Food and Chemical Toxicology 55 (2013) 290–296

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Food and Chemical Toxicology

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Antioxidant potentials and anticholinesterase activities of methanolic andaqueous extracts of three endemic Centaurea L. species

Abdurrahman Aktumsek a,⇑, Gokhan Zengin a, Gokalp Ozmen Guler b, Yavuz Selim Cakmak c,Ahmet Duran a

a Department of Biology, Science Faculty, Selcuk University, Konya, Turkeyb Department of Biological Education, Ahmet Kelesoglu Education Faculty, Necmettin Erbakan University, Konya, Turkeyc Deparment of Biotechnology and Molecular Biology, Faculty of Science and Arts, Aksaray University, Aksaray, Turkey

a r t i c l e i n f o

Article history:Received 17 November 2012Accepted 8 January 2013Available online 26 January 2013

Keywords:CentaureaAntioxidantPhenolicsAnticholinesterase activity

0278-6915/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fct.2013.01.018

⇑ Corresponding author. Tel.: +90 332 223 18 66; fE-mail address: aktumsek@selcuk.edu.tr (A. Aktum

a b s t r a c t

The methanol and aqueous extracts of three endemic Centaurea species (C. polypodiifolia var. pseudobehen,C. pyrrhoblephara and C. antalyense) were investigated for their antioxidant and cholinesterase inhibitoryactivities. The antioxidant activities of these extracts were evaluated by in vitro models including, phosp-homolybdenum assay, free radical scavenging assays (DPPH and ABTS), b-carotene/linoleic acid test sys-tem, metal chelating assay, FRAP assay, ferric and cupric reducing power. Cholinesterase inhibitoryactivities were examined using Ellman’s colorimetric method. Total phenol, flavonoid, and saponin con-tents were also measured. Among the six Centaurea extracts evaluated, the highest antioxidant abilitieswere obtained from C. polypodiifolia var. pseudobehen. Methanolic extracts from C. polypodiifolia var.pseudobehen and C. antalyense had a noticeable inhibition towards AChE and BChE. These findings suggestthat Centaurea species could be an anticholinesterase agent and antioxidant resource in some industries,such as food, pharmacology, and cosmetics.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Free radicals, especially oxygen free radicals (OFRs) or reactiveoxygen species (ROSs) (e.g. superoxide, hydroxyl and hydrogenperoxide) are active oxygen compounds generated by oxidationreactions of exogenous factors (Halliwell and Gutteridge, 1990;Cerutti, 1991). These reactive species may oxidize proteins, lipidsor DNA and can initiate degenerative/chronic diseases including,cancer, diabetes and cardiovascular disease (Lee et al., 2004; Man-tena et al., 2008). Antioxidants are substances that when present atlow concentrations with respect to oxidizable substrates, inhibit ordelay the oxidation process (Sikorski, 2001). Therefore, antioxi-dants have a vital role in the maintenance of human health andprevention of disease caused by free radicals. Due to the benefitsof antioxidants, food and pharmaceutical products are normallyenriched with synthetic antioxidants such as BHA, BHT and PG.However, application of these synthetic antioxidants might leadto toxic effects such as carcinogen (Barlow and Schlatter, 2010).Hence, stronger restrictions have been mandated for their applica-tion and there is a trend to substitute synthetic antioxidants withnatural antioxidants.

ll rights reserved.

ax: +90 332 241 01 06.sek).

Alzheimer’s disease (AD) is the most common form of neurode-generative disease and characterized by memory dysfunction.Reduction of acetylcholine levels in the brain is the most notablebiochemical change in AD (Loizzo et al., 2009). Therefore, AD canbe treated by use of agents which restore the level of acetylcholinethrough inhibition of both two major form of cholinesterase: ace-tylcholinesterase (AChE) and butrylcholinesterase (BChE) (Jaenet al., 1996). Tacrine, rivastigmine and galanthamine are used ascholinesterase inhibitors for treatment of AD. However, these com-pounds have been reported to have their adverse effects such as,hepatotoxicity and gastrointestinal disturbances (Lee et al., 2011;Schulz, 2003; Melzer, 1998). Therefore, safe and active AChE inhib-itors particularly from natural products have been gaining moreattention in recent decades.

