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Keywords:Black chokeberryBlueberryCultivars

wasberrry a

DPPH(2,2-diphenyl-1-picrylhydrazyl), ABTS(2,2-azino-bis-(3-ethylenebenzothiozoline-6-sulphonic acid))radical-scavenging activity, FRAP(ferric-reducing antioxidant power) and reducing power. The major

includgen pe

Typical natural antioxidants include tocopherols, carotenoids,avonoids, and polyphenolic compounds (Amro, Aburjai, & Al-Kha-lil, 2002) that can potentially provide protection against the devel-opment of certain oxidation-linked chronic diseases (kerget et al.,2005). Regular consumption of bioactive compounds from plantand fruit may be associated with protecting against oxidative dam-age and lowering the risk of chronic diseases, such as cancer, heart

xidant vitaminsle, 2007). Amongnd bluebeerage ingr

in Korea. In addtion, black chokeberries (Aronia melanocarphave received considerable attention, because they are knfunction as chemopreventive agents against oxidative d(Pool-Zobel, Bub, Schrder, & Rechkemmer, 1999). Black choke-berry is very rich in phenolic compounds from the anthocyaninsubclass, namely cyanidin-3-O-galactoside, cyanidin-3-O-arabino-side, cyanidin-3-O-xyloside and cyanidin-3-O-glucoside (Oszmian-ski & Sapis, 1988) that have been reported to possess antioxidativeactivities (Noda, Kaneyuki, Mori, & Packer, 2002; Tsuda, Shiga,Ohshima, Kawakishi, & Osawa, 1996; Wang et al., 1999). Althoughthe antioxidant activity of black chokeberry cultivated in North

Corresponding author. Tel.: +82 33 250 6456; fax: +82 82 33 241 0508.

Food Chemistry 146 (2014) 7177

Contents lists availab

he

lseE-mail address: jongdai@kangwon.ac.kr (J.D. Kim).To inhibit the physiological damage caused by excess ROS, severalenzymatic and non-enzymatic endogenous antioxidant defencesystems have been evolved to compensate the generation of ROS(Fridovich, 1997; Sies, 2005).

pounds, avonoids, anthocyanidins and antio(Kammerer, Schillmller, Maier, Schieber, & Carthe berries, high demand for black chokeberry aincreased for their use in foodstuffs and as bev0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.09.035rry hasedientsa) Ellotown toamageradical (ROO), singlet oxygen (1O2), and peroxynitrite (ONOO),can be generated from unbalanced pro-oxidant and antioxidant en-zyme response systems (Bursal & Glin, 2011). Furthermore, dif-ferent environmental stress factors, such as pollution, drought,temperature, excessive light intensities and nutritional limitation,increase the production of ROS (Ehling-Schulz & Scherer, 1999).

protective effects due to their antioxidant properties (La Casa,Villegas, Alarcon de La Lastra, Motilva, & Martn Calero, 2000;Martin et al., 1998).

Berries are well known as good natural antioxidants (Benvenuti,Pellati, Melegari, & Bertelli, 2004; Garzn, Riedl, & Schwartz, 2009;Meyers, Watkins, Pritts, & Liu, 2003), which contain phenolic com-Antioxidant activitiesCyanidin-3-galactoside

1. Introduction

Reactive oxygen species (ROS),(O2), hydroxyl radical (HO), hydrophenolic compounds, including cyanidin-3-galactoside, cyanidin-3-arabinoside, neochlorogenic acid,procyanidin B1, were analysed by HPLC with a photodiode array detector. Results showed that totalphenol, avonoid and proanthocyanidin contents of black chokeberry extract were higher than thoseof blueberry extract. In addition, black chokeberry extract exhibited higher free radical-scavengingactivity and reducing power than did blueberry extract. Cyanidin-3-galactoside was identied as a majorphenolic compound, with considerable content in black chokeberry, that correlated with its higherantioxidant and radical-scavenging effects. These results suggest that black chokeberry extracts couldbe considered as a good source of natural antioxidants and functional food ingredients.

