The genus Centaurea L. belongs to Asteraceae family which comprises of more than 500-600 species that are widespread all over the world, particularly around the Mediterranean and western Asia [1]. In Anatolia, the genus comprises about 207 taxa including 134 endemic species classified in 4 sections with endemism 64 % [1]. Centaurea species are often called zerdali dikeni, timur dikeni and peygamber çiçegi in Anatolia [2]. In Turkish traditional medicine, these species are widely used as expectorant, antidiabetic, antipyretic and antidiarrhoeal [2]. Centaurea species are known to have various biological activities such as antimicrobial [4], antifungal [5], antiinflammatory [6], antiulcerogenic [7], antioxidant [8], antiplasmoidal [9], antiprotozoal [4], cytotoxic [9,10] and anticancer [10].

In previous studies, sesquiterpene lactones, flavonoids and phenolic compounds have been isolated from the plants [3]. These isolates include 10 flavonoids (myricetin, fisetin, quercetin, naringenin, hesperetin, luteolin, kaempferol, apigenin, rhamnetin, chrysin), 3 flavonoid glycosides (rutin, hesperidin, hyperoside), 9 phenolic acids (gallic, chlorogenic, protocatechuic, tannic, tr-caffeic, p-coumaric, rosmarinic, 4-OH benzoic and salycylic acids), one phenolic aldehyde (vanillin), one coumarin and other 3 organic acids (quinic, malic and tr-aconitic acids). Some of the compounds obtained from other Centaurea species include quercetin from C. omphalotricha [11], luteolin and apigenin and kaempferol from C. urvillei subsp. urvillei [12], rutin and chlorogenic acid from C. calolepis [13], protocatechuic acid from C. isaurica [14], chlorogenic acid from C. cadmea [15],  and C. isaurica [14],  vanillin from  C. sadleriana [16].  To the best of our knowledge, none of these earlier studies used LC-MS/MS in determination of phytochemical compositions of the plant species.

Therefore the purpose of this study was to determine the chemical profiles of Centaurea species using liquid chromatography coupled to tandem electrospray mass spectrometry and as well as the biological activities of the plants.

Chemicals

Butylated hydrox-ytoluene (BHT) and 2,2’-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Merck (Darmstadt, Germany). Malic acid, quercetin, protocatechuic acid, chrysin, rutin, hesperetin, naringenin, rosmarinic acid, vanillin, p-coumaric acid,caffeic acid, chlorogenic acid, hyperoside, myricetin, coumarin, kaempferol, formicacid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 5,5-dithiobis-(2-nitro benzoic acid) (DTNB), copper (II) chloride dihydrate (CuCl₂.2H₂O), neocuproine(2,9-dimethyl-1,10-phenanthroline), EDTA (Ethylenedi-aminetetraacetic acid), acetylcholinesterase (AChE: fromelectric eel) (Type-VI-S, EC 3.1.1.7, 425.84 U/mg), and butyryl-cholinesterase (BChE: from horse serum) (EC 3.1.1.8, 11.4 U/mg) were obtained from Sigma (Steinheim,  Germany). Quinic acid, tr-aconitic acid, 4-hydroxybenzoic acid, fisetin, α-tocopherol and acetylthiocholine iodide were from Aldrich (Steinheim, Germany). Gallic acid, tannic acid, salicylic acid, and galanthamine hydrobromide were purchased from Sigma–Aldrich (Steinheim, Germany). Folin Ciocalteu Phenol reagent was from Applichem (Darmstadt, Germany) while hesperidin, luteolin, apigenin, rhamnetin and butyrylthiocholineiodide were from Fluka (Steinheim, Germany). All solvents were of LC-MS and analytical grade.

Plant material

The whole plants of Centaurea balsamita Lam., Centaurea depressa Bieb. and Centaurea lycopifolia Boiss et Kotschy were collected from southeastern Turkey (Diyarbakır, Malatya and Maraş, respectively) in July 2012 by Dr A Ertaş, and identified by Dr Y Yeşil. These specimens were stored at the Herbarium of Istanbul University (ISTE 97140, ISTE 97664 and ISTE 97138).

