characterisation of anthocyanins and proanthocyanidins of...

Characterisation of anthocyanins andproanthocyanidins of adzuki bean extracts andtheir antioxidant activity

Kyu-Ho Han a Tomoko Kitano-Okada ab Jeong-Min Seo c Sun-Ju Kim cKeiko Sasaki d Ken-ichiro Shimada a Michihiro Fukushima aa Department of Food Science Obihiro University of Agriculture and Veterinary Medicine Obihiro Hokkaido080-8555 Japanb Cosmo Foods Co Ltd Tokyo 103-0001 Japanc Department of Bio-Environmental Chemistry Chungnam National University Daehak-ro 99 Yusung-guDaejon 305-764 Republic of Koread Hokkaido Tokachi Area Regional Food Processing Technology Center Obihiro Hokkaido 080-2462 Japan

A R T I C L E I N F O

Article history

Received 1 September 2014

Received in revised form 6 February

2015

Accepted 9 February 2015

Available online

A B S T R A C T

A novel extract powder purified from the boiled water of adzuki bean paste production was

developed to better utilize this resource The compounds contributing to pigmentation of

purified adzuki bean extract powders were investigated in order to compare their antioxi-

dant activity in vitro with (+)-catechin When a normal extract was exposed to the air under

heat treatment the colour of adzuki bean extract became more strongly reddish which was

associated with polyphenol polymerization Anthocyanins also contributed to the pigmen-

tation of the purified adzuki bean extracts Especially two anthocyanin compounds peonidin-

3-rutinoside and malvidin-3-O-glucoside were newly identified in the adzuki bean extract

The reducing powder iron chelating activity and free-radical scavenging capacity of the adzuki

bean extract were greater than that of (+)-catechin while its total antioxidant value was lower

Thus adzuki bean extract powders are promising alternatives to replace synthetic antioxi-

dants and potential dyes

copy 2015 Elsevier Ltd All rights reserved

Keywords

Vigna angularis

Anthocyanins

Proanthocyanidins

Pigments

Antioxidants

1 Introduction

Legumes are a staple food in many countries and an excel-lent source of natural bioactive compounds Among them redadzuki bean (Vigna angularis) is cultivated throughout East Asiawhere it is traditionally used for making bean pastes for usein confectioneries As high polyphenol content in kidney bean(Phaseolus vulgaris L) seed coats have been observed (Chen et al

2014) the adzuki bean seed coat is also high in polyphenols(Lin amp Lai 2006) these polyphenols have been dominantly iden-tified to contain catechin glycosides quercetin glycosidesmyricetin 3-rhamnoside anthocyanin and procyanidin dimers(Amarowicz Estrella Hernandez amp Troszynaska 2008 ArigaKoshiyama amp Fukushima 1988) The bioactive compounds inthe adzuki bean seed coat have received significant interestbecause of their health-promoting antioxidant properties (Linamp Lai 2006) Aroma extracts from adzuki bean seed coat are

Corresponding author Department of Food Science Obihiro University of Agriculture and Veterinary Medicine Obihiro Hokkaido 080-8555 Japan Tel +81 155 49 5557 fax +81 155 49 5577

E-mail address fukushimobihiroacjp (M Fukushima)Chemical compounds Peonidin-3-rutinoside (CID 44256842) Pelargonidin-3-O-glucoside (CID 443648) Malvidin-3-O-glucoside (CID 443652)

(+)-catechin (CID 9064)httpdxdoiorg101016jjff2015020181756-4646copy 2015 Elsevier Ltd All rights reserved

j o u rna l o f f un c t i ona l f o od s 1 4 ( 2 0 1 5 ) 6 9 2 ndash 7 0 1

Available online at wwwsciencedirectcom

ScienceDirect

journal homepage wwwelseviercom locate j ff

reported to inhibit the formation of malonaldehyde (LeeMitchell amp Shibamoto 2000) which is a maker for oxidativestress A decrease in vascular oxidative stress and inflamma-tion has also been reported in rats fed with the polyphenol-containing adzuki bean seed coat (Mukai amp Sato 2011)Furthermore Kitano-Okada et al (2012) have shown in vitro thatextracts from adzuki bean seed coat inhibit the activity of pan-creatic lipase These inhibitions would explain the results ofa clinical trial that suggests that the consumption of the adzukibean is linked to a reduced risk of lifestyle-related diseases inhumans (Maruyama et al 2008)

To manufacture the adzuki bean paste the beans are boiledin water which is then generally discarded after completingthe boiling process However the pigments contained in thiswater are receiving increased interest because of the need tomaximize the utilization of natural resources in order to de-crease the associated carbon footprint Moreover the foodindustry is experiencing an increasing demand for natural al-ternatives to replace synthetic food additives (eg antioxidants)(Amarowicz Naczk amp Shahidi 2000) In addition adzuki beanextract is a potential source of pigments (food colourants)because the water used to boil the beans becomes strongly redor purple Therefore a novel extract powder purified from thewater of adzuki bean paste production was developed and in-troduced in Japan to better utilize the resource (Adzuki-no-moto Cosmo Foods Co Ltd Tokyo Japan) The extract powderis highly purified and rich in natural polyphenols (Kitano-Okadaet al 2012) Interestingly hot air-exposed purified adzuki beanextract rather maintains consistent colour strength than thenormal purified adzuki bean extract because polyphenols inthe adzuki bean extract were polymerized by oxidation andboth the extracts are stable to light heat and pH changesHowever information on responsible compounds for pigmen-tation as well as the potent antioxidative activity of the differentpurified adzuki bean extract powders is very limited

Therefore the objective of this investigation was to comparethe different purified adzuki bean extract powders from in-dustrial residue in order to reveal their bioactive compoundsfor pigmentation and in vitro functional attributes such as re-ducing power and radical scavenging activity assay For thisthe antioxidant activity was compared with (+)-catechin as stan-dard polyphenol

2 Materials and methods

21 Reagents chemicals and standards

Calibrations were performed by using standard compounds (cat-echin cyanidin-3-O-glucoside pelargonidin-3-O-glucosidepeonidin-3-O-glucoside and malvidin-3-O-glucoside) fromSigma-Aldrich (St Louis MO USA) Solvents (ethanol butanolacetonitrile and formic acid) and concentrated hydrochloric acid(HCl) were purchased from Wako Chemical Co Ltd (TokyoJapan) Hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) malondialdehyde 22-azobis(2-methylpropionamide)dihydrochloride (AAPH) 22-diphenyl-1-picrylhydrazyl (DPPH)thiobarbituric acid and FolinndashCiocalteu phenol reagent wereobtained from Sigma-Aldrich Ferric ammonium sulphate andferrozine were purchased from Kishida Chemical Co Ltd

