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Chemical composition, antimicrobial and antioxidant activities of Centaurea ensiformis Hub.-Mor. (Asteraceae), a species endemic to Mugla (Turkey)
Aysel Ugur a; Mehmet Emin Duru b; Ozgur Ceylan a; Nurdan Sarac c; Omer Varol a; Ibrahim Kivrak b a Department of Biology, Faculty of Arts and Sciences, Mugla University, Turkey b Department of Chemistry, Faculty of Arts and Sciences, Mugla University, Turkey c Medical Laboratory Program, Vocational School of Health Services, Mugla University, Turkey Online Publication Date: 01 January 2009

To cite this Article Ugur, Aysel, Duru, Mehmet Emin, Ceylan, Ozgur, Sarac, Nurdan, Varol, Omer and Kivrak, Ibrahim(2009)'Chemical

composition, antimicrobial and antioxidant activities of Centaurea ensiformis Hub.-Mor. (Asteraceae), a species endemic to Mugla (Turkey)',Natural Product Research,23:2,149 167
To link to this Article: DOI: 10.1080/14786410801915770 URL: http://dx.doi.org/10.1080/14786410801915770

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Natural Product Research Vol. 23, No. 2, 20 January 2009, 149167

Chemical composition, antimicrobial and antioxidant activities of Centaurea ensiformis Hub.-Mor. (Asteraceae), a species endemic to Mugla (Turkey)
Aysel Ugura*, Mehmet Emin Durub, Ozgur Ceylanc, Nurdan Saracd, Omer Varola and Ibrahim Kivrakb
Department of Biology, Faculty of Arts and Sciences, Mugla University, Kotekli-Mugla, Turkey; Department of Chemistry, Faculty of Arts and Sciences, Mugla University, Kotekli-Mugla, Turkey; cDepartment of Biology, Faculty of Arts and Sciences, Mugla University, Kotekli-Mugla, Turkey; dMedical Laboratory Program, Vocational School of Health Services, Mugla University, Marmaris-Mugla, Turkey
b a

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(Received 19 June 2007; final version received 16 January 2008) The n-hexane, chloroform, ethyl acetate and ethyl alcohol extracts of the aerial parts of Centaurea ensiformis, endemic to Turkey, have been assessed for antimicrobial and antioxidant activities. The chemical composition of the hexane extract of this plant was determined. The antimicrobial activities on microorganisms, including multiple antibiotic resistant bacteria, were evaluated using the disc diffusion method. The antioxidant activities were determined by using four complementary in vitro assays: inhibition of 2,2-diphenyl-1-picrylhydrazyl radical, total antioxidant activity, and the total amount of phenolic and flavonoid compounds. The structure of the active extract was elucidated by: thin layer chromatography, column chromatography, gas chromatography or gas chromatography-mass spectrometry. The various extracts of C. ensiformis inhibited the growth of tested bacteria. All the C. ensiformis extracts had no effect on the yeasts. The total antioxidant activity increased with the increasing amount of the extracts (20, 40, 80 and 160 mg), which contained linoleic acid emulsion. The major compounds of the hexane extract of the plant were caryophyllene oxide (28.72%), spathulenol (17.81%), eudesmol (13.03%) and bourbonene (8.51%). Keywords: Centaurea ensiformis; chemical composition; antimicrobial activity; antioxidant activity

1. Introduction The genus Centaurea L. (Asteraceae) is represented in Turkish flora by 179 species, of which 111 are endemic (Erik, 2004). This genus, with the rate of 61, is among the richest genera in terms of endemic species (Wagenitz, 1975). The aerial parts of the plant are known as peygamber cicegi, zerdali dikeni, coban kaldiran, timur dikeni in Turkey (Baytop, 1999; David, 1997; Wagenitz, 1975). Many species of the genus Centaurea have

*Corresponding author. Email: ayselugur@hotmail.com

ISSN 14786419 print/ISSN 10292349 online 2009 Taylor & Francis DOI: 10.1080/14786410801915770 http://www.informaworld.com

