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

Food and Chemical Toxicology 55 (2013) 290–296

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Antioxidant potentials and anticholinesterase activities of methanolic and aqueous 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 b c

Department of Biology, Science Faculty, Selcuk University, Konya, Turkey Department of Biological Education, Ahmet Kelesoglu Education Faculty, Necmettin Erbakan University, Konya, Turkey 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 2012 Accepted 8 January 2013 Available online 26 January 2013 Keywords: Centaurea Antioxidant Phenolics Anticholinesterase activity

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 inhibitory activities. The antioxidant activities of these extracts were evaluated by in vitro models including, phosphomolybdenum assay, free radical scavenging assays (DPPH and ABTS), b-carotene/linoleic acid test system, metal chelating assay, FRAP assay, ferric and cupric reducing power. Cholinesterase inhibitory activities were examined using Ellman’s colorimetric method. Total phenol, flavonoid, and saponin contents were also measured. Among the six Centaurea extracts evaluated, the highest antioxidant abilities were 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 suggest that Centaurea species could be an anticholinesterase agent and antioxidant resource in some industries, such as food, pharmacology, and cosmetics. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Free radicals, especially oxygen free radicals (OFRs) or reactive oxygen species (ROSs) (e.g. superoxide, hydroxyl and hydrogen peroxide) are active oxygen compounds generated by oxidation reactions of exogenous factors (Halliwell and Gutteridge, 1990; Cerutti, 1991). These reactive species may oxidize proteins, lipids or DNA and can initiate degenerative/chronic diseases including, cancer, diabetes and cardiovascular disease (Lee et al., 2004; Mantena et al., 2008). Antioxidants are substances that when present at low concentrations with respect to oxidizable substrates, inhibit or delay the oxidation process (Sikorski, 2001). Therefore, antioxidants have a vital role in the maintenance of human health and prevention of disease caused by free radicals. Due to the benefits of antioxidants, food and pharmaceutical products are normally enriched with synthetic antioxidants such as BHA, BHT and PG. However, application of these synthetic antioxidants might lead to toxic effects such as carcinogen (Barlow and Schlatter, 2010). Hence, stronger restrictions have been mandated for their application and there is a trend to substitute synthetic antioxidants with natural antioxidants.

⇑ Corresponding author. Tel.: +90 332 223 18 66; fax: +90 332 241 01 06. E-mail address: [email protected] (A. Aktumsek). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.01.018

Alzheimer’s disease (AD) is the most common form of neurodegenerative disease and characterized by memory dysfunction. Reduction of acetylcholine levels in the brain is the most notable biochemical change in AD (Loizzo et al., 2009). Therefore, AD can be treated by use of agents which restore the level of acetylcholine through inhibition of both two major form of cholinesterase: acetylcholinesterase (AChE) and butrylcholinesterase (BChE) (Jaen et al., 1996). Tacrine, rivastigmine and galanthamine are used as cholinesterase inhibitors for treatment of AD. However, these compounds 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 inhibitors particularly from natural products have been gaining more attention in recent decades. The genus Centaurea (Asteraceae) is a large genus and comprises approximately 500 species distributed in old World (Dittrich, 1977). In Anatolia peninsula, the genus is represented by about 190 species with more than 100 endemic species (Davis, 1988; Güner et al., 2000). Some Centaurea species are used as herbal remedies in Anatolian folk medicine to treat fever, diabetes and hemoroid and peptid ulcer (Kargioglu et al., 2008; Honda et al., 1996). Pharmacological and phytochemical studies on many Centaurea species have reported antioxidant, antimicrobial and antipyretic activities (Tepe et al., 2006; Zengin et al., 2011; Tekeli et al., 2011; Koca et al., 2009). Nonetheless, to our best knowledge,

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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 antioxidant and cholinesterase inhibitory effect of methanol and aqueous extracts obtained from three Centaurea species, as well as their phenolic, flavonoid and saponin contents. Thus, the present study might supply valuable information on the phytochemical properties of the Centaurea species for nutraceutical and pharmacological industry.