2013 Elsevier Ltd. All rights reserved.

ing superoxide anionroxide (H2O2), peroxyl

disease, and cerebrovascular disease (Block, Patterson, & Subar,1992; Hung et al., 2004; Liu et al., 2000; Willis, Shukitt-Hale, &Joseph, 2009). Moreover, several authors have recently reportedthat phytochemicals, including phenolic compounds, have gastro-Accepted 5 September 2013Available online 14 September 2013

tent, total proanthocyanidin content, and antioxidative activities, using various in vitro assays, such asRadical-scavenging-linked antioxidant acchokeberry and blueberry cultivated in K

Seok Joon Hwang a, Won Byong Yoon a, Ok-Hwan LeaDepartment of Food Science and Biotechnology, Kangwon National University, Chunchb Legal Personality Icheon Beks & Cornus Co. Ltd, Icheon 467-822, South Korea

a r t i c l e i n f o

Article history:Received 8 March 2013Received in revised form 28 June 2013

a b s t r a c t

The objective of this studyextracts from black chokepared from black chokeber

Food C

journal homepage: www.eities of extracts from blackea

, Seung Ju Cha b, Jong Dai Kim a,200-701, South Korea

to investigate the radical-scavenging-linked antioxidant properties of they and blueberry cultivated in Korea. The 70% ethanol extracts were pre-nd blueberry, and evaluated for total phenolic content, total avonoid con-

le at ScienceDirect

mistry

vier .com/locate / foodchem

(2,2-diphenyl-1-picrylhydrazy) radical-scavenging activity, ABTS

The following compoundswere used as standards for analysis by

blueberry

(1981). The sample solution (1 ml) was placed in a test tube with

A : absorbance value of testing solution

hemFolinCiocalteau reagent (1 ml) and sodium carbonate solution(1 ml). After incubation for 1 h at 25 C, the absorbance was mea-sured at 750 nm and total phenolic content was calculated as gallicacid equivalents (mg GAE/g).

2.4. Determination of total avonoid contents

The total avonoid contents of extracts from black chokeberryand blueberry were determined according to the method ofMoreno, Isla, Sampietro, and Vattuone (2000). The sample solution(0.5 ml) was mixed with 1.5 ml of ethanol (95%), followed by0.1 ml of aluminium chloride (10%), 0.1 ml of potassium acetateThe black chokeberry and blueberry were collected from Legalpersonality Icheon beks & cornus of Korea and kept in a deep free-zer (70 C). The black chokeberry and blueberry were extractedwith 10 vol (v/w) of 70% ethanol at 70 C for 3 h, and extractionwas repeated three times. The extracts were ltered throughWhatman lter paper (No. 2), concentrated with a vacuum evapo-rator, and completely dried with a freeze-drier. The freeze-driedpowder was dissolved in 50% DMSO (dimethyl sulfoxide) and l-tered through a membrane lter (0.45 lm) and used for antioxi-dant activity.

2.3. Determination of total phenol contents

The total phenolic contents of extracts from black chokeberryand blueberry were determined by a modied method of GutngerHPLC-DAD: cyanidin 3-galactoside and cyanidin 3-arabinosidewereobtained from Polyphenols Laboratories AS (Sandnes, Norway);neochlorogenic acid and procyanidin B1 were also obtained fromChengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, China).FolinCiocalteau reagent, gallic acid, rutin and catechin were ob-tained from SigmaAldrich (St. Louis, MO, USA). The chemicals usedfor antioxidant activities, such as DPPH (2,2-diphenyl-1-pic-rylhyd-razy), ABTS (2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid), TPTZ (2,4,6-tripyridyl-s-triazine), trichloroacetic acid,ascorbic acid, were purchased from SigmaAldrich (St. Louis, MO,USA). All other chemicals were of reagent grade.

2.2. Preparation of 70% ethanol extract of black chokeberry and(2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay,ferric reducing ability (FRAP) assay, major phenolic compoundsof black chokeberry and blueberry (Vaccinium corymbosum L.)cultivated in Korea by HPLC with a photodiode array detector.

2. Materials and methods

2.1. ChemicalsAmerica and Europe has been extensively investigated(Oszmianski & Wojdylo, 2005; Skupien & Oszmianski, 2007; Wu,Gu, Ronald, & McKay, 2004; Zheng and Wang, 2003), there are noreports documenting radical-scavenging-linked antioxidant activ-ity of black chokeberry cultivated in Korea.