(Osaka Japan) All other chemicals were purchased from KantoChemical Co Ltd (Tokyo Japan)

22 Adzuki bean extract powder (AEP)

The adzuki bean extract powder (AEP also known as Adzuki-no-moto) made from the simmering water obtained during thesweetened adzuki bean paste production was kindly sup-plied by Cosmo Foods The extract powder was made by thefollowing procedure first the bean was boiled and cooled Thewater was collected from supernatant adjusted at pH 40treated with 0005 pectinase HL (Yakult Pharmaceutical In-dustry Co Ltd Tokyo Japan) and passed through a 50 meshsieve to separate undigested materials It was further appliedto an ultrafiltration device to remove polymeric componentsand adjusted at pH 85Then two different types of adzuki beanextract powder were made depending on the process with andwithout oxidative polymerization of polyphenols One was ster-ilized (115 degC for 90 min) and spray-dried (AEP-1)The other wasexposed to the air under heat treatment (90 degC for 5 h) andspray-dried (AEP-2)

23 Total polyphenols and proanthocyanidins analyses

The total concentration of polyphenols in the AEP samples wasdetermined according to the FolinndashCiocalteursquos methodusing (+)-catechin as a standard (Singleton Orthofer ampLamuela-Raventos 1999) The absorbance was read at 750 nmusing a spectrophotometer (1600-UV Shimadzu Kyoto Japan)The results were expressed in mg of (+)-catechin equivalentsto per gram of bean extract powder All tests were performedin triplicate

For assay of total proanthocyanidins concentration in theAEP samples the acid butanol method was employed (PorterHrstich amp Chan 1986) In brief 60 mL of the butanolndashHClreagent (955 vv) were added to 10 mL of suitably dilutedextract (1 mgmL) in a screw cap test tubeThen 02 mL of ferricreagent (2 ferric ammonium sulphate in 2M HCl) was addedto the solution The container was capped mixed and boiledfor 30 min in a water bath Similarly a standard curve was pre-pared using procyanidin B-2 (Sigma-Aldrich) ranging from 0to 025 mgmL After cooling the absorbance was read at 550 nmusing a spectrophotometer The results were expressed in mgof procyanidin B-2 equivalents to per gram of bean extractpowder

For quantification of oligomeric proanthocyanidins the AEPsample was dissolved in water (1 mgmL) and filtered using a045 microm polytetrafluoroethylene (PTFE) hydrophilic syringe filter(Advantec Dismic-13HP Toyo Roshi Co Ltd Tokyo Japan) Thefiltrate was loaded into a Shimadzu Prominence HPLC systemequipped with an LC-20AD pump (Shimadzu)The analytes wereseparated using a TSKgel ODS-80Ts column (46 mm times 250 mmTosoh Tokyo Japan) with photodiode array detector (SPD-M20A Shimadzu) Absorbance was monitored at 210ndash600 nmThe column oven temperature was set at 40 degC The injectionvolume was 10 microL and the flow rate was 10 mLminThe mobilephases were made of 005 trifluoroacetic acid (TFA) in water(vv mobile phase A) and 005 TFA in 90 acetonitrile (vvmobile phase B) Elution was used as a linear gradient from 5to 35 B in 13 min from 35 to 70 B in 20 min and then 70

693j o u rna l o f f un c t i ona l f o od s 1 4 ( 2 0 1 5 ) 6 9 2 ndash 7 0 1

B in 25 min Proanthocyanidin oligomers were quantified bycomparing the HPLC peak area with that of flavangenol (ToyoShinyaku Co Ltd Saga Japan)

The molecular weight (Mw) of AEP was determined by gelsize exclusion chromatography The filtrate (10 microL) was loadedinto a Shimadzu gel permeation chromatography (GPC) systemequipped with an LC-20AD pump and a refractive index de-tector (RID-10A Shimadzu) The analytical double columns(76 mm times 250 mm Shodex OHpak SB-806MHQ and OHpak SB-8025HQ Tokyo Japan) equipped with a security guard column(Shodex OHpak SB-G 60 mm times 50 mm) were eluted with 20 mMlithium bromide in dimethylformamide at a flow rate of 06 mLmin under the maximum operating pressure of 42 MPa Thecolumn oven temperature was set at 40 degC The standard curveof Mw was obtained using the following materials catechin(Mw 290) procyanidin B-2 (Mw 578) and polystyrenes (Mws 780ndash20000 Shodex SL 105 Tokyo Japan) The average number-average molecular weight (Mn) and number-average degree ofpolymerization (DP) of the oligomers from the AEP sample wasdetermined using GPC Software (LCsolution GPC Ver 121Shimadzu)

24 Anthocyanin monomer analysis

The anthocyanins present in each sample were extracted andanalyzed according to Chun et al (2013) In brief 5 formic acidin water (vv 2 mL) was added to sample tubes containing AEP(100 mg) The solution in each tube was vigorously mixed for5 min sonicated for 20 min and centrifuged at 9200 times g for15 min at 4 degC The supernatant was filtered using a 045 micromPTFE hydrophilic syringe filter (Advantec Dismic-13HP)The an-thocyanin containing filtrate was analyzed with an AgilentTechnologies 1200 series HPLC (Palo Alto CA USA)The analyteswere separated using a Synergitrade 4 microm Polar-RP 80A column(46 mm times 250 mm Phenomenex Torrance CA USA) equippedwith a Security Guard Cartridges Kit AQ C18 column(30 mm times 40 mm Phenomenex) The UV detector was set at310 nm and the column oven temperature was set at 30 degCTheinjection volume was 10 microL and the flow rate was 10 mLmin The mobile phases were made of waterformic acid (955vv mobile phase A) and acetonitrileformic acid (955 vvmobile phase B) A linear gradient programme was used asfollows AB (955 vv) to AB (7030 vv) after 20 min and thenAB (955) for an additional 10 min (modified from Wu amp Prior2005)The anthocyanin components present in the AEP sampleswere identified by comparing the retention time (RT) of the au-thentic standards of the compounds (cyanidin-3-O-glucosidepelargonidin-3-O-glucoside peonidin-3-O-glucoside andmalvidin-3-O-glucoside) Some compounds were also identi-fied by liquid chromatographyndashtandem mass spectrometry (LCndashMSMS) with a 4000 Qtrap LCndashMSMS system (AppliedBiosystems Foster City CA USA) using electrospray ioniza-tion (ESI) operating in the positive ion mode [M+H] + The ESI-MS was performed at a capillary temperature of 550 degC an ionspray voltage of 55 kV a curtain gas pressure of 20 psi and aspectra range (mz) 100ndash1300 (scan time 48 s) Nitrogen wasused as a nebulizer gas with a pressure of 50 psi The identi-fied anthocyanin components present in the AEP samples werequantified by comparing the HPLC peak area with that of theauthentic standards Unidentified anthocyanin components

were quantified by comparing the HPLC peak area with thatof the pelargonidin-3-O-glucoside because such anthocyanidinis known to be a main component in adzuki bean (Wu amp Prior2005)

