Food and Chemical Toxicology 49 (2011) 2914–2920

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Food and Chemical Toxicology

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Screening for in vitro antioxidant properties and fatty acid profiles of fiveCentaurea L. species from Turkey flora

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

a Department of Biology, Science Faculty, Selcuk University, Konya, Turkeyb Department of Biological Education, Ahmet Kelesoglu Education Faculty, Selcuk University, Konya, Turkey

a r t i c l e i n f o

Article history:Received 29 June 2011Accepted 16 August 2011Available online 22 August 2011

Keywords:Antioxidant capacityCentaureaNatural antioxidantPharmaceutical applicationUnsaturated fatty acids

0278-6915/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.fct.2011.08.016

⇑ 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

Centaurea species are used for the treatment of various ailments in the popular medicine in some coun-tries. This study was designed to examine antioxidant potentials and fatty acid profiles of five Centaureaspecies from Turkey flora. Antioxidant properties of methanolic extracts from these species were evalu-ated by six different methods: phosphomolybdenum assay, free radical scavenging assay, b-carotene/lin-oleic acid test system, metal chelating activity, ferric and cupric reducing power. Total phenolic andflavonoid concentrations of each extract were also determined using the Folin–Ciocalteu reagent and alu-minum chloride. The results of these assay showed a significant antioxidant capacity in all researchedextracts. Centaurea cheirolopha extract, with the highest amount of total phenolic and flavonoids, showedthe highest antioxidant activities in all assay, except for metal chelating. Fatty acid profiles of these spe-cies were examined by GC–FID and 30 fatty acids were identified. Palmitic, linoleic, oleic, and linolenicacid were detected as the main components. The results of the study indicated that the Centaurea speciescan be considered as a source of new natural antioxidants and unsaturated fatty acids for food, cosmeticand pharmaceutical industries.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Free radicals, such as superoxide ðO��2 Þ, hydroxyl (OH�) and per-oxyl (ROO�), are responsible for many chronic and degenerativediseases such as cancer, atherosclerosis, diabetes, neurodegenera-tive disorders and aging (Halliwell and Gutteridge, 1999; Yu,1994). The free radicals are generated by the normal metabolicprocess of the body and external factors such as pollution, radia-tion, and smoking. Overproduction of these radicals can lead toabove mentioned disorders and this situation is called as ‘‘oxida-tive stress/oxidative damage’’. Antioxidant compounds are effec-tive in human body against oxidative damage caused by theseradicals (Issa et al., 2006; Kaliora et al., 2006). Many natural com-pounds, especially phenolic compounds, possess significant antiox-idant properties such as metal chelation, scavenging of freeradicals and inhibition of lipid peroxidation. Potent synthetic anti-oxidants such as BHA (butylated hydroxyanisole), BHT (butylatedhydroxytoluene), TBHQ (tertiary butylhydroquinone) and PG (pro-pyl gallate) are widely used for inhibition of lipid oxidation in foodindustry. However, use of these synthetic antioxidants is begun to

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ax: +90 332 2410106.sek).

be restricted due to their toxicity (Ito et al., 1986; Sasaki et al.,2002). Therefore, there is a great deal of interest in the isolationand identification of natural antioxidant of plant origin.

The genus Centaurea is belonging to the Asteraceae family, andcomprises 400–700 species of annual, biennial and perennialgrassy plants distributed in Europe and Asia (Dittrich, 1977;Wagenitz and Hellwig, 1996). In Turkey, this genus is representedby a very large number of spices (more than 180 species), many ofwhich are endemic (more than 100 species) (Davis, 1988). Ethno-pharmacological studies shown that many Centaurea species areused for the treatment of various ailments in the popular medicinein some countries. For example, Centaurea cyanus and Centaureascabiosa are used as diuretic and tonic in Scottish medicine(Kumarasamy et al., 2007) Centaurea pulchella, Centaurea drabifolia,and Centaurea solstitialis are used to treat abcesses, hemorrhoids,pepticulcers, and the common cold in Turkish folk medicine(Honda et al., 1996; Sezik et al., 2001). Owing to their potentialuse in different purposes, several studies focused on secondarymetabolites and biological activities of Centaurea species(Karamenderes et al., 2007a,b, 2006; Koca et al., 2009; Milosevicet al., 2010; Shoeb et al., 2005, 2006a,b). A literature surveyshowed no previous reports of antioxidant capacity and fatty acidprofile of five used Centaurea species (C. kurdica, C. rigida,C. amanicola, C. cheirolopha and C. ptosimopappoides) in the present

A. Aktumsek et al. / Food and Chemical Toxicology 49 (2011) 2914–2920 2915

study. However, antimicrobial activity of C. amanicola, C. kurdicaand C. ptosomipappoides were reported by Güven et al. (2005).Thus, this study is the first report on antioxidant properties andfatty acid profile of the species, three of them are endemic toTurkey flora. The objectives of this study were: (1) to evaluateantioxidant activities using different tests including phosphomo-lybdenum assay, free radical scavenging activity, b-carotene/linoleic acid system, metal chelating activity, ferric and cupricreducing power, (2) to determine total phenolic and flavonoidcontent, (3) to identify fatty acid composition, and (4) to assesswhether this species could be a source of natural antioxidants forpharmaceutical and food applications.

