Food Chemistry 141 (2013) 282–288

Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Isolation and characterization of a cyanidin-catechin pigment fromadzuki bean (Vigna angularis)

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.02.113

⇑ Corresponding author. Fax: +81 93 582 6000.E-mail address: takahama@kyu-dent.ac.jp (U. Takahama).

Umeo Takahama a,⇑, Ryo Yamauchi b, Sachiko Hirota c

a Department of Bioscience, Kyushu Dental University, Kitakyushu 803-8580, Japanb Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japanc Department of Nutrition, Kyushu Women’s University, Kitakyushu 807-8586, Japan

a r t i c l e i n f o

Article history:Received 5 November 2012Received in revised form 7 January 2013Accepted 26 February 2013Available online 7 March 2013

Keywords:Anthocyanidin–flavanol adductColoration of foodspH effectsVigna angularis

a b s t r a c t

Adzuki bean is used to prepare many kinds of foods in east Asia, and the seed coat contains water-solubleanthocyanins, catechins, and flavonols. In the present study, ethyl acetate-soluble purplish pigmentswere isolated from adzuki bean. Pigments of soaked adzuki bean were extracted with 1% HCl in methanol.Ethyl acetate-soluble purple pigments were obtained from the methanol soluble components. Purple pig-ments 1 and 2 were purified from the ethyl acetate-soluble pigments by Sephadex LH-20 column chro-matography and preparative reversed-phase HPLC. NMR and mass spectra suggested that pigment 1 wasa condensation product of cyanidin and (+)-catechin, in which 5-hydroxy and C-4 positions of the cyani-din moiety were substituted by the addition of 5-hydroxy and C-6 positions of the (+)-catechin moiety,respectively. Pigment 2 was an isomer of pigment 1. It is suggested that pigments 1 and 2 contribute tothe purplish-red colour of foods prepared using adzuki bean.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Adzuki bean, Vigna angularis (Willd.) Ohwi et Ohashi, is used forvarious dishes in east Asia. The paste prepared from adzuki bean isan important ingredient for Japanese and Chinese sweets, the col-our of which is purplish-red or dark purple. In addition, adzukibean is cooked together with glutinous rice to prepare purplish-red rice in Japan. The preparation of purplish-red rice is tradition-ally common in lucky events, such as festivals or birthdays. As pig-ments of adzuki bean paste and glutinous rice, anthocyanins are inthe seed coat (Sasanuma, Takeda, & Hayashi, 1996; Yoshida et al.,1996). However, anthocyanins in adzuki bean and those extractedfrom the bean may be broken down during cooking, becauseanthocyanin is degraded during thermal processing (Buckow, Kas-tell, Terefe, & Versteeg, 2010; Patras, Brunton, O’Donnell, & Tiwari,2010).

It has been reported that blocking of the C-4 position of antho-cyanins prevents their bleaching by bisulfite (Bakker & Timberlake,1997; Wrolstad, Durst, & Lee, 2005). Thus, we postulated that col-our-stability of adzuki bean might be increased if it contained theabove anthocyanin-derived compounds. Pyranoanthocyanins areincluded in such anthocyanin-derived compounds, in which the5-hydroxy and C-4 positions of an anthocyanin are combined withanother component to form the pyran ring structure. These com-

pounds have been isolated from Dasymaschalon sootepense (Sinz,Matusch, Santisuk, Suttiporn Chaichana, & Vichai Reutrakul,1998), red onion (Fossen, Rayyan, & Andersen, 2004), rose (Fukui,Kusumi, Masuda, Iwashita, & Nomoto, 2002), strawberry (Fossen,Slimestad, & Andersen, 2003), and red wine (Andersen, Fossen,Torskangerpoll, Fossen, & Hauge, 2004; Bakker & Timberlake,1997; Fulcrand, dos Santos, Sarni-Manchado, Cheynier, & Favre-Bonvin, 1996). Another group of compounds of the anthocyanin-derived family are anthocyanin�flavanol adducts, in which the C-4 position of an anthocynin is combined with the C-8 position offlavanol. Such adducts have been isolated from red wine (Fulcrand,Dueñas, Salas, & Cheynier, 2006).

In addition, compounds with carbon–carbon linkage betweenthe C-8 of anthocyanin and the C-4 of flavanol may also contributeto the colour-stability of adzuki pigments. Such compounds havebeen found in black soybean (Lee J. H. et al., 2009), fig (Dueñas,Pérez-Alonso, Santos-Buelga, & Escribano-Bailón, 2008), Guatema-lan beans (Macz-Pop, González-Paramás, Pérez-Alonso, & Rivas-Gonzalo, 2006), pomegranate juice (Sentandreu, Navarro, & Sendro,2010; Sentandreu, Navarro, & Sendro, 2012), and purple corn (Gon-zález-Manzano et al., 2008), as well as red wine (Fulcrand et al.,1996; Fulcrand et al., 2006; Lee, Swinny, & Jones, 2004; Mateus, Sil-va, Santos-Buelga, Rivas-Gonzalo, & de Freitas, 2002).