2.7. Evaluation of total antioxidant capacity by phosphomolybdate assay

2. Materials and methods

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

2.1. Plant materials Centaurea species were collected from different localities in Turkey during vegetation season of 2010. The plants were identified by botanist Professor. Dr. Ahmet Duran. The voucher specimens were deposited in Selcuk University Science Faculty Herbarium, Konya. Taxonomical information’s, localities, collection periods and voucher 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 plant materials were ground to a fine powder using a laboratory mill. Fifteen grams of powdered plant were mixed with 250 mL methanol and extracted in a Soxhlet apparatus for 6–8 h. The extracts concentrated under vacuum at 40 °C by using a rotary evaporator. To obtain water extracts, air dried powdered plants were boiled with 250 mL of distilled water for 30 min. The aqueous extracts were filtered and lyophilized (80 °C, 48 h). Extracts were stored at +4 °C in dark until use.

The total antioxidant capacities of extracts were evaluated by phosphomolybdenum method according to Prieto et al. (1999). 0.3 mL of extracts (2 mg mL1) were combined with 3 mL reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95 °C for 90 min. Then the absorbance of the solution was measured at 695 nm against a blank. The antioxidant capacity of extracts was expressed as equivalents of ascorbic acid (mg AE g1 extract). 2.8. Scavenging activity on DPPH

Ið%Þ ¼ ðA0  A1 Þ=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 scavenging activity The scavenging capacity against ABTS assay was measured according to the method of Re et al. (1999). Briefly, ABTS radical cation was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the 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 at 734 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 at 734 nm. The results were reported as percentage inhibition and calculated according to Eq. (1). 2.10. b-Carotene–linoleic acid assay

2.3. Chemicals Potassium ferricyanide, ferric chloride, Folin–Ciocalteu’s reagent, trichloroacetic acid, methanol, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and methanol were purchased from Merck (Darmstadt, Germany). 2,2-Diphenyl1-picrylhydrazyl (DPPH), b-carotene, linoleic acid and Tween 40 were purchased from Sigma Chemical Co. (Sigma–Aldrich GmbH, Sternheim, Germany). All other chemicals and solvents were analytical grade. 2.4. Assay for total phenolics Total phenolic constituents of the extracts were determined by employing the methods given in the literature (Slinkard and Singleton, 1977) involving Folin– Ciocalteu reagent and gallic acid as standard. One milliliter of extract solution containing 2 mg extract was added to a volumetric flask. Forty-five milliliter distilled water and 1 mL Folin–Ciocalteu reagent was added and flask was shaken vigorously. After 3 min, 3 mL of Na2CO3 (2%) solution was added and the mixture was allowed to stand for 2 h by intermittent shaking. Absorbance was measured at 760 nm (Shimadzu-UV1800). The total phenolic content was determined as gallic acid equivalents (mg GAE g1 extract).

In this assay antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation (Dapkevicius et al., 1998). A stock solution of b-carotene–linoleic acid mixture was prepared as following: 0.5 mg b-carotene was dissolved in 1 mL of chloroform (HPLC grade) 25 lL linoleic acid and 200 mg Tween 40 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 the extracts (2 mg mL1) were added and the emulsion system was incubated for up to 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 was measured at 490 nm. Measurement of absorbance was continued until the color of b-carotene disappeared. The bleaching rate (R) of b-carotene was calculated according 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 to the control using Eq. (3).

AA ¼ ½ðRControl  RSample Þ=RControl   100

ð3Þ

2.5. Assay for total flavonoids The total flavonoid content in extracts was determined spectrophotometrically according 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 (at 2 mg mL1 concentration). The absorbance values of the reaction mixtures were determined at 415 nm after 10 min duration against a blank. Rutin was used as the standard and the total flavonoids content of the extracts was expressed as mg rutin equivalents per gram of extract (mg RE g1 extract). 2.6. Determination of total saponins The total saponins contents of extracts were determined by the vanillin-sulfuric acid method (Hiai et al., 1976). These extracts were reacted with vanillin (8%) and sulfuric acid (72%). The mixture was incubated at 60 °C for 10 min. Then the mixture was cooled for another 15 min, followed by absorbance measurement at 538 nm. Quillaja saponin was used as a standard and the content of total saponins was expressed as Quillaja equivalents (mg QAE g1 extract).