The objective of this study was to investigate the totalphenolics, total avonoid proanthocyanidin contents, DPPH

72 S.J. Hwang et al. / Food C(1 M) and distilled water. After incubation at room temperaturefor 30 min, the absorbance was measured at 415 nm and totalavonoids content was calculated as rutin equivalents (mg RE/g).B : absorbance value of control solution

2.7. ABTS radical-scavenging activity

The antioxidant activity was determined using ABTS free radi-cal, as previously described by Re et al. (1999). Before analysis,the stock solution was prepared by stirring ABTS+ (7 mM) andpotassium persulfate (2.45 mM) in water at room temperaturefor 16 h. The ABTS+ solution was diluted with ethanol to achievean absorbance of 0.75 0.025 at 750 nm. Then, 1 ml ABTS+ solutionwas added to 10 ll of different concentrations (10500 lg/ml) ofblack chokeberry and blueberry extracts. These samples were vor-texed and incubated in the dark for 6 min. ABTS radical-scavengingactivities were measured by spectrophotometer at 750 nm, werecalculated and expressed as a percentage using the followingformula:

ABTS radical-scavenging activity % 1 A=B 100

A : absorbance value of testing solution

B : absorbance value of control solution

2.8. FRAP assay

The FRAP assay is based on the ability of the antioxidant to re-duce Fe3+ to Fe2+ in the presence of TPTZ, forming an intense blueFe2+TPTZ complex with an absorbance maximum at 593 nm,which is pH-dependent. The absorbance decrease is proportionalto the antioxidant activity (Benzie & Strain, 1996). The FRAP valuesof black chokeberry and blueberry extract were obtained by using2.6. The antioxidant activity of extracts by DPPH radical

In the DPPH assay, the natural and synthetic antioxidants wereable to reduce the stable radical DPPH to the yellow-colouredDPPH. This method is based on the reduction of DPPH in alcoholicsolution in the presence of a hydrogen-donating antioxidant due tothe formation of the non-radical form DPPH-H in the reaction. Theuse of DPPH provides an easy way to evaluate antioxidant activity(Oyaizu, 1986). The antioxidant activity of these extracts was mea-sured on basis of their electron-donating ability (EDA) of the stableDPPH as previously described (Chu, Chang, & Hsu, 2000) withslight modication. Different concentrations (10500 lg/ml) ofblack chokeberry and blueberry were prepared. Then, 1 ml of eth-anolic DPPH solution (4 104 M) was added to the samples.These samples were vortexed and incubated in the dark for10 min at room temperature. DPPH radical-scavenging activitieswere measured by spectrophotometer at 490 nm and were calcu-lated and expressed as a percentage using the following formula:

DPPH radical-scavenging activity % 1 A=B 1002.5. Determination of proanthocyanidin contents

The proanthocyanidin contents of black chokeberry and blue-berry were evaluated by the method of Sun, Ricardo-da-Silva, &Spranger (1998). The sample solutions (0.5 ml) were mixed with3 ml of vanillin (4%) and 1.5 ml of conc-HCl. After incubation for15 min, the absorbance was measured at 490 nm and proanthocy-anidin content was calculated as catechin (mg CE/g).

istry 146 (2014) 7177the method of Benzie and Strain, (1996) with modication. FRAPreagent was prepared from 300 mM acetate buffer (pH 3.6),10 mM TPTZ in 40 mM HCl and 20 mM iron chloride in proportions

and Sakariah (2001). Different concentrations (10500 lg/ml) of

3.2. Antioxidant activities of black chokeberry and blueberry extracts,

of black chokeberry cultivated in Poland to be 38.1%. Moreover,Bermdez-Soto and Toms-Barbern (2004) determined that theantioxidant activity of chokeberry juice cultivated in Spain was60.0 1.2 mg TEAC (trolox equivalent antioxidant capacity)/ml.

Another effective method to measure radical-scavenging activ-ity is the ABTS radical cation decolorisation assay, which showedresults similar to those obtained in the DPPH reaction. The ABTSradical-scavenging activities of black chokeberry, blueberry andascorbic acid were in the following order: ascorbic acid > blackchokeberry extract > blueberry extract at a concentration of500 lg/ml (Fig. 1B) The ABTS radical-scavenging percentages ofvarious concentrations (10500 lg/ml) of black chokeberry extractwere 4.6%, 10.3% and 46.3%, respectively. In contract, the ABTS rad-ical-scavenging percentages at concentrations of 10, 50 and

hemblack chokeberry and blueberry extracts in distilled water(0.5 ml) were mixed with 2.5 ml of sodium phosphate buffer (pH6.6) and 2.5 ml of potassium ferricyanide (1%). The mixtures wereincubated at 50 C for 20 min. 2.5 ml of trichloroacetic acid (10%)were then added and the mixture was centrifuged 1790g for10 min. Then, 2.5 ml of the supernatant were withdrawn andmixed with 2.5 ml of distilled water. After adding 0.5 ml of iron(III) chloride (0.1%), the absorbance was spectrophotometricallymeasured at 750 nm.