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 Education Fac-ulty Herbarium, Konya. The complete list of the plant materials with taxonomicalinformation, location, voucher number and section was shown in Table 1.

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. 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, Tween 40, ferrozine (3-(2-pyri-dyl)-5-6-diphenly-1,2,4-triazine-40 ,400-disulfonic acid sodium salt) and FeCl2 werepurchased from Sigma Chemical Co. (Sigma–Aldrich GmbH, Sternheim, Germany).All other chemicals and solvents were analytical grade.

2.4. Assay for total phenolics

The total phenolic content of the extracts was determined using the Folin–Cio-calteu reagent (Slinkard and Singleton, 1977). 0.2 lL of extract solution (2 mg mL�1)were mixed with 1 mL Folin–Ciocalteu reagent and 2 mL Na2CO3 (7.5%). The finalmixture volume was brought to 7 mL with deionized water. After 2 h incubationat room temperature, the absorbance was measured at 765 nm with spectropho-tometer (Shimadzu, UV-1800). Gallic acid was used as standard for calibrationcurve. The total phenolic content was determined as gallic acid equivalents (mgGAE 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).

Table 1Localities, voucher numbers and sections of five Centaurea species.

Centaurea species Localities

Centaurea kurdica Reichardt (E) Sanliurfa: Sanliurfa-Viransehir road, SCentaurea rigida Banks et Sol. Sanliurfa: Viransehir-Siverek road, KaCentaurea amanicola Hub.-Mor. (E) Osmaniye: Osmaniye-Yarpuz road, PiCentaurea cheirolopha (Fenzl) Wagenitz Osmaniye: Osmaniye-Yarpuz road, PiCentaurea ptosimopappoides Wagenitz (E) Adana: between Akdag-Kizildam, und

E: endemic.

2.6. 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 sample extracts(2 mg mL�1) were combined with 3 mL reagent solution (6 M sulfuric acid,28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containingthe reaction solution were incubated at 95 �C for 90 min. Then the absorbance ofthe solution was measured at 695 nm against a blank. The antioxidant capacity ofextracts was expressed as equivalents of ascorbic acid (mg AE g�1 extract).

2.7. Scavenging activity on DPPH

The free radical scavenging activity of plant extracts were determined by slightmodifications of the method described by Sarikurkcu et al. (2008). 0.5 mL of variousconcentrations of the extracts (50–400 lg mL�1) and BHT (3.125–200 lg mL�1) inthe methanol were added into 3 mL (6 � 10�5 M) of a methanol solution of DPPH.The solution was incubated for 30 min in the dark at room temperature. After theincubation, the mixture absorbance was measured at 517 nm. Inhibition activitywas calculated in following way:

Ið%Þ ¼ ðA0 � A1Þ=A0 � 100

where A0 is the absorbance of the control, A1 is the absorbance of the extract/stan-dard. Fifty percent of free radical inhibition (IC50) of extract was calculated. The low-er the IC50 value indicates high antioxidant capacity.

2.8. b-Carotene–linoleic acid bleaching assay

The antioxidant activity was performed by slight modifications of the proceduredescribed by Eminagaoglu et al. (2007). Briefly, 0.5 mg b-carotene was dissolved in1 mL of chloroform, 25 lL linoleic acid and 200 mg Tween 40 was added. Chloro-form was removed by rotary vacuum evaporator and 100 mL distilled water satu-rated with oxygen were added with vigorous shaking. 2.5 mL of this reactionmixture dispensed into test tubes and 350 lL portion (2 mg mL�1) of the extractswere added. The absorbance was measured at 490 nm immediately. The reactionmixture was incubated at room temperature for 48 h and the absorbance was mea-sured again. The same procedure was repeated with synthetic antioxidant, BHT andBHA, as positive control, and a blank. Inhibition ratio of linoleic acid oxidation wascalculated for test samples.