Antioxidative activities of the extracts were compared with those of BHT and BHA at 2.0 mg mL1. 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 reaction was initiated by adding 200 lL of 5 mM ferrozine. Total volume was adjusted to 2 ml with methanol. After 10 min incubation at room temperature, the absorbance at 562 nm was recorded. EDTA was used as a standard chelating agent and results were expressed as mg EDTA equivalents g1 extract. 2.12. Reducing power activity (Iron(III) to iron(II) reduction) The ferric reducing power applied with slight modifications of the method of Oyaizu (1986). Various concentrations of the extracts (2.5 mL) were mixed with 2.5 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% trichloroacetic acid was added. 2.5 mL of the reaction mixture was mixed with 2.5 mL distilled water and 0.5 mL of 0.1% ferric chloride. The solution absorbance was measured at 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 the method of Apak et al. (2006). One milliliter each of 10 mM CuCl2, 7.5 mM neocuproine, 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 was brought up to 4.1 mL with deionized water. The mixture absorbance was recorded against 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 and Strain (1996) with slight modifications. The FRAP reagent was prepared freshly by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ and 20 mM ferric chloride in a ratio of 10:1:1 (v/v/v). Then, 2 mL reagent and 200 lL samples were added to test tubes and incubated at 30 °C for 30 min. Absorbance was measured at 593 nm. Trolox was used as standard and results were reported as Trolox equivalents per gram of extract.

2.15. Cholinesterase inhibitory activity Cholinesterase inhibitory activity of tested Centaurea species was assessed by the spectrophotometric method developed by Ellman et al. (1961). Into a 96-well plate 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 mL1). Absorbance was measured at 405 nm every 45 s, three times consecutively. Thereafter, 25 lL of AChE or BChE (0.026 U mL1) was added to the wells and the absorbance was recorded again on a microplate reader (Rayto-3100). Galanthamine and methanol were used as positive and negative controls, respectively. The percentage inhibition was calculated by using Eq. (1). The results were also evaluated galanthamine equivalents per gram of extract (mg GALE g1 extract).

3. Results and discussion Several chemical tests were selected in this study, based on the ability of antioxidants to act as reducing agents (ferric and cupric ions) and as radical scavengers (DPPH and ABTS scavenging assays. The assessment of the capacity of pure antioxidant compounds and plant extracts have been the subjects of extensive studies and arguments. A number of methods and variations have been developed and applied for the assessment of antioxidant capacity (Niki, 2011). However, evaluation of antioxidant potentials of plant products or antioxidant compounds cannot be carried out accurately by any single universal method. Thus, we applied several antioxidant assays that would provide a better insight into the true antioxidant potential 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 human body. The phytochemicals that are frequently associated with human health are polyphenols, carotenoids and tocopherols. Polyphenols are the major secondary metabolites in most plants reported to possess antioxidant and free radical scavenging activity. There are several studies on dietary polyphenol use to decrease the risk of coronary heart disease (Williams and Elliot, 1997). Likewise, the compounds are used as anticarcinogenic and anti-inflammatory agents (Carrol et al., 1999; Maeda-Yamamoto et al., 1999). For instance, ellagic acid was the phenolic component primarily associated to the chemopreventive effects, appearing to function as an anticarcinogen at the initiation and postinitiation stages of tumor development in in vitro and in vivo experiments (Losso et al., 2004; Giampieri et al., 2012). Thus, measurements of polyphenols content in plants have become important tools in