The genus Centaurea (Asteraceae) is a large genus and com-prises approximately 500 species distributed in old World (Dit-trich, 1977). In Anatolia peninsula, the genus is represented byabout 190 species with more than 100 endemic species (Davis,1988; Güner et al., 2000). Some Centaurea species are used as her-bal remedies in Anatolian folk medicine to treat fever, diabetes andhemoroid and peptid ulcer (Kargioglu et al., 2008; Honda et al.,1996). Pharmacological and phytochemical studies on many Cen-taurea species have reported antioxidant, antimicrobial and anti-pyretic activities (Tepe et al., 2006; Zengin et al., 2011; Tekeliet al., 2011; Koca et al., 2009). Nonetheless, to our best knowledge,

A. Aktumsek et al. / Food and Chemical Toxicology 55 (2013) 290–296 291

no data on phytochemical studies of the Centaurea species (C.polypodiifolia var. pseudobehen, C. pyrrhoblephara and C. antalyanse)are available to date. This study aims to evaluate the antioxidantand cholinesterase inhibitory effect of methanol and aqueous ex-tracts obtained from three Centaurea species, as well as their phe-nolic, flavonoid and saponin contents. Thus, the present studymight supply valuable information on the phytochemical proper-ties of the Centaurea species for nutraceutical and pharmacologicalindustry.

2. Materials and methods

2.1. Plant materials

Centaurea species were collected from different localities in Turkey during veg-etation season of 2010. The plants were identified by botanist Professor. Dr. AhmetDuran. The voucher specimens were deposited in Selcuk University Science FacultyHerbarium, Konya. Taxonomical information’s, localities, collection periods andvoucher numbers of endemic Centaurea species studied were given below:

1. C. polypodiifolia var. pseudobehen (Boiss) Wagenitz: Sivas-Erzincan road, 11 km,14.07.2010, 1310 m, AD9113.

2. C. pyrrhoblephara (Boiss): Erzincan, between Sakaltutan and Erzincan,14.07.2010, 2045 m, AD9117.

3. C. antalyanse: Antalya, Akseki, Guzelsu, Otluk Mountain, Kuyuonu station,23.07.2010, 1640 m, AD9219.

2.2. Preparation of the extracts

The aerial plant materials were dried at the room temperature. The dried plantmaterials were ground to a fine powder using a laboratory mill. Fifteen grams ofpowdered plant were mixed with 250 mL methanol and extracted in a Soxhletapparatus for 6–8 h. The extracts concentrated under vacuum at 40 �C by using arotary evaporator. To obtain water extracts, air dried powdered plants were boiledwith 250 mL of distilled water for 30 min. The aqueous extracts were filtered andlyophilized (�80 �C, 48 h). Extracts were stored at +4 �C in dark until use.

2.3. Chemicals

Potassium ferricyanide, ferric chloride, Folin–Ciocalteu’s reagent, trichloroaceticacid, methanol, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA)and methanol were purchased from Merck (Darmstadt, Germany). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), b-carotene, linoleic acid and Tween 40 were purchasedfrom Sigma Chemical Co. (Sigma–Aldrich GmbH, Sternheim, Germany). All otherchemicals and solvents were analytical grade.

2.4. Assay for total phenolics

Total phenolic constituents of the extracts were determined by employing themethods given in the literature (Slinkard and Singleton, 1977) involving Folin–Ciocalteu reagent and gallic acid as standard. One milliliter of extract solutioncontaining 2 mg extract was added to a volumetric flask. Forty-five milliliterdistilled water and 1 mL Folin–Ciocalteu reagent was added and flask was shakenvigorously. After 3 min, 3 mL of Na2CO3 (2%) solution was added and the mixturewas allowed to stand for 2 h by intermittent shaking. Absorbance was measuredat 760 nm (Shimadzu-UV1800). The total phenolic content was determined as gallicacid equivalents (mg GAE g�1 extract).

2.5. Assay for total flavonoids

The total flavonoid content in extracts was determined spectrophotometricallyaccording to Arvouet-Grand et al. (1994). Briefly, 1 mL of 2% aluminum trichloride(AlCl3) methanolic solution was mixed with the same volume of extract solution (at2 mg mL�1 concentration). The absorbance values of the reaction mixtures weredetermined at 415 nm after 10 min duration against a blank. Rutin was used asthe standard and the total flavonoids content of the extracts was expressed as mgrutin equivalents per gram of extract (mg RE g�1 extract).

2.6. Determination of total saponins

The total saponins contents of extracts were determined by the vanillin-sulfuricacid method (Hiai et al., 1976). These extracts were reacted with vanillin (8%) andsulfuric acid (72%). The mixture was incubated at 60 �C for 10 min. Then the mix-ture was cooled for another 15 min, followed by absorbance measurement at538 nm. Quillaja saponin was used as a standard and the content of total saponinswas expressed as Quillaja equivalents (mg QAE g�1 extract).