2.9. Reducing power activity (iron(III) to iron(II) reduction)

The ferric reducing power method applied with slight modifications of themethod of Oyaizu (1986). Various concentrations of the extracts (2.5 mL) weremixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassiumferricyanide. The mixture was incubated at 50 �C for 20 min. After 2.5 mL of 10% tri-chloroacetic acid was added. 2.5 mL of the reaction mixture was mixed with 2.5 mLdistilled water and 0.5 mL of 0.1% ferric chloride. The solution absorbance was mea-sured at 700 nm. The reducing power of samples increased with the absorbance va-lue. The same procedure was applied with BHA and BHT. The EC50 value (theeffective concentration at which the absorbance was 0.5) was calculated for extract,BHA and BHT.

2.10. 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. Ascorbicacid was used as a standard antioxidant. The results of the assay were evaluatedby using EC50 values.

Voucher numbers Sections

iverek district, road side, 605 m AD 9015 Cynaroidesrakecili district, 806 m AD 9016 Microlophusnus brutia and Quercus forest, 835 m AD 9020 Cynaroidesnus brutia and Quercus forest, 835 m AD 9028 Pseudoseridaer forest, 890 m AD 9042 Ptosimpappus

Table 2Extraction yield, total antioxidant capacity, total phenolic and flavonoid content of five Centaurea species.

Species Yield (%) TPCa TFCb TACc

C. kurdica 10.73 135.707 ± 0.463d 165.209 ± 2.214 178.326 ± 9.177C. rigida 8.25 113.586 ± 2.018 76.252 ± 2.214 188.780 ± 3.374C. amanicola 10.11 104.596 ± 7.674 134.252 ± 0.615 180.144 ± 3.374C. cheirolopha 7.26 175.404 ± 3.908 245.035 ± 5.042 234.765 ± 0.919C. ptosimopappoides 13.01 82.273 ± 4.762 156.165 ± 0.738 141.280 ± 4.444

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 Values expressed are means ± S.D.

2916 A. Aktumsek et al. / Food and Chemical Toxicology 49 (2011) 2914–2920

2.11. Metal chelating activity

The chelating ability of metal ion was measured according to the method re-ported by Dinis et al. (1994). Briefly, 2 mL solution containing extract (400 lg mL�1)was mixed 0.05 mL of 2 mM of FeCl2 and 0.2 mL of 5 mM ferrozine. Then, total vol-ume was adjusted to 5 mL with methanol. The mixture was shaken well and incu-bated at room temperature for 10 min. Absorbance of the solution was measured at562 nm against a blank. EDTA was used as positive control. The metal chelatingactivity was calculated by the formula:

Chelating activity ð%Þ ¼ ½ðAcontrol � AsampleÞ=Acontrol� � 100

where Acontrol was absorbance of control (the control contained ferrozine and FeCl2)and Asample was absorbance of the test sample.

2.12. Extraction of oils

The oil extraction of dried and powdered aerial plants (10 g) was carried out atboiling point (34 �C) for 6 h by Soxhlet extractor using diethyl ether as a solvent.The solvent was evaporated by rotary evaporator. The oil was esterified to deter-mine fatty acid composition.

2.13. Fatty acids methyl esters (FAMEs) preparation

The fatty acids in the oil were esterified into methyl esters by saponificationwith 0.5 N methanolic NaOH and transesterified with 14% BF3 (v/v) in methanol(IUPAC, 1979).

2.14. Gas chromatographic analysis

FAMEs were analyzed on a HP (Hewlett Packard) Agilent 6890 N model gaschromatograph (GC), equipped with a flame ionization detector (FID) and fittedto a HP-88 capillary column (100 m length, 0.25 mm i.d. and 0.2 lm thickness).Injector and detector temperatures were set at 240 and 250 �C, respectively. Theoven was held at 160 �C for 2 min. Thereafter the temperature was increased upto 185 �C at rate of 4 �C min�1 then increased at up to 200 �C at rate of 1 �C min�1

and held at 200 �C for 46.75 min. Total run time was 70 min. Helium was used ascarrier gas (1 mL min�1).

Identification of fatty acids was carried out by comparing sample FAME peakrelative retention times with those obtained for Alltech and Accu standards. Resultswere expressed as FID response area in relative percentages. Each reported result isgiven in the average value of three GC analyses. The results are offered asmeans ± S.D. Atherogenic index (AI) and thrombogenicity index (TI) were calculatedaccording to Ulbricht and Southgate (1991). AI = [12:0 + (4 � 14:0) + 16:0]/[(x6 + x3) PUFA + 18:1 + other MUFA]; TI = [14:0 + 16:0 + 18:0]/[0.5 � 18:1 + 0.5� other MUFA + 0.5 �x6 PUFA + 3 �x3 PUFA + (x3 PUFA/x6 PUFA)]. The clusteranalysis of data on fatty acid profiles of the studied samples were performed withWard’s method using Statistica 8.0.