25 Total antioxidant activity

For stock solutions an equivalent amount of polyphenols (6 mgof (+)-catechin) to that contained in the AEP samples was dis-solved in distilled water (10 mL) The total antioxidant activity(TAA) was measured using a Randox kit (Randox Laborato-ries Ltd Antrim UK) according to the manufacturerrsquosinstructions The results are expressed as millimole Trolox pergram of polyphenols

26 Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity of the samples wasdetermined according to 2-deoxyribose oxidation method de-scribed by Chung Osawa and Kawashiki (1997) All solutionsexcept FeSO4ndashethylenediaminetetraacetic acid (EDTA) were dis-solved in 01 M sodium phosphate buffer (pH 74) In brief thestock solution (100 microL containing 600 microgmL of polyphenols)was added to a freshly prepared mixture of 10 mM2-deoxyribose 10 mM FeSO4 and 10 mM EDTA (111 vvv)(600 microL) A sodium phosphate buffer (1 mL) and 10 mM H2O2

(200 microL) were added to the previous solution and put into awater bath at 37 degC Exactly 4 h after incubation 28 trichlo-roacetic acid (133 mL vv) and 10 thiobarbituric acid (670 microLwv) were added to the solution The mixtures were then in-cubated at 80 degC for 10 min After cooling the absorbance (A)of the solution was measured at 532 nm using a spectropho-tometer The pentose 2-deoxyribose is oxidized by hydroxylradical that is generated by Fe2+ndashEDTA to yield TBAmalondialdehyde adduct (Chung et al 1997) The hydroxylradical scavenging activity of the AEP samples was calcu-lated using the following formula

Inhibition () = [1 minus (Asample minus Asample blank)(Acontrol minusAcontrol blank)] times 100 where Asample is the absorption of the samplesolution and Acontrol is the absorption of the control solutionBoth Ablanks are for the blank solutions not containing H2O2

27 Peroxyl radical scavenging activity

The peroxyl radical scavenging activity of the samples was mea-sured by using the method described by Loacutepez-Alarcoacuten and Lissi(2005) In brief the stock solution (100 microL containing 600 microgmL of polyphenols) was mixed with 06 M AAPH (15 microL) and60 mM pyrogallol red in the PBS buffer (pH 70) containing 30ethanol (1 mL)The resulting solution was incubated in a waterbath at 37 degC for 2 h and the absorbance was then measuredat 540 nm using a spectrophotometerThe peroxyl radical scav-enging activity of the AEP samples was then calculated usingthe following formula

Inhibition () = [1 minus (Asample minus Asample blank)(Acontrol minusAcontrol blank)] times 100 where Asample is the absorption of the samplesolution and Acontrol is the absorption of the control solution(not containing the sample) Both Ablanks are for the blank so-lutions not containing AAPH

694 j o u rna l o f f un c t i ona l f o od s 1 4 ( 2 0 1 5 ) 6 9 2 ndash 7 0 1

28 DPPH radical scavenging activity

A diluted stock solution (300 microL containing 0ndash108 microgmL of poly-phenols) was mixed with 04 mM DPPH (300 microL) and 02 M MESbuffer (pH 60) containing 20 ethanol (600 microL) The standardsolution used was 02 mM Trolox The absorbance of the so-lution was measured at 520 nm and the radical scavengingactivity was calculated using the following formula(Brand-Williams Cuvelier amp Berset 1995)

DPPH radical scavenging activity () = (1 minus AsampleAcontrol) times 100where Asample is the absorption of the sample solution and Acontrol

is the absorption of the control solution (not containing thesample)

29 Reducing power

The reducing capacity of the samples was measured using themethod described by Oyaizu (1986) Briefly a diluted stock so-lution (25 mL containing 0ndash150 microgmL of total polyphenols) wasadded to a freshly prepared solution of 02 M PBS buffer (pH 66)and 1 potassium ferricyanide (11 wv) The mixture was in-cubated at 50 degC for 20 min and then 10 trichloroacetic acid(25 mL vv) was added The resulting mixture was centri-fuged at 650 times g for 10 min The upper layer of the solution(25 mL) was separated and mixed with distilled water (25 mL)and 01 FeCl3 (05 mL wv)The absorbance was read at 520 nmusing a spectrophotometer

210 Chelating ability of ferrous ions

The chelating ability of ferrous ions in the samples was de-termined by using the method of Dinis Madeira and Almeida(1994) A diluted stock solution (740 microL containing 0ndash600 microgmL of polyphenols) was mixed with 2 mM FeCl2 (20 microL) Thereaction was initiated by adding 5 mM ferrozine (40 microL) and themixture was shaken vigorously After exactly 10 min the ab-sorbance of the solution was measured at 562 nm using aspectrophotometer The blank sample was prepared withoutferrozine for background subtraction

Chelating activity () = [1 minus (Asample minus Asample blank)Acontrol] times 100where Asample is the absorption of the sample solution and Acontrol

is the absorption of the control solution (not containing thesample) Asample blank is for background subtraction

211 Statistical analysis

The results reported here are the means of at least three mea-surements with standard error (SE) The data were analyzedusing the general linear model procedure and the signifi-cance of differences was determined by the Tukeyrsquos multiple-range test (SPSS 17 version SPSS Institute Armonk NY USA)A P-value of less than 001 was considered statisticallysignificant

3 Results and discussion

31 Total polyphenols and proanthocyanidins

The polyphenols in Vigna species of legume have been mainlyidentified as catechin quercetin myricetin anthocyanin andprocyanidin dimers (Amarowicz et al 2008) However the poly-phenols content in organic solvent extracts of adzuki beanvaries Sreerama Takahashi and Yamaki (2012) reported thatthe total polyphenols content in the methanol extract ob-tained from whole adzuki bean ranges from 35 to 73 mg of pergram dry weight equivalent of gallic acid FurthermoreAmarowicz et al (2008) reported that the total polyphenols con-centration in the acetone extract of adzuki beans wasapproximately 90 (ww) In this study the total polyphe-nols content (equivalent to (+)-catechin) in samples AEP-1 andAEP-2 from the boiled water of adzuki bean paste productionwere 281 and 108 mgg dry weight respectively (Table 1) whichsuggests that the extract powders were highly purified and richin polyphenols