2.7. Evaluation of total antioxidant capacity by phosphomolybdate assay

The total antioxidant capacities of extracts were evaluated by phosphomolyb-denum method according to Prieto et al. (1999). 0.3 mL of extracts (2 mg mL�1)were combined with 3 mL reagent solution (0.6 M sulfuric acid, 28 mM sodiumphosphate and 4 mM ammonium molybdate). The tubes containing the reactionsolution were incubated at 95 �C for 90 min. Then the absorbance of the solutionwas measured at 695 nm against a blank. The antioxidant capacity of extractswas expressed as equivalents of ascorbic acid (mg AE g�1 extract).

2.8. Scavenging activity on DPPH

The hydrogen atoms or electrons donation ability of the corresponding extractsand were measured from the bleaching of purple colored methanol solution ofDPPH. The effect of Centaurea extracts on DPPH radical was estimated accordingto Sarikurkcu et al. (2009). One milliliter of various concentrations of the extractswas added to a 4 mL of a 0.004% methanol solution of DPPH. The mixture was sha-ken vigorously and allowed standing for 30 min; the absorbance of the resultingsolution was measured at 517 nm with a spectrophotometer. Inhibition activitywas calculated in following way (Eq. (1)):

Ið%Þ ¼ ðA0 � A1Þ=A0 � 100 ð1Þ

where A0 is the absorbance of the control, A1 is the absorbance of the extract/standard.

2.9. ABTS (2,2 azino-bis (3-ethylbenzothiazloine-6-sulfonic acid)) radical scavengingactivity

The scavenging capacity against ABTS assay was measured according to themethod of Re et al. (1999). Briefly, ABTS radical cation was produced directly byreacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowingthe mixture to stand for 12–16 in dark at the room temperature. Prior to assay,the solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at734 nm. Extracts (1 mL) were then added to 2 mL of ABTS solution and mixed.The mixture was left at room temperature for 30 min and absorbance was read at734 nm. The results were reported as percentage inhibition and calculated accord-ing to Eq. (1).

2.10. b-Carotene–linoleic acid assay

In this assay antioxidant capacity is determined by measuring the inhibition ofthe volatile organic compounds and the conjugated diene hydroperoxides arisingfrom linoleic acid oxidation (Dapkevicius et al., 1998). A stock solution of b-caro-tene–linoleic acid mixture was prepared as following: 0.5 mg b-carotene wasdissolved in 1 mL of chloroform (HPLC grade) 25 lL linoleic acid and 200 mg Tween40 was added. Chloroform was completely evaporated using a vacuum evaporator.Then 100 mL of oxygenated distilled water was added with vigorous shaking;2.5 mL of this reaction mixture was dispersed to test tubes and 0.35 mL of theextracts (2 mg mL�1) were added and the emulsion system was incubated for upto 2 h at 50 �C. The same procedure was repeated with the positive control BHT,BHA and a blank. After this incubation period, absorbance of the mixtures wasmeasured at 490 nm. Measurement of absorbance was continued until the colorof b-carotene disappeared. The bleaching rate (R) of b-carotene was calculatedaccording to the following equation:

R ¼ lnða=bÞ=t ð2Þ

where ln = natural log, a = absorbance at time 0, b = absorbance at time t (120 min).The antioxidant activity (AA) was calculated in terms of percent inhibition relative tothe control using Eq. (3).

AA ¼ ½ðRControl � RSampleÞ=RControl� � 100 ð3Þ

Antioxidative activities of the extracts were compared with those of BHT andBHA at 2.0 mg mL�1.

2.11. Metal chelating activity

The chelating activity of iron(II) ions was studied as described by Dinis et al.(1994). Initially, 500 lL extract was added to 100 lL of 2 mM FeCl2. The reactionwas initiated by adding 200 lL of 5 mM ferrozine. Total volume was adjusted to2 ml with methanol. After 10 min incubation at room temperature, the absorbanceat 562 nm was recorded. EDTA was used as a standard chelating agent and resultswere expressed as mg EDTA equivalents g�1 extract.

2.12. Reducing power activity (Iron(III) to iron(II) reduction)

The ferric reducing power applied with slight modifications of the method ofOyaizu (1986). Various concentrations of the extracts (2.5 mL) were mixed with2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide.

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The mixture was incubated at 50 �C for 20 min. After 2.5 mL of 10% trichloroaceticacid was added. 2.5 mL of the reaction mixture was mixed with 2.5 mL distilledwater and 0.5 mL of 0.1% ferric chloride. The solution absorbance was measuredat 700 nm. The reducing power of samples increased with the absorbance value.