This paper deals with the isolation of a purple pigment from ad-zuki bean to determine its structure, the effects of solvents and pHon absorption spectra of the isolated pigment, and its presence inboiled adzuki bean and boiling water. The results indicate that

U. Takahama et al. / Food Chemistry 141 (2013) 282–288 283

the isolated purple pigment is a novel compound that containscyanidin and (+)-catechin moieties in the molecule, and it is dis-cussed that the pigment could contribute to make foods pur-plish-red, when cooked with adzuki bean.

2. Materials and methods

2.1. Plant materials and reagents

Adzuki bean (cultivar Erimoshozu) was obtained from Kawaka-mi Co., Ltd. (Kitakyushu, Japan). Dimethylsulfoxide (DMSO)-d6 andtetramethylsilane were from Sigma–Aldrich Japan Co. LLC (Tokyo,Japan). Quercetin and all other reagents were from Wako PureChemical Industries, Ltd (Osaka, Japan).

2.2. Extraction of purple pigments

Water was added to 900 g of dried adzuki bean to make the to-tal volume of 3 L, and then left at room temperature (about 25 �C)overnight. After removing water by decantation, pigments ofsoaked bean were extracted with 1.5 L of 1% HCl in methanol for1–2 h (Fig. 1). The extract (about 1.2 L) was concentrated to 400–450 ml at ca. 40 �C using a rotary evaporator. The concentratedsolution was filtered through a filter paper under reduced pressure.The filtrate was extracted 3 times with 200 ml of ethyl acetate. Theethyl acetate extracts were combined, and then washed 3 timeswith 200 ml of 0.1% HCl in water to make the colour of ethyl ace-tate layer turn purple by removing water-soluble reddish pig-ments. Petroleum ether (100 ml) was added to the purple ethylacetate solution (about 500 ml), and then the mixture of petroleum

Extraction with 1% HCl in MeOH (1.5 L)

Extracted with EtOAc.Washed with

0.1% HCl in H2O

Adzuki bean (900 g)

Sephadex LH-20Solvent: MeOH

Purplish fraction

Preparative HPLC (2 times)Solvents: (1) MeOH/25 mM KH2PO4

(2) CH3CN/25 mM KH2PO4

Soaked bean

Filtrate Precipitates

Purplish pigment

Soaked in water (3 L)

Extract

Concentrated to be ca. 400 mL

Dissolved in EtOAc.Washed with

0.1% HCl in H2O

Pigment 1 Pigment 2

Ste

p 1

x 2

Ste

p 2

Fig. 1. Isolation processes for pigments 1 and 2 from adzuki bean. For details, seetext. MeOH, methanol; EtOAc, ethyl acetate.

ether and ethyl acetate was washed 3 times again with 200 ml of0.1% HCl in water.

The purple precipitate obtained by filtration was extracted with100 ml of ethyl acetate for 2 h at room temperature (Fig. 1). Petro-leum ether (25 ml) was added to the purplish ethyl acetate extract,and then the mixture of ethyl acetate and petroleum ether waswashed 3 times with 100 ml of 0.1% HCl in water.

Purple organic solutions, which were prepared from the filtrateand the precipitate as described above, were combined and dehy-drated with anhydrous sodium sulfate. The above extraction pro-cess (Step 1 in Fig. 1) was repeated twice.

2.3. Column chromatography and preparative HPLC

The purple residue, which was obtained from the dehydratedorganic solution, was dissolved in 2 ml of methanol. To prepare apurple fraction from the methanol solution, column chromatogra-phy was carried out on a Sephadex LH-20 column (30 � 2 cm i. d.).The eluent used was methanol. After evaporation of methanol ofthe purple fraction, the residue was dissolved in 8 ml of a mixtureof methanol and 25 mM KH2PO4 (2:1, v/v). Two purple pigmentswere isolated from the solution by preparative HPLC as describedbelow.

Preparative HPLC was performed using a Shim-pack PREP-ODS(H) kit (25 cm � 20 mm i. d.; particle size, 5 lm; pore size,10 nm) (Shimadzu, Kyoto, Japan) combined with a 1 ml sampleloop. The mobile phase was a mixture of methanol and 25 mMKH2PO4 (2:1, v/v) and the flow rate was 9 ml min–1. Componentsseparated by the preparative HPLC were detected at 210, 360,and 570 nm using a spectrophotometric detector with a photodi-ode array (SPD-M10Avp; Shimadzu). Peaks 1 and 2 had retentiontimes of about 19 and 25 min, respectively (Fig. 2, top). Ultravio-let–visible (UV–vis) absorption spectrum of the component of peak1 (pigment 1) had an absorption maximum at 568 nm in the

568

417278

372254

1

2

a

200 300 400 500 600 700Wavelength (nm)

10 20 min0

1& 2

Quercetin

a

Abso

rban

ce(a

rbitr

ary

unit)

A 360

= 0.

5

Fig. 2. Preparative HPLC. (Top) HPLC profile at 360 nm. Sample for HPLC analysiswas prepared using Sephadex LH-20 column chromatography. (Bottom) Absorptionspectra of peaks a (quercetin), 1 (pigment 1), and 2 (pigment 2) in the mobile phasefor preparative HPLC.