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long been used in traditional medicine to cure various ailments (Sarker, Savchenko, Whiting, Sik, & Dinan, 1997) and also many members of the genus are reported to be used in Anatolian folk medicine (Baytop, 1999; Sarker et al., 1997; Sezik et al., 2001; Yesilada et al., 1995, 1999). Various biological activities of the members of this genus were reported elsewhere; cytogenetic (Radic, Prolic, Pavlica, & Pevalek-Kozlina, 2005), antimicrobial (Barbour et al., 2004; Buruk, Sokmen, Aydin, & Erturk, 2006; Garbacki et al., 1999; Karamenderes, Khan, Tekwani, Jacob, & Khan, 2006; Kose, Iscan, Demirci, Baser, & Celik, 2007; Kumarsamy et al., 2002, 2003; Skaltsa et al., 2000; Skliar, Toribio, & Oriani, 2005; Yayli et al., 2005), antifungal (Barrero et al., 1997, 2000; Koukoulitsa, 2005; Skaltsa et al., 2000; Vajs, 1999), anti-inflammatory (Garbacki et al., 1999; Negrete et al., 1993), antiulcerogenic activities (Yesilada et al., 1999), antioxidant properties (Oncel, Yurdakulol, Keles, Kurt, & Yildiz, 2004; Severino, Stich, & Soja, 2007; Tepe, Sokmen, Akpulat, Yumrutas, & Sokmen, 2006), antiviral (Rusak, Krajacic, & Plese, 1997), anti-Helicobacter pylori (Stamatis et al., 2003), antiplasmodial (Medjroubi, Benayache, & Bermejo, 2005), antiprotozoal (Karamenderes et al., 2006), anti-colon cancer (Shoeb et al., 2006) and cytotoxic activity (Medjroubi et al., 2005; Shoeb et al., 2006). Chemical compositions of the members of this genus were also reported by some studies (Dural et al., 2003; Yayli et al., 2005). Centaurea ensiformis P.H. Davis occurs on serpentine in open Pinus nigra subsp. pallasiana forest on Sandras Mountain, Mugla, Turkey. The plant is locally endemic to Sandras Mountain and it is an East-Mediterranean element (Wagenitz, 1975). Resistance to antimicrobial agents has become an increasingly important and pressing global problem (Alexandria, 2004; Cushnie & Lamb, 2005). In recent years, there has been an alarming increase in nosocomial staphylococcal infections by strains with multiple drug resistance (MDR) (Al-Masaudi, Day, & Russell, 1991; Hiramatsu, 1997; Kloos & Bannerman, 1995; Lyon & Skurray, 1987). At present, this situation is leading to evaluation of staphylococcal pathogens as potentially resistant to any available antibiotic (Huycke, Sahm, & Gilmore, 1998; Lyon & Skurray, 1987; Noble, Virani, & Cree, 1992; Rubin et al., 1999). Stenotrophomonas maltophilia is intrinsically resistant to many commonly used antimicrobial agents and a rapid selection of high-level multidrugresistant isolates in clinical strains has also been observed (Fass, Barnishan, Solomon, & Ayers, 1996; Krueger, Clark, & Nix, 2001). Stenotrophomonas maltophilia may be responsible for a wide spectrum of diseases. The respiratory tract, however, is the most common site of infection, and the incidence of these infections is on the increase (from 2.5% in 1998 to 4.1% in 1999) (Spencer, 1996; Gales et al., 2001). Over the past decade, however, S. maltophilia has emerged as an important nosocomial pathogen (Calza, Manfredi, & Chiodo, 2003; Denton & Kerr, 1998) with patient morbidity and mortality similar to that observed with other gram negative infections (Hanes et al., 2002; Morrison, Hoffmann, & Wenzel, 1986; Senol, DesJardin, Stark, Barefoot, & Snydman, 2002). Nowadays, the development of resistance by a pathogen to many of the commonly used antibiotics provides an impetus for further attempts to search for new antimicrobial agents to combat infections and overcome problems of resistance and the side effects of the currently available antimicrobial agents (Ali-Shtayeh, Yaghmour, Faidi, Salem, & Al-Nuri, 1998; Primo et al., 2001). New chemical entities with such activities may be identified through a variety of approaches, one of them being screening of natural products (Bedoya, Sanchez-Palomino, Abad, Bermejo, & Alcami, 2001). Natural products can be selected for biological screening based on ethnomedical use of plants, because many

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infectious diseases are known to have been treated with herbal remedies throughout the history of mankind. Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries (Zakaria, 1991; Sokmen, Jones, & Erturk, 1999). Natural products have traditionally been a rich source of antimicrobial agents (Silver & Bostian, 1990) and flavonoids, a group of heterocyclic organic compounds that occur widely in the plant kingdom (Havsteen, 1983), are increasingly becoming the subject of medical research (Harborne & Williams, 2000). Flavonoids have been proven to display a wide range of pharmacological and biochemical actions such as antimicrobial, antithrombotic, antimutagenic and anticarcinogenic activities (Cook & Samman, 1996; Harborne & Baxter, 1999; Harborne & Williams, 2000; Havsteen, 1983; Kandasawami & Middleton, 1997; Sahu & Green, 1997). One of the largest classes of naturally-occurring polyphenolic compounds are the flavonoids (Geissman & Crout, 1969). Many pharmacological effects of flavonoids are linked to their ability to act as strong antioxidants and free radical scavengers, to chelate metals, and to interact with enzymes, adenosine receptors, and biomembranes (Mills & Bone, 2000). It was reported that the antioxidant activity of plant materials was well correlated with the content of their phenolic compounds (Velioglu, Mazza, Gao, & Oomah, 1998). This study aimed to screen the chemical composition, antimicrobial and antioxidant activities of C. ensiformis. We can conclude that it may be beneficial to pharmacy, and it deserves further study.