2.13. Cupric ion reducing antioxidant capacity (CUPRAC assay)

The cupric ion reducing capacity of extracts was determined according to themethod of Apak et al. (2006). One milliliter each of 10 mM CuCl2, 7.5 mM neocupr-oine, and NH4Ac buffer (1 M, pH 7.0) solutions were added into a test tube. Then,0.5 mL different concentrations of the extracts were mixed and total volume wasbrought up to 4.1 mL with deionized water. The mixture absorbance was recordedagainst a blank at 450 nm after 30 min incubation at room temperature.

2.14. FRAP assay

The FRAP assay was determined through a method described by Benzie andStrain (1996) with slight modifications. The FRAP reagent was prepared freshlyby mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ and 20 mM ferric chloridein a ratio of 10:1:1 (v/v/v). Then, 2 mL reagent and 200 lL samples were added totest tubes and incubated at 30 �C for 30 min. Absorbance was measured at593 nm. Trolox was used as standard and results were reported as Trolox equiva-lents per gram of extract.

2.15. Cholinesterase inhibitory activity

Cholinesterase inhibitory activity of tested Centaurea species was assessed bythe spectrophotometric method developed by Ellman et al. (1961). Into a 96-wellplate was placed: 25 lL of 1.5 mM ATCI or BTCI, 125 lL of 0.3 mM DTNB in Buffer(50 mM Tris–HCl pH 8) and 50 lL of tested extracts (2 mg mL�1). Absorbance wasmeasured at 405 nm every 45 s, three times consecutively. Thereafter, 25 lL ofAChE or BChE (0.026 U mL�1) was added to the wells and the absorbance was re-corded again on a microplate reader (Rayto-3100). Galanthamine and methanolwere used as positive and negative controls, respectively. The percentage inhibitionwas calculated by using Eq. (1). The results were also evaluated galanthamineequivalents per gram of extract (mg GALE g�1 extract).

3. Results and discussion

Several chemical tests were selected in this study, based on theability of antioxidants to act as reducing agents (ferric and cupricions) and as radical scavengers (DPPH and ABTS scavenging assays.The assessment of the capacity of pure antioxidant compounds andplant extracts have been the subjects of extensive studies andarguments. A number of methods and variations have been devel-oped and applied for the assessment of antioxidant capacity (Niki,2011). However, evaluation of antioxidant potentials of plant prod-ucts or antioxidant compounds cannot be carried out accurately byany single universal method. Thus, we applied several antioxidantassays that would provide a better insight into the true antioxidantpotential of the extracts.

3.1. Total phenolic, flavonoid and saponin contents

Phytochemicals are increasingly accepted as health promoting,maintaining and repairing agents in cells, tissues, or the whole hu-man body. The phytochemicals that are frequently associated withhuman health are polyphenols, carotenoids and tocopherols. Poly-phenols are the major secondary metabolites in most plants re-ported to possess antioxidant and free radical scavengingactivity. There are several studies on dietary polyphenol use to de-crease the risk of coronary heart disease (Williams and Elliot,1997). Likewise, the compounds are used as anticarcinogenic andanti-inflammatory agents (Carrol et al., 1999; Maeda-Yamamotoet al., 1999). For instance, ellagic acid was the phenolic componentprimarily associated to the chemopreventive effects, appearing tofunction as an anticarcinogen at the initiation and postinitiationstages of tumor development in in vitro and in vivo experiments(Losso et al., 2004; Giampieri et al., 2012). Thus, measurementsof polyphenols content in plants have become important tools in

understanding the values of plants for human health (Amarowiczet al., 2010).

Many reports demonstrate that, besides polyphenolics, plantscontaining biological important substances such as vitamin C,carotenoids and tocopherols have beneficial effects on humanhealth. For example, vitamin C in the strawberry is responsiblefor more than 30% of the total antioxidant capacity (Tulipaniet al., 2008).

The total phenolic and flavonoid contents in the Centaurea ex-tracts are summarized in Table 1. Among studied Centaurea extracts,C. polypodiifolia var. pseudobehen had the highest polyphenol con-tents. The total phenolic contents in the other water extracts werefound as 227.12 mg GAE g�1 extract. The methanolic extracts con-tained similar levels of polyphenolic contents.