284 U. Takahama et al. / Food Chemistry 141 (2013) 282–288

mobile phase, and the spectrum was the same as that of the com-ponent of peak 2 (pigment 2) (Fig. 2, bottom). The fraction of eachpeak was collected, and the pigments were extracted from the mo-bile phase with ethyl acetate after removing methanol.

The purple residue was obtained from each ethyl acetate extractafter evaporating the solvent. Each residue was dissolved in 8 ml ofa mixture of acetonitrile and 25 mM KH2PO4 (1:2, v/v) to furtherpurify pigments 1 and 2 using the above HPLC column. The mobilephase was a mixture of acetonitrile and 25 mM KH2PO4 (1:2, v/v),and its flow rate was 9 ml min–1. Purified pigments 1 and 2 (reten-tion time, about 17 and 23 min, respectively) were extracted afterremoving acetonitrile from the mobile phase. The residue obtainedfrom each ethyl acetate extract was dissolved in 1 ml of 1% HCl inethanol, and then water was added to each acidic ethanol solutionto make a final volume of 10 ml. A purple precipitate was gener-ated after each solution was left in the dark at 4 �C overnight.The precipitate was collected by centrifugation (5,000g, 3 min),washed with 10 ml of water by centrifugation, and then lyophi-lized. From 1.8 kg of dried adzuki bean, 2.6 mg of pigment 1 and0.6 mg of pigment 2 were obtained. Pigment 1 was used for struc-tural analysis.

2.4. Analytical HPLC and liquid chromatography-mass spectra (LC-MS)

The purity of isolated pigments 1 and 2 were examined using aShim-pack CLC-ODS column (15 � 6 mm i. d.) (Shimadzu) com-bined with the above spectrophotometric detector with a photodi-ode array. The mobile phase was a mixture of methanol and 25 mMKH2PO4 (3:2 or 2:1, v/v) and the flow rate was 1 ml min–1.

Electrospray ionisation (ESI) and atmosphere-pressure chemicalionisation (APCI) mass spectra were obtained with a LCMSQP8000a quadrupole mass spectrometer (Shimadzu). LC was donewith a TSKgel-ODS 80TS column (15 cm � 2 mm i. d.) (Tosoh, To-kyo, Japan). The mobile phase was a 15-min linear gradient from65% to 100% methanol (containing 0.2% formic acid) and the flowrate was 0.2 ml min–1. Positive-ion mass spectra were obtained atan ESI or APCI prove voltage of +4.5 kV.

2.5. Nuclear magnetic resonance (NMR) spectra

1H and 13C NMR spectra were recorded at room temperaturewith an ECX-400P FT-NMR spectrometer (JEOL, Ltd., Tokyo, Japan)with DMSO-d6 as the solvent and tetramethylsilane as the internalstandard. 1H NMR was performed at 399.78 MHz, and the 1H–1Hchemical shift correlated (COSY) technique was employed to assign1H shifts and couplings. 13C NMR was at 100.53 MHz with protondecoupling. Heteronuclear multiple-bond correlation (HMBC) andheteronuclear multiple-quantum coherence (HMQC) techniqueswere used to assign correlations between 1H and 13C signals.

2.6. Presence of pigment 1 in boiled adzuki bean

Water was added to 30 g of adzuki bean to make the total vol-ume of 100 ml, and then boiled for 20 min. After cooling, adzukibean was separated from the brownish-red boiling water. As pig-ment 1 was extractable from the water solution by the use of ethylacetate (see above) and the direct extraction of the pigment byethyl acetate avoided its acid hydrolysis, both the boiled adzukibean (43 g) and the boiling water fraction (about 60 ml, pH 5.9)were extracted with 30 and 50 ml of ethyl acetate, respectively.The boiling enhanced the release of pigment 1 from adzuki beanand increased the extractability of pigment 1 by ethyl acetate fromthe adzuki bean. Each residue obtained after evaporating ethyl ace-tate was dissolved in 0.5 ml of a mixture of methanol and 25 mMKH2PO4 (2:1, v/v), and analysed by the above analytical HPLCsystems.

2.7. Spectrophotometric studies

Effects of organic solvent and pH on UV–vis absorption spectraof pigment 1 were studied using a spectrophotometer (UV-2450,Shimadzu) equipped with an integrating sphere (ISR-240A, Shima-dzu). The path length of the measuring beam was 4 mm. Isolatedpigment 1 was dissolved in DMSO at a concentration of 10 mM,and the concentration in each organic solvent was 20 lM. Buffersolutions of pH 1–3 and pH 3–9 were prepared using 50 mMKCl–HCl and 0.1 M H3PO4-0.1 M NaH2PO4-0.1 M Na2HPO4, respec-tively, and the concentration of pigment 1 in the buffer solutionswere also 20 lM.