Proanthocyanidins which are oligomers or polymers ofpolyhydroxyflavan-3-ol units are largely found in plant kingdomand food materials as pigment (Hosseinian amp Mazza 2009 Lee

Table 1 ndash Total polyphenols anthocyanins and proanthocyanidins contents in the purified extract of adzuki bean seedcoat (n = 3)

Peaka Trivial names (anthocyanin) RT (min) AEP-1 AEP-2

mgg dry mgg dry

1 Unknown 67 261b 2762 Unknown 83 620 1933 Unknown 100 455 ndash4 Unknown 103 564 ndash5 Peonidin-3-rutinoside 109 438 1746 Pelargonidin-3-O-glucoside 137 790 1657 Malvidin-3-O-glucoside 150 589 4078 Rutin or peonidin-3-(p-coumaroyl)glucoside 159 207 ndashAnthocyanins (mgg dry) 97 28Proanthocyanidins (mg proanthocyanidin B2 equivalent g dry) 157 ndashOPC (mg flavangenol equivalentg dry) ndash 69Total phenolics (mg catechin equivalentsg dry) 281 109

a The elution order of anthocyanin in HPLC chromatogram (see Fig 1)b Expressed as mg of anthocyanin per g of dry weightAEP-1 normal adzuki bean extracts powder AEP-2 polymerized adzuki bean extracts powder and OPC oligomeric proanthocyanidin complexes


2013) It was reported that some proanthocyanidins as dimericprocyanidins (B-type) are responsible for pigments of adzukibean (Amarowicz et al 2008 Ariga et al 1988 Sreerama et al2012) In this study the total proanthocyanidins content (equiva-lent to procyanidin B-2) in the AEP-1 measured by the acidbutanol assay was 157 mgg dry weight (Table 1) However wecould not determine the total proanthocyanidins in the AEP-2by this assay because the colour of reagents containing AEP-2sample after heating was lower than the colour before heatingAlthough this assay is largely used for determining con-densed tannins in foods (Porter et al 1986) it is limited toquantify some interflavan bonded or doubly linked procyanidinoligomers (A-type) owing to them not being hydrolyzed uponheating (Hemingway 1989 Watterson amp Butler 1983) Thus itwas hypothesized that the procyanidin dimers in the puri-fied adzuki bean extract was more condensed and stable againstacid and heat and simultaneously became strongly reddishwhen an oxidative polymerization process was employed tomake the AEP-2 sample As expected the polyphenols in theAEP-2 sample were polymerized with average DP 79 (exclud-ing monomeric polyphenols) and the oligomericproanthocyanidin complexes (equivalent to flavagenol) was69 mgg dry weight (Fig 1) Therefore the different polyphe-nols contents (or ratio of monomer to oligomer) in both theAEP samples might be explained by the polyphenols com-pounds being polymerized when a normal purified adzuki beanextract was exposed to the air under heat treatment

32 Anthocyanins

Anthocyanins are also responsible for the pigment of severalplants However only trace amounts of anthocyanins have beenidentified as representative pigments in the adzuki bean seedcoat The anthocyanin in the adzuki bean was first identified

by Yoshida et al (1996) using HPLC as cyanidin-3-O-(β-D-glucopyranosyl)-5-O-(β-glucopyranosyl) with a concentrationof less than 01 mgg of dried powder Wu and Prior (2005) iden-tified three anthocyanins such as cyanidin-3-glucosidepelargonidin-3-glucoside and pelargonidin-3-sambubioside atlow concentrations (67 mg per 100 g of fresh weight) (Wu et al2006) Sreerama et al (2012) reported that anthocyanin con-tents (mg cyanidin-3-glucoside equivalents per g of defattedflour) in the seed coat of adzuki bean are in the range 314ndash794 mgg dry weight In contrast our findings revealed thattotal anthocyanins in the purified adzuki bean extracts wereconsiderably higher than those found in others The total an-thocyanins content in samples AEP-1 and AEP-2 were 973 and278 mgg dry weight respectively (Table 1) HPLC chromato-gram showed that the adzuki bean extract powder containseight (Fig 2A) and five peaks (Fig 2B) for the AEP-1 and AEP-2samples respectively The anthocyanin peaks were eluted at67 83 100 103 109 137 150 and 159 min respectively(Table 1) The authentic standard of cyanidin-3-glucoside hada RT of 132 min however cyanidin-3-glucoside in the adzukibean extract powder could not be identified when comparedwith an authentic standard (Fig 2) Peaks one to four are thoseof unknown compounds while peak five was identified aspeonidin-3-rutinoside ([M+H]+ mz 577179) by mass spectros-copy (Fig 3A) Peaks six and seven were identified aspelargonidin-3-O-glucoside and malvidin-3-O-glucoside re-spectively by comparing with the RT of authentic standardsIdentification of peak eight not found in the AEP-2 sample wascomplicated by its similar molecular weight between quercetin-3-rutinside (rutin) and peonidin 3-(p-coumaroyl) glucosideshowing to have MS data ([M+H]+ mz 609301) (Fig 3B) To ourknowledge no published data are available for malvidin-3-O-glucoside and peonidin-3-rutinoside as pigments in the adzukibean seed coat Furthermore these chromatograms clearly

Standard MW RT (min)

Polystyrene 1 20000 23027




Procyanidin B-2 578 27594

Catechin 290 28389Peaks 1-3 (OPC Mn 2290)

12 3

4

Peak 4 (Monomer Mn 274)

00 50 100 150 200 250 300 350 400 450 500 550 min

0

5000

10000

15000

20000

25000

uV

Fig 1 ndash GPC profiles of monomer (peak 4) and oligomers (peaks 1ndash3) from the purified extract of adzuki bean powder-2 Peak1 RT 23579 peak 2 RT 25208 peak 3 RT 26858 and peak 4 RT 27742 RT retention time (min) Mn the average number-average molecular weight OPC oligomeric proanthocyanidin complexes


demonstrate that the class of anthocyanin compounds con-tained in the AEP samples differs depending on the processwith and without oxidative polymerization which was carriedout by exposing extracts to the air under heat treatment