Flavonoids comprise the most common group of plant polyphe-nols. Epidemiological studies yield unequivocal results: consump-tion of plants rich in secondary metabolites, especially flavonoidsis associated with a reduced risk of atherosclerosis, cancer, and neu-rodegenerative diseases (Chiva-Blanch and Visioli, 2012; Chonget al., 2010; Nobili et al., 2009). Though the bioactivity of flavonoidsappears to be mediated through a variety of mechanisms, particularattention has been focused on their direct and indirect antioxidantactions. The antioxidant properties are conferred on flavonoids bythe phenolic hydroxyl groups attached to ring structures and theycan act as free radical scavenger, reducing agents and metal chela-tors (Carocho and Ferreira, 2013). Total flavonoids in the Centaureaextracts were determined using spectrophotometric method withaluminum chloride and obtained results varied from 29.43 to55.75 mg RE g�1 extract. All methanol extracts (C. pyrrhoblephara(55.75 mg RE g�1) > C. antalyense (46.76 mg RE g�1) > C. polypodiifo-lia var. pseudobehen (45.94 mg RE g�1)) possessed highest content offlavonoid compared to water extracts (Table 1).

Saponins are a vast group of glycosides, widely distributed inhigher plants. They have pharmacological properties and are usedin phytotheraphy. These compounds are believed to from the mainconstituents of many plant drugs and are reported many pharma-cological properties. Total saponin contents of studied Centurea ex-tracts are presented in Table 1. The highest saponin content wasobtained from methanolic extract of C. pyrrhoblephara with159.87 mg QAE g�1. Total saponin contents in the Centaurea ex-tracts followed the same trend as the total flavonoid content. Nostudies have so far been reported total saponin content of Centau-rea species. Thus, the present study is the first report on saponincontent of Centaurea species.

3.2. Evaluation of antioxidant capacity by phosphomolybdenummethod

The phosphomolybdenum method based on reduction ofMo(VI) to Mo(V) by antioxidant substance and subsequent forma-tion of a green phosphate Mo(V) compounds at acidic pH with anabsorbance at 695 nm (Prieto et al., 1999). Using this method,the result indicated that the water extract of C. polypodiifolia var.pseudobehen had highest antioxidant capacity with a value of549.48 mg AE g�1 extract and this activity may be due to the pres-ence of phenolic compounds. The lowest total antioxidant capacitywas found in the methanol extract of C. pyrrhoblephara with a va-lue of 226.15 mg AE g�1 extract. All water extracts displayed rela-tively higher total antioxidant activity in comparison to methanolextracts (Table 1).

3.3. Radical scavenging potentials (DPPH and ABTS assay)

The stable organic radical DPPH have been widely used in anti-oxidant capacity studies of plant extracts or antioxidant com-pounds. In this assay, the purple radical (picrylhydrazyl) is

Table 1Total phenolic, flavonoid, saponin content and antioxidant capacity of three Centaurea species.

Centaurea species Extracts Total phenolic content(mg GAE g�1)a

Total flavonoid content(mg RE g�1)b

Total antioxidant capacity(mg AE g�1)c

Total saponin content(mg QAE g�1)d

C. polypodiifolia var.pseudobehen

Methanol 269.58 ± 14.34e 45.94 ± 1.77 266.33 ± 6.55 139.38 ± 13.90Water 349.29 ± 19.00 40.38 ± 1.32 549.48 ± 6.29 84.85 ± 0.66

C. pyrrhoblephara Methanol 232.78 ± 2.33 55.75 ± 1.37 226.15 ± 18.86 159.87 ± 1.85Water 227.12 ± 1.00 29.43 ± 1.20 371.33 ± 15.71 51.90 ± 3.44

C. antalyense Methanol 207.78 ± 5.00 46.76 ± 1.43 248.74 ± 11.00 147.71 ± 13.48Water 227.12 ± 1.00 30.76 ± 1.87 528.56 ± 1.83 77.84 ± 4.96

a Total phenolic content (TPC) expressed as gallic acid equivalent (mg GAE g�1 extract).b Total flavonoid content (TFC) expressed as rutin equivalent (mg RE g�1 extract).c Total antioxidant capacity (TAC) expressed as ascorbic acid equivalent (mg AE g�1 extract).d Total saponins was expressed as Quillaja equivalents (mg QAE g�1 extract).e Values expressed are means ± SD.

Table 2Scavenging activity (%) of three Centaurea species on DPPH radical.

Centaurea species Extracts Concentration (lg mL�1)

100 200 500 1000

C. polypodiifolia var. pseudobehen Methanol 19.38 ± 2.41a 36.85 ± 0.82 76.09 ± 5.70 93.53 ± 0.14Water 20.53 ± 1.14 51.58 ± 0.05 87.81 ± 0.62 89.12 ± 0.01

C. pyrrhoblephara Methanol 13.97 ± 2.81 29.06 ± 0.01 56.27 ± 5.10 90.06 ± 2.21Water 20.07 ± 1.39 37.91 ± 1.12 76.14 ± 0.15 88.16 ± 2.11

C. antalyense Methanol 12.29 ± 0.99 23.37 ± 0.62 52.57 ± 1.45 85.07 ± 0.60Water 22.97 ± 0.22 43.46 ± 2.36 80.74 ± 2.03 86.11 ± 0.05

a Values expressed are means ± SD.