3. Results and discussion

3.1. Total phenolic (TPC) and total flavonoid (TFC) content

Plant phenolics constitute one of the major groups acting as pri-mary antioxidants (Rice-Evans et al., 1997). Therefore, the amountof phenolics in the studied extracts was measured by Folin–Ciocal-teu method. Table 2 shows gallic acid equivalents of total phenolicsof Centaurea species. Content of phenolic compounds in theextracts varied from 82.273 mg GAE g�1 in C. ptosimopappoides to175.404 mg GAE g�1 in C. cheirolopha. As displayed in Table 2, thetotal phenolic content (TPC) of the studied Centaurea species can

be ranked as: C. cheirolopha > C. kurdica > C. rigida > C. amanicola >C. ptosimmopappoides. A similar level of diversity in phenoliccontents was seen by Karamenderes et al. (2007a,b) who examinedeight Centaurea species collected from Aegean region ofTurkey. Phenolic content of three Centaurea species (C. patula,C. tchihatcheffeii and C. pulchella at 1 mg ml�1 concentration) wasalso reported by Zengin et al. (2010) as 25.61, 22.27, and55.00 mg GAE g�1, respectively. Total phenolic content ofC. ammocyanus, which was collected from Jordan, observed as10.9 mg GAE g�1 (Alali et al., 2007).

Flavonoids are the major components of the phenolic com-pounds. The total flavonoid contents (TFC) of studied species wereestimated by using aluminum chloride colorimetric assay. Thetotal flavonoids ranged from 76.252 to 245.035 mg RE g�1.C. cheirolopha had the highest TFC value (245.035 mg RE g�1), fol-lowed by C. kurdica (165.209 mg RE g�1), C. ptosimopappoides(156.165 mg RE g�1), C. amanicola (134. 252 mg RE g�1) andC. rigida (76.252 mg RE g�1) (Table 2). Apparently, C. cheirolophahas almost four times more total flavonoid content than C. rigida.Literature is scarce about the levels of flavonoids in Centaureaspecies (Zengin et al., 2011a; Hosseinimehr et al., 2007).

3.2. Total antioxidant capacity

Total antioxidant capacity of the extracts was assessed by phos-phomolybdate assay. Recently, the assay is widely used to deter-mine antioxidant capacity because it is simply and cheap. Thetest system based on reduction of Mo(VI) to Mo(V) by antioxidantcompounds and subsequent formation of a green phosphate/Mo(V)complex at acid pH (Prieto et al., 1999). Total antioxidant capacityof studied Centaurea extract was expressed as the number of equiv-alents of ascorbic acid (Table 2). C. cheirolopha showed the greatestantioxidant efficacy, followed by C. rigida and C. amanicola. Valueswere 234.765, 188.780, and 180.144 mg AE g�1, respectively. Thevalues obtained from the test system comparable to the values re-ported in the literature for other Centaurea species, for example C.pulchella (67.89 mg AE g�1 at 1 mg mL�1 concentration) (Zenginet al., 2010). In the ranking of the antioxidant capacity of obtainedby phosphomolybdate assay, C. cheirolopha showed a greater anti-oxidant capacity than that of other Centaurea species. The strongantioxidant capacity of C. cheirolopha extract may be attributedto presence of phenolic and flavonoid compounds (Falleh et al.,2008).

3.3. Free radical scavenging activity (DPPH assay) and b-carotene/linoleic acid test system

DPPH assay is often used to evaluate the antioxidant ability ofplant extracts. DPPH, a stable radical with purple color, changesinto a stable yellow compound (diphenyl-picrylhydrazine) onreacting with an antioxidant. DPPH scavenging activity of the ex-tracts and synthetic antioxidant (BHT) was assessed by using IC50

0 50 100 150 200 250 300 350 400 450

Concentration (µg/mL)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Abs

orba

nce

(700

nm

)

C. kurdica C.rigida C.amanicola C. cheirolopha C. ptosimopappoides

Fig. 1. Ferric reducing power of five Centaurea species at different concentrations.