33 Total antioxidant activity (TAA)

Due to the difficulties in measuring the antioxidant capacityof individual compounds in a complex mixture several esti-mations including the Trolox equivalent (TE) value have beenbroadly used to represent the antioxidant capacity of foodsbeverages and supplements The TAA which is expressed asTE value of leguminous pigment extracts from whole beanpowder ranged from 030 to 176 mmol Trolox equivalent to per

gram bean extract (Amarowicz Troszynska Barylko-Pikielnaamp Shahidi 2005) Especially a higher TAA has been reportedfor the extracts of polyphenols from adzuki bean which TEvalue is higher than those obtained from others (Amarowiczet al 2005) The TE values in this study were extrapolated tothose determined by Amarowicz et al (2005) with AEP-1 andAEP-2 samples having approximately 0908 and 0359 mmolTroloxg of powder respectively The different TAA betweenAEP-1 and AEP-2 samples was owing to the presence of dif-ferent concentrations of polyphenols in the AEP samplesAlthough the total amount of total polyphenols was higher inthe AEP-1 sample than the AEP-2 sample (Table 1) a higher TAAwas measured for AEP-2 than AEP-1 when the TE value wasbased on the amounts of polyphenols in the AEP samples 330

Fig 2 ndash HPLC profiles of anthocyanins isolated from the purified extract of adzuki bean seed coat Authentic standardcompounds (A) cyanidin-3-O-glucoside (S1) pelargonidin-3-O-glucoside (S2) peonidin-3-O-glucoside (S3) and malvidin-3-O-glucoside (S4) AEP-1 (B) normal adzuki bean extracts powder AEP-2 (C) polymerized adzuki bean extracts powder


versus 323 mmol Troloxg of polyphenol respectively (Table 2)This difference strongly implies that the polyphenols presentin the extract powder were not degraded by the oxidationprocess used for AEP-2 but rather condensed in their activeforms These results are in agreement with a recent study (Liet al 2015) that found a higher TAA for polymerized polyphe-nols (average DPs of approximately 3ndash6) than dimer polyphenols

34 Radical scavenging activity

Peroxyl radical inhibition was determined using the AAPHmethod (Loacutepez-Alarcoacuten amp Lissi 2005) AAPH is a water-solubleazo compound that is used extensively as a free-radical gen-erator in the characterization of antioxidants As shown in theTable 2 peroxyl radical inhibition was significantly (P lt 001)

higher in the order AEP-1 gt AEP-2 gt (+)-catechin On the otherhand samples with the same equivalent concentration of poly-phenols had a relatively lower hydroxyl-radical scavengingactivity than peroxyl radical inhibition no significant differ-ence was observed between the trials (Table 2) In addition theradial-scavenging activity of the extracts was examined usingthe free radical DPPH As shown in Fig 4 the DPPH radical scav-enging activity increased directly with the concentration ofpolyphenols (from 0 to 108 microg per assay)The DPPH radical scav-enging activity at the highest concentration (108 microg) wassignificantly higher (P lt 001) in the order AEP-2 gt AEP-1 gt (+)-catechin The DPPH radical assay revealed that both the AEPsamples showed higher antioxidant capacities than (+)-catechin with AEP-2 showing the highest value This findingis in accordance with the findings of previous studies that

Fig 3 ndash LCndashMS spectrums of peak five (A) and peak eight (B) in adzuki bean extract power Peak (A) presents peonidin-3-rutinoside and peak (B) presents rutin or peonidin-3-(p-coumaroyl) glucoside


reported that the polymerized fraction of the adzuki beanextract exhibited a greater radical scavenging activity (DPPHradical) than the low-molecular-weight fraction of polyphe-nols (Amarowicz et al 2008 Sulaiman Ibrahim Kassim ampSheh-Hong 2011) A possible explanation for these results re-cently reported by several researchers (Kurisawa Chung Uyamaamp Kobayashi 2003 Sulaiman et al 2011) is that condensedphenols such as proanthocyanin are better free radical inhibi-tors (primary antioxidants) than monomeric phenols such as(+)-catechin

35 Reducing capacity

The reducing capacity of the samples is based on the reduc-tion of the Fe3+ ion where antioxidants are the reducing agentsthus the reducing capacity is associated with antioxidant ac-tivity (Benzie amp Strain 1999) Compounds that are capable ofdonating a single electron or hydrogen atom for reduction mightreduce oxidized intermediates In this assay the presence ofan antioxidant in the extracts reduced the Fe3+ferricyanidecomplex to Fe2+ ion (Fig 5A) The reducing capacity of a varietyof plant extracts including those of fruits tea and legumes

has been extensively studied (Li et al 2015 Lin amp Lai 2006Xiao et al 2014) In a study by Lin and Lai (2006) the reduc-ing power of various legumes was shown to be dependent onthe content of phenolic compounds However in our study thepolyphenols concentration of the bean extract was adjustedso that it was equivalent to (+)-catechin therefore no corre-lation was found These results (Fig 5A) show that theabsorbance values were remarkably higher for AEP-2 than forAEP-1 samples and (+)-catechin strongly suggesting that oligo-meric proanthocyanidins in the AEP-2 sample were moreefficient reductants than low-molecular-weight polyphenols inthe AEP-1 sample Polymerized polyphenols (eg condensedtannin) exhibit a stronger reducing capacity than that of low-molecular-weight andor monomeric phenols (Pulido Bravoamp Saura-Calixto 2000) and the results are in agreement withthe findings of our study

Interestingly a comparison of the reducing capacity andperoxyl radical inhibitory capacity reveals that these proper-ties are not correlated to the state of polymerization Onepossible explanation for this observation can be drawn fromthe study of Simic and Jovanovic (1994) Their study demon-strated that lower redox potentials correlated to higherantioxidant efficiency against free radicals such as peroxyl orhydroxyl radicals (Simic amp Jovanovic 1994) This finding mayexplain the opposite observation between the reducing ca-pacity and the peroxyl radical inhibitory capacity in AEP-2samples compared to that of AEP-1 samples These resultsprovide important insights into the properties of polyphe-nols depending on their polymerization state

36 Ferrous ion chelating activity

Fig 5B shows the Fe2+ ion chelating activity () of AEPs and(+)-catechin at six concentration levels (0ndash450 microgassay) wherebythe activity increased depending on the concentration for allsamples investigated At all concentrations tested the ferrousion chelating activity of the AEP samples was far superior tothat of (+)-catechin At a concentration of 100 microgassay boththe AEP samples showed approximately 96 chelating activ-ity however (+)-catechin showed only 16 chelating activityAndjelkovic et al (2006) showed that the ability of phenoliccompounds to chelate ferrous ions is far lower than that ofEDTA In contrast Sreerama et al (2012) showed that the abilityof the adzuki bean extract to chelate ferrous ions was com-parable to that of EDTA However some researchers have arguedthat metal chelation plays a smaller role in the overall

Table 2 ndash Total antioxidant activity (TAA) and hydroxyl and peroxyl radical scavenging activities of adzuki bean extractspowder