2.10. Analysis of phenolic compounds using HPLC-DAD

HPLC analysis of black chokeberry and blueberry extracts wasdone as described by Dyrby, Westergaard, and Stapelfeldt (2001).10 mg quantities of extracts were dissolved in 10 ml of water/for-mic acid (90/10, v/v,%) and ltered with a Millipore membrane l-ter (0.45 lm) prior to HPLC analysis. The HPLC equipment was anAgilent 1260 with photodiode array detector at 280 nm. HPLCanalysis was carried out using a C18 column (Agilent Technologies,Palo Alto, CA, USA) (4.6 150 mm, 3.5 lm). The mobile phaseconsisted of water/formic acid (90/10, v/v,%) (A) and water/acetonitrile/formic acid (40/50/10, v/v/v,%) (B). The ow rate was1.0 ml/min with the following gradient programme: 0 min88%A + 12%B, 26 min: 70%A + 30%B, 29 min: 0%A + 100%B, 30 min:88%A + 12%B. The standards were cyanidin 3-galactoside, cyanidin3-arabinoside, neochlorogenic acid, procyanidin B1.

2.11. Statistical analysis

All measurements were repeated three times. The results werestatistically analysed by ANOVA and Duncans multiple range tests.Statistical signicance was accepted at a level of p < 0.05.

3. Results

3.1. Total phenolic, avonoid and proanthocyanidin contents ofextracts from black chokeberry and blueberry

The extraction yields of black chokeberry and blueberry were14.2 and 8.7%, respectively. In addition, the total phenolic contentsof chokeberry and blueberry, determined using the regressionequation of the calibration curve (y = 14.826 + 0.0337, R2: 0.9985)and expressed in gallic acid equivalents, were found to be110 5.6 mg GAE/g, and 27.4 7.4 mg GAE/g, respectively(Table 1). Our results are slightly different from another study byKhknen, Hopia, and Heinonen (2001), in which total phenoliccontent of black chokeberry was 4210 100 mg/100 g. Benvenutiof 10:1:1(v/v), respectively. Different concentrations (10500 lg/ml)of sample solution (50 ll) were mixed with distilled water (150 ll)and FRAP reagent (1.5 ml) was added. The absorbance of the reac-tion mixture was then measured at 595 nm after 4 min.

2.9. Reducing power

In this reducing power assay, the presence of reducers (namely,black chokeberry and blueberry extract) converted the Fe3+/ferricy-anide complex present in the assay system to the ferrous form. Bymeasuring the absorbance at 750 nm to determine the Fe2+ con-centration, it is possible to estimate the reducing power of thereducers. The reducing power of black chokeberry and blueberryextracts were evaluated by the method of Jayaprakasha, Singh,

S.J. Hwang et al. / Food Cet al. (2004) also showed that total phenolic content of blackchokeberry extract was 690 8.8 mg/100 g. The total avonoidcontents of chokeberry and blueberry extracts, determined usingusing in vitro model

The DPPH radical-scavenging activities of black chokeberry,blueberry extracts and ascorbic acid were in the following order:ascorbic acid > black chokeberry extract > blueberry extract at thesame concentration of test samples (500 lg/ml) (Fig. 1A). TheDPPH radical-scavenging percentages of various concentrations(10, 50 and 500 lg/ml) of black chokeberry extract were 31.1%,37.0% and 72.7%, respectively. In addition, the DPPH radical-scav-enging activities of various concentrations (10, 50 and 500 lg/ml)of blueberry extract were 29.4%, 29.6% and 40.6%, respectively.The black chokeberry exhibited markedly higher DPPH free radi-cal-scavenging activity than did blueberry extract. Benvenutiet al. (2004) reported that the EC50 (DPPH radical-scavengingactivity) of chokeberry cultivated in Italy was 1.8 mg. Oszmianskiand Wojdylo, (2005) also showed the DPPH radical-inhibitionthe regression equation of the calibration curve (y = 2.0637 +0.05, R2: 0.9996) and expressed in rutin equivalents, were foundto be 5.3 0.8 mg RE/g, and 1.6 0.3 mg RE/g, respectively(Table 1).

The proanthocyanidins constitute a complex mixture of mono-mers, oligomers and polymers which generally consist of (+)-cate-chin, ()-epicatechin, (+)-gallocatechin, ()-epigallocatechin andtheir 3-O-gallic acid esters (Prieur, Rigaud, Cheynier, & Moutounet,1994). They are also an important sensory component, and areresponsible for the antioxidant efciency of berries products (Kan-ner, Frankel, Granit, German, & Kinsella, 1994). The proanthocyani-din contents of chokeberry and blueberry extracts, determinedusing the regression equation of the calibration curve(y = 1.5811 + 0.0343, R2: 0.9985) and expressed in catechin equivalents, were found to be 107 6.6 mg CE/g and 11.8 5.0 mgCE/g, respectively (Table 1).

