Boga et al Tropical Journal of Pharmaceutical Research September 2016; 15 (9): 1865-1875 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v15i9.8 Original Research Article Phytochemical profile and some biological activities of three Centaurea species from Turkey Mehmet Boğa1*, Hüseyin Alkan2, Abdulselam Ertaş3, Elif Varhan Oral4, Mustafa A Yılmaz5, Yeter Yeşil6, Ahmet C Gören7, Hamdi Temel8 and Ufuk Kolak9 1Department of Pharmaceutical Technology, 2Department of Biochemistry, 3Department of Pharmacognosy, 4Department of Analytical Chemistry, Faculty of Pharmacy, 5Research and Application of Science and Technology Center (DUBTAM), 21280 Dicle University, Diyarbakır, 6Department of Pharmaceutical Botany, Faculty of Pharmacy, 34116, Istanbul University, Istanbul, 7TÜBITAK UME, National Metrology Institute, Chemistry Group, Organic Chemistry Laboratory, 4147, Kocaeli, 8Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Dicle University, 21280 Diyarbakır, 9Department of General and Analytical Chemistry, Faculty of Pharmacy, Istanbul University, 34116 Beyazıt, Istanbul, Turkey *For correspondence: Email: [email protected], [email protected]; Tel: +90 412 2411000/7542 Received: 18 December 2015 Revised accepted: 19 June 2016 Abstract Purpose: To characterise the phytochemical profile of whole plants of Centaurea balsamita, C. depressa and C. lycopifolia with LC-ESI-MS/MS, and as well as their antioxidant, anticholinesterase and antimicrobial activities. Methods: Organic and aqueous extracts of the three Centaurea species were evaluated for DPPH free radical, ABTS cation radical scavenging and cupric reducing antioxidant capacity (CUPRAC). Acetyl- and butyryl-cholinesterase enzyme inhibition abilities of the extracts using petroleum ether, acetone, methanol and water were studied to determine anticholinesterase activity, while antimicrobial activity was determined by disc diffusion method using appropriate antimicrobial standards and organisms. The phytochemical components of the methanol extracts were assessed by LC-MS/MS. Results: The methanol extract of C. balsamita exhibited much higher DPPH free and ABTS cation radicals scavenging activities (with IC50 of 62.65 ± 0.97 and 24.21 ± 0.70 mg/ml, respectively) than the other extracts. The petroleum ether extracts of the plant species exhibited moderate inhibitory activity against butyrylcholinesterase enzymes while the acetone extract of C. balsamita showed good antifungal activity against Candida albicans. Quinic acid (17513 ± 813 µg/g, 63874 ± 3066 µg/g and 108234 ± 5195 µg/g) was the major compound found in the methanol extracts of C. balsamita, C. depressa and C. Lycopifolia, respectively. Conclusion: These results indicate quinic acid is the major compound in the three plant species and that Centaurea balsamita has significant antioxidant, anticholinesterase and antimicrobial properties. Further studies to identify the compounds in the extracts responsible for the activities are required. Keywords: Centaurea, LC-ESI-MS/MS, Anticholinesterase, Antioxidant, Antimicrobial Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION western Asia [1]. In Anatolia, the genus comprises about 207 taxa including 134 endemic The genus Centaurea L. belongs to Asteraceae species classified in 4 sections with endemism 64 family which comprises of more than 500-600 % [1]. Centaurea species are often called zerdali species that are widespread all over the world, dikeni, timur dikeni and peygamber çiçegi in particularly around the Mediterranean and Anatolia [2]. In Turkish traditional medicine, these Trop J Pharm Res, September 2016; 15(9): 1865 Boga et al species are widely used as expectorant, Sigma (Steinheim, Germany). Quinic acid, tr- antidiabetic, antipyretic and antidiarrhoeal [2]. aconitic acid, 4-hydroxybenzoic acid, fisetin, α- Centaurea species are known to have various tocopherol and acetylthiocholine iodide were from biological activities such as antimicrobial [4], Aldrich (Steinheim, Germany). Gallic acid, tannic antifungal [5], antiinflammatory [6], acid, salicylic acid, and galanthamine antiulcerogenic [7], antioxidant [8], antiplasmoidal hydrobromide were purchased from Sigma– [9], antiprotozoal [4], cytotoxic [9,10] and Aldrich (Steinheim, Germany). Folin Ciocalteu anticancer [10]. Phenol reagent was from Applichem (Darmstadt, Germany) while hesperidin, luteolin, apigenin, In previous studies, sesquiterpene lactones, rhamnetin and butyrylthiocholineiodide were from flavonoids and phenolic compounds