0.7

0.8

0.9

1.0 n

m)

C. kurdica C.rigida C.amanicola C. cheirolopha C. ptosimopappoides

A. Aktumsek et al. / Food and Chemical Toxicology 49 (2011) 2914–2920 2917

values (Table 3). A low IC50 value indicates strong antioxidantactivity in a sample. Among the studied Centurea species, the mostpotent radical scavenger was C. cheirolopha (227.448 lg mL�1), fol-lowed by C. kurdica (367.711 lg mL�1), C. rigida (475.056 lg mL�1),C. amanicola (581.358 lg mL�1) and finally C. ptosimopappoides(745.133 lg mL�1). However, BHT showed excellent scavengingability (34.06 lg mL�1) when compared to the extracts. To the bestof our knowledge, several studied were fulfilled on radical scaveng-ing activity of the members of the genus Centaurea, such asC. cyanus, C. scabiosa (Kumarasamy et al., 2007), C. calolepis andC. cadmea (Karamenderes et al., 2007a,b). However, the DPPH as-says used for those studies were different (concentration of DPPHsolution) for each species, so the results are no directlycomparable.

b-Carotene/linoleic acid test system based on discoloration ofb-carotene in the absence of antioxidants. The presence of anantioxidant can hinder the extent of b-carotene degradation byneutralizing the linoleate free radical and any other free radicalsformed within the system (Kamath and Rajini, 2007). As shownin Table 3, C. cheirolopha had the highest antioxidant activity(79.515%) in the studied Centaurea extracts. In term of b-carotenebleaching effect, the Centaurea species exhibited the following or-der: C. cheirolopha > C. kurdica > C. ptosimopappoides > C. rigida >C. amanicola. BHA and BHT investigated under same conditions,exhibited 94.005% and 93.350% inhibition of discoloration ofb-carotene, hence, the activities of the synthetic antioxidants werestrong. At same concentration (2 mg mL�1) of the methanol extractof C. mucronifera, ethylacetate and chloroform extract ofC. ensiformis exhibited 35.2%, 85.15%, and 72.51% antioxidantactivity, respectively (Ugur et al., 2009; Tepe et al., 2006).

0 50 100 150 200 250 300 350 400 450

Concentration (µg/mL)

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orba

nce

(450

Fig. 2. Cupric reducing antioxidant capacity (CUPRAC) of five Centaurea species atdifferent concentrations.

3.4. Ferric and cupric reducing power

The reducing power of a compound is related to its electrontransfer ability which is an important mechanism of antioxidant ac-tion. Many reports revealed that there is a direct correlation be-tween antioxidant activities and reducing power of plant extract(Yildirim et al., 2001; Koleva et al., 2002). Fig. 1 illustrates the ferricreducing power of the researched Centaurea extracts. EC50 values ofeach extracts also were calculated and the values were demon-strated in Table 3. A lower EC50 value indicates greater reducingpower. Therefore, C. cheirolopha, which contained the highestamount of total phenolics and flavonoids, displayed the highestreductive capability in the Centaurea species. Under similar condi-tions, BHT (EC50 = 23.348 lg mL�1) was the most potent reducingagent. At concentration of 50, 100, 200, 400 lg mL�1, BHT showedabsorbance with 0.773, 1.162, 1.781, and 2.818, respectively. Theabsorbance values of BHT were also higher when compared withthe studied Centaurea extracts. Moreover, reducing powers of other

Table 3Free radical scavenging, linoleic acid inhibition, ferric and cupric ion reducing power activ

Species IC50 (lg ml�1)a Inhibitio

C. kurdica 367.711 ± 2.652e 75.273 ±C. rigida 475.056 ± 0.345 69.344 ±C. amanicola 581.358 ± 3.033 65.635 ±C. cheirolopha 227.448 ± 2.136 79.515 ±C. ptosimopappoides 745.133 ± 20.904 70.448 ±BHA – 94.005 ±BHT 34.061 ± 0.381 94.350 ±Ascorbic acid – –

a Results of free radical scavenging activity (DPPH assay).b Results of b-carotene/linoleic acid test system.c Results of ferric ion reducing power.d Results of cupric ion reducing power.e Values expressed are means ± S.D.

Centaurea species were close to that of each other. The reducingpower observed in the present study can be ranked as: BHT > C. chei-rolopha > C. rigida > C. amanicola > C. kurdica > C. ptosimopappoides.

ity of five Centaurea species.

n (%)b EC50 (lg ml�1)c EC50 (lg ml�1)d

0.043 541.917 ± 11.196 339.347 ± 17.1720.027 482.000 ± 60.811 334.400 ± 7.7080.148 540.867 ± 74.293 339.107 ± 13.9590.207 205.929 ± 2.424 154.534 ± 5. 6430.256 646.750 ± 3.889 518.883 ± 8.0850.069 – –0.318 24.348 ± 7.852 –

– 13.725 ± 0.478

Fig. 3. Comparison of metal chelation effect of five Centaurea species and EDTA at 400 lg/ml.