understanding the values of plants for human health (Amarowicz et al., 2010). Many reports demonstrate that, besides polyphenolics, plants containing biological important substances such as vitamin C, carotenoids and tocopherols have beneficial effects on human health. For example, vitamin C in the strawberry is responsible for more than 30% of the total antioxidant capacity (Tulipani et al., 2008). The total phenolic and flavonoid contents in the Centaurea extracts are summarized in Table 1. Among studied Centaurea extracts, C. polypodiifolia var. pseudobehen had the highest polyphenol contents. The total phenolic contents in the other water extracts were found as 227.12 mg GAE g1 extract. The methanolic extracts contained similar levels of polyphenolic contents. Flavonoids comprise the most common group of plant polyphenols. Epidemiological studies yield unequivocal results: consumption of plants rich in secondary metabolites, especially flavonoids is associated with a reduced risk of atherosclerosis, cancer, and neurodegenerative diseases (Chiva-Blanch and Visioli, 2012; Chong et al., 2010; Nobili et al., 2009). Though the bioactivity of flavonoids appears to be mediated through a variety of mechanisms, particular attention has been focused on their direct and indirect antioxidant actions. The antioxidant properties are conferred on flavonoids by the phenolic hydroxyl groups attached to ring structures and they can act as free radical scavenger, reducing agents and metal chelators (Carocho and Ferreira, 2013). Total flavonoids in the Centaurea extracts were determined using spectrophotometric method with aluminum chloride and obtained results varied from 29.43 to 55.75 mg RE g1 extract. All methanol extracts (C. pyrrhoblephara (55.75 mg RE g1) > C. antalyense (46.76 mg RE g1) > C. polypodiifolia var. pseudobehen (45.94 mg RE g1)) possessed highest content of flavonoid compared to water extracts (Table 1). Saponins are a vast group of glycosides, widely distributed in higher plants. They have pharmacological properties and are used in phytotheraphy. These compounds are believed to from the main constituents of many plant drugs and are reported many pharmacological properties. Total saponin contents of studied Centurea extracts are presented in Table 1. The highest saponin content was obtained from methanolic extract of C. pyrrhoblephara with 159.87 mg QAE g1. Total saponin contents in the Centaurea extracts followed the same trend as the total flavonoid content. No studies have so far been reported total saponin content of Centaurea species. Thus, the present study is the first report on saponin content of Centaurea species. 3.2. Evaluation of antioxidant capacity by phosphomolybdenum method The phosphomolybdenum method based on reduction of Mo(VI) to Mo(V) by antioxidant substance and subsequent formation of a green phosphate Mo(V) compounds at acidic pH with an absorbance 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 of 549.48 mg AE g1 extract and this activity may be due to the presence of phenolic compounds. The lowest total antioxidant capacity was found in the methanol extract of C. pyrrhoblephara with a value of 226.15 mg AE g1 extract. All water extracts displayed relatively higher total antioxidant activity in comparison to methanol extracts (Table 1). 3.3. Radical scavenging potentials (DPPH and ABTS assay) The stable organic radical DPPH have been widely used in antioxidant capacity studies of plant extracts or antioxidant compounds. In this assay, the purple radical (picrylhydrazyl) is

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A. Aktumsek et al. / Food and Chemical Toxicology 55 (2013) 290–296 Table 1 Total phenolic, flavonoid, saponin content and antioxidant capacity of three Centaurea species.

a b c d e

Centaurea species

Extracts

Total phenolic content (mg GAE g1)a

Total flavonoid content (mg RE g1)b

Total antioxidant capacity (mg AE g1)c

Total saponin content (mg QAE g1)d

C. polypodiifolia var. pseudobehen

Methanol Water

269.58 ± 14.34e 349.29 ± 19.00

45.94 ± 1.77 40.38 ± 1.32

266.33 ± 6.55 549.48 ± 6.29

139.38 ± 13.90 84.85 ± 0.66

C. pyrrhoblephara

Methanol Water

232.78 ± 2.33 227.12 ± 1.00

55.75 ± 1.37 29.43 ± 1.20

226.15 ± 18.86 371.33 ± 15.71

159.87 ± 1.85 51.90 ± 3.44

C. antalyense

Methanol Water

207.78 ± 5.00 227.12 ± 1.00

46.76 ± 1.43 30.76 ± 1.87

248.74 ± 11.00 528.56 ± 1.83

147.71 ± 13.48 77.84 ± 4.96

Total phenolic content (TPC) expressed as gallic acid equivalent (mg GAE g1 extract). Total flavonoid content (TFC) expressed as rutin equivalent (mg RE g1 extract). Total antioxidant capacity (TAC) expressed as ascorbic acid equivalent (mg AE g1 extract). Total saponins was expressed as Quillaja equivalents (mg QAE g1 extract). Values expressed are means ± SD.