2. Materials and methods 2.1. Plant material Centaurea ensiformis, locally endemic to the Sandras Mountain, Mugla, Turkey, and naturally growing plants belonging to Asteraceae (Compositae) family were collected at the flowering stage in July 2003 from Mugla, Turkey. A voucher specimen (Herbarium No: O.V. 4394) has been deposited in the Herbarium of the Faculty of Arts and Science, University of Mugla, Turkey. The plant was identified immediately after collection and air-dried at room temperature for later analysis.

2.2. Preparation of the organic extracts The air-dried and powdered aerial parts of C. ensiformis (200 g) were extracted successively with n-hexane, chloroform, ethyl acetate and ethyl alcohol in a soxhlet apparatus until the last portion of the extract became colourless. Solvents of all the extracts were removed under low vacuum using rotary evaporation. Crude extracts were maintained at 4 C until used. Crude extracts were investigated for antimicrobial and antioxidant activity (DPPH assay, -carotenelinoleic acid assay, total phenolic compounds, flavonoid content).

2.3. Antimicrobial assay 2.3.1. Microorganisms and condition for cultivation Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Streptococcus mutans CNCTC 8/77,

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Micrococcus luteus NRRL B-4375, Enterobacter aerogenes RSKK 720, Candida albicans ATCC 10239 and Candida tropicalis RSKK 665 were used as test microorganisms. Also, Staphylococcus aureus MU 38, MU 40, MU 46, Staphylococcus xylosus MU 34, MU 35, MU 37, MU 42, Staphylococcus capitis MU 27, Staphylococcus epidermidis MU 30, Staphylococcus lentus MU 43, Staphylococcus sp. MU 28, and Strenotrophomonas maltophilia MU 23, MU 25, MU 52, MU 53, MU 63, MU 64, MU 69, MU 94, MU 99, MU 136, MU 137, multiple antibiotic resistant bacteria, were used. The strains of coded MU were obtained from the Mugla University Culture Collection. The above mentioned bacteria, except S. mutans, were cultured in Nutrient Broth (NB) (Difco); S. mutans was cultured in Brain Heart Infusion Broth (BHIB) (Difco), C. albicans and C. tropicalis cultured in Sabouraud Dextrose Broth (SDB) (Difco). P. aeruginosa and S. maltophilia strains and the fungi were incubated at 30 0.1 C for 1824 h and 2448 h, respectively. Other bacteria strains were incubated at 37 0.1 C for 2448 h. Inocula were prepared by adjusting the turbidity of the medium to match the 0.5 Mcfarland Standard Dilutions of this suspension, in 0.1% peptone (w/v) solution in sterile water inoculated on NB, BHIB, SDB to check the viability of the preparation. The cultures of the microorganisms were maintained in their appropriate agar slants at 4 C throughout the study and used as stock cultures. 2.3.2. Disc diffusion method The antimicrobial activities of the extracts were assayed by the standard disc diffusion method (Collins, Lyne, & Grange, 1995; Murray, Baron, & Pfaller, 1995). Each crude extract was dissolved in its own organic solvent. Twenty-five microlitres of each extract containing 25 mg per 100 mL crude extract were injected into discs of 6 mm in diameter (Schleicher & Schuell). Mueller Hinton Agar (MHA) (Difco) and Sabouraud Dextrose Agar (SDA) (Difco) sterilised in a flask and cooled to 4550 C were distributed to sterilised petri dishes with a diameter of 9 cm (15 mL) after injecting cultures of bacteria and yeast (0.1 mL) and distributing the medium in petri dishes homogeneously. Dishes injected with the above mentioned materials were located on the solid agar medium by pressing slightly. Petri dishes were incubated at the appropriate temperature and time for microorganisms, as mentioned above. Antimicrobial activity was evaluated by measuring the zone of inhibition against the tested microorganisms. Discs of n-hexane, chloroform, ethyl alcohol, and ethyl acetate (Merck) were used as controls. Studies were performed in triplicate.

2.4. Antioxidant activity 2.4.1. DPPH assay The hydrogen atom or electron donating abilities of the corresponding extracts and some pure compounds were measured by the bleaching of the purple-coloured methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). This spectrophotometric assay uses the stable radical DPPH as a reagent (Burits & Bucar, 2000; Cuendet, Hostettmann, & Potterat, 1997). Two hundred microlitres of various concentrations of the extracts in ethanol were added to 5 mL of a 0.004% methanol solution of DPPH. After a 30 min incubation period at room temperature, the absorbance was read against a blank at