A. Aktumsek et al. / Food and Chemical Toxicology 55 (2013) 290–296 293

reduced by antioxidant compounds to the corresponding pale yel-low hydrazine (picrylhydrazine). The discoloration indicates freeradical scavenging activity of tested sample.

The antioxidant activities obtained by the DPPH method for theCentaurea extracts are displayed in Table 2. The studied extractsshowed scavenging activity in a concentration-dependent manner.Among methanol and water extracts, the best results were ob-tained with C. polypodiidolia var. pseudobehen. The DPPH radicalscavenging activities at 1000 lg mL�1 concentration in the studiedextracts were in order: C. antalyense methanol < C. antalyensewater < C. pyrrhoblephara water < C. polypodiifolia var. pseudobehenwater < C. pyrrhoblephara methanol < C. polypodiifolia var. pseud-obehen methanol. Also, at our previous studies, the DPPH scaveng-ing potentials were reported for some Centaurea species, such as C.cheirolopha (Aktumsek et al., 2011), C. kotschyi var. persica (Zenginet al., 2011) and C. patula (Zengin et al., 2010).

ABTS is generally used for testing the preliminary radical scav-enging activity of antioxidant compounds or plant extracts. TheABTS�+, generated from oxidation of ABTS by potassium persulfate,is presented as an excellent tool for determining the antioxidantactivity of hydrogen-donating antioxidants and chain-breakingantioxidants (Leong and Shui, 2002). The ABTS scavenging capacityof plant extracts were expressed as inhibition capacity and the re-sults are shown in Table 3. Similar to DPPH assay, the methanol ex-tract from C. polypodiifolia var. pseudobehen had high ABTSscavenging capacity (93.42% at 50 lg mL�1). All tested concentra-tions; the studied methanol extracts had the strongest antioxidantactivity in ABTS method. As far as our literature survey could ascertain, there is no study concerning the ABTS scavenging abilityof Centaurea species. Therefore, this study is the first in this area.

3.4. Ferric and cupric reducing power

Literature reports suggest that the reducing properties are gen-erally associated with the presence of reductones (Pin-Der Duh,1998). The reductones have been shown to exert antioxidant

activity by breaking the free radical chain by donating a hydrogenatom. Therefore, in the present study was evaluated measuring theconversion of a Fe3+ and Cu2+ to the Fe2+ and Cu+ form, respectively.Both ferric and cupric reducing power increased with increase inthe sample concentration.

In this ferric reducing power, the presence of antioxidantscauses the conversion of the Fe3+/ferricyanide complex used tothe ferrous form. The ferrous ion can be monitored by measuringthe formation of Perl’s Prussian blue at 700 nm. Ferric reducingpowers obtained for all the Centaurea extracts were excellent;at 800 lg mL�1 they were above 0.5. As shown in Table 4, thehighest ferric reducing power activities were exhibited by the ex-tracts from methanol. Methanol extract obtained from C. polypo-diifolia var. pseudobehen showed maximum absorbance (at700 nm) and hence maximum ferric reducing capacity assessmentamong investigated Centaurea extracts. The presence of high con-centrations of polyphenols in the methanol extract may explainthe high ferric reducing power abilities. The other methanol ex-tract exhibited similar reducing power activity at all testedconcentration.

The CUPRAC assay has been used by many researchers to deter-mine cupric reducing power activities of plant extracts or antioxi-dant compounds (Ozturk et al., 2007; Bursal and Gulcin, 2011). Theassay based on reduction of Cu2+ to Cu1+ by antioxidant com-pounds in the presence of neocuproine. The test system has dis-tinct advantages such as, simplicity, clarity of end point andmechanism, readily available instrumentation, good intra- and in-ter-assay reproducibility (Prior et al., 2005). According to result ofthis study, reducing power of the extracts (both methanol andwater extracts) decreased in order of C. polypodiifolia var. pseudobe-hen > C. antalyense > C. pyrrhoblephara, in the presence of800 lg mL�1 extract (Table 5). In recent studies, reducing poweractivities (cupric and ferric) were reported for some Centaurea spe-cies such as, C. solsititialis (Tekeli et al., 2008), C. drabifolia subsp.detonsa (Zengin et al., 2012) and C. kotschyi var. persica (Zenginet al., 2011).

Table 3Scavenging activity (%) of three Centaurea species on ABTS radical.