(+)-Catechin AEP-1 AEP-2

TAA (mmol Troloxg polyphenol1) 334 plusmn 001a 323 plusmn 001b 330 plusmn 001a

Hydroxyl radical inhibition2 () 287 plusmn 071a 840 plusmn 177a 492 plusmn 049a

Peroxyl radical inhibition2 () 145 plusmn 03c 619 plusmn 05a 488 plusmn 04b

1 Polyphenol concentration was equivalent to (+)-catechin2 Amount of extract was prepared at an equivalent catechin concentration as 06 mgmLAEP-1 normal adzuki bean extracts powder and AEP-2 polymerized adzuki bean extracts powderValues are mean plusmn standard error of 3 replicates Means within the same rows bearing different superscripts are significantly different (P lt 001)by analysis using Tukey test

Contents (microgassay)

DP

PH

rad

ical

sca

veng

ing

activ

ity (

)

a

b

c

0

10

20

30

40

50

60

0 30 60 90 120 150 180

AEP-1 AEP-2 Catechin

Fig 4 ndash Scavenging effect of adzuki bean extract powder onthe DPPH radical as measured by changes in absorbance at520 nm The experiments were performed in triplicateMeans with different letters (andashc) at each concentrationlevel are significantly different (P lt 001) by analysis usingTukey test P lt 001 versus AEP-1 AEP-1 normal adzukibean extracts powder AEP-2 polymerized adzuki beanextracts powder


antioxidant activities of some polyphenols such as (+)-catechin(Rice-Evans Miller amp Paganga 1996) Furthermore some pro-teins (Saiga Soichi amp Nishimura 2003) and oligosaccharides(Wang et al 2007) can chelate metal ions Thus we could notconclude that polyphenols compounds such as anthocyaninsand proanthocyanidins in the AEP samples were solely re-sponsible for metal chelation because AEP samples are acomplex mixture of food ingredients (Kitano-Okada et al 2012)Nevertheless the results of this study indicate that AEP itselfmay serve as a potential source of chelating agents

4 Conclusions

This study determined that proanthocyanidins as well as an-thocyanins were responsible for the pigment properties of theadzuki bean extract powders The new anthocyanins in theadzuki bean extract powder were identified as malvidin-3-O-glucoside and peonidin-3-rutinoside One of the more significantfindings of this study was that the antioxidant capacity of boththe AEP samples was superior to that of (+)-catechin with regardto ferrous ion chelating activity although it is not conclusivewhether only polyphenols were responsible for metal chela-tionThe reducing capacity of the AEP-2 sample was higher thanthat of (+)-catechin or AEP-1 sample Furthermore our find-ings suggest that the antioxidant activities of polyphenolscontaining oligomeric proanthocyanidins in the AEP-2 weregreater than that of AEP-1 Thus this study shows that theadzuki bean extract powder especially AEP-2 which is gen-erally discarded is a valuable resource for naturally derivedfood additives with significant antioxidant activityThus adzukibean extract powders are promising alternatives to syntheticantioxidants and dyes which are currently used in the foodindustry

Acknowledgments

This work was supported by a grant from the programme Co-operation of Innovative Technology and Advanced Research inthe Evolution Area (CITY AREA Development Stage) of the Min-istry of Education Culture Sports Science and Technology ofJapan

Appendix Supplementary material

Supplementary data to this article can be found online atdoi101016jjff201502018

R E F E R E N C E S

Amarowicz R Estrella I Hernandez T amp Troszynaska A(2008) Antioxidant activity of extract of adzuki bean and itsfractions Journal of Food Lipids 15(1) 119ndash136

Amarowicz R Naczk M amp Shahidi F (2000) Antioxidantactivity of crude tannins of canolar and rapeseed hullsJournal of the American Oil Chemistsrsquo Society 77(9) 957ndash961

Amarowicz R Troszynska A Barylko-Pikielna N amp Shahidi F(2005) Polyphenolics extracts from legume seedsCorrelations between total antioxidant activity totalphenolics content tannins content and astringency Journal ofFood Lipids 11(4) 278ndash286

Andjelkovic M Camp J V Meulenaer B D Depaemelaere GSocaciu C Verloo M amp Verhe R (2006) Iron-chelationproperties of phenolic acids bearing catechol and galloylgroups Food Chemistry 98(1) 23ndash31

Ariga T Koshiyama I amp Fukushima D (1988) Antioxidativeproperties of proanthocyanindins B-1 and B-3 from azuki

00

05

10

15

20

25

30

0 50 100 150



Red

ucin

g ca

paci

ty (

Abs

orba

nce

at 7

00 n

m)

(A)

0

20

40

60

80

100

0 50 100 150 200 250 300 350 400 450



Fer

rous

ion

chel

atin

g ac

tivity

(

)

(B)

Fig 5 ndash Reducing capacity (A) and ferrous ion chelating activity (B) The experiments were performed in triplicate P lt 001versus AEP-1 AEP-1 normal adzuki bean extracts powder AEP-2 polymerized adzuki bean extracts powder


beans in aqueous systems Agricultural Biological Chemistry52(11) 2717ndash2722

Benzie I F F amp Strain J J (1999) Ferric reducing (antioxidant)power as a measure of antioxidant capacity The FRAP assayIn L Packer (Ed) Oxidants and antioxidants 299 of methods inenzymology (pp 15ndash27) Orlando Academic Press

Brand-Williams W Cuvelier M E amp Berset C (1995) Use of afree radical method to evaluate antioxidant activity LWT ndashFood Science and Technology 28(1) 25ndash30

Chen P X Bozzo G G Freixas-Coutin J A Marcone M FPauls P K Tang Y Zhang B Liu R amp Tsao R (2014) Freeand conjugated phenolic compounds and their antioxidantactivities in regular and non-darkening cranberry bean(Phaseolus vulgaris L) seed coats Journal of Functional Foodsdoi101016jjff201410032

Chun J H Jang I H Arasu M V Al-Dhabi N ADuraipandiyan V Lee D H Lee S amp Kim S J (2013)Isolation and identification of alkaloids and anthocyaninsfrom flower and bulb of Lycoris radiata using HPLC and LC-ESI-MS Journal of Agricultural Chemistry and Environment 2(1)22ndash26

Chung S K Osawa T amp Kawashiki S (1997) Hydroxyl radical-scavenging effects of spices and scavengers from brownmustard (Brassica nigra) Bioscience Biotechnology andBiochemistry 61(1) 118ndash123

Dinis T C P Madeira V M C amp Almeida L M (1994) Action ofphenolic derivatives (acetaminophen salicylate and5-aminosalicylate) as inhibitors of membrane lipidperoxidation and as peroxyl radical scavengers Archives ofBiochemistry and Biophysics 315(1) 161ndash169