Preparation of plant extracts for LC-ESI-MS/MS

The plants were dried, powdered and 10 g of each was extracted with MeOH for 24 h at room temperature. Resultant extracte were filtered and evaporated under vacuum. Dry filtrate was diluted to 250 mg/L and passed through the microfiber filter (0.2 µm) for LC-ESI MS/MS.

Preparation of plant extracts for biological activities

Whole plant materials were dried, powdered and 100 g of each was sequentially macerated three times with petroleum ether, acetone, methanol and water (250 mL) for 24 h at room temperature. After filtration, the solvents were evaporated to obtain the crude extracts. The yields of the petroleum ether extracts of the three plants studied were obtained as: CBP (Centaurea balsamita petroleum ether extract) 0.7 %, CDP (Centaurea depressa petroleum ether extract) 0.6 %, CLP (Centaurea lycopifolia petroleum ether extract) 0.8 %; the acetone extracts as CBA (Centaurea balsamita acetone extract) 2.1 %, CDA (Centaurea depressa acetone extract) 1.3 %, CLA (Centaurea lycopifolia acetone extract) 2.6 %, the methanol extracts as CBM (Centaurea balsamita methanol extract) 7.2 %, CDM (Centaurea depressa methanol extract) 5.2 %, CLM (Centaurea lycopifolia methanol extract) 8.5 %, and the water extracts as CBW (Centaurea balsamita water extract) 2.3 %, CDW (Centaurea depressa water extract) 1.8 %, CLW (Centaurea lycopifolia water extract) 3.1 % (w/w).

Phenolic compound identification and quantification

LC-MS/MS analysis of the phenolic compounds were performed using a Shimadzu Nexera model UHPLC instrument coupled to a tandem MS instrument. The liquid chromatograph was equipped with LC-30AD binary pumps, DGU-20A3R degasser, CTO-10ASvp column oven and SIL-30AC autosampler. Chromatographic separation of plant extract samples was performed on a C18 reversed-phase Inertsil ODS-4 (150 mm × 4.6 mm, 3 µm) analytical column with the column temperature fixed at 40 ^oC. The elution gradient consisted of mobile phase (A) water (5 mM ammonium formate and 0.1 % formic acid) and (B) methanol (5 mM ammonium formate and 0.1 % formic acid). Gradient elution using the proportions of solvent B of 40 %, 90 %, 90 %, 40 % and 40 % at 0, 20, 23.99, 24 and 29 min, respectively was applied. The solvent flow rate was maintained at 0.5 mL/min and injection volume was 4 µL. MS detection was performed using Shimadzu LCMS 8040 model triple quadrupole mass spectrometer equipped with an ESI source operating in both positive and negative ionization mode (Shimadzu, Kyoto, Japan). LC-ESI-MS/MS data were collected and processed using LabSolutions software (Shimadzu, Kyoto, Japan). The multiple reaction monitoring (MRM) mode was used to quantify the analytes: the assay of phenolic compounds was performed following two or three transitions per compound, the first one for quantitative purposes and the second and/or the third one for confirmation.

Optimization of LC-ESI-MS/MS method

Subsequent to several combinations of trials, a gradient of methanol (5 mM ammonium formate and 0.1 % formic acid) and water (5 mM ammonium formate and 0.1 % formic acid) system was concluded to be the best mobile phase solution. ESI source was chosen instead of APCI (Atmospheric Pressure Chemical Ionization) and APPI (Atmospheric Pressure Photoionization) sources as the phenolic compounds were small and relatively polar molecules. Tandem mass spectrometry was used for the current study since this system is commonly used for its fragmented ion stability [18]. The working conditions were determined as interface temperature of 350 ^oC, DL temperature of 250 ^oC, heat block temperature of 400 ^oC, nebulizing gas flow using Nitrogen, 3 L/min and drying gas flow (nitrogen) of 15 L/min.