Centaurea species Extracts Concentration (lg mL�1)

12.5 25 50

C. polypodiifolia var. pseudobehen Methanol 66.22 ± 0.81a 77.77 ± 4.45 93.42 ± 4.72Water 16.32 ± 7.15 38.65 ± 7.96 73.86 ± 2.43

C. pyrrhoblephara Methanol 76.81 ± 0.41 81.87 ± 0.01 91.13 ± 3.37Water 17.65 ± 4.45 31.30 ± 4.32 61.83 ± 2.16

C. antalyense Methanol 67.37 ± 2.70 78.05 ± 1.350 90.65 ± 1.35Water 21.37 ± 5.40 36.74 ± 2.56 76.24 ± 6.61

a Values expressed are means ± SD.

Table 4Ferric reducing power (absorbance at 700 nm) of three Centaurea species at different concentrations.

Centaurea species Extracts Concentration (lg mL�1)

200 400 800

C. polypodiifolia var. pseudobehen Methanol 0.27 ± 0.04a 0.57 ± 0.02 1.17 ± 0.03Water 0.24 ± 0.01 0.47 ± 0.01 0.89 ± 0.02

C. pyrrhoblephara Methanol 0.22 ± 0.03 0.42 ± 0.01 0.84 ± 0.08Water 0.17 ± 0.01 0.32 ± 0.01 0.60 ± 0.02

C. antalyense Methanol 0.22 ± 0.03 0.45 ± 0.04 0.85 ± 0.03Water 0.17 ± 0.01 0.34 ± 0.01 0.67 ± 0.01

a Values expressed are means ± SD.

Table 5Cupric reducing power (absorbance at 450 nm) of three Centaurea species at different concentrations.

Centaurea species Extracts Concentration (lg mL�1)

200 400 800

C. polypodiifolia var. pseudobehen Methanol 0.31 ± 0.03a 0.67 ± 0.04 1.31 ± 0.11Water 0.39 ± 0.01 0.77 ± 0.02 1.48 ± 0.03

C. pyrrhoblephara Methanol 0.27 ± 0.05 0.57 ± 0.04 0.97 ± 0.16Water 0.25 ± 0.02 0.52 ± 0.02 1.01 ± 0.03

C. antalyense Methanol 0.32 ± 0.08 0.59 ± 0.06 1.03 ± 0.127Water 0.34 ± 0.02 0.63 ± 0.02 1.20 ± 0.01

a Values expressed are means ± SD.

Fig. 1. Antioxidant activity (%) of the Centaurea species by b-carotene/linoleic acidtest system.

Fig. 2. Antioxidant activities of three Centaurea species on their abilities to reducethe ferric ion–TPTZ complex.

294 A. Aktumsek et al. / Food and Chemical Toxicology 55 (2013) 290–296

3.5. b-Carotene/linoleic acid bleaching assay

In this method, the linoleic acid free radical formed attacks thehighly unsaturated b-carotene molecules and in the absence of anantioxidant rapidly bleached the orange color of b-carotene. Theextent of discoloration is monitored spectrophotometrcially at490 nm (Jayaprakasha et al., 2001). The lowest b-carotene discolor-

ation rate exhibited the highest antioxidant activity. Fig. 1 showsthe inhibition values for b-carotene/linoleic acid assay obtainedfrom each Centaurea species. The inhibition capacities measuredin the methanol extracts were better than that from water extracts.The inhibition value reached 81.10% in methanolic extract of C.polypodiifolia var. pseudobehen. However, synthetic antioxidants

Table 6Metal chelating and anticholinesterase activities of three Centaurea species.

Centaurea species Solvent Metal chelating activity (mg EDTA g�1) AChE BChE

Inhibition (%) mg GALE g�1 Inhibition (%) mg GALE g�1

C. polypodiifolia var. pseudobehen Methanol 17.04 ± 0.71a 24.54 ± 2.23a 1.29 ± 0.12 45.50 ± 9.62 105.42 ± 22.30Water 64.72 ± 0.67 14.49 ± 2.07 0.72 ± 0.1 31.14 ± 6.62 72.37 ± 14.45

C. pyrrhoblephara Methanol 25.67 ± 0.60 14.31 ± 1.54 0.71 ± 0.08 na –Water 73.81 ± 9.49 na – 5.67 ± 0.80 13.72 ± 1.93

C. antalyense Methanol 16.31 ± 0.13 21.25 ± 1.94 1.1 ± 0.1 37.14 ± 8.17 86.18 ± 18.97Water 73.71 ± 11.58 – na 36.18 ± 4.72 83.97 ± 10.45

na: not activity.a Values expressed are means ± SD.