Table 1Extraction yields, total phenol, avonoid and proanthocyanidin contents of 70%ethanol extracts from black chokeberry and blueberry cultivated in Korea.

Black chokeberry Blueberry

Extraction yields (%) 14.2 8.7Total phenol content(mg GAE/g) 110 5.6 27.4 7.4Total avonoids content(mg RE/g) 5.3 0.8 1.6 0.3Proanthocyanidin content(mg CE/g) 107 6.6 11.8 5.0

Signicantly different at p < 0.05 by student t-test. The values are means standarddeviation from three replications.GAE: Gallic acid equivalents.RE: Rutin equivalents.CE: Catechin equivalents.

istry 146 (2014) 7177 73500 lg/ml of blueberry extract were 2.3%, 4.2% and 8.6%, respec-tively. These results show that black chokeberry extract had signif-icantly higher ABTS radical-scavenging activity than had blueberry

cti b b

a

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)

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40

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ChokeberryBlueberryAscorbic acid

edd

bb

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hemistry 146 (2014) 7177d

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74 S.J. Hwang et al. / Food Cextract. Moreover, these results are in good agreement withBermdez-Soto and Toms-Barbern (2004) who reported thatchokeberry juice exhibited strong ABTS radical-scavenging activity(103 2.2 mg TEAC/ml).

In the present study, the trend for FRAP of black chokeberry,blueberry extracts and ascorbic acid are shown in Fig. 1C. For blackchokeberry and blueberry extract, the absorbance was increaseddue to the formation of the Fe2+TPTZ complex with increasingconcentration (Fig. 1C). The black chokeberry extract, at a concen-tration of 500 lg/ml, had FRAP value similar to 50 lg/ml of ascor-bic acid and higher than that of blueberry extract.

The reducing powers of the black chokeberry and blueberry ex-tracts were increased along with the treatment concentrations(Fig. 1D). The black chokeberry extract, at a concentration of500 lg/ml, had reducing power (about 0.71 at 750 nm) similar toa concentration of 50 lg/ml of ascorbic acid and higher than thatof blueberry extract.

3.3. Correlation among the antioxidant characteristics

Total phenolic, total avonoids and proanthocyanidin contentshave been reported to be responsible for the antioxidant activitiesof botanical extracts. DPPH, ABTS, FRAP, and reducing power activ-ities have been used to measure antioxidant activity and these re-sults should correlate with those of total phenolic, total avonoids

Abs

orba

nce

(595

nm

0.0

0.5

a

c

bb

dcdcddd

10 50 500Concentration (g/mL)

Fig. 1. Antioxidant activities of black chokeberry and blueberry extracts. (A) DPPH radreducing power. aeValues are means standard deviation (n = 3); means in the same comultiple range test.)

0.8

ChokeberryBlueberryAscorbic acid

d)and proanthocyanidin contents. A recent report (Zheng and Wang,2001) demonstrated that some bioactive compounds present inmedical plants possessed high total antioxidant activity, whichwas due to the presence of phenolics, carotenoids and avonoids.Therefore, we evaluated the correlation coefcient between

c

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ical-scavenging activity, (B) ABTS radical-scavenging activity, (C) FRAP assay, (D)lumn not sharing a common letter are signicantly different (p < 0.05) by Duncans

Table 2Correlations (ra) between different antioxidant capacity parameters (by DPPH, ABTS,FRAP, reducing power activity) and total phenolic contents, total avonoid contents orproanthocyanidin contents of extracts from black chokeberry.

TPCb TFCc PCd DPPHe ABTSf FRAPg RPAh

TPC 0.9999 0.9999 0.9953 0.9960 0.9997 0.9995TFC 0.9999 0.9964 0.9970 0.9994 0.9998PC 0.9964 0.9970 0.9994 0.9998DPPH 0.9999 0.9928 0.9979ABTS+ 0.9937 0.9984FRAP 0.9984RPA

a r, correlation coefcient.b TPC, total phenolic content.c TFC, total avonoid content.d PC, proanthocyanidin content.e DPPH radical-scavenging activity.f ABTS, ABTS radical-scavenging activity.g FRAP, ferric reducing ability of plasma.h RPA, reducing power activity.