Table 4Fatty acid composition of five Centaurea species (%).

Fatty acids C. kurdica C. rigida C. amanicola C. cheirolopha C. ptosimopappoides

C 8:0 0.04 ± 0.01a 0.12 ± 0.04 0.07 ± 0.01 0.04 ± 0.01 0.09 ± 0.01C 10:0 0.07 ± 0.01 0.13 ± 0.05 0.86 ± 0.01 0.22 ± 0.01 0.15 ± 0.01C 11:0 0.09 ± 0.01 0.14 ± 0.01 0.04 ± 0.01 0.03 ± 0.01 0.07 ± 0.01C 12:0 2.97 ± 0.01 0.54 ± 0.08 4.69 ± 0.29 1.56 ± 0.01 3.03 ± 0.02C 13:0 0.32 ± 0.05 0.12 ± 0.01 0.25 ± 0.02 0.15 ± 0.01 0.18 ± 0.01C 14:0 2.20 ± 0.06 1.14 ± 0.04 1.66 ± 0.16 2.33 ± 0.07 1.42 ± 0.01C 15:0 1.03 ± 0.01 0.39 ± 0.04 0.61 ± 0.01 0.10 ± 0.02 0.35 ± 0.01C 16:0 37.59 ± 0.05 29.70 ± 0.18 36.68 ± 0.55 19.84 ± 0.01 26.78 ± 0.03C 17:0 1.02 ± 0.03 1.29 ± 0.01 1.15 ± 0.02 0.46 ± 0.01 0.76 ± 0.02C 18:0 5.22 ± 0.01 9.15 ± 0.10 6.72 ± 0.12 4.83 ± 0.01 3.88 ± 0.02C 19:0 0.25 ± 0.01 0.05 ± 0.01 0.15 ± 0.01 0.04 ± 0.01 0.10 ± 0.02C 20:0 0.14 ± 0.01 0.11 ± 0.06 0.14 ± 0.02 0.13 ± 0.02 0.25 ± 0.01C 21:0 0.82 ± 0.04 0.30 ± 0.08 0.45 ± 0.06 0.05 ± 0.01 0.24 ± 0.01C 22:0 0.38 ± 0.02 0.97 ± 0.08 0.47 ± 0.04 0.18 ± 0.01 0.39 ± 0.01RSFAb 52.14 ± 0.08 44.15 ± 0.13 53.94 ± 0.76 29.96 ± 0.16 37.69 ± 0.01C 14:1 x5 0.30 ± 0.01 0.37 ± 0.03 0.21 ± 0.04 0.73 ± 0.04 1.05 ± 0.03C 15:1 x5 0.21 ± 0.10 0.15 ± 0.08 0.21 ± 0.11 0.07 ± 0.01 0.10 ± 0.02C 16:1 x7 0.31 ± 0.01 0.32 ± 0.01 0.80 ± 0.18 0.66 ± 0.44 0.40 ± 0.01C 17:1 x8 0.14 ± 0.01 0.32 ± 0.01 0.17 ± 0.01 0.08 ± 0.01 0.27 ± 0.01C 18:1 x9 5.61 ± 0.06 9.52 ± 0.08 4.95 ± 0.05 10.85 ± 0.02 11.50 ± 0.01C 18:1 x7 1.44 ± 0.02 5.39 ± 0.05 3.05 ± 0.35 3.96 ± 0.01 4.17 ± 0.06C 20:1 x9 1.07 ± 0.04 0.26 ± 0.03 0.29 ± 0.04 0.14 ± 0.01 0.22 ± 0.02C 22:1 x9 0.18 ± 0.01 0.04 ± 0.01 0.10 ± 0.01 0.04 ± 0.01 0.10 ± 0.01RMUFAb 9.26 ± 0.03 16.37 ± 0.17 9.78 ± 0.59 16.53 ± 0.01 17.81 ± 0.04C 18:2 x6 13.90 ± 0.04 11.69 ± 0.16 14.79 ± 0.26 21.85 ± 0.01 18.75 ± 0.02C 18:3 x3 14.26 ± 0.05 8.31 ± 0.49 9.37 ± 0.22 19.99 ± 0.01 14.64 ± 0.01C 18:3 x6 3.61 ± 0.01 7.46 ± 0.08 4.77 ± 0.78 5.33 ± 0.09 4.55 ± 0.05C 20:2 x6 0.32 ± 0.03 0.59 ± 0.16 0.34 ± 0.01 0.05 ± 0.01 0.30 ± 0.01C 20:4 x6 3.99 ± 0.04 9.08 ± 0.10 4.26 ± 0.04 4.38 ± 0.03 4.01 ± 0.01C 20:5 x3 0.58 ± 0.09 0.82 ± 0.04 0.94 ± 0.05 0.24 ± 0.01 0.24 ± 0.03C 22:2 x6 0.12 ± 0.01 0.49 ± 0.02 0.10 ± 0.01 0.17 ± 0.01 0.08 ± 0.01C 22:6 x3 1.82 ± 0.03 1.04 ± 0.03 1.80 ± 0.04 1.50 ± 0.24 1.93 ± 0.10RPUFAb 38.60 ± 0.06 39.48 ± 0.03 36.37 ± 0.16 53.51 ± 0.17 44.50 ± 0.04RUFAb 47.86 ± 0.09 55.85 ± 0.14 46.15 ± 0.75 70.04 ± 0.16 62.31 ± 0.01REFA 28.16 ± 0.09 20.00 ± 0.23 24.16 ± 0.48 41.84 ± 0.01 33.39 ± 0.01Rx3 16.66 ± 0.01 10.17 ± 0.08 12.11 ± 0.31 21.73 ± 0.23 16.81 ± 0.08Rx6 21.94 ± 0.08 29.31 ± 0.06 24.26 ± 0.47 31.78 ± 0.06 27.69 ± 0.04x3/x6 0.76 ± 0.01 0.35 ± 0.01 0.50 ± 0.02 0.68 ± 0.01 0.61 ± 0.01x6/x3 1.32 ± 0.01 2.88 ± 0.04 2.00 ± 0.09 1.46 ± 0.02 1.65 ± 0.01AIc 1.03 ± 0.01 0.62 ± 0.01 1.04 ± 0.02 0.44 ± 0.01 0.57 ± 0.01TIc 0.68 ± 0.01 0.74 ± 0.01 0.84 ± 0.01 0.30 ± 0.01 0.43 ± 0.01Oil content 1.22 1.35 1.19 1.89 2.06