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517 nm. Inhibition of free radicals by DPPH in percent (I%) was calculated by the following:   Ablank Asample I% 100, Ablank where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated from the graph plot of inhibition percentage against extract concentration. Tests were carried out in triplicate. 2.4.2. -Carotenelinoleic acid assay In this assay, antioxidant capacities were determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation (Dapkevicius, Venskutonis, Van Beek, & Linssen, 1998). A stock solution of -carotenelinoleic acid mixture was prepared as follows: 0.5 mg -carotene was dissolved in 1 mL of chloroform (HPLC grade), and 25 ml linoleic acid and 200 mg Tween 40 were added. Chloroform was completely evaporated using a vacuum evaporator. Then, 100 mL distilled water saturated with oxygen (30 min, 100 mL min1) was added through vigorous shaking. Four thousand microlitres of this reaction mixture were dispensed into test tubes and 200 ml portions of the extracts, prepared at 2 gL1 concentrations, were added and the emulsion system was incubated for 2 h at 50 C temperature. The same procedure was repeated with synthetic antioxidant, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), -tocoferol, as positive control, and a blank. After this incubation period, absorbances of the mixtures were measured at 490 nm. Antioxidative capacities of the extracts were compared with those of BHT, BHA, -tocoferol and blank. 2.4.3. Determination amount of total phenolic compounds Total soluble phenolics in the C. ensiformis extracts were determined with FolinCiocalteu reagent according to the method of Slinkard (1977), using pyrocatechol as a standard. Briefly, 1 mL of extract solution (contains 2000 mg) in a volumetric flask diluted glassdistilled water (46 mL). FolinCiocalteu reagent (1 mL) was added and the contents of the flask were mixed thoroughly. After 3 min, 3 mL of Na2CO3 (2%) was added, then the mixture was allowed to stand for 2 h with intermittent shaking. The absorbance was measured at 760 nm. The concentration of total phenolic compounds in the C. ensiformis extracts determined as a microgram of pyrocatechol equivalent by using an equation that was obtained from the standard pyrocatechol graph, given as: Absorbance 0:00246 mg pyrocatechol 0:00325 R2 : 0:9996 2.4.4. Determination of the amount of total flavonoid compounds Flavonoid concentration was determined as follows: C. ensiformis extract solution (1 mL) was diluted with 4.3 mL of 80% aqueous ethanol and test tubes were added containing 0.1 mL of 10% aluminum nitrate and 0.1 mL of 1 M aqueous potassium acetate. After 40 min at room temperature, the absorbance was determined spectrophotometrically

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at 415 nm. Total flavonoid concentration was calculated using quercetin as standard (Park, Koo, Ikegaki, & Contado, 1997). Absorbance 0:002108 mg quercetin 0:01089 R2 : 0:9999 2.5. Chemical compositions of active extract 2.5.1. Column chromatography (CC) Firstly, CC was performed to highlight the structure of the active extract. For CC, silicagel 60 (70230 mesh) as adsorbent in a column with 2 80 cm measurements was used and mobile phases were, respectively, 95: 5, 90: 10 and 85: 15 hexane: acetone systems. Under these conditions, the hexane extract was purified from hydrocarbons and the active parts separated their fractions. Both mixture and compounds separated at TLC, controlled with the indicators and determined the compound classes. After the separation of compounds, pigments and chlorophylls were with high polarity in the column, first, the chromatograms of fractions with terpenoid content was obtained in GC, then their mass spectrums were individually obtained in GC-MS and then finally their characterisation was achieved with the help of libraries. 2.5.2. Gas chromatography (GC) The GC analysis of the extract was performed using a Shimadzu GC-17 AAF, V3, 230V LV series GC, equipped with a FID and a DB-5 fused silica capillary column (30 m 0.32 id., film thickness 0.25 mm); the initial oven temperature was held at 100 C for 5 min, then programmed to 240 C at 3 C min1 and held at this temperature for 30 min; injector temperature and detector temperature was 250 and 270 C, respectively; carrier gas was He at a flow rate of 1.4 mL min1; sample size, 1.0 mL; split ratio, 50: 1. The percentage composition of the essential oil was determined with the Class-GC 10 computer program. 2.5.3. Gas chromatography-mass spectrometry (GC-MS) Around 50 mg mL1 of solution of the hexane extract obtained from a C. ensiformis plant through different solvents and which was bio-active was prepared from GC and GC-MS analyses. The analysis of the extract was performed using a Varian Saturn 2100 (E.I Quadrupole), equipped with a DB-5 MS fused silica capillary column (30 m 0.32 mm i.d., film thickness 0.25 mm). For GC-MS detection, an electron ionisation system with ionisation energy of 70 eV was used. Carrier gas was helium at a flow rate of 1.7 mL min1. Injector and MS transfer line temperatures were set at 220 C and 290 C, respectively. The oven temperature was held at 100 C for 5 min, then increased up to 240 C with 3 C min1 increases and held at this temperature for 25 min. Diluted samples (1/100, v/v, in methylene chloride) of 1.0 mL were injected manually in the splitless mode. The relative percentage of the extract constituents were expressed as percentages by peak area normalisation. The identification of components of the extract was done by computer matching of mass spectra with those of standards (NIST 2002, Wiley library data of GC-MS systems and a personal library of 320 spectra), as well as by comparison with the fragmentation patterns of the mass spectra with those reported in the literature (Adams, 2001) and, whenever possible, by co-injection with authentic compounds.

