In Vitro Biological Evaluation and Phytochemical Contents of Three Centaurea L. Species Growing from Eastern Anatolia in Turkey

Centaurea L. species were used as medicinal plants among the people for treatment of the common cold, abscesses, peptic ulcers, hemorrhoid and diabetes etc.. In the present study, antiradical properties, phytochemical contents, antimicrobial and antiproliferative activities of three Centaurea species were investigated. Centaurea saligna (K.Koch) Wagenitz methanol (99.94%), Centaurea virgata Lam. methanol (98.23%) and water (98.10%) extracts were showed higher ABTS scavenging than trolox (96.79%). Centaurea kurdica Reichardt extracts showed lower activity than trolox for all the antiradical assays. Centaurea extracts exhibited antimicrobial activity against to some microorganisms. It was determined that these Centaurea species contain high amount of total flavonoid, phenolic and proanthocyanidin, phenolic acids, phytosterols and unsaturated fatty acids. Also, three Centaurea extracts showed very high antiproliferative property on LNCaP, HCT-116, MCF-7 cancer cell lines. Research Article Article History Received : 09.07.2019 Accepted : 26.09.2019


INTRODUCTION
Plant-derived antimicrobials possess great therapeutic potentials and have been used for many years for the treatment of various infectious diseases (Iwu et al., 1999). Natural products can provide countless opportunities for the discovery of a new drug as pure compounds or herbal extracts owing to the fact that chemical diversity of these products have a very high potential. Recently, researchers have been looking for new ways to develop more effective drugs against microbial infections. Phytochemical compounds have antimicrobial effects and can be used in treating of microbial infections (Modi et al., 2012).
It was considered that plants are the oldest drugs used in the cancer therapy. The various reports indicated that the anticancer activity of medicinal plants caused by them contain antioxidant compounds. Indeed, the medicinal plants have lower costs, and easily available when compared to modern synthetic drugs. Therefore, the world of science is working hard for determining of the anticancer properties of plant-derived natural products, and their direct isolation and characterization of these natural products (Pandey and Madhuri, 2009;Prema et al., 2011;Wen et al., 2011).
The Centaurea genus is located in the Asteraceae family, and is represented by about 700 species. These genus members are annual, biennial and/or perennial herbaceous plants (Dittrich, 1977;Wagenitz and Hellwig, 1996). There are more than 180 Centaurea species in Turkey, and about 120 species of them are endemic (Davis, 1988). It is specified that a lot of Centaurea species are used in the treatment of common cold, abscesses, peptic ulcers, hemorrhoid and diabetes, and fresh shoots of some species are consumed as food among the people. In addition, many ethnopharmacological studies have shown that Centaurea species have antioxidant, antiradical, antibacterial, antimicrobial, antipyretic, antirheumatic, and antiinflammatory properties (Arif et al., 2004;Formisano et al., 2008;Ugur et al., 2009;Tekeli et al., 2010;Aktumsek et al., 2011;Zengin et al. 2012;Aktumsek et al., 2013a;Aktumsek et al., 2013b;Bruno et al., 2018).
As far as we know, there is no report on the antiradical and antiproliferative properties of Centaurea saligna (K.Koch) Wagenitz and Centaurea virgata Lam. species. Yet, there is more information about antiradical (Aktumsek et al., 2011), antimicrobial (Guven et al., 2005 and phytochemical properties (Aktumsek et al., 2011) of Centaurea kurdica Reichardt, the antimicrobial properties (Tekeli et al., 2008) of Centaurea virgata Lam. in the literature.
The aim of the present study was to investigate i) the antiradical activities; ii) the antimicrobial properties; iii) the antiproliferative properties; iv) phytochemical compositions of C. virgata, C. kurdica, C. saligna water, ethanol, methanol and acetone extracts.

Chemicals and standards
All standards and chemical compounds were purchased from Sigma-Aldrich.

Extraction procedures
Centaurea kurdica Reichardt, Centaurea virgata Lam. and Centaurea saligna (K.Koch) Wagenitz flowers were collected in June-September of 2016 from Elazig, Turkey. Voucher specimen numbers were Turkoglu 4865, 4866 and 4867, respectively. Voucher specimen was stored in the herbarium of Firat University, Science Faculty, Department of Biology, Elazig, Turkey. The flowers were dried at dark and room temperature. Flowers were pulverized using a mechanic grinder, and then 100 g of the powdered samples was extracted with 1000 mL of solvent (water, ethanol, methanol and acetone). These were centrifuged at 5000 rpm. After centrifuging and filtrating of solvents, the supernatants were concentrated with a rotary evaporator. All extractions were repeated three times. The standard antioxidants and extracts were dissolved in DMSO (for HPLC grade) at the concentration of 1000 μg/mL (Keser, 2014).