3. Results and discussion

3.1. Structural analysis of pigments 1 and 2

HPLC analysis of isolated pigment 1 showed a single peak in awavelength range from 200 to 800 nm, and the absorption spec-trum was the same as that of peak 1 in Fig. 2. The positive ESI-MS of pigment 1 showed the molecular ion (M+) at m/z 557.1.The positive APCI-MS showed fragment ions at m/z 435.1 ([M –C7H6O2]+), 405.1 ([M – C8H8O3]+), and 393.1 ([M – C9H8O3]+), inaddition to the molecular ion at m/z 557.1 (Fig. 3). An ion at m/z557 has been reported to be the major fragment ion of anthocya-nin–flavanol pigments by the ESI-MSn analyses (Macz-Pop et al.,2006; Sentandreu et al., 2010; Sentandreu et al., 2012). Accordingto the references, the fragment ion at m/z 557 seems to be gener-ated by deglycosidation and dehydration of anthocyanin–flavanolpigments. If we assume that the ion detected by ESI- and APCI-MS (m/z 557.1) is the molecular ion of pigment 1, then it probablyrepresents an anthocyanidin–flavanol adduct. The following NMRdata of pigment 1 support this ion as the molecular ion.

The structural assignment of pigment 1 was confirmed by 1H-and 3C NMR analyses (Table 1). The 1H NMR spectrum indicatesthe presence of cyanidin moiety [d 6.69 (d, J = 1.8 Hz, 1H, H-6),6.85 (d, J = 2.3 Hz, 1H, H-8), 6.92 (d, J = 8.7 Hz, 1H, H-50), 8.02 (dd,J = 2.4, 8.7 Hz, 1H, H-60), and 8.15 (d, J = 2.3 Hz, 1H, H-20)] (Chirol& Jay, 1995), and flavanol moiety [d 2.47 (dd, J = 16.5, 6.9 Hz, 1H,H-4⁄ax), 2.66 (dd, J = 16.4, 4.6 Hz, 1H, H-4⁄eq), 3.97 (dt, J = 5.5,6.8 Hz, 1H, H-3⁄), 4.80 (d, J = 6.4 Hz, 1H, H-2⁄), 6.12 (s, 1H, H-8⁄),6.62 (dd, J = 1.8, 8.2 Hz, 1H, H-6⁄0), 6.73 (d, J = 8.6 Hz, 1H, H-5⁄0),and 6.74 (d, J = 2.3 Hz, 1H, H-2⁄0)] (Shen, Chang & Ho, 1993). Onthe 1H–1H COSY spectrum of the flavanol moiety, cross peaks ap-peared between resonances at d 2.47 and 2.66, at d 2.47 and3.97, at d 2.66 and 3.97, and at d 3.97 and 4.80 (data not shown).The 1H NMR data of H-2⁄, H-3⁄ and H-4⁄ in the flavanol moietyresembled to those in (+)-catechin rather than (�)-epicatechin:the relative 2,3-sterochemistry was trans, as the proton signal atd 4.80 (H-2⁄) appeared as a doublet (J2,3 = 6.4 Hz) correspondingto (+)-catechin (Shen, Chang & Ho, 1993).

The 13C NMR spectrum showed 30 carbon signals includingflavanol carbons at d 27.4 (C-4), 65.8 (C-3), and 81.8 (C-2). Thestructural assignment was supported by HMBC cross peaks asshown in Table 1: the correlations were observed between the pro-ton signal at d 6.69 (H-6) and carbon signals at d 105.1 (C-4a), 150.1(C-5), 162.1 (C-7), and 96.3 (C-8); between the proton signals at d6.85 (H-8) and carbon signals at d 105.1 (C-4a), 98.5 (C-6), 162.1(C-7), and 150.8 (C-8a); between the proton signals at d 8.15 (H-20) and carbon signals at d 152.0 (C-2), 145.0 (C-30), 149.4 (C-40),and 122.5 (C-60); between the proton signals at d 6.92 (H-50) andcarbon signals at d 122.9 (C-10), 145.0 (C-3), and 149.4 (C-40); be-tween the proton signals at d 8.02 (H-60) and carbon signals at d152.0 (C-2), 116.5 (C-20), and 149.4 (C-40); between the proton sig-nals at d 4.80 (H-2⁄) and carbon signals at d 65.8 (C-3⁄), 27.4 (C-4⁄),

m/z 557m/z 435

m/z 405

m/z 393

200 300 400 500 600 m/z0

50

100 557.1(M+)

393.2

435.1405.1R

elat

ive

abun

danc

e (%

)

Fig. 3. APCI-MS of pigment 1 and scheme of fragmentation.