Rice-Evans, Miller, & Paganga, 1996). It was reasonable to experi-

Table 3Correlations (ra) between different antioxidant capacity parameters (by DPPH, ABTS+,FRAP, reducing power activity) and total phenolic contents, total avonoid contents orproanthocyanidin contents of extracts from blueberry.

TPCb TFCc PCd DPPHe ABTSf FRAPg RPAh

TPC 0.9999 0.9999 0.9977 0.9454 0.9771 0.7895TFC 0.9999 0.9968 0.9492 0.9796 0.7965PC 0.9968 0.9492 0.9796 0.7965DPPH 0.9214 0.9605 0.7489ABTS+ 0.9930 0.9431FRAP 0.8981RPA

a

Table 4Phenolic composition of extracts from black chokeberry and blueberry.

Phenolic compounds Black chokeberry extract(mg/g)

Blueberry extract(mg/g)

Cyanidin 3-galactoside

93.3.0.1 6.5 0.0

Cyanidin 3-arabinoside

37.1 0.6 2.6 0.1

Neochlorogenic acid 35.7 0.1 N.D.Procyanidin B1 25.4 0.3 3.0 0.1Total 192 1.1 12.1 0.2

N.D.: Not detected.

S.J. Hwang et al. / Food Chemistry 146 (2014) 7177 75in vitro antioxidant activities and antioxidant compounds in blackchokeberry and blueberry, respectively (Tables 2 and 3). Our re-sults show that high correlation coefcients were found betweenthe in vitro antioxidant activities (DPPH, ABTS, FRAP, and reducingpower activity) and antioxidant compounds (total phenolics, totalavonoids and proanthocyanidin contents). These results suggestthat strong antioxidant and radical-scavenging activities of blackchokeberry and blueberry extracts can be attributable to high lev-els of the antioxidant compounds (total phenolics, total avonoidsand proanthocyanidin contents) present in these extracts, protect-ing against damage from reactive oxygen radicals.

3.4. HPLC analysis of phenolic compounds

We further investigated the contents of cyanidin 3-galactoside,cyanidin 3-arabinoside, neochlorogenic acid, procyanidin B1, usingHPLC (Fig. 2). The levels of cyanidin 3-galactoside, cyanidin 3-ara-binoside, neochlorogenic acid and procyanidin B1 in black choke-berry extract were 93.3 0.1 mg/g, 37.1 0.6 mg/g, 35.7 0.1 mg/g, 25.4 0.3 mg/g, respectively (Table 4). Among the four standardcontents of black chokeberry and blueberry extracts, the cyanidin3-galactoside was present in the largest amounts, followed bycyanidin 3-arabinoside, neochlorogenic acid and procyanidin B1.

r, correlation coefcient.b TPC, total phenolic content.c TFC, total avonoid content.d PC, proanthocyanidin content.e DPPH radical-scavenging activity.f ABTS, ABTS radical-scavenging activity.g FRAP, ferric reducing ability of plasma.h RPA, reducing power activity.Our results show that total content of the four standards in blackchokeberry extract was higher than that of blueberry extract.These results are in good agreement with Pool-Zobel et al.(1999), who reported that the contents of both cyanidin-3-galacto-side and cyandidin-3-arabinoside of black chokeberry were 166and 39 mg/g, respectively. Bijak et al. (2011) reported that cyanidin3-galactoside, cyanidin 3-arabinoside, neochlorogenic acid, procy-anidin B1 contents of black chokeberry were 64.0 3.53,

ment with their total amount in the selected vegetables or fruits.

Fig. 2. HPLC chromatograms of major phenolic compounds in black chokeberry and blueneochlorogenic acid; STD 4-procyanidin B1. 0 min 88%A (90/10, v/v) + 12%B (40/50/10,In addition, avonoids also possess antioxidant activity (Lotito &Frei, 2004), and are the most common type of polyphenolic com-pound in the plant (Bursal & Glin, 2011) and are capable of che-lating Fe3+, Fe2+ (Moridani, Pourahmad, Bui, Siraki, & OBrien,23.4 1.12, 24.1 0.94, and 10.3 0.06 mg/g, respectively. Fur-thermore, Oszmianski and Wojdylo (2005) indicated that cyanidin3-galactoside, cyanidin 3-arabinoside and neochlorogenic acid ofblack chokeberry were 1,282, 582, and 291 mg/100 g, respectively.