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

a

Concentration (lg mL1)

Centaurea species

Extracts

100

200

500

1000

C. polypodiifolia var. pseudobehen

Methanol Water

19.38 ± 2.41a 20.53 ± 1.14

36.85 ± 0.82 51.58 ± 0.05

76.09 ± 5.70 87.81 ± 0.62

93.53 ± 0.14 89.12 ± 0.01

C. pyrrhoblephara

Methanol Water

13.97 ± 2.81 20.07 ± 1.39

29.06 ± 0.01 37.91 ± 1.12

56.27 ± 5.10 76.14 ± 0.15

90.06 ± 2.21 88.16 ± 2.11

C. antalyense

Methanol Water

12.29 ± 0.99 22.97 ± 0.22

23.37 ± 0.62 43.46 ± 2.36

52.57 ± 1.45 80.74 ± 2.03

85.07 ± 0.60 86.11 ± 0.05

Values expressed are means ± SD.

reduced by antioxidant compounds to the corresponding pale yellow hydrazine (picrylhydrazine). The discoloration indicates free radical scavenging activity of tested sample. The antioxidant activities obtained by the DPPH method for the Centaurea extracts are displayed in Table 2. The studied extracts showed scavenging activity in a concentration-dependent manner. Among methanol and water extracts, the best results were obtained with C. polypodiidolia var. pseudobehen. The DPPH radical scavenging activities at 1000 lg mL1 concentration in the studied extracts were in order: C. antalyense methanol < C. antalyense water < C. pyrrhoblephara water < C. polypodiifolia var. pseudobehen water < C. pyrrhoblephara methanol < C. polypodiifolia var. pseudobehen methanol. Also, at our previous studies, the DPPH scavenging potentials were reported for some Centaurea species, such as C. cheirolopha (Aktumsek et al., 2011), C. kotschyi var. persica (Zengin et al., 2011) and C. patula (Zengin et al., 2010). ABTS is generally used for testing the preliminary radical scavenging activity of antioxidant compounds or plant extracts. The ABTS+, generated from oxidation of ABTS by potassium persulfate, is presented as an excellent tool for determining the antioxidant activity of hydrogen-donating antioxidants and chain-breaking antioxidants (Leong and Shui, 2002). The ABTS scavenging capacity of plant extracts were expressed as inhibition capacity and the results are shown in Table 3. Similar to DPPH assay, the methanol extract from C. polypodiifolia var. pseudobehen had high ABTS scavenging capacity (93.42% at 50 lg mL1). All tested concentrations; the studied methanol extracts had the strongest antioxidant activity in ABTS method. As far as our literature survey could as certain, there is no study concerning the ABTS scavenging ability of 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 generally 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 hydrogen atom. Therefore, in the present study was evaluated measuring the conversion of a Fe3+ and Cu2+ to the Fe2+ and Cu+ form, respectively. Both ferric and cupric reducing power increased with increase in the sample concentration. In this ferric reducing power, the presence of antioxidants causes the conversion of the Fe3+/ferricyanide complex used to the ferrous form. The ferrous ion can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. Ferric reducing powers obtained for all the Centaurea extracts were excellent; at 800 lg mL1 they were above 0.5. As shown in Table 4, the highest ferric reducing power activities were exhibited by the extracts from methanol. Methanol extract obtained from C. polypodiifolia var. pseudobehen showed maximum absorbance (at 700 nm) and hence maximum ferric reducing capacity assessment among investigated Centaurea extracts. The presence of high concentrations of polyphenols in the methanol extract may explain the high ferric reducing power abilities. The other methanol extract exhibited similar reducing power activity at all tested concentration. The CUPRAC assay has been used by many researchers to determine cupric reducing power activities of plant extracts or antioxidant compounds (Ozturk et al., 2007; Bursal and Gulcin, 2011). The assay based on reduction of Cu2+ to Cu1+ by antioxidant compounds in the presence of neocuproine. The test system has distinct advantages such as, simplicity, clarity of end point and mechanism, readily available instrumentation, good intra- and inter-assay reproducibility (Prior et al., 2005). According to result of this study, reducing power of the extracts (both methanol and water extracts) decreased in order of C. polypodiifolia var. pseudobehen > C. antalyense > C. pyrrhoblephara, in the presence of 800 lg mL1 extract (Table 5). In recent studies, reducing power activities (cupric and ferric) were reported for some Centaurea species such as, C. solsititialis (Tekeli et al., 2008), C. drabifolia subsp. detonsa (Zengin et al., 2012) and C. kotschyi var. persica (Zengin et al., 2011).