U. Takahama et al. / Food Chemistry 141 (2013) 282–288 285

161.9 (C-8⁄a), 129.6 (C-1⁄0), 114.2 (C-2⁄0), and 118.1 (C-6⁄0); be-tween the proton signals at d 3.97 (H-3⁄) and carbon signals at d106.2 (C-4⁄a); between the proton signals at d 2.47 (H-4⁄ax) andcarbon signals at d 81.8 (C-2⁄), 65.8 (C-3⁄), 106.2 (C-4⁄a), 167.4(C-5⁄), and 161.9 (C-8⁄a); between the proton signals at d 2.66(H-4⁄eq) and carbon signals at d 106.2 (C-4⁄a), between the protonsignals at d 6.12 (H-8⁄) and carbon signals at d 106.2 (C-4⁄a), 103.3(C-6⁄), 153.6 (C-7⁄), and 161.9 (C-8⁄a); between the proton signalsat d 6.74 (H-2⁄0) and carbon signals at d 81.8 (C-2⁄), 145.0 (C-3⁄0),145.1 (C-4⁄0), and 118.1 (C-6⁄0); between the proton signals at d6.73 (H-5⁄0) and carbon signals at d 129.6 (C-1⁄0), 145.0 (C-3⁄0),and 145.1 (C-4⁄0); and between the proton signals at d 6.62 (H-6⁄0) and carbon signals at d 81.8 (C-2⁄), 114.2 (C-2⁄0), and 145.1(C-4⁄0). From these spectral data, we assigned that pigment 1 wasa condensation product of cyanidin and (+)-catechin, in whichthe 5-hydroxy and C-4 positions of the cyanidin moiety weresubstituted by the addition of the 5-hydroxy and C-6 positions ofthe (+)-catechin moiety, respectively (Fig. 4). We named this cyani-din�(+)-catechin adduct vignacyanidin, based on the generic nameof the adzuki plant.

The direct condensation between anthocyanins and flavanolstakes place during processing and storage of red wines (Fulcrandet al., 1996; Fulcrand et al., 2006; Lee et al., 2004; Mateus et al.,2002). Such anthocyanin�flavanol adducts have been synthesizedin model systems (Dueñas, Fulcrand, & Cheynier, 2006; Es-Safi,Cheynier, & Moutounet, 2002). In the synthesized adducts, the 7-hydroxy and C-8 positions of the catechin moiety substituted by

the addition of 5-hydroxy and C-4 positions of malvidin 3-O-gluco-side moiety is included (Dueñas et al., 2006). In addition, smallamounts of anthocyanin–flavanol condensed pigments have beenfound in some plants, as described in the introduction. In theseanthocyanin–flavanol adducts, the C-8 position of the anthocyaninmoieties bind to the C-4 position of flavonol moieties.

Pigment 2 showed the molecular ion (M+) at m/z 557.1 with thesame fragmentation pattern as vignacyanidin: ESI-MS, m/z 557.1(M+, 100%); APCI-MS, m/z 393.1 ([M – C9H8O3]+, 88%), 405.1 ([M– C8H8O3]+, 3%), and 435.1 ([M – C7H6O2]+, 17%) and 557.1 (M+,100%). Furthermore, the UV–vis absorption spectrum of pigment2 was the same as that of vignacyanidin (Fig. 2). These data suggestthat pigment 2 is an isomer of vignacyanidin, such as a cyani-din�(�)-epicatechin adduct, where the (+)-catechin moiety is re-placed by the (�)-epicatechin moiety. Since 2R,3S compounds[such as (+)-catechin] elute earlier than 2R,3R compounds [suchas (�)-epicatechin] in reversed-phase columns (Fossen et al.,2004), the order of elution in reversed-phase HPLC supports thatpigment 2 might be a cyanidin�(�)-epicatechin adduct. In thepresent study, we could not resolve the detailed structure of pig-ment 2 due to its low yield.

Anthocyanins are normally present as glycosides in plants, andanthocyanin�flavanol adducts found in red wine and plants arealso glycosides (Dueñas et al., 2008; Fulcrand et al., 2006; Macz-Pop et al., 2006; Sentandreu et al., 2010; Sentandreu et al., 2012),but vignacyanidin and its isomer isolated in the present study wereaglycones. The use of acidic solutions during the isolation of vigna-

Table 11H and 13C NMR spectral data and HMBC and HMQC correlations of pigment 1a.

1H 13CPosition db J (Hz)c db HMBC HMQC

Cyanidinmoiety

2 152 H-20 , -60

3 1444 1414a 105 H-6, -85 150 H-66 6.69 d, 1.8 98.5 H-8 H-67 162 H-6, -88 6.85 d, 2.3 96.3 H-6 H-88a 151 H-810 123 H-50