Determination of Antiradical Activities
The ABTS •+ , hydroxyl and DPPH radical scavenging activities (RSAs) were determined by the methods of Re et al. (1999), Halliwell et al. (1987) and Brand-Williams et al. (1995), respectively. The antiradical activity tests were done at 500 μg/mL concentration for the extracts and standard antioxidant. All tests were repeated thrice and the average values were computed.
The radical scavenging activity percentages (RSA%) for each sample was estimated by the following equation: RSA% = [(A0 -A1)/A0] x 100 A0 and A1 are the absorbance of control and the sample, respectively.

Determination of Phytochemical Compounds
Total Phenolic Contents These contents were determined according to Slinkard and Singleton's method (1977). The results were expressed as gallic acid equivalent.

Total Flavonoid Content
The total flavonoid contents were performed according to Kim et al.'s method (2003). The catechin was used as a standard.

Proanthocyanidin Content
The proanthocyanidin contents were determined according to method described by Amaeze et al. (2011). The catechin was used as a standard.

Flavonoids and Phenolic Acids Analyses
The flavonoids and phenolic acids in the Centaurea extracts were done using according to the method of Zu et al. (2006). The results of the analyses were expressed as mg/g.

Fatty Acids Analyses
Fatty acids in the Centaurea extracts were analyzed by GC according to Christie's method (1992). The results were expressed as percent.

Vitamins and Phytosterols Analyses
The phytosterols and vitamins were extracted from  Collins and Lyne's method (1989) were used for the antimicrobial tests using the disc diffusion method. All the antimicrobial tests were repeated three times. All the results were compared with nystatin (30 mg/disc) and streptomycin sulfate (10 mg/disc) used as standards.

Determination of Antiproliferative Properties
The prostate cancer (LNCaP), colon cancer (HCT-116) and breast cancer (MCF-7) cell lines were used in the present study. These cell lines were retrieved from American Type Culture Collection (ATCC).
Statistical Analyses SPSS Statistics software was used for statistical analysis. The antiradical results were evaluated using the analysis of variance and the means were compared by Duncan's multiple range tests. For antiproliferative activity tests, normal distribution was obtained using Kolmogorov Smirnov test (p<0.05). The IC50 values were calculated by using % cell viabilities of extracts.

Antimicrobial Properties
The antimicrobial property results of C. virgata, C. kurdica and C. saligna water, ethanol, methanol and acetone extracts are summarized in Tables 3-5.
It was observed that C. kurdica water extract has an antimicrobial activity on only P. aeruginosa, P. vulgaris, S. aureus bacteria and C. albicans yeast; ethanol and methanol extracts have an antimicrobial activity on P. vulgaris, E. coli, L. monocytogenes, P. aeruginosa, S. aureus, K. pneumoniae, B. subtilis and B. megaterium bacteria, and C. albicans yeast; the acetone extract has an antimicrobial activity only P. vulgaris, E. coli, B. subtilis, P. aeruginosa, S. aureus and B. megaterium bacteria, and C. albicans yeast. It was determined that C. virgata water, ethanol, methanol and acetone extracts have an antimicrobial property on only P. aeruginosa, P. vulgaris, B. megaterium, S. aureus and B. subtilis bacteria, and C. albicans yeast. Table 3. The antimicrobial activities of C. kurdica extracts (mm zone) Tablo 3. C. kurdica ekstraktlarının antimikrobiyal aktiviteleri (mm zone) Streptomycin sulfate (10 mg/disc) and Nystatin (30 mg/disc) were used as standard antibiotic discs. The diameter of the paper discs was 6 mm. nd: not determined Table 4. The antimicrobial activities of C. virgata extracts (mm zone) Tablo 4. C. virgata ekstraktlarının antimikrobiyal aktiviteleri (mm zone) Streptomycin sulfate (10 mg/disc) and Nystatin (30 mg/disc) were used as standard antibiotic discs. The diameter of the paper discs was 6 mm. nd: not determined

Antiproliferative Properties
The antiproliferative property results of C. virgata, C. kurdica and C. saligna water, ethanol, methanol and acetone extracts on the LNCaP, HCT-116 and MCF-7 cancer cell lines are shown in Tables S7-S15.
The IC50 values of all the extracts are presented in Table 6 and Figure 1 for the antiproliferative activity.