ANTIBACTERIAL ACTIVITIES OF EXTRACTS FROM TWELVE CENTAUREA SPECIES FROM TURKEY

Members of the genus Centaurea (Asteraceae) have been used in traditional plant-based medicine. The methanol extracts of twelve Centaurea species, of which five are endemic to Turkey flora, were screened for antibacterial activity against four bacteria (Escherichia coli, Bacillus cereus, Salmonella enteritidis, Staphylococcus aureus). The antibacterial activity was evaluated by the microdilution method and the minimum inhibition concentrations (MIC) of the extracts were determined. C. cariensis subsp. microlepis exhibited an antimicrobial effect on all tested microorganisms. The extracts from eight Centaurea species (C. balsamita, C. calolepis, C. cariensis subsp. maculiceps, C. cariensis subsp. microlepis, C. kotschyi var. kotschyi, C. solstitialis subsp. solstitialis, C. urvillei subsp. urvillei and C. virgata) possessed antibacterial activity against several of the tested microorganisms.


INTRODUCTION
Plant-derived drugs remain an important resource, especially in developing countries, to combat serious diseases.Approximately 60-80% of the world's population still relies on traditional medicines for the treatment of common illness (WHO, 2002;Zhang, 2004).Plants contain several compounds that have potent biological activity (Mokbel and Hashinaga, 2006).For example, phenolic compounds and essential oil play a vital role as powerful natural biological agent (Cutter, 2000;Hao et al., 1998;Lis-Balchin and Deans, 1997;Puupponen-Pimia et al., 2001).Antibiotic use has led to the emergence of infectious bacteria that are resistant to one or more antibiotics (Falagas and Bliziotis, 2007).The situation has brought about the failure of the treatments of many microbial diseases.A number of previous investigations have indicated that many medicinal plant extracts constitute a class of potent natural antimicrobial agent (Rayne and Mazza, 2007;Sufferidini et al., 2004).Therefore, many researchers have focused on the investigation of aromatic and medicinal plants as a source of new antimicrobial substances (Recio and Rios, 1989;Silver and Bostian, 1993).
Turkey's flora is comprised of over 3000 aromatic plant species (Baser, 2002), but there is no detailed information about most of these species.The Asteraceae plant family has ±2500 species and 1500 genera in the world (Wagstaff and Breitwieser, 2002) Some Asteraceae species have been used in many fields, including nutrition and medicinal industries (Roig, 1965).The genus Centaurea comprises more than 500-600 species that are widespread all over the world, in particular around the Mediterranean and western Asia area (Mabberley, 1997); 179 species of the genus Centaurea are found in Turkey, among which 109 species are endemic (Davis et al., 1988;Guner et al., 2000).Some members of the genus are known by various local names such as "peygamber çiceği", "zerdali dikeni", "çoban kaldiran", "Timur dikeni" and "boga dikeni" in Anatolia (Baytop, 1999;Sezik et al., 2001;Wagenitz, 1975).Many Centaurea species have long been used in traditional medicine to cure various ailments, such as hemorrhoids, abscess and the common cold (Baytop, 1999;Kargıoglu et al., 2008;Kargıoglu et al., 2010;Sezik et al., 2001).Due to the above-mentioned pharmacological importance of Centaurea species; it is worthwhile to determine the antibacterial properties of Centaurea.Recently, some Centaurea species were examined for biological properties such as antioxidant capacity, antimicrobial activity and essential oil composition.The aim of this study was to determine the antibacterial activity of twelve Centaurea species growing in Turkey's flora.

Preparation of methanolic extracts
Air-dried plant material was finely powdered using a laboratory mill.15 g of each sample was exhaustively extracted with 300 ml methanol at room temperature under stirring and the extracts were filtered through a Whatman blue filter.After evaporation of the solvent at 40°C in rotary evaporator, the residues were stored at 4°C until further analysis.

Microorganisms
The antibacterial activities of the Centaurea species were assessed against four bacteria species: Escherichia coli ATCC 25922, Bacillus cereus ATCC 14579, Salmonella enteritidis ATCC 13076 and Staphylococcus aureus ATCC 25923.Gentamicin was used as a positive control.

Microdilution method
Minimum inhibition concentration (MIC) values were determined using 96-well microtiter plates by dissolving the sample in DMSO.Serial dilutions were made to obtain concentrations ranging from 8 to 0.0625 mg/ml.Suspensions of standard microorganisms were inoculated onto the microplates.The growth of the microorganisms was observed by using a microplate photometer (Thermo Scientific Multiskan).The MIC values were defined as the lowest concentrations of the plant extracts to inhibit the growth of microorganisms.