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

a

Concentration (lg mL1)

Centaurea species

Extracts

12.5

25

50

C. polypodiifolia var. pseudobehen

Methanol Water

66.22 ± 0.81a 16.32 ± 7.15

77.77 ± 4.45 38.65 ± 7.96

93.42 ± 4.72 73.86 ± 2.43

C. pyrrhoblephara

Methanol Water

76.81 ± 0.41 17.65 ± 4.45

81.87 ± 0.01 31.30 ± 4.32

91.13 ± 3.37 61.83 ± 2.16

C. antalyense

Methanol Water

67.37 ± 2.70 21.37 ± 5.40

78.05 ± 1.350 36.74 ± 2.56

90.65 ± 1.35 76.24 ± 6.61

Values expressed are means ± SD.

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

a

Concentration (lg mL1)

Centaurea species

Extracts

200

400

800

C. polypodiifolia var. pseudobehen

Methanol Water

0.27 ± 0.04a 0.24 ± 0.01

0.57 ± 0.02 0.47 ± 0.01

1.17 ± 0.03 0.89 ± 0.02

C. pyrrhoblephara

Methanol Water

0.22 ± 0.03 0.17 ± 0.01

0.42 ± 0.01 0.32 ± 0.01

0.84 ± 0.08 0.60 ± 0.02

C. antalyense

Methanol Water

0.22 ± 0.03 0.17 ± 0.01

0.45 ± 0.04 0.34 ± 0.01

0.85 ± 0.03 0.67 ± 0.01

Values expressed are means ± SD.

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

a

Centaurea species

Extracts

Concentration (lg mL1) 200

400

800

C. polypodiifolia var. pseudobehen

Methanol Water

0.31 ± 0.03a 0.39 ± 0.01

0.67 ± 0.04 0.77 ± 0.02

1.31 ± 0.11 1.48 ± 0.03

C. pyrrhoblephara

Methanol Water

0.27 ± 0.05 0.25 ± 0.02

0.57 ± 0.04 0.52 ± 0.02

0.97 ± 0.16 1.01 ± 0.03

C. antalyense

Methanol Water

0.32 ± 0.08 0.34 ± 0.02

0.59 ± 0.06 0.63 ± 0.02

1.03 ± 0.127 1.20 ± 0.01

Values expressed are means ± SD.

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

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

3.5. b-Carotene/linoleic acid bleaching assay In this method, the linoleic acid free radical formed attacks the highly unsaturated b-carotene molecules and in the absence of an antioxidant rapidly bleached the orange color of b-carotene. The extent of discoloration is monitored spectrophotometrcially at 490 nm (Jayaprakasha et al., 2001). The lowest b-carotene discolor-

ation rate exhibited the highest antioxidant activity. Fig. 1 shows the inhibition values for b-carotene/linoleic acid assay obtained from each Centaurea species. The inhibition capacities measured in 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

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A. Aktumsek et al. / Food and Chemical Toxicology 55 (2013) 290–296 Table 6 Metal chelating and anticholinesterase activities of three Centaurea species. Centaurea species

Solvent

Metal chelating activity (mg EDTA g1)

AChE

BChE

Inhibition (%) a

a

mg GALE g

1

Inhibition (%)