20 8.15 d, 2.3 117 H-60 H-20

30 145 H-20 , -50

40 149 H-20 , -50 , -60

50 6.92 d, 8.7 116 H-50

60 8.02 dd, 2.4, 8.7 123 H-20 H-60

Catechin moiety2⁄ 4.80 d, 6.4 81.8 H-4⁄ax, -2⁄0 , -6⁄0 H-2⁄

3⁄ 3.97 dt, 5.5, 6.8 65.8 H-2⁄, -4⁄ax H-3⁄

4⁄ 2.47 dd, 16.5, 6.9 27.4 H-2⁄ H-4⁄ax

2.66 dd, 16.4, 4.6 27.4 H-2⁄ H-4⁄eq

4⁄a 106 H-3⁄, -4⁄ax, -4⁄eq, -8⁄

5⁄ 167 H-4⁄ax

6⁄ 103 H-8⁄

7⁄ 154 H-8⁄

8⁄ 6.12 s 92.7 H-8⁄

8⁄a 162 H-2⁄, -4⁄ax, -8⁄

1⁄0 130 H-2⁄, -5⁄0

2⁄0 6.74 d, 2.3 114 H-2⁄, -6⁄0 H-2⁄0

3⁄0 145 H-2⁄0 , -5⁄0

4⁄0 145 H-2⁄0 , -5⁄0 , -6⁄0

5⁄0 6.73 d, 8.6 115 H-5⁄0

6⁄0 6.62 dd, 1.8, 8.2 118 H-2⁄, -2⁄0 H-6⁄0

a Measured in DMSO-d6.b Shifts in parts per million downfield relative to tetramethylsilane.c Multiplicity: s, singlet; d, doublet; t, triplet.

Cyanidin

(+)-Catechin

Fig. 4. Proposed structure of pigment 1 (vignacyanidin).

286 U. Takahama et al. / Food Chemistry 141 (2013) 282–288

cyanidin and its isomer, might suggest these were produced fromtheir glycosides by hydrolysis. This possibility was excluded bythe following results. Namely, both pigments were extracted with

ethyl acetate from boiling-water extract of adzuki bean (pH 5.9)and boiled adzuki bean (data not shown), which indicates (i) thatvigancyanidin and its isomer are not compounds formed by acidhydrolysis nor enzymatic hydrolysis and (ii) that these pigmentscan remain in foods prepared using adzuki bean. Two mechanismsare possible for the synthesis of vignacyanidin and its isomer inseed coat of adzuki bean; one is the reaction of (epi)catechins withcyanidin aglycones, and the other is the removal of sugars from theglycosides of vignacyanidin and its isomer, which are formed from(epi)catechins and cyanidin glycosides. Further studies are re-quired to elucidate the mechanism of synthesis of vignacyanidinand its isomer in seed coat of adzuki bean during ripening.

In addition to vignacyanidin and its isomer, the quercetin agly-cone was found as an ethyl acetate-soluble component (Fig. 2, peaka). Quercetin was identified by comparing the retention time andthe absorption spectrum with those of standard quercetin. Sincequercetin is formed from its glycosides during drying of onionscales (Takahama & Hirota, 2000) and quercetin glycosides arepresent in the adzuki bean (Yoshida, Kondo, Ito, & Kondo, 2005),quercetin in adzuki bean may also be formed from quercetin glyco-sides in the seed coat during ripening. An aglycone with a xanthy-lium skeleton has been isolated from the adzuki bean (Yanase,Nishimoto, Kamatari, & Nakatsuka, 2012), suggesting that phenolicaglycones can coexist with their glycosides in the dried seed coat ofthe adzuki bean. Furthermore, an aglycone of pyranocyanidin hasbeen reported to be present in rose petals (Fukui et al., 2002).

3.2. Effects of solvent and pH on absorption spectra of vignacyanidin

UV–vis absorption spectra of vignacyanidin (20 lM) were re-corded in methanol, 1-butanol, and ethyl acetate. Absorption max-ima (kmax) of the spectra were observed at 572, 581, and 591 nm inmethanol, 1-butanol, and ethyl acetate, respectively, indicatingthat kmax shifted to longer wavelength with the increase of solventhydrophobicity. Molar extinction coefficients at their kmax in theabove solvents were similar to each other, and the value in meth-anol was calculated to be 31 mM�1 cm�1. The value of cyanidin 3-O-glucoside dissolved in various acidic solutions such as 1% HCl inmethanol and 0.1 M HCl in aqueous ethanol has been reported torange from 19 to 34 mM�1 cm�1 (Lee, Durst, & Wrolstad, 2005;Yoshida et al., 1996).

It is well known that the absorption of anthocyanin is depen-dent on pH. UV–vis absorption spectrum of vignacyanidin had akmax at 551 nm and a shoulder at approximately 600 nm at pH 1(Fig. 5, top). The kmax shifted to 520 nm when pH was increasedto 5 without affecting the absorbance significantly. Accompanyingthe increase in pH from 6 to 9, kmax shifted to a longer wavelength,increasing the kmax absorbance (Fig. 5, bottom). Such changes inkmax and absorbance with the increase in pH from 6 to 9 have beenreported for pelargonidin 3-glucopyranoside and 5-carboxypyrano-pelargonidin 3-glucopyranoside isolated from strawberry (Fragariaananassa) (Andersen et al., 2004), supporting the presence of theanthocyanin structure in vignacyanidin.