4. Discussion

Phenolics have a common structure composed of an aromatichydroxyl nucleus and approximately 8000 known in nature (Kar-aman, Ttem, Szgen Baskan, & Apak, 2010). Furthermore, the phe-nolic compounds are plant secondary metabolites extensivelyspread throughout the plant kingdom (Andreasen, Christensen,Meyer, & Hansen, 2000). The recent focus of interest on phenoliccompounds stems from their potential protective role, throughingestion of fruits and vegetables, against oxidative damage dis-eases (coronary heart disease, stroke, and cancers) (Othman, Is-mail, Abdul Ghani, & Adenan, 2007; Robbins, 2003). Theavonoids are the most common group of polyphenolic com-pounds in the human diet and are commonly found in fruits andvegetables. They can prevent coronary heart disease and have anti-oxidant properties (Pietta, 2000).

Plant polyphenols and avonoids are multifunctional in thatthey can act as reducing agents, hydrogen atom donors, and singletoxygen scavengers. They are also effective as antioxidants, capableof chelating transition metal ions, which may induce Fenton-typeoxidation reactions in their free states (Karaman et al., 2010;

Signicantly different at p < 0.05 by student t-test. The values are means standarddeviation from three replications.2003).Recently, many experimental studies have been carried out on

black chokeberries from the United States and Europe, owing to

berry extracts. STD 1-cyanidin 3-galactoside; STD 2-cyanidin 3-arabinoside; STD 3-v/v/v), 26 min: 70%A + 30%B, 29 min: 0%A + 100%B, 30 min: 88%A + 12%B.

Fridovich, I. (1997). Superoxide anion radical (O2), superoxide dismutases, andrelated matters. Journal of Biological Chemistry, 272(30), 1851518517.

Herms, D. A., & Mattson, W. J. (1992). The dilemma of plants to grow or defend.Quarterly Review of Biology, 283335.

hemtheir biological activities (Skupien and Oszmianski, 2007;Valcheva-Kuzmanova et al., 2005; Wu et al., 2004; Zheng andWang, 2003). In the present study, we attempted to assess antiox-idant activities of black chokeberry and blueberry extracts culti-vated in Korea. The present study shows that the total phenol,avonoids, and proanthocyanidin contents of chokeberry extractwere signicantly higher than those of blueberry extract. Khknenet al. (2001) reported that total phenolic content of black choke-berry was 4,210 100 mg/100 g. Benvenuti et al. (2004) alsoshowed that total phenolic content was 690 8.8 mg/100 g,whereas our results indicated that the total phenolic contents ofblack chokeberry and blueberry extract cultivated in Korea were110 5.6 and 27.4 7.4 mg GAE/g, respectively. These resultssuggest that different total phenol contents of black chokeberry ex-tract from diverse countries are inuenced by environmental fac-tors such as soil, climate and temperature. Environmental factorsmay account for the differences in phenolic content among previ-ous reports (Benvenuti et al., 2004; Herms & Mattson, 1992).

We also examined antioxidant activity of black chokeberry andblueberry extracts by various in vitro methods (DPPH, ABTS+,FRAP, and reducing power). The DPPH and ABTS+ are widely usedradical-scavenging methods because of their ease and convenience(Brand-Williams, Cuvelier, & Berset, 1995). They offer a rapidscreening for radical-scavenging activity of various natural sub-stances. DPPH is scavenged by bioactive compounds throughdonation of hydrogen (Matthus, 2002). ABTS+ assay involves anelectron transfer process. The FRAP and reducing power are alsoantioxidant assays. These methods reect the electron donationcapacity of antioxidant compounds and are associated with antiox-idant activity. Natural antioxidants can be reductants and inacti-vate oxidants. Both FRAP and reducing power are designed tomeasure overall antioxidant activity, or reducing potential (Glin,Bursal, Sehitoglu, Bilsel, & Gren, 2010). Our results show that theantioxidant activity of black chokeberry, blueberry extracts andascorbic acid possess effective radical-scavenging ability. At a con-centration of 500 lg/ml, black chokeberry extract was markedlyhigher in free radical-scavenging activity than was blueberry ex-tract, and indicated values similar to vitamin C at a concentrationof 50 lg/ml. These antioxidant activities may depend on theamount of total phenolics, avonoids and proanthocyanidin con-tents. Furthermore, the antioxidant activity of both berries couldbe due to the individual phenolic compounds. Therefore, we fur-ther investigated cyanidin-3-galactoside, cyanidin-3-arabinoside,neochlorogenic acid and procyanidin B1, using HPLC with a photo-diode array detector. Cyanidin-3-galactoside (93.3 mg/g) in blackchokeberry extract was found to be a major phenolic compound.The total amount of these phenolic compounds in black chokeberryextract was higher than that in blueberry extract (P < 0.05). Thephenolic compounds in black chokeberry are known to have strongantioxidant activities and may also be responsible for the pharma-ceutical effects of black chokeberry extract. However, since theblack chokeberry and blueberry were evaluated on a dry basisand black choke berry has higher antioxidant activity than hasblueberry, further studies on fresh black chokeberry and blueberrywith respect to antioxidant properties are needed.