The linearity of the phenolic standards was affirmed in the range of: 0.025 to 10 mg/L (). Regression coefficient of each calibration graph was found to be higher than 0.99. Limit of detection (LOD) and limit of quantitation (LOQ) of the method reported in this study were dependent on the calibration curve established from six measurements. LOD and LOQ were determined using the equations, 3S/N and 10S/N, respectively where S/N is the signal (S) to noise (N) ratio) (). For different compounds, LOD ranged from 0.05 to 25.8 µg/L and LOQ ranged from 0.17 to 85.9 µg/L (). Furthermore, the recovery of the phenolic compounds standards ranged from 96.9 % to 106.2 %.

Determination of total phenolic and flavonoid contents

Total phenolic and flavonoid amounts in the crude methanol extracts were determined as previously described and expressed as pyrocatechol and quercetin equivalents using the following equations [19]:

Absorbance = 0.0251 pyrocatechol (μg) + 0.0445   (R² = 0.9945)

Absorbance = 0.0301 quercetin (μg) + 0.0553         (R² = 0.9984)

Antioxidant activity of the extracts

We used the DPPH free radical, ABTS cation radical scavenging activity and cupric reducing antioxidant capacity (CUPRAC) methods to determine the antioxidant activity [19].

Anticholinesterase activity of the extracts

A spectrophotometric method developed by Ellman et al [19] was used to test the acetyl- and butyryl-cholinesterase inhibitory activities.

Antimicrobial activity of the extracts

Five different microorganisms including Gram positive bacteria (Streptococcus pyogenes ATCC19615 and Staphylococcus aureus ATCC 25923), Gram negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922) and yeast (Candida albicans ATCC10231) which were purchased from Refik Saydam Sanitation Center (Turkey) were used for detecting the antimicrobial activity of the extracts. The disc diffusion method was employed for this purpose and the minimum inhibitory concentrations (MICs) were determined by the broth macrodilution method according to NCCLS [20]. Ampicillin and fluconazole were used as positive controls for bacteria and yeast, respectively.

Statistical analysis

The results of the antioxidant and anticholinesterase activity assays were expressed as means ± SD. Unpaired Student’s t-test and ANOVA were used for data comparison. A 2-tailed p values less than 0.05 was considered to be significant.

Quantitative analysis of phenolic compounds

Methanolic extracts of C. balsamita, C. depressa and C. lycopifolia were found to contain. 27 compounds including 10 flavonoids, 3 flavonoid glycosides, 9 phenolic acids, 1 phenolic aldehyde, 1 coumarin and 3 other organic acids (A). These compounds were monitored by the transition from the specific deprotonated molecular ions [M-H⁺] to the corresponding fragment ions [M-H⁺-X]. The molecular ions and fragments observed in MS/MS, related collision energies for these fragments and the quantified results for the three Centaurea species are shown in .

Fisetin and kaempferol were not detected by this method from C. balsamita. Similarly, fisetin and hesperetin were not detected from C. depressa. Myricetin, fisetin, hesperetin, quercetin, naringenin and kaempferol were equally detected from the extracts of C. lycopifolia. However, the quamtities of quinic acid found in C. balsamita (17513.16 ± 813.48 µg/g), C. depressa (63874.33 ± 3065.97 µg/g) and C. lycopifolia (108233.80 ± 5195.22 µg/g) were more than the quantities of other compounds. About 10 % of the methanol extract of C. lycopifolia was quinic acid. C. balsamita and C. lycopifolia were found to be rich in chlorogenic acid (10770.70 ± 365.66 µg/g and 15782.64 ± 463.45 µg/g, respectıvely) while C. depressa was found to have high amount of malic acid (22587.95 ± 1524.52 µg/g) (, B-D).

Antioxidant activity assays and total phenolic and flavonoid content

The methanol extract of C. balsamita had the highest amount of total phenolic while the water extract of the same plant species had the highest content of total flavonoid content ().

As shown in , CBM, CDM and CLM extracts had 62.65 ± 0.97, 82.21 ± 0.92 and 81.45 ± 1.32 µg/mL IC₅₀ values in the DPPH free radical scavenging activity assay, respectively. The other extracts showed weak or no activity.

In the ABTS cation radical scavenging assay, CBA, CBM, CBW and CLW extracts exhibited 47.50 ± 0.63, 24.21 ± 0.70, 44.89 ± 1.09 and 35.03 ± 0.80 µg/mL IC₅₀ values, respectively. The CBM, and CLW extracts exhibited strong ABTS cation radical scavenging activity. CBA and CBW extracts showed moderate activity with 70.96, 65.91 and 79.49 % inhibitions, respectively ().