have been Fluka (Steinheim, Germany). All solvents were of isolated from the plants [3]. These isolates LC-MS and analytical grade. include 10 flavonoids (myricetin, fisetin, quercetin, naringenin, hesperetin, luteolin, Plant material kaempferol, apigenin, rhamnetin, chrysin), 3 flavonoid glycosides (rutin, hesperidin, The whole plants of Centaurea balsamita Lam., hyperoside), 9 phenolic acids (gallic, chlorogenic, Centaurea depressa Bieb. and Centaurea protocatechuic, tannic, tr-caffeic, p-coumaric, lycopifolia Boiss et Kotschy were collected from rosmarinic, 4-OH benzoic and salycylic acids), southeastern Turkey (Diyarbakır, Malatya and one phenolic aldehyde (vanillin), one coumarin Maraş, respectively) in July 2012 by Dr A Ertaş, and other 3 organic acids (quinic, malic and tr- and identified by Dr Y Yeşil. These specimens aconitic acids). Some of the compounds obtained were stored at the Herbarium of Istanbul from other Centaurea species include quercetin University (ISTE 97140, ISTE 97664 and ISTE from C. omphalotricha [11], luteolin and apigenin 97138). and kaempferol from C. urvillei subsp. urvillei [12], rutin and chlorogenic acid from C. calolepis Preparation of plant extracts for LC-ESI- [13], protocatechuic acid from C. isaurica [14], MS/MS chlorogenic acid from C. cadmea [15], and C. isaurica [14], vanillin from C. sadleriana [16]. To The plants were dried, powdered and 10 g of the best of our knowledge, none of these earlier each was extracted with MeOH for 24 h at room studies used LC-MS/MS in determination of temperature. Resultant extracte were filtered and phytochemical compositions of the plant species. evaporated under vacuum. Dry filtrate was diluted to 250 mg/L and passed through the Therefore the purpose of this study was to microfiber filter (0.2 µm) for LC-ESI MS/MS. determine the chemical profiles of Centaurea species using liquid chromatography coupled to Preparation of plant extracts for biological tandem electrospray mass spectrometry and as activities well as the biological activities of the plants. Whole plant materials were dried, powdered and EXPERIMENTAL 100 g of each was sequentially macerated three times with petroleum ether, acetone, methanol Chemicals and water (250 mL) for 24 h at room temperature. After filtration, the solvents were evaporated to Butylated hydrox-ytoluene (BHT) and 2,2’- obtain the crude extracts. The yields of the Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) petroleum ether extracts of the three plants diammonium salt (ABTS) were purchased from studied were obtained as: CBP (Centaurea Merck (Darmstadt, Germany). Malic acid, balsamita petroleum ether extract) 0.7 %, CDP quercetin, protocatechuic acid, chrysin, rutin, (Centaurea depressa petroleum ether extract) 0.6 hesperetin, naringenin, rosmarinic acid, vanillin, %, CLP (Centaurea lycopifolia petroleum ether p-coumaric acid,caffeic acid, chlorogenic acid, extract) 0.8 %; the acetone extracts as CBA hyperoside, myricetin, coumarin, kaempferol, (Centaurea balsamita acetone extract) 2.1 %, formicacid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), CDA (Centaurea depressa acetone extract) 1.3 5,5-dithiobis-(2-nitro benzoic acid) (DTNB), %, CLA (Centaurea lycopifolia acetone extract) copper (II) chloride dihydrate (CuCl2.2H2O), 2.6 %, the methanol extracts as CBM (Centaurea neocuproine(2,9-dimethyl-1,10-phenanthroline), balsamita methanol extract) 7.2 %, CDM EDTA (Ethylenedi-aminetetraacetic acid), (Centaurea depressa methanol extract) 5.2 %, acetylcholinesterase (AChE: fromelectric eel) CLM (Centaurea lycopifolia methanol extract) 8.5 (Type-VI-S, EC 3.1.1.7, 425.84 U/mg), and %, and the water extracts as CBW (Centaurea butyryl-cholinesterase (BChE: from horse serum) balsamita water extract) 2.3 %, CDW (Centaurea (EC 3.1.1.8, 11.4 U/mg) were obtained from Trop J Pharm Res, September 2016; 15(9): 1866 Boga et al depressa water extract) 1.8 %, CLW (Centaurea lycopifolia water extract) 3.1% (w/w). The linearity of the phenolic standards was affirmed in the range of: 0.025 to 10 mg/L (Table Phenolic compound identification and 1). Regression coefficient of each calibration quantification graph was found to be higher than 0.99. Limit of detection (LOD) and limit of quantitation (LOQ) of LC-MS/MS analysis of the phenolic compounds the method reported in this study were were performed using a Shimadzu Nexera model dependent on the calibration curve established UHPLC instrument coupled to a tandem MS from six measurements. LOD and LOQ