The absorbance around 500 nm of the pelargonidin glucosidesat pH 5.1 is less than one tenth and one fifth of that at pH 3 and6, respectively (Andersen et al., 2004), which is normally commonfor anthocyanins (Wrolstad et al., 2005), whereas the absorbancearound 500 nm of the carboxypyranopelargonidin glucoside isnot largely affected when pH is decreased from 6 to 1 (Andersenet al., 2004). The effect of pH on the absorbance for the carboxypy-ranopelargonidin glucoside and vigancyanidin was similar (Fig. 5),supporting the presence of a pyran structure in vignacyanidin.Absorption maximum of vignacyanidin was observed around600 nm at pH 9, whereas 5-carboxypyranopelargonidin 3-glucopy-ranoside had a kmax around 560 nm at pH 8.9. The difference mightbe explained by the difference in the number of conjugated double

50 mM KCl HCl50 mM KCl-HCl

523 (pH 3)

e

0.1 530 (pH 2)

banc

419

sorb 419

Ab 551

(pH 1)(p )

0

0.2Phosphate buffer

598542 (pH 7)

(pH 9)

579520 (pH 4 & 5)

( H 8)

ance

579 (pH 8)

0.1

orba 437 (pH 9)

Abs

oA

534(pH 6)

417

(pH 6)

0

800600400

Wavelength (nm)Wavelength (nm)

Fig. 5. Effect of pH on absorption spectra of vignacyanidin. The concentration ofvignacyanidin was 20 lM. (Top) pH 1–3; (bottom) pH 3–9.

U. Takahama et al. / Food Chemistry 141 (2013) 282–288 287

bonds and the number of hydroxyl groups in B ring of the anthocy-anindin moiety.

3.3. Concluding remarks

In this study, two cyanidin-catechin adducts were isolated fromthe adzuki bean. In pigment 1 (vignacyanidin), the 5-hydroxy andC-4 positions of cyanidin were substituted by the addition of 5-hy-droxy and C-6 positions of (+)-catechin, respectively. Pigment 2was an isomer of vignacyanidin. The detection of vignacyanidinand its isomer in the boiling water extract of adzuki bean andthe boiled adzuki bean suggests that these pigments can contributeto the coloration of the adzuki bean paste and glutinous rice. Thecoloration is probably due to the binding of the pigments to starchin the adzuki bean paste and glutinous rice. The binding of vigna-cyanidin and its isomer to starch is deduced from the report thatflavonoids such as quercetin and epicatechin-O-(4,5-dimethyl)-gallate can bind to starch (Takahama & Hirota, 2010). Becausethe binding of quercetin and epicatechin-O-(4,5-dimethyl)-gallateto starch results in suppression of a-amylase-catalysed digestionof starch, the next study will deal with the interactions betweenvignacyanidin and starch under various conditions.

Acknowledgements

Part of this study was supported by Grants-in-Aid for ScientificResearch (22500790 and 23500986) from the Japan Society for thePromotion of Science.

References

Andersen, Ø. M., Fossen, T., Torskangerpoll, K., Fossen, A., & Hauge, U. (2004).Anthocyanin from strawberry (Fragaria ananassa) with the novel aglycone, 5-carboxypyranopelargonidin. Phytochemistry, 65, 405–410.

Bakker, J., & Timberlake, C. F. (1997). Isolation, identification, and characterizationof new color-stable anthocyanins occurring in some red wines. Journal ofAgricultural and Food Chemistry, 45, 35–43.

Buckow, R., Kastell, A., Terefe, N. S., & Versteeg, C. (2010). Pressure and temperatureeffects on degradation kinetics and storage stability of total anthocyanins inblueberry juice. Journal of Agricultural and Food Chemistry, 58, 10076–10084.

Chirol, N., & Jay, M. (1995). Acylated anthocyanins from flowers of Begonia.Phytochemistry, 40, 275–277.

Dueñas, M., Fulcrand, H., & Cheynier, V. (2006). Formation of anthocyanin–flavanoladducts in model solutions. Analytica Chimica Acta, 563, 15–25.

Dueñas, M., Pérez-Alonso, J. J., Santos-Buelga, C., & Escribano-Bailón, T. (2008).Anthocyanin composition of fig (Ficus carica L.). Journal of Food Composition andAnalysis, 21, 107–115.

Es-Safi, N.-E., Cheynier, V., & Moutounet, M. (2002). Interactions between cyanidin3-O-glucoside and furfural derivatives and their impact on food color. Journal ofAgricultural and Food Chemistry, 50, 5586–5595.

Fossen, T., Rayyan, S., & Andersen, Ø. M. (2004). Dimeric anthocyanins fromstrawberry (Fragaria ananassa) consisting of pelargonidin 3-glucosidecovalently linked to four flavan-3-ols. Phytochemistry, 65, 1421–1428.

Fossen, T., Slimestad, R., & Andersen, Ø. M. (2003). Anthocyanins from red onion,Allium cepa, with novel aglycone. Phytochemistry, 62, 1217–1220.

Fukui, T., Kusumi, T., Masuda, K., Iwashita, T., & Nomoto, K. (2002). Structure ofrosacyanin B, a novel pigment from the petal of Rosa hybrid. Tetrahedron Letters,43, 2637–2639.

Fulcrand, H., dos Santos, P.-J. C., Sarni-Manchado, P., Cheynier, V., & Favre-Bonvin, J.(1996). Structure of new anthocyanin-derived wine pigments. Journal ofChemical Society, Perkin Transaction, 1, 735–739.