a Values reported are means ± S.D.b SFA: Saturated fatty acids, MUFA: Monounsaturated fatty acids, PUFA: Polyunsaturated fatty acids, UFA: Unsaturated fatty acids, EFA: Essential fatty acids.c AI: Atherogenic index, TI: Thrombogenicity index.

2918 A. Aktumsek et al. / Food and Chemical Toxicology 49 (2011) 2914–2920

CUPRAC (cupric reducing antioxidant capacity) method wasdeveloped for antioxidant measurement of plant material. Themethod is based on the measurement of absorbance at 450 nmby the formation of a stable complex between neocuproine andcopper(I), the latter is formed by the reduction of copper(II) inthe presence of neocuproine (Apak et al., 2006). Lower EC50 valueand higher absorbance (at 450 nm) indicated greater cupric reduc-ing power (Fig. 2). Therefore, C. cheirolopha extract was signifi-cantly better cupric reducing activity than other extracts with

EC50 value 154.534 lg mL�1, while C. ptosimmopappoides had thelowest cupric reducing activity with EC50 518.883 lg mL�1 (Table3). However, ascorbic acid concentration required to reduce cupricion was lower than C. cheirolopha extract, indicating a better cupricreducing activity. Moreover, absorbance of ascorbic acid was foundto be higher than Centaurea extracts (0.902, 1762, 2847 and 3.855at concentration of 50, 100, 200, 400 lg mL�1, respectively). Theorder in reductive activity was similar in the reducing power assay.These results are in agreement with a previous study by Ozturk

Ward`s methodEuclidean distances

0 10 20 30 40 50 60 70

Linkage Distance

C. ptosimopappoides

C. cheirolopha

C. rigida

C. amanicola

C. kurdica

Fig. 4. Dendrogram derived from fatty acid analysis of five Centaurea species.

A. Aktumsek et al. / Food and Chemical Toxicology 49 (2011) 2914–2920 2919

et al. (2007) who reported that a correlation between ferric reduc-ing power and CUPRAC assay. As far as our literature survey couldas certain, data on ferric and cupric reducing power of Centaureaspecies are very few (Zengin et al., 2011a,b).

3.5. Metal chelating activity

Transition metal ions are known to generate free radicals throughthe Fenton and Haber–Weiss reaction (Halliwell and Gutteridge,1990). The metal ion chelating activity of an antioxidant preventsfree radical generation and the consequent oxidative damage (Duhet al., 1999). The ion chelating ability (%) of Centaurea extracts andEDTA are summarized in Fig. 3. C. rigida extract showed the highestferrous iron chelating activity (at 400 lg mL�1, 70.720 ± 0.650%). Onthe other hand, other Centaurea extracts exhibited close chelatingactivity. For instance, the chelating activities of C. kurdica and C. ama-nicola were 57.282 ± 0.722% and 56.566 ± 0.578%, respectively.However, at the same concentration, synthetic chelator (EDTA)showed an excellent chelating ability (93.323 ± 3.987%). Untilnow, to the best of our knowledge, there is no report on metal che-lating activity of members of Centaurea. Therefore, the present studyis the first report about chelating activity of Centaurea species.