In conclusion, the black chokeberry cultivated in Korea waseffective in scavenging radicals and containing various phenoliccompounds. These radical-scavenging activities are likely due tototal phenol, avonoids, proanthocyanidin and phenolic com-pounds present in the black chokeberry extracts. Therefore, our re-sults strongly suggest that black chokeberry extract is a goodsource of natural antioxidants and is a protective ingredient foroxidative stress-related chronic diseases. However, the extent of

76 S.J. Hwang et al. / Food Cin vivo antioxidant and protective effects of black chokeberry de-pend on their bioavailability. The apparent absorption of severalanthocyanidins after black chokeberry and blueberry consumptionHung, H. C., Joshipura, K. J., Jiang, R., Hu, F. B., Hunter, D., Smith-Warner, S. A., et al.(2004). Fruit and vegetable intake and risk of major chronic disease. Journal ofthe National Cancer Institute, 96(21), 15771584.

Jayaprakasha, G., Singh, R., & Sakariah, K. (2001). Antioxidant activity of grape seed(Vitis vinifera) extracts on peroxidation models in vitro. Food Chemistry, 73(3),285290.

Khknen, M. P., Hopia, A. I., & Heinonen, M. (2001). Berry phenolics and theirantioxidant activity. Journal of Agricultural and Food Chemistry, 49(8),40764082.

Kammerer, D. R., Schillmller, S., Maier, O., Schieber, A., & Carle, R. (2007). Colourstability of canned strawberries using black carrot and elderberry juiceconcentrates as natural colourants. European Food Research and Technology,224(6), 667679.Garzn, G., Riedl, K., & Schwartz, S. (2009). Determination of anthocyanins, totalphenolic content, and antioxidant activity in Andes berry (Rubus glaucusBenth). Journal of Food Science, 74(3), 227232.

Glin, _I., Bursal, E., Sehitoglu, M. H., Bilsel, M., & Gren, A. C. (2010). Polyphenolcontents and antioxidant activity of lyophilized aqueous extract of propolisfrom Erzurum Turkey. Food and Chemical Toxicology, 48(8), 22272238.

Gutnger, T. (1981). Polyphenols in olive oils. Journal of the American Oil ChemistsSociety, 58(11), 966968.has been reported in animal models and clinical tests (Kaume,Howard, & Devareddy, 2012; Zafra-Stone et al., 2007). Pedersenet al. (2000) reported that consumption of cranberry juice resultedin a signicant increase in the ability of plasma to reduce oxidativestress of healthy female volunteers, whereas consumption of blue-berry juice had no such effect. These studies strongly indicate thatthe antioxidant activity of black chokeberry is mainly due to theirrich antioxidant compounds (total phenolics, avonoid and pro-anthocyanidin contents) and bioavailability.

Acknowledgements

This research was supported by Basic Science Research Programthrough the National Research Foundation of Korea (NRF) fundedby the Ministry of Education, Science and Technology (NO2011-0022812) to J-D Kim.

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Radical-scavenging-linked antioxidant activities of extracts from black chokeberry and blueberry cultivated in Korea1 Introduction2 Materials and methods2.1 Chemicals2.2 Preparation of 70% ethanol extract of black chokeberry and blueberry2.3 Determination of total phenol contents2.4 Determination of total flavonoid contents2.5 Determination of proanthocyanidin contents2.6 The antioxidant activity of extracts by DPPH radical2.7 ABTS radical-scavenging activity2.8 FRAP assay2.9 Reducing power2.10 Analysis of phenolic compounds using HPLC-DAD2.11 Statistical analysis

3 Results3.1 Total phenolic, flavonoid and proanthocyanidin contents of extracts from black chokeberry and blueberry3.2 Antioxidant activities of black chokeberry and blueberry extracts, using in vitro model3.3 Correlation among the antioxidant characteristics3.4 HPLC analysis of phenolic compounds

4 DiscussionAcknowledgementsReferences