Fulcrand, H., Dueñas, M., Salas, E., & Cheynier, V. (2006). Phenolic reactions duringwinemaking and aging. American Journal of Enology and Viticulture, 57,283–297.

González-Manzano, S., Pérez-Alonso, J. J., Salinas-Moreno, Y., Mateus, N., Silva, A. M.S., de Freitas, V., et al. (2008). Flavanol–anthocyanin pigments in corn: NMRcharacterization and presence in different purple corn varieties. Journal of FoodComposition and Analysis, 21, 521–526.

Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Determination of total monomericanthocyanin pigment content of fruit juices, beverages, natural colorant, andwine by the pH difference method: collaborative study. Journal of AOACInternational, 88, 1269–1278.

Lee, J. H., Kang, N. S., Shin, S.-O., Shin, S.-H., Lim, S.-G., Suh, D.-Y., Baek, I.-Y., Park, K.-Y., & Ha, T. J. (2009). Characterisation of anthocyanins in the black soybean(Glycine max L.) by HPLC-DAD-ESI/MS analysis. Food Chemistry, 112, 226–231.

Lee, D. F., Swinny, E. E., & Jones, G. P. (2004). NMR identification of ethyl-linkedanthocyanin–flavanol pigments formed in model wine ferments. TetrahedronLetters, 45, 1671–1674.

Macz-Pop, G. A., González-Paramás, A. M., Pérez-Alonso, J. J., & Rivas-Gonzalo, J. C.(2006). New flavanol–anthocyanin condensed pigments and anthocyanincomposition in Guatemalan beans (Phaseolus spp.). Journal of Agricultural andFood Chemistry, 54, 536–542.

Mateus, N., Silva, A. M. S., Santos-Buelga, C., Rivas-Gonzalo, J. C., & de Freitas, V.(2002). Identification of anthocyanin–flavanol pigments in red wines by NMRand mass spectrometry. Journal of Agricultural and Food Chemistry, 50,2110–2116.

Patras, A., Brunton, N. P., O’Donnell, C., & Tiwari, B. K. (2010). Effect of thermalprocessing on anthocyanin stability in foods; mechanisms and kinetics ofdegradation. Trends in Food Science and Technology, 21, 3–11.

Sasanuma, S., Takeda, K., & Hayashi, K. (1996). Black red pigment of ‘‘adzuki bean’’studies on anthocyanins LV. Botanical Magazine Tokyo, 79, 807–810.

Sentandreu, E., Navarro, J. L., & Sendro, J. M. (2010). LC-DAD-ESI/MSn determinationof direct condensation flavanol–anthocyanin adducts in pressure extractedpomegranate (Punica granatum L.) juice. Journal of Agricultural and FoodChemistry, 58, 10560–10567.

Sentandreu, E., Navarro, J. L., & Sendro, J. M. (2012). Identification of new colouredanthocyanin–flavanol adducts in pressure-extracted pomegranate (Punicagranatum L.) juice by HPLC/ESI mass spectrometry. Food Analytical Methods, 5,702–709.

Shen, C. C., Chang, Y. S., & Ho, L. K. (1993). Nuclear magnetic resonance studies of5,7-dihydroxyflavonoids. Phytochemistry, 34, 843–845.

Sinz, A., Matusch, R., Santisuk, T., Suttiporn Chaichana, S., & Vichai Reutrakul, V.(1998). Flavonol glycosides from Dasymaschalon sootepense. Phytochemistry, 47,71393–71396.

Takahama, U., & Hirota, S. (2000). Deglucosidation of quercetin glucosides to theaglycone and formation of antifungal agents by peroxidase-dependentoxidation of quercetin on browning of onion scales. Plant and Cell Physiology,41, 1021–1029.

Takahama, U., & Hirota, S. (2010). Fatty acids, epicathchin-dimethylgallate, andrutin interact with buckwheat starch inhibiting its digestion by amylase:implication for the decrease in glycemic index by buckwheat flour. Journal ofAgricultural and Food Chemistry, 58, 12431–12439.

Wrolstad, R. E., Durst, R. W., & Lee, J. (2005). Tracking color and pigment changes inanthocyanin products. Trends in Food Science and Technology, 16, 423–428.

288 U. Takahama et al. / Food Chemistry 141 (2013) 282–288

Yanase, E., Nishimoto, K., Kamatari, Y. O., & Nakatsuka, S.-I. (2012). Isolation of anew xanthylium-related pigment from adzuki beans Vigna angularis. Bioscience,Biotechnology, and Bioscience, 76, 1571–1572.

Yoshida, K., Kondo, T., Ito, M., & Kondo, T. (2005). Analysis of polyphenols in waterextract of red adzuki bean, Vigna angularis. ITE Letters, 6, 226–231.

Yoshida, K., Sato, Y., Okuno, R., Kameda, K., Isobe, M., & Kondo, T. (1996). Structuralanalysis and measurement of anthocyanins from colored seed coats of Vigna,Phaseolus, and Glycine legumies. Bioscience, Biotechnology, and Bioscience, 60,589–593.