A. Aktumsek et al. / Food and Chemical Toxicology 55 (2013) 290–296 295

(BHA and BHT) showed very strong inhibition ability (over 90%). Inthe same test system, inhibition values of linoleic oxidation werefound to be 79.52% in C. cheirolopha (Aktumsek et al., 2011),63.60% in C. pulchella (Zengin et al., 2010), 85.15% in ethyl acetateextract of C. ensiformis (Ugur et al., 2009) and 35.2% in C. mucronif-era (Tepe et al., 2006).

3.6. FRAP assay

The assay based on the ability of antioxidant compounds to re-duce complex (Fe(III)–TPTZ) to (Fe(II)–TPTZ). The Fe(II)–TPTZ com-plex gives a blue color with an absorbance maximum at 593 nm.The results of FRAP assay are presented in Fig. 2. In this assay,the highest activities were noted for water extracts. The water ex-tract obtained from C. polypodiifolia var. pseudobehen(122.75 lM TE g�1 extract) had highest activity followed by C.antalyense (89.50 lM TE g�1) and C. pyrrhoblephara(81.95 lM TE g�1), respectively. The presence of high concentra-tions of polyphenols in water extracts may explain the high FRAPactivities. FRAP assay was used by several authors for the evalua-tion of antioxidant capacity of some plants (Jing et al., 2012; Saj-eesh et al., 2011; Nithiyanantham et al., 2012). However, nostudy on the FRAP activities of Centaurea species have beendocumented.

3.7. Transition metal chelating activity

Transition metal chelating activity depends on the ability ofsamples to chelate transition metals (Fe2+ or Cu+). The ability playsa vital role in antioxidant mechanism since it reduces the concen-tration of the catalyzing transition metal in lipid peroxidationmechanism (Du et al., 2009; Duh et al., 1999). The Fe2+ chelatingability of studied Centaurea extracts was determined by measuringthe iron–ferrozine complex and results are summarized in Table 6.Among the extracts, water extracts showed better metal chelatingactivity compared to methanol extracts. The chelating capacity ofwater extracts was ranged from 64.72 to 73.81 mg EDTA equiva-lents g�1 extracts. The methanol extracts displayed a similar fer-rous ion chelating activities (16.31–25.67 mg EDTA equivalentsg�1 extracts). In our previous study, the metal chelating activityof Centaurea species was reported as inhibition value for the firsttime (Aktumsek et al., 2011).

3.8. Cholinesterase inhibitory activity

AChE inhibitors are well utilized for the management of mild tomoderate Alzheimer’s disease, and there are several researchers fo-cused on the search of new AChE inhibitors from the herbal re-sources (Giacobini, 2004). This study examined theanticholinesterase activity of Centaurea species for the first time.The Centaurea extracts were screened for AChE inhibitory activity

using Ellman’s colorimetric method in a 96-well plate. The resultsof AChE and BChE inhibitory activities of tested Centaurea specieswere expressed as percentage of inhibition and galanthamineequivalents (mg GALE g�1) (Table 6). The methanol extracts hadbetter activity against AChE than water extracts. At 2 mg mL�1,water extracts of C. pyrrhoblephara and C. antalyense did not exhibitany activity against AChE, while the most active extract was foundto be the methanol extract of C. polypodiifolia var. pseudobehen(24.54%), followed by C. antalyense (21.25%) and C. pyrrhoblephara(14.31%). Also, the galanthamine equivalent values in Centaurea ex-tracts varied between 0.71 and 1.29 mg g�1 extract. As to BChE, ex-pect for methanolic extract of C. pyrrhoblephara, all extractsdisplayed activity inhibition on BChE. Among the extractsscreened, the highest inhibition percentage against both of the en-zymes was observed for methanolic extract of C. polypodiifolia var.pseudobehen (45.50% inhibition). Although the water extracts of C.pyrrhoblephara and C. antalyense displayed no activity on AChE,these extracts showed inhibition activity against BChE.

4. Conclusion

From this study it can be conducted that tested Centaurea ex-tracts have potential antioxidant properties. Moreover, methanolicextracts of C. pyrrhoblephara and C. antalyense exhibited moderateinhibitory activity on AChE and BChE. This study is the first timeanticholinesterase property is reported for members of the genusCentaurea. Indeed, there is a current need for availability of newplant derived bioactive molecules, thus these plant extracts maybe a great natural source for antioxidants and anticholinesteraseagents in some applications including food and medicinal. Furtherstudies are required to identity of bioactive compounds in theseextracts responsible for observed antioxidant and ACE inhibitoryactivities.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported financially as a project (11401066).The authors thank Selcuk University Scientific Research Founda-tion (BAP) for providing financial support for this study. Theauthors also want to thank Dr. Harun Simsek for proofreadingthe present manuscript.

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