3, GC/MS analysis resulted in the identification of antioxidative capacity than the positive control, butylated
64 compounds representing 92.24% of the oil. A high hydoxytoluene (BHT), which had a very low IC 50 value number of oxygenated monoterpenes characterized the (19.3 mg/ml) in the DPPH assay. Similarly, in β- oil composition. Piperitone (31.06%), camphor carotene/linoleic acid assay, the samples were not (12.46%), and eucalyptol (10.98%) were the main effectively able to inhibit the linoleic acid oxidation; components, comprising 54.5% of the essential oil (Table exhibiting only 22.7% (the extract) and 16% (the 3). Compositions of the essential oils of A. biebersteinii, essential oil) inhibitions at 2 mg/ml, respectively; being collected from different localities, have been reported far below than that of BHT (97.0%). As shown in Table before. As far as the existence of a major component, 4, our results demonstrate that neither the extract nor piperitone, is taken into account, our findings are the essential oil has powerful antioxidants. Weaker identical to those reported for the plants collected from antioxidative capacity of the extract could be attributed to Sivas (Turkey) (8) Ankara (Turkey) (16) and from Iran the presence of polar phenolics as they were found in the (17), whereas ascaridole (18) and eucalyptol (1,8- extract at low concentration [51 mg/mg (5.1%, w/w, as cineole)(19) were given as major constituents in the other gallic acid equivalent)]. For the essential oil, on the other reports. Camphor, eucalyptol and/or borneol have been hand, the weak activity could inevitably be expected, as found as major compounds in some Achillea species the main constituents were piperitone, camphor and (20,21). The richness of these monoterpenes may be due eucalyptol, none of which have been proven to be strong to the collection of plant material during the flowering antioxidant agents as emphasized elsewhere (22). As far period and therefore may not be used to characterize as DPPH results are concerned, the results mentioned in species belonging to different sections. the previous report (8) are 2-fold higher than our The antimicrobial activities of A. biebersteinii essential findings. oils and methanol extracts assayed against a wide range of human, plant-associated and food-borne Conclusions microorganisms and their potency was qualitatively and quantitatively assessed by evaluating the presence of This result suggests that Achillea biebersteinii inhibition zones, zone diameter, and MIC values. essential oil possesses compounds with antimicrobial Methanol extract of A. biebersteinii did not exert any properties that can be used as especially antifungal and antimicrobial activity against any test microorganism anticandidal agents in new drugs for treating infectious selected. However, the essential oil showed antibacterial diseases in humans, and plant diseases.
70 Ö. BARIfi, M. GÜLLÜCE, F. fiAH‹N, H. ÖZER, H. KILIÇ, H. ÖZKAN, M. SÖKMEN, T. ÖZBEK
Table 3. Chemical composition of Achillea biebersteinii essential oil (GC-MS).