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3.1. Antimicrobial activity The antimicrobial activities of four extracts of C. ensiformis were tested in vitro by using the disc diffusion method with the microorganisms, as seen in Table 1. In this study, 14 gram negative bacteria (E. aerogenes RSKK 720, P. aeruginosa ATCC 27853, E. coli ATCC 25922 and 11 various multiple antibiotic resistant S. maltophilia strains); 15 gram positive test bacteria (M. luteus NRRL B-4375, B. subtilis ATCC 6633, S. mutans CNCTC 8/77, S. aureus ATCC 25923 and 11 multiple antibiotic resistant strains of various species of Staphylococcus) and 2 yeasts (C. albicans ATCC 10239 and C. tropicalis RSKK 665) were used. The antibiotic resistance patterns of the multiple antibiotic resistant strains of S. maltophilia and Staphylococcus spp. were shown in Tables 2 and 3, respectively.

Table 1. Antimicrobial activities of C. ensiformis extracts.

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Inhibition zone (mm) Strains Hexane extract 13 10 10 13 13 13 9 10 9 14 10 9 9 11 11 10 12 10 Chloroform extract 10 12 11 9 10 9 10 9 9 9 9 9 Ethyl alcohol extract 20 17 15 16 16 14 23 23 10 13 13 21 20 11 12 9 12 10 13 15 13 15 14 13 12 19 11 17 16 Ethyl acetate extract 9 11 10 9 10 16 14 9 15 12 15 11 13 11 10 16 11 11 16 11 14 13 14 18 11 17 14 17 -

E. aerogenes RSKK 720 P. aeruginosa ATCC 27853 E. coli ATCC 25922 S. maltophila MU 23 S. maltophila MU 25 S. maltophila MU 52 S. maltophila MU 53 S. maltophila MU 63 S. maltophila MU 64 S. maltophila MU 69 S. maltophila MU 94 S. maltophila MU 99 S. maltophila MU 136 S. maltophila MU 137 M. luteus NRRL B23 B. subtilis ATCC 6633 S. aureus ATCC 25923 S. mutans CNCTC 8/77 S. capitis MU 27 Staphylococcus sp. MU 28 S. epidermidis MU 30 S. xylosus MU 34 S. xylosus MU 35 S. xylosus MU 37 S. aureus MU 38 S. aureus MU 40 S. xylosus MU 42 S. lentus MU 43 S. aureus MU 46 C. albicans ATCC 10239 C. tropicalis RSKK 665 Note: (-): No activity.

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The ethyl alcohol, hexane, chloroform and ethyl acetate extracts obtained from the aerial parts of C. ensiformis demonstrated various antimicrobial activities on tested microorganisms. The extracts showed antibacterial activity against gram positive and gram negative bacteria, but no antifungal activity was observed against the two yeasts. The ethyl alcohol extract inhibited the growth of all bacteria tested and the inhibition zones ranged between 923 mm. The hexane extract produced 914 mm, the ethyl acetate extract produced 918 mm and the chloroform extract produced 912 mm inhibition zones on the bacteria. The chloroform extract of C. ensiformis exhibited the weakest antibacterial activity. The hexane, chloroform and ethyl alcohol extracts showed better antimicrobial activity against the gram negative bacteria when compared to the gram positive bacteria. All of the extracts inhibited S. maltophilia MU 69 and MU 94. The ethyl alcohol extract inhibited all of the strains of S. maltophilia tested. The ethyl alcohol and ethyl acetate extracts showed antibacterial activity against all of the gram positive bacteria and gram negative bacteria tested, except S. maltophilia MU 63. No antimicrobial activity of the ethyl acetate extract was observed against S. maltophilia MU 63. However, it is interesting to note that the strains of S. maltophilia, S. aureus and some of the

Table 2. Antibiotic resistance patterns of S. maltophilia strains. Strains S. maltophilia MU 23 S. maltophilia MU 25 S. maltophilia MU 52 S. maltophilia MU 53 S. maltophilia MU 63 S. maltophilia MU 64 S. maltophilia MU 69 S. maltophilia MU 94 S. maltophilia MU 99 S. maltophilia MU 136 S. maltophilia MU 137 Resistance patterns MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, TVA, AM, PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, TVA, AM, PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, TVA, AM PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, NOR, C, TVA, AM, PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, TVA, AM, PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, SXT, TVA, AM, PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, SXT, TVA, AM, PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, TVA, AM, PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, SXT, TVA, AM, PRL, ATM,SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, TVA, AM, PRL, ATM, SAM, AMC MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, TVA, AM, PRL, ATM, SAM, AMC

Note: MEZ: Mezlocillin (75 mcg), TIM: Ticarcillin clavulanic acid (75 10 mcg), CAZ: Ceftazidime (30 mcg), FEP: Cephepim (30 mcg), CRO: Ceftriaxone (30 mcg), CTX: Cefotaxime (30 mcg), KF: Cephalothin (30 mcg), IPM: Imipenem (10 mcg), P: Penicillin (10 U), AK: Amikacin (30 mcg), TOB: Tobramycin (10 mcg), NET: Netilmicin (30 mcg), CN: Gentamicin (10 mcg), TE: Tetracycline (30 mcg), NOR: Norfloxacin (10 mcg), C: Chloramphenicol (30 mcg), TVA: Trovafloksasin (10 mcg), AM: Ampicillin (10 mcg), PRL: Piperacillin (100 mcg), ATM: Aztreonam (30 mcg), SAM: Sulbactam Ampicillin (10 10 mcg), AMC: Amoxicillin Clavulanic acid (20 10 mcg), CIP: Ciprofloxacin (5 mcg), SXT: Trimetoprimsulfamethoxazole (1.25 23.75 mcg).

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coagulase-negative staphylococci (CNS), which are resistant to antibiotics, were found to be active at test concentrations in our study. Staphylococci are among the most commonly encountered pathogens in clinical practice. S. aureus is a major cause of nosocomial infections, food poisoning, osteomyelitis, pyoarthritis, endocarditis, toxic shock syndrome and a broad spectrum of other disorders (Hajjeh et al., 1999; Rubin et al., 1999; Todd, 1998; Willett, 1992). In recent years, several species of CNS have been recognised as opportunistic pathogens and have been implicated in human infections and disease, especially in immunocompromised and seriously ill patients (Kloos & Bannerman, 1995; Thornsberry, 1985). In recent years, there has been an alarming increase in nosocomial staphylococcal infections by strains with MDR (Al-Masaudi et al., 1991; Hiramatsu et al., 1997; Kloos & Bannerman, 1995; Lyon & Skurray, 1987). At present, this situation is leading to the evaluation of staphylococcal as pathogens as potentially resistant to any available antibiotics (Lyon & Skurray, 1987; Rubin et al., 1999; Huycke et al., 1998; Noble et al., 1992). S. maltophilia (Palleroni & Bradbury, 1993) (Xanthomonadaceae), previously known as Pseudomonas maltophilia (Hugh & Ryschenkow, 1961) (Pseudomonadaceae), and subsequently as Xanthomonas maltophilia (Swings, Devos, Van der Mooter, & De Ley, 1983) (Xanthomonadaceae), has received much attention in the last decade because of its role as a pathogenic microorganism in an increasing number of clinical syndromes (Robin & Janda, 1996), such as bacteremia, infections of the respiratory and urinary tracts, skin and soft tissue infections, biliary tract infection, meningitis, serious wound infections, conjunctivitis and endocarditis (Denton & Kerr, 1998; Fisher, Long, Roberts, Dun, & Babara, 1981). The treatment of infections caused by this microorganism is difficult because S. maltophilia is frequently resistant to most of the widely used antibiotics

Table 3. Antibiotic resistance patterns of Staphylococcus spp. Strains S. xylosus MU 34 MU 35 MU 37 MU 42 S. aureus MU 38 MU 40 MU 46 Staphylococcus sp. MU 28 S. capitis MU 27 S. epidermidis MU 30 S. lentus MU 43 Resistance patterns P, P, P, P, AK, DA, AK, AK, DA, E, CN, OX, TEC E, C, OX, TE DA, E, CN, TEC, TE DA, CN, OX, TE

P, AK, DA, CN, ME, TEC, TE, OX P, AK, CN, C, ME, OX, TE P, AK, DA, E, CN, TE, OX P, AK, DA, E, CN, TE P, AK, DA, E, CN, TE P, AK, DA, CN, OX, TEC, TE P, AK, DA, CN, OX, TE

Note: P: Penicillin-G (10 U); AK: Amikacin (30 mg); DA: Clindamycin (2 mg); E: Erythromycin (15 mg); CN: Gentamicin (10 mg); C: Chloramphenicol (30 mg); ME: Methicillin (5 mg); OX: Oxacillin (1 mg); TEC: Teicoplanin (30 mg); TE: Tetracycline (30 mg).

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(Krueger et al., 2001; Liu et al., 1995; Skaehill, 2000; Vartivarian, Anaissie, Bodey, Sprigg, & Rolston, 1994). In conclusion, various extracts of C. ensiformis showed strong activities against S. maltophilia and S. aureus and CNS which are resistant to various antibiotics. There are possible alternative therapies for these strains. Such therapies are very important because the types of organisms that have been emerged as most problematic for patients within the Intensive Care Unit include: methicillin resistant S. aureus (MRSA), vancomycin resistant Enterococcus (VRE), extended spectrum beta-lactamases (ESBL), stably derepressed AmpC enzyme producers among Enterobacteriaceae, and MDR, non-fermentative gram negative bacilli, principally P. aeruginosa, Acinetobacter spp., and S. maltophilia (Fluit, Verhoef, & Schmitz, 2001; Fridkin, Hill, & Volkova, 2002). Furthermore, the dosages of extracts we used were much lower than the dosages of antibiotics. This in vitro study provides evidence that this plant is potentially a rich source of antibacterial agents against the multidrug resistant bacteria which were tested. Hence, the extracts of C. ensiformis may be useful as alternative antimicrobial agents for multiple antibiotic resistant S. maltophilia, S. aureus and some CNS.

3.2. Antioxidant activity Various extracts were subjected to screening for their possible antioxidant activities. Four complementary test systems, namely DPPH free radical scavenging, total antioxidant activity, and the amount of total phenolic and flavonoid compounds were used for the analysis. DPPH, a stable free radical with a characteristic absorption at 517 nm, was used to study the radical scavenging effects of the extracts. As antioxidants donate protons to these radicals, the absorption decreases. The decrease in absorption is taken as a measure of the extent of radical scavenging. The free radical scavenging capacities of the extracts, measured by DPPH assay, are shown in Figure 1. The ethyl alcohol (94.97%) and ethyl acetate (91.75%) extracts obtained from C. ensiformis were observed to have scavenged the most radicals. With the increase in concentration, radical removal activities of all the extracts increase. When we compare the radical removal activity of the ethanol extract with that of -tocopherol, it was found that 160 mg of the C. ensiformis extract scavenges

120 100 % Inhibition 80 60 40 20 0 Hexane Chloroform Ethyl acetate Extracts Ethanol -Tocopherol
20 g 40 g 80 g 160 g

Figure 1. Free radical scavenging capacities of the extracts measured in DPPH assay.

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more radical than 40 mg of -tocopherol. This radical scavenging capacity exhibited by the extract obtained from the extraction with ethanol, when compared with the -tocopherol, which is known to be a very effective antioxidant, shows that this capacity is a high and important capacity for a natural extract (Figure 1). It is seen that the total antioxidant activity of the extracts through solvents with increasing polarities is remarkably high (Figure 2). In the linoleic acid system, the inhibition value of the ethyl acetate extract was found to be higher than other extracts. The ethyl acetate extract shows 85.15% inhibition, which is close to the synthetic antioxidant reagent -tocopherol (96.48%). Inhibition was also observed in the ethyl acetate extract, hexane extract, ethanolic extract and chloroform extract; however, the lowest inhibition value was found in the chloroform extract (72.51%). Although DPPH radical scavenging activity of the ethanolic extract was higher than that of the other extracts, total antioxidant activity of the ethyl acetate and hexane extracts were seen to be higher than that of the ethanol extract (Figure 2). The various extracts were investigated through screening for their amount of total phenolic and flavonoid compounds. Polyphenolic compounds have an important role in stabilising lipid oxidation and are associated with antioxidant activity (Turkoglu, Duru, Mercan, Kivrak, & Gezer, 2007; Yen, Duh, & Tsai, 1993). In some literature, it has been reported that polyphenolic compounds are important in preventing lipid oxidation, as they are potential antioxidants, and in line with the increase seen in their amounts, antioxidant activity increases (Ramarathnam, Osawa, & Namiki, 1986; Velioglu et al., 1998). It is suggested that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans, when up to 1.0 g is ingested daily from a diet rich in fruits and vegetables (Tanaka, Kuei, Nagashima, & Taguchi, 1998). Hence, it is important (or necessary) to know the amount of the total phenolic substances to determine antioxidant activity. Based on the absorbance values of the various extract solutions, reacting with FolinCiocalteu reagent and compared with the standard solutions of pyrocatechol

120

100

80 % Inhibition
20g 40g 80g 160g

60

40

20

0 Hexane Chloroform E. Acetate Extracts Ethanol

-Tocopherol

Figure 2. Total antioxidant activity of -tocopherol and different doses of C. ensiformis extracts in the linoleic acid emulsion.

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equivalents, as described above, results of the colorimetric analysis of total phenolics are given in Table 4. The amount of total phenolic compounds in hexane, chloroform, ethyl acetate and ethyl alcohol extracts of C. ensiformis were 18.04, 16.01, 54.89 and 64.61 mg [PK], and the amount of total flavonoids were 8.35, 25.69, 109.64 and 29.50 mg [QE], respectively (Table 4). The development of the antioxidant supplement industry has created a need for reliable antioxidant assays to measure the real antioxidant activity of a product. Flavonoid structured compounds have been used recently in the protection of the heart and as an antioxidant substance. It is known that flavonoids increase the blood flow to the brain, arms and legs, and that this is beneficial for the elderly (Hunter & Fletcher, 2002). The ethyl alcohol extract (64.61 mg mL1 equivalent pyrocatechol) and ethyl acetate extract (54.89 mg mL1 equivalent pyrocatechol), including the highest amount of phenolic compound, is effective on both gram positive and gram negative bacteria. The ethyl

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Table 4. Amount of total phenolic and flavonoid compounds in C. ensiformis extracts. Total phenolic compound amounts mg pyrocatechol (mg[PK]) 18.04 0.01 16.01 0.04 54.89 0.18 64.61 0.09 Total flavonoid amounts mg quercetin (mg[QE]) 8.35 0.12a 25.69 0.15 109.64 0.13 29.50 0.04

Extracts Hexane Chloroform Ethyl acetate Ethyl alcohol

Note: Data expressed as mean SEM of the three samples analysed separately. a Standard deviation.

Table 5. Chemical composition of C. ensiformis hexane extract. Compoundsa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Rose oxide -Bourbonene Spathulenol Caryophyllene oxide Eudesmol trans-(Z)--Bisabolene epoxide Hexahydro farnesyl acetone Heneicosane n-Docosane -Amyrin Lupeol n-Tricosanol -Amyrin acetate [3]-12-Oleanen-3yl-acetate []-Lup-20(29)-en-3-ol acetate % 4.57 8.51 17.81 28.72 13.03 1.82 3.71 2.15 1.06 5.62 3.74 2.96 3.92 1.43 0.95 Methods a, a, a, a, a, b a, a, b b a, b b b b b b b b b b b b

Note: a: Co-injection with authentic compounds; b: MS. a In DB-1 fused silica capillary column.

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acetate extract, including the highest amount of flavonoid compound amount, is effective on both gram positive and gram negative bacteria. In this study, high total antioxidant activities were seen in the ethyl acetate and hexane extracts. Especially the high total antioxidant activity of the ethyl acetate extract was thought to be attributable to being rich in flavonoid compounds. In many studies conducted up to this point, it has been found that total antioxidant capacity of extracts rich in flavonoids is high (Cakir et al., 2003; Hollman & Katan, 1997; Klimczak, Malecka, wiglo, 2007; Middleton & Kandaswami, 1993; Ozturk, ska-S Szlachta, & Gliszczyn Aydogmus-Ozturk, Duru, & Topcu, 2007; Prior & Cao 2000a; Prior & Cao, 2000b; s-Barberan & Clifford, 2000). The reason that total Kaur & Kapoor, 2001; Toma antioxidant activity of ethyl acetate extract is higher than that of other extracts can be related to its rich flavonoid content. One study related to the antioxidant activity of C. ensiformis was detected in the literature. In the study, total phenolic contents, free radical scavenging activities and inhibitory effects on the activation of NF-kappa B of eight Centaurea L. species were determined. Methanol extracts (0.25 mg per 4 mL) of C. urvillei, C. cadmea and C. ensiformis showed strong antioxidant activity, with 90.41, 86.66 and 86.19% FRSA, respectively (Karamenderes, Konyaliyoglu, Khan, & Khan, 2007).

3.3. Chemical composition of active extract In spite of the fact that the DPPH radical scavenging activity of the hexane extract was low; the total antioxidant activity of this extract was comparable to the total antioxidant activity of -tocopherol. Also, the hexane extract showed antibacterial activity. The compounds of the hexane extract were determined. To our knowledge, no study has been reported on the chemical composition of C. ensiformis. A total 15 compounds were detected in the hexane extract of this plant, which was rendered bio-active by using GC and GC-MS methods. Detected compounds were identified by using reference substances and NIST 2002, Wiley library and a special library established in our laboratory. As can be seen in Table 5, the major compounds of the hexane extract of C. ensiformis were caryophyllene oxide (28.72%), spathulenol (17.81%), eudesmol (13.03%), -bourbonene (8.51%), amyrin (5.62%) and rose oxide (4.57%). In this study, the rate of oxygen carrying sesquiterpene in the hexane extract was high. Other studies on the chemical composition of some species of Centaurea also determined that the main component was oxygen carrying sesquiterpene. Some of the major compounds of Centaurea mucronifera and Centaurea chrysantha, plants endemic to Turkey, caryophyllene oxide (5.2 and 9.5%) and spathulenol (1.5 and 3.8%) were detected, respectively (Dural et al., 2003). Numerous publications have dealt with the secondary metabolites of the large genus Centaurea, sesquiterpene lactones being the most characteristic constituents (Nowak, Drozdzg, & Holub, 1996). Chemical investigations of various Centaurea species have revealed mainly sesquiterpene lactones (Gousiadou & Skaltsa, 2003), flavonoids (Akal, Benayache, Medjroubi, Tillequin, & kovic , Tes evic , Milosavljevic , Vajs, & Marin, 2004). Seguin, 2003) and lignans (Janac Secondary metabolites of the Centaurea species are mainly sesquiterpene lactones with ` ne et al., 2005; Marco, Sanzguaiane, germacrane, and eudesmane skeletons (Bentame n, & Carda, 2005). Cervera, Yuste, Sanceno

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In conclusion, the results indicate that the extracts of C. ensiformis have a capacity to inhibit the growth of the pathogenic bacteria tested. In addition, due to the antioxidant activities of the various extracts of C. ensiformis, these extracts can be used for natural antioxidants in food protective and pharmaceutical studies. The antimicrobial and antioxidant activities of the extracts must be due to their phenolic and especially flavonoid compounds. Therefore, they could be suitable for use as antimicrobial and antioxidative agents.

Acknowledgements
This work was supported by Mugla University Research Funds.

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