3.6. Oil content and fatty acid composition

The fatty acid composition of investigated Centaurea specieswas determined using GC–FID. Results are presented in Table 4and are expressed as percentage of total fatty acids. The GC analy-sis showed 30 fatty acids in the species. Their oil content rangedfrom 1.19% to 2.06%. Except for C. cheirolopha oil, palmitic acid (C16:0) was identified as the major component in the remainingspecies. This result was in agreement with a previous study onfatty acid profile of 16 Centaurea species (Yildirim et al., 2009). Thisfatty acid varied between 19.84% in C. cheirolopha and 37.59% in C.kurdica. Generally, palmitic, linoleic (C 18:2 x6), oleic (C 18:1 x9)and linolenic acid (C 18:3 x3) were the most abundant fatty acids.Linoleic acid was detected in the greatest proportion in C. cheirolo-pha oil and this fatty acid accounted for 21.85% of total fatty acids.Linolenic acid was also found to be the highest level in the species.These fatty acids (linoleic and linolenic acid) are called ‘‘essentialfatty acid’’ because the human body is not able to produce them.Therefore, C. cheirolopha oil is considered as a source of essentialfatty acids. Similarly, linoleic acid was reported as major fatty acidof C. pulchella by Zengin et al. (2010), C. calolepis and C. iberica byTekeli et al. (2010). The main monounsaturated fatty acid is oleicwhich contributes more than 50% of the total monounsaturated

fatty acids. C. ptosimopappoides had the highest percentage of oleicacid but found to be at its lowest level in C. amanicola. The fattyacid compositions of studied Centaurea species ranged from29.96% to 53.94 % saturated fatty acids (SFAs), 9.26–17.81% mono-unsaturated fatty acids (MUFAs) and 36.37–53.51% PUFAs. The to-tal content of unsaturated fatty acid (UFA) was higher and that ofSFA was lower in oil samples from three Centaurea species (C. rig-ida, C. cheirolopha and C. ptosimopappoides). The levels of unsatu-rated fatty acids reached 70.04% in C. cheirolopha oil.

The ratio of x3/x6 fatty acids is a reliable indicator for nutri-tional value of lipids. The highest x3/x6 value was observed inC. kurdica oil (0.76), followed by C. cheirolopha (0.68), C. ptosimop-appoides (0.61), C. amanicola (0.50) and C. rigida (0.35). Atherogenic(AI) and thrombogenicity index (TI) were also used to assess thenutritional quality of the oils. Their lower values are in the favorof good health. With regard to AI and TI, C. cheirolopha oil(AI = 0.44, TI = 0.30) showed the superior nutritional quality.

The dendrogram obtained from the fatty acids analysis is shownin Fig. 4. The dendrogram revealed two major groups. The firstgroup included C. kurdica and C. amanicola which contained highproportion of saturated fatty acids. The result may be considerableinterest for chemotaxonomic analysis because these species areclassified in section Cynaroides. The second group consisted oftwo species (C. cheirolopha and C. ptosimopappoides). In contrastto the first group, the group was characterized with the greatestproportion of unsaturated fatty acids.

4. Conclusion

Presented results indicated significant antioxidant activity in allinvestigated Centaurea extracts. These species had large amountsof phenolic compounds and flavonoids. C. cheirolopha proved tobe the richest in phenolics and flavonoids. It also exhibited thehighest total antioxidant capacity, DPPH scavenging ability, inhibi-tion of linoleic acid oxidation (b-carotene/linoleic acid test system),ferric and cupric reducing power whereas C. rigida showed thegreatest ferrous chelating activity. Moreover, the Centaurea oilespecially C. cheirolopha and C. ptosimopappoides, contained thehigh concentrations of unsaturated fatty acids. Results of the pres-ent study suggested that the Centaurea extracts and oils could beutilized as a source of natural antioxidants and unsaturated fattyacids for nutritional and pharmacological applications.

Conflict of Interest

The authors declare that there are no conflicts of interest.

2920 A. Aktumsek et al. / Food and Chemical Toxicology 49 (2011) 2914–2920

Acknowledgements

This study was supported financially as a project. The authorsthank Selcuk University Scientific Research Foundation (BAP) forproviding financial support for this study.

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