b No R › tR(min) Components Composition (%)
1 921 10.24 α-Pinene 0.15 2 944 10.97 Camphene 0.05 3 975 12.08 Sabinene 0.07 4 981 12.33 β-Pinene 0.15 5 1015 13.69 α-Phellandrene 0.75 6 1027 14.19 α-Terpinene 0.36 7 1037 14.65 p-cymene 0.81 8 1044 14.99 1,8-Cineole 10.93 9 1069 16.19 γ-Terpinene 0.21 10 1083 16.91 trans-Sabinenhydrate 0.16 11 1094 17.50 Terpinolene 0.05 12 1109 18.30 Linalool 0.45 13 1115 18.67 Nonanal 0.04 14 1131 19.59 cis-Ment-2-en-1-ol 3.18 15 1134 19.80 α-Camholenal 0.27 16 1136 19.89 1,3,4-trimethyl-3-cyclohexene-1-carboxaldehyde 0.16 17 1146 20.52 trans-Ment-2-en-1ol 3.28 18 1148 20.70 trans-Verbenol 0.89 19 1152 20.90 Camphor 12.46 20 1160 21.46 Pinocamphone 1.61 21 1162 21.57 Pinocarvone 2.82 22 1164 21.74 Safranal 0.10 23 1168 22.00 Borneol 1.74 24 1173 22.33 4-Terpineol 1.44 25 1180 22.80 p-Cymen-8-ol 0.13 26 1181 22.89 Crypton 0.24 27 1184 23.09 α-Terpineol 2.61 28 1185 23.20 Myrtenal 0.22 29 1192 23.70 trans-3-methyl-6-(1-methylethyl)-2-cyclohexen-1-ol 1.58 30 1199 24.23 trans-Carveol 0.17 31 1211 25.15 Pulegone 1.27 32 1213 25.26 Ascaridole 0.49 33 1216 25.49 Cuminyl aldehyde 0.34 34 1223 26.10 Piperitone 31.06 35 1235 27.05 Bornyl acetate 1.28 36 1237 27.23 Sabinyl acetate 0.38 37 1241 27.54 Thymol 0.41 38 1245 27.90 Carvacrol 0.21 39 1249 28.28 Isoascaridole 0.77 40 1261 29.30 p-Mentha-1,4-dien-7-ol 0.11 41 1266 29.81 Piperitenone 0.34 42 1271 30.29 Eugenol 0.11 43 1276 30.77 Piperitenone oxide 0.28 44 1278 30.94 α-Copaene 0.29
71 Biological Activities of the Essential Oil and Methanol Extract of Achillea biebersteinii Afan. (Asteraceae)
Table 3. Continued.
b No R › tR(min) Components Composition (%)
45 1282 31.30 β-Bourbonene 0.12 46 1284 31.54 β-Elemene 0.27 47 1290 32.09 cis-Jasmone 0.21 48 1291 32.21 Nepetalactone 0.55 49 1298 32.89 trans-Caryophyllene 1.52 50 1300 33.15 α-Ionone 0.02 51 1302 33.33 β-Gurjunene 0.07 52 1313 34.45 β-Humulene 0.25 53 1323 35.52 Germacrene D 1.47 54 1326 35.90 β-Selinene 0.08 55 1328 36.13 Germacrene B 0.19 56 1332 36.54 γ-Bisabolene 0.19 57 1336 37.03 δ-Cadinene 0.28 58 1343 37.84 n-Terpinenyl ester of isopentanoic acid 0.70 59 1349 38.54 Bornyl ester of n-pentanoic acid 0.17 60 1362 40.13 Spathulenol 0.74 61 1364 40.42 Caryophyllene oxide 1.01 62 1401 45.28 β-Eudesmol 0.34 63 1429 49.40 7-Ethyl-1,4-dimethyl azulene 1.0 64 1445 51.96 Hexahydrofarnesyl acetone 0.21
Total 92.24
a Retention time (as minutes), b Compounds listed in order of elution from a HP-5 MS column, c trace ≤ 0.06%
Table 4. Amounts of total phenolic and effects of the essential oil and metanol extract of Achillea biebersteinii on the in vitro free radical (DPPH) scavenging and β-Carotene/Linoleic Acid Assaysa.
Sample Total phenolicsb DPPHc β-carotene/linoleic acidd
Essential oil 8900.00 ± 20 16 ± 1.0 MeOH 5.1 ± 0.6 89.90 ± 6.7 22.7 ± 1.0 BHT (positive control) 19.3 ± 0.4 97 ± 1.0
a Results are presented as mean ± standard deviation b Amounts of total phenolics are given as gallic acid equivalent (µg/mg) c IC50 values of DPPH assay (as µg/ml) d Given as percentage inhibition of the linoleic acid.