mg GALE g1

C. polypodiifolia var. pseudobehen

Methanol Water

17.04 ± 0.71 64.72 ± 0.67

24.54 ± 2.23 14.49 ± 2.07

1.29 ± 0.12 0.72 ± 0.1

45.50 ± 9.62 31.14 ± 6.62

105.42 ± 22.30 72.37 ± 14.45

C. pyrrhoblephara

Methanol Water

25.67 ± 0.60 73.81 ± 9.49

14.31 ± 1.54 na

0.71 ± 0.08 –

na 5.67 ± 0.80

– 13.72 ± 1.93

C. antalyense

Methanol Water

16.31 ± 0.13 73.71 ± 11.58

21.25 ± 1.94 –

1.1 ± 0.1 na

37.14 ± 8.17 36.18 ± 4.72

86.18 ± 18.97 83.97 ± 10.45

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

(BHA and BHT) showed very strong inhibition ability (over 90%). In the same test system, inhibition values of linoleic oxidation were found to be 79.52% in C. cheirolopha (Aktumsek et al., 2011), 63.60% in C. pulchella (Zengin et al., 2010), 85.15% in ethyl acetate extract of C. ensiformis (Ugur et al., 2009) and 35.2% in C. mucronifera (Tepe et al., 2006). 3.6. FRAP assay The assay based on the ability of antioxidant compounds to reduce complex (Fe(III)–TPTZ) to (Fe(II)–TPTZ). The Fe(II)–TPTZ complex 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 extract obtained from C. polypodiifolia var. pseudobehen (122.75 lM TE g1 extract) had highest activity followed by C. antalyense (89.50 lM TE g1) and C. pyrrhoblephara (81.95 lM TE g1), respectively. The presence of high concentrations of polyphenols in water extracts may explain the high FRAP activities. FRAP assay was used by several authors for the evaluation of antioxidant capacity of some plants (Jing et al., 2012; Sajeesh et al., 2011; Nithiyanantham et al., 2012). However, no study on the FRAP activities of Centaurea species have been documented. 3.7. Transition metal chelating activity Transition metal chelating activity depends on the ability of samples to chelate transition metals (Fe2+ or Cu+). The ability plays a vital role in antioxidant mechanism since it reduces the concentration of the catalyzing transition metal in lipid peroxidation mechanism (Du et al., 2009; Duh et al., 1999). The Fe2+ chelating ability of studied Centaurea extracts was determined by measuring the iron–ferrozine complex and results are summarized in Table 6. Among the extracts, water extracts showed better metal chelating activity compared to methanol extracts. The chelating capacity of water extracts was ranged from 64.72 to 73.81 mg EDTA equivalents g1 extracts. The methanol extracts displayed a similar ferrous ion chelating activities (16.31–25.67 mg EDTA equivalents g1 extracts). In our previous study, the metal chelating activity of Centaurea species was reported as inhibition value for the first time (Aktumsek et al., 2011). 3.8. Cholinesterase inhibitory activity AChE inhibitors are well utilized for the management of mild to moderate Alzheimer’s disease, and there are several researchers focused on the search of new AChE inhibitors from the herbal resources (Giacobini, 2004). This study examined the anticholinesterase 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 results of AChE and BChE inhibitory activities of tested Centaurea species were expressed as percentage of inhibition and galanthamine equivalents (mg GALE g1) (Table 6). The methanol extracts had better activity against AChE than water extracts. At 2 mg mL1, water extracts of C. pyrrhoblephara and C. antalyense did not exhibit any activity against AChE, while the most active extract was found to 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 extracts varied between 0.71 and 1.29 mg g1 extract. As to BChE, expect for methanolic extract of C. pyrrhoblephara, all extracts displayed activity inhibition on BChE. Among the extracts screened, the highest inhibition percentage against both of the enzymes 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 extracts have potential antioxidant properties. Moreover, methanolic extracts of C. pyrrhoblephara and C. antalyense exhibited moderate inhibitory activity on AChE and BChE. This study is the first time anticholinesterase property is reported for members of the genus Centaurea. Indeed, there is a current need for availability of new plant derived bioactive molecules, thus these plant extracts may be a great natural source for antioxidants and anticholinesterase agents in some applications including food and medicinal. Further studies are required to identity of bioactive compounds in these extracts responsible for observed antioxidant and ACE inhibitory activities. 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 Foundation (BAP) for providing financial support for this study. The authors also want to thank Dr. Harun Simsek for proofreading the present manuscript. References Aktumsek, A., Zengin, G., Guler, G.O., Cakmak, Y.S., Duran, A., 2011. Screening for in vitro antioxidant properties and fatty acid profiles of Five Centaurea L. species from Turkey Flora. Food Chem. Toxicol. 49, 2914–2920.

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