Hemingway R W (1989) Structural variations inproanthocyanidins and their derivatives In R W Hemingwayamp J J Karchesy (Eds) Chemistry and significance of condensedtannins (pp 83ndash107) New York Plenum Press

Hosseinian F S amp Mazza G (2009) Triticale bran and strawPotential new sources of phenolic acids proanthocyanidinsand lignans Journal of Functional Foods 1(1) 57ndash64

Kitano-Okada T Ito A Koide A Nakamura Y Han K HShimada K Sasaki K amp Michihiro S F (2012) Anti-obesityrole of adzuki bean extract containing polyphenols In vivoand in vitro effects Journal of the Science of Food and Agriculture92(13) 2644ndash2651

Kurisawa M Chung J E Uyama H amp Kobayashi S (2003)Enzymatic synthesis and antioxidant properties of poly(rutin) Biomacromolecules 4(5) 1394ndash1399

Lee J (2013) Proanthocyanidin A2 purification and quantificationof American cranberry (Vaccinium macrocarpon Ait) productsJournal of Functional Foods 5(1) 144ndash153

Lee K G Mitchell A E amp Shibamoto T (2000) Determination ofantioxidant properties of aroma extracts from various beansJournal of Agricultural and Food Chemistry 48(10) 4817ndash4820

Li W Liu J Guan R Chen J Yang D Zhao Z amp Wang D(2015) Chemical characterization of procyanidins fromSpatholobus suberectus and their antioxidative and anticanceractivities Journal of Functional Foods 12 468ndash477

Lin P Y amp Lai H M (2006) Bioactive compounds in legumes andtheir germinated products Journal of Agricultural and FoodChemistry 54(11) 3807ndash3814

Loacutepez-Alarcoacuten C amp Lissi E (2005) Interaction of pyrogallol redwith peroxyl radicals A basis for a simple methodology forthe evaluation of antioxidant capabilities Free RadicalResearch 39(7) 729ndash736

Maruyama C Araki R Kawamura M Kondo N Kigawa MKawai Y Takanami Y Miyashita K amp Shimomitsu T (2008)Azuki bean juice lowers serum triglyceride concentrations inhealthy young women Journal of Clinical Biochemistry andNutrition 43(1) 19ndash25

Mukai Y amp Sato S (2011) Polyphenol-containing azuki bean(Vigna angularis) seed coats attenuate vascular oxidativestress and inflammation in spontaneously hypertensive ratsJournal of Nutritional Biochemistry 22(1) 16ndash21

Oyaizu M (1986) Studies on product of browning reactionAntioxidative activities of products of browning reactionprepared from glucosamine Japanese Journal of Nutrition 44(6)307ndash315

Porter L J Hrstich L N amp Chan B G (1986) The conversion ofprocyanidins and prodephinidins to cyanidin anddelphinidin Phytochemistry 25(1) 223ndash230

Pulido R Bravo L amp Saura-Calixto F (2000) Antioxidantactivity of dietary polyphenols as determined by a modifiedferric reducingantioxidant power assay Journal of Agriculturaland Food Chemistry 48(8) 3396ndash3402

Rice-Evans C A Miller N J amp Paganga G (1996) Structure-antioxidant activity relationships of flavonoids and phenolicacids Free Radical Biology and Medicine 20(7) 933ndash956

Saiga A Soichi T amp Nishimura Y (2003) Antioxidant activity ofpeptides from porcine myofibrillar proteins by proteasetreatment Journal of Agricultural and Food Chemistry 51(12)3661ndash3667

Simic M G amp Jovanovic S V (1994) Inactivation of oxygenradicals by dietary phenolic compounds inanticarcinogenesis In C T Ho T Osawa M-T Huang amp R TRosen (Eds) Food phytochemicals for cancer prevention IIWashington DC American Chemical Society

Singleton V L Orthofer R amp Lamuela-Raventos R (1999)Analysis of total phenols and other oxidation substrates andantioxidants by means of Folin-Ciocalteu reagent Methods inEnzymology 299 152ndash178

Sreerama Y N Takahashi Y amp Yamaki K (2012) Phenolicantioxdiants in some Vigna species of legumes and theirdistinct inhibitory effect on α-glucosidase and pancreaticlipase activities Journal of Food Science 77(9) C927ndashC933

Sulaiman S Ibrahim D Kassim J amp Sheh-Hong L (2011)Antimicrobial and antioxidant activities of condensed tanninfrom Rhizophora apiculata barks Journal of Chemical andPharmaceutical Research 3(4) 436ndash444

Wang P Jiang X Jiang Y Hu X Mou H Li M amp Guan H(2007) In vitro antioxidative activities of three marineoligosaccharides Natural Product Research 21(7) 646ndash654

Watterson J J amp Butler J G (1983) Occurrence of an unusualleucoanthocyanidin and absence of proanthocyanidins insorghum leaves Journal of Agricultural and Food Chemistry31(1) 41ndash45

Wu X Beecher G R Holden J M Haytowitz D B Gebhardt SE amp Prior R L (2006) Concentrations of anthocyanins incommon foods in the United States and estimation of normalconsumption Journal of Agricultural and Food Chemistry 54(11)4069ndash4075

Wu X amp Prior R L (2005) Identification and characterization ofanthocyanins by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry incommon foods in the United States Vegetables nuts andgrains Journal of Agricultural and Food Chemistry 53(8) 3101ndash3113

Xiao Y Xing G Rui X Li W Chen X Jiang M amp Dong M(2014) Enhancement of the antioxidant capacity of chickpeasby solid state fermentation with Cordyceps militaris SN-18Journal of Functional Foods 10 201ndash222

Yoshida K Sato Y Okuno R Kameda K Isobe M amp Kondo T(1996) Structural analysis and measurement of anthocyaninsfrom colored seed coats of Vigna Phaseolus and Glycinelegumes Bioscience Biotechnology and Biochemistry 60(4) 589ndash593


Characterisation of anthocyanins and proanthocyanidins of adzuki bean extracts and their antioxidant activity

Introduction

Materials and methods

Reagents chemicals and standards

Adzuki bean extract powder (AEP)

Total polyphenols and proanthocyanidins analyses

Anthocyanin monomer analysis

Total antioxidant activity

Hydroxyl radical scavenging activity

Peroxyl radical scavenging activity

DPPH radical scavenging activity

Reducing power

Chelating ability of ferrous ions

Statistical analysis

Results and discussion

Total polyphenols and proanthocyanidins

Anthocyanins

Total antioxidant activity (TAA)