Acknowledgments Corresponding author: This study was supported by a grant from the Özlem BARIfi Research Funds appropriated to Atatürk University. Atatürk University, Faculty of Arts and Science, Department of Biology, 25240, Erzurum, Turkey E-mail: [email protected]
72 Ö. BARIfi, M. GÜLLÜCE, F. fiAH‹N, H. ÖZER, H. KILIÇ, H. ÖZKAN, M. SÖKMEN, T. ÖZBEK
References
1. Cheers G. Botanica. Könemann-Verlag, Köln; 1999. pp. 1020. 12. Sahin F, Karaman I, Gulluce M et al. Evaluation of antimicrobial activities of Satureja hortensis L. Journal of Ethnopharmacology, 2. Skocibusic M, Bezic N, Dunkic V et al. Antibacterial activity of 87: 61-65, 2003. Achillea clavennae essential oil against respiratory tract pathogens. Fitoterapia, 75: 733-736, 2005. 13. Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytotheraphy Research, 14: 323-328, 2000. 3. Senatore F, Napolitano F, Arnold NA et al. Composition and antimicrobial activity of the essential oil of Achillea falcata L. 14. Dapkevicius A, Venskutonis R, Van Beek TA et al. Antioxidant (Asteraceae). Flavour and Fragrance Journal. 20: 291-294, activity of extracts obtained by different isolation procedures 2005. from some aromatic herbs grown in Lithuania. J of the Science of Food and Agriculture, 77: 140-146, 1998. 4. Huber-Morath A. Achillea L. In: Davis PH. (Ed.), Flora of Turkey and the East Aegean Islands, Edinburgh University Press, 15. Chandler SF, Dodds JH. The effect of phosphate, nitrogen and Edinburgh 1975. sucrose on the production of phenolics and solasidine in callus cultures of Solanum lacinitum. Plant Cell Reports, 2: 105, 1983. 5. Duman H. Achillea L. In: Guner A, Ozhatay N, Ekim T, Baser KHC. (Eds.), Flora of Turkey and the East Aegean Islands, vol. 11. 16. Küsmeno¤lu S, Bafler KHC, Özek T et al. Constituents of the Edinburgh University Press, Edinburgh 2000, pp. 158-159. essential oil of Achillea biebersteinii Afan. J Essent Oil Res 7: 527- 528, 1995. 6. Baytop T. Türkçe Bitki Adları Sözlü¤ü, A Dictionary of Vernacular Names of Wild, Türk Dil Kurumları Yayınları , Ankara, 578: pp 17. Jaimand K, Rezaee MB. Comparative study of the essential oils of 163-238, 1997. three Achillea species from Iran. J Essent Oil Res 13: 354-356, 2001. 7. Sezik E, Yeflilada E, Honda G et al. Traditional medicine in Turkey X. Folk medicine in Central Anatolia. J Ethnopharmacol 75: 95- 18. Rustaiyan A, Komeilizadeh H, Shariatpanahi MS. Comparative 115, 2001. study of the essential oils of three Achillea Species from Iran. J Essent Oil Res 10: 207-209, 1998. 8. Sokmen A, Sokmen M, Daferera D et al. The in vitro antioxidant and antimicrobial activities of the essential oil and methanol 19. Chialva F, Monguzzi F, Manitto P et al. Essential oil constituents extracts of Achillea biebersteini Afan. (Asteraceae). Phytother. of Achillea biebersteinii Afan. J Essent Oil Res 5: 87-88, 1993. Res. 18: 451-456, 2004. 20. Weyerstahl P, Marschall H, Seelmann I et al. Constituents of the 9. Sökmen A, Jones BM, Ertürk M. The in vitro antibacterial essential oil of Achillea eriophora DC. Flavour Fragrance J 12: 71- activities of Turkish medicinal plants. J Ethnopharmacol. 67: 79- 78, 1997. 86, 1999. 21. Chalchat JC, Gorunovic MS, Petrovic SD. Aromatic plants of 10. Adams RP. Identification of Essential Oils components by Gas Yugoslavia. I. chemical composition of the oils of Achillea Chromatography/ Quadrupole Mass Spectroscopy, Allured millefolium L. ssp. pannonica (Scheele) Hayak, A. Crithmifolia W. Publishing Corporation. Illinois, USA, 2001. et K., A. serbica Nym. and A. tanacetifolia All. J Essent Oil Res 11: 306-310, 1999. 11. Gulluce M, Sokmen M, Daferera D et al. The in vitro antibacterial, antifungal and antioxidant activities of the essential oil and 22. Ruberto G, Barrata MT. Antioxidant activity of selected essential methanol extracts of herbal parts and callus cultures of Satureja oil components in two lipid model systems. Food Chemistry, 69: hortensis L. Journal of Agricultural and Food Chemistry, 51: 167-174, 2000. 3958-3965, 2003.
73