Anticholinesterase and antimicrobial activities

As provided in , all the extracts had no activity against acetylcholinesterase enzyme, while CBP, CBA, CDP, CLP and CLA extracts showed moderate activity (56.69, 57.46, 48.15, 53.11 and 47.19 % inhibition, respectively) against butyrylcholinesterase enzyme at 200 µg/mL. The acetone extracts of the plants were active against all microorganisms tested with different levels of inhibition: weak (inhibition zone <12) and moderate (inhibition zone <20-12) (). Among the tested microorganisms, C. albicans was the most susceptible microorganism against acetone extracts. The extracts exhibited moderate antimicrobial activity against C. albicans and S. pyogenes.   For a more reliable assessment of antimicrobial activity, a broth dilution assay was carried out. The susceptibility of the test microorganisms against active extracts was evaluated and results are shown as MIC (). The lowest MIC value was recorded by C. balsamita against C. albicans (45 µg mL-1) in conformity with the result of disc diffusion method. MIC values of C. balsamita against E. coli and other Gram (-) bacteria were more than 1000 μg mL^-1.

Present study has revealed that C. balsamita has antioxidant, anticholinesterase and antimicrobial properties. Several studies are present in literature reporting the use of liquid chromatography electrospray ionization tandem mass spectrometry to perform quantitative analyses [18,19]. In this study, an accurate method on a mass spectrometer equipped with a triple quadrupole analyzer was developed for the analyses of 27 compounds in Centaurea species. Quinic acid was found to be the most abundant among the compounds and about 10 % of methanol extract of C. lycopifolia was quinic acid. For the first time, mrycetin, fisetin, naringenin, hesperetin, rhamnetin, chrysin, hesperidin, hyperoside, gallic acid, tannic acid, caffeic acid, p-coumaric acid, rosmarinic acid, 4-hydroxybenzoic acid, salicylic acid, malic acid and tr-aconitic acid were detected in the three plant species using LC-MS-MS. Also, flavonoids, flavonoid glycosides, phenolic acids and other organic acids constituents of Centaurea species have been detected with LC-MS-MS.

The antibacterial effect of C. balsamita seen in this study has been reported previously in eight Centaurea species [4,9]. Sımilarly, the antifungal activity of C. balsamita has been reported in Candida krusei [10].  As in an earlier report, the methanol extract of the three Centaurea species exhibited the highest effect in DPPH free and ABTS cation radicals scavenging activities just like C. pulcherrima var. pulcherrima [21]. The ABTS cation radical scavenging activity in our study may be caused by higher quercetin contents of C. balsamita than other investigated plants. In CUPRAC assay, Centaurea extracts showed very weak activity that verify the CUPRAC assay results of Centaurea species reported by Aktümsek et al [21].

In the report of Aktümsek et al [21], water extracts of C. pyrrhoblephara and C. antalyense were not active against AChE as in our results but their methanol extracts exhibited weak activity against AChE. However, all the tested extracts displayed more or less inhibition on BChE, except for methanolic extract of C. pyrrhoblephara [21] while our results of CBP, CBA, CDP, CLP and CLA extracts showed moderate antibutyrylcholinesterase activity. Acetone extract of Centaurea balsamita has good antifungal activity against Candida albicans. Inhibition zones and MIC values of the acetone extracts of Centaurea species on the yeast Candida albicans compared to that of positive control (fluconazole) indicated their moderate antifungal properties.

Some compounds including mrycetin, fisetin, naringenin, hesperetin, rhamnetin, chrysin, hesperidin, hyperoside, gallic acid, tannic acid, caffeic acid, p-coumaric acid, rosmarinic acid, 4-hydroxybenzoic acid, salicylic acid, malic acid and tr-aconitic acid have been detected for the first time in Centaurea species using LC-MS-MS. Centaurea balsamita has antioxidant, anticholinesterase and antimicrobial properties. Further studies to identify the compounds in the extracts responsible for the activities are needed.