Radical scavenging activity

Reducing capacity

Ferrous ion chelating activity

Conclusions

Acknowledgments

Supplementary material

References

reported to inhibit the formation of malonaldehyde (LeeMitchell amp Shibamoto 2000) which is a maker for oxidativestress A decrease in vascular oxidative stress and inflamma-tion has also been reported in rats fed with the polyphenol-containing adzuki bean seed coat (Mukai amp Sato 2011)Furthermore Kitano-Okada et al (2012) have shown in vitro thatextracts from adzuki bean seed coat inhibit the activity of pan-creatic lipase These inhibitions would explain the results ofa clinical trial that suggests that the consumption of the adzukibean is linked to a reduced risk of lifestyle-related diseases inhumans (Maruyama et al 2008)

To manufacture the adzuki bean paste the beans are boiledin water which is then generally discarded after completingthe boiling process However the pigments contained in thiswater are receiving increased interest because of the need tomaximize the utilization of natural resources in order to de-crease the associated carbon footprint Moreover the foodindustry is experiencing an increasing demand for natural al-ternatives to replace synthetic food additives (eg antioxidants)(Amarowicz Naczk amp Shahidi 2000) In addition adzuki beanextract is a potential source of pigments (food colourants)because the water used to boil the beans becomes strongly redor purple Therefore a novel extract powder purified from thewater of adzuki bean paste production was developed and in-troduced in Japan to better utilize the resource (Adzuki-no-moto Cosmo Foods Co Ltd Tokyo Japan) The extract powderis highly purified and rich in natural polyphenols (Kitano-Okadaet al 2012) Interestingly hot air-exposed purified adzuki beanextract rather maintains consistent colour strength than thenormal purified adzuki bean extract because polyphenols inthe adzuki bean extract were polymerized by oxidation andboth the extracts are stable to light heat and pH changesHowever information on responsible compounds for pigmen-tation as well as the potent antioxidative activity of the differentpurified adzuki bean extract powders is very limited

Therefore the objective of this investigation was to comparethe different purified adzuki bean extract powders from in-dustrial residue in order to reveal their bioactive compoundsfor pigmentation and in vitro functional attributes such as re-ducing power and radical scavenging activity assay For thisthe antioxidant activity was compared with (+)-catechin as stan-dard polyphenol

2 Materials and methods

21 Reagents chemicals and standards

Calibrations were performed by using standard compounds (cat-echin cyanidin-3-O-glucoside pelargonidin-3-O-glucosidepeonidin-3-O-glucoside and malvidin-3-O-glucoside) fromSigma-Aldrich (St Louis MO USA) Solvents (ethanol butanolacetonitrile and formic acid) and concentrated hydrochloric acid(HCl) were purchased from Wako Chemical Co Ltd (TokyoJapan) Hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) malondialdehyde 22-azobis(2-methylpropionamide)dihydrochloride (AAPH) 22-diphenyl-1-picrylhydrazyl (DPPH)thiobarbituric acid and FolinndashCiocalteu phenol reagent wereobtained from Sigma-Aldrich Ferric ammonium sulphate andferrozine were purchased from Kishida Chemical Co Ltd

(Osaka Japan) All other chemicals were purchased from KantoChemical Co Ltd (Tokyo Japan)

22 Adzuki bean extract powder (AEP)

The adzuki bean extract powder (AEP also known as Adzuki-no-moto) made from the simmering water obtained during thesweetened adzuki bean paste production was kindly sup-plied by Cosmo Foods The extract powder was made by thefollowing procedure first the bean was boiled and cooled Thewater was collected from supernatant adjusted at pH 40treated with 0005 pectinase HL (Yakult Pharmaceutical In-dustry Co Ltd Tokyo Japan) and passed through a 50 meshsieve to separate undigested materials It was further appliedto an ultrafiltration device to remove polymeric componentsand adjusted at pH 85Then two different types of adzuki beanextract powder were made depending on the process with andwithout oxidative polymerization of polyphenols One was ster-ilized (115 degC for 90 min) and spray-dried (AEP-1)The other wasexposed to the air under heat treatment (90 degC for 5 h) andspray-dried (AEP-2)

23 Total polyphenols and proanthocyanidins analyses

The total concentration of polyphenols in the AEP samples wasdetermined according to the FolinndashCiocalteursquos methodusing (+)-catechin as a standard (Singleton Orthofer ampLamuela-Raventos 1999) The absorbance was read at 750 nmusing a spectrophotometer (1600-UV Shimadzu Kyoto Japan)The results were expressed in mg of (+)-catechin equivalentsto per gram of bean extract powder All tests were performedin triplicate

For assay of total proanthocyanidins concentration in theAEP samples the acid butanol method was employed (PorterHrstich amp Chan 1986) In brief 60 mL of the butanolndashHClreagent (955 vv) were added to 10 mL of suitably dilutedextract (1 mgmL) in a screw cap test tubeThen 02 mL of ferricreagent (2 ferric ammonium sulphate in 2M HCl) was addedto the solution The container was capped mixed and boiledfor 30 min in a water bath Similarly a standard curve was pre-pared using procyanidin B-2 (Sigma-Aldrich) ranging from 0to 025 mgmL After cooling the absorbance was read at 550 nmusing a spectrophotometer The results were expressed in mgof procyanidin B-2 equivalents to per gram of bean extractpowder

For quantification of oligomeric proanthocyanidins the AEPsample was dissolved in water (1 mgmL) and filtered using a045 microm polytetrafluoroethylene (PTFE) hydrophilic syringe filter(Advantec Dismic-13HP Toyo Roshi Co Ltd Tokyo Japan) Thefiltrate was loaded into a Shimadzu Prominence HPLC systemequipped with an LC-20AD pump (Shimadzu)The analytes wereseparated using a TSKgel ODS-80Ts column (46 mm times 250 mmTosoh Tokyo Japan) with photodiode array detector (SPD-M20A Shimadzu) Absorbance was monitored at 210ndash600 nmThe column oven temperature was set at 40 degC The injectionvolume was 10 microL and the flow rate was 10 mLminThe mobilephases were made of 005 trifluoroacetic acid (TFA) in water(vv mobile phase A) and 005 TFA in 90 acetonitrile (vvmobile phase B) Elution was used as a linear gradient from 5to 35 B in 13 min from 35 to 70 B in 20 min and then 70





















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mgg dry mgg dry





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4 Conclusions


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R E F E R E N C E S






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Conclusions

Acknowledgments


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32 Anthocyanins










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00 50 100 150 200 250 300 350 400 450 500 550 min

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DP

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activ

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R E F E R E N C E S






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32 Anthocyanins










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R E F E R E N C E S






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DP

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characterisation of anthocyanins and proanthocyanidins of...

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