RESULTS AND DISCUSSION
The available screening methods for the detection of antibacterial potency of plant extracts can be classified as diffusion and dilution methods.The diffusion methods are qualitative methods, while dilution methods are quantitative assays (with minimal inhibition concentration (MIC) (Vanden Berghe and Vlietinck, 1991)).The antibacterial activities of the methanolic extracts of twelve Centau-rea species were evaluated by using the MIC values in a micro-dilution method.Table 1 displays the in vitro antibacterial activities of the Centaurea species.The results show that eight plants exhibited antibacterial activity, while four species (C.carduiformis subsp.carduiformis, C. iberica, C. pterocaula and C. triumfettii) did not have any effect on all the tested bacteria.The extract of C. cariensis subsp.microlepis had an antibacterial effect on all tested microorganisms.C. cariensis subsp.maculiceps extract inhibited the bacteria with the exception of one bacterium (S. enteritidis).S. enteritidis was inhibited by three of the studied Centaurea species, namely, C. calolepis, C. cariensis subsp.microlepis and C. virgata, while seven Centaurea species had an antibacterial effect on E. coli.S. aureus is the cause of many diseases, such as food poisoning, osteomyelitis, polyarthritis, endocarditis (Hajjeh et al., 1999;Rubin et al., 1999;Willet, 1992).Five studied Centaurea species exhibited a strong antibacterial effect on S. aureus and C. solsitialis subsp.solsitialis had the highest activity on the bacteria with a 0.5 mg/ml concentration.Therefore, C. solstialis subsp.solstialis may be used as an antibiotic for S. aureus infections.However, a lower concentration of gentamicin (0.001 mg/ml) had a strong antibacterial effect on the bacteria.Bacillus species, especially B. cereus, are responsible for foodborne diseases (Hillard et al., 2003).B. cereus was inhibited by two of the studied Centaurea species (C.cariensis subsp.maculiceps and C. cariensis subsp.microlepis) with 4 mg/ml concentration.In a previous report, the antimicrobial activities of C. calolepis, C. urvillei subsp.urvillei were investigated by Karamenderes et al. (2006) who found that the species had an antimicrobial effect on two microorganisms (Candida krusei and Cryptococcus neoformans).In the present study, C. calolepis showed activity on Salmonella enteritidis, while C. urvillei subsp.urvillei was active on Escherichia coli and Staphylococcus aureus.Cansaran et al. (2010) examined the antimicrobial activity of various extracts of C. cankiriense.Both the ethyl acetate and methanol extracts of this species inhibited the growth of 13 bacteria and the MIC values of the ethyl acetate extracts were determined as 250 µg/ml for E. coli and 62.5 µg/ml for S. aureus.According to the results, all examined Centaurea species showed weaker antibacterial activity than the ethyl acetate extract of C. cankiriense.C. bornmuelleri, C. hubermorathii and C. schiskinii did not show any effect on E. coli and B. cereus in the micro-dilution assay (Sarker et al., 2005).However, our results demonstrated that some studied Centaurea species were active against E. coli and B. cereus.
The antibacterial effect of the methanolic extract of dry heads of C. diffusa was much lower than some of the studied Centaurea species (Skliar et al., 2005).Guven et al. (2005) found a significant antimicrobial effect of five Centaurea species (C. ptosomipappoides, C. odyssei, C. ptosomipappa, C. amonicola and C. kurdica) on some microorganisms, including S. aureus and B. cereus.Extracts of C. appendicigera and C. helenioides did not display antimicrobial activity on E. coli and the results were reported by Buruk et al. (2006).Ugur et al. (2010) reported that the chloroform and ethyl alcohol extracts of C. cariensis subsp.niveo-tomentosa exhibited strong antibacterial activities on many resistant bacteria, especially Staphylococcus strains.In a previous study, the methanolic extract of C. polyclada had a lower sensitivity than some other studied Centaurea species (especially, C. cariensis subsp.maculiceps and C. cariensis subsp.microlepis) on B. cereus and S. aureus (Granger et al., 2009).The antibacterial effects of some Yemeni medicinal plants were investigated and the methanolic extract of C. pseudosinaica displayed a potent antibacterial effect on S. aureus (Montana et al., 2010).The methanol extract of C. ainetensis flowers showed efficacy against 88% of the tested microorganisms.In the same study, C. erengoides exhibited a low antibacterial effect on the microorganisms (Barbour et al., 2004).Some authors focused on the antimicrobial activity of the essential oil of Centaurea species.For example, Yayli et al. (2009) investigated the antimicrobial effect of the essential oils from two Centaurea species (C.appendicigera and C. helenioides) and demonstrated a moderate antibacterial activity on Gram-positive and Gram-negative.Similar results were obtained for the essential oils of C. sessilis and C. armena (Yayli et al., 2005).The essential oil of C. aladagensis had an antibacterial effect against seven human pathogenic microorganisms (Kose et al., 2007) However, the essential oils of C. nicaeensis and C. parlatoris showed low activity on fourteen selected microorganisms (Senatore et al., 2008).

CONCLUSIONS
In general, the present study revealed that eight Centaurea extracts (C. balsamita, C. calolepis, C. cariensis subsp. maculiceps, C. cariensis subsp. microlepis, C. kotschyi var. kotschyi, C. solsitialis subsp. solsitialis, C. urvillei subsp. urvillei and C. virgata) possess antibacterial activity against several tested microorganisms.Therefore, the extracts can be used as a source of natural antibiotics Antibioticresistant bacteria are a seriouns problem and these extracts point to potential novel drugs for treating infectious diseases.Further studies should be performed in order to determine the